• The Interactive Fly

    Genes regulating behavior

    Behavioral paradigms

  • Major areas of study
  • Additional areas of research

    The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3 Drosophila larvae

    Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the Drosophila larva is simple enough to be tractable, yet complex enough to be interesting. It features about 10,000 neurons and is capable of various taxes, kineses and Pavlovian conditioning. All its neurons are currently being mapped into a light-microscopical atlas, and Gal4 strains are being generated to experimentally access neurons one at a time. In addition, an electron microscopic reconstruction of its nervous system seems within reach. Notably, this electron microscope-based connectome is being drafted for a stage 1 larva. This study undertook a survey the behaviour of stage 1 larvae. In a community-based approach called the Ol1mpiad, stage 1 Drosophila larvae were probed for free locomotion, feeding, responsiveness to substrate vibration, gentle and nociceptive touch, burrowing, olfactory preference and thermotaxis, light avoidance, gustatory choice of various tastants plus odour-taste associative learning, as well as light/dark-electric shock associative learning (see Artificial Intelligence Helps Build Brain Atlas of Fly Behavior). Quantitatively, stage 1 larvae show lower scores in most tasks, arguably because of their smaller size and lower speed. Qualitatively, however, stage 1 larvae perform strikingly similar to stage 3 larvae in almost all cases. These results bolster confidence in mapping brain structure and behaviour across developmental stages (Almeida-Carvalho, 2017).

    Action-based attention in Drosophila melanogaster

    The mechanism of action selection is a widely shared fundamental process required by animals to interact with the environment and adapt to it. A key step in this process is the filtering of many "distracting" sensory inputs which may disturb action selection. Because it has been suggested that, beyond sharing common mechanisms, action selection may also be processed by shared circuits in vertebrates and invertebrates, it was asked whether invertebrates showed the ability to filter out "distracting" stimuli to maintain a goal-directed action, as seen in vertebrates. In this experiment action selection was studied in wild-type Drosophila melanogaster, by investigating their reaction to the abrupt appearance of a visual distractor during an ongoing locomotor action directed to a specific visual target. Flies tended to shift the original trajectory towards the distractor, thus acknowledging it's presence, but did not appear to commit to it, suggesting that an inhibition process took place in order to continue to carry out the original goal-directed action. To some extent flies appeared to take into account the level of salience of the abrupt distractor appearance as a basis for the ensuing motor program. However, they did not engage in a complete change in their initial motor program in favour of the distractor. These results provide interesting insights into the selection-for-action mechanism, in a context requiring action-centered attention which might have appeared rather early in the course of evolution (Frighetto, 2019).

    Genetics of cocaine and methamphetamine consumption and preference in Drosophila melanogaster

    Illicit use of psychostimulants, such as cocaine and methamphetamine, constitutes a significant public health problem. Whereas neural mechanisms that mediate the effects of these drugs are well-characterized, genetic factors that account for individual variation in susceptibility to substance abuse and addiction remain largely unknown. Drosophila melanogaster can serve as a translational model for studies on substance abuse, since flies have a dopamine transporter that can bind cocaine and methamphetamine, and exposure to these compounds elicits effects similar to those observed in people, suggesting conserved evolutionary mechanisms underlying drug responses. This study used the D. melanogaster Genetic Reference Panel to investigate the genetic basis for variation in psychostimulant drug consumption, to determine whether similar or distinct genetic networks underlie variation in consumption of cocaine and methamphetamine, and to assess the extent of sexual dimorphism and effect of genetic context on variation in voluntary drug consumption. Quantification of natural genetic variation in voluntary consumption, preference, and change in consumption and preference over time for cocaine and methamphetamine uncovered significant genetic variation for all traits, including sex-, exposure- and drug-specific genetic variation. Genome wide association analyses identified both shared and drug-specific candidate genes, which could be integrated in genetic interaction networks. The effects were assessed of ubiquitous RNA interference (RNAi) on consumption behaviors for 34 candidate genes: all affected at least one behavior. Finally, RNAi knockdown in the nervous system was used to implicate dopaminergic neurons and the mushroom bodies as part of the neural circuitry underlying experience-dependent development of drug preference (Highfill, 2019).

    Single serotonergic neurons that modulate aggression in Drosophila

    Monoamine serotonin (5HT) has been linked to aggression for many years across species. However, elaboration of the neurochemical pathways that govern aggression has proven difficult because monoaminergic neurons also regulate other behaviors. There are approximately 100 serotonergic neurons in the Drosophila nervous system, and they influence sleep, circadian rhythms, memory, and courtship. In the Drosophila model of aggression, the acute shut down of the entire serotonergic system yields flies that fight less, whereas induced activation of 5HT neurons promotes aggression. Using intersectional genetics, the population of 5HT neurons that can be reproducibly manipulated were restricted to identify those that modulate aggression. Although similar approaches were used recently to find aggression-modulating dopaminergic and Fru(M)-positive peptidergic neurons, the downstream anatomical targets of the neurons that make up aggression-controlling circuits remain poorly understood. This study identified a symmetrical pair of serotonergic PLP neurons (5HT-PLP neurons) that are necessary for the proper escalation of aggression. Silencing these neurons reduced aggression in male flies, and activating them increased aggression in male flies. GFP reconstitution across synaptic partners (GRASP) analyses suggested that 5HT-PLP neurons form contacts with 5HT1A receptor-expressing neurons in two distinct anatomical regions of the brain. Activation of these 5HT1A receptor-expressing neurons, in turn, caused reductions in aggression. These studies, therefore, suggest that aggression may be held in check, at least in part, by inhibitory input from 5HT1A receptor-bearing neurons, which can be released by activation of the 5HT-PLP neurons (Alekseyenko, 2014).

    Displays of appropriate levels of aggression rely on the ability of an animal to analyze many factors, including the following: the correct identification and evaluation of the abilities of potential competitors; the evaluation of the value of a territory and the likelihood of acquiring it; and the physiological state of the animal. Multiple sensory systems and circuits will be utilized in making such evaluations. The fixed number of neurons and neuronal circuits in nervous systems might limit the abilities of an animal to evaluate such a multiplicity of factors, but great flexibility is introduced into the system by the availability of neuromodulators. These have the capability of rapidly, efficiently, and reversibly reconfiguring the networks of neurons without changing the 'hardwiring.' The current studies illustrate the modulation by 1-2 pairs of serotonergic neurons that enhance aggression. Other modulatory neurons and systems that influence aggression have been identified previously in Drosophila, including dopaminergic neurons, FruM-positive octopamine neurons that influence the behavioral choice between courtship and aggression, FruM-positive tachykinin neurons that enhance aggression, and neuropeptide F circuits that decrease aggression. The arbors of processes of the 5HT-PLP neurons examined in this study densely innervate several integrative centers in the fly brain, but thus far, they do not seem to overlap with the processes of the other reported aggression-influencing neuromodulatory neurons. The 5HT-PLP neurons do not coexpress FruM or Dsx. Thus, the modulatory control of the male-specific higher-level aggression appears to involve both sex-specific regulatory factors and other as-yet-unidentified control elements. The current studies further suggest that going to higher-intensity levels in fights may be held in check by inhibition, which can be released by activation of the 5HT-PLP neurons. Learning more about the neurons and neuronal circuits involved with a suggested downstream aggression-suppressing system and with the sensory systems that trigger aggression in the first place will be essential steps in further unraveling the complex circuitry that controls the release of aggression in Drosophila (Alekseyenko, 2014).

    In summary, using a Drosophila model system and an intersectional genetic strategy, this study identified a pair of serotonergic neurons in the PLP cluster that modulate aggressive behavior. These neurons arborize through several neuropil regions in the central brain, where they influence the escalation of aggression, at least in part, via 5HT1A receptor-bearing neurons and also independently influence locomotion and sleep. The single-cell resolution in identification of neuronal connections and explorations of their functions in behaving animals provides an entry point into unraveling the circuitry associated with complex behaviors like aggression (Alekseyenko, 2014).

    Aggression and discrimination among closely versus distantly related species of Drosophila

    Fighting between different species is widespread in the animal kingdom, yet this phenomenon has been relatively understudied in the field of aggression research. Particularly lacking are studies that test the effect of genetic distance, or relatedness, on aggressive behaviour between species. This study characterized male-male aggression within and between species of fruit flies across the Drosophila phylogeny. Male Drosophila are shown to discriminate between conspecifics and heterospecifics and show a bias for the target of aggression that depends on the genetic relatedness of opponent males. Specifically, males of closely related species treated conspecifics and heterospecifics equally, whereas males of distantly related species were overwhelmingly aggressive towards conspecifics. This is the first study to quantify aggression between Drosophila species and to establish a behavioural bias for aggression against conspecifics versus heterospecifics. The results suggest that future study of heterospecific aggression behaviour in Drosophila is warranted to investigate the degree to which these trends in aggression among species extend to broader behavioural, ecological and evolutionary contexts (Gupta, 2019).

    A small number of cholinergic neurons mediate hyperaggression in female Drosophila

    In the Drosophila model of aggression, males and females fight in same-sex pairings, but a wide disparity exists in the levels of aggression displayed by the 2 sexes. A screen of Drosophila Flylight Gal4 lines by driving expression of the gene coding for the temperature sensitive dTRPA1 channel, yielded a single line (GMR26E01-Gal4) displaying greatly enhanced aggression when thermoactivated. Targeted neurons were widely distributed throughout male and female nervous systems, but the enhanced aggression was seen only in females. No effects were seen on female mating behavior, general arousal, or male aggression. The enhancement was quantified by measuring fight patterns characteristic of female and male aggression and confirmed that the effect was female-specific. To reduce the numbers of neurons involved, an intersectional approach was used with a library of enhancer trap flp-recombinase lines. Several crosses reduced the populations of labeled neurons, but only 1 cross yielded a large reduction while maintaining the phenotype. Of particular interest was a small group (2 to 4 pairs) of neurons in the approximate position of the pC1 cluster important in governing male and female social behavior. Female brains have approximately 20 doublesex (dsx)-expressing neurons within pC1 clusters. Using dsx (FLP) instead of 357 (FLP) for the intersectional studies, it was found that the same 2 to 4 pairs of neurons likely were identified with both. These neurons were cholinergic and showed no immunostaining for other transmitter compounds. Blocking the activation of these neurons blocked the enhancement of aggression (Palavicino-Maggio, 2019).

    Dietary supplementation with the ketogenic diet metabolite beta-hydroxybutyrate ameliorates post-TBI aggression in young-adult male Drosophila

    Traumatic brain injury (TBI), caused by repeated concussive head trauma can induce chronic traumatic encephalopathy (CTE), a neurodegenerative disease featuring behavioral symptoms ranging from cognitive deficits to elevated aggression. In a Drosophila model, this study used a high-impact trauma device to induce TBI-like symptoms and to study post-TBI behavioral outcomes. Following TBI, aggression in banged male flies was significantly elevated as compared with that in unbanged flies. Various forms of dietary therapy, especially the high-fat, low-carbohydrate ketogenic diet (KD), have recently been explored for a wide variety of neuropathies. It is thus hypothesized that putatively neuroprotective dietary interventions might be able to suppress post-traumatic elevations in aggressive behavior in animals subjected to head-trauma-inducing strikes, or "bangs". A normal high-carbohydrate Drosophila diet was supplemented with the KD metabolite beta-hydroxybutyrate (beta-HB)-a ketone body (KB). Banged flies raised on a KB-supplemented diet exhibited a marked reduction in aggression, whereas aggression in unbanged flies was equivalent whether dieted with KB supplements or not. Pharmacological blockade of the ATP-sensitive potassium (KATP) channel abrogated KB effects reducing post-TBI aggression while pharmacological activation mimicked them, suggesting a mechanism by which KBs act in this model. KBs did not significantly extend lifespan in banged flies, but markedly extended lifespan in unbanged flies. This study has developed a functional model for the study of post-TBI elevations of aggression. Further, It is concluded that dietary interventions may be a fruitful avenue for further exploration of treatments for TBI- and CTE-related cognitive-behavioral symptoms (Lee, 2019).

    The neuropeptide Drosulfakinin regulates social isolation-induced aggression in Drosophila

    Social isolation strongly modulates behavior across the animal kingdom. This study utilized the fruit fly Drosophila melanogaster to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide Drosulfakinin (Dsk), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors. Dsk knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of Dsk neurons each similarly increased isolation-driven aggression. The results suggest a U-shaped dependence of social isolation-induced aggressive behavior on Dsk signaling, similar to the actions of many neuromodulators in other contexts (Agrawal, 2020).

    Alcohol potentiates a pheromone signal in flies

    For decades, numerous researchers have documented the presence of the fruit fly or Drosophila melanogaster on alcohol-containing food sources. Although fruit flies are a common laboratory model organism of choice, there is relatively little understood about the ethological relationship between flies and ethanol. This study finds that when male flies inhabit ethanol-containing food substrates they become more aggressive. A possible mechanism was identified for this behavior. The odor of ethanol potentiates the activity of sensory neurons in response to an aggression-promoting pheromone. Finally, it was observed that the odor of ethanol also promotes attraction to a food-related citrus odor. Understanding how flies interact with the complex natural environment they inhabit can provide valuable insight into how different natural stimuli are integrated to promote fundamental behaviors (Park, 2020).

    Serotonin Signals Overcome Loser Mentality in Drosophila

    Traumatic experiences generate stressful neurological effects in the exposed persons and animals. Previous studies have demonstrated that in many species, including Drosophila, the defeated animal has a higher probability of losing subsequent fights. However, the neural basis of this "loser effect" is largely unknown. This study reports that elevated serotonin (5-HT) signaling helps a loser to overcome suppressive neurological states. Coerced activation of 5-HT neurons increases aggression in males and promotes losers to both vigorously re-engage in fights and even defeat the previous winners and regain mating motivation. P1 neurons act upstream and 5-HT1B neurons in the ellipsoid body act downstream of 5-HT neurons to arouse losers. These results demonstrate an ancient neural mechanism of regulating depressive behavioral states after distressing events (Hu, 2020).

    Cell types and neuronal circuitry underlying female aggression in Drosophila

    Aggressive social interactions are used to compete for limited resources and are regulated by complex sensory cues and the organism's internal state. While both sexes exhibit aggression, its neuronal underpinnings are understudied in females. This study identified a population of sexually dimorphic aIPg neurons in the adult Drosophila melanogaster central brain whose optogenetic activation increased, and genetic inactivation reduced, female aggression. Analysis of GAL4 lines identified in an unbiased screen for increased female chasing behavior revealed the involvement of another sexually dimorphic neuron, pC1d, and implicated aIPg and pC1d neurons as core nodes regulating female aggression. Connectomic analysis demonstrated that aIPg neurons and pC1d are interconnected and suggest that aIPg neurons may exert part of their effect by gating the flow of visual information to descending neurons. This work reveals important regulatory components of the neuronal circuitry that underlies female aggressive social interactions and provides tools for their manipulation (Schretter, 2020).

    Social hierarchy is established and maintained with distinct acts of aggression in male Drosophila melanogaster

    Social interactions pivot on an animal's experiences, internal states and feedback from others. This complexity drives the need for precise descriptions of behavior to dissect the fine detail of its genetic and neural circuit bases. In laboratory assays, male Drosophila melanogaster reliably exhibit aggression, and its extent is generally measured by scoring lunges, a feature of aggression in which one male quickly thrusts onto his opponent. This study introduces an explicit approach to identify both the onset and reversals in hierarchical status between opponents and observe that distinct aggressive acts reproducibly precede, concur or follow the establishment of dominance. Lunges were found to be insufficient for establishing dominance. Rather, lunges appear to reflect the dominant state of a male and help in maintaining his social status. Lastly, this study characterized the recurring and escalating structure of aggression that emerges through subsequent reversals in dominance. Collectively, this work provides a framework for studying the complexity of agonistic interactions in male flies, enabling its neurogenetic basis to be understood with precision (Simon, 2020).

    A circuit logic for sexually shared and dimorphic aggressive behaviors in Drosophila

    Aggression involves both sexually monomorphic and dimorphic actions. How the brain implements these two types of actions is poorly understood. This study has identified three cell types that regulate aggression in Drosophila: one type is sexually shared, and the other two are sex specific. Shared common aggression-promoting (CAP) neurons mediate aggressive approach in both sexes, whereas functionally downstream dimorphic but homologous cell types, called male-specific aggression-promoting (MAP) neurons in males and fpC1 in females, control dimorphic attack. These symmetric circuits underlie the divergence of male and female aggressive behaviors, from their monomorphic appetitive/motivational to their dimorphic consummatory phases. The strength of the monomorphic → dimorphic functional connection is increased by social isolation in both sexes, suggesting that it may be a locus for isolation-dependent enhancement of aggression. Together, these findings reveal a circuit logic for the neural control of behaviors that include both sexually monomorphic and dimorphic actions, which may generalize to other organisms (Chiu, 2020).

    Long-Term Dietary Restriction Leads to Development of Alternative Fighting Strategies

    In competition for food, mates and territory, most animal species display aggressive behavior through visual threats and/or physical attacks. Such naturally-complex social behaviors have been shaped by evolution. Environmental pressure, such as the one imposed by dietary regimes, forces animals to adapt to specific conditions and ultimately to develop alternative behavioral strategies. The quality of the food resource during contests influence animals' aggression levels. However, little is known regarding the effects of a long-term dietary restriction-based environmental pressure on the development of alternative fighting strategies. To address this, two lines were employed of the wild-type Drosophila melanogaster Canton-S (CS)which originated from the same population but raised under two distinct diets for years. One diet contained both proteins and sugar, while the second one was sugar-free. Male-male aggression assays were set up using both CS lines; differences were found in aggression levels and the fighting strategies employed to establish dominance relationships. CS males raised on a sugar-containing diet started fights with a physical attack and employed a high number of lunges for establishing dominance but displayed few wing threats throughout the fight. In contrast, the sugar-free-raised males favored wing threats as an initial aggressive demonstration and used fewer lunges to establish dominance, but displayed a higher number of wing threats. This study demonstrates that fruit flies that have been raised under different dietary conditions have adapted their patterns of aggressive behavior and developed distinct fighting strategies: one favoring physical attacks, while the other one favoring visual threats (Legros, 2020).

    Masculinized Drosophila females adapt their fighting strategies to their opponent

    Many animal species show aggression to gain mating partners and to protect territories and other resources from competitors. Both male and female fruit flies of the species Drosophila melanogaster exhibit aggression in same-sex pairings, but the strategies used are sexually dimorphic. The biological basis for the differing aggression strategies, and the cues promoting one form of aggression over the other, are being explored. This study describes a line of genetically masculinized females that switch between male and female aggression patterns based on the sexual identity of their opponents. When these masculinized females are paired with more aggressive opponents, they increase the amount of male-like aggression they use, but do not alter the level of female aggression. This suggests that male aggression may be more highly responsive to behavioral cues than female aggression. Although the masculinized females of this line show opponent-dependent changes in aggression and courtship behavior, locomotor activity and sleep are unaffected. Thus, the driver line used may specifically masculinize neurons involved in social behavior. A discussion of possible different roles of male and female aggression in fruit flies is included in this paper. These results can serve as precursors to future experiments aimed at elucidating the circuitry and triggering cues underlying sexually dimorphic aggressive behavior (Monyak, 2021).

    Sex ratio and the evolution of aggression in fruit flies

    Aggressive behaviours are among the most striking displayed by animals, and aggression strongly impacts fitness in many species. Aggression varies plastically in response to the social environment, but direct tests of how aggression evolves in response to intra-sexual competition are lacking. This study investigated how aggression in both sexes evolves in response to the competitive environment, using populations of Drosophila melanogaster that were experimentally evolved under female-biased, equal, and male-biased sex ratios. After evolution in a female-biased environment-with less male competition for mates-males fought less often on food patches, although the total frequency and duration of aggressive behaviour did not change. In females, evolution in a female-biased environment-where female competition for resources is higher-resulted in more frequent aggressive interactions among mated females, along with a greater increase in post-mating aggression. These changes in female aggression could not be attributed solely to evolution either in females or in male stimulation of female aggression, suggesting that coevolved interactions between the sexes determine female post-mating aggression. Evidence was found consistent with a positive genetic correlation for aggression between males and females, suggesting a shared genetic basis. This study demonstrates the experimental evolution of a behaviour strongly linked to fitness, and the potential for the social environment to shape the evolution of contest behaviours (Bath, 2021).

    Temporal and genetic variation in female aggression after mating

    Aggression between individuals of the same sex is almost ubiquitous across the animal kingdom. Winners of intrasexual contests often garner considerable fitness benefits, through greater access to mates, food, or social dominance. In females, aggression is often tightly linked to reproduction, with females displaying increases in aggressive behavior when mated, gestating or lactating, or when protecting dependent offspring. In the fruit fly, Drosophila melanogaster, females spend twice as long fighting over food after mating as when they are virgins. However, it is unknown when this increase in aggression begins or whether it is consistent across genotypes. This study shows that aggression in females increases between 2 to 4 hours after mating and remains elevated for at least a week after a single mating. In addition, this increase in aggression 24 hours after mating is consistent across three diverse genotypes, suggesting this may be a universal response to mating in the species. This study also reports the first use of automated tracking and classification software to study female aggression in Drosophila and assess its accuracy for this behavior. Dissecting the genetic diversity and temporal patterns of female aggression assists in better understanding its generality and adaptive function, and will facilitate the identification of its underlying mechanisms (Bath, 2020).

    Gut microbiome modulates Drosophila aggression through octopamine signaling

    Gut microbiome profoundly affects many aspects of host physiology and behaviors. This study reports that gut microbiome modulates aggressive behaviors in Drosophila. Germ-free males showed substantial decrease in inter-male aggression, which could be rescued by microbial re-colonization. These germ-free males are not as competitive as wild-type males for mating with females, although they displayed regular levels of locomotor and courtship behaviors. it was further found that Drosophila microbiome interacted with diet during a critical developmental period for the proper expression of octopamine and manifestation of aggression in adult males. These findings provide insights into how gut microbiome modulates specific host behaviors through interaction with diet during development (Jia, 2021).

    A circuit node that integrates convergent input from neuromodulatory and social behavior-promoting neurons to control aggression in Drosophila

    Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. A systematic, unbiased screen identified OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. The results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM(+) neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals (Watanabe, 2017).

    A rich behavioral literature has implicated OA in the control of invertebrate aggression, although the direction of its effects differs between species. Classic studies in lobsters have shown that injection of OA into the hemolymph promotes a subordinate-like posture, while injection of serotonin (5HT) produces a dominant-like posture. In contrast, hemolymph injections of OA in crickets restore aggressiveness to subordinated animals, mimicking the arousing effects of episodes of free flight. OA has also been suggested to play a role in aggressive motivation restored to defeated crickets by residency in a shelter. In Drosophila, null mutations of TβH strongly suppressed aggressiveness, suggesting a positive-acting role for OA in flies as in crickets. Interestingly, intra-hypothalamic infusion of NE in mammals can also enhance aggression. However, little is known about the neurons on which these amines act directly to influence aggression, in any organism (Watanabe, 2017).

    This study applied a novel, unbiased approach to identify OARNs relevant to aggression in Drosophila. Importantly this screen was based not on mutations in OAR genes, but rather upon genetic silencing of neurons that express GAL4 under the control of different OAR gene cis-regulatory modules (CRMs). This screen was agnostic with respect to which OAR gene is involved, or in which neurons that OAR is expressed. It yielded a small population of male-specific, FruM+ OA-sensitive neurons, called aSP2, the activity of which is required for normal levels of aggressiveness. No significant change in UWEs (male-male courtship) was observed when these neurons were activated or silenced. Nevertheless, neuronal silencing in the parental R47A04-GAL4 line increased male-male courtship, perhaps reflecting an inhibitory role for non-aSP2 neurons in that line. Therefore, while it is not possible to completely exclude a role for aSP2 neurons to suppress male-male courtship, the evidence does not strongly support it (Watanabe, 2017).

    Multiple lines of evidence suggest that R47A04aSP2 neurons are indeed OA responsive, likely via OAMB. First, these neurons are labeled by a CRM from the Oamb gene. Second, RNAi-mediated knockdown of Oamb in R47A04 neurons reduced aggression, phenocopying the effects of an Oamb null allele. (However, knockdown using the split-GAL4 R47A04aSP2driver only yielded a trend to reduced aggression that did not reach significance, perhaps reflecting a floor effect in this assay.) Third, overexpression of Oamb cDNAs in these neurons using R47A04-GAL4 rescued the Oamb null mutant and enhanced the effect of OA feeding to promote aggression. Fourth,R47A04aSP2 neurons were activated by bath-applied OA in brain explants, and this effect was also blocked by RNAi-mediated knockdown of Oamb. Taken together, these data strongly suggest that aSP2 neurons respond directly to OA to mediate its effects on aggression, although they do not exclude a role for other OA-responsive non-aSP2 neurons in line R47A04. While it was not possible to definitively establish which of the 27 different classes of OANs in Drosophila provide functional OA input to aSP2 cells, some candidate OA neurons labeled in retrograde PA-GFP experiments (VUM and VPM) have previously been implicated in aggression (Watanabe, 2017).

    In Drosophila OA, like NE in vertebrates, is thought to promote arousal. Consistent with such a function, OAergic fibers are broadly distributed across the entire Drosophila CNS, as are NE fibers in vertebrates. Thus OARNs could enhance aggression by increasing arousal, and there is evidence for such a function in crickets. However, manipulations of R47A04aSP2neurons that increased or decreased aggression did not affect locomotion, circadian activity, or sleep. This suggests that these neurons influence aggression directly and specifically, rather than by increasing generalized arousal. Other classes of OARNs not investigated in this study have been implicated in sleep-wake arousal (Watanabe, 2017).

    Does OA promote aggression in a permissive or instructive manner? While it is clear that OA synthesis and release are required for aggression in Drosophila, whether increasing OA suffices to promote aggression is less clear. It was reported that NaCh Bac-mediated activation of Tdc2-GAL4 neurons enhanced aggression, but the current study neither this manipulation, nor activation of Tdc2 neurons using dTrpA1 or Chrimson, yielded consistent effects. Thus, while OA is essential for normal levels of aggression, it is not clear whether it plays an instructive role to promote this behavior (Watanabe, 2017).

    In principle, OA RNs could act directly in command-like neurons that mediate aggression, or rather in cells that play a modulatory role. It was found that aggression was increased by tonically enhancing the excitability of R47A04aSP2 neurons using NaChBac, but not by phasically activating them optogenetically, arguing against a command-like function. Furthermore, the influence of TK FruM neurons, which do promote aggression when phasically activated, was not dependent on the activity of R47A04aSP2 neurons, indicating that the latter are not functionally downstream of the former. Together, these data argue against a role for R47A04aSP2 cells as command-like neurons, or as direct outputs of command neurons, for aggression. Rather, these cells exert a modulatory influence on agonistic behavior (Watanabe, 2017).

    In searching for neurons that may interact with R47A04aSP2 cells in their modulatory capacity, P1 neurons, a FruM+ population of 20 neurons/hemibrain was identified that controls male courtship, but which can also promote aggression when activated. It has been argued that this aggression-promoting effect is due to a subset of FruM neurons in the GAL4 line used in these studies, R71G01-GAL4. However, this study shows that conditional expression of FLP-ON Chrimson in a subset of neurons within the R71G01-GAL4 population using Fru-FLP. Nevertheless, these data do not exclude that the aggression-promoting neurons in the P1 cluster expressed Fru-FLP only transiently during development, nor do they exclude the possibility that different subpopulations of neurons within line R71G01 control courtship versus aggression; further studies will be required to resolve these issues (Watanabe, 2017).

    The P1 cluster is known to project to downstream cells that are specific for courtship . The present study provides the first evidence that cells in this cluster also functionally activate (and physically contact) aggression-specific neurons. However, due to limitations of the genetic reagents employed, it is not certain that the behavioral, physiological, and anatomical interactions with aSP2 cells demonstrated in this study are all mediated by the same subset of neurons in the P1 cluster. With this caveat in mind, these data suggest that aSP2 neurons are functionally downstream of both a subset(s) of P1 neurons, as well as of OA neurons (Watanabe, 2017).

    Feeding flies OA potentiated the activation of R47A04aSP2 neurons by P1 neuron stimulation, in brain explants. Furthermore, activation of aggression by P1 stimulation was enhanced and suppressed by pharmacologically increasing or decreasing OA signaling, respectively. While some off-target effects of the drugs, or an action on non-aSP2 neurons expressing OARs, cannot be excluded these pharmacologic effects were overridden by opposite-direction genetic manipulations of R47A04aSP2neuronal activity. Whether P1 neurons and OANs normally activate aSP2 neurons in vivo, simultaneously or sequentially, is not yet clear. Nevertheless it is striking that P1 and Tdc2 putative inputs occupy adjacent regions of aSP2 dendrites. Taken together, these findings suggest that aSP2 cells may serve as a node through which OA can bias output from a multifunctional social behavior network involving P1 neurons, in a manner that favors aggression. However, aSP2 neurons themselves do not appearto control directly the choice between mating and fighting (Watanabe, 2017).

    Male-specific cuticular hydrocarbons such as 7-tricosene (7-T) are known to be required for aggression. Interestingly, it has recently been shown that gustatory neurons expressing Gr32a, which encodes a putative 7-T receptor, innervate OANs in the SEZ; these OANs are activated by 7-T in a Gr32a-dependent manner. SEZ-innervating OANs include the VPM/VUM subsets seen in PA-GFP retrograde labeling experiments. These data raise the possibility that R47A04aSP2 neurons might be targets of VPM/VUM OANs activated by 7-T. If so, then they could provide a potential link between the influence of male-specific pheromones, OA, and central aggression circuitry. Studies of NE neurons in vertebrates have led to a prevailing view that this neuromodulator is released in a diffuse, 'sprinkler system'-like manner to control brain-wide states like arousal. Recent studies in Drosophila indicate that the broad, brain-wide distribution of OAergic fibers reflects the superposition of close to 30 anatomically distinct subclasses of OANs). The data presented in this study reveal a high level of circuit specificity for OARNs that mediate the effects of OA on aggression, mirroring the anatomical and functional specificity of OANs reported to control this behavior. If this anatomical logic is conserved, then such circuit specificity may underlie the actions of NE in mammals to a greater extent than is generally assumed (Watanabe, 2017).

    Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons

    Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both 'classical' and 'modulatory' signals that could produce diverse and/or complementary effects in associated circuits. This study establishes that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identifed the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. These findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network (Sherer, 2020).

    Addressing the functional complexities of 'one neuron, multiple transmitters' is critical to understanding how neuron communication, circuit computation, and behavior can be regulated by a single neuron. Over many decades, significant progress has been made elucidating the functional properties of neurons co-expressing neuropeptides and small molecule neurotransmitters, where the neuropeptide acts as a co-transmitter and modulates the action of the neurotransmitter. Only recently have studies begun to examine the functional significance of co-transmission by a fast-acting neurotransmitter and a slow-acting monoamine (Sherer, 2020).

    This study has demonstrated that OA neurons express dVGLUT and has utilized a new genetic tool to remove dVGLUT in OA-glutamate neurons. Quantifying changes in the complex social behaviors of aggression and courtship revealed that dVGLUT in brain OGNs is required to promote aggressive behavior and a specific behavioral pattern, the lunge. In contrast, males deficient for dVGLUT function do not exhibit an increase in inter-male courtship. These results establish a previously undetermined role for dVGLUT in OA neurons located in the adult brain and reveal glutamate uncouples aggression from inter-male courtship. It has been suggested that classical neurotransmitters and monoamines present in the same neuron modulate each other's packaging into synaptic vesicles or after release via autoreceptors. For example, a reduction of dVGLUT in DA-glutamate neurons resulted in decreased AMPH-stimulated hyperlocomotion in Drosophila and mice suggesting a key function of dVGLUT is the mediation of vesicular DA content. In this study, the independent behavioral changes suggests enhancing the packaging of OA into vesicles is not the sole function of dVGLUT co-expression and suggests differences in signaling by OA from OGNs on courtship-related circuitry (Sherer, 2020).

    Co-transmission can generate distinct circuit-level effects via multiple mechanisms. One mechanism includes spatial segregation; the release of two neurotransmitters or a neurotransmitter and monoamine from a single neuron occurring at different axon terminals or presynaptic zones. Recent studies examining this possible mechanism have described (1) the release of GLU and DA from different synaptic vesicles in midbrain dopamine neurons and (2) the presence of VMAT and VGLUT microdomains in a subset of rodent mesoaccumbens DA neurons. This study expressed a new conditionally expressed epitope-tagged version of VMAT in OGNs and visualized endogenous dVGLUT via antibody labeling. Within OGNs, the colocalization of VMAT and dVGLUT puncta was not complete suggesting the observed behavioral phenotype differences may be due to spatial differences in OA signaling (Sherer, 2020).

    A second mechanism by which co-transmission may generate unique functional properties relies on activating distinct postsynaptic receptors. In Drosophila, recent work has identified a small population of male-specific neurons that express the alpha-like adrenergic receptor, OAMB, as aggression-promoting circuit-level neuronal targets of OA modulation independent of any effect on arousal and separately knockdown of the Rdl GABAa receptor in a specific doublesex+ population stimulated male aggression (Watanabe, 2017). Future experiments identifying downstream targets that express both glutamate and octopamine receptors would be informative, as well as using additional split-Gal4 lines to determine if segregation of transporters is a hallmark of the majority of OGNs. Finally, a third possible mechanism is Glu may be co-released from OGNs and act on autoreceptors to regulate presynaptic OA release (Sherer, 2020).

    Deciphering the signaling complexity that allows neural networks to integrate external stimuli with internal states to generate context-appropriate social behavior is a challenging endeavor. Neuromodulators including monoamines are released to signal changes in an animal's environment and positively or negatively reinforce network output. In invertebrates, a role for OA in responding to external chemosensory cues as well as promoting aggression has been well-established. In terms of identifying specific aggression circuit-components that utilize OA, previous results determined OA neurons directly receive male-specific pheromone information and the aSP2 neurons serve as a hub through which OA can bias output from a multi-functional social behavior network towards aggression. The ability of OA to bias behavioral decisions based on positive and negative reinforcement was also recently described for food odors. In vertebrates, it has been proposed that DA-GLU cotransmission in the NAc medial shell might facilitate behavioral switching. The finding that the majority of OA neurons are glutamatergic, suggests that the complex social behavior of aggression may rely on small subsets of neurons that both signal the rapid temporal coding of critical external stimuli as well as the frequency coding of such stimuli resulting in the enhancement of this behavioral network. One implication of the finding regarding the separable OA-dependent inhibition of inter-male courtship is the possibility of identifying specific synapses or axon terminals that when activated gate two different behavioral outcomes. A second implication is that aggressive behavior in other systems may be modified by targeting GLU function in monoamine neurons (Sherer, 2020).

    Finally, monoamine-expressing neurons play key roles in human behavior including aggression and illnesses that have an aggressive component such as depression, addiction, anxiety, and Alzheimer's. While progress is being made in addressing the functional complexities of dual transmission, the possible pathological implications of glutamate co-release by monoamine neurons remains virtually unknown. Analyzing the synaptic vesicle and release properties of monoamine-glutamate neurons could offer new possibilities for therapeutic interventions aimed at controlling out-of-context aggression (Sherer, 2020).

    GABA transmission from mAL interneurons regulates aggression in Drosophila males

    Aggression is known to be regulated by pheromonal information in many species. But how central brain neurons processing this information modulate aggression is poorly understood. Using the fruit fly model of Drosophila melanogaster, this study systematically characterize the role of a group of sexually dimorphic GABAergic central brain neurons, popularly known as mAL, in aggression regulation. The mAL neurons are known to be activated by male and female pheromones. This report shows that mAL activation robustly increases aggression, whereas its inactivation decreases aggression and increases intermale courtship, a behavior considered reciprocal to aggression. GABA neurotransmission from mAL is crucial for this behavior regulation. Exploiting the genetic toolkit of the fruit fly model, a small group of approximately three to five GABA(+) central brain neurons were found with anatomical similarities to mAL. Activation of the mAL resembling group of neurons is necessary for increasing intermale aggression. Overall, these findings demonstrate how changes in activity of GABA(+) central brain neurons processing pheromonal information, such as mAL in Drosophila melanogaster, directly modulate the social behavior of aggression in male-male pairings (Sengupta, 2022).

    Mating pair drives aggressive behavior in female Drosophila.

    Aggression is an adaptive set of behaviors that allows animals to compete against one another in an environment of limited resources. Typically, males fight for mates and food, whereas females fight for food and nest sites. Although the study of male aggression has been facilitated by the extravagant nature of the ritualized displays involved and the remarkable armaments sported by males of many species the subtler and rarer instances of inter-female aggression have historically received much less attention. In Drosophila, females display high levels of complex and highly structured aggression on a food patch with conspecific females. Other contexts of female aggression have not been explored. Indeed, whether females compete for mating partners, as males do, has remained unknown so far. In the present work, it is reported that Drosophila melanogaster females reliably display aggression toward mating pairs. This aggressive behavior is regulated by mating status and perception of mating opportunities and relies heavily on olfaction. Furthermore, food odor in combination with OR47b-dependent fly odor sensing is required for proper expression of aggressive behavior. Taken together, this study describes a social context linked to reproduction in which Drosophila females aspiring to mate produce consistent and stereotyped displays of aggression. These findings open the door for further inquiries into the neural mechanisms that govern this behavior (Gaspar, 2002).

    Antibiotics increase aggression behavior and aggression-related pheromones and receptors in Drosophila melanogaster

    Aggression is a behavior common in most species; it is controlled by internal and external drivers, including hormones, environmental cues, and social interactions, and underlying pathways are understood in a broad range of species. To date, though, effects of gut microbiota on aggression in the context of gut-brain communication and social behavior have not been completely elucidated. This study examined how manipulation of Drosophila melanogaster microbiota affects aggression as well as the pathways that underlie the behavior in this species. Male flies treated with antibiotics exhibited significantly more aggressive behaviors. Furthermore, they had higher levels of cVA and (Z)-9 Tricosene, pheromones associated with aggression in flies, as well as higher expression of the relevant pheromone receptors and transporters OR67d, OR83b, GR32a, and LUSH. These findings suggest that aggressive behavior is, at least in part, mediated by bacterial species in flies (Grinberg, 2022)

    The genetic basis of variation in sexual aggression: Evolution versus social plasticity

    Male sexual aggression towards females is a form of sexual conflict that can result in increased fitness for males through forced copulations (FCs) or coercive matings at the cost of female lifetime fitness. This study used male fruit flies (Drosophila melanogaster) as a model system to uncover the genomic contributions to variation in FC, both due to standing variation in a wild population, and due to plastic changes associated with variation in social experience. RNAseq was used to analyse whole-transcriptome differential expression (DE) in male head tissue associated with evolved changes in FC from lineages previously selected for high and low FC rate and in male flies with varying FC rates due to social experience. Hundreds of genes were identified associated with evolved and plastic variation in FC, however only a small proportion (27 genes) showed consistent DE due to both modes of variation. This trend of low concordance in gene expression effects across broader sets of genes was confirmed to be significant in either the evolved or plastic analyses using multivariate approaches. The gene ontology terms neuropeptide hormone activity and serotonin receptor activity were significantly enriched in the set of significant genes. Of seven genes chosen for RNAi knockdown validation tests, knockdown of four genes showed the expected effect on FC behaviours. Taken together, these results provide important information about the apparently independent genetic architectures that underlie natural variation in sexual aggression due to evolution and plasticity (Scott, 2022).

    A Neural Circuit Controlling Virgin Female Aggression Induced by Mating-related Cues in Drosophila

    Females increase aggression for mating opportunities and for acquiring reproductive resources. Although the close relationship between female aggression and mating status is widely appreciated, whether and how female aggression is regulated by mating-related cues remains poorly understood. This study reports an interesting observation that Drosophila virgin females initiate high-frequency attacks toward mated females. 11-cis-vaccenyl acetate (cVA), a male-derived pheromone transferred to females during mating, was shown to promote virgin female aggression. A cVA-responsive neural circuit was subsequently reveal consisting of four orders of neurons, including Or67d, DA1, aSP-g, and pC1 neurons, that mediate cVA-induced virgin female aggression. It was also determined that aSP-g neurons release acetylcholine (ACh) to excite pC1 neurons via the nicotinic ACh receptor nAChRα7. Together, beyond revealing cVA as a mating-related inducer of virgin female aggression, these results identify a neural circuit linking the chemosensory perception of mating-related cues to aggressive behavior in Drosophila females (Wan, 2023).

    Drosophila tachykininergic neurons modulate the activity of two groups of receptor-expressing neurons to regulate aggressive tone

    Neuropeptides influence animal behaviors through complex molecular and cellular mechanisms, the physiological and behavioral effects of which are difficult to predict solely from synaptic connectivity. Many neuropeptides can activate multiple receptors, whose ligand affinity and downstream signaling cascades are often different from one another. Although it is known that the diverse pharmacological characteristics of neuropeptide receptors form the basis of unique neuromodulatory effects on distinct downstream cells, it remains unclear exactly how different receptors shape the downstream activity patterns triggered by a single neuronal neuropeptide source. This study uncovered two separate downstream targets that are differentially modulated by Tachykinin, an aggression-promoting neuropeptide in Drosophila. Tachykinin from a single male-specific neuronal type recruits two separate downstream groups of neurons. One downstream group, synaptically connected to the tachykinergic neurons, expresses the receptor TkR86C and is necessary for aggression. In this case, tachykinin supports cholinergic excitatory synaptic transmission between the tachykinergic and TkR86C downstream neurons. The other downstream group expresses the TkR99D receptor and is recruited primarily when tachykinin is over-expressed in the source neurons. Differential activity patterns in the two groups of downstream neurons correlate with levels of male aggression triggered by the tachykininergic neurons. These findings highlight how the amount of neuropeptide released from a small number of neurons can reshape the activity patterns of multiple downstream neuronal populations. The results lay the foundation for further investigations into the neurophysiological mechanism by which a neuropeptide controls complex behaviors (Wohl, 2023).

    Inbreeding-Driven Innate Behavioral Changes in Drosophila melanogaster

    Drosophila melanogaster has long been used to demonstrate the effect of inbreeding, particularly in relation to reproductive fitness and stress tolerance. In comparison, less attention has been given to exploring the influence of inbreeding on the innate behavior of D. melanogaster. In this study, multiple replicates of six different types of crosses were set in pair conformation of the laboratory-maintained wild-type D. melanogaster. This resulted in progeny with six different levels of inbreeding coefficients. Larvae and adult flies of varied inbreeding coefficients were subjected to different behavioral assays. In addition to the expected inbreeding depression in the-egg to-adult viability, noticeable aberrations were observed in the crawling and phototaxis behaviors of larvae. Negative geotactic behavior as well as positive phototactic behavior of the flies were also found to be adversely affected with increasing levels of inbreeding. Interestingly, positively phototactic inbred flies demonstrated improved learning compared to outbred flies, potentially the consequence of purging. Flies with higher levels of inbreeding exhibited a delay in the manifestation of aggression and courtship. In summary, these findings demonstrate that inbreeding influences the innate behaviors in D. melanogaster, which in turn may affect the overall biological fitness of the flies (Amanullah, 2023).

    Drosophila mutants lacking the glial neurotransmitter-modifying enzyme Ebony exhibit low neurotransmitter levels and altered behavior

    Inhibitors of enzymes that inactivate amine neurotransmitters (dopamine, serotonin), such as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), are thought to increase neurotransmitter levels and are widely used to treat Parkinson's disease and psychiatric disorders, yet the role of these enzymes in regulating behavior remains unclear. This study investigated the genetic loss of a similar enzyme in the model organism Drosophila melanogaster. Because the enzyme Ebony modifies and inactivates amine neurotransmitters, its loss is assumed to increase neurotransmitter levels, increasing behaviors such as aggression and courtship and decreasing sleep. This study quantitatively confirmed that ebony mutants exhibited increased aggressive behaviors such as boxing but also decreased courtship behaviors and increased sleep. Through tissue-specific knockdown, the role of ebony in these behaviors was found to be specific to glia. Unexpectedly, direct measurement of amine neurotransmitters in ebony brains revealed that their levels were not increased but reduced. Thus, increased aggression is the anomalous behavior for this neurotransmitter profile. It was further found that ebony mutants exhibited increased aggression only when fighting each other, not when fighting wild-type controls. Moreover, fights between ebony mutants were less likely to end with a clear winner than fights between controls or fights between ebony mutants and controls. In ebony vs. control fights, ebony mutants were more likely to win. Together, these results suggest that ebony mutants exhibit prolonged aggressive behavior only in a specific context, with an equally dominant opponent (Pantalia, 2023).

    Genomic regions influencing aggressive behavior in honey bees are defined by colony allele frequencies

    For social animals, the genotypes of group members affect the social environment, and thus individual behavior, often indirectly. This study used genome-wide association studies (GWAS) to determine the influence of individual vs. group genotypes on aggression in honey bees. Aggression in honey bees arises from the coordinated actions of colony members, primarily nonreproductive "soldier" bees, and thus, experiences evolutionary selection at the colony level. This study shows that individual behavior is influenced by colony environment, which in turn, is shaped by allele frequency within colonies. Using a population with a range of aggression, individual whole genomes were sequenced and for genotype-behavior associations were looked for within colonies in a common environment. There were no significant correlations between individual aggression and specific alleles. By contrast, strong correlations were found between colony aggression and the frequencies of specific alleles within colonies, despite a small number of colonies. Associations at the colony level were highly significant and were very similar among both soldiers and foragers, but they covaried with one another. One strongly significant association peak, containing an ortholog of the Drosophila sensory gene dpr4 (see Dips and Dprs) on linkage group (chromosome) 7, showed strong signals of both selection and admixture during the evolution of gentleness in a honey bee population. Links were thus found between colony genetics and group behavior and also, molecular evidence was found for group-level selection, acting at the colony level. It is concluded that group genetics dominates individual genetics in determining the fatal decision of honey bees to sting (Avalos, 2020).

    A neuropeptide regulates fighting behavior in Drosophila melanogaster

    Aggressive behavior is regulated by various neuromodulators such as neuropeptides and biogenic amines. This study found that the neuropeptide Drosulfakinin (Dsk) modulates aggression in Drosophila melanogaster. Knock-out of Dsk or Dsk receptor CCKLR-17D1 reduced aggression. Activation and inactivation of Dsk-expressing neurons increased and decreased male aggressive behavior, respectively. Moreover, data from transsynaptic tracing, electrophysiology and behavioral epistasis reveal that Dsk-expressing neurons function downstream of a subset of P1 neurons (P1(a)-splitGAL4) to control fighting behavior. In addition, winners show increased calcium activity in Dsk-expressing neurons. Conditional overexpression of Dsk promotes social dominance, suggesting a positive correlation between Dsk signaling and winning effects. The mammalian ortholog CCK has been implicated in mammal aggression, thus this work suggests a conserved neuromodulatory system for the modulation of aggressive behavior (Wu, 2020).

    Aggression is a common innate behavior in most vertebrate and invertebrate species and a major driving force for natural and sexual selections. It is a critical behavior for defense against conspecifics to obtain food resources and mating partners (Wu, 2020).

    Aggressive behavior of fruit flies was first reported by Alfred Sturtevant. Since then, a number of ethological and behavioral studies in flies pave the way for using Drosophila as a genetic system to study aggression. Drosophila provides an excellent system to manipulate genes and genetically defined populations of neurons, leading to the identification of multiple genes and neural circuits that control aggression. The neural circuits of aggression involve the peripheral sensory systems that detect male-specific pheromones and auditory cues necessary for aggression, a subset of P1 neurons, pCd in the central brain controlling aggressive arousal, and AIP neurons controlling threat displays. Aggression is modulated by various monoamines and neuropeptides. Octopamine, serotonin and dopamine are important neuromodulators for fly aggression and the specific aminergic neurons that control aggression have been identified. Neuropeptides such as tachykinin and neuropeptide F are required for normal male aggression. Cholecystokinin (CCK) is a neuropeptide that is linked to a number of psychiatric disorders and involved in various emotional behaviors in humans and other mammals. Infusion of CCK induces panic attack in humans. Enhanced CCK level is detected in a rat model of social defeat. CCK is implicated to act in the periaqueductal gray to potentiate defensive rage behavior in cats. In addition, CCK is a satiety signal in a number of species. Silencing CCK-like peptide Drosulfakinin could decrease satiety signaling and increase intake of food in flies. Co-injection of nesfatin-1 and CCK8 decreased food intake in Siberian sturgeon (Acipenser baerii) (Wu, 2020).

    This study investigated the roles of cholecysokinin-like peptide Drosulfakinin (Dsk) in Drosophila aggression. Knock-outs and GAL4 knock-ins were generated for Dsk and candidate Dsk receptors. Loss-of-function in either Dsk or Dsk receptor CCKLR-17D1 reduces aggression. Thermogenetic activation of DskGAL4 neurons promotes aggression, while silencing these neurons suppresses aggression. Transsynaptic tracing, electrophysiology and behavioral epistasis experiments were performed to illustrate that Dsk-expressing neurons are functionally connected with a subset of P1 neurons (P1a-splitGAL4, 8 ~ 10 pairs of P1 Neurons) and act downstream of a subset of P1 neurons to control fighting behavior. Furthermore, this study found that winners show increased calcium activity in Dsk-expressing neurons and that conditional overexpression of Dsk promotes winning effects, implicating an important role of the Dsk system in the establishment of social hierarchy during fly fighting. Previously the mamalian ortholog CCK has been implicated in aggression, thus this work suggests a potentially conserved neural pathway for the modulation of aggressive behavior (Wu, 2020).

    This study has systematically dissected the neuromodulatory roles of the Dsk system in fly aggression. At the molecular level, Dsk neuropeptide and its receptor CCKLR-17D1 are important for fly aggression. At the circuit level, Dsk-expressing neurons function downstream of a subset of P1 neurons (P1a-splitGAL4, 8 ~ 10 pairs of P1 Neurons) to control aggression. Furthermore, winners show increased calcium activity of Dsk-expressing neurons. Conditional overexpression of Dsk promotes winner effects, suggesting that Dsk is closely linked to the establishment of dominance. Taken together, these results elucidate the molecular and circuit mechanism underlying male aggression and suggest that cholecystokinin-like neuropeptide is likely to be evolutionarily conserved for the neuromodulation of aggression (Wu, 2020).

    A neural circuitry controlling aggression should be composed of multiple modules that extend from sensory inputs to motor outputs. A variety of peptidergic and aminergic neurons are implicated in fly aggression, but it is not clear how these modulatory neurons integrate input signals from other neural circuits to signal specific physiological states. The current data from circuit tracing, functional connectivity and behavioral epistasis suggest that Dsk-expressing neurons function downstream of a subset of P1 neurons and likely summate inputs from a subset of P1 neurons to signal an internal state of aggression. Activation of a subset of P1 neurons triggers both aggression and courtship. Interestingly, while the aggression-promoting effect of activating a subset of P1 neurons is dramatically suppressed by the loss of the Dsk gene, the courtship-promoting effect remains intact in the ΔDsk mutant background. On the other hand, recent study suggested that Dsk neurons might function to antagonize P1 neurons on regulating male courtship. This dissociation suggests that while a subset of P1 neurons signal an arousal state facilitating both aggression and courtship, the Dsk system acts downstream of a subset of P1 neurons specifically required for aggression. It worth mentioning that the P1a-splitGAL4 used in those studies not only labeled a small subset of Fru+ neurons but also several Fru- neurons, and previous study on pC1 neurons suggested that Fru+ pC1 neurons promote courtship and Fru- pC1 neurons promote aggression, so further studies are needed to characterize whether different subset of P1a-splitGAL4 labeled neurons are function differently on aggression and how Dsk system are involved. In addition, it remains unknown whether the Dsk system is responsible for integrating the sensory inputs and arousal state related to aggression, and how it connects to other components of the aggression circuitry, such as Tk neurons and AIP neurons (Wu, 2020).

    As a caveat, it has been reported that Dsk is involved in feeding behavior. The current experiment also reproduced the result that ΔDsk mutants show increased food consumption in the Capillary Feeder (CAFE) essay. Previous studies reported a positive correlation between the body size of flies and the aggression level, suggesting that the modulational effects of DSK neurons on aggression and feeding can be separated. Further research is required to disentangle the relationship between DSK neurons modulating aggression and those regulating feeding (Wu, 2020).

    This study classified the eight DSK neurons into three subtypes (Type I, II and III) based on the morphology of the neurites or two subtypes (DSK-M and DSK-L) based on the location of the cell bodies. Interestingly, these subtypes also show functional difference in modulating aggression and differential connectivity with the a subset of P1 neurons. Note that Type I and II neurons correspond to DSK-M and Type III neurons correspond to DSK-L. The finding that DSK-M neurons showed stronger responses to a subset of P1 neurons activation is consistent with the behavioral results of the flip-out experiment, in which Type I and II neurons, but not Type III, are critical to aggression. In future research, it would be interesting to use intersect method to more specifically label and manipulate the DSK neuron subtypes (Wu, 2020).

    Previous study implicated that the cholecystokinin system is closely linked with various human psychiatric disorders, such as bipolar disorder and panic attacks. Interestingly, verbal aggression is promoted by the administration of cholecystokinin tetrapeptide in human subjects. In cats, cholecystokinin agonists potentiate the defensive rage behavior while the cholecystokinin antagonists suppress it. These results reveal that cholecystokinin-like peptide Dsk and Dsk receptor CCKLR-17D1 are important for Drosophila aggression. In addition, increased calcium activity in Dsk-expressing neurons coincides with winner states. Thus, the cholecystokinin system is linked to aggressive behavior in a variety of species and is likely to be an evolutionarily conserved pathway for modulating aggressiveness (Wu, 2020).

    It has long been noticed that hierarchical relationships could be established during fly fights, with winners remaining highly aggressive and winning the subsequent encounters, and losers retreating and losing second fights. The winner state is perceived as a reward signal while losing experience is aversive (Kim, 2018). The establishment of social hierarchy is only observed in males, and this male-specific feature of fly aggression is specified by fruitless. However, neural correlates of dominance have not been reported. In this study, Using a transcriptional reporter of intracellular calcium (TRIC), it was found that winners display increased calcium activity in the median Dsk-expressing neurons. Moreover, conditional overexpression of Dsk specifically in the adult stage increases the flies' aggressiveness and makes them more likely to win against opponents without Dsk overexpression. Thus, both the enhanced Dsk signaling in the brain and the winning-promoting effect of conditional overexpression supported that the Dsk system may be involved in the establishment of social hierarchy during fly aggression (Wu, 2020).

    The neuropeptide Drosulfakinin regulates social isolation-induced aggression in Drosophila

    Social isolation strongly modulates behavior across the animal kingdom. This study utilized the fruit fly Drosophila melanogaster to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide Drosulfakinin (Dsk), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors. Dsk knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of Dsk neurons each similarly increased isolation-driven aggression. The results suggest a U-shaped dependence of social isolation-induced aggressive behavior on Dsk signaling, similar to the actions of many neuromodulators in other contexts (Agrawal, 2020).

    This study has shown that knockdown of the neuropeptide Dsk or its receptor CCKLR-17D1 in the pars intercerebralis (PI) increases social isolation-driven aggression of male flies. Moreover, Dsk appears to act in a U-shaped fashion, with both knockdown (the current results) and overexpression (Williams, 2014) increasing aggression. Dsk neuronal activity follows a similar trend, with both activation and silencing increasing aggression. Williams (2014) overexpressed the Dsk transcript in the PI region, which resulted in increased aggression; furthermore, activation of PI neurons was also shown to increase aggression in a separate study. Taken together, this suggests that the primary role of these neurons in this context is indeed production and secretion of Dsk. Transcription factors in the fly PI neurons regulating aggression were recently identified, and it was shown that activation of PI neurons increases aggression. However, the downstream neuropeptides were not known. The current findings identify Dsk as a key neuropeptide expressed in the PI region that regulates aggression. Further work will be required to delineate the aggression-modulating functions, if any, of other neuropeptides also secreted from the PI region (Agrawal, 2020).

    A recent neural activation screen (Asahina, 2014) explored the role of neuropeptides in aggression in Drosophila, but investigated only group-housed flies. Intriguingly, Asahina (2014) identified tachykinin signaling in the lateral protocerebrum and did not find increased aggression in group-housed (GH) flies upon activation of Dsk neurons. Thus, male-male aggression in GH and solitary-housed (SH) flies appears to be controlled by different neuropeptides in different brain regions. The absence of Dsk neurons from the screen results in GH flies (Asahina, 2014), combined with the results showing suppressed aggression in GH flies regardless of Dsk transcription or neural activity, suggests a mechanism that overrides Dsk function (Agrawal, 2020).

    Downregulation of the Dsk receptor CCKLR-17D1 in Dsk/Dilp2 neurons also increased aggression, consistent with the observation that some neuropeptidergic neurons, e.g. those for neuropeptide F, neuropeptide Y and FMRFamide, have receptors to modulate their signaling in an autocrine manner. However, pan-neuronal downregulation of CCKLR-17D1 receptor did not affect aggression, suggesting potential antagonistic effects outside Dsk/Dilp2 neurons (Agrawal, 2020).

    To address potential developmental effects of Dsk signaling, it would be useful to temporally restrict neural perturbation. However,efforts to conditionally silence Dsk+ neurons only in the adult using temperature-sensitive UAS-Kir2.1-GAL80ts were inconclusive, because prolonged exposure of flies (including controls) to the permissive temperature (30°C) affected their basal locomotion and aggression. To address potential off-target targets of the TRiP Dsk RNAi line, another RNAi line against Dsk (VDRC 14201) was tested but no significant reduction in Dsk levels were observed. It would be useful to test other Dsk loss-of-function alleles in future. However, the current conclusions about the involvement of Dsk in isolation-mediated aggression are supported by the similar effects from knockdown of its receptor CCKLR-17D1, as well as silencing and activation of Dsk-secreting neurons (Agrawal, 2020).

    The U-shaped ('hormetic') response of the aggression phenotype to both Dsk levels and Dsk+ neuronal activity is similar to such responses seen for NPF and dopamine neurons in Drosophila aggression. Such effects are not unexpected, given the ubiquity of such hormetic responses in neuromodulator signaling pathways and receptors in general. At the level of individual G-protein coupled receptors, such U-shaped responses (low-dose agonism, high-dose antagonism) arise directly from equations considering receptor expression level and the effects of receptor activation on downstream signaling pathways. At the circuit level, it is thought that such U-shaped responses help to maintain neuronal activity patterns, and the resulting behaviors, near homeostatic optima, with deviations resulting in negative feedback (Agrawal, 2020).

    There have been a number of prior studies on the genetic basis of aggression in Drosophila, many of them performed with DNA microarrays rather than with RNA-seq, that record counts for specific transcripts of interest. These studies counted all transcripts within cells. Four such studies have been performed in recent years, each identifying a large number of putative aggression-related genes. Given that the involvement of Dsk in aggression is quite context-specific. Asahina (2014) explicitly ruled out involvement of Dsk in aggression of group-housed flies. Therefore, it is perhaps unsurprising that it was not found in several of the screens. In fact, the only one of these four studies to uncover Dsk was the one that utilized socially isolated flies, strengthening the notion that Dsk specifically links social isolation to aggression. It was this link with social behavior that drew attention to Dsk, and indeed the current experiments bear out that this function is mediated through activity in the brain. The PI region has been shown to be the seat of regulation of many other social and sexually dimorphic behaviors (Agrawal, 2020).

    In mammals, the Dsk homologue cholecystokinin (CCK) and its receptors regulate aggression, anxiety and social-defeat responses. For instance, intravenous injection of the smallest isoform, CCK-4, in humans reliably induces panic attack and is often used to screen anxiolytic drug candidates. However, in other contexts, such as in mating and juvenile play, CCK encodes strong positive valence. CCK colocalizes with dopamine in the ventral striatum, and microinjection of CCK into the rat nucleus accumbens phenocopies the effects of dopamine agonists, increasing attention and reward-related behaviors, further supporting its role in positive valence encoding. CCK actions differ across brain regions, in a context-dependent manner. For instance, time pinned (negative valence) during rough-and-tumble play correlated with increased CCK levels in the posterior cortex and decreased levels in hypothalamus. However, lower hypothalamic CCK also correlated with positive-valence play aspects including dorsal contacts and 50 kHz ultrasonic vocalizations. Thus, CCK can encode both positive- and negative-valence aspects of complex behaviors differentially across the brain. As with many neuromodulators, CCK appears to act in a U-shaped fashion, with increases and decreases of signaling from baseline levels often producing similar phenotypes (Agrawal, 2020).

    Taken together, the results suggest an evolutionarily conserved role for neuropeptide signaling through the Drosulfakinin pathway (homologue of cholecystokinin) in promoting aggression. Intriguingly, this pathway only seems active in socially isolated flies; in socially enriched flies, aggression is controlled by tachykinin (a.k.a. Substance P) signaling. The PI region, in which the Dsk/Dilp2 neurons reside, has considerable similarities with the hypothalamus, a brain region crucial for regulating aggression in mammals, with the most relevant activity localized to the ventrolateral subdivision of the ventromedial hypothalamus, where CCK neurons reside. Thus, the predominant aggression-regulating mechanism in rodents bears strong homology to the fly pathway regulating aggression of socially deprived, but not socially enriched, individuals (Agrawal, 2020).

    Aggression and social experience: genetic analysis of visual circuit activity in the control of aggressiveness in Drosophila

    Animal aggressiveness is controlled by genetic and environmental factors. Among environmental factors, social experience plays an important role in modulating aggression in vertebrates and invertebrates. In Drosophila, pheromonal activation of olfactory neurons contributes to social suppression of aggression. While it was reported that impairment in vision decreases the level of aggression in Drosophila, it remains unknown if visual perception also contributes to the modulation of aggression by social experience. This study investigated the role of visual perception in the control of aggression in Drosophila. Several genetic approaches were taken to examine the effects of blocking visual circuit activity on fly aggressive behaviors. In wild type, group housing greatly suppresses aggressiveness. Loss of vision by mutating the ninaB gene does not affect social suppression of fly aggression. Similar suppression of aggressiveness by group housing is observed in fly mutants carrying a mutation in the eya gene leading to complete loss of eyes. Chronic visual loss does not affect the level of aggressiveness of single-housed flies that lack social experience prior to behavioral tests. When visual circuit activity is acutely blocked during behavioral test, however, single-housed flies display higher levels of aggressiveness than that of control flies. It is concluded that visual perception does not play a major role in social suppression of aggression in Drosophila. For single-housed individuals lacking social experience prior to behavioral tests, visual perception decreases the level of aggressiveness (Ramin, 2014).

    P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila

    How brains are hardwired to produce aggressive behavior, and how aggression circuits are related to those that mediate courtship, is not well understood. This large-scale screen for aggression-promoting neurons in Drosophila identifies several independent hits that enhance both inter-male aggression and courtship. Genetic intersections reveal that P1 interneurons, previously thought to exclusively control male courtship, are responsible for both phenotypes. The aggression phenotype is fly-intrinsic, and requires male-specific chemosensory cues on the opponent. Optogenetic experiments indicate that P1 activation promotes aggression vs. wing extension at low vs. high thresholds, respectively. High frequency photostimulation promotes wing extension and aggression in an inverse manner, during light ON and OFF, respectively. P1 activation enhances aggression by promoting a persistent internal state, which could endure for minutes prior to social contact. Thus P1 neurons promote an internal state that facilitates both aggression and courtship, and can control these social behaviors in a threshold-dependent manner (Hoopfer, 2015).

    This study describes the first large-scale neuronal activation screen for aggression neurons in Drosophila. Using the thermosensitive ion channel dTrpA1, a collection of over 3,000 GAL4 lines was screened for flies that exhibited increased fighting following thermogenetic neuronal activation. Among ~20 hits obtained, three exhibited both increased aggression and male-male courtship behavior. Intersectional refinement of expression patterns using split-GAL4 indicated that both social behaviors are controlled, in all three hits, by a subpopulation of ~8-10 P1 neurons per hemibrain. P1 cells are male-specific, FruM+ interneurons that integrate pheromonal and visual cues to promote male courtship behavior. The results indicate, surprisingly, that at least a subset of P1 neurons, previously thought to control exclusively courtship, can promote male aggression as well. Moreover, it was shown that they exert this influence by inducing a persistent fly-intrinsic state, lasting for minutes, that enhances these behaviors. These data define a sexually dimorphic neural circuit node that may link internal states to the control of mating and fighting, and identify a potentially conserved circuit 'motif' for the control of social behaviors (Hoopfer, 2015).

    Genetic variation in social environment construction influences the development of aggressive behavior in Drosophila melanogaster

    Individuals are not merely subject to their social environments; they choose and create them, through a process called social environment (or social niche) construction. This study identified multiple mechanisms of social environment construction that differ among natural genotypes of Drosophila melanogaster and investigated their consequences for the development of aggressive behavior. Male genotypes differed in the group sizes that they preferred and in their aggressive behavior; both of these behaviors influenced social experience, demonstrating that these behaviors function as social environment-constructing traits. Further, the effects of social experience-as determined in part by social environment construction-carried over to affect focal male aggression at a later time and with a new opponent. These results provide manipulative experimental support for longstanding hypotheses in psychology, that genetic variation in social environment construction has a causal role in behavioral development. More broadly, these results imply that studies of the genetic basis of complex traits should be expanded to include mechanisms by which genetic variation shapes the environments that individuals experience (Saltz, 2016).

    Comparative analysis of the brain transcriptome in a hyper-aggressive fruit fly

    Aggressive behavior is observed in many animals, but its intensity differs between species. In a model animal of genetics, Drosophila melanogaster, genetic basis of aggressive behavior has been studied intensively, including transcriptome analyses to identify genes whose expression level was associated with intra-species variation in aggressiveness. However, whether these genes are also involved in the evolution of aggressiveness among different species has not been examined. De novo transcriptome analysis was performed in this study in the brain of Drosophila prolongata to identify genes associated with the evolution of aggressiveness. Males of D. prolongata were hyper-aggressive compared with closely related species. Comparison of the brain transcriptomes identified 21 differentially expressed genes in males of D. prolongata. They did not overlap with the list of aggression-related genes identified in D. melanogaster, suggesting that genes involved in the evolution of aggressiveness were independent of those associated with the intra-species variation in aggressiveness in Drosophila. Although females of D. prolongata were not aggressive as the males, expression levels of the 21 genes identified in this study were more similar between sexes than between species (Kudo, 2017).

    Putative transmembrane transporter modulates higher-level aggression in Drosophila

    By selection of winners of dyadic fights for 35 generations, this study generated a hyperaggressive Bully line of flies that almost always win fights against the parental wild-type Canton-S stock. Maintenance of the Bully phenotype is temperature dependent during development: the phenotype is lost when flies are reared at 19 °C. No similar effect is seen with the parent line. This difference served as the basis for RNA-seq experiments which identify a limited number of genes that are differentially expressed by twofold or greater in the Bullies; one of these is a putative transmembrane transporter, CG13646, which shows consistent and reproducible twofold down-regulation in Bullies. The causal effect of this gene on the phenotype was examined with a mutant line for CG13646, and with an RNAi approach. In all cases, reduction in expression of CG13646 by approximately half leads to a hyperaggressive phenotype partially resembling that seen in the Bully flies. This gene is a member of a very interesting family of solute carrier proteins (SLCs), some of which have been suggested as being involved in glutamine/glutamate and GABA cycles of metabolism in excitatory and inhibitory nerve terminals in mammalian systems (Chowdhury, 2017).

    SlgA, the homologue of the human schizophrenia associated PRODH gene, acts in clock neurons to regulate Drosophila aggression

    Mutations in proline dehydrogenase (PRODH) are linked to behavioral alterations in schizophrenia and as part of DiGeorge and velo-cardio-facial syndromes, but the role of PRODH in their etiology remains unclear. This study established a Drosophila model to study the role of PRODH in behavioral disorders. The distribution was determined of the Drosophila PRODH homolog slgA in the brain, and knock-down and overexpression of human PRODH and slgA in the lateral neurons ventral (LNv) were shown to lead to altered aggressive behavior. SlgA acts in an isoform-specific manner and is regulated by casein kinase II (CkII). The data suggest that these effects are, at least partially, due to effects on mitochondrial function. It is thus shown that precise regulation of proline metabolism is essential to drive normal behavior and Drosophila aggression is a model behavior relevant for the study of mechanisms impaired in neuropsychiatric disorders (Zwarts, 2017).

    Genomic analysis of genotype by social environment interaction for Drosophila aggressive behavior

    Human psychiatric disorders such as schizophrenia, bipolar disorder and attention-deficit/hyper-activity disorder often include adverse behaviors including increased aggressiveness. Individuals with psychiatric disorders often exhibit social withdrawal, which can further increase the probability of conducting a violent act. This study used the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP) to investigate the genetic basis of variation in male aggressive behavior for flies reared in a socialized and socially isolated environment. Genetic variation was identified for aggressive behavior, as well as significant genotype by social environmental interaction (GSEI); i.e., variation among DGRP genotypes in the degree to which social isolation affected aggression. Genome-wide association (GWA) analyses was performed to identify genetic variants associated with aggression within each environment. Genomic prediction was used to partition genetic variants into gene ontology (GO) terms and constituent genes, and GO terms and genes were identified with high prediction accuracies in both social environments and for GSEI. The top predictive GO terms significantly increased the proportion of variance explained, compared to prediction models based on all segregating variants. Genomic prediction was performed across environments, and genes in common were identified between the social environments which turned to be enriched for genome-wide associated variants. A large proportion of the associated genes have previously been associated with aggressive behavior in Drosophila and mice. Further, many of these genes have human orthologs that have been associated with neurological disorders, indicating partially shared genetic mechanisms underlying aggression in animal models and human psychiatric disorders (Rohde, 2017).

    Behavioral, transcriptomic and epigenetic responses to social challenge in honey bees

    Understanding how social experiences are represented in the brain and shape future responses is a major challenge in the study of behavior. This problem was addressed by studying behavioral, transcriptomic and epigenetic responses to intrusion in honey bees. Previous research showed that initial exposure to an intruder provokes an immediate attack; this study now shows that this also leads to longer-term changes in behavior in the response to a second intruder, with increases in the probability of responding aggressively and the intensity of aggression lasting 2 and 1 h, respectively. Previous research also documented the whole-brain transcriptomic response; it is now shown that in the mushroom bodies (MBs) there are 2 waves of gene expression, the first highlighted by genes related to cytoskeleton remodeling, and the second highlighted by genes related to hormones, stress response and transcription factors (TFs). Overall, 16 of 37 (43%) of the TFs whose cis-motifs were enriched in the promoters of the differentially expressed genes (DEGs) were also predicted from transcriptional regulatory network analysis to regulate the MB transcriptional response, highlighting the strong role played by a relatively small subset of TFs in the MB's transcriptomic response to social challenge. Whole brain histone profiling showed few changes in chromatin accessibility in response to social challenge; most DEGs were 'ready' to be activated. These results show how biological embedding of a social challenge involves temporally dynamic changes in the neurogenomic state of a prominent region of the insect brain that are likely to influence future behavior (Shpigler, 2017).

    Isolation of aggressive behavior mutants in Drosophila using a screen for wing damage

    Aggression is a complex social behavior that is widespread in nature. To date only a limited number of genes that affect aggression have been identified, in large part because the complexity of the phenotype makes screening difficult and time consuming regardless of the species that is studied. Aggressive group-housed Drosophila melanogaster males inflict damage on each other's wings; wing damage negatively affects their ability to fly and mate. Using this wing-damage phenotype, males from ~1,400 chemically mutagenized strains were screened and ~40 mutant strains were found with substantial wing damage. Five of these mutants also had increased aggressive behavior. To identify the causal mutation in one of the top aggressive strains, whole genome sequencing and genomic duplications rescue strategies were used. A novel mutation was identified in the voltage-gated potassium channel Shaker (Sh) and a nearby previously identified Sh mutation was also shown to exhibit increased aggression. This simple screen can be used to dissect the molecular mechanisms underlying aggression (Davis, 2017).

    Distinct activity-gated pathways mediate attraction and aversion to CO2 in Drosophila

    Carbon dioxide is produced by many organic processes and is a convenient volatile cue for insects that are searching for blood hosts, flowers, communal nests, fruit and wildfires. Although Drosophila melanogaster feed on yeast that produce CO2 and ethanol during fermentation, laboratory experiments suggest that walking flies avoid CO2. This study resolved this paradox by showing that both flying and walking Drosophila find CO2 attractive, but only when they are in an active state associated with foraging. Their aversion to CO2 at low-activity levels may be an adaptation to avoid parasites that seek CO2, or to avoid succumbing to respiratory acidosis in the presence of high concentrations of CO2 that exist in nature. In contrast to CO2, flies are attracted to ethanol in all behavioural states, and invest twice the time searching near ethanol compared to CO2. These behavioural differences reflect the fact that ethanol is a unique signature of yeast fermentation, whereas CO2 is generated by many natural processes. Using genetic tools, it was determined that the evolutionarily conserved ionotropic co-receptor IR25a is required for CO2 attraction, and that the receptors necessary for CO2 avoidance are not involved in this attraction. This study lays the foundation for future research to determine the neural circuits that underlie both state- and odorant-dependent decision-making in Drosophila (van Breugel, 2018).

    D. melanogaster feed, mate and deposit eggs on rotting fruit. Between 10 and 14 days later, the next generation of flies must locate a fresh ferment. Because of the high volatility of CO2, the emission of CO2 is greatest near the start of fermentation, whereas ethanol emission increases more slowly. Other odours associated with fermentation (for example, acetic acid and ethyl acetate) form later, when bacteria break down ethanol. In trap assays, Drosophila show a preference for two-day-old apple juice ferments compared to older solutions, which suggests that they might be attracted to CO2. Although it is difficult to estimate concentrations of CO2 in wild ferments, the CO2 concentration in bottles commonly used to rear flies has been determined to be 0.5-1% (van Breugel, 2018).

    This evidence that CO2 might attract Drosophila contradicts previous studies conducted using small chambers. To study how flies respond to odours under more-ethological conditions, the flight trajectories was recorded of flies in a wind tunnel that contained a landing platform, which was programmed to periodically release plumes of CO2 or ethanol. Both odours elicited approaches, landings and explorations of a conspicuous visual feature, which is consistent with previous experiments with flies and mosquitoes. Flies were more likely to approach the platform or dark spot in the presence of ethanol compared to CO2, but were equally likely to land in response to either odour (van Breugel, 2018).

    To quantify the behaviour of flies after they land, a platform was designed that is suitable for automated tracking. At a flow rate of 60 ml min-1 CO2, the CO2 concentration near the surface of the platform was approximately 3%. After landing near a source of CO2, ethanol or apple cider vinegar, flies exhibited a local search behaviour that was similar to so-called 'dances'. Flies spent twice the amount of time exploring platforms that emitted ethanol compared to CO2 or vinegar. Flies approached a source that emitted both ethanol and CO2 more frequently than they approached vinegar, or either odour alone. Vinegar elicited smaller local searches and slightly fewer approaches compared to CO2, consistent with the hypothesis that vinegar might indicate a less favourable, late-stage ferment. Flies spent significantly less time standing still on the platform in the presence of CO2 compared to any other odour, with a mean walking speed > 2 mm s-1 (van Breugel, 2018).

    One previous study showed that Drosophila are attracted to CO2 while flying on a tether. The current results confirm this observation in freely flying flies; however, it was also found that flies remain attracted to CO2 after they land, which contradicts previous studies. One potential explanation is that flies in constrained walking chambers might behave differently to those that arrived on the open wind tunnel platform after tracking the odour plume and landing. To test this hypothesis, an enclosed arena was built in which flies were unable to fly, and they were presented with pulses of 5% CO2. Groups of 10 starved flies presented with CO2 after acclimating to the arena for 10 min exhibited aversion, as previously reported. However, if allowed to acclimate in the chamber for two hours, the flies exhibited attraction to CO2 (van Breugel, 2018).

    To study the response of these flies in more detail, the behaviour of flies was recorded for 20 h, while providing 10-min presentations of CO2 from alternating sides of the arena every 40 min. To control for humidity, 20 ml min-1 of H2O-saturated air was continuously pumped through the odour ports on both sides of the chamber. The flies exhibited a clear circadian rhythm within the chamber, as indicated by their mean walking speed. At times of peak activity-near dusk and dawn-flies showed a strong initial attraction to CO2, which decayed stereotypically during the 10-min presentation. At times of low activity-at mid-day and during the night-flies exhibited a mild aversion to CO2. Starving flies for 24 h before the experiment changed their activity profile, resulting in a slightly elevated attraction during the night. Ethanol, by contrast, elicited sustained attraction regardless of baseline activity (van Breugel, 2018).

    To probe this relationship between activity and CO2 attraction, the temperature was increased and the wind speed-manipulations that are known to elevate and depress activity were elevated, respectively. When wthe bulk-flow rate was increased to 100 ml min-1, flies exhibited a peak walking speed of about 1.5 mm s-1 at dusk-nearly half the speed measured at a flow rate of 20 ml min-1. Instead of showing attraction, these flies exhibited aversion to 5% CO2, although they were still attracted to ethanol. This result helps to explain why previous studies that used higher flows (100-1,000 ml min-1) to present CO2 observed aversion. To further explore the effect of wind, the aristae of the flies, which destroys their primary means of detecting airflow but does not interfere with the detection of odours, were clipped. The flies without aristae exhibited the same walking speed and attraction to CO2 at the high flow rate as was exhibited by normal flies at the low flow rate. Warming flies with intact aristae to 32°C also increased their baseline activity and recovered their attraction to CO2 at the higher flow rate. Pooling data across all experimental conditions, it was found that flies were attracted to CO2 when they had a baseline walking speed that was above about 2.4 mm s-1. This value is similar to the walking speed that was observed in the wind tunnel assay, which was higher for CO2 than the other odours. To confirm that activity-dependent attraction to CO2 is not a function of social interactions, 29 single flies, which behaved similarly to the cohorts of 10, were tested. Three concentrations of CO2 (1.7%, 5% and 15%) were also tested and found that the 5% concentration elicited the strongest response, consistent with wind tunnel experiments (van Breugel, 2018).

    Although the responses of flies to ethanol and CO2 were similar at stimulus onset, attraction to ethanol was more sustained. The time course of behaviour was notably similar in the walking arena and wind tunnel, which suggests that the behavioural dynamics of olfactory attraction are robust to the stimulus environment and may represent an adaptation for using information that broad (CO2) and more specific (ethanol) odorants provide (van Breugel, 2018).

    Previous research shows that CO2 aversion is mediated by Gr63a and Gr21a receptors; high concentrations of CO2 are also detected by an acid-sensitive ionotropic receptor, IR64a10. In the current assay, mutant flies that lack the IR64a receptor showed no significant change in their behaviour compared to wild type. Consistent with previous work, mutants that lack the Gr63a receptor exhibited no aversion to CO2; however, they were still attracted to CO2 when active. Mutant flies that are homozygous for both Gr63a and IR64a behaved similarly to the Gr63a mutants. It is noteworthy that the characteristic decaying time course of attraction was unaffected in Gr63a mutants, even though these flies showed no aversion. Thus, the decay in attraction to CO2 is not caused by an increase in aversion over time (van Breugel, 2018).

    Given that CO2 attraction is not mediated by Gr63a, Gr21a or IR64a, it was of interest to confirm that the attraction is indeed a chemosensory response. To determine whether CO2 attraction is mediated by either an olfactory or ionotropic receptor, a mutant was tested that lacks the olfactory and ionotropic co-receptors (Orco, IR25a and IR8a) as well as Gr63a. These near-anosmic mutants exhibited no detectable behavioural response to CO2. Flies in which the third antennal segment was surgically removed showed no response to CO2, despite normal levels of activity. Together with the arista ablations, these experiments show that CO2 attraction is mediated by receptors on the third antennal segment. To further confirm this, each co-receptor mutant was tested individually, and it was found that mutants that lack IR25a did not exhibit wild-type CO2 attraction, whereas Orco and IR8a mutants did. Mutant flies that lack Orco, IR8a and Gr63a also exhibit wild-type attraction to CO2, confirming that the only required co-receptor is IR25a. IR25a has previously been implicated in a wide range of behaviours, including temperature and humidity sensation. The temperature in the arena near the CO2 port was measured, and no change was found in temperature as a result of the stimulus. To eliminate the possibility of a humidity artifact, an IR40a mutant, which still exhibited attraction to CO2, was tested. In summary, these experiments show that CO2 attraction is mediated by a separate chemosensory pathway from that which governs aversion, and that CO2 attraction requires the IR25a co-receptor. IR25a is the most highly conserved olfactory receptor among insects. It is possible that other insect species that lack Gr63a26 but that still respond to CO2 use the same IR25a-dependent pathway. Unfortunately, the GAL4 driver for the IR25a promoter is expressed only in about half of the endogenous IR25a-expressing neurons, which makes imaging experiments that aim to identify which glomerulus is involved difficult at this time (van Breugel, 2018).

    The finding that active flies are attracted to CO2 makes ethological sense, given that CO2 is generated by yeast-the preferred food of these flies. Why it might be that Drosophila avoid CO2 when in a low-activity state was considered. Flies do not exhibit this state-dependent reaction to ethanol and vinegar; perhaps the aversion to CO2 at low activity is an adaptation that minimizes encounters with parasites that seek CO2. Alternatively, the behaviour may help flies to avoid respiratory acidosis when near high concentrations of CO2 within the environment. Previous studies have suggested that CO2 serves as an aversive pheromone by which stressed flies signal others to flee a local environment. However, an alternative explanation is that agitated flies release CO2 not as a social signal but simply because it is present in their tracheal system owing to their process of discontinuous respiration. Further work on this state-dependent reaction to CO2 will require experiments that carefully consider the natural ethology of the flies (van Breugel, 2018).

    Self-regulation and the foraging gene (PRKG1) in humans

    Foraging is a goal-directed behavior that balances the need to explore the environment for resources with the need to exploit those resources. In Drosophila melanogaster, distinct phenotypes have been observed in relation to the foraging (for) gene, labeled the rover and sitter. Adult rovers explore their environs more extensively than do adult sitters. This study explored whether this distinction would be conserved in humans. A distinction was used from regulatory mode theory between those who "get on with it," so-called locomotors, and those who prefer to ensure they "do the right thing," so-called assessors. In this logic, rovers and locomotors share similarities in goal pursuit, as do sitters and assessors. Genetic variation in PRKG1, the human ortholog of for, is associated with preferential adoption of a specific regulatory mode. Next, participants performed a foraging task to see whether genetic differences associated with distinct regulatory modes would be associated with distinct goal pursuit patterns. Assessors tended to hug the boundary of the foraging environment, much like behaviors seen in Drosophila adult sitters. In a patchy foraging environment, assessors adopted more cautious search strategies maximizing exploitation. These results show that distinct patterns of goal pursuit are associated with particular genotypes of PRKG1, the human ortholog of for (Struk, 2019).

    Softness sensing and learning in Drosophila larvae

    Mechanosensation provides animals with important sensory information in addition to olfaction and gustation during feeding behavior. This study used Drosophila larvae to investigate the role of softness sensing in behavior and learning. In the natural environment, Drosophila larvae need to dig into soft foods for feeding. Finding foods that are soft enough to dig into is likely to be essential for their survival. This study reports that Drosophila larvae can discriminate between different agar concentrations and prefer softer agar. Interestingly, it was shown that larvae on a harder place search for a softer place using memory associated with an odor and that they evaluate foods by balancing softness and sweetness. These findings suggest that Drosophila larvae integrate mechanosensory information with chemosensory input while foraging. Moreover, it was found that the larval preference for softness is affected by genetic background (Kudow, 2019).

    Diverse food-sensing neurons trigger idiothetic local search in Drosophila

    Foraging animals may benefit from remembering the location of a newly discovered food patch while continuing to explore nearby. For example, after encountering a drop of yeast or sugar, hungry flies often perform a local search. That is, rather than remaining on the food or simply walking away, flies execute a series of exploratory excursions during which they repeatedly depart and return to the resource. Fruit flies, Drosophila melanogaster, can perform this food-centered search behavior in the absence of external landmarks, instead relying on internal (idiothetic) cues. This path-integration behavior may represent a deeply conserved navigational capacity in insects, but its underlying neural basis remains unknown. This study used optogenetic activation to screen candidate cell classes and found that local searches can be initiated by diverse sensory neurons. Optogenetically induced searches resemble those triggered by actual food, are modulated by starvation state, and exhibit key features of path integration. Flies perform tightly centered searches around the fictive food site, even within a constrained maze, and they can return to the fictive food site after long excursions. Together, these results suggest that flies enact local searches in response to a wide variety of food-associated cues and that these sensory pathways may converge upon a common neural system for navigation. Using a virtual reality system, this study demonstrated that local searches can be optogenetically induced in tethered flies walking on a spherical treadmill, laying the groundwork for future studies to image the brain during path integration (Corfas, 2019).

    To discover sensory pathways triggering local search, the behavior of individual female flies was tracked as they explored a circular arena with a featureless optogenetic activation zone at its center. The assay consists of an initial 10-min baseline control period followed by a 30-min period during which animals receive a 1-s pulse of red light (628 nm) whenever they enter the activation zone. For flies expressing the light-sensitive channel CsChrimson in food-sensing neurons, the activation zone should act as a patch of fictive food, potentially able to elicit a local search. Using this setup, gustatory, olfactory, and reward-signaling neurons were screened to identify cell classes that trigger local search. Aside from the light pulses used for optogenetic activation, the animals are in complete darkness and must rely on internal cues to navigate the open-field portion of the arena. To examine whether flies were conducting local search, trajectories were analyzed, beginning at the activation zone and ending at the arena edge. Prior to testing, flies were subjected to 33-42 h of starvation, during which they had access to water only (Corfas, 2019).

    It is known that flies perform local searches after discovering a drop of sucrose, suggesting that sweet-sensing neurons may be sufficient to initiate this behavior. To test this, Gr43a-GAL4 >UAS-CsChrimson flies were used to activate fructose-sensing neurons whenever the flies entered the activation zone. Activation of these gustatory neurons triggered local searches remarkably similar to those previously observed in response to actual food, consisting of a series of excursions from and returns to the fictive food site. Unlike parental controls, Gr43a-GAL4 >UAS-CsChrimson flies extensively searched the area surrounding the activation zone (~30 cm2) after receiving a light pulse. These search trajectories were highly centered at the activation zone and consisted of numerous revisits to the activation zone-both features of local searches shown to require path integration. During local search, flies cumulatively walked ~30-300 cm (approximately 100-1,000 body lengths) before eventually straying to the arena edge. Prior studies, using another Gr43a-GAL4 line, suggest that Gr43a is expressed in neurons of the pharynx and brain that measure post-ingestive sugar levels as well as in sugar-sensing neurons in the periphery. However, this study found that nearly identical local searches are triggered by activation using the Gr5a-GAL4 driver, which only labels peripheral sugar-sensing neurons. Therefore, non-pharyngeal sugar sensors are capable of eliciting local search. This result is in disagreement with recent experiments suggesting that only pharyngeal sugar sensors can trigger local search; however, that study did not examine the effect of activating specific subsets of sugar-sensing neurons. The use of fictive food in the current experiments provides further evidence that flies are in fact using idiothetic path integration during local search rather than relying on external (allothetic) cues coming from an actual drop of food, such as visual appearance, odor or humidity gradients, or tracks of food residue deposited during prior search excursions (Corfas, 2019).

    Previous work has shown that, compared to sucrose-triggered searches, searches triggered by a drop of 5% yeast solution elicits search trajectories that are even longer and include more revisits to the food, suggesting that proteinaceous food components may also initiate this behavior. Amino acids present in yeast are a coveted source of nutrition for mated females, which require a protein source to produce eggs. The ionotropic receptor Ir76b has been implicated in the detection of the taste of yeast, amino acids, carbonation, and other important nutrients, such as salt, polyamines, and fatty acids. Ir76b-GAL4>UAS-CsChrimson flies were tested; activation of these amino-acid sensors resulted in a modest increase in residence near the activation zone, due largely to the animals ceasing locomotor activity. However, the activation did not trigger a local search-the trajectories covered little distance and rarely included a revisit to the activation site. The failure to elicit local searches via activation of Ir76b-GAL4 may be due to the fact that this line labels a large population of neurons associated with diverse sensory functions. Indeed, whereas silencing of these neurons disrupts preference for feeding on yeast, direct activation of Ir76b-GAL4 neurons has never been shown to trigger feeding behavior (Corfas, 2019).

    Food odorants also trigger search behavior in insects. In flight, for example, encounters with an odor plume elicit the stereotyped cast and surge maneuvers that enable insects to localize the source of an advected odor. Recent studies have demonstrated that this also occurs during walking-flies increase their turn rate when they exit a plume of apple cider vinegar (ACV) odor. Attraction to the smell of ACV in Drosophila is mediated primarily by neurons expressing the olfactory receptor Or42b. Optogenetic activation of Or42b-GAL4 neurons produces attraction behavior in flies, as does activation of Or59b-GAL4 neurons, which respond to acetate esters found in food odors. Simultaneous optogenetic activation of nearly all the olfactory receptor neurons via Orco-GAL4 also produces attraction in flies. This study tested whether these three classes of olfactory neurons could trigger a local search and found that activation of Orco- and Or59b-GAL4 neurons did not elicit searches. Activation of ACV-odor-sensing Or42b-GAL4 neurons resulted in increased residence near the activation zone, much like the results from searches triggered by activation of Ir76b-GAL4, but it did not produce local searches, according to the metrics used in this study (Corfas, 2019).

    The water content of food drops might be enough to evoke local search. In Drosophila, water sensation is mediated by the osmosensitive ion channel ppk28, a member of the degenerin/epithelial sodium channel family. Activation of water-sensing ppk28-GAL4 neurons in food-deprived flies did not result in local search. This result is in agreement with previous behavioral experiments showing that Drosophila do not produce local search bouts after encountering a drop of pure water (Corfas, 2019).

    It was hypothesized that reward-signaling neurons of the central nervous system might also trigger local searches. Neuropeptide F (NPF) is a highly conserved hunger-signaling neuropeptide that stimulates a variety of Drosophila behaviors, including feeding. NPF-GAL4 labels neurons in the posterior region of the Drosophila brain, and activation of these cells is rewarding in the context of olfactory conditioning. Much like Ir76b-GAL4 and Or42b-GAL4, activation of NPF-GAL4 neurons was shown to result in a modest increase in residence near the activation zone but not statistically significant local searches. Another set of reward-signaling neurons are the dopaminergic protocerebral anterior medial (PAM) neurons, which are activated by sugar ingestion and innervate the mushroom body, a structure critical for forming associative memories. Activation of PAM neurons via R58E02-GAL4 is known to mediate reward during olfactory conditioning, and silencing PAM neurons inhibits food occupancy during foraging. However,activation of R58E02-GAL4 neurons did not produce search behavior (Corfas, 2019).

    Because local searches are initiated by activation of sugar receptors, it was hypothesized that starvation state may influence the extent of optogenetically induced searches. The influence of starvation has been observed for sucrose-induced searches in Drosophila as well as for protein- and water-induced searches in the blowfly (Phormia regina). Until this point, all of the experiments were conducted with animals allowed access only to water for 33-42 h preceding the trial. To examine the importance of starvation in promoting local search, activation of sugar-sensing Gr43a-GAL4 neurons was tested in flies that were reared continuously on food or starved for only 9-18 h. As expected, longer starvation times result in more extensive searches, with longer trajectories and more revisits to the activation zone (Corfas, 2019).

    Tests were performed to see whether additional food deprivation could produce searches triggered by sensory pathways that had weak behavioral effects in the original screen. For these experiments, flies were starved for 7 days: 5 days with access to sucrose solution followed by 2 days with access to only water. Even in 7-day-starved animals, activation of amino-acid-sensing Ir76b-GAL4 neurons did not elicit substantial local search, despite the fact that protein-deprived mated females are known to develop a strong preference for amino-acid-containing food. However, activation of ACV-odor-sensing Or42b-GAL4neurons in 7-day-starved animals resulted in extensive and centralized local searches, comparable to those triggered by sugar-sensing neurons. This finding is consistent with work showing that starvation promotes food search behavior in Drosophila and that this effect is mediated by neuropeptidergic modulation of Or42b-GAL4 neuron activity. Local search was found to be triggered by activation of NPF-GAL4 neurons in 7-day-starved animals. This effect may be related to previous work showing that NPF-GAL4 neurons are activated by food odors in a starvation-dependent manner. Finally, it was found that nutritional deprivation can even produce searches triggered by water sensation-activation of ppk28-GAL4 neurons elicited robust local searches in animals subjected to a desiccating environment without food or water (Corfas, 2019).

    Collectively, these results show that optogenetic activation of a variety of food-associated sensors can trigger searches and that this behavior is influenced by the internal nutritional state, much like searches triggered by actual food. Previous work has shown that flies regulate their consumption of sugar and yeast depending on whether they are deficient in that specific nutrient. When a fly finds a patch of yeast, for example, its decision to stay or leave depends strongly on whether it is deficient in amino acids. Thus, it may be that flies only perform local search when they experience a food cue associated with a nutrient they currently need. To test this hypothesis further, animals were starved on a synthetic food medium that allowed deprivation of flies specifically of either sugar or protein while supplying them with an otherwise complete and balanced diet. Activation of Gr43a-GAL4 sugar sensors produced robust local searches in animals subjected to sugar deprivation but not in animals subjected to long-term protein deprivation, suggesting that sugar-sensation-triggered searches are a response to a specific nutritional need. However, activation of ACV-odor-sensing Or42b-GAL4 neurons did not elicit searches in protein-deprived flies and elicited modest searches in sugar-deprived flies. However, activation of Or42b-GAL4 neurons does elicit robust searches in 7-day-starved animals subjected to a combination of sugar and protein deprivation. One possible functional explanation for these differing results is that substances sensed via contact, such as sugar or water, produce local search conditional on the specific internal state of that nutrient. In contrast, because the detection of volatile compounds is a less reliable indicator of the nutritional content of nearby food, the potency with which an odor can elicit local search may depend on the general nutritional state of the animal. Determining the ethological connection between food-triggered search and nutrient homeostasis will require further investigation (Corfas, 2019).

    In his initial description of food-induced local search, it has been demonstrated that when a hungry blowfly discovers a drop of food, it performs a proboscis extension response (PER)-a reflex associated with appetitive cues. To explore the role of proboscis extension in optogenetically induced local search, tests were performed to see whether activation of each of these neuron classes elicits PER. As has been previously reported, activation of sugar-sensing Gr5a-GAL4 neurons elicits PER. This study found that activation of Gr43a-GAL4 neurons also elicits PER in a starvation-dependent manner, indicating that fructose triggers a feeding reflex similar to that triggered by other sugars. Activation of water sensors via ppk28-GAL4 neurons also resulted in PER, even in animals that had not been subjected to dry starvation. Activation of hunger-signaling neurons via NPF-GAL4 was found to elicit strong PER , demonstrating a novel function for these neurons. However, none of the other neuron classes in the screen consistently triggered PER, including Or42b-GAL4 neurons, indicating that local search can be initiated by receptors that do not by themselves elicit PER (Corfas, 2019).

    Together, these results suggest that local searches are triggered by both contact chemosensory cues that signal that the fly is on food (e.g., water or sugar) as well as volatile cues that indicate food is nearby (e.g., the odor of ACV). It appears that flies even initiate local searches around a location associated with a rewarding stimulus (i.e., activation of NPF-GAL4 neurons) without accompanying activation of peripheral chemosensors. Although searches triggered by sugar, water, odor, and reward signaling appear broadly similar in these experiments, it is likely that their underlying behavioral structure differs. For example, it was found that whereas activation of Gr43a-, Gr5a-, Ir76b-, ppk28-, or NPF-GAL4 results in decreased locomotion or complete stopping, activation of ACV-odor-sensing Or42b-GAL4 neurons only elicits a brief startle response, similar to controls. The absence of slowing at the initiation of searches triggered by Or42b-GAL4 neurons is consistent with the interpretation that these searches are related to the casting behaviors elicited by loss of an odor plume. Future studies using the current paradigm may determine whether local searches triggered by distinct food-associated stimuli are stereotyped or are instead accomplished through diverse behavioral strategies (Corfas, 2019).

    The results show that optogenetically induced local searches resemble those evoked by actual food, suggesting that flies are using idiothetic path integration to keep track of their position relative to the activation zone. Unlike in previous studies using real food, it was possible to monitor every occasion at which the fly senses the fictive food and can easily reinforce the memory of its location. Using the data from Gr43a-GAL4>UAS-CsChrimson animals in the original screen, the search trajectories occurring after each optogenetic stimulation were examined. Many of these trajectories lasted only a few seconds and covered only a few centimeters before the fly returned to the activation zone, thus receiving another optogenetic pulse. In many cases, however, flies performed a centered local search lasting minutes and covering hundreds of body lengths without an intervening optogenetic stimulation. This implies that a persistent internal representation of space underlies this behavior-without sustaining a centered search, flies would quickly stray to the arena edge. It was also observed that flies can update the center of their search upon discovering another activation zone, as has been found with searches around real food. Moreover, flies repeatedly shift the center of their search between activation zones, resembling experiments in which flies foraged among an array of food patches. In those experiments, flies were found to execute local searches around food sites but also discovered new food sites by exploring further-a behavior dependent on the internal nutrient state of the fly (Corfas, 2019).

    The ability to execute a sustained search centered around a fictive food site in complete darkness, and moreover to carry this out in an environment with arbitrary geometric constraints, strongly suggests that flies can keep track of their location relative to the activation zone. This feat of idiothetic path integration has previously been compared to other insect behaviors, such as the foraging excursions of desert ants (Cataglyphis fortis), which routinely embark on long and winding runs through featureless terrain and yet are able to return to their nest in a direct path. To accomplish this, these ants keep track of both the distance and the direction of their travel, enabling them to integrate their position relative to a point of origin. During food-triggered searches, Drosophila may be using the same computational strategies as Cataglyphis and thus may be relying on the same highly conserved brain structures. In particular, studies point to the importance of the central complex-a sensorimotor hub of the insect brain that processes numerous aspects of locomotion, navigation, and decision making. Wedge neurons of the ellipsoid body encode azimuthal heading, potentially serving as a compass for path integration, celestial navigation, and other behaviors. Whereas less is known about how insects monitor odometry, it is thought that step counting can be achieved by using proprioceptive feedback or efferent copies of motor commands to integrate the distance traveled (Corfas, 2019).

    It is proposed that optogenetic activation of Gr43a- and Gr5a-GAL4 sugar sensors may be a potent tool in future experiments seeking to characterize the neural implementation of path integration. Among the sensory pathways studied, these sweet-sensing neurons are the most reliable triggers of local search. However, the comparatively weaker searches elicited by activation of other neural pathways in this study may be a consequence of differences in the levels or anatomical depth of transgene expression rather than a reflection of their contribution to search behavior. Regardless of this experimental limitation, the fact that so many sensory modalities can trigger local searches suggests a convergence of these pathways onto the set of brain structures underlying navigation. This is consistent with anatomical studies of the central complex showing that it receives a variety of indirect sensory inputs as well as direct innervation by a large subset of NPF-GAL4 neurons (Corfas, 2019).

    Elucidating the function of these circuits in path integration would require the ability to record neural activity in the Drosophila brain during local search. To this end, a preparation was developed to elicit local searches in a tethered fly walking on an air-suspended spherical treadmill. Similar setups have been successfully used to examine the path integration behavior of Cataglyphis ants. The fly's fictive path was reconstructed in real time using the FicTrac machine vision system, and a closed-loop program controlled optogenetic stimulation to present fictive food sites in the virtual 2D environment. As the fly walked, it would at certain points receive optogenetic stimulation; this virtual location became a fictive food site with an activation zone, thus mimicking the free-walking experiments. If the fly strayed far away from the activation zone, the fictive food site was abolished, and a new fictive food site was spawned soon after at the fly's new position. In this manner, flies experienced numerous virtual fictive food sites, and this study later examined whether they performed local searches in each case. Trials included an initial baseline period and a final post-experimental period during which mock fictive food sites were created, but the fly received no activation (Corfas, 2019).

    Activation of Gr43a-GAL4 sugar sensors triggered local searches in the virtual environment that resembled searches in free-walking flies. The spatial scale of the searches was smaller than that of free-walking flies, perhaps due to increased error accumulation in idiothetic path integration caused by a mismatch between the fly's intended locomotion and the machine-reconstructed fictive path. Nevertheless, compared to the baseline and post conditions, and unlike parental controls, these searches covered greater distances, consisted of numerous revisits to the activation zone, and were highly centered at the fictive food site, suggesting that flies walking on the treadmill apparatus are capable of performing idiothetic path integration. As in free-walking flies, activation of Gr43a-GAL4 neurons in tethered flies elicited a reduction in walking speed and proboscis extension, accompanied by a strong startle response. These results are consistent with a recent report, using a similar setup in which flies explore a virtual environment with visual features. That study found that activation of sugar receptors triggers local search in a virtual landscape and that visual landmarks do not contribute to this behavior, supporting the hypothesis that flies are performing idiothetic path integration. Adapting these setups for use with a 2-photon microscope may permit future studies to examine how sensory stimuli, reward signals, spatial information, and memory are encoded and integrated to produce path integration (Corfas, 2019).

    In summary, this study found that hungry flies initiate a sustained local search when they experience a fictive food stimulus. This search behavior appears to constitute a generalized foraging response, as it can be triggered by multiple types of food-associated neurons, including water-, sugar-, and vinegar-odor-sensing neurons, as well as by hunger-signaling neurons of the central nervous system. Like local searches triggered by real food, optogenetically induced local searches are modulated by internal nutritional state and show key features of idiothetic path integration. The results suggest that flies are able to keep track of their spatial position relative to a fictive food stimulus, even within a constrained maze. Long-lasting local search bouts can be initiated repeatedly by the brief activation of specific sets of neurons, and a system was developed to reconstitute this behavior in a tethered fly, thus establishing a promising entry point to tracing the neural pathways underlying path integration in insects (Corfas, 2019).

    Learning a spatial task by trial and error in Drosophila

    The ability to use memory to return to specific locations for foraging is advantageous for survival. Although recent reports have demonstrated that the fruit flies Drosophila melanogaster are capable of visual cue-driven place learning and idiothetic path integration, the depth and flexibility of Drosophila's ability to solve spatial tasks and the underlying neural substrate and genetic basis have not been extensively explored. This study shows that Drosophila can remember a reward-baited location through reinforcement learning and do so quickly and without requiring vision. This study found that both sighted and blind flies can learn-by trial and error-to repeatedly return to an unmarked location where a brief stimulation of the 0273-GAL4 neurons was available for each visit. Optogenetic stimulation of these neurons enabled learning by employing both a cholinergic pathway that promoted self-stimulation and a dopaminergic pathway that likely promoted association of relevant cues with reward. Lastly, inhibiting activities of specific neurons time-locked with stimulation of 0273-GAL4 neurons showed that mushroom bodies (MB) and central complex (CX) both play a role in promoting learning of the task. This work uncovered new depth in flies' ability to learn a spatial task and established an assay with a level of throughput that permits a systematic genetic interrogation of flies' ability to learn spatial tasks (Stern, 2019).

    Social foraging extends associative odor-food memory expression in an automated learning assay for Drosophila melanogaster

    Animals socially interact during foraging and share information about the quality and location of food sources. The mechanisms of social information transfer during foraging have been mostly studied at the behavioral level, and its underlying neural mechanisms are largely unknown. Fruit flies have become a model for studying the neural bases of social information transfer, because they provide a large genetic toolbox to monitor and manipulate neuronal activity, and they show a rich repertoire of social behaviors. Fruit flies aggregate, they use social information for choosing a suitable mating partner and oviposition site, and they show better aversive learning when in groups. However, the effects of social interactions on associative odor-food learning have not yet been investigated. This paper presents an automated learning and memory assay for walking flies that allows the study of the effect of group size on social interactions and on the formation and expression of associative odor-food memories. Both inter-fly attraction and the duration of odor-food memory expression were found to increase with group size. This study opens up opportunities to investigate how social interactions during foraging are relayed in the neural circuitry of learning and memory expression (Sehdev, 2019).

    Cooperative foraging during larval stage affects fitness in Drosophila

    Cooperative behavior can confer advantages to animals. This is especially true for cooperative foraging which provides fitness benefits through more efficient acquisition and consumption of food. This study has taken advantage of an experimental model system featuring cooperative foraging behavior in Drosophila. Under crowded conditions, fly larvae form coordinated digging groups (clusters). where individuals are linked together by sensory cues and group membership requires prior experience. However, fitness benefits of Drosophila larval clustering remain unknown. This study demonstrates that animals raised in crowded conditions on food partially processed by other larvae experience a developmental delay presumably due to the decreased nutritional value of the substrate. Intriguingly, same conditions promote the formation of cooperative foraging clusters which further extends larval stage compared to non-clustering animals. Remarkably, this developmental retardation also results in a relative increase in wing size, serving an indicator of adult fitness. Thus, this study finds that the clustering-induced developmental delay is accompanied by fitness benefits. Therefore, cooperative foraging, while delaying development, may have evolved to give Drosophila larvae benefits when presented with competition for limited food resources (Dombrovski, 2020).

    Desiccation Stress Acts as Cause as well as Cost of Dispersal in Drosophila melanogaster

    Environmental stress is one of the important causes of biological dispersal. At the same time, the process of dispersal itself can incur and/or increase susceptibility to stress for the dispersing individuals. Therefore, in principle, stress can serve as both a cause and a cost of dispersal. These potentially contrasting roles of a key environmental stress (desiccation) were studied using Drosophila melanogaster. By modulating water and rest availability, it was asked whether (a) dispersers are individuals that are more susceptible to desiccation stress, (b) dispersers pay a cost in terms of reduced resistance to desiccation stress, (c) dispersal evolution alters the desiccation cost of dispersal, and (d) females pay a reproductive cost of dispersal. Desiccation was was found to be a clear cause of dispersal in both sexes, as both male and female dispersal propensity increased with increasing duration of desiccation. However, the desiccation cost of dispersal was male biased, a trend unaffected by dispersal evolution. Instead, females paid a fecundity cost of dispersal. The complex relationship between desiccation and dispersal, which can lead to both positive and negative associations, were discussed. Furthermore, the sex differences highlighted here may translate into differences in movement patterns, thereby giving rise to sex-biased dispersal patterns (Mishra, 2022).

    cAMP signaling mediates behavioral flexibility and consolidation of social status in Drosophila aggression

    Social rituals, such as male-male aggression in Drosophila, are often stereotyped and the component behavioral patterns modular. The likelihood of transition from one behavioral pattern to another is malleable by experience and confers flexibility to the behavioral repertoire. Experience-dependent modification of innate aggressive behavior in flies alters fighting strategies during fights and establishes dominant-subordinate relationships. Dominance hierarchies resulting from agonistic encounters are consolidated to longer-lasting, social-status-dependent behavioral modifications, resulting in a robust loser effect. This study shows that cAMP dynamics regulated by the calcium-calmodulin-dependent adenylyl cyclase, Rut, and the cAMP phosphodiesterase, Dnc, but not the Amn gene product, in specific neuronal groups of the mushroom body and central complex, mediate behavioral plasticity necessary to establish dominant-subordinate relationships. rut and dnc mutant flies were unable to alter fighting strategies and establish dominance relationships during agonistic interactions. This real-time flexibility during a fight was independent of changes in aggression levels. Longer-term consolidation of social status in the form of a loser effect, however, required additional Amn-dependent inputs to cAMP signaling and involved a circuit-level association between the alpha/beta and gamma neurons of the mushroom body. These findings implicate cAMP signaling in mediating the plasticity of behavioral patterns in aggressive behavior and in the generation of a temporally stable memory trace that manifests as a loser effect (Chouhan, 2017).

    Repetitive aggressive encounters generate a long-lasting internal state in Drosophila melanogaster males

    Multiple studies have investigated the mechanisms of aggressive behavior in Drosophila; however, little is known about the effects of chronic fighting experience. This study investigated if repeated fighting encounters would induce an internal state that could affect the expression of subsequent behavior. Wild-type males were trained to become winners or losers by repeatedly pairing them with hypoaggressive or hyperaggressive opponents, respectively. As described previously, it was observed that chronic losers tend to lose subsequent fights, while chronic winners tend to win them. Olfactory conditioning experiments showed that winning is perceived as rewarding, while losing is perceived as aversive. Moreover, the effect of chronic fighting experience generalized to other behaviors, such as gap-crossing and courtship. It is proposed that in response to repeatedly winning or losing aggressive encounters, male flies form an internal state that displays persistence and generalization; fight outcomes can also have positive or negative valence. Furthermore, it was shown that the activities of the PPL1-gamma1pedc dopaminergic neuron and the MBON-gamma1pedc>α/β mushroom body output neuron are required for aversion to an olfactory cue associated with losing fights (Kim, 2018).

    A brain module for scalable control of complex, multi-motor threat displays

    Threat displays are a universal feature of agonistic interactions. Whether threats are part of a continuum of aggressive behaviors or separately controlled remains unclear. Threats were analyzed in Drosophila; they are triggered by male cues and visual motion, and comprised of multiple motor elements that can be flexibly combined. A cluster of approximately 3 neurons was isolated whose activity is necessary for threat displays but not for other aggressive behaviors, and whose artificial activation suffices to evoke naturalistic threats in solitary flies, suggesting that the neural control of threats is modular with respect to other aggressive behaviors. Artificially evoked threats suffice to repel opponents from a resource in the absence of contact aggression. Depending on its level of artificial activation, this neural threat module can evoke different motor elements in a threshold-dependent manner. Such scalable modules may represent fundamental "building blocks" of neural circuits that mediate complex multi-motor behaviors (Duistermars, 2018).

    The peacefulness gene promotes aggression in Drosophila

    Natural aggressiveness is commonly observed in all animal species, and is displayed frequently when animals compete for food, territory and mating. Aggression is an innate behaviour, and is influenced by both environmental and genetic factors. However, the genetics of aggression remains largely unclear. This study identified the peacefulness (pfs) gene as a novel player in the control of male-male aggression in Drosophila. Mutations in pfs decreased intermale aggressiveness, but did not affect locomotor activity, olfactory avoidance response and sexual behaviours. pfs encodes for the evolutionarily conserved molybdenum cofactor (MoCo) synthesis 1 protein (Mocs1), which catalyzes the first step in the MoCo biosynthesis pathway. Neuronal-specific knockdown of pfs decreased aggressiveness. By contrast, overexpression of pfs greatly increased aggressiveness. Knocking down Cinnamon (Cin) catalyzing the final step in the MoCo synthesis pathway, caused a pfs-like aggression phenotype. In humans, inhibition of MoCo-dependent enzymes displays anti-aggressive effects. Thus, the control of aggression by Pfs-dependent MoCo pathways may be conserved throughout evolution (Ramin, 2019).

    Serotonergic modulation of aggression in Drosophila involves GABAergic and cholinergic opposing pathways

    Pathological aggression is commonly associated with psychiatric and neurological disorders and can impose a substantial burden and cost on human society. Serotonin (5HT) has long been implicated in the regulation of aggression in a wide variety of animal species. In Drosophila, a small group of serotonergic neurons selectively modulates the escalation of aggression. This study has identified downstream targets of serotonergic input-two types of neurons with opposing roles in aggression control. The dendritic fields of both neurons converge on a single optic glomerulus LC12, suggesting a key pathway linking visual input to the aggression circuitry. The first type is an inhibitory GABAergic neuron: its activation leads to a decrease in aggression. The second neuron type is excitatory: its silencing reduces and its activation increases aggression. RNA sequencing (RNA-seq) profiling of this neuron type identified that it uses acetylcholine as a neurotransmitter and likely expresses 5HT1A, short neuropeptide F receptor (sNPFR), and the resistant to dieldrin (RDL) category of GABA receptors. Knockdown of RDL receptors in these neurons increases aggression, suggesting the possibility of a direct crosstalk between the inhibitory GABAergic and the excitatory cholinergic neurons. These data show further that neurons utilizing serotonin, GABA, ACh, and short neuropeptide F interact in the LC12 optic glomerulus. Parallel cholinergic and GABAergic pathways descending from this sensory integration area may be key elements in fine-tuning the regulation of aggression (Alekseyenko, 2019).

    Identification of a putative binding site critical for general anesthetic activation of TRPA1

    General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors (see Drosophila Rdl). In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. This study exploited the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, this study showed that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, the combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels (Ton, 2017).

    Isoflurane impairs low-frequency feedback but leaves high-frequency feedforward connectivity intact in the fly brain

    Hierarchically organized brains communicate through feedforward (FF) and feedback (FB) pathways. In mammals, FF and FB are mediated by higher and lower frequencies during wakefulness. FB is preferentially impaired by general anesthetics in multiple mammalian species. This suggests FB serves critical functions in waking brains. The brain of Drosophila melanogaster is also hierarchically organized, but the presence of FB in these brains is not established. This study examined FB in the fly brain, by simultaneously recording local field potentials (LFPs) from low-order peripheral structures and higher-order central structures. The data was analyzed using Granger causality (GC), the first application of this analysis technique to recordings from the insect brain. The analysis revealed that low frequencies (0.1-5 Hz) mediated FB from the center to the periphery, while higher frequencies (10-45 Hz) mediated FF in the opposite direction. Further, isoflurane anesthesia preferentially reduced FB. The results imply that the spectral characteristics of FF and FB may be a signature of hierarchically organized brains that is conserved from insects to mammals. It is speculated that general anesthetics may induce unresponsiveness across species by targeting the mechanisms that support FB (Cohen, 2018).

    Genetic variability affects absolute and relative potencies and kinetics of the anesthetics isoflurane and sevoflurane in Drosophila melanogaster

    Genetic variability affects the response to numerous xenobiotics but its role in the clinically-observed irregular responses to general anesthetics remains uncertain. To investigate the pharmacogenetics of volatile general anesthetics (VGAs), a Serial Anesthesia Array apparatus was developed to expose multiple Drosophila melanogaster samples to VGAs, and behavioral assays were carried out to determine pharmacokinetic and pharmacodynamic properties of VGAs. The VGAs isoflurane and sevoflurane were studied in four wild type strains from the Drosophila Genetic Reference Panel, two commonly used laboratory strains (Canton S and w1118), and a mutant in Complex I of the mitochondrial electron transport chain (ND2360114). In all seven strains, isoflurane was more potent than sevoflurane, as predicted by their relative lipid solubilities, and emergence from isoflurane was slower than from sevoflurane, reproducing cardinal pharmacokinetic and pharmacodynamic properties in mammals. In addition, ND2360114 flies were more sensitive to both agents, as observed in worms, mice, and humans carrying Complex I mutations. Moreover, substantial variability was found among the fly strains both in absolute and in relative pharmacokinetic and pharmacodynamic profiles of isoflurane and sevoflurane. These data indicate that naturally occurring genetic variations measurably influence cardinal pharmacologic properties of VGAs and that flies can be used to identify relevant genetic variations (Olufs, 2018).

    The Effect of General Anaesthesia on Circadian Rhythms in Behaviour and Clock Gene Expression of Drosophila melanogaster

    General anaesthesia (GA) is implicated as a cause of postoperative sleep disruption and fatigue with part of the disturbance being attributed to a shift of the circadian clock. In this study, Drosophila melanogaster was used as a model to determine how Isoflurane affects the circadian clock at the behavioural and molecular levels. The response of the clock was measured at both of these levels caused by different durations and different concentrations of Isoflurane at circadian time 4 (CT4). Once characterized, the duration and concentration constants (at 2% in air for 6 h) were held and the phase responses were calculated over the entire circadian cycle in both activity and period expression. Phase advances in behaviour were observed during the subjective day, whereas phase delays were associated with subjective night time GA interventions. The corresponding pattern of gene expression preceded the behavioural pattern by approximately four hours. The implications of this effect for clinical and research practice are discussed (Li, 2020).

    Trapping of Syntaxin1a in presynaptic nanoclusters by a clinically relevant general anesthetic

    Propofol is the most commonly used general anesthetic in humans. Understanding of its mechanism of action has focused on its capacity to potentiate inhibitory systems in the brain. However, it is unknown whether other neural mechanisms are involved in general anesthesia. This study demonstrates that the synaptic release machinery is also a target. Using single-particle tracking photoactivation localization microscopy, it was shown that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility. Removing the interaction with the t-SNARE partner SNAP-25 abolishes propofol-induced Syntaxin1A confinement, indicating that Syntaxin1A and SNAP-25 together form an emergent drug target. Impaired Syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated Syntaxin1A construct that interacts with SNAP-25. These results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional Syntaxin1A nanoclusters (Bademosi, 2018).

    This study demonstrates that clinical concentrations of a commonly used GABA-acting general anesthetic, propofol, also restrict syntaxin1A mobility on the plasma membrane. The contrast seen with the effect of propofol analogs is particularly striking, with the non-anesthetic analogs significantly increasing syntaxin1A mobility instead. These results indicate that propofol acts like its non-anesthetic analogs when the interaction between syntaxin1A and SNAP-25 is lost, suggesting that propofol targets this interaction to immobilize syntaxin1A. It seems plausible that syntaxin1A confinement to nanoclusters could lead to impaired neurotransmission, which was also observed under propofol. However, more work is needed to establish causality here. How exactly propofol impairs syntaxin1A mobility remains unclear, although the requirement for SNAP-25 interaction suggests the nanoclusters are composed of syntaxin1A/SNAP-25 heterodimers that have been blocked from proceeding to a subsequent step in SNARE complex formation due to the presence of the general anesthetic. It is also unclear how a truncated syntaxin1A protein might preserve this process against the effects of propofol on syntaxin1A mobility and exocytosis. The finding that the truncated syntaxin1A molecule simultaneously interacts with both SNAP-25 and wild-type syntaxin1A suggests occupancy of a site that might otherwise be targeted by propofol. In this regard, future experiments with other truncation constructs employing propofol resistance as a readout will be helpful toward determining whether the effects on syntaxin1A mobility and exocytosis are indeed correlated (Bademosi, 2018).

    In addition to identifying an alternative target process for this widely used sedative, the current findings may provide a more complete understanding of general anesthesia. Every neuron communicates with other neurons by way of syntaxin1A-mediated neurotransmission, which is highly conserved from worms to humans. Although these experiments were focused on the intravenous drugs propofol and etomidate, it will be interesting to see in future studies whether other general anesthetics have the same effect on syntaxin1A mobility. There is already considerable evidence that a broader range of general anesthetics affect synaptic release mechanisms, and a previous study using nuclear magnetic resonance found that clinical concentrations of these drugs interact with syntaxin1A and SNAP-25, but not VAMP2, which is consistent with the conclusion that propofol acts before completed SNARE formation. One hypothesis consistent with these findings would be that a general anesthetic target emerges only when syntaxin1A and SNAP-25 interact on the plasma membrane and that the association of propofol with this emergent target interferes with subsequent steps in SNARE formation. This would lead to a 'traffic jam' of syntaxin1A/SNAP-25 heterodimers (or another pre-SNARE moiety), which would manifest as syntaxin1A nanoclusters in this analysis. Another explanation for the decrease in syntaxin1A mobility could be that propofol promotes its recruitment into nonfunctional SNARE complexes that do not promote vesicle fusion. Whereas the data suggest interactions in the membrane, this need not be the only explanation for altered syntaxin1A mobility. An alternative possibility is that anesthetics might alter syntaxin1A mobility by more specifically interfering with other key protein interactions leading to SNARE formation, such as between syntaxin1A/SNAP-25 and Munc-13, which is a crucial mediator in forming the final tetrameric complex with VAMP2. Future experiments testing the effects of mutating candidate residues in the syntaxin1A SNARE motif should reveal the exact nature of this propofol-binding target, as has been revealed for other propofol targets, such as GABAA receptors (Bademosi, 2018).

    Like sleep, general anesthesia resembles a reversible switch, and the search for mechanisms of anesthesia has justifiably focused on proteins that exert major effects on neuronal excitability, such as inhibitory GABAA receptors, which are indeed targets of many general anesthetics. However, the current results and the work of others show that clinically relevant concentrations of general anesthetics also compromise neurotransmitter release, and the current set of results with intravenous drugs suggests this may be consequence of effects on syntaxin1A mobility in the plasma membrane. However, general anesthetics do not abolish neurotransmission; they only decrease quantal content. So how could this be relevant to the behavioral endpoint that is general anesthesia? With most animal brains comprising anywhere between millions and trillions of synapses, it seems plausible that normal brain functions would be compromised if syntaxin1A mobility became globally restricted across a variety of synapses following exposure to general anesthetics. While a decrease in quantal content may not significantly impair some muscular (or spinal cord) functions, it is likely that a similar effect on central synapses would dramatically change temporal dynamics in the brain, leading to a loss of functional connectivity. In support of this view, recent electroencephalogram (EEG) and fMRI studies have shown that functional connectivity throughout the brain is significantly altered in patients undergoing general anesthesia. Thus, other manipulations that compromise presynaptic communication, including effects on presynaptic excitability , might fall into the same category of anesthetic mechanisms as the syntaxin1A-mediated effects described in this study, that may be considered a class of effects that is distinct from GABAergic sleep-related mechanisms. One possibility, which has been raised previously, is that GABAergic processes (e.g., sedation and loss of consciousness) are induced at lower drug doses (e.g., < 1 µM propofol), while the presynaptic processes discussed in this study are affected at the slightly higher concentrations required for surgery. It remains unknown however whether other general anesthetics target presynaptic mechanisms. A recent study using hippocampal cultures found that isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx rather than Ca2+-exocytosis coupling. In contrast, the current results suggest that propofoland etomidate-mediated presynaptic effects might be directly coupled to the exocytosis machinery. Whether this is a difference between intravenous and volatile anesthetics is unclear. Nevertheless, a set of distinct presynaptic mechanisms linked to exocytosis might explain why recovery from general anesthesia appears to involve a different process than anesthesia induction; re-establishing functional connectivity after neurotransmission has returned to normal levels across the brain would likely involve a different process than falling asleep or waking up. It will be interesting in future research to use transgenic syntaxin1A animals to link the local effects found at the presynapse with consequent changes in global readouts, such as whole-brain connectivity and coherence (Bademosi, 2018).

    Syntaxin1A neomorphic mutations promote rapid recovery from isoflurane anesthesia in Drosophila melanogaster

    Syntaxin1A is a presynaptic molecule that plays a key role in vesicular neurotransmitter release. Mutations of syntaxin1A result in resistance to both volatile and intravenous anesthetics. Truncated syntaxin1A isoforms confer drug resistance in cell culture and nematode models of anesthesia Resistance to isoflurane anesthesia can be produced by transiently expressing truncated syntaxin1A proteins in adult Drosophila flies. Electrophysiologic and behavioral studies in Drosophila show that mutations in syntaxin1A facilitate recovery from isoflurane anesthesia. These observations suggest that presynaptic mechanisms, via syntaxin1A-mediated regulation of neurotransmitter release, are involved in general anesthesia maintenance and recovery Mutations in the presynaptic protein syntaxin1A modulate general anesthetic effects in vitro and in vivo. Coexpression of a truncated syntaxin1A protein confers resistance to volatile and intravenous anesthetics, suggesting a target mechanism distinct from postsynaptic inhibitory receptor processes. Hypothesizing that recovery from anesthesia may involve a presynaptic component, whether synatxin1A mutations facilitated recovery from isoflurane anesthesia in Drosophila melanogaster was tested. The same neomorphic syntaxin1A mutation that confers isoflurane resistance in cell culture and nematodes also produces isoflurane resistance in Drosophila. Resistance in Drosophila is, however, most evident at the level of recovery from anesthesia, suggesting that the syntaxin1A target affects anesthesia maintenance and recovery processes rather than induction. The absence of truncated syntaxin1A from the presynaptic complex suggests that the resistance-promoting effect of this molecule occurs before core complex formation (Troup, 2019).

    There is growing evidence that general anesthetics target presynaptic mechanisms in addition to postsynaptic receptors. For example, clinical concentrations of both intravenous and volatile anesthetics have been found to impair neurotransmission. Syntaxin1A plays a key role in neurotransmission, presenting a crucial endpoint for synaptic vesicle release, without which neurotransmission could not occur. Mutations in this protein produce both hypersensitivity and resistance to volatile anesthetics in nematode worms and Drosophila flies, suggesting that the protein may be proximal to a presynaptic target for these drugs. Coexpression of a truncated syntaxin1A protein has been shown to produce resistance to volatile anesthetics in nematode worms and mammalian neurosecretory cells,mas well as resistance to the intravenous anesthetic propofol in mammalian cells. How exactly this neomorphic syntaxin1A protein protects synaptic release from the effect of general anesthetics remains unclear, although an interaction with other presynaptic-related proteins seems likely (Troup, 2019).

    Most explanations of general anesthesia relate to postsynaptic targets. However, should general anesthesia comprise at least two distinct target domains, one postsynaptic and one presynaptic, this is likely to be reflected in the different kinetics of anesthesia induction and recovery. Many general anesthetics have rapid induction and slower recovery kinetics. This recovery inertia has been proposed as evidence that different processes might be involved during induction and recovery. One idea that has been proposed is that induction is rapid because it primarily reflects the sedative properties of these drugs, and the loss of consciousness associated with sleep is a rapid process. However, recovery time can vary significantly, and some patients report incomplete recovery for days or even months after the procedure. It has been have proposed that recovery inertia reflects in part presynaptic processes, in contrast to the rapid induction kinetics which are understood to reflect postsynaptic processes. Therefore, manipulations of presynaptic proteins that preserve neurotransmission under isoflurane anesthesia in vitro should reduce the recovery time required after the procedure, in vivo. This study tested this in an animal model, Drosophila melanogaster (Troup, 2019).

    General anesthesia is fundamentally a behavioral endpoint, and understanding its mechanisms of action requires methods to probe behavioral responsiveness in behaving animals. Because the sedative component of general anesthesia most likely engages sleep-promoting pathways in the brain, it was decided to use Drosophila, which have sleep-promoting neurons that have been found to be involved in isoflurane anesthesia (Troup, 2013). Drosophila is an established model to study general anesthesia. Assays for probing sleep intensity or behavioral responsiveness are also well developed for Drosophila, providing an effective way of assessing the role of syntaxin1A in isoflurane induction and recovery. A tagged, truncated version of syntaxin1A was developed could be expressed in fly neurons, to determine how this affected isoflurane induction and recovery for behavioral endpoints as well as for neurotransmission. It was hypothesized that syntaxin1A effects on recovery from isoflurane anesthesia would be reflected across these different levels of investigation (Troup, 2019).

    This study found that deletion mutations in syntaxin1A, when coexpressed alongside wild-type syntaxin1A in Drosophila melanogaster, significantly reduce the recovery time after isoflurane anesthesia. Whereas resistance to isoflurane was also evident during anesthesia induction, this effect was weaker compared with effects on recovery, in adult flies. This suggests that recovery from isoflurane anesthesia depends at least in part on syntaxin1A function. It was surprising how long adult wild-type flies required to regain normal levels of behavioral responsiveness after the procedure, compared with regaining locomotion; behavioral responsiveness still remained impaired even after two hours. In contrast, the syntaxin1A mutants could recover behavioral responsiveness after 10min. Delayed recovery of behavioral responsiveness in Drosophila may therefore be a promising model for studying cognitive impairments following general anesthesia in humans, which also often follow a longer time course than simply regaining consciousness. A full restoration of presynaptic functions across the brain is probably a complex problem in any animal, and the extremely conserved nature of synaptic release mechanisms suggests this might be a common mechanism (Troup, 2019).

    syntaxin1A manipulations improved anesthesia recovery times across entirely different levels of analysis. Examination of effects on neurotransmission at the fly neuromuscular junction corroborated behavioral findings: recovery of quantal content occurred within 5min in the syntaxin1A mutant animals. Although it is unknown what neurotransmission recovery dynamics might be like in adult brain synapses, it seems likely that similar effects on recovery might be present because the same syntaxin1A protein is involved in the brain as at the larval neuromuscular junction, or in all animal synapses. Mutant Drosophila larvae also recovered faster than controls behaviorally (within 5 min), indicating an effect that transcends brains at different levels of complexity (the larval brain has an order of magnitude fewer neurons than the adult fly brain). It remains unknown, however, whether adult brain synapses are affected in the same way as motor synapses. Behavioral recovery dynamics in adult mutant animals remain more sluggish than recovery of quantal content at the larval neuromuscular junction. This suggests that brain synapses might recover function differently than motor nerve terminals in larvae (Troup, 2019).

    One limitation of this study was the use of only female flies for behavioral experiments in adults. Sex-specific effects in Drosophila have been observed during recovery from anesthesia, although these effects generalize to cold-shock and oxygen deprivation anesthesia, which are likely unrelated to the presynaptic mechanisms described in this study. Given the lack of sexual dimorphism in synaptic proteins, it is unlikely that the phenotypes described in this study would be different using male flies, although this remains to be tested experimentally. In the larval studies both male and female animals were used. Despite any potential sexual differentiation in larval motor nerve terminals, significant effects were still found in the larval isoflurane experiments with expression of the truncated syntaxin1A protein (Troup, 2019).

    How might a coexpressed syntaxin1A truncation construct be conferring a rapid recovery from isoflurane anesthesia? This same manipulation has now been shown to produce resistance to diverse general anesthetics across a variety of systems, in vitro and in vivo. Because syntaxin1A is a key player in SNARE-mediated exocytosis, it was therefore surprising to find that the HA-tagged truncated protein syx227 was not present in soluble nethylmaleimide sensitive factor attachment protein receptor complexes, at least in Drosophila. Because syx227 has been shown to interact with synaptosomal associated protein 25 (SNAP25) and wild-type syntaxin1A, this suggests an effect immediately before SNARE formation, meaning the truncated protein is probably ejected upon full soluble n-ethylmaleimide sensitive factor attachment protein receptor formation (when vesicle-associated membrane protein 2 links with syntaxin1A and SNAP25 to form a release ready tetrameric complex). This would imply that the protective effect of this protein is required before SNARE formation, and accordingly that the anesthetic effect on syntaxin1A function is also prior to soluble nethylmaleimide sensitive factor attachment protein receptor formation. This view is consistent with recent findings using super-resolution microscopy to track the mobility of single syntaxin1A molecules under propofol anesthesia (Troup, 2019).

    It was found that clustering of syntaxin1A caused by propofol was dependent on an interaction with SNAP25, but not with vesicle-associated membrane protein 2, thereby suggesting a mechanism of action (for propofol) immediately before full SNARE formation. Work in other systems also suggests an interaction between volatile anesthetics and syntaxin1A/SNAP25 suggesting that this might indeed by a general anesthetic target. On the other hand, work in Caenorhabditis elegans, where the effect of the truncated syntaxin1A was discovered, points to unc-13 as a likely mediator of this resistance-promoting mechanism. unc-13 is understood to be associated with presynaptic active zones, where the SNAREs ultimately reside, so one interpretation of these diverse findings is that the drug-mediated clustering of syntaxin1A/SNAP25 occurs at these active zones, and that these pre-SNARE complexes are prevented from transforming into full SNAREs because unc-13 is less able to catalyze the next step. In this regard, it will be especially interesting to investigate what role unc-13 plays in this process; unc-13 has been shown to keep syntaxin1A in a closed conformation, until interaction with unc-13 opens syntaxin1A to promote complete full SNARE formation. One hypothesis consistent with this model is that general anesthetics promote a closed syntaxin1A conformation, by for example impairing the capacity of unc-13 to catalyze SNARE formation. One hypothesis for how syx227 affords resistance then is that the truncated (or deletion) proteins might promote open syntaxin1A moieties, and in this way remove an emergent target (the closed syntaxin1A-unc-18 complex). Interactions with vesicle-bound vesicle-associated membrane protein 2 would then lead to an energetically more favorable ternary complex, effectively ejecting syx227 upon SNARE formation. Future biochemical experiments should determine whether syx227 promotes an open syntaxin1A conformation, and to what level unc-13 and unc-18 are involved in this process (Troup, 2019).

    One of the most striking observations in this this study is the prolonged duration of recovery from isoflurane anesthesia in wild-type flies, and how syntaxin1A mutations significantly reduce this recovery time. If syx227 is acting before SNARE formation, then how might this lead to faster recovery? One possibility following from the hypothesis proposed above is that syx227 provides more efficient access to already open pre-SNARE complexes that are ready to be incorporated into fully formed SNAREs. If general anesthetics produce a traffic jam of nonfunctional pre-SNARE nanoclusters, as suggested by single-molecule imaging experiments, then the time required for dissolving this proteinaceous traffic jam might take longer than clearance of the anesthetic drugs themselves. Consistent with this view, imaging work showed that expression of syx227 in mammalian cells prevented the syntaxin1A clustering effects of another general anesthetic, propofol. In contrast to these sluggish presynaptic recovery effects, the postsynaptic effects of general anesthetics such as isoflurane and propofol are most likely rapid, as they primarily linked to gamma-aminobutyric acid receptor function. General anesthesia induction is a rapid process, as this probably engages potent inhibitory systems in the brain that are designed to promote a rapid loss of consciousness. However, a rapid reversal of the effect on gamma-aminobutyric acid receptors after removal of these drugs might have little consequence on recovery from anesthesia until presynaptic processes across the brain have been fully restored. The data on syntaxin1A fly mutants exposed to isoflurane support this view of general anesthesia, with the largest effects seen for recovery rather than induction. However, the fact that these mutants are also resistant to isoflurane upon induction suggests that presynaptic effects might play a role during anesthesia induction as well. It will be interesting in future experiments to combine genetic manipulations that promote anesthetic resistance at both a pre and postsynaptic levels, in animals that have both target mechanisms (i.e., sleep/wake pathways and SNAREs). Such experiments will allow better dissecting of the relative contributions of either target process, and to determine whether some circuits or neurotransmitter systems are more affected by the presynaptic mechanisms this study has uncovered (Troup, 2019).

    Integrated information structure collapses with anesthetic loss of conscious arousal in Drosophila melanogaster

    The physical basis of consciousness remains one of the most elusive concepts in current science. One influential conjecture is that consciousness is to do with some form of causality, measurable through information. The integrated information theory of consciousness (IIT) proposes that conscious experience, filled with rich and specific content, corresponds directly to a hierarchically organised, irreducible pattern of causal interactions; i.e. an integrated informational structure among elements of a system. This study tested this conjecture in a simple biological system (fruit flies), estimating the information structure of the system during wakefulness and general anesthesia. Consistent with this conjecture, it was found that integrated interactions among populations of neurons during wakefulness collapsed to isolated clusters of interactions during anesthesia. Classification analysis to quantify the accuracy of discrimination between wakeful and anesthetised states, and found that informational structures inferred conscious states with greater accuracy than a scalar summary of the structure, a measure which is generally championed as the main measure of IIT. In stark contrast to a view which assumes feedforward architecture for insect brains, especially fly visual systems, rich information structures were found, which cannot arise from purely feedforward systems, occurred across the fly brain. Further, these information structures collapsed uniformly across the brain during anesthesia. The results speak to the potential utility of the novel concept of an "informational structure" as a measure for level of consciousness, above and beyond simple scalar values (Leung, 2021).

    'Hangry' Drosophila: food deprivation increases male aggression

    Aggressive interactions are costly, such that individuals should display modified aggression in response to environmental stress. Many organisms experience frequent periods of food deprivation, which can influence an individual's capacity and motivation to engage in aggression. However, because food deprivation can simultaneously decrease an individual's resource-holding potential and increase its valuation of food resources, its net impact on aggression is unclear. This study tested the influence of increasingly prolonged periods of adult food deprivation on inter-male aggression in pairs of fruit flies, Drosophila melanogaster. Males displayed increased aggression following periods of food deprivation longer than a day. Increased aggression in food-deprived flies occurred despite their reduced mass. This result is probably explained by an increased attraction to food resources, as food deprivation increased male occupancy of central food patches, and food patch occupancy was positively associated with aggression. These findings demonstrate that aggressive strategies in male D. melanogaster are influenced by nutritional experience, highlighting the need to consider past nutritional stresses to understand variation in aggression (Edmunds, 2021).

    Mutations in Complex I of the Mitochondrial Electron-Transport Chain Sensitize the Fruit Fly (Drosophila melanogaster) to Ether and Non-Ether Volatile Anesthetics

    The mitochondrial electron transport chain (mETC) contains molecular targets of volatile general anesthetics (VGAs), which places carriers of mutations at risk for anesthetic complications. The ND-2360114 and mt:ND2del1 lines of fruit flies (Drosophila melanogaster) that carry mutations in core subunits of Complex I of the mETC replicate numerous characteristics of Leigh syndrome (LS) caused by orthologous mutations in mammals and serve as models of LS. ND-2360114 flies are behaviorally hypersensitive to volatile anesthetic ethers and develop an age- and oxygen-dependent anesthetic-induced neurotoxicity (AiN) phenotype after exposure to isoflurane but not to the related anesthetic sevoflurane. The goal of this paper was to investigate whether the alkane volatile anesthetic halothane and other mutations in Complex I and in Complexes II-V of the mETC cause AiN. It was found that (1) ND-2360114 and mt:ND2del1 were susceptible to toxicity from halothane; (2) in wild-type flies, halothane was toxic under anoxic conditions; (3) alleles of accessory subunits of Complex I predisposed to AiN; and (iv) mutations in Complexes II-V did not result in an AiN phenotype. It is concluded that AiN is neither limited to ether anesthetics nor exclusive to mutations in core subunits of Complex I (Borchardt, 2023)

    Long-term sevoflurane exposure resulted in temporary rather than lasting cognitive impairment in Drosophila

    Sevoflurane is the primary inhaled anesthetic used in pediatric surgery. It has been the focus of research since animal models studies found that it was neurotoxic to the developing brain two decades ago. However, whether pediatric general anesthesia can lead to permanent cognitive deficits remained a subject of heated debate. Therefore, this study aims to determine the lifetime neurotoxicity of early long-time sevoflurane exposure using a short-life-cycle animal model, Drosophila melanogaster. To investigate this question, the lifetime changes of two-day-old flies' learning and memory abilities after anesthesia with 3 % sevoflurane for 6 h by the T-maze memory assay. Apoptosis, levels of ATP and ROS, and related genes were evaluated in the fly head. The results suggest that 6 h 3 % sevoflurane exposure at a young age can only induce transient neuroapoptosis and cognitive deficits around the first week after anesthesia. But this brain damage recedes with time and vanishes in late life. It was also found that the mRNA level of caspases and Bcl-2, ROS level, and ATP level increased during this temporary neuroapoptosis process. And mRNA levels of antioxidants, such as SOD2 and CAT, increased and decreased simultaneously with the rise and fall of the ROS level, indicating a possible contribution to the recovery from the sevoflurane impairment. In conclusion, these results suggest that one early prolonged sevoflurane-based general anesthesia can induce neuroapoptosis and learning and memory deficit transiently but not permanently in Drosophila (Liu, 2023).

    Neural Ensemble Fragmentation in the Anesthetized Drosophila Brain

    General anesthetics cause a profound loss of behavioral responsiveness in all animals. In mammals, general anesthesia is induced in part by the potentiation of endogenous sleep-promoting circuits, although "deep" anesthesia is understood to be more similar to coma. Surgically relevant concentrations of anesthetics, such as isoflurane and propofol, have been shown to impair neural connectivity across the mammalian brain, which presents one explanation why animals become largely unresponsive when exposed to these drugs. It remains unclear whether general anesthetics affect brain dynamics similarly in all animal brains, or whether simpler animals, such as insects, even display levels of neural connectivity that could be disrupted by these drugs. This study used whole-brain calcium imaging in behaving female Drosophila flies to investigate whether isoflurane anesthesia induction activates sleep-promoting neurons, and then inquired how all other neurons across the fly brain behave under sustained anesthesia. It was possible to track the activity of hundreds of neurons simultaneously during waking and anesthetized states, for spontaneous conditions as well as in response to visual and mechanical stimuli. Whole-brain dynamics and connectivity were compared under isoflurane exposure to optogenetically induced sleep. Neurons in the Drosophila brain remain active during general anesthesia as well as induced sleep, although flies become behaviorally inert under both treatments. This study identified surprisingly dynamic neural correlation patterns in the waking fly brain, suggesting ensemble-like behavior. These become more fragmented and less diverse under anesthesia but remain wake-like during induced sleep (Troup, 2023).

    Pathogenic bacteria enhance dispersal through alteration of Drosophila social communication

    Pathogens and parasites can manipulate their hosts to optimize their own fitness. For instance, bacterial pathogens have been shown to affect their host plants' volatile and non-volatile metabolites, which results in increased attraction of insect vectors to the plant, and, hence, to increased pathogen dispersal. Behavioral manipulation by parasites has also been shown for mice, snails and zebrafish as well as for insects. This study shows that infection by pathogenic bacteria alters the social communication system of Drosophila melanogaster. More specifically, infected flies and their frass emit dramatically increased amounts of fly odors, including the aggregation pheromones methyl laurate, methyl myristate, and methyl palmitate, attracting healthy flies, which in turn become infected and further enhance pathogen dispersal. Thus, olfactory cues for attraction and aggregation are vulnerable to pathogenic manipulation, and the alteration of social pheromones can be beneficial to the microbe while detrimental to the insect host. Behavioral manipulation of host by pathogens has been observed in vertebrates, invertebrates, and plants. This study shows that in Drosophila, infection with pathogenic bacteria leads to increased pheromone release, which attracts healthy flies. This process benefits the pathogen since it enhances bacterial dispersal, but is detrimental to the host (Keesey, 2017).

    Robust manipulation of the behavior of Drosophila melanogaster by a fungal pathogen in the laboratory

    Many microbes induce striking behavioral changes in their animal hosts, but how they achieve this is poorly understood, especially at the molecular level. Mechanistic understanding has been largely constrained by the lack of an experimental system amenable to molecular manipulation. A strain of the behavior-manipulating fungal pathogen Entomophthora muscae infects wild Drosophila, and methods were established to infect D. melanogaster in the lab. Lab-infected flies manifest the moribund behaviors characteristic of E. muscae infection: hours before death, they climb upward, extend their proboscides, affixing in place, then raise their wings, clearing a path for infectious spores to launch from their abdomens. E. muscae was found to invade the nervous system, suggesting a direct means by which the fungus could induce behavioral changes. Given the vast molecular toolkit available for D. melanogaster, this new system will enable rapid progress in understanding how E. muscae manipulates host behavior (Elya, 2018).

    Statistical modelling of navigational decisions based on intensity versus directionality in Drosophila larval phototaxis

    Many species are able to share information about their environment by communicating through auditory, visual, and olfactory cues. In Drosophila melanogaster, exposure to parasitoid wasps leads to a decline in egg laying, and exposed females communicate this threat to naive flies, which also depress egg laying. This study found that species across the genus Drosophila respond to wasps by egg laying reduction, activate cleaved caspase in oocytes, and communicate the presence of wasps to naive individuals. Communication within a species and between closely related species is efficient, while more distantly related species exhibit partial communication. Remarkably, partial communication between some species is enhanced after a cohabitation period that requires exchange of visual and olfactory signals. This interspecies "dialect learning" requires neuronal cAMP signaling in the mushroom body, suggesting neuronal plasticity facilitates dialect learning and memory. These observations establish Drosophila as genetic models for interspecies social communication and evolution of dialects (Kacsoh, 2018).

    Social environment mediates cancer progression in Drosophila

    The influence of oncogenic phenomena on the ecology and evolution of animal species is becoming an important research topic. Similar to host-pathogen interactions, cancer negatively affects host fitness, which should lead to the selection of host control mechanisms, including behavioral traits that best minimize the proliferation of malignant cells. Social behavior is suggested to influence tumor progression. While the ecological benefits of sociality in gregarious species are widely acknowledged, only limited data are available on the role of the social environment on cancer progression. This study exposed adult Drosophila, with colorectal-like tumors, to different social environments. Subtle variations in social structure have dramatic effects on the progression of tumor growth. Finally, it is revealed that flies can discriminate between individuals at different stages of tumor development and selectively choose their social environment accordingly. This study demonstrates the reciprocal links between cancer and social interactions and how sociality may impact health and fitness in animals and its potential implications for disease ecology (Dawson, 2018).

    A simple computer vision pipeline reveals the effects of isolation on social interaction dynamics in Drosophila

    Isolation profoundly influences social behavior in all animals. Longer-term analysis of large groups of flies is hampered by the lack of effective and reliable tools. In this study a new imaging arena was built and the existing tracking algorithm was improved to reliably follow a large number of flies simultaneously. Next, based on the automatic classification of touch and graph-based social network analysis, an algorithm was designed to quantify changes in the social network in response to prior social isolation. It was observed that isolation significantly and swiftly enhanced individual and local social network parameters depicting near-neighbor relationships. The genome-wide molecular correlates of these behavioral changes were explored, and it was found that whereas behavior changed throughout the six days of isolation, gene expression alterations occurred largely on day one. These changes occurred mostly in metabolic genes, and the metabolic changes were varified by showing an increase of lipid content in isolated flies. In summary, this study describes a highly reliable tracking and analysis pipeline for large groups of flies that were use to unravel the behavioral, molecular and physiological impact of isolation on social network dynamics in Drosophila (Liu, 2018).

    A plastic visual pathway regulates cooperative behavior in Drosophila larvae

    Cooperative behavior emerges in biological systems through coordinated actions among individuals. Although widely observed across animal species, the cellular and molecular mechanisms underlying the establishment and maintenance of cooperative behaviors remain largely unknown. To characterize the circuit mechanisms serving the needs of independent individuals and social groups, this study investigated cooperative digging behavior in Drosophila larvae. Although chemical and mechanical sensations are important for larval aggregation at specific sites, an individual larva's ability to participate in a cooperative burrowing cluster relies on direct visual input as well as visual and social experience during development. In addition, vision modulates cluster dynamics by promoting coordinated movements between pairs of larvae. To determine the specific pathways within the larval visual circuit underlying cooperative social clustering, larval photoreceptors (PRs) and the downstream local interneurons (lOLPs) were examined using anatomical and functional studies. The results indicate that rhodopsin-6-expressing-PRs (Rh6-PRs) and lOLPs are required for both cooperative clustering and movement detection. Remarkably, visual deprivation and social isolation strongly impact the structural and functional connectivity between Rh6-PRs and lOLPs, while at the same time having no effect on the adjacent rhodopsin-5-expressing PRs (Rh5-PRs). Together, these findings demonstrate that a specific larval visual pathway involved in social interactions undergoes experience-dependent modifications during development, suggesting that plasticity in sensory circuits could act as the cellular substrate for social learning, a possible mechanism allowing an animal to integrate into a malleable social environment and engage in complex social behaviors (Dombrovski, 2019).

    Neural circuitry of social learning in Drosophila requires multiple inputs to facilitate inter-species communication

    Drosophila species communicate the threat of parasitoid wasps to naive individuals. Communication of the threat between closely related species is efficient, while more distantly related species exhibit a dampened, partial communication. Partial communication between D. melanogaster and D. ananassae about wasp presence is enhanced following a period of cohabitation, suggesting that species-specific natural variations in communication 'dialects' can be learned through socialization. This study identified six regions of the Drosophila brain essential for dialect training. Subgroups of neurons in these regions were identified, including motion detecting neurons in the optic lobe, layer 5 of the fan-shaped body, the D glomerulus in the antennal lobe, and the odorant receptor Or69a, where activation of each component is necessary for dialect learning. These results reveal functional neural circuits that underlie complex Drosophila social behaviors, and these circuits are required for integration several cue inputs involving multiple regions of the Drosophila brain (Kacsoh, 2019).

    Emergence of social cluster by collective pairwise encounters in Drosophila

    Many animals exhibit an astonishing ability to form groups of large numbers of individuals. The dynamic properties of such groups have been the subject of intensive investigation. The actual grouping processes and underlying neural mechanisms, however, remain elusive. This study established a social clustering paradigm in Drosophila to investigate the principles governing social group formation. Fruit flies spontaneously assembled into a stable cluster mimicking a distributed network. Social clustering was exhibited as a highly dynamic process including all individuals, which participated in stochastic pair-wise encounters mediated by appendage touches. Depriving sensory inputs resulted in abnormal encounter responses and a high failure rate of cluster formation. Furthermore, the social distance of the emergent network was regulated by ppk-specific neurons, which were activated by contact-dependent social grouping. Taken together, these findings revealed the development of an orderly social structure from initially unorganised individuals via collective actions (Jiang, 2020).

    Individual, but not population asymmetries, are modulated by social environment and genotype in Drosophila melanogaster

    Theory predicts that social interactions can induce an alignment of behavioral asymmetries between individuals (i.e., population-level lateralization), but evidence for this effect is mixed. To understand how interaction with other individuals affects behavioral asymmetries, this study systematically manipulated the social environment of Drosophila melanogaster, testing individual flies and dyads (female-male, female-female and male-male pairs). In these social contexts individual and population asymmetries in individual behaviors (circling asymmetry, wing use) and dyadic behaviors (relative position and orientation between two flies) were measured in five different genotypes. It was reasoned that if coordination between individuals drives alignment of behavioral asymmetries, greater alignment at the population-level should be observed in social contexts compared to solitary individuals. It was observed that the presence of other individuals influenced the behavior and position of flies but had unexpected effects on individual and population asymmetries: individual-level asymmetries were strong and modulated by the social context but population-level asymmetries were mild or absent. Moreover, the strength of individual-level asymmetries differed between strains, but this was not the case for population-level asymmetries. These findings suggest that the degree of social interaction found in Drosophila is insufficient to drive population-level behavioral asymmetries (Versace, 2020).

    Aralar Sequesters GABA into Hyperactive Mitochondria, Causing Social Behavior Deficits

    Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. This study shows that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. The regulation of GABA availability by mitochondrial activity was identified as a biologically relevant mechanism, and its contribution to social behavior was identified. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. Aralar was identified as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions (Kanellopoulos, 2020).

    Behavioral and environmental contributions to drosophilid social networks

    Animals interact with each other in species-specific reproducible patterns. These patterns of organization are captured by social network analysis, and social interaction networks (SINs) have been described for a wide variety of species including fish, insects, birds, and mammals. The aim of this study is to understand the evolution of social organization in Drosophila. Using a comparative ecological, phylogenetic, and behavioral approach, the different properties of SINs formed by 20 drosophilids were compared. Whether drosophilid network structures arise from common ancestry, a response to the species' past climate, other social behaviors, or a combination of these factors was investigated. This study shows that differences in past climate predicted the species' current SIN properties. The drosophilid phylogeny offered no value to predicting species' differences in SINs through phylogenetic signal tests. This suggests that group-level social behaviors in drosophilid species are shaped by divergent climates. However, it was found that the social distance at which flies interact correlated with the drosophilid phylogeny, indicating that behavioral elements of SINs have remained largely unchanged in their evolutionary history. A significant correlation was found of leg length to social distance, outlining the interdependence of anatomy and complex social structures. Although SINs display a complex evolutionary relationship across drosophilids, this study suggests that the ecology, and not common ancestry, contributes to diversity in social structure in Drosophila (Jezovit, 2020).

    Drosophila melanogaster behaviour changes in different social environments based on group size and density

    Many organisms, when alone, behave differently from when they are among a crowd. Drosophila similarly display social behaviour and collective behaviour dynamics within groups not seen in individuals. In flies, these emergent behaviours may be in response to the global size of the group or local nearest-neighbour density. This study investigated i) which aspect of social life flies respond to: group size, density, or both and ii) whether behavioural changes within the group are dependent on olfactory support cells. Behavioural assays demonstrate that flies adjust their interactive behaviour to group size but otherwise compensate for density by achieving a standard rate of movement, suggesting that individuals are aware of the number of others within their group. Olfactory support cells are necessary for flies to behave normally in large groups. These findings shed insight into the subtle and complex life of Drosophila within a social setting (Rooke, 2020).

    Early Life Experience Shapes Male Behavior and Social Networks in Drosophila

    Living in a group creates a complex and dynamic environment in which behavior of individuals is influenced by and affects the behavior of others. Although social interaction and group living are fundamental adaptations exhibited by many organisms, little is known about how prior social experience, internal states, and group composition shape behavior in groups. This study presents an analytical framework for studying the interplay between social experience and group interaction in Drosophila melanogaster. The complexity of interactions in a group was simplified using a series of experiments in which the social experience and motivational states of individuals were controlled to compare behavioral patterns and social networks of groups under different conditions. Social enrichment promotes the formation of distinct group structure that is characterized by high network modularity, high inter-individual and inter-group variance, high inter-individual coordination, and stable social clusters. Using environmental and genetic manipulations, this study showed that visual cues and cVA-sensing neurons are necessary for the expression of social interaction and network structure in groups. Finally, the formation of group behavior and structure was exploited in heterogenous groups composed of flies with distinct internal states, and emergent structures were documented that are beyond the sum of the individuals that constitute it. These results demonstrate that fruit flies exhibit complex and dynamic social structures that are modulated by the experience and composition of different individuals within the group. This paves the path for using simple model organisms to dissect the neurobiology of behavior in complex social environments (Bentzur, 2020).

    Social competition stimulates cognitive performance in a sex-specific manner

    Social interactions are thought to be a critical driver in the evolution of cognitive ability. Cooperative interactions, such as pair bonding, rather than competitive interactions have been largely implicated in the evolution of increased cognition. This is despite competition traditionally being a very strong driver of trait evolution. Males of many species track changes in their social environment and alter their reproductive strategies in response to anticipated levels of competition. This study predicts this to be cognitively challenging. Using a Drosophila melanogaster model, it was possible to distinguish between the effects of a competitive environment versus generic social contact by exposing flies to same-sex same-species competition versus different species partners, shown to present non-competitive contacts. Males increase olfactory learning/memory and visual memory after exposure to conspecific males only, a pattern echoed by increased expression of synaptic genes and an increased need for sleep. For females, largely not affected by mating competition, the opposite pattern was seen. The results indicate that specific social contacts dependent on sex, not simply generic social stimulation, may be an important evolutionary driver for cognitive ability in fruit flies (Rouse, 2020).

    The neural basis for a persistent internal state in Drosophila females

    Sustained changes in mood or action require persistent changes in neural activity, but it has been difficult to identify the neural circuit mechanisms that underlie persistent activity and contribute to long-lasting changes in behavior. This study shows that a subset of Doublesex+ pC1 neurons in the Drosophila female brain, called pC1d/e, can drive minutes-long changes in female behavior in the presence of males. Using automated reconstruction of a volume electron microscopic (EM) image of the female brain, all inputs and outputs to both pC1d and pC1e were mapped. This reveals strong recurrent connectivity between, in particular, pC1d/e neurons and a specific subset of Fruitless+ neurons called aIPg. This study additionally found that pC1d/e activation drives long-lasting persistent neural activity in brain areas and cells overlapping with the pC1d/e neural network, including both Doublesex+ and Fruitless+ neurons. This work thus links minutes-long persistent changes in behavior with persistent neural activity and recurrent circuit architecture in the female brain (Deutsch, 2020).

    Social attraction in Drosophila is regulated by the mushroom body and serotonergic system

    Sociality is among the most important motivators of human behaviour. However, the neural mechanisms determining levels of sociality are largely unknown, primarily due to a lack of suitable animal models. This study reports the presence of a surprising degree of general sociality in Drosophila. A newly-developed paradigm to study social approach behaviour in flies reveal that social cues perceive through both vision and olfaction converged in a central brain region, the γ lobe of the mushroom body, which exhibit activation in response to social experience. The activity of these γ neurons control the motivational drive for social interaction. At the molecular level, the serotonergic system is critical for social affinity. These results demonstrate that Drosophila are highly sociable, providing a suitable model system for elucidating the mechanisms underlying the motivation for sociality (Sun, 2020).

    Transcriptome Analysis of NPFR Neurons Reveals a Connection Between Proteome Diversity and Social Behavior

    Social behaviors are mediated by the activity of highly complex neuronal networks, the function of which is shaped by their transcriptomic and proteomic content. Contemporary advances in neurogenetics, genomics, and tools for automated behavior analysis make it possible to functionally connect the transcriptome profile of candidate neurons to their role in regulating behavior. This study used Drosophila melanogaster to explore the molecular signature of neurons expressing receptor for neuropeptide F (NPF), the fly homolog of neuropeptide Y (NPY). By comparing the transcription profile of NPFR neurons to those of nine other populations of neurons, this study discovered that NPFR neurons exhibit a unique transcriptome, enriched with receptors for various neuropeptides and neuromodulators, as well as with genes known to regulate behavioral processes, such as learning and memory. By manipulating RNA editing and protein ubiquitination programs specifically in NPFR neurons, this study demonstrated that the proper expression of their unique transcriptome and proteome is required to suppress male courtship and certain features of social group interaction. The results highlight the importance of transcriptome and proteome diversity in the regulation of complex behaviors and pave the path for future dissection of the spatiotemporal regulation of genes within highly complex tissues, such as the brain (Ryvkin, 2021).

    The Drosophila melanogaster foraging gene affects social networks

    Drosophila melanogaster displays social behaviors including courtship, mating, aggression, and group foraging. Recent studies employed social network analyses (SNAs) to show that D. melanogaster strains differ in their group behavior, suggesting that genes influence social network phenotypes. Aside from genes associated with sensory function, few studies address the genetic underpinnings of these networks. The foraging gene (for) is a well-established example of a pleiotropic gene that regulates multiple behavioral phenotypes and their plasticity. In D. melanogaster, there are two naturally occurring alleles of for called rover and sitter that differ in their larval and adult food-search behavior as well as other behavioral phenotypes. It was hypothesized that for affects behavioral elements required to form social networks and the social networks themselves. These effects are evident when gene dosage was manipulated. Flies of the rover and sitter strains were found to exhibit differences in duration, frequency, and reciprocity of pairwise interactions, and they form social networks with differences in assortativity and global efficiency. Consistent with other adult phenotypes influenced by for, rover-sitter heterozygotes show intermediate patterns of dominance in many of these characteristics. Multiple generations of backcrossing a rover allele into a sitter strain showed that many but not all of these rover-sitter differences may be attributed to allelic variation at for. These findings reveal the significant role that for plays in affecting social network properties and their behavioral elements in Drosophila melanogaster (Alwash, 2021).

    Social perception of young adults prolongs the lifespan of aged Drosophila

    Lifespan is modulated at distinct levels by multiple factors, including genetic backgrounds, the environment, behavior traits, metabolic status, and more interestingly, sensory perceptions. However, the effects of social perception between individuals living in the same space remain less clear. This study used the Drosophila model to study the influences of social perception on the lifespan of aged fruit flies. The lifespan of aged Drosophila was found to be markedly prolonged after being co-housed with young adults of the same gender. Moreover, the changes of lifespan were affected by several experimental contexts: (1) the ratios of aged and young adults co-housed, (2) the chronological ages of two populations, and (3) the integrity of sensory modalities. Together, it is hypothesize the chemical/physical stimuli derived from the interacting young adults are capable of interfering with the physiology and behavior of aged flies, ultimately leading to the alteration of lifespan (Cho, 2021).

    Social environment drives sex and age-specific variation in Drosophila melanogaster microbiome composition and predicted function

    The composition of the microbiome (the assemblage of symbiotic microorganisms within a host) is determined by environmental factors and the host's immune, endocrine and neural systems. The social environment could alter host microbiomes extrinsically by affecting transmission between individuals. Alternatively, intrinsic effects arising from interactions between the microbiome and host physiology (the microbiota-gut-brain axis) could translate social stress into dysbiotic microbiomes, with consequences for host health. This study investigated how manipulating social environments during larval and adult life-stages altered the microbiome composition of Drosophila melanogaster fruit flies. Social contexts that particularly alter the development and lifespan of males were used, predicting that any intrinsic social effects on the microbiome would therefore be sex-specific. The presence of adult males during the larval stage significantly altered the microbiome of pupae of both sexes. In adults, same-sex grouping increased bacterial diversity in both sexes. Importantly, the microbiome community structure of males was more sensitive to social contact at older ages, an effect partially mitigated by housing focal males with young rather than coaged groups. Functional analyses suggest that these microbiome changes impact ageing and immune responses. This is consistent with the hypothesis that the substantial effects of the social environment on individual health are mediated through intrinsic effects on the microbiome, and provides a model for understanding the mechanistic basis of the microbiota-gut-brain axis (Leech, 2021).

    Comparing single- and mixed-species groups in fruit flies: differences in group dynamics, but not group formation

    Mixed-species groups describe active associations among individuals of 2 or more species at the same trophic level. Mixed-species groups are important to key ecological and evolutionary processes such as competition and predation, and research that ignores the presence of other species risks ignoring a key aspect of the environment in which social behavior is expressed and selected. Despite the defining emphasis of active formation for mixed-species groups, surprisingly little is known about the mechanisms by which mixed-species groups form. Furthermore, insects have been almost completely ignored in the study of mixed-species groups, despite their taxonomic importance and relative prominence in the study of single-species groups. In this study group formation processes were measured in Drosophila melanogaster and its sister species, Drosophila simulans. Each species was studied alone, and together, and one population of D. melanogaster was also studied both alone and with another, phenotypically distinct D. melanogaster population, in a nested-factorial design. This approach differs from typical methods of studying mixed-species groups in that group formation could be quantitatively compared between single-population, mixed-population, and mixed-species treatments. Surprisingly, no differences were found between treatments in the number, size, or composition of groups that formed, suggesting that single- and mixed-species groups form through similar mechanisms of active attraction. However, it was found that mixed-species groups showed elevated interspecies male-male interactions, relative to interpopulation or intergenotype interactions in single-species groups. These findings expand the conceptual and taxonomic study of mixed-species groups while raising new questions about the mechanisms of group formation broadly (Girardeau, 2021).

    A genetic screen for Drosophila social isolation mutants and analysis of sex pistol

    Prolonged periods of forced social isolation is detrimental to well-being, yet little is known about which genes regulate susceptibility to its effects. In the fruit fly, Drosophila melanogaster, social isolation induces stark changes in behavior including increased aggression, locomotor activity, and resistance to ethanol sedation. To identify genes regulating sensitivity to isolation, A collection of sixteen hundred P-element insertion lines was screened for mutants with abnormal levels of all three isolation-induced behaviors. The screen identified three mutants whose affected genes are likely central to regulating the effects of isolation in flies. One mutant, sex pistol (sxp), became extremely aggressive and resistant to ethanol sedation when socially isolated. sxp also had a high level of male-male courtship. The mutation in sxp reduced the expression of two minor isoforms of the actin regulator hts (adducin), as well as mildly reducing expression of CalpA, a calcium-dependent protease. As a consequence, sxp also had increased expression of the insulin-like peptide, dILP5. Analysis of the social behavior of sxp suggests that these minor hts isoforms function to limit isolation-induced aggression, while chronically high levels of dILP5 increase male-male courtship (Eddison, 2021).

    Chronic social isolation signals starvation and reduces sleep in Drosophila

    Social isolation and loneliness have potent effects on public health. Research in social psychology suggests that compromised sleep quality is a key factor that links persistent loneliness to adverse health conditions. Although experimental manipulations have been widely applied to studying the control of sleep and wakefulness in animal models, how normal sleep is perturbed by social isolation is unknown. This study reports that chronic, but not acute, social isolation reduces sleep in Drosophila. Quantitative behavioural analysis and transcriptome profiling were used to differentiate between brain states associated with acute and chronic social isolation. Although the flies had uninterrupted access to food, chronic social isolation altered the expression of metabolic genes and induced a brain state that signals starvation. Chronically isolated animals exhibit sleep loss accompanied by overconsumption of food. This resonates with anecdotal findings of loneliness-associated hyperphagia in humans. Chronic social isolation reduces sleep and promotes feeding through neural activities in the peptidergic fan-shaped body columnar neurons of the fly. Artificial activation of these neurons causes misperception of acute social isolation as chronic social isolation and thereby results in sleep loss and increased feeding. These results present a mechanistic link between chronic social isolation, metabolism, and sleep, addressing a long-standing call for animal models focused on loneliness (Li, 2021).

    Fruit flies are social animals, and exhibit dynamic social network structures and collective behaviours, that contribute to environmental sensing, foraging, feeding, fighting, mating, oviposition, circadian time setting and even the existence of 'culture'. These important aspects of social interactions imply that insects can provide suitable models for studying how the objective absence of social relationships is perceived and represented in the brain (Li, 2021).

    Social experience affects sleep need in Drosophila. This study revisited this finding by exploring how social isolation affects sleep in flies that have prior social experience. Sleep behaviour was tested after maintaining 1, 2, 5, 25 or 100 male flies in a food vial for 7 days. Group-housed flies, regardless of group size (2, 5, 25 or 100), exhibited similar sleep profiles. By contrast, flies housed in isolation displayed a significant loss of sleep, mainly distributed during the daytime (Li, 2021).

    The duration of social isolation was manipulated: flies were either isolated or housed in a group of 25 flies for 1, 3, 5, or 7 days, before sleep was measured in a Drosophila activity monitor (DAM). Sleep profiles, which display the proportion of time spent sleeping in consecutive 30-min segments over 24 h, showed that chronic social isolation (5 or 7 days) changed sleep architecture primarily during the daytime and especially during an interval of several hours following dawn (lights on). Although short durations of social isolation (1 or 3 days) did not induce sleep loss, chronic social isolation (5 or 7 days) significantly reduced daily total sleep, daytime sleep and sleep between Zeitgeber time (ZT) 0 and ZT4 (corresponding to the first 4 h after lights-on in a light-dark (LD) cycle) (Li, 2021).

    To assess how social isolation alters daytime sleep, all daytime sleep bouts were pooled from all animals tested for a given condition and their distributions were plotted as cumulative relative fractions for bout lengths. Acute social isolation (1 day) produced sleep bout distributions that were indistinguishable from those of 1-day group-housed flies. Flies that were socially isolated for 3 days showed slightly different sleep bout distributions from their group-housed counterparts. However, there was no deficit in total daily sleep, daytime sleep or ZT0-4 sleep in these flies. Flies that were isolated for longer periods (5 or 7 days) had sleep distributions that were significantly different from those of their group-housed counterparts. Cumulative relative frequency curves of daytime sleep bouts from chronically isolated flies climbed faster than those of their group-housed counterparts as shorter sleep bouts accumulated (5 or 7 days). Over seven days of isolation, daily total sleep, daytime sleep and ZT0-4 sleep all decreased progressively (Li, 2021).

    Age-matched flies were used to rule out the possibility that chronic social isolation induced sleep loss because the flies were older. Chronic social isolation (7 days) induced sleep loss consistently in various isogenic strains, in aged (4-week-old) wild-type flies, and in sleep inbred panel (SIP) strains with different baseline levels of sleep (Li, 2021).

    RNA sequencing (RNA-seq) libraries were prepared for three conditions: socially enriched flies (group treated, Grp), chronically isolated flies (isolated for 7 days, Iso_7D) and acutely isolated flies (isolated for 1 day, Iso_1D). Raster plots demonstrate sleep bouts of individual animals during a 24-h period measured immediately after group enrichment or social isolation. Daytime sleep was reduced and much more fragmented in chronically isolated flies than in group-housed or acutely isolated flies. Fly heads were collected between ZT0.5 and ZT2, a window of time within ZT0-4 that immediately preceded significant loss of daytime sleep in chronically isolated flies. Using differential gene expression analyses, intersectional and clustering strategies, candidate genes were identified for the sleep loss induced by chronic social isolation. These 214 candidate genes showed differences in expression in chronically isolated flies compared with both acutely isolated and group-housed flies and underwent unidirectional changes during the process of chronic social isolation. Gene ontology enrichment analysis suggested that these 214 genes are enriched for biological pathways associated with oxidation-reduction processes, one-carbon metabolic processes and carbohydrate metabolic processes. The rest of the gene ontology of biological pathways showed a strong preference for metabolic functions, such as fatty acid, pyruvate, glucose and amino acid metabolic processes. Consistent with the sleep loss phenotype, sleep was also among the most overrepresented gene ontologies for biological pathways (Li, 2021).

    Among the top 20 genes in this list, two genes stood out: Limostatin (Lst, CG8317), expression of which increased 1.67-fold after chronic isolation, and Drosulfakinin (Dsk), expression of which decreased 2.03-fold after chronic isolation. Limostatin is a decretin hormone that is induced by starvation and suppresses insulin release. Drosulfakinin, a satiety-inducing cholecystokinin-like peptide, is secreted when the animal is fed. As a signal of satiety, drosulfakinin is depleted under starvation conditions. A third gene, target of brain insulin (tobi), also showed significantly increased expression (1.76-fold) during chronic social isolation. tobi encodes an α-glucosidase that is regulated by Drosophila insulin and glucagon analogues. In addition, 7 of these top 20 genes and 32 of the total 214 candidate genes were previously identified as being regulated in Drosophila heads after 24 h of starvation. Thus, from a transcriptomic perspective, the brain of a fly maintained in chronic social isolation closely resembles the brain of a starving fly, despite continuous access to food. It was reasoned that such a 'starvation brain state' might broadly affect gene expression associated with metabolic processes. Massive changes in mitochondrial functions and oxidation-reduction processes could be direct consequences of starvation and/or elevated feeding (Li, 2021).

    The activity recording capillary feeder (ARC) assay, a video recording capillary feeder (CAFE) assay that monitors sleep and feeding behaviours simultaneously and continuously in individual Drosophila, was used. ARC assays validated the isolation-induced sleep loss phenotype previously observed with DAM assays: daily total sleep, daytime sleep and ZT0-4 sleep were reduced significantly after 7 days of social isolation. In addition, nighttime sleep was also reduced, probably owing to higher sensitivity in detecting movements using the positional tracking method, or differences in chamber shape and food source between the ARC and DAM systems. As predicted from the gene expression profiling results, increased feeding was observed in socially isolated animals compared to their group-treated counterparts. Flies isolated for 7 days showed significant increases in total food consumption, daytime food consumption, nighttime food consumption and ZT0-4 food consumption in comparison to flies that were group-housed for 7 days. Thus, chronic social isolation induces a starvation state in Drosophila at the levels of both gene expression in the brain and behaviour (Li, 2021).

    The altered feeding pattern produced by chronic social isolation is not merely a consequence of sleep loss, because several classic sleep mutants all exhibited normal feeding behaviour. In addition, acutely isolated flies did not show a strong increase in food consumption (Li, 2021).

    The candidate gene Lst is normally induced by nutrient restriction in endocrine neurons in the corpora cardiaca. However, the RNA profiling experiment suggested that there could be a previously unknown brain source for LST production. A resource of high-resolution transcriptomes of 100 GAL4 driver lines suggested that cells labelled by the driver line NPF-GAL4 (NPF, neuropeptide F [the fly homologue of neuropeptide Y)] are likely to express LST. Using a monoclonal antibody against LST, co-localized LST immunoreactivity and NPF-GAL4-driven GFP signals were detected. Among six known neuronal clusters that express NPF, LST immunoreactivity appeared to be co-localized with NPF-GAL4-driven GFP signals at the dorsal stratum of the fan-shaped body (dorsal fan-shaped body, dFB) and in a cluster of small cell bodies in the dorsal brain. Neurons without known function that comprise this cluster of NPF cells were previously named P2. A recent study used a split-GAL4 driver, SS0020-split-GAL4 (abbreviated as P2-GAL4 below), to strongly label the majority of P2 neurons that showed positive immunoreactivity for LST and NPF22 (Li, 2021).

    Notably, the projections of the P2 neurons overlapped with the axonal projections of the dFB neurons labelled by R23E10-GAL4, which suggests that P2 neurons might signal to sleep-promoting dFB neurons. At the cell body level, P2 neurons differ from the R23E10-GAL4 labelled cells. A MultiColor FlpOut (MCFO) approach was used to stochastically decorate individual neurons labelled by P2-GAL4, and it was found that they are fan-shaped body columnar neurons. The hemibrain connectome allowed determination that P2 neurons include, as a dominant constituent, the hDeltaK cell type—a columnar cell class, where each neuron has a stereotypical dendritic input in the ellipsoid body (EB) in addition to the FB innervation. hDeltaK cells exhibit extensive synaptic connections with a known subset of R23E10-GAL4-labelled sleep-promoting dFB neurons27 . On the basis of the above connectome data and existing evidence that NPF/NPY is involved in animal metabolism and stress responses, focus was placed on P2 neurons (Li, 2021).

    To test whether P2 neurons contribute to sleep loss induced by chronic social isolation, these neurons were chronically silenced by expressing the inward-rectifying potassium channel Kir2.1 under the control of P2-GAL4. In flies carrying both P2-GAL4 and UAS-Kir2.1, chronic isolation no longer induced an altered sleep profile when compared to their group-housed counterparts. The cumulative relative frequency curve of daytime sleep bouts for socially isolated animals no longer climbed faster than that of group-reared animals. Raster plots of sleep bouts in individual flies showed little difference between chronically isolated and group-housed flies. No difference was found between isolated and group-housed animals for daily total sleep, daytime sleep, or ZT0-4 sleep. By contrast, in heterozygous parental control animals carrying either the P2-GAL4 or the UAS-Kir2.1 transgene, chronic social isolation robustly induced sleep loss. Temporally silencing P2 neurons using UAS-shibirets1 during group enrichment or social isolation did not block social isolation-induced sleep loss. Although isolated flies carrying both P2-GAL4 and UAS-Kir2.1 still showed some overconsumption of food, they no longer showed excessive food consumption for ZT0-4, and the total increase in daytime food consumption was much smaller than in parental controls (Li, 2021).

    Using [Ca2+] imaging, it was found that the activity of P2 neurons was correlated with locomotor activity in both group-housed and isolated flies. One might expect that P2 neurons would be tonically more active in isolated flies than in group-housed flies, but this effect in baseline [Ca2+] levels could not be detected. Alternatively, it can be hypothesize that locomotion drives more P2 neuron total activity during 7 days of isolation than during 1 day of isolation (Li, 2021).

    Therefore whether boosting activity in P2 neurons during acute social isolation (1 day) is sufficient to promote behavioural changes that resemble the effects of chronic social isolation (7 days) was measured. To activate P2 neurons, a Drosophila warmth-gated cation channel, UAS-dTRPA1, was expressed with P2-GAL4. The P2-GAL4-labelled neurons were activated by treating the flies at 28 °C during acute social isolation or group enrichment (1 day). Control experiments, using flies of the same genotype, were conducted by treating the flies at 22 °C during acute social isolation or group enrichment (1 day). Following these treatments, all flies were subsequently maintained at 22 °C for measurement of sleep or feeding behaviour. In animals carrying both P2-GAL4 and UAS-dTRPA1, there were significant interactions between temperature treatment and isolation status for total, daytime, and ZT0-4 sleep and food consumption: activation of P2 neurons during acute social isolation promoted significant sleep loss and excessive feeding, whereas activation of P2 neurons in group housing did not alter either sleep or feeding behaviour. In control experiments, no evidence was found for interactions between temperature treatment and isolation status in the heterozygous parental flies (Li, 2021).

    Notably, P2 neurons are connected to the dFB neurons that are known to regulate sleep homeostasis and couple energy metabolism to sleep. Artificial activation of P2 neurons can produce a behavioural state that resembles the effects of chronic isolation after social isolation for a single day; however, activation of P2 neurons failed to produce these behaviours in the complete absence of social isolation. This indicates that both activity in P2 neurons and a status of being socially isolated are required to induce reduced sleep and increased feeding. Social isolation might be sensed by P2 neurons or elsewhere in the brain, but in either case appears to cause the activity of P2 neurons to be interpreted differently and thereby to generate novel behaviours. Downregulation of a secreted cytokine in a non-neural tissue, the fat body, suppresses sleep and promotes feeding in Drosophila. It would be interesting to determine whether these behavioural responses also depend on P2 neuronal activity (Li, 2021).

    Modifications of feeding circuits appear to be crucial for the evolution of complex social behaviours. For example, in C. elegans, a single-residue difference in the neuropeptide Y receptors of naturally occurring strains determines whether the strains exhibit solitary or social feeding behaviour. As antibodies to neuropeptide F, the fly homologue of neuropeptide Y, label P2 neurons, future work may ascertain whether Drosophila's P2 neurons influence social patterns of feeding behaviour as well as mediating feeding and sleep responses to social isolation (Li, 2021).

    In humans, social isolation promotes new emotional states that intensify with the passage of time. Sleep loss in Drosophila is a faithful readout of the duration of social isolation, and this allowed identification of specific patterns of gene and behavioural states that emerge as social isolation becomes chronic. This unexpected association between social isolation, sleep and metabolism in an insect model is reminiscent of the connection observed by social psychologists between loneliness, sleep difficulties and hyperphagia. Such robust findings in Drosophila suggest that studies of animal models might identify conserved brain states, genes, and neural circuits that are associated with social isolation (Li, 2021).

    Social facilitation of long-lasting memory is mediated by CO(2) in Drosophila

    How social interactions influence cognition is a fundamental question, yet rarely addressed at the neurobiological level. It is well established that the presence of conspecifics affects learning and memory performance, but the neural basis of this process has only recently begun to be investigated. In the fruit fly Drosophila melanogaster, the presence of other flies improves retrieval of a long-lasting olfactory memory. This study demonstrates that this is a composite memory composed of two distinct elements. One is an individual memory that depends on outputs from the α'β' Kenyon cells (KCs) of the mushroom bodies (MBs), the memory center in the insect brain. The other is a group memory requiring output from the αβ KCs, a distinct sub-part of the MBs. Social facilitation of memory increases with group size and is triggered by CO(2) released by group members. Among the different known neurons carrying CO(2) information in the brain, this study established that the bilateral ventral projection neuron (biVPN), which projects onto the MBs, is necessary for social facilitation. Moreover, it was demonstrated that CO(2)-evoked memory engages a serotoninergic pathway involving the dorsal-paired medial (DPM) neurons, revealing a new role for this pair of serotonergic neurons. Overall, this study identified both the sensorial cue and the neural circuit (biVPN>αβ>DPM>αβ) governing social facilitation of memory in flies. This study provides demonstration that being in a group recruits the expression of a cryptic memory and that variations in CO(2) concentration can affect cognitive processes in insects (Maria, 2021).

    The ability of an individual to form distinct memories and refer to past experiences contributes to the survival of many species. Sensory stimuli from the environment are processed and integrated during memory formation and retrieval, sometimes impacting animal physiology over the very long term. In so-called social species, conspecifics are part of each individual's environment and constitute an important source of information that can lead to social learning. Although social learning has been widely examined in the literature, the influence of social context on memory retrieval has been poorly addressed, as most memory protocols are carried out on isolated individuals. This is not the case for the fruit fly Drosophila melanogaster, for which memory studies are generally carried out on groups and thus measure memory expression in a social context (Maria, 2021).

    Despite a small brain of about 100,000 neurons, Drosophila can learn to associate and memorize different stimuli. A protocol leading to a measurable aversive olfactory memory is widely used in the literature. When exposed to one odor (conditioned stimulus plus; CS+) associated with electric shocks versus another odor (conditioned stimulus minus; CS-) without electric shock, flies learn the association between the CS+ odor and electrical shocks and form an aversive associative olfactory memory. Memory is then scored using a T maze offering a choice between two compartments enriched in the previously negatively reinforced CS+ odor versus the non-reinforced CS- odor. Memory is thus revealed by a selective avoidance of the CS+. After a single training protocol, this memory is short lasting. However, repeated training cycles generate a long-lasting memory that is measurable at least 24 h after training. Multiple training cycles without any resting period (i.e., massed training) form a consolidated memory that persists for at least 24 h and is independent of de novo protein synthesis. So far, this form of consolidated memory has been characterized as anesthesia-resistant memory (ARM) because it is resistant to a cold-shock anesthesia. Interestingly, memory after massed training is socially facilitated, as flies tested in groups perform better than individuals tested alone, which is not the case for short-lasting memory. After massed training, only flies that express ARM are influenced by the social context during memory retrieval, which implies that ARM formed after massed conditioning is required to reveal this socially facilitated memory (hereafter SFM). Another form of consolidated memory can be generated by multiple training cycles performed with a 15-min resting period between each cycle (i.e., spaced training), which leads to a robust memory dependent at least partly on de novo protein synthesis and defined as long-term memory (LTM). Recent work proposed that spaced training leads to a dual memory composed of a safety memory for the CS-, identified as the de novo protein synthesis LTM, and an aversive memory for the CS+, which displays similarities with ARM generated by a massed training.9 Unlike memory generated after massed conditioning, individual memory (i.e., memory performance of a fly tested alone) is much higher and not sensitive to the social context. The lack of influence of the social context after spaced training could be explained by the high individual memory, which would have reached a ceiling effect. Alternatively, the ARM generated by spaced conditioning might be different from that formed by massed training and not be subject to SFM or, although sharing similarities with ARM, the CS+ memory measured after spaced training might not be ARM as formally described in other studies. In any case, only memory formed after massed training is predisposed to SFM, for which memory performance increases in a social context. Although social facilitation of memory retrieval has been reported in humans, the increased memory performance of Drosophila tested in groups constitutes the first example of this phenomenon in invertebrates. Understanding the mechanisms underlying SFM could lead to insight into how social interactions influence cognition (Maria, 2021).

    This study has shown that CO2 can act as a facilitating cue leading to an improvement in memory retrieval. Moreover, it was demonstrated that such improvement relies on the expression of ARM formed after a massed training, which is expressed distinctly from individual memory, and the neural network supporting the expression of this additional CO2-sensitive memory was identified. Memory retrieval within a group relies on the recruitment of a second neural network in addition to the one required when flies are tested alone. SFM is not a simple improvement of the expression of an individual memory but constitutes a memory expression in its own right. Therefore, the memory revealed in a social context is actually a composite memory consisting of two previously encoded memories, ASM and ARM, whose expression relies on distinct neural structures. Expression of these memories is indeed independent and additive given that the inhibition of one memory during the retrieval phase does not impair the expression of the other. Thus, this work has provided evidence that ASM is the memory expressed when flies are tested individually and is independent of CO2, whereas SFM has been characterized as the additional expression of ARM in a social context (Maria, 2021).

    The predictability of an unconditioned stimulus (US) by an originally neutral stimulus becomes higher upon repetition of the stimulus pairing over extended periods. In Drosophila, two types of aversive long-lasting memories have been characterized. On the one hand, the composite memory described in the present study arises after massed training and is independent of protein synthesis. On the other hand, another form of consolidated memory occurs after spaced training and is dependent on de novo protein synthesis (LTM). Recently, this consolidated memory has been defined as the addition of LTM and ARM, an aversive memory independent of protein synthesis. ARM potentially generated by spaced training and the socially facilitated ARM generated by massed training would involve distinct molecular processes, as suggested by the distinct pathways recruited by spaced and massed trainings. Indeed, serotonin synthesis inhibitor para-chlorophenylalanine (pCPA) treatment, the Drk mutation, or the biVPN blockade (this study) impairs the memory formed after massed training but not the memory generated by spaced training. Like ARM measured after massed conditioning, the CS+ memory measured after spaced training is Radish dependent, which led to its characterization as ARM. However, the memory generated by spaced conditioning does not seem to share the other ARM characteristics detailed above and it should be considered that this CS+ memory would not be ARM in the classical sense, as supported by other studies. In any case, memory formed after spaced training is the most stable form of memory reported in Drosophila and can last up to 7 days post-training. It enables high individual retrieval performances but requires, at least in part, de novo protein synthesis (LTM) involving metabolically costly processes, which can occur at the expense of an animal's fitness under stressful conditions. Similar to aversive LTM formed after spaced training, long-lasting appetitive memory depends on de novo protein synthesis. Interestingly, neither aversive nor appetitive memory dependent on protein synthesis is socially facilitated. The SFM mechanism, purely independent of protein synthesis, would then allow flies to behave appropriately while reducing the costs of learning. Surprisingly, social context does not influence the formation of SFM but rather only its retrieval. This suggests that CO2 possibly released by flies during training does not foster individual learning. this would indicate that the training procedure used in this study generated sufficiently high levels of learning for the influence of the social context to become negligible. Because CO2 is not necessary for the retrieval of memory formed after aversive spaced training, it is concluded that CO2 does not play a general role as a memory enhancer. This aspect deserves further investigation (Maria, 2021).

    Besides Drosophila, an influence of the social context on memory retrieval has been highlighted in humans, first addressed by Kenneth Spence in 1956 and summarized by the Drive theory. According to this theory, an individual's performance is potentiated by the presence of other individuals provided that the task performed has been correctly learned beforehand. Social facilitation of memory in Drosophila is consistent with this theory. Yet, because the studies in humans have focused only on short-term restitution, the influence of social context on long-lasting retrieval evinced in the current work remains to be addressed in other taxa, such as rodents or insects. Memory tests are typically conducted on individuals because the characterization of memory refers to an individual's acquisition, storage, and retrieval of information. Yet, in light of the current findings, it would be interesting to determine to what extent social context affects memory retrieval in other animal species (Maria, 2021).

    This study showed that CO2 recruits additional circuits leading to the socially facilitated ARM expression. Flies emit and process more CO2 in a group, possibly integrating CO2 as a marker of stress. Therefore, CO2 can be conceived as a stress cue enhancing a fly's attention, changing its representation of the environment, and mediating the expression of an additive memory. Indeed, this study has provided evidence that exposure to CO2 alters the CS- response in DPM neurons, which could stimulate flies' awareness to the CS+ memory trace by inhibiting the responses to the irrelevant CS- stimulus. In vertebrates, moderate stress can promote aversive long-lasting memory. Although memory mechanisms described for vertebrates differ from those in the current model, the benefits of moderate stress on memory seem to be common across species (Maria, 2021).

    So far, the role of CO2 in insect behavior has been mostly limited to naive avoidance and attraction. This study reveals an important role for CO2 as a facilitator of olfactory memory. In natural environments, CO2 is a ubiquitous cue, including within the nest of eusocial insects such as ants, termites, or bees8 that can be potentially significant and attractive. It is an attractive cue for insects at food sources and oviposition sites and also plays a key role in host detection for hematophagous insects such as tsetse flies or mosquitoes. Olfactory learning plays a significant role in host preference and disease transmission in blood-feeding insects. Thus, exploring the impact of CO2 on memory processes in these insects would be interesting to develop and improve control strategies to reduce the risk of disease transmission. These findings suggest that CO2 may have an unsuspected impact on the cognition of a broad spectrum of insect species (Maria, 2021).

    Birth temperature followed by a visual critical period determines cooperative group membership>

    Cooperative behavior often arises when a common exploitable resource is generated. Cooperation can provide equitable distribution and protection from raiding of a common resource such as processed food. Under crowded conditions in liquid food, Drosophila larvae adopt synchronized feeding behavior which provides a fitness benefit. A key for this synchronized feeding behavior is the visually guided alignment of a 1-2 s locomotion stride between adjacent larvae in a feeding cluster. The locomotion stride is thought to be set by embryonic incubation temperature. This raises a question as to whether sib larvae will only cluster efficiently if they hatch at the same temperature. To test this, larvae were first collected and incubated in outdoor conditions. Morning hatched lower temperature larvae move slower than their afternoon higher temperature sibs. Both temperature types synchronize but tend to exclude the other type of larvae from their clusters. In addition, fitness, as measured by adult wing size, is highest when larvae cluster with their own temperature type. Thus, the temperature at which an egg is laid sets a type of behavioral stamp or password which locks in membership for later cooperative feeding (Williamson, 2021).

    Expansion and application of dye tracers for measuring solid food intake and food preference in Drosophila

    The Drosophila model is used to investigate the effects of diet on physiology as well as the effects of genetic pathways, neural systems and environment on feeding behavior. Previous work showed that Blue 1 works well as a dye tracer to track consumption of agar-based media in Drosophila in a method called Consumption-Excretion (Con-Ex. This study describes Orange 4 as a novel dye for use in Con-Ex studies that expands the utility of this method. Con-Ex experiments using Orange 4 detect the predicted effects of starvation, mating status, strain, and sex on feeding behavior in flies. Orange 4 is consumed and excreted into vials linearly with time in Con-Ex experiments, the number of replicates required to detect differences between groups when using Orange 4 is comparable to that for Blue 1, and excretion of the dye reflects the volume of consumed dye. In food preference studies using Orange 4 and Blue 1 as a dye pair, flies decreased their intake of food laced with the aversive tastants caffeine and NaCl as determined using Con-Ex or a more recently described modification called EX-Q. These results indicate that Orange 4 is suitable for Con-Ex experiments, has comparable utility to Blue 1 in Con-Ex studies, and can be paired with Blue 1 to assess food preference via both Con-Ex and EX-Q (Shell, 2021).

    Central and Peripheral Clock Control of Circadian Feeding Rhythms

    Many behaviors exhibit ~24-h oscillations under control of an endogenous circadian timing system.Most circadian research in Drosophila has focused on the generation of locomotor activity rhythms, but a fundamental question is how the circadian clock orchestrates multiple distinct behavioral outputs. This study has investigated the cells and circuits mediating circadian control of feeding behavior. This study shows that the presence of feeding rhythms requires molecular clock function in the ventrolateral clock neurons of the central brain. This study further demonstrate that the speed of molecular clock oscillations in these neurons dictates the free-running period length of feeding rhythms. In contrast to the effects observed with central clock cell manipulations, This study shows that genetic abrogation of the molecular clock in the fat body, a peripheral metabolic tissue, is without effect on feeding behavior. Under these conditions, the period of feeding rhythms tracks with molecular oscillations in central brain clock cells, consistent with a primary role of the brain clock in dictating the timing of feeding behavior. Finally, despite a lack of effect of fat body selective manipulations, this study found that flies with simultaneous disruption of molecular clocks in multiple peripheral tissues (but with intact central clocks) exhibit decreased feeding rhythm strength and reduced overall food intake. This study concluded that both central and peripheral clocks contribute to the regulation of feeding rhythms, with a particularly dominant, pacemaker role for specific populations of central brain clock cells (Fulgham, 2021).

    dFRAME: A Video Recording-Based Analytical Method for Studying Feeding Rhythm in Drosophila

    Animals, from insects to humans, exhibit obvious diurnal rhythmicity of feeding behavior. Serving as a genetic animal model, Drosophila has been reported to display feeding rhythms; however, related investigations are limited due to the lack of suitable and practical methods. This study presents a video recording-based analytical method, namely, Drosophila Feeding Rhythm Analysis Method (dFRAME). Using this newly developed computer program, FlyFeeding, the movement track of individual flies was extracted, and their food-approaching behavior was characterized. To distinguish feeding and no-feeding events, high-magnification video recording was used to optimize the method by setting cut-off thresholds to eliminate the interference of no-feeding events. Furthermore, it was verified that this method is applicable to both female and male flies and for all periods of the day. Using this method, long-term feeding status of wild-type and period mutant flies was analyzed. The results recaptured previously reported feeding rhythms and revealed detailed profiles of feeding patterns in these flies under either light/dark cycles or constant dark environments. Together, the dFRAME method enables a long-term, stable, reliable, and subtle analysis of feeding behavior in Drosophila. High-throughput studies in this powerful genetic animal model will gain great insights into the molecular and neural mechanisms of feeding rhythms (Niu, 2021).

    Alteration in information flow through a pair of feeding command neurons underlies a form of Pavlovian conditioning in the Drosophila brain

    Pavlovian conditioning is a broadly used learning paradigm where defined stimuli are associated to induce behavioral switching. To define a causal relationship between activity change in a single neuron and behavioral switching, this study took advantage of a 'command neuron' that connects cellular function to behavior. To examine the cellular and molecular basis of Pavlovian conditioning, previous work identified a pair of feeding command neurons termed 'feeding neurons' in the adult Drosophila brain using genetic screening and opto- and thermo-genetic techniques. The feeding neuron is activated by sweet signals like sucrose and induces the full complement of feeding behaviors, such as proboscis extension and food pumping. Ablation or inactivation of the pair of feeding neurons abolishes feeding behavior, suggesting that this single pair of neurons is indispensable for natural feeding behaviors. This study describes a novel conditioning protocol to associate a signal-mediating rod removal from legs (conditioned stimulus [CS]) to feeding behavior induced by sucrose stimulation (unconditioned stimulus [US]). Calcium imaging of the feeding neuron demonstrated it acquires responsiveness to CS during conditioning, with inactivation of the feeding neuron during conditioning suppressing plasticity. These results suggest conditioning alters signals flowing from the CS into the feeding circuit, with the feeding neuron functioning as a key integrative hub for Hebbian plasticity (Sakurai, 2021).

    This study demonstrate Pavlovian conditioning between tactile (CS) and gustatory (US) stimuli results in altered information processing by a pair of command neurons that control the Drosophila feeding circuit. This conditioning paradigm creates CS-induced excitement of the feeding neuron that commands feeding behavior in this animal, with the conditioned response requiring activity of the feeding neuron during pairing. Pioneering studies by Kandel and colleagues demonstrated the first synaptic and cellular mechanism underlying classical conditioning using the Aplysia gill withdrawal response. In Aplysia, the presynaptic terminal of a sensory neuron innervating the motor neuron was modulated by serotonin. Presynaptic modulation as a mechanism to generate Drosophila valence behaviors has been extensively studied, and recent progress indicates presynaptic terminals innervating mushroom body output neurons are modulated by dopaminergic neurons to establish Drosophila valence through appetitive and aversive olfactory association. Neither Aplysia plasticity nor Drosophila valence in these paradigms requires postsynaptic activity during learning. In contrast, Hebb proposed general principles to explain mechanisms for memory formation that better match results from commonly used mammalian experimental models, such as hippocampal long-term potentiation (LTP).Hebb postulated sequential firing of a presynaptic neuron and postsynaptic partner strengthens their connection. The requirement of feeding neuron activity for the conditioned response observed in this study fits well to a Hebbian mechanism if the underlying change is manifested in altered synaptic properties (although it cannot be excluded that inactivation of the feeding neuron and subsequent behavioral changes also alter neuromodulation, influencing memory formation). During association, CS-conveying neurons and the feeding neuron driven by sucrose stimulation would now fire together, resulting in strengthened connection between CS-conveying neurons and the feeding neuron according to a Hebbian mechanism. The response to US, however, did not change during conditioning, suggesting that connections between US-conveying neurons and the feeding neuron were not altered. Thus, one can hypothesize that the CS-feeding neuron circuit was newly established, whereas the pre-existing US-feeding neuron connection was not changed. These results suggest that Pavlovian conditioning is established through a change in information processing by the command neuron, which functions as the integrative hub of the feeding circuit (Sakurai, 2021).

    This Pavlovian conditioning mechanism can also accommodate presynaptic modulation as demonstrated in Aplysia plasticity and Drosophila valence if reward signals are coupled to Hebbian plasticity through presynaptic neuromodulation. For Drosophila valance memory, reward signals consist of both sweet sensing and nutrition. Similar reward signals are likely to be relevant in vivo for Pavlovian conditioning, although the nutrition reward is eliminated in the current study due to removal of the esophagus from the preparation and application of a sucrose-wet paper strip only to the sensilla of the proboscis. Therefore, reward signals are likely constant between the groups that were tested, even for different US responses in the halorhodopsin experiments. Thus, differences in reward signal can be excluded from the altered conditioned responses observed between the groups. It is hypothesized that inactivation of the feeding neuron results in weaker memory due to postsynaptic activity in this neuron contributing to memory formation independent of changes in the reward signal. It is speculated that reward signals in the current model may also be mediated by dopamine, octopamine, or serotonin, similar to their role as reward signals in the mushroom bodies for Drosophila valence memory. In Aplysia, presynaptic adenylyl cyclase, which synthesizes cAMP, is believed to associate CS and US in this conditioning paradigm through US-driven serotonin modulation of the presynaptic terminal of the CS-conveying neuron. Adenylyl cyclase is encoded by rut, while dnc encodes a cAMP phosphodiesterase that degrades cAMP. As demonstrated in Aplysia and Drosophila, cAMP functions as a signal to modulate synaptic transmission. Given its role in LTP, cAMP is likely to play a critical role in Hebbian plasticity as well, consistent with the disruption of CS-US pairing in rut and dnc mutants. Considering the involvement of postsynaptic cells in Hebbian plasticity, retrograde signals from the postsynaptic cell can also be coupled to presynaptic cAMP signaling, as demonstrated previously at the Drosophila neuromuscular junction (Sakurai, 2021).

    In the original experiment conducted by Pavlov, it is speculated there are groups of neurons that command feeding behavior in the dog. CS/US association may change responsiveness of a subset of those neurons that result in sound-induced saliva secretion, even in the absence of food signals. Electrophysiological studies have shown neural responses to CS are altered after Pavlovian conditioning in cat red nucleus and rabbit cerebellum, although how this kind of plastic change leads to alterations in command neuron function is unknown. Neurons with command function have been identified across many species. A command neuron is pivotally located within the sensorimotor watershed of a neuronal circuit and triggers a behavioral program after integrating numerous sensory inputs. Command neurons were first identified in crayfish through experiments where electrical stimulation of a certain neuron switched on or off behaviors, such as rhythmical movement of the swimmeret or escape responses. After identification of command neurons in invertebrate CNSs Mauthner cells were demonstrated to command escape behavior in fish. Recently, a group of neurons commanding feeding behavior have been identified in the mouse brain. Therefore, the scheme shown in Figure 4D may represent a common mechanism underlying Pavlovian conditioning across species, given the role of command neurons as an integrative hub within the sensorimotor watershed of neuronal circuits (Sakurai, 2021).

    The feeding neuron in the Drosophila brain functions as a single command neuron pair that triggers the entire feeding program.3 This feature allowed reliable demonstration that CS-induced activation of the feeding neuron after conditioning was as robust as US-induced activation, suggesting the CS-induced activation of the feeding neuron can trigger the conditioned behavior. Thus, neurophysiological changes can be unambiguously correlated with behavioral change, making the causal relationship clear and allowing reliable manipulation. The current results are consistent with the assumption that both the CS signal and the US signal converge at a single identified neuron through a Hebbian mechanism. Taking advantage of the defined circuit with the feeding neuron at the center, it is not possible to define the cellular and molecular mechanisms for synaptic plasticity using this experimentally accessible neuron within the CNS. This approach, coupled with real-time live imaging, may allow tracking of changes in the structure or activity of identified synapses responsible for memory formation once CS-conveying neurons are defined in the experimental system. If so, it may be possible to directly observe pre- and/or postsynaptic changes mediating memory formation on the dendrite of the feeding neuron. Whether a new circuit is generated by strengthening a rudimentary pre-existing connection or a new connection forms de novo during associative conditioning will require future analysis. Molecular and cellular mechanisms underlying this plastic change can be investigated in detail as previously characterized at neuromuscular junctions. Taken together, the study of synaptic plasticity in the feeding neuron provides a model system to characterize basic principles of memory formation at the single-cell level (Sakurai, 2021).

    A neural circuit integrates pharyngeal sensation to control feeding

    Swallowing is an essential step of eating and drinking. However, how the quality of a food bolus is sensed by pharyngeal neurons is largely unknown. This study finds that mechanical receptors along the Drosophila pharynx are required for control of meal size, especially for food of high viscosity. The mechanical force exerted by the bolus passing across the pharynx is detected by neurons expressing the mechanotransduction channel NOMPC (no mechanoreceptor potential C) and is relayed, together with gustatory information, to IN1 neurons in the subesophageal zone (SEZ) of the brain. IN1 (ingestion neurons) neurons act directly upstream of a group of peptidergic neurons that encode satiety. Prolonged activation of IN1 neurons suppresses feeding. IN1 neurons receive inhibition from DSOG1 (descending subesophageal neurons) neurons, a group of GABAergic neurons that non-selectively suppress feeding. These results reveal the function of pharyngeal mechanoreceptors and their downstream neural circuits in the control of food ingestion (Yang, 2021).

    Overconsumption is harmful for animals. Although the drive to ingest can be overwhelming for a hungry animal in the initial stage of a meal, inhibition becomes more dominant with the processes of food intake. This study found that food flowing across the pharynx accumulates the satiety state in the brain, demonstrating that multiple strategies are used by the nervous systems to avoid overeating. These pharyngeal sensory neurons are sensitive to sugar and mechanical force, serving as a flow meter that monitors food quality and amount so that the brain knows how much food is ingested even before the food reaches the intestine. This circuit may coordinate with other satiety signals, such as those conveyed by mechanical feedback from the intestine, to control feeding (Yang, 2021).

    Gustatory and mechanosensory neurons are well separated on the fly labellum before their axons reach the SEZ, where they interact with each other to regulate the perception of food quality. In contrast, the sensory neurons in the pharynx seem to adapt a different coding mechanism. Some of the pharyngeal neurons are polymodal because they respond to chemical and mechanical stimuli, with PM neurons being an example. A 'generalist' versus 'specialist strategy has been found in other sensory organs too. Being able to evaluate multiple properties of a bolus in the pharynx allow the animals to effectively control the feeding amount. There are sensory neurons in the pharynx that may be tuned to gustatory or mechanosensory cues. For example, the R41E11-GAL4 and nompC-QF labeled approximately 10 pairs of neurons in LSOs along the pharynx, similar to the number observed for mechanosensory neurons. Most of those neurons are likely 'generalist' and are tuned to mechanical stimuli only. It would be valuable to determine the full repertoire of these sensory neurons to understand how the swallowing maneuver is initiated, sustained, and terminated (Yang, 2021).

    It has been proposed that IN1 neurons may function as a key node of the feeding control circuits to govern rapid feeding decisions. Previous studies have revealed that IN1 neurons are directly downstream of pharyngeal GRNs and that activation of IN1 neurons to sugar stimulation is correlated with a fly's motivation to feed. Because activation of IN1 neurons triggers proboscis extension to food, they are likely upstream of the motor circuit that controls feeding. IN1 neurons thus appear to function as a hub that integrates sensory information to initiate food ingestion. This study found that IN1 neurons' activity is under control of the fly's feeding states. IN1 neurons are directly downstream of DSOG1 neurons that non-selectively suppress ingestion. In fed flies, DSOG1 neurons impart inhibition on IN1 neurons, resulting in a transient and moderate response to a sugar sip that triggers a robust and sustained calcium response in fasted flies (Yang, 2021).

    It has been proposed that DSOG1 neurons impart constant inhibition on the neuronal circuits that initiate food ingestion. However, the upstream circuit of DSOG1 neurons has not been identified. A cohort of neuropeptide receptor genes has been screened, but none of them seemed to function on DSOG1 neurons in feeding control. This study found that interrupting signaling of the neuropeptide MIP phenocopied overfeeding in flies with silenced DSOG1 neurons. It is tantalizing to hypothesize that MIP neurons are upstream of the DSOG1 circuit, either directly or indirectly. Because the receptors of MIP have not yet been identified, further experiments are need to differentiate between the two possibilities (Yang, 2021).

    Besides PM neurons, there are many NOMPC-expressing mechanosensory neurons along the fly pharynx. Because of the lack of specific driver lines and the technique to record a single neuron's activity during feeding, their roles in feeding regulation are interesting open questions and await in-depth investigation. Moreover, the receptors of MIP peptide have not been identified, especially the ones involved in feeding regulation, making it difficult to establish a connection between MIP neurons and DSOG1 neurons (Yang, 2021).

    Drosophila TRPgamma is required in neuroendocrine cells for post-ingestive food selection

    The mechanism through which the brain senses the metabolic state, enabling an animal to regulate food consumption, and discriminate between nutritional and non-nutritional foods is a fundamental question. Flies choose the sweeter non-nutritive sugar, L-glucose, over the nutritive D-glucose if they are not starved. However, under starvation conditions, they switch their preference to D-glucose, and this occurs independent of peripheral taste neurons. This study found that eliminating the TRPgamma channel impairs the ability of starved flies to choose D-glucose. This food selection depends on trpgamma expression in neurosecretory cells in the brain that express Diuretic hormone 44 (DH44). Loss of trpgamma increases feeding, alters the physiology of the crop, which is the fly stomach equivalent, and decreases intracellular sugars and glycogen levels. Moreover, survival of starved trpgamma flies is reduced. Expression of trpgamma in DH44 neurons reverses these deficits. These results highlight roles for TRPgamma in coordinating feeding with the metabolic state through expression in DH44 neuroendocrine cells (Dhakal, 2022).

    Fitness benefits of dietary restriction

    Dietary restriction (DR) improves survival across a wide range of taxa yet remains poorly understood. The key unresolved question is whether this evolutionarily conserved response to temporary lack of food is adaptive. Recent work suggests that early-life DR reduces survival and reproduction when nutrients subsequently become plentiful, thereby challenging adaptive explanations. A new hypothesis maintains that increased survival under DR results from reduced costs of overfeeding. This study tested the adaptive value of DR response in an outbred population of Drosophila melanogaster fruit flies. DR females did not suffer from reduced survival upon subsequent re-feeding and had increased reproduction and mating success compared to their continuously fully fed (FF) counterparts. The increase in post-DR reproductive performance was of sufficient magnitude that females experiencing early-life DR had the same total fecundity as continuously FF individuals. These results suggest that the DR response is adaptive and increases fitness when temporary food shortages cease (Sultanova, 2021).

    Excessive energy expenditure due to acute physical restraint disrupts Drosophila motivational feeding response

    To study the behavior of Drosophila, it is often necessary to restrain and mount individual flies. This requires removal from food, additional handling, anesthesia, and physical restraint. A strong positive correlation was found between the length of time flies are mounted and their subsequent reflexive feeding response, where one hour of mounting is the approximate motivational equivalent to ten hours of fasting. In an attempt to explain this correlation, anesthesia side-effects, handling, additional fasting, and desiccation were ruled out. Respirometric and metabolic techniques coupled with behavioral video scoring were used to assess energy expenditure in mounted and free flies. A specific behavior was isolated capable of exerting large amounts of energy in mounted flies, and it was identified as an attempt to escape from restraint. A model is presented where physical restraint leads to elevated activity and subsequent faster nutrient storage depletion among mounted flies. This ultimately further accelerates starvation and thus increases reflexive feeding response. In addition, it was shown that the consequences of the physical restraint profoundly alter aerobic activity, energy depletion, taste, and feeding behavior, and suggest that careful consideration is given to the time-sensitive nature of these highly significant effects when conducting behavioral, physiological or imaging experiments that require immobilization (Gordon, 2021).

    DIlp7-Producing Neurons Regulate Insulin-Producing Cells in Drosophila

    Cellular Insulin signaling (IS) shows a remarkable high molecular and functional conservation. Insulin-producing cells respond directly to nutritional cues in circulation and receive modulatory input from connected neuronal networks. Neuronal control integrates a wide range of variables including dietary change or environmental temperature. Although it is shown that neuronal input is sufficient to regulate Insulin-producing cells, the physiological relevance of this network remains elusive. In Drosophila melanogaster, Insulin-like peptide7-producing neurons are wired with Insulin-producing cells. The former cells regulate the latter to facilitate larval development at high temperatures, and to regulate systemic Insulin signaling in adults feeding on calorie-rich food lacking dietary yeast. These results demonstrate a role for neuronal innervation of Insulin-producing cells important for fruit flies to survive unfavorable environmental conditions (Prince, 2021).

    This study has analyzed the role of dIlp7-producing neurons in different thermal treatments. D7Ns are active on yeast diets, but show no activity in animals kept on yeast-free corn food (CF). Activated D7Ns are required to respond to heat stress. In addition, dIlp7 produced by D7Ns regulates dIlp2/dIlp3-induced Insulin signaling (IS) on CF, and yeast products are able to supplement efficiently for the loss of this neuropeptide (Prince, 2021).

    The generative cycle of Drosophila is divided into feeding and non-feeding stages. Due to the absence of food intake during embryonic and pupal development these stages highly rely on internal energy stores. In contrast, larvae and adults need to absorb food to survive and develop. The insulin signaling cascade is one metabolic circuit to regulate the absorption and internal turnover of macronutrients. In addition, the cascade is essential to provide thermal resistance for ectothermic insects. All feeding stages of Drosophila express four neuronal Insulin-like peptides, namely dIlp 2, 3, 5, and 7 . Larvae with functionally compromised Insulin-producing cells (IPCs) kept on yeast diets are heat sensitive, slow in development and small in size (Prince, 2021).

    Dietary yeast increase intracellular Ca2+ levels of IPCs, elevate systemic IS and support survival at high temperatures. This study found that IPCs with high Ca2+ are not sufficient to rescue larval survival at high temperatures on yeast-free CF. Therefore, it was speculated that yeast products likely activate additional neurons involved in heat stress responses. It was shown that animals kept on yeast increase Ca2+ in D7Ns (Linneweber, 2014). D7Ns connect to IPCs and are able to stimulate the latter. This study shows that, on CF, D7Ns are low on Ca2+ with respect to yeast-fed animals and that induced Ca2+ levels in D7Ns improve larval heat resistance on CF. In addition, larvae with inactivated D7Ns kept on yeast show poor survival at high temperatures. Thus, D7Ns are one integral part of the heat response and it is speculated that these neurons directly communicate with IPCs. D7Ns secrete a multitude of neuropeptides including dIlp7. DIlp7 mutants kept on yeast food (YF) are slightly heat sensitive, and due to such relative high survival rates, is is deemed unlikely that dIlp7 is one main cue crucial to withstand thermal treatments (Prince, 2021).

    D7Ns are inactive on CF and attempts were made to identify dIlp candidates responsible for IS on yeast-free diets. Interestingly, dIlp2, dIlp3, and dIlp7 were identified as essential for larval development. Moreover, genetic interactions revealed that δdIlp2,3 double mutants are unable to survive on CF. In stark contrast, δdilp2,7 and δdilp2-3,7 animals rescued the lethality shown by single mutants. These findings indicate a new metabolic link between dIlp7 and dIlp2 essential for larval development in yeast-free environments. However, wild larvae grow in microbe-rich environments, such as rotting fruits, and have likely access to dietary yeast. Adult flies sometimes feed on yeast-poor diets or avoid yeast in response to cold. Therefore, adults kept on CF were sampled. Adult δdilp7 flies show reduced IS levels and higher lethality rates with respect to genetic controls. Moreover, the combined absence of dIlp2 and dIlp7 pronounced the observed adult lethality on CF. Thus, larval and adult dIlp7 signaling is likely very different (Prince, 2021).

    It was reported that dIlp7 is expressed in the subesophageal ganglion region of the brain and suggested that D7Ns regulate the feeding behavior (Cognigni, 2011). Therefore, reduced feeding of dIlp7 mutants could explain the lower IS levels on CF. This study has shown that, on CF, δdilp2, and δdilp7 mutants ingest food faster, have a longer retention time of the ingested material and are able to absorb macronutrients. Therefore, the idea that these flies are starving on CF is not favored. It is more likely that dIlp7 is required to stimulate IPCs to maintain basic dIlp levels in circulation. To test for this possibility, wthe predicted target receptor of dIlp7, the G-protein-coupled rector Lgr3 was knocked down. The loss of Lgr3 results in low IS levels on CF. In contrast, on YF, all tested genotypes show IS comparable to controls. Taken together, it is concluded that neuronal dIlp7/Lgr3 signaling controls IPCs in adults kept on yeast-free diets. As such dIlp7 secures a basic amount of systemic IS and therefore, likely contributes to thermal resistance of adult flies. However, required adult tracking on CF at low temperatures appeared impractical to confirm this idea (Prince, 2021).

    Neuronal innervation of IPCs is established in many animals and modulates metabolic signals. The current findings indicate that food products can overwrite such neuronal stimulation. In Drosophila, a dual role for D7Ns was found: (1) these neurons facilitate the heat response of larvae feeding on yeast and (2) they form a metabolic circuit that enables adult flies to thrive on yeast-free diets if required. In mice and humans, pancreatic islets are directly innervated; however, the role of this neuronal stimulation in response to dietary cues is not well understood. This study has identified the importance of D7Ns and their product, dIlp7, in regulating IS in response to dietary quality. These findings provide new insights into the neuronal stimulation of IPCs within a given ecological context and provide a model to study neuronal innervation of insulin producing cells (Prince, 2021).

    A neural circuit linking two sugar sensors regulates satiety-dependent fructose drive in Drosophila

    In flies, neuronal sensors detect prandial changes in circulating fructose levels and either sustain or terminate feeding, depending on internal state. This study describes a three-part neural circuit that imparts satiety-dependent modulation of fructose sensing. Dorsal fan-shaped body neurons display oscillatory calcium activity when hemolymph glucose is high, and these oscillations require glutamatergic input from SLP-AB or 'Janus' neurons projecting from the protocerebrum to the asymmetric body. Suppression of activity in this circuit, either by starvation or by genetic silencing, promotes specific drive for fructose ingestion. This is achieved through neuropeptidergic signaling by tachykinin, which is released from the fan-shaped body when glycemia is high. Tachykinin, in turn, signals to Gr43a-positive fructose sensors to modulate their response to fructose. Together, these results demonstrate how a three-layer neural circuit links the detection of two sugars to produce precise satiety-dependent control of feeding behavior (Musso, 2021).

    Regulation of energy intake is a complex process involving food search, an animal's internal state, and the sensory qualities of food. In flies, fructose, either consumed directly or rapidly metabolized from precursors, promotes feeding through activation of a brain fructose sensor called Gr43a. This study describes how a neuronal network composed of neurons in the FB and asymmetric body contributes to energy homeostasis by detecting satiety-dependent changes in hemolymph glucose and modulating fructose drive (Musso, 2021).

    The central complex, which is composed of the FB, the protocerebral bridge (PB), the ellipsoid body, and the noduli, is regarded as a center for sensorimotor integration that functions in goal-directed behavior. The FB is organized in nine horizontal layers and nine vertical columns. FB large field neurons of layers 1 to 3, and inputs to these layers from the PB, encode flight direction and general sensory orientation. FB layers 6 and 7 are well known to regulate sleep and arousal, locomotor control, courtship, visual memory, and decision-making related to taste. Layer 6 also plays a role in avoiding conditioned odors, while layers 1, 2, 4, and 5 respond to electric stimuli and are required for innate odor avoidance. However, the function of the most dorsal FB layers (8 and 9), mostly innervated local tangential neurons and AB-FBl8 (or vΔA_a), remained poorly understood. The results demonstrate a role for these layers in feeding regulation (Musso, 2021).

    dFB oscillations were found to be require glutamatergic input from Janus neuron projections to the asymmetric body. Described for the first time in 2004, very little is known about AB function; 92.4% of flies display asymmetry in the AB, with the body present only in the right hemisphere, while 7.6% also have a body on the left side. It is noted that oscillations in the dFB display a tendency to be faster on the right side, with clearly asynchronous activity between the two sides that may reflect their asymmetric input from Janus neurons. The small proportion of flies displaying symmetry in the AB have defects in LTM, a process that is known to require energy. It is speculated that these symmetric flies may have a dysfunctional Janus neurons-to-dFB connection, resulting in impaired Tk release. This could affect LTM either directly or through changes in feeding. A role for TK in memory has been demonstrated in honeybees and mammals, and TkR86C appears to be expressed in serotonergic paired neurons known to interact with MB-MP1 neurons required for LTM formation. Tk also acts through TkR99D to modulate activity in neurons producing insulin-like peptides, which affect LTM formation (Musso, 2021).

    Modulation of dFB oscillations by Janus neurons requires glutamatergic signaling through a group of glutamate receptors including KaiR1D, NmdaR1, NmdaR2, and GluClα, but not AMPA receptors. Both KaiR1D receptors, which are homomeric, and N-methyl-D-aspartate (NMDA) receptors, which are heteromeric complexes between subunits 1 and 2, pass Ca2+ current. NMDA receptors (NMDAR) are well known for their role in mediating synaptic plasticity and can also trigger oscillatory activity. NMDAR function as molecular coincidence detectors, requiring simultaneous ligand binding and membrane depolarization for activation. It is possible that dFB neuron oscillations are triggered by the coincident detection of glutamate from Janus neurons and glucose from the hemolymph; however, because the FB are receiving many inputs from other brain region, it is suspect that dFB oscillations require additional inputs as well. The chloride channel GluClα is also required for dFB oscillations. GluClα has been previously implicated in on/off responses of the visual system of flies and memory retention in honeybees, demonstrating a role in regulating cell excitability. Perhaps, GluClα functions in repolarization of the dFB neurons between calcium bursts. Further study will be required to fully understand how the suite of glutamate receptors function together to drive oscillations, along with the source of input to Janus neurons in the protocerebrum (Musso, 2021).

    Because glucose is the primary circulating energy source, one might intuitively expect that enhancing feeding in response to postingestive glucose detection would be the most efficient means of optimizing energy uptake. However, using elevation of hemolymph glucose as a signal to continue feeding is problematic because glucose levels are tightly regulated and elevated glucose serves as a signal of satiety. On the other hand, internal fructose can vary widely in response to ingestion and can therefore be a more reliable indicator of recent sugar intake. Thus, the separation of glucose as a satiety indicator and fructose as marker of sugar consumption removes the potential ambiguity of each as a signal. Moreover, fructose typically coexists with other nutritive sugars in common food sources. Therefore, it may not be the case that flies specifically benefit from fructose intake but rather that fructose serves as an effective proxy for general carbohydrate ingestion. By using fructose and the narrowly tuned Gr43a fructose receptor to survey sugar consumption, flies can effectively benefit from both a fructose-mediated positive feedback loop and glucose-mediated negative feedback to co-operatively ensure appropriate energy intake (Musso, 2021).

    The finding that dFB glucose sensing modulates fructose feeding via Gr43a brain neurons fits with the established model of Gr43a brain neurons as central fructose sensors. For this mechanism to effectively sustain feeding on a rich sugar source, ingested sugars must rapidly increase fructose signaling to Gr43a brain neurons, which then must acutely promote feeding. While the precise kinetics of internal fructose elevation after sugar consumption have not been quantified, fructose levels in the head rapidly increase 10-fold after fructose feeding and then return to baseline. The role of direct fructose sensing by Gr43a brain neurons is highlighted by the observation that Gr43a knockdown in those neurons results in markedly lower relative intake of fructose compared to glucose. Unexpectedly, knockdown of Gr64a, another sugar receptor expressed in the same neurons, produced the opposite effect. This could be because Gr64a contributes to modulation of Gr43a brain neurons by other sugar cues, and the absence of this activity makes Gr43a-mediated fructose responses more pronounced. Alternatively, Gr43a may be expressed more strongly after Gr64a knockdown, leading to an increased fructose response (Musso, 2021).

    Little is known about the mechanisms downstream of Gr43a brain neurons that promote feeding. All Gr43a brain neurons express the peptide Crz, but knockdown of Crz expression produced no significant effect on fructose preference over glucose. This suggests an important functional role for another neurotransmitter, although it is also possible that the RNAi knockdown was not effective. Irrespective of mechanism, two experiments support the idea that activation of Gr43a neurons acutely enhances feeding. First, silencing of dFB neurons by genetic manipulation or prolonged starvation produces Gr43a-dependent fructose preference within the first 10 min of a flyPAD assay. Second, closed-loop optogenetic activation of Gr43a brain neurons was sufficient to produce a strong positive preference within 10 min in the STROBE (Musso, 2021).

    The separable functions of glucose and fructose sensing in flies bear notable resemblance to the differential effects of these two sugars in the mammalian hypothalamus. In particular, AMPK expression in the arcuate nucleus of the hypothalamus is known to link energy levels to food drive. When glycemia is low, AMPK is activated and thereby promotes feeding through orexigenic AgRP/NPY neuron activity. Glucose administration suppresses activity in these peptidergic neurons, while fructose can have the opposite effect and promote further feeding. The first description of fly Gr43a neurons noted their orexinegenic activity and suggested a potential functional homology with the hypothalamus. In the present study, a multilayered neural system centered on a brain energy sensor (dFB), was uncovered whose activation by glucose leads to anorexigenic behavior through inhibition of the brain fructose sensor Gr43a. Thus, the results are consistent with at least partial functional homology between the mammalian hypothalamus and brain Gr43a neurons of the fly (Musso, 2021).

    Taste cues elicit prolonged modulation of feeding behavior in Drosophila

    Taste cues regulate immediate feeding behavior, but their ability to modulate future behavior has been less well studied. Pairing one taste with another can modulate subsequent feeding responses through associative learning, but this requires simultaneous exposure to both stimuli. This study investigated whether exposure to one taste modulates future responses to other tastes even when they do not overlap in time. Using Drosophila, it was found that brief exposure to sugar enhanced future feeding responses, whereas bitter exposure suppressed them. This modulation relies on neural pathways distinct from those that acutely regulate feeding or mediate learning-dependent changes. Sensory neuron activity was required not only during initial taste exposure but also afterward, suggesting that ongoing sensory activity may maintain experience-dependent changes in downstream circuits. Thus, the brain stores a memory of each taste stimulus after it disappears, enabling animals to integrate information as they sequentially sample different taste cues that signal local food quality (Deere, 2022).

    Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae

    The feeding of pests is one of the important reasons for losses of agricultural crop yield. This study aimed to reveal how juvenile hormone participates in larval feeding regulation of the Asian corn borer Ostrinia furnacalis. Larvae of O. furnacalis exhibit a daily circadian rhythm on feeding, with a peak at ZT18 and a trough at ZT6 under both photoperiod (LD) and constant dark (DD) conditions, which may be eliminated by application of fenoxycarb, a juvenile hormone (JH) active analogue. JH negatively regulates larval feeding as a downstream factor of neuropeptide F (NPF), in which knocking down JH increases larval feeding amount along with body weight and length. The production of JH in the brain-corpora cardiaca-corpora allata (brain-CC-CA) is regulated by the brain NPF rather than gut NPF, which was demonstrated in Drosophila larvae through GAL4/UAS genetic analysis. In addition, feeding regulation of JH is closely related to energy homeostasis in the fat body by inhibiting energy storage and promoting degradation. The JH analogue fenoxycarb is an effective pesticide to O. furnacalis that controls feeding and metabolism. The brain NPF system regulates JH, with functions in food consumption, feeding rhythms, energy homeostasis and body size. This study provides an important basis for understanding the feeding mechanism and potential pest control (Yu, 2022).

    Fast-Scan Cyclic Voltammetry (FSCV) Reveals Behaviorally Evoked Dopamine Release by Sugar Feeding in the Adult Drosophila Mushroom Body

    Drosophila melanogaster, the fruit fly, is an excellent model organism for studying dopaminergic mechanisms and simple behaviors, but methods to measure dopamine during behavior are needed. This study developed fast-scan cyclic voltammetry (FSCV) to track in vivo dopamine during sugar feeding. First, acetylcholine stimulation was used to evaluate the feasibility of in vivo measurements in an awake fly. Next, sugar feeding was tested by placing sucrose solution near the fly proboscis. In the mushroom body medial tip, 1 pmol acetylcholine and sugar feeding released 0.49±0.04 μM and 0.31#177;0.06 μM dopamine, respectively but sugar-evoked release lasted longer than with acetylcholine. Administering the dopamine transporter inhibitor nisoxetine or D2 receptor antagonist flupentixol significantly increased sugar-evoked dopamine. This study develops FSCV to measure behaviorally evoked release in fly, enabling Drosophila studies of neurochemical control of reward, learning, and memory behaviors (Shin, 2022).

    Effects of Food Availability Cycles on Phase and Period of Activity-rest Rhythm in Drosophila melanogaster

    Foraging and feeding are indispensable for survival and their timing depends not only on the metabolic state of the animal but also on the availability of food resources in their environment. Since both these aspects are subject to change over time, these behaviors exhibit rhythmicity in occurrence. As the locomotor activity of an organism is related to its disposition to acquire food, and peak feeding in fruit flies has been shown to occur at a particular time of the day, it was asked if cyclic food availability can entrain their rhythmic activity. By subjecting flies to cyclic food availability, that is, feeding-starvation (FS) cycles, food cues were provided contrasting to the preferred activity times, and whether this imposed cycling in food availability could entrain the activity-rest rhythm was studied. Phase control, which is a property integral to entrainment, was not achieved despite increasing starvation duration of FS cycles (FS 12:12, FS 10:14, and FS 8:16). It was also found that flies subjected to T21 and T26 FS zeitgeber cycles were unable to match period of the activity rhythm to short or long T-cycles. Taken together, these results show that external food availability cycles do not entrain the activity-rest rhythm of fruit flies. However, it was found that starvation-induced hyperactivity causes masking which results in phase changes. In addition, T-cycle experiments resulted in minor period changes during FS treatment. These findings highlight that food cyclicity by itself may not be a potent zeitgeber but may act in unison with other abiotic factors like light and temperature to help flies time their activity appropriately (Singh, 2022).

    Serotonergic control of feeding microstructure in Drosophila

    To survive, animals maintain energy homeostasis by seeking out food. Compared to freely feeding animals, food-deprived animals may choose different strategies to balance both energy and nutrition demands, per the metabolic state of the animal. Serotonin mediates internal states, modifies existing neural circuits, and regulates animal feeding behavior, including in humans and fruit flies. However, an in-depth study on the neuromodulatory effects of serotonin on feeding microstructure has been held back for several technical reasons. Firstly, most feeding assays lack the precision of manipulating neuronal activity only when animals start feeding, which does not separate neuronal effects on feeding from foraging and locomotion. Secondly, despite the availability of optogenetic tools, feeding in adult fruit flies has primarily been studied using thermogenetic systems, which are confounded with heat. Thirdly, most feeding assays have used food intake as a measurement, which has a low temporal resolution to dissect feeding at the microstructure level. To circumvent these problems, OptoPAD assay, which provides the precision of optogenetics to control neural activity contingent on the ongoing feeding behavior, was utilized. Manipulating the serotonin circuit optogenetically affects multiple feeding parameters state-dependently. Food-deprived flies with optogenetically activated and suppressed serotonin systems feed with shorter and longer sip durations and longer and shorter inter-sip intervals, respectively. It was further shown that serotonin suppresses and enhances feeding via 5-HT1B and 5-HT7 receptors, respectively (Banu, 2022).

    A dedicate sensorimotor circuit enables fine texture discrimination by active touch

    Active touch facilitates environments exploration by voluntary, self-generated movements. However, the neural mechanisms underlying sensorimotor control for active touch are poorly understood. During foraging and feeding, Drosophila gather information on the properties of food (texture, hardness, taste) by constant probing with their proboscis. This study identified a group of neurons (sd-L neurons) on the fly labellum< that are mechanosensitive to labellum displacement and synapse onto the sugar-sensing neurons via axo-axonal synapses to induce preference to harder food. These neurons also feed onto the motor circuits that control proboscis extension and labellum spreading to provide on-line sensory feedback critical for controlling the probing processes, thus facilitating ingestion of less liquified food. Intriguingly, this preference was eliminated in mated female flies, reflecting an elevated need for softer food. These results propose a sensorimotor circuit composed of mechanosensory, gustatory and motor neurons that enables the flies to select ripe yet not over-rotten food by active touch (Yu, 2023).

    Hunger- and thirst-sensing neurons modulate a neuroendocrine network to coordinate sugar and water ingestion

    Consumption of food and water is tightly regulated by the nervous system to maintain internal nutrient homeostasis. Although generally considered independently, interactions between hunger and thirst drives are important to coordinate competing needs. In Drosophila, four neurons called the Interoceptive Subesophageal zone Neurons (ISNs) respond to intrinsic hunger and thirst signals to oppositely regulate sucrose and water ingestion. This study investigated the neural circuit downstream of the ISNs to examine how ingestion is regulated based on internal needs. Utilizing the recently available fly brain connectome, this study found that the ISNs synapse with a novel cell type Bilateral T-shaped neuron (BiT) that projects to neuroendocrine centers. In vivo neural manipulations revealed that BiT oppositely regulates sugar and water ingestion. Neuroendocrine cells downstream of ISNs include several peptide-releasing and peptide-sensing neurons, including insulin producing cells (IPC), crustacean cardioactive peptide (CCAP) neurons, and CCHamide-2 receptor isoform RA (CCHa2R-RA) neurons. These neurons contribute differentially to ingestion of sugar and water, with IPCs and CCAP neurons oppositely regulating sugar and water ingestion, and CCHa2R-RA neurons modulating only water ingestion. Thus, the decision to consume sugar or water occurs via regulation of a broad peptidergic network that integrates internal signals of nutritional state to generate nutrient-specific ingestion (Lez-Segarra, 2023).

    Monochromatic visible lights modulate the timing of pre-adult developmental traits in Drosophila melanogaster

    Light exposure impacts several aspects of Drosophila development including the establishment of circadian rhythms, neuroendocrine regulation, life-history traits, etc. Introduction of artificial lights in the environment has caused almost all animals to develop ecological and physiological adaptations. White light which comprises different lights of differing wavelengths shortens the lifespan in fruit flies Drosophila melanogaster. The wavelength-specific effects of white light on Drosophila development remains poorly understood. This study shows that different wavelengths of white light differentially modulate Drosophila development in all its concomitant stages when maintained in a 12-h light: 12-h dark photoperiod. It was observed that exposure to different monochromatic lights significantly alters larval behaviours such as feeding rate and phototaxis that influence pre-adult development. Larvae grown under shorter wavelengths of light experienced an altered feedingrate. Similarly, larvae were found to avoid shorter wavelengths of light but were highly attracted to the longer wavelengths of light. Most of the developmental processes were greatly accelerated under the green light regime while in other light regimes, the effects were highly varied. Interestingly, pre-adult survivorship remained unaltered across all light regimes but light exposure was found to show its impact on sex determination. This study for the first time reveals how different wavelengths of white light modulate Drosophila development which in the future might help in developing non-invasive therapies and effective pest measures (Ramakrishnan, 2022).

    Neprilysin 4: an essential peptidase with multifaceted physiological relevance

    Neprilysins are highly conserved ectoenzymes that hydrolyze and thus inactivate signaling peptides in the extracellular space. This study focused on Neprilysin 4 from Drosophila melanogaster and evaluate the existing knowledge on the physiological relevance of the peptidase. Particular attention is paid to the role of the neprilysin in regulating feeding behavior and the expression of insulin-like peptides in the central nervous system. In addition, this study assessed the function of the peptidase in controlling the activity of the sarcoplasmic and endoplasmic reticulum Ca(2+) ATPase in myocytes, as well as the underlying molecular mechanism in detail expression evolution structure and function (Buhr, 2023)

    A STIM dependent dopamine-neuropeptide axis maintains the larval drive to feed and grow in Drosophila

    Appropriate nutritional intake is essential for organismal survival. In holometabolous insects such as Drosophila melanogaster, the quality and quantity of food ingested as larvae determines adult size and fecundity. This study has identified a subset of dopaminergic neurons (THD') that maintain the larval motivation to feed. Dopamine release from these neurons requires the ER Ca2+ sensor STIM. Larvae with loss of STIM stop feeding and growing, whereas expression of STIM in THD' neurons rescues feeding, growth and viability of STIM null mutants to a significant extent. Moreover STIM is essential for maintaining excitability and release of dopamine from THD' neurons. Optogenetic stimulation of THD' neurons activated neuropeptidergic cells, including median neuro secretory cells that secrete insulin-like peptides. Loss of STIM in THD' cells alters the developmental profile of specific insulin-like peptides including ilp3. Loss of ilp3 partially rescues STIM null mutants and inappropriate expression of ilp3 in larvae affects development and growth. In summary this study has identified a novel STIM-dependent function of dopamine neurons that modulates developmental changes in larval feeding behaviour and growth (Kasturacharya, 2023).

    In Drosophila, as in other holometabolous insects, growth is restricted to the larval stages. In early stages of larval development cells exit mitotic quiescence and re-enter mitosis resulting in organismal growth. This change is accompanied by an increase in the feeding rate of the organism so as to provide sufficient nutrition for the accompanying growth in organismal size. In STIMKO larvae a loss of this ability to feed persistently was observed starting from early second instar larvae. The focus of this feeding deficit lies in a subset of central dopaminergic neurons that require STIM function to maintain excitability. Importantly, these dopaminergic neurons communicate with multiple neuropeptidergic cells in the brain to regulate appropriate changes in larval feeding behaviour. The identified dopaminergic cells also communicate with ilp producing neuropeptidergic cells, the MNSc, through which they appear to impact larval growth (Kasturacharya, 2023).

    The THD' cells were identified as critical for larval feeding from their inability to function in the absence of the store-operated Ca2+ entry (SOCE) regulator STIM. Loss of excitability and the absence of dopamine release from THD' cells in STIMKO larvae suggests that voltage-dependent receptor activity is required to maintain growth in early 2nd instar larvae. Changes in expression of ion channels and presynaptic components have been observed earlier upon knockdown of STIM in Drosophila and mammalian neurons. Moreover, loss of STIM-dependent SOCE in Drosophila neurons effects their synaptic release properties. Partial rescue of viability in STIMKO organisms by over-expression of a bacterial sodium channel NaChBac and restoration of dopamine release upon rescue by STIM+ supports the idea that STIM-dependant SOCE maybe required for appropriate function and/or expression of ion channels and synaptic components in THD' neurons. Changes in ER-Ca2+ suggest that STIM is also required to maintain neuronal Ca2+ homeostasis (Kasturacharya, 2023).

    While mechanisms that regulate developmental progression of Drosophila larvae have been extensively studied, neural control of essential changes in feeding behaviour that need to accompany each larval developmental stage have not been identified previously. Artificial manipulation of activity in the central dopaminergic neuron subset examined in this study (THD'), either by expression of an inward rectifying potassium channel (Kir2.1) or the bacterial sodium channel NaChBac, suggests an important role for THD' neurons during larval development. This idea is supported by the altered dynamics of muscarinic acetylcholine receptor (mAChR) stimulated Ca2+ release observed in THD' neurons between early, mid and late third instar larvae when larval feeding slows down and ultimately stops and re-iterates that signaling in and from these neurons drives larval feeding whereas lower carbachol-induced Ca2+ responses signal cessation of feeding. A weaker rescue of STIMKO larvae is also obtained from STIM+ expression in the THC' neuron subset. Taken together these observations suggest a neuromodulatory role for dopamine, where DA release from THD' neurons has a greater influence on feeding than the DA release from THC' neurons, possibly due to the DL1 and DL2 cluster (among THD' marked neurons) receiving more feeding and metabolic inputs. A role for cells other than THD', in maintaining kinetics of dopamine release required for feeding behaviour are also indicated because expression of STIM+ in THD' neurons did not revert kinetics of dopamine release to wild type levels. The prolonged dopamine release observed in wild-type THD' neurons may arise from synaptic/modulatory inputs to THD' neurons from other neurons that require STIM function (Kasturacharya, 2023).

    Though the cells that provide cholinergic inputs to THD' cells have not been identified it is possible that such neurons sense the nutritional state. In this context, two pairs of cells in the THD' subset also motivate the search for food in hungry adult Drosophila. Starved flies with knock down of the mAChR on THD' neurons exhibit a decrease in food seeking behaviour. Cholinergic inputs to THD' neurons for sensing nutritional state/hunger may thus be preserved between larval and adult stages (Kasturacharya, 2023).

    Interestingly, dopamine is also required for reward-based feeding, initiation, and reinforcement of feeding behaviour in adult mice. These findings parallel past studies where prenatal mice genetically deficient for dopamine (DA-/-), were unable to feed and died from starvation. Feeding could however be initiated upon either enforced supplementation or injection with L-DOPA allowing them to survive. More recent findings show that dopaminergic neurons in the ventral tegmental area (VTA), and not the substantia nigra, drive motivational behaviour and facilitate action initiation for feeding in adult mice (Kasturacharya, 2023).

    Both activation and inhibition of specific classes of neuropeptidergic cells by optogenetic activation of THD' cells suggests a dual role for dopamine possibly due to the presence of different classes of DA receptors. The Drosophila genome encodes four DA receptors referred to as Dop1R1, Dop1R2, DD2R and a non-canonical DopEcR [66]. Dop1R1, Dop1R2 and DopEcR activate adenylate cyclase and stimulate cAMP signaling whereas DD2R is inhibitory. Cell specific differences among dopamine receptors have been observed in adults. Down regulation of Dop1R1 on AstA and NPF cells shifted preference towards sweet food whereas down regulation of DopEcR in DH44 cells shifted preference towards bitter food. In third instar larvae a dopaminergic-NPF circuit, arising from central dopaminergic DL2 neurons, two cells of which are marked by THD'GAL4, motivates feeding in presence of appetitive odours. The dopamine-neuropeptide axis identified in this study demonstrates a broader role for dopamine in regulating neuropeptide release and/or synthesis, in the context of larval feeding behaviour, perhaps similar to the mammalian circuit described above (Kasturacharya, 2023).

    Of specific interest is the untimely upregulation of ilp3 transcripts in STIMKO larvae. Rescue of lethality in STIMKO larvae either by bringing back activity to THD' neurons or by reducing ilp3 levels suggests an interdependence of Dopamine-Insulin signaling that is likely conserved across organisms. Thr data suggest that ilp3 expression is suppressed during the feeding and growth stages of larvae, and once enough nutrition accumulates expression of ilp3 is up-regulated, concurrent with a reduction in carbachol-induced Ca2+ signals in THD' neurons, possibly followed by upregulation and release of ilp3. The idea of ilp3 as a metabolic signal whose expression is antagonistic to larval growth is supported by the observation that knock-down of ilp3 in the MNSc leads to larger pupae in wild type animals and larger larvae in STIMKO. This is the first report of ilp3 as a larval signal that is antagonistic to growth. Given that Drosophila encode a single Insulin receptor for ilp2, ilp3 and ilp5 the cellular mechanism of ilp3 action remains to be elucidated. Possibly, ilps with different affinity for the insulin receptor stimulate different cellular subsets and/or different intracellular signaling mechanisms, including ecdysone signaling that is essential for larval transition to pupae. Interestingly, in STIMKO larval brains there is a significant increase in expression of the Insulin Receptor. Further studies are needed to fully understand ilp3 function in larvae (Kasturacharya, 2023).

    Expression of other neuropeptides did not show significant changes in STIMKO larval brains, suggesting that for neuropeptidergic cells in the LNC and SEZ, dopamine signals alter release properties rather than synthesis. However, it was not possible to to identify specific neuropeptides for cells in the LNC and the SEZ that responded upon activation of THD' (Kasturacharya, 2023).

    The importance of dopamine for multiple aspects of feeding behaviour is well documented in juvenile and adult mice. Of interest are more recent findings linking dysregulation of dopamine-insulin signaling with the regulation of energy metabolism and the induction of binge eating. The identification of a simple neuronal circuit where dopamine-insulin signaling regulates feeding and growth could serve as a useful model for investigating new therapeutic strategies targeted towards the treatment of psychological disorders for obesity and metabolic syndrome (Kasturacharya, 2023).

    Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila

    Feeding behavior is essential for growth and survival of animals; however, relatively little is known about its intrinsic mechanisms. This study demonstrates that Gart is expressed in the glia, fat body, and gut and positively regulates feeding behavior via cooperation and coordination. Gart in the gut is crucial for maintaining endogenous feeding rhythms and food intake, while Gart in the glia and fat body regulates energy homeostasis between synthesis and metabolism. These roles of Gart further impact Drosophila lifespan. Importantly, Gart expression is directly regulated by the CLOCK/CYCLE heterodimer via canonical E-box, in which the CLOCKs (CLKs) in the glia, fat body, and gut positively regulate Gart of peripheral tissues, while the core CLK in brain negatively controls Gart of peripheral tissues. This study provides insight into the complex and subtle regulatory mechanisms of feeding and lifespan extension in animals (He, 2023).

    Feeding is a necessary behavior for animals to grow and survive, with a characteristic of taking food regularly. The quality and quantity of feeding directly impact the normal growth and development of animals. Time-restricted feeding or fasting is beneficial for preventing obesity, alleviating inflammation, and attenuating cardiac diseases and even has antitumor effects. Metabolic syndrome has become a global health problem. Shift work and meal irregularity disrupt circadian rhythms, with an increased risk of developing metabolic syndrome. The maintenance of the daily feeding rhythm is very important in metabolic homeostasis.Irregular feeding perturbs circadian metabolic rhythms and results in adverse metabolic consequences and chronic diseases (He, 2023).

    Most behaviors in animals are synchronized to a ~24 h (circadian) rhythm induced by circadian clocks in both the central nervous system and peripheral tissues. Circadian rhythmic behaviors, such as feeding and locomotion, are involved in complex connections and specific output pathways under the control of the circadian clock. Although the core clock feedback loop has been well established in recent decades, the crucial genes responsible for rhythmic feeding regulation as well as for the interrelation between the core clocks and feeding are still unclear (He, 2023).

    To increase the understanding of how the circadian clock regulates feeding and metabolism, this study sought to identify the output genes in the circadian feeding and metabolism control network, in which the model animal Drosophila provides special advantages to explore the mechanistic underpinnings and the complex integration of these primitive responses. Previous studies confirmed that one of juvenile hormone receptors, methoprene tolerance (Met), is important for the control of neurite development and sleep behavior in Drosophila. Many genes related to metabolic regulation have attracted attention in the transcriptome data from Met27, a Met-deficient fly line, in which this study focused on the target genes regulated by CLOCK/CYCLE (CLK/CYC). As a basic Helix-Loop-Helix-Per-ARNT-Sim (bHLH-PAS) transcription factor with a canonical binding site “CACGTG," the CLK/CYC heterodimer is a crucial core oscillator that regulates circadian rhythms (He, 2023).

    The Gart trifunctional enzyme, a homologous gene of adenosine-3 in mammals, is a trifunctional polypeptide with the activities of phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase (Tiong, 1990). Gart in astrocytes of vertebrates plays a role in the lipopolysaccharide-induced release of proinflammatory factors (Zhang, 2014), and Gart expressed in the liver and heart is required for de novo purine synthesis. However, there is no information yet for Gart's functions in feeding rhythm. In this study, Gart was identified as a candidate that was controlled by the core clock gene CLK/CYC heterodimer and was found to be related to feeding behavior in Drosophila. Thus this study focused Gart studies on the role of feeding rhythms and further regulatory mechanisms. This study provides a critical foundation for understanding the complex feeding mechanism. (He, 2023).

    In animals, hundreds of genes exhibit daily oscillation under clock regulation, and some of them are involved in metabolic functions. This study identified a CLK/CYC-binding gene, Gart, which is involved in feeding rhythms and energy metabolism independent of locomotor rhythms. Previous research reported that blocking CLK in different tissues yields different phenotypes. This study found that MET, like CYC, can combine with CLK to regulate the transcription of Gart. Knocking down Gart in different tissues exhibits different phenotypes, and Gart in different tissues can rescue the phenotype caused by CLK deletion; thus, the phenomenon caused by CLK deletion is due to the change in Gart (He, 2023).

    CLK regulates the feeding rhythms of Drosophila, and its loss can cause disorders of feeding rhythms and abnormal energy storage. Tim01, Cry01, and Per01 mutants have significantly lower levels of truactkglycerides (TAGs). The maintenance of energy homeostasis is achieved by a dynamic balance of energy intake (feeding), storage, and expenditure (metabolic rate), which are crucial factors for longevity and resistance to adverse environments in Drosophila. Additionally, studies have shown that mutations of Timeless and per shorten the adult lifespan of Drosophila. This study further reveals that peripheral CLKs control the oscillation of Gart among different peripheral tissues; however, core CLKs in the brain can negatively regulate Gart expression in peripheral tissues, indicating that a complex and refined network regulatory system exists between CLK and Gart in the brain and in different peripheral tissues to coordinate feeding behavior and energy homeostasis in Drosophila and that it further affects sensitivity to starvation and longevity. These novel findings enrich the network of regulatory mechanisms by the clocks-Gart pathway on feeding, energy homeostasis, and longevity (He, 2023).

    Glial cells have vital functions in neuronal development, activity, plasticity, and recovery from injury. This study reveals that flies lacking Gart in glial cells display a significant decline in the viability of Drosophila under starvation, caused by a decrease in energy storage that puts flies under a state of energy deficit. This discovery extends the functions of glial cells in feeding, energy storage, and starvation resistance controlled by Gart (He, 2023).

    The fat body is the primary energy tissue for the storage of fuel molecules, such as TAG and glycogen, which play an important role in the regulation of metabolic homeostasis and provide the most energy during starvation. Indeed, functional defects of the fat body increase starvation sensitivity in Drosophila. In this study, flies lacking Gart in the fat body led to decreased energy storage, which reduces the survival rate and the survival time under starvation conditions. However, flies lacking gut Gart still maintain normal energy storage, which is not sensitive to food shortage or starvation. In addition, this study found that although high temperature can stimulate the food intake of Drosophila, which is consistent with previous reports, it does not affect the feeding rhythm (He, 2023).

    This study reveals that Gart in the glia and the fat body collectively regulate the homeostasis of energy intake, storage, and expenditure, thereby influencing the viability of flies under starvation stress. Although Gart in the gut strongly influences feeding behavior, it does not play similar functions as the glia and the fat body in adversity resistance. This occurs possibly because the gut has vital roles in digestion and absorption, while the fat body has crucial functions in energy metabolism. In addition, Gart in the glia and the fat body has biased roles in the synthesis of glycogen and TAG, despite having similar functions in energy storage. The biased role of the glia and the fat body may be coordinated to provide effective energy homeostasis. These findings provide new insight into how specific circadian coordination of various tissues modulates adversity resistance and aging (He, 2023).

    Such robust findings in Drosophila suggest that a decrease in lifespan and an increase in sensitivity to starvation in Drosophila is a faithful readout of disordered feeding rhythms and abnormal metabolism. Gart effects on metabolism in both glia cells and the fat body indicate the intricacy of the circadian network and energy homeostasis. It is crucial for animals to have a well-organized network to coordinate and ensure that these various tissue regions are in a normal state (He, 2023).

    This study has demonstrated that CLK regulates feeding, energy homeostasis, and longevity via Gart. Even though attempts were made to explore more comprehensively how Gart coordinates and regulates the physiological functions in different tissues of D. melanogaster, there are still some limitations. For instance, it is still unclear that how Gart achieves functional differentiation in different tissues, as well as whether Gart regulates lifespan through autophagy and/or bacterial content or not, which are two critical factors related to lifespan. These future studies are of great significance for understanding the relationship between feeding and longevity regulated by Gart (He, 2023).

    Evolution of sociability by artificial selection

    There has been extensive research on the ecology and evolution of social life in animals that live in groups. Less attention, however, has been devoted to apparently solitary species, even though recent research indicates that they also possess complex social behaviors. To address this knowledge gap, this study artificially selected on sociability, defined as the tendency to engage in nonaggressive activities with others, in fruit flies. The goal was to quantify the factors that determine the level of sociability and the traits correlated with this feature. After 25 generations of selection, the high-sociability lineages showed sociability scores about 50% higher than did the low-sociability lineages. Experiments using the evolved lineages indicated that there were no differences in mating success between flies from the low and high lineages. Both males and females from the low lineages, however, were more aggressive than males and females from the high lineages. Finally, the evolved lineages maintained their sociability scores after 10 generations of relaxed selection, suggesting no costs to maintaining low and high sociability, at least under the settings used in this study. Sociability is a complex trait, which is currently being assessed through genomic work on the evolved lineages (Scott, 2021).

    Indirect genetic effects for social network structure in Drosophila melanogaster

    The position an individual holds in a social network is dependent on both its direct and indirect social interactions. Because social network position is dependent on the actions and interactions of conspecifics, it is likely that the genotypic composition of individuals within a social group impacts individuals' network positions. However, little is known about whether social network positions have a genetic basis, and even less about how the genotypic makeup of a social group impacts network positions and structure. With ample evidence indicating that network positions influence various fitness metrics, studying how direct and indirect genetic effects shape network positions is crucial for furthering understanding of how the social environment can respond to selection and evolve. Using replicate genotypes of Drosophila melanogaster fruit flies, social groups were created that varied in their genotypic makeup. Social groups were videoed, and networks were generated using motion-tracking software. It was found that both an individual's own genotype and the genotypes of conspecifics in its social group affect its position within a social network. These findings provide an early example of how indirect genetic effects and social network theory can be linked, and shed new light on how quantitative genetic variation shapes the structure of social groups (Wice, 2023).

    Modulation of social space by dopamine in Drosophila melanogaster, but no effect on the avoidance of the Drosophila stress odorant

    Appropriate response to others is necessary for social interactions. Yet little is known about how neurotransmitters regulate attractive and repulsive social cues. Using genetic and pharmacological manipulations in Drosophila melanogaster, this study shows that dopamine is contributing the response to others in a social group, specifically, social spacing, but not the avoidance of odours released by stressed flies (dSO). Interestingly, this dopamine-mediated behaviour is prominent only in the day-time, and its effect varies depending on tissue, sex and type of manipulation. Furthermore, alteration of dopamine levels has no effect on dSO avoidance regardless of sex, which suggests that a different neurotransmitter regulates this response (Fernandez, 2017).

    Drosophila melanogaster Stress Odorant (dSO) Displays the Characteristics of an Interspecific Alarm Cue

    Organisms depend on visual, auditory, and olfactory cues to signal the presence of danger that could impact survival and reproduction. Drosophila melanogaster emits a volatile olfactory alarm signal, termed the Drosophila stress odorant (dSO), in response to mechanical agitation or electric shock. While it has been shown that conspecifics avoid areas previously occupied by stressed individuals, the contextual underpinnings of the emission of, and response to dSO, have received little attention. Using a binary choice assay, it was determined that neither age and sex of emitters, nor the time of the day, affected the emission or avoidance of dSO. However, both sex and mating status affected the response to dSO. It was also demonstrated that while D. melanogaster, D. simulans, and D. suzukii, have different dSO profiles, its avoidance was not species-specific. Thus, dSO should not be considered a pheromone but a general alarm signal for Drosophila. However, the response levels to both intra- and inter-specific cues differed between Drosophila species and possible reasons for these differences are discussed (Yost, 2021).

    Thermal fluctuations affect the transcriptome through mechanisms independent of average temperature

    Terrestrial ectotherms are challenged by variation in both mean and variance of temperature. Phenotypic plasticity (thermal acclimation) might mitigate adverse effects, however, there is lack in fundamental understanding of the molecular mechanisms of thermal acclimation and how they are affected by fluctuating temperature. This study investigated the effect of thermal acclimation in Drosophila melanogaster on critical thermal maxima (CTmax) and associated global gene expression profiles as induced by two constant and two ecologically relevant (non-stressful) diurnally fluctuating temperature regimes. Both mean and fluctuation of temperature contribute to thermal acclimation and affect the transcriptome. The transcriptomic response to mean temperatures comprises modification of a major part of the transcriptome, while the response to fluctuations affects a much smaller set of genes, which is highly independent of both the response to a change in mean temperature and to the classic heat shock response. Although the independent transcriptional effects caused by fluctuations are relatively small, they are likely to contribute to the understanding of thermal adaptation. It was also found that environmental sensing, particularly phototransduction, is a central mechanism underlying the regulation of thermal acclimation to fluctuating temperatures. Thus, genes and pathways involved in phototransduction are likely of importance in fluctuating climates (Sørensen, 2016).

    Canalization of gene expression is a major signature of regulatory cold adaptation in temperate Drosophila melanogaster

    Transcriptome analysis may provide means to investigate the underlying genetic causes of shared and divergent phenotypes in different populations and help to identify potential targets of adaptive evolution. Applying RNA sequencing to whole male Drosophila melanogaster from the ancestral tropical African environment and a very recently colonized cold-temperate European environment at both standard laboratory conditions and following a cold shock, this study sought to uncover the transcriptional basis of cold adaptation. In both the ancestral and the derived populations, the predominant characteristic of the cold shock response is the swift and massive upregulation of heat shock proteins and other chaperones. Although ~25 % of the genome was found to be differentially expressed following a cold shock, only relatively few genes (n = 16) are up- or down-regulated in a population-specific way. Intriguingly, 14 of these 16 genes show a greater degree of differential expression in the African population. Likewise, there is an excess of genes with particularly strong cold-induced changes in expression in Africa on a genome-wide scale. The analysis of the transcriptional cold shock response most prominently reveals an upregulation of components of a general stress response, which is conserved over many taxa and triggered by a plethora of stressors. Despite the overall response being fairly similar in both populations, there is a definite excess of genes with a strong cold-induced fold-change in Africa. This is consistent with a detrimental deregulation or an overshooting stress response. Thus, the canalization of European gene expression might be responsible for the increased cold tolerance of European flies (von Heckel, 2016).

    A switch in thermal preference in Drosophila larvae depends on multiple rhodopsins

    Drosophila third-instar larvae exhibit changes in their behavioral responses to gravity and food as they transition from feeding to wandering stages. Using a thermal gradient encompassing the comfortable range (18°C to 28°C), this study found that third-instar larvae exhibit a dramatic shift in thermal preference. Early third-instar larvae prefer 24°C, which switches to increasingly stronger biases for 18°C-19°C in mid- and late-third-instar larvae. Mutations eliminating either of two rhodopsins, Rh5 and Rh6, wipe out these age-dependent changes in thermal preference. In larvae, Rh5 and Rh6 are thought to function exclusively in the light-sensing Bolwig organ. However, the Bolwig organ was found to be dispensable for the thermal preference. Rather, Rh5 and Rh6 are required in trpA1-expressing neurons in the brain, ventral nerve cord, and body wall. Because Rh1 contributes to thermal selection in the comfortable range during the early to mid-third-instar stage, fine thermal discrimination depends on multiple rhodopsins (Sokabe, 2016).

    It is concluded that third-instar Drosophila larvae undergo an age-dependent change in their thermal preference, and this behavioral modification requires. Rh5 and Rh6 were the most important, given that the stage-dependent alteration in temperature selection was eliminated in either rh5 and rh6 mutant flies. Several observations support the conclusion that the thermotaxis exhibited by the rh5 and rh6 mutants are not secondary consequences of developmental defects or motor problems. The percentage of larvae that entered the third-instar larval stage at 74 hr AEL was similar to controls, as were the times to pupation. Furthermore, the morphology of the peripheral trpA1-positive neurons that normally express rh5 and rh6 were indistinguishable between the rh5 and rh6 mutants and controls. In addition, the movement speeds of the rh5 and rh6 mutants were not reduced, and they were able to choose 18°C over 28°C normally in two-way choice assays (Sokabe, 2016).

    The requirements for Rh5 and Rh6 were light independent, since the thermotaxis occurred equally well in the light or dark and was not dependent on the Bolwig organ, which is the rhodopsin expressing light-sensitive tissue in larvae. Rhodopsins are composed of the protein subunit, opsin and a vitamin-A-derived chromophore, which senses light. In Drosophila photoreceptor cells, the chromophore also functions as a molecular chaperone to facilitate transport of the opsin out of the endoplasmic reticulum. This study found that thermotaxis in late third-instar larvae was impaired in a mutant that disrupts chromophore. However, it is suggested that this phenotype is due to the second function of the chromophore as a molecular chaperone (Sokabe, 2016).

    The findings lead to the conclusion that Rh5 and Rh6 function upstream of a Gq/PLC/TRPA1 signaling cascade, which allows late third-instar larvae to select their favorite temperature in the comfortable range. It is proposed that this pathway enables the animals to sense minute temperature differences over a shallow thermal gradient through signal amplification, similar to the role of these proteins in phototransduction. If the perfect option is not available in the thermal landscape, the thermosensory signaling cascade may facilitate adaptation to hospitable temperatures that deviate slightly from their preferred temperature (Sokabe, 2016).

    Because of the exquisite effectiveness of rhodopsin in photon capture, it is suggested that Rh5 and Rh6 are expressed outside the Bolwig organ at extremely low levels to prevent light from interfering with temperature sensation. Nevertheless, expression of the rh5 and rh6 reporters was observed in a subset of trpA1-CD neurons in the body wall. Using the GAL4/UAS system, evidence is provided that rh5 and rh6 both function in trpA1-CD- as well as trpA1-AB-expressing neurons outside of the Bolwig organ. In addition, rh5 GAL4-mediated RNAi knockdown of rh6 and rh6 GAL4-mediated knockdown of rh5 resulted in defects in 18°C selection. RNAi-based knockdown of trpA1 with either of the rh5- and rh6-GAL4 drivers caused similar thermotaxis defects. Although these drivers are expressed at very low levels, it is suggested that they are still effective, since trpA1 is also expressed at very low levels in the periphery. The effects of the rh5- and rh6-GAL4 drivers in suppressing trpA1 were not non-specific, as no thermotaxis phenotype was observed using the trp-GAL4 driver. It was also found that the rh5- and rh6-GAL4s silenced the thermosensory neurons in combination with UAS-kir2.1. It is proposed that this was effective, since small increases in hyperpolarization due to slight elevation of Kir2.1 cannot be overcome by the slight depolarization mediated by the low levels of TRPA1 (Sokabe, 2016).

    The combination of these findings indicates that both rh5 and rh6 are co-expressed and function in the same, or overlapping, subsets of neurons required for thermotaxis. These findings raise the possibility that Rh5 and Rh6 may form heterodimers in vivo. Another key question is whether rhodopsins are direct thermosensors, an issue that remains unresolved due to challenges inherent in expressing these and most invertebrate rhodopsins in vitro (Sokabe, 2016).

    The observation that multiple rhodopsins function in thermotaxis in Drosophila raise the question as to whether rhodopsin-dependent thermosensory signaling cascades are used in other animals, including mammals. It is suggested that mammalian cells that undergo thermotaxis over very small temperature gradients may rely on opsin-coupled amplification cascades. Intriguing possibilities include leukocytes, which thermotax to sites of inflammation, and mammalian sperm, which undergo thermotaxis to the egg over temperature gradients of ~1°C and require PLC for this cellular behavior. Intriguingly, mammalian TRP channels and non-visual rhodopsins appear to be expressed in sperm and have been suggested to function in sperm thermotaxis (Sokabe, 2016).

    Chronic dietary salt stress mitigates hyperkalemia and facilitates chill coma recovery in Drosophila melanogaster

    Chill susceptible insects like Drosophila lose the ability to regulate water and ion homeostasis at low temperatures. This loss of hemolymph ion and water balance drives a hyperkalemic state that depolarizes cells, causing cellular injury and death. The ability to maintain ion homeostasis at low temperatures and/or recover ion homeostasis upon rewarming is closely related to insect cold tolerance. It was hypothesized that changes to organismal ion balance, which can be achieved in Drosophila through dietary salt loading, could alter whole animal cold tolerance phenotypes. Flies were put in the presence of diets highly enriched in NaCl, KCl, xylitol (an osmotic control) or sucrose (a dietary supplement known to impact cold tolerance) for 24h. Independently of their osmotic effects, NaCl, KCl, and sucrose supplementation all improved the ability of flies to maintain K+ balance in the cold, which allowed for faster recovery from chill coma after 6h at 0 ° C. These supplements, however, also slightly increased the CTmin and had little impact on survival rates following chronic cold stress (24h at 0 ° C), suggesting that the effect of diet on cold tolerance depends on the measure of cold tolerance assessed. In contrast to prolonged salt stress, brief feeding (1.5h) on diets high in salt slowed coma recovery, suggesting that the long-term effects of NaCl and KCl on chilling tolerance result from phenotypic plasticity, induced in response to a salty diet, rather than simply the presence of the diet in the gut lumen (Yerushalmi, 2016).

    Linear ubiquitination by LUBEL has a role in Drosophila heat stress response

    The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. This study provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila. This study identified Drosophila CG11321, which was renamed Linear Ubiquitin E3 ligase (LUBEL), and found that it catalyzes linear ubiquitination in vitro. Endogenous linear ubiquitin chain-derived peptides were detected by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, linear ubiquitination-defective flies were established by mutating residues essential for the catalytic activity of LUBEL. Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies (Asaoka, 2016).

    Local adaptation of reproductive performance during thermal stress

    Considerable evidence exists for local adaptation of critical thermal limits in ectotherms following adult temperature stress, but fewer studies have tested for local adaptation of sublethal heat stress effects across life-history stages. In organisms with complex life cycles, such as holometabolous insects, heat stress during juvenile stages may severely impact gametogenesis, having downstream consequences on reproductive performance that may be mediated by local adaptation, although this is rarely studied. This study tested how exposure to either benign or heat stress temperature during juvenile and adult stages, either independently or combined, influences egg-to-adult viability, adult sperm motility and fertility in high- and low-latitude populations of Drosophila subobscura. Both population- and temperature-specific effects on survival and sperm motility were found- juvenile heat stress decreases survival and subsequent sperm motility and each trait is lower in the northern population. An interaction between population and temperature on fertility following application of juvenile heat stress was observed; although fertility is negatively impacted in both populations, the southern population is less affected. When the adult stage was subjected to heat stress, the southern population was found to exhibit positive carry-over effects whereas the northern population's fertility remained low. Thus, the northern population is more susceptible to sublethal reproductive consequences following exposure to juvenile heat stress. This may be common in other organisms with complex life cycles and current models predicting population responses to climate change, which do not take into account the impact of juvenile heat stress on reproductive performance, may be too conservative (Porcelli, 2016).

    Inducing Cold-Sensitivity in the Frigophilic Fly Drosophila montana by RNAi

    Cold acclimation is a critical physiological adaptation for coping with seasonal cold. By increasing their cold tolerance individuals can remain active for longer at the onset of winter and can recover more quickly from a cold shock. In insects, despite many physiological studies, little is known about the genetic basis of cold acclimation. Recently, transcriptomic analyses in Drosophila virilis and D. montana revealed candidate genes for cold acclimation by identifying genes upregulated during exposure to cold. This study tested the role of myo-inositol-1-phosphate synthase (Inos), in cold tolerance in D. montana using an RNAi approach. D. montana has a circumpolar distribution and overwinters as an adult in northern latitudes with extreme cold. Cold tolerance of dsRNA knock-down flies was tested using two metrics: chill-coma recovery time (CCRT) and mortality rate after cold acclimation. Injection of dsRNAInos did not alter CCRT, either overall or in interaction with the cold treatment, however it did induced cold-specific mortality, with high levels of mortality observed in injected flies acclimated at 5 degrees C but not at 19 degrees C. Overall, injection with dsRNAInos induced a temperature-sensitive mortality rate of over 60% in this normally cold-tolerant species. qPCR analysis confirmed that dsRNA injection successfully reduced gene expression of Inos. Thus, these results demonstrate the involvement of Inos in increasing cold tolerance in D. montana. The potential mechanisms involved by which Inos increases cold tolerance are also discussed (Vigoder, 2016).

    The role of PDF neurons in setting preferred temperature before dawn in Drosophila

    Animals have sophisticated homeostatic controls. While mammalian body temperature fluctuates throughout the day, small ectotherms, such as Drosophila, achieve a body temperature rhythm (BTR) through their preference of environmental temperature. This study demonstrates that pigment dispersing factor (PDF) neurons play an important role in setting preferred temperature before dawn. Amall lateral ventral neurons (sLNvs), a subset of PDF neurons, activate the dorsal neurons 2 (DN2s), the main circadian clock cells that regulate temperature preference rhythm (TPR). The number of temporal contacts between sLNvs and DN2s peak before dawn. The data suggest that the thermosensory Anterior Cells (ACs) likely contact sLNvs via serotonin signaling. Together, the ACs-sLNs-DN2s neural circuit regulates the proper setting of temperature preference before dawn. Given that sLNvs are important for sleep and that BTR and sleep have a close temporal relationship, these data highlight a possible neuronal interaction between body temperature and sleep regulation (Tang, 2017).

    Large scale phosphoprotein profiling to explore Drosophila cold acclimation regulatory mechanisms

    The regulatory mechanisms involved in the acquisition of thermal tolerance are unknown in insects. Reversible phosphorylation is a widespread post-translational modification that can rapidly alter proteins function(s). A large-scale comparative screening was conducted of phosphorylation networks in adult Drosophila flies that were cold-acclimated versus control. Using a modified SIMAC method followed by a multiple MS analysis strategy, a large collection of phosphopeptides (about 1600) and phosphoproteins (about 500) was identified in both groups, with good enrichment efficacy (80%). The saturation curves from the four biological replicates revealed that the phosphoproteome was rather well covered under the experimental conditions. Acclimation evoked a strong phosphoproteomic signal characterized by large sets of unique and differential phosphoproteins. These were involved in several major GO superclusters of which cytoskeleton organization, positive regulation of transport, cell cycle, and RNA processing were particularly enriched. Data suggest that phosphoproteomic changes in response to acclimation were mainly localized within cytoskeletal network, and particularly within microtubule associated complexes. This study opens up novel research avenues for exploring the complex regulatory networks that lead to acquired thermal tolerance (Colinet, 2017).

    Feeding-state-dependent modulation of temperature preference requires insulin signaling in Drosophila warm-sensing neurons

    Starvation is life-threatening and therefore strongly modulates many aspects of animal behavior and physiology. In mammals, hunger causes a reduction in body temperature and metabolism, resulting in conservation of energy for survival. However, the molecular basis of the modulation of thermoregulation by starvation remains largely unclear. Whereas mammals control their body temperature internally, small ectotherms, such as Drosophila, set their body temperature by selecting an ideal environmental temperature through temperature preference behaviors. This study demonstrates in Drosophila that starvation results in a lower preferred temperature, which parallels the reduction in body temperature in mammals. The insulin/insulin-like growth factor (IGF) signaling (IIS) pathway is involved in starvation-induced behaviors and physiology and is well conserved in vertebrates and invertebrates. Insulin-like peptide 6 (Ilp6) in the fat body (fly liver and adipose tissues) is responsible for the starvation-induced reduction in preferred temperature (Tp). Temperature preference behavior is controlled by the anterior cells (ACs), which respond to warm temperatures via transient receptor potential A1 (TrpA1). This study demonstrated that starvation decreases the responding temperature of ACs via insulin signaling, resulting in a lower Tp than in nutrient-rich conditions. Thus, this study shows that hunger information is conveyed from fat tissues via Ilp6 and influences the sensitivity of warm-sensing neurons in the brain, resulting in a lower temperature set point. Because starvation commonly results in a lower body temperature in both flies and mammals, it is proposed that insulin signaling is an ancient mediator of starvation-induced thermoregulation (Umezaki, 2018).

    Redefining reproductive dormancy in Drosophila as a general stress response to cold temperatures

    Organisms regularly encounter unfavorable conditions and the genetic adaptations facilitating survival have been of long-standing interest to evolutionary biologists. Despite dormancy being a well-studied adaptation to facilitate overwintering, there is still considerable controversy about the distribution of dormancy among natural populations and between species in Drosophila. The current definition of dormancy as developmental arrest of oogenesis at the previtellogenic stage (stage 7) distinguishes dormancy from general stress related block of oogenesis at early vitellogenic stages (stages 8 - 9). In an attempt to resolve this, reproductive dormancy in D. melanogaster and D. simulans was scrutinized. WDormancy shows the same hallmarks of arrest of oogenesis at stage 9, as described for other stressors and propose a new classification for dormancy. Applying this modified classification, this study showed that both species express dormancy in cosmopolitan and African populations, further supporting that dormancy uses an ancestral pathway induced by environmental stress. While significant differences were found between individuals and the two Drosophila species in their sensitivity to cold temperature stress, it is also noted that extreme temperature stress (8 degrees C) resulted in very strong dormancy incidence, which strongly reduced the differences seen at less extreme temperatures. It is concluded that dormancy in Drosophila should not be considered a special trait, but is better understood as a generic stress response occurring at low temperatures (Lirakis, 2018).

    Effects of cold acclimation and dsRNA injections on Gs1l gene splicing in Drosophila montana

    Alternative splicing, in which one gene produce multiple transcripts, may influence how adaptive genes respond to specific environments. A newly produced transcriptome of Drosophila montana shows the Gs1-like (Gs1l) gene to express multiple splice variants and to be down-regulated in cold acclimated flies with increased cold tolerance. Gs1l's effect on cold tolerance was further tested by injecting cold acclimated and non-acclimated flies from two distantly located northern and southern fly populations with double stranded RNA (dsRNA) targeting Gs1l. While both populations had similar cold acclimation responses, dsRNA injections only effected the northern population. The nature of splicing expression was then investigated in the northern population by confirming which Gs1l variants are present, by comparing the expression of different gene regions and by predicting the protein structures of splices using homology modelling. Different splices of Gs1l not only appear to have independent impacts on cold acclimation but also elicit different effects in populations originating from two very different environments. Also, at the protein level, Gs1l appears homologous to the human HDHD1A protein and some splices might produce functionally different proteins though this needs to be verified in future studies by measuring the particular protein levels. Taken together, Gs1l appears to be an interesting new candidate to test how splicing influences adaptations (Hopkins, 2018).

    Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance

    For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization, and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of Capability (CAPA) (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been previously demonstrated to cause anti-diuresis in insects, including dipterans. This study provides evidence that low (fM) and high (microM) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K(+) clearance rates and reduce chill tolerance, high doses facilitate K(+) clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, the maximum achievable hormonal titres of CAPA was estimated, and evidence was further found that CAPA may function as an anti-diuretic hormone in Drosophila melanogaster. Evidence is provided of a neuropeptide that can negatively affect cold tolerance in an insect, and further evidence of CAPA functioning as an anti-diuretic peptide in this ubiquitous insect model (MacMillan, 2018).

    Using Drosophila behavioral assays to characterize terebrid venom-peptide bioactivity

    The number of newly discovered peptides from the transcriptomes and proteomes of animal venom arsenals is rapidly increasing, resulting in an abundance of uncharacterized peptides. There is a pressing need for a systematic, cost effective, and scalable approach to identify physiological effects of venom peptides. To address this discovery-to-function gap, a sequence driven:activity-based hybrid approach was developed for screening venom peptides that is amenable to large-venom peptide libraries with minimal amounts of peptide. Using this approach, the physiological and behavioral phenotypes of two peptides were characterized from the venom of predatory terebrid marine snails, teretoxins Tv1 from Terebra variegata and Tsu1.1 from Terebra subulata. The results indicate that Tv1 and Tsu1.1 have distinct bioactivity. Tv1 (100 microM) had an antinociceptive effect in adult Drosophila using a thermal nociception assay to measure heat avoidance. Alternatively, Tsu1.1 (100 microM) increased food intake. These findings describe the first functional bioactivity of terebrid venom peptides in relation to pain and diet and indicate that Tv1 and Tsu1.1 may, respectively, act as antinociceptive and orexigenic agents. Tv1 and Tsu1.1 are distinct from previously identified venom peptides, expanding the toolkit of peptides that can potentially be used to investigate the physiological mechanisms of pain and diet (Eriksson, 2018).

    Correcting locomotion dependent observation biases in thermal preference of Drosophila

    Sensing environmental temperatures is essential for the survival of ectothermic organisms. One drawback of gradients is that small ectothermic animals are susceptible to cold-trapping: a physiological inability to move at the cold area of the gradient. Often cold-trapping cannot be avoided, biasing the resulting temperature preference (TP) to lower temperatures. Two mathematical models were previously developed to correct for cold-trapping. These models, however, focus on group behaviour which can lead to overestimation of cold-trapping due to group aggregation. This study presents a mathematical model that simulates the behaviour of individual Drosophila in temperature gradients. The model takes the spatial dimension and temperature difference of the gradient into account, as well as the rearing temperature of the flies. Furthermore, it allows the quantification of cold-trapping and reveals unbiased TP. Additionally, the model reveals that flies have a range of tolerable temperatures, and this measure is more informative about the behaviour than commonly used TP (Giraldo, 2019).

    Cold acclimation triggers major transcriptional changes in Drosophila suzukii

    Insects have the capacity to adjust their physiological mechanisms during their lifetime to promote cold tolerance and cope with sublethal thermal conditions, a phenomenon referred to as thermal acclimation. The spotted wing drosophila, Drosophila suzukii, is an invasive fruit pest that, like many other species, enhances its thermotolerance in response to thermal acclimation. This study promoted flies' cold tolerance by gradually increasing acclimation duration (i.e. pre-exposure from 2 h to 9 days at 10 ° C), and then compared transcriptomic responses of cold hardy versus cold susceptible phenotypes using RNA sequencing. Cold tolerance of D. suzukii increased with acclimation duration; the longer the acclimation, the higher the cold tolerance. Cold-tolerant flies that were acclimated for 9 days were selected for transcriptomic analyses. RNA sequencing revealed a total of 2908 differentially expressed genes: 1583 were up- and 1325 were downregulated in cold acclimated flies. Functional annotation revealed many enriched GO-terms among which ionic transport across membranes and signaling were highly represented in acclimated flies. Neuronal activity and carbohydrate metabolism were also enriched GO-terms in acclimated flies. Results also revealed many GO-terms related to oogenesis which were underrepresented in acclimated flies. It is concluded that involvement of a large cluster of genes related to ion transport in cold acclimated flies suggests adjustments in the capacity to maintain ion and water homeostasis. These processes are key mechanisms underlying cold tolerance in insects. Down regulation of genes related to oogenesis in cold acclimated females likely reflects that females were conditioned at 10 ° C, a temperature that prevents oogenesis (Enriquez, 2019).

    Three quantitative trait loci explain more than 60% of variation for chill coma recovery time in a natural population of Drosophila ananassae
    Ectothermic species such as insects are particularly vulnerable to climatic fluctuations. Nevertheless, many insects that evolved and diversified in the tropics have successfully colonized temperate regions all over the globe. To shed light on the genetic basis of cold tolerance in such species, a quantitative trait locus (QTL) mapping experiment for chill coma recovery time (CCRT) was conducted in Drosophila ananassae, a cosmopolitan species that has expanded its range from tropical to temperate regions. Using a hierarchical mapping approach that combined standard interval mapping and a multiple-QTL model, three QTL were mapped which altogether explained 64% of the phenotypic variance. For two of the identified QTL, evidence was found of epistasis. To narrow down the list of cold tolerance candidate genes, the QTL intervals was cross-referenced with genes that had previously been identified as differentially expressed in response to cold in D. ananassae, and with thermotolerance candidate genes of D. melanogaster. Among the 58 differentially expressed genes that were contained within the QTL, GF15058 showed a significant interaction of the CCRT phenotype and gene expression. Further, the orthologs of four D. melanogaster thermotolerance candidate genes, MtnA, klarsicht, CG5246 (D.ana/GF17132) and CG10383 (D.ana/GF14829) were identified as candidates for cold tolerance in D. ananassae (Koniger, 2019).

    Robustness and plasticity in Drosophila heat avoidance

    Simple innate behavior is often described as hard-wired and largely inflexible. This study shows that the avoidance of hot temperature, a simple innate behavior, contains unexpected plasticity in Drosophila. First, it was demonstrate that hot receptor neurons of the antenna and their molecular heat sensor, Gr28B.d, are essential for flies to produce escape turns away from heat. High-resolution fly tracking combined with a 3D simulation of the thermal environment shows that, in steep thermal gradients, the direction of escape turns is determined by minute temperature differences between the antennae (0.1°-1 °C). In parallel, live calcium imaging confirms that such small stimuli reliably activate both peripheral thermosensory neurons and central circuits. Next, based on these measurements, a fly/vehicle model with two symmetrical sensors and motors (a "Braitenberg vehicle") was evolved which closely approximates basic fly thermotaxis. Critical differences between real flies and the hard-wired vehicle reveal that fly heat avoidance involves decision-making, relies on rapid learning, and is robust to new conditions, features generally associated with more complex behavior (Simoes, 2021).

    Identification of a neural basis for cold acclimation in Drosophila

    Low temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors), but it is unknown how the nervous system might participate in cold tolerance. This study describes cold-nociceptive behavior among 11 drosophilid species; the predominant cold-evoked larval response was found to be a head-to-tail contraction behavior, which is likely inherited from a common ancestor, but is unlikely to be protective. Therefore the hypothesis that cold nociception functions to protect larvae by triggering cold acclimation was tested. Drosophila melanogaster Class III nociceptors were found to be sensitized by and critical to cold acclimation and that cold acclimation can be optogenetically evoked, sans cold. Collectively, these findings demonstrate that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation (Himmel, 2021).

    Cardiac performance in heat-stressed flies of heat-susceptible and heat-resistant Drosophila melanogaster

    Thermotolerance is a complex trait that can greatly differ between heat-susceptible (HS) and heat-adapted populations of small insects including Drosophila, with short-term effects after a sub-lethal level of heat stress on many physiological functions. Cardiac performance could accordingly be more robust in heat-resistant (HR) than in HS individuals under heat stress. This study tested heart performance under heat-stress effects in two recombinant inbred lines (RIL) of Drosophila melanogaster that dramatically differ in heat knockdown resistance. Heart rate did not strongly differ between heat-susceptible and heat-tolerant flies after a sub-lethal heat stress. Instead, heat-susceptible flies showed a much higher arrhythmia incidence, a longer duration of each heartbeat, and a larger amount of bradycardia than heat-tolerant flies. The highly conserved cardiac proteins SERCA, RyR and NCX that participate in the excitation/contraction coupling, did not differ in activity level between HR and HS flies. Available information for both RIL suggests that heart performance under heat stress may be linked, at least partially, to candidate genes of previously identified quantitative trait loci (QTL) for thermotolerance. This study indicates that HR flies can be genetically more robust in their heart performance than HS flies under even sub-lethal levels of heat stress (Rodriguez, 2021).

    Evolution of sex-specific heat stress tolerance and larval Hsp70 expression in populations of Drosophila melanogaster adapted to larval crowding

    The ability to tolerate temperature stress is an important component of adult fitness. In holometabolous insects like Drosophila melanogaster, adult stress resistance can be affected by growth conditions experienced during the larval stages. Although evolution under crowded larval conditions is known to lead to the correlated evolution of many adult traits, its consequences on adult heat stress tolerance have not been investigated. Therefore, the present study assessed the adult heat stress tolerance in populations of D. melanogaster adapted to a stressful larval crowding environment. Replicate populations of D. melanogaster, selected for adaptation to larval crowding stress (MCUs), were used for more than 230 generations, and their respective controls (MBs). Larvae from selected and control populations were grown under crowded and uncrowded conditions, and their adult heat shock resistance at two different temperatures was measured. Further, Hsp70 expression was compared in crowded and uncrowded larvae of both populations and also measured the Hsp70 expression after a mild heat treatment in adults of selected and control populations. The results showed that adaptation to larval crowding leads to the evolution of Hsp70 gene expression in larval stages and improves adult heat stress tolerance ability in males, but not in females (Kapila, 2021).

    A unifying model to estimate thermal tolerance limits in ectotherms across static, dynamic and fluctuating exposures to thermal stress

    Temperature tolerance is critical for defining the fundamental niche of ectotherms and researchers classically use either static (exposure to a constant temperature) or dynamic (ramping temperature) assays to assess tolerance. The use of different methods complicates comparison between studies and this study presents a mathematical model (and R-scripts) to reconcile thermal tolerance measures obtained from static and dynamic assays. The model uses input data from several static or dynamic experiments and is based on the well-supported assumption that thermal injury accumulation rate increases exponentially with temperature (known as a thermal death time curve). The model also assumes thermal stress at different temperatures to be additive and using experiments with Drosophila melanogaster, this study validates these central assumptions by demonstrating that heat injury attained at different heat stress intensities and durations is additive. In a separate experiment it was demonstrated that the model can accurately describe injury accumulation during fluctuating temperature stress and further the model was validated by successfully converting literature data of ectotherm heat tolerance (both static and dynamic assays) to a single, comparable metric (the temperature tolerated for 1 h). The model presented in this study has many promising applications for the analysis of ectotherm thermal tolerance and potential pitfalls that should be considered and avoided using this model are discussed (Jorgensen, 2021).

    Characterization of a novel stimulus-induced glial calcium wave in Drosophila larval peripheral segmental nerves and its role in PKG-modulated thermoprotection

    Insects, as poikilotherms, have adaptations to deal with wide ranges in temperature fluctuation. Allelic variations in the foraging gene that encodes a cGMP dependent protein kinase, were discovered to have effects on behavior in Drosophila by Dr. Marla Sokolowski in 1980. This single gene has many pleiotropic effects and influences feeding behavior, metabolic storage, learning and memory and has been shown to affect stress tolerance. PKG regulation affects motoneuronal thermotolerance in Drosophila larvae as well as adults. While the focus of thermotolerance studies has been on the modulation of neuronal function, other cell types have been overlooked. Because glia are vital to neuronal function and survival, this study determine if glia play a role in thermotolerance as well. In this investigation, a novel calcium wave was discovered at the larval NMJ and the wave's dynamics and the potential mechanism underlying the wave prior was characterized to determining what effect, if any, PKG modulation has on the thermotolerance of glia cells. Using pharmacology, it was determined that calcium buffering mechanisms of the mitochondria and endoplasmic reticulum play a role in the propagation of the novel glial calcium wave. By coupling pharmacology with genetic manipulation using RNA interference (RNAi), it was found that PKG modulation in glia alters thermoprotection of function as well as glial calcium wave dynamics (Krill, 2021).

    Gomez-Llano, M., Scott, E. and Svensson, E. I. (2021). The importance of pre- and postcopulatory sexual selection promoting adaptation to increasing temperatures. Curr Zool 67(3): 321-327. PubMed ID: 34616924

    The importance of pre- and postcopulatory sexual selection promoting adaptation to increasing temperatures

    Global temperatures are increasing rapidly affecting species globally. Understanding if and how different species can adapt fast enough to keep up with increasing temperatures is of vital importance. One mechanism that can accelerate adaptation and promote evolutionary rescue is sexual selection. Two different mechanisms by which sexual selection can facilitate adaptation are pre- and postcopulatory sexual selection. However, the relative effects of these different forms of sexual selection in promoting adaptation are unknown. This study presents the results from an experimental study in which fruit flies Drosophila melanogaster were exposed to either no mate choice or 1 of 2 different sexual selection regimes (pre- and postcopulatory sexual selection) for 6 generations, under different thermal regimes. Populations showed evidence of thermal adaptation under precopulatory sexual selection, but this effect was not detected in the postcopulatory sexual selection and the no choice mating regime. This study further demonstrates that sexual dimorphism decreased when flies evolved under increasing temperatures, consistent with recent theory predicting more sexually concordant selection under environmental stress. These results suggest an important role for precopulatory sexual selection in promoting thermal adaptation and evolutionary rescue (Gomez-Llano, 2021).

    No evidence for short-term evolutionary response to a warming environment in Drosophila

    Adaptive evolution is key in mediating responses to global warming and may sometimes be the only solution for species to survive. Such evolution will expectedly lead to changes in the populations' thermal reaction norm and improve their ability to cope with stressful conditions. Conversely, evolutionary constraints might limit the adaptive response. This study tests these expectations by performing a real-time evolution experiment in historically differentiated Drosophila subobscura populations. The phenotypic change was addressed after nine generations of evolution in a daily fluctuating environment with average constant temperature, or in a warming environment with increasing average and amplitude temperature across generations. The results showed that (1) evolution under a global warming scenario does not lead to a noticeable change in the thermal response; (2) historical background appears to be affecting responses under the warming environment, particularly at higher temperatures; and (3) thermal reaction norms are trait dependent: although lifelong exposure to low temperature decreases fecundity and productivity but not viability, high temperature causes negative transgenerational effects on productivity and viability, even with high fecundity. These findings in such an emblematic organism for thermal adaptation studies raise concerns about the short-term efficiency of adaptive responses to the current rising temperatures (Santos, 2021).

    Developmental temperature affects thermal dependence of locomotor activity in Drosophila

    In their natural environments, animals have to cope with fluctuations in numerous abiotic and biotic factors, and phenotypic plasticity can facilitate survival under such variable conditions. However, organisms may differ substantially in the ability to adjust their phenotypes in response to external factors. This study investigated how developmental temperature affects the thermal performance curve for locomotor activity in adult fruit flies (Drosophila melanogaster). The thermal dependence was examined of spontaneous activity in individuals originating from two natural populations (from tropical (India) and temperate climate zone (Slovakia)) that developed at three different temperatures (19 °C, 25 °C, and 29 °C). Firstly, developmental temperature was found to have a significant impact on overall activity - flies that developed at high temperature (29 °C) were, on average, less active than individuals that developed at lower temperatures. Secondly, developmental acclimation had a population-specific effect on the thermal optimum for activity. Whereas the optimal temperature was not affected by thermal conditions experienced during development in flies from India, developmental temperature shifted thermal optimum in flies from Slovakia. Thirdly, high developmental temperature broadened performance breadth in flies from the Indian population but narrowed it in individuals from the Slovak population. Finally, no consistent effect of acclimation temperature was detected on circadian rhythms of spontaneous activity. Altogether, these results demonstrate that developmental temperature can alter different parameters (maximum performance, thermal optimum, performance breadth) of the thermal performance curve for spontaneous activity. Since adult fruit flies are highly vagile, this sensitivity of locomotion to developmental conditions may be an important factor affecting fitness in changing environments (Klepsatel, 2022).

    The genetic basis and adult reproductive consequences of developmental thermal plasticity

    Increasing temperature and thermal variability generates profound selection on populations. Given the fast rate of environmental change, understanding the role of plasticity and genetic adaptation in response to increasing temperatures is critical. This may be especially true for thermal effects on reproductive traits in which thermal fertility limits at high temperatures may be lower than for survival traits. A panel of Drosophila (the Drosophila Genetic Reference Panel; DGRP) was used in which male fertility performance was previously defined as either showing relatively little (status = "high" performing lines) or substantial ("low" performing lines) decline when exposed to increasing developmental temperatures. Developmental thermal stress impacted the means and thermal reaction norms of all reproductive traits except offspring sex-ratio. Mating success declined as temperature increased with no difference between high and low lines whereas increasing temperature resulted in declines for both male and female fertility and productivity but depended on line status. Fertility and offspring number were positively correlated within and between the sexes, but males were more affected than females. 933 SNPs were identified with significant evolved genetic differentiation between high and low lines. 54 of these lie within genomic windows of overall high differentiation, have significant effects of genotype on the male thermal reaction norm for productivity and are associated with 16 genes enriched for phenotypes affecting reproduction, stress responses and autophagy in Drosophila and other organisms. These results illustrate considerable plasticity in male thermal limits on several reproductive traits following development at high temperature, and differentiated loci with relevant phenotypic effects were identified that may contribute to this population variation (Rodrigues, 2022).

    Dorsal clock networks drive temperature preference rhythms in Drosophila

    Animals display a body body temperature rhythm (BTR). Little is known about the mechanisms by which a rhythmic pattern of BTR is regulated and how body temperature is set at different times of the day. As small ectotherms, Drosophila exhibit a daily temperature preference rhythm (TPR), which generates BTR. This study demonstrates dorsal clock networks that play essential roles in TPR. Dorsal neurons 2 (DN2s) are the main clock for TPR. It was found that DN2s and posterior DN1s (DN1ps) contact and the extent of contacts increases during the day and that the silencing of DN2s or DN1ps leads to a lower temperature preference. The data suggest that temporal control of the microcircuit from DN2s to DN1ps contributes to TPR regulation. This study also identified anterior DN1s (DN1as) as another important clock for TPR. Thus, this study shows that the DN networks predominantly control TPR and determine both a rhythmic pattern and preferred temperatures (Chen, 2022).

    Molecular mechanisms underlying plasticity in a thermally varying environment

    Adaptation to environmental variability is a prerequisite for species' persistence in their natural environments. With climate change predicted to increase the frequency and severity of temperature fluctuations, ectothermic organisms may increasingly depend on acclimation capacity to accommodate thermal variability. To elucidate the molecular basis of fluctuating temperature induced phenotypic plasticity, this study investigated heat tolerance and the mechanisms induced by acclimation to thermal variability as compared to those seen at constant temperature. Genome-wide transcriptomic analysis was carried out on Drosophila melanogaster subjected to acclimation at constant (19 ± 0°C) and fluctuating (19 ± 8°CC) temperatures and contrasted the induction of molecular mechanisms in adult males, adult females, and larvae. Life stage and sex specific dynamics of the acclimation responses to fluctuating temperatures were found. Adult flies exposed to temperature fluctuations showed a constitutive improvement in heat tolerance while heat tolerance of larvae tracked thermal fluctuations. A constitutive down-regulation of gene expression was observed for several genes in the case of larvae exposed to fluctuations. These results for adult females showed that, for several genes, fluctuating temperature acclimation resulted in canalization of gene expression. Both transcriptional and post-transcriptional machinery were greatly affected by fluctuations in the case of adult males. Gene ontology analysis showed enrichment of heat stress response involving several major heat shock proteins in both larvae and adults exposed to fluctuating temperatures, even though fluctuations were in a benign range of temperatures. Finally, molecular mechanisms related to environmental sensing seem to be an important component of insect response to thermal variability (Salachan, 2022).

    Thermal boldness: Volunteer exploration of extreme temperatures in fruit flies

    A dominant perception is that small and motile ectothermic animals must use behavior to avoid exposure to critical or sub-critical temperatures impairing physiological performance. Concomitantly, volunteer exploration of extreme environments by some individuals may promote physiological adjustments and enhance ecological opportunity. This study introduces to the literature a Thermal Decision System (TDS) which is fully modular, thermally stable, versatile, and adaptable to study navigation through thermal landscapes in insects and other small motile animals. A specific setting of the TDS was used to investigate volunteer navigation through critical cold and hot temperatures in Drosophila melanogaster. It was demonstrated that a thermally bold behavior (volunteer crossings through a Critical Temperature Zone, CTZ) characterized a fraction of flies in a sample; such a fraction was higher in an outbred population relative to isofemale lines. As set, the TDS generated a thermal gradient within the cold and hot CTZs, and the exploration of this gradient by flies did not relate simply with a tendency to be thermally bold. Mild fasting affected thermal exploration and boldness in complex manners, but thermal boldness was evident in both fasted and fed flies. Also, thermal boldness was not associated with individual critical temperatures. Finally, some flies showed consistent thermal boldness, as flies that performed an extreme thermal cross were more likely to perform a second cross compared with untested flies. It is hypothesized that a simple 'avoidance principle' is not the only behavioral drive for D. melanogaster facing extreme temperatures over space, and that this pattern may characterize other small motile ectothermic animals with analogous natural history. The physiological correlates, genetic architecture, and interspecific variation of thermal boldness deserve further consideration (Navas, 2022).

    Female fruit flies cannot protect stored sperm from high temperature damage

    Recently, it has been demonstrated that heat-induced male sterility is likely to shape population persistence as climate change progresses. However, an under-explored possibility is that females may be able to successfully store and preserve sperm at temperatures that sterilise males, which could ameliorate the impact of male infertility on populations. This study tested whether females from two fruit fly species can protect stored sperm from a high temperature stress. Sperm carried by female Drosophila virilis are almost completely sterilised by high temperatures, whereas sperm carried by female Zaprionus indianus show only slightly reduced fertility. Heat-shocked D. virilis females can recover fertility when allowed to remate, suggesting that the delivered heat-shock is damaging stored sperm and not directly damaging females in this species. The temperatures required to reduce fertility of mated females are substantially lower than the temperatures required to damage mature sperm in males, suggesting that females are worse than males at protecting mature sperm. This suggests that female sperm storage is unlikely to ameliorate the impacts of high temperature fertility losses in males, and instead exacerbates fertility costs of high temperatures, representing an important determinant of population persistence during climate change (Walsh, 2022).

    A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat

    Small poikilotherms such as the fruit fly Drosophila depend on absolute temperature measurements to identify external conditions that are above (hot) or below (cold) their preferred range and to react accordingly. Hot and cold temperatures have a different impact on fly activity and sleep, but the circuits and mechanisms that adjust behavior to specific thermal conditions are not well understood. This study use patch-clamp electrophysiology to show that internal thermosensory neurons located within the fly head capsule (the AC neurons(1)) function as a thermometer active in the hot range. ACs exhibit sustained firing rates that scale with absolute temperature-but only for temperatures above the fly's preferred ~25°C (i.e., "hot" temperature). ACs were identified in the fly brain connectome and demonstrate that they target a single class of circadian neurons, the LPNs.(2) LPNs receive excitatory drive from ACs and respond robustly to hot stimuli, but their responses do not exclusively rely on ACs. Instead, LPNs receive independent drive from thermosensory neurons of the fly antenna via a new class of second-order projection neurons (TPN-IV). Finally, silencing LPNs blocks the restructuring of daytime "siesta" sleep, which normally occurs in response to persistent heat. Previous work described a distinct thermometer circuit for cold temperature.(3) Together, the results demonstrate that the fly nervous system separately encodes and relays absolute hot and cold temperature information, show how patterns of sleep and activity can be adapted to specific temperature conditions, and illustrate how persistent drive from sensory pathways can impact behavior on extended temporal scales (Alper, 2022).

    Reserpine and PCPA reduce heat tolerance in Drosophila melanogaster

    Drosophila melanogaster is a model organism to study molecular mechanisms and the role of the genes and proteins involved in thermal nociception. Monoamines (i.e. dopamine) have been involved in temperature preference behavior in D. melanogaster. Therefore, this study investigated whether the monoamines, particularly dopamine and serotonin, participate in the response to thermal nociceptive stimuli in D. melanogaster. Flies were treated with reserpine (an inhibitor of vesicular monoamines transporter, 3-300 μM), 3-Iodo-L-tyrosine (3-I-T, an inhibitor of tyrosine hydroxylase, 16.28-65.13 mM), and para-Chloro-DL-phenylalanine (PCPA, an inhibitor of tryptophan hydroxylase, 20-80 mM); then, the flies were subjected to tests of thermal tolerance and avoidance of noxious heat. Climbing behavior was used as a test to evaluate locomotor activity. Reserpine reduces the thermal tolerance profile of the D. melanogaster, as well as the avoidance of noxious heat and locomotor activity depending on the concentration. PCPA, but not 3-I-T, decreased heat tolerance and avoidance of noxious heat. These data suggest that monoamines, particularly serotonin, are associated with the impaired avoidance of noxious heat which could be related to the reduction of heat tolerance in D. melanogaster (Bressan, 2023).

    Temperature change exerts sex-specific effects on behavioural variation

    Temperature is a key factor mediating organismal fitness and has important consequences for species' ecology. While the mean effects of temperature on behaviour have been well-documented in ectotherms, how temperature alters behavioural variation among and within individuals, and whether this differs between the sexes, remains unclear. Such effects likely have ecological and evolutionary consequences, given that selection acts at the individual level. This study investigated the effect of temperature on individual-level behavioural variation and metabolism in adult male and female Drosophila melanogaster (n = 129), by taking repeated measures of locomotor activity and metabolic rate at both a standard temperature (25°C) and a high temperature (28°C). Males were moderately more responsive in their mean activity levels to temperature change when compared to females. However, this was not true for either standard or active metabolic rate, where no sex differences in thermal metabolic plasticity were found. Furthermore, higher temperatures increased both among- and within-individual variation in male, but not female, locomotor activity. Given that behavioural variation can be critical to population persistence, it is suggest edthat future studies test whether sex differences in the amount of behavioural variation expressed in response to temperature change may result in sex-specific vulnerabilities to a warming climate (Brand, 2023). Although containing genes important for sex determination, genetic variation within the Y chromosome was traditionally predicted to contribute little to the expression of sexually dimorphic traits. This prediction was shaped by the assumption that the chromosome harbours few protein-coding genes, and that capacity for Y-linked variation to shape adaptation would be hindered by the chromosome's lack of recombination and holandric inheritance. Consequently, most studies exploring the genotypic contributions to sexually dimorphic traits have focused on the autosomes and X chromosome. Yet, several studies have now demonstrated that the Y chromosome harbours variation affecting male fitness, moderating the expression of hundreds of genes across the nuclear genome. Furthermore, emerging results have shown that expression of this Y-linked variation may be sensitive to environmental heterogeneity, leading to the prediction that Y-mediated gene-by-environment interactions will shape the expression of sexually dimorphic phenotypes. This study tested this prediction, investigating whether genetic variation across six distinct Y chromosome haplotypes affects the expression of locomotor activity, at each of two temperatures (20 and 28 °C) in male fruit flies (Drosophila melanogaster). Locomotor activity is a sexually dimorphic trait in this species, previously demonstrated to be under intralocus sexual conflict. This study demonstrated Y haplotype effects on male locomotor activity, but the rank order and magnitude of these effects were unaltered by differences in temperature. This study contributes to a growing number of studies demonstrating Y-linked effects moderating expression of traits evolving under sexually antagonistic selection, suggesting a role for the Y chromosome in shaping outcomes of sexual conflict.

    Y chromosome-linked variation affects locomotor activity in male Drosophila melanogaster and is robust to differences in thermal environment

    Although containing genes important for sex determination, genetic variation within the Y chromosome was traditionally predicted to contribute little to the expression of sexually dimorphic traits. This prediction was shaped by the assumption that the chromosome harbours few protein-coding genes, and that capacity for Y-linked variation to shape adaptation would be hindered by the chromosome's lack of recombination and holandric inheritance. Consequently, most studies exploring the genotypic contributions to sexually dimorphic traits have focused on the autosomes and X chromosome. Yet, several studies have now demonstrated that the Y chromosome harbours variation affecting male fitness, moderating the expression of hundreds of genes across the nuclear genome. Furthermore, emerging results have shown that expression of this Y-linked variation may be sensitive to environmental heterogeneity, leading to the prediction that Y-mediated gene-by-environment interactions will shape the expression of sexually dimorphic phenotypes. This study tested this prediction, investigating whether genetic variation across six distinct Y chromosome haplotypes affects the expression of locomotor activity, at each of two temperatures (20 and 28 °C) in male fruit flies (Drosophila melanogaster). Locomotor activity is a sexually dimorphic trait in this species, previously demonstrated to be under intralocus sexual conflict. This study demonstrated Y haplotype effects on male locomotor activity, but the rank order and magnitude of these effects were unaltered by differences in temperature. This study contributes to a growing number of studies demonstrating Y-linked effects moderating expression of traits evolving under sexually antagonistic selection, suggesting a role for the Y chromosome in shaping outcomes of sexual conflict (Lay, 2023).

    The structure of behavioral variation within a genotype

    Individual animals vary in their behaviors. This is true even when they share the same genotype and were reared in the same environment. Clusters of covarying behaviors constitute behavioral syndromes, and an individual's position along such axes of covariation is a representation of their personality. Despite these conceptual frameworks, the structure of behavioral covariation within a genotype is essentially uncharacterized and its mechanistic origins unknown. Passing hundreds of inbred Drosophila individuals through an experimental pipeline that captured hundreds of behavioral measures, sparse but significant correlations were found among small sets of behaviors. Thus, the space of behavioral variation has many independent dimensions. Manipulating the physiology of the brain, and specific neural populations, altered specific correlations. It was also observed that variation in gene expression can predict an individual's position on some behavioral axes. This work represents the first steps in understanding the biological mechanisms determining the structure of behavioral variation within a genotype (Werkhoven, 2021).

    Mitochondrial polymorphism shapes intrapopulation behavioural variation in wild Drosophila

    Intrapopulation variation in behaviour, including activity, boldness and aggressiveness, is becoming more widely recognized and is hypothesized to substantially affect ecological and evolutionary dynamics. Although previous studies used candidate-gene approaches and genome-wide association analyses to identify genes correlated with variations in activity and aggressiveness, behavioural variation may not be fully captured in the nuclear genome, as it does not account for mitochondrial genomes. Mitochondrial genes encode products that are key regulators of the cellular energy-producing pathways in metabolic processes and are thought to play a significant role in life-history and reproductive traits. This study considered many isofemale lines of Drosophila immigrans established from two wild populations to investigate whether intrapopulation variation in the mitochondrial genome affected activity level within this species. Two major haplogroups in these populations, and activity levels in both larvae and adults differed significantly between the two haplogroups. This result indicated that intrapopulation variation in activity level may be partially controlled by mitochondrial genes, along with the interaction between nuclear and mitochondrial genes and the age of individual organisms (Ueno, 2021).

    A disinhibitory mechanism biases Drosophila innate light preference

    Innate preference toward environmental conditions is crucial for animal survival. Although much is known about the neural processing of sensory information, how the aversive or attractive sensory stimulus is transformed through central brain neurons into avoidance or approaching behavior is largely unclear. This study shows that Drosophila larval light preference behavior is regulated by a disinhibitory mechanism. In the disinhibitory circuit, a pair of GABAergic neurons exerts tonic inhibition on one pair of contralateral projecting neurons that control larval reorientation behavior. When a larva enters the light area, the reorientation-controlling neurons are disinhibited to allow reorientation to occur as the upstream inhibitory neurons are repressed by light. When the larva exits the light area, the inhibition on the downstream neurons is restored to repress further reorientation and thus prevents the larva from re-entering the light area. It is suggested that disinhibition may serve as a common neural mechanism for animal innate preference behavior (Zhao, 2019).

    Quantification of visual fixation behavior and spatial orientation memory in Drosophila melanogaster

    Drosophila melanogaster has been shown to exhibit short-term orientation memory by fixating on orientations toward previously displayed visual landmarks. However, the fixation behavior varies and is often mixed with other types of movement. Therefore, carefully designed statistical measures are required in order to properly describe the characteristics of the fixation behavior and to quantify the orientation memory exhibited by the fruit flies. To this end, this paper proposes a set of analytical methods. First, the deviation angle, which is used to quantify the deviation of the fruit fly's heading from the landmark positions, is defined. The deviation angle is defined based on the fruit fly's perspective and is able to reveal more task-relevant movement patterns than the commonly used definition which is based on the "observer's perspective." A temporal deviation angle plot is introduced that visually presents the complex movement pattern as a function of time. Next, a fixation index is defined that tolerates fluctuation in the movement and performs better in quantifying the level of fixation behavior, or the orientation memory, than the conventional method (Yen, 2019).

    Intraspecific competition affects the pupation behavior of spotted-wing Drosophila (Drosophila suzukii)

    In Drosophila, intraspecific competition (IC) may cause stress, cannibalism, and affect survival and reproduction. By migrating to less crowded environments, individuals can escape IC. Larvae of spotted-wing drosophila (SWD, Drosophila suzukii) are often exposed to IC. They are known to pupate either attached to or detached from their hosts. This study hypothesized that SWD pupates detached from the larval host as a means to escape IC and increase their survival and fitness. Under laboratory conditions, IC resulted in increased pupation detached from the larval host in both cornmeal medium and blueberry fruit. Males were more prone to detached pupation than females. In blueberry, IC-exposed larvae pupated farther away from the fruit relative to singly-developed individuals. Detached pupation was associated to survival and fitness gains. For example, larvae that displayed detached pupation showed shorter egg-pupa development times, higher pupa-adult survival, and larger adult size relative to fruit-attached individuals. These findings demonstrate that SWD larvae select pupation sites based on IC, and that such a strategy is associated with improved survival and fitness. This information contributes to a better understanding of SWD basic biology and behavior, offering insights to the development of improved practices to manage this pest in the field (Bezerra Da Silva, 2019).

    Plasticity in male mating behavior modulates female life history in fruit flies

    In many species, intense male-male competition for the opportunity to sire offspring has led to the evolution of selfish reproductive traits that are harmful to the females they mate with. In the fruit fly, Drosophila melanogaster, males modulate their reproductive behavior based on the perceived intensity of competition in their premating environment. Specifically, males housed with other males subsequently transfer a larger ejaculate during a longer mating compared to males housed alone. Although the potential fitness benefits to males from such plasticity are clear, its effects on females are mostly unknown. Hence, this study tested the long-term consequences to females from mating with males with distinct social experiences. First, it was verified that competitive experience influences male mating behavior and it was found that males housed with rivals subsequently have shorter mating latencies and longer mating durations. Then, females were exposed every other day for 20 days to males that were either housed alone or with rivals, and subsequently their fitness was measured. Females mated to males housed with rivals were found to produce more offspring early in life but fewer offspring later in life and have shorter lifespans but similar intrinsic population growth rates. These results indicate that plasticity in male mating behavior can influence female life histories by altering females' relative allocation to early versus late investment in reproduction and survival (Filice, 2020).

    Male mating success evolves in response to increased levels of male-male competition

    Male-biased operational sex ratios can increase male-male competition and can potentially select for both increased pre- and postcopulatory male success. In the present study, using populations of Drosophila melanogaster evolved under male-biased (M) or female-biased (F) sex ratios, it was asked whether (a) male mating success can evolve, (b) males are better at mating females that they have coevolved with, (c) males mating success is affected by female mating status, and (d) male mating success is correlated with their courtship effort. M and F males were directly competed for mating with (a) virgin ancestral (common) females, (b) virgin females from the M and F populations, and (c) singly mated females from the M and F populations. The courtship frequency was assessed of the males when paired with mated M or F females. The results show that M males, evolving under an increased level of male-male competition, have higher mating success than F males irrespective of the female evolutionary history. However, the difference in mating success is more pronounced if the females had mated before. M males also have a higher courtship frequency than F males, but no correlation was found between mating success and courtship frequency (Chechi, 2022).

    The transcriptomic signature of responses to larval crowding in Drosophila melanogaster

    Intraspecific competition at the larval stage is an important ecological factor affecting life-history, adaptation and evolutionary trajectory in holometabolous insects. However, the molecular pathways underpinning these ecological processes are poorly characterised. Drosophila melanogaster were reared at three egg densities (5, 60 and 300 eggs/mL), and the transcriptomes were sequenced of pooled third-instar larvae. Emergence time, egg-to-adult viability, adult mass and adult sex-ratio were also measured at each density. Medium crowding had minor detrimental effects on adult phenotypes compared to low density and yielded 24 differentially expressed genes (DEGs) including several chitinase enzymes. In contrast, high crowding had substantial detrimental effects on adult phenotypes and yielded 2107 DEGs. Among these, upregulated gene sets were enriched in sugar, steroid and amino acid metabolism as well as DNA replication pathways, whereas downregulated gene sets were enriched in ABC transporters, Taurine, Toll/Imd signalling and P450 xenobiotics metabolism pathways. Overall, these findings show that larval crowding has a large consistent effect on several molecular pathways (i.e., core responses) with few pathways displaying density-specific regulation (i.e., idiosyncratic responses). This provides important insights into how holometabolous insects respond to intraspecific competition during development (Morimoto, 2022).

    Drosophila female reproductive glands contribute to mating plug composition and the timing of sperm ejection

    Reproductive traits that influence female remating and competitive fertilization rapidly evolve in response to sexual selection and sexual conflict. One such trait, observed across diverse animal taxa, is the formation of a structural plug inside the female reproductive tract (FRT), either during or shortly after mating. In Drosophila melanogaster, male seminal fluid forms a mating plug inside the female bursa, which has been demonstrated to influence sperm entry into storage and latency of female remating. Processing of the plug, including its eventual ejection from the female's reproductive tract, influences the competitive fertilization success of her mates and is mediated by female x male genotypic interactions. However, female contributions to plug formation and processing have received limited attention. Using developmental mutants that lack glandular FRT tissues, this study revealed that these tissues are essential for mating plug ejection. Proteomics was used to demonstrate that female glandular proteins, and especially proteolytic enzymes, contribute to mating plug composition and have a widespread impact on plug formation and composition. Together, these phenotypic and molecular data identify female contributions to intersexual interactions that are a potential mechanism of post-copulatory sexual selection (McDonough-Goldstein, 2022).

    Density-dependent selection in Drosophila: evolution of egg size and hatching time

    Many different laboratory studies of adaptation to larval crowding in Drosophila spp. have all yielded the evolution of preadult competitive ability, even though the ecological context in which crowding was experienced varied across studies. However, the evolution of competitive ability was achieved through different suites of traits in studies wherein crowding was imposed in slightly different ways. This study reports results from a study in which egg size and hatching time were assayed on three sets of populations adapted to larval crowding experienced in slightly different ways, as well as their low density ancestral control populations. Egg size and hatching time are traits that may provide larvae with initial advantages under crowding through increased starting larval size and a temporal head-start, respectively. In each set of populations adapted to some form of larval crowding, the evolution of longer and wider eggs was seen, compared to controls, thus making egg size the first consistent correlate of the evolution of increased larval competitive ability across Drosophila populations experiencing crowding in slightly different ways. Among the crowding-adapted populations, those crowded at the lowest overall eggs/food density, but the highest density of larvae in the feeding band, showed the largest eggs, on an average. All three sets of crowding-adapted populations showed shorter average egg hatching time than controls, but the difference was significant only in the case of populations experiencing the highest feeding band density. These results underscore the importance of considering factors other than just eggs/food density when studying the evolution of competitive ability, as also the advantages of having multiple selection regimes within one experimental set up, allowing for a more nuanced understanding of the subtlety with which adaptive evolutionary trajectories can vary across even fairly similar selection regimes (Venkitachalam, 2022).

    Interaction and integration among behaviors of adult Drosophila in nature

    Living in environments whose ecologies vary in periods as short as 24 h is a challenge for animals as Drosophila species that inhabit pear and apple orchards. These orchards have sunny and shady sections. The size and shape of these habitats change daily according to the position of the sun in the sky. Sunny areas are related to dryness and water loss, and shady places have lower temperatures and higher humidity. The presence of heterospecific flies may lead to competition for space and food. In sunny habitats adult Drosophila were not found. In shady sections conspecific groups D. melanogaster, D. simulans, D. immigrans, D. subobscura, and the Chilean endemic D. pavani were found perched on grasses and herbs at 8-10 cm from fruits that had fallen on the ground. In the fruits, 99% of the adults were females and they were not grouped. The way in which daily changes in the size and shape of shady habitats together with the presence of heterospecific adults influence the selection of places to live is poorly understood in Drosophila. These experiments show that adults of the five species prefer dark areas. The experimental results show that the odors of each species: 1) influence conspecifics to select similar perch sites and decrease mobility, and 2) increase mobility in heterospecific adults and modify their perch site preferences. Attractions between conspecifics, the repulsions between species, and preferences for shaded areas matter in choosing a place to live in the five Drosophila species. These behaviors seem to have evolved as coordinated routines, contributing to the coexistence of the five Drosophila species in the apple and pear orchards examined (Silva-Lopez, 2023).

    Cooperative behavior emerges among Drosophila larvae
    This paper describes a model experimental system of cooperative behavior involving Drosophila larvae. While foraging in liquid food, larvae are observed to align themselves and coordinate their movements in order to drag a common air cavity and dig deeper. Large-scale cooperation is required to maintain contiguous air contact across the posterior breathing spiracles. On the basis of a directed genetic screen, it was found that vision plays a key role in cluster dynamics. The experiments show that blind larvae form fewer clusters and dig less efficiently than wild-type and that socially isolated larvae behave as if they were blind. Furthermore, it was observed that blind and socially isolated larvae do not integrate effectively into wild-type clusters. Behavioral data indicate that vision and social experience are required to coordinate precise movements between pairs of larvae, therefore increasing the degree of cooperativity within a cluster. Hence, it is hypothesized that vision and social experience allow Drosophila larvae to assemble cooperative digging groups leading to more effective feeding and potential evasion of predators. Most importantly, these results indicate that control over membership of such a cooperative group can be regulated (Dombrovski, 2017).

    Kin recognition and co-operative foraging in Drosophila melanogaster larvae

    A long-standing goal for biologists and social scientists is to understand the factors that lead to the evolution and maintenance of co-operative behaviour between conspecifics. To that end, the fruit fly, Drosophila melanogaster, is becoming an increasingly popular model species to study sociality, however, most of the research to date has focused on adult behaviours. This study set out to examine group feeding behaviour by larvae and to determine whether the degree of relatedness between individuals mediates the expression co-operation. In a series of assays, the average degree of relatedness was manipulated in groups of third instar larvae that were faced with resource scarcity, and measured the size, frequency and composition of feeding clusters, as well as the fitness benefits associated with co-operation. The results suggest that larval D. melanogaster are capable of kin recognition (something that has not been previously described in this species), as clusters were more numerous, larger and involved more larvae, when more closely related kin were present in the social environment. These findings are discussed in the context of the correlated fitness-associated benefits of co-operation, the potential mechanisms by which individuals may recognize kin, and how that kinship may play an important role in facilitating the manifestation of this co-operative behaviour (Khodaei, 2019).

    Addition of saturated and trans-fatty acids to the diet induces depressive and anxiety-like behaviors in Drosophila melanogaster

    This study aimed to evaluate the effects of the addition of saturated fat and hydrogenated vegetable fat (HVF) to the diet on depressive and anxiety-like behaviors in Drosophila melanogaster. Flies were exposed to experimental diets: regular diet (RD). or HVF in the concentrations of the substitute (SHVF). HVF 10% and HVF 20%, or Lard (L) in the concentrations of the substitute (SL). L 10% and L 20%, during seven days. The results showed that flies fed with the HVF diet presented similar behaviors to depression, anxiety, and a higher number of aggressive events. Flies exposed to L showed only depressive-like behavior. Regarding serotonin levels (5HT), there was a significant reduction in the flies exposed to SHVF, HVF 10%, HVF 20%, and L 20%. Regarding the levels of octopamine (OA), there was a significant reduction in the flies exposed to both HVF and L rich diets when compared with the RD group. Also, there was a significant negative correlation between 5HT or OA levels and behaviors of aggressiveness, negative geotaxis, immobility time, light/dark, and grooming in the flies. This study shows that Drosophila melanogaster can serve as a valuable model for understanding psychiatric disorders and that the type of fatty acid (FA) offered in the diet can influence these disorders. This demonstrates the importance of the composition of the FAs in the neural pathways, being able to influence the signaling of neurotransmitters, such as 5HT and OA, and thus, cause behavioral changes (Meichtry, 2020).

    A single-cell transcriptomic atlas tracking the neural basis of division of labour in an ant superorganism

    Ant colonies with permanent division of labour between castes and highly distinct roles of the sexes have been conceptualized to be superorganisms, but the cellular and molecular mechanisms that mediate caste/sex-specific behavioural specialization have remained obscure. This study characterized the brain cell repertoire of queens, gynes (virgin queens), workers and males of Monomorium pharaonis by obtaining 206,367 single-nucleus transcriptomes. In contrast to Drosophila, the mushroom body Kenyon cells are abundant in ants and display a high diversity with most subtypes being enriched in worker brains, the evolutionarily derived caste. Male brains are as specialized as worker brains but with opposite trends in cell composition with higher abundances of all optic lobe neuronal subtypes, while the composition of gyne and queen brains remained generalized, reminiscent of solitary ancestors. Role differentiation from virgin gynes to inseminated queens induces abundance changes in roughly 35% of cell types, indicating active neurogenesis and/or programmed cell death during this transition. Insemination-induced cell changes were identified probably associated with the longevity and fecundity of the reproductive caste, including increases of ensheathing glia and a population of dopamine-regulated Dh31-expressing neurons. It is concluded that permanent caste differentiation and extreme sex-differentiation induced major changes in the neural circuitry of ants (Li, 2022).

    This study generated a superorganismal brain cell atlas by profiling all brain cells of the full panel of adult phenotypes that typically make up an ant colony. The ant mushroom body KCs are abundant and transcriptionally diverse relative to the KCs of Drosophila. Conserved optic lobe (OL) neurons were identified that probably play crucial roles in visual courtship behaviour and circadian rhythm regulation in ants. The results are consistent with advanced brain-level division of labour in superorganismal colonies and shed new light on neural mechanisms associated with the lifespan differences between workers and queens (Li, 2022).

    It was expected that the major evolutionary transition to superorganismal colony organization in ancestral ants should have selected for specialization of neural circuitry rather than bigger brains per se. This study provides direct evidence to support this hypothesis, with high degrees of specialization being detectable in the brain cellular composition of all four adult phenotypes of M. pharaonis ants. 41 out of 43 cell types could be detected in all four brain phenotypes, albeit in different abundances. In particular, workers and males have evolved extreme forms of brain specialization and with almost opposite cell-type preferences. Worker brains had the most abundant KCs and optic nerves (OPNs) and the least abundant optic lobe (OL) neurons, all biases that were opposite in male brains. These cellular differences were consistent with anatomical brain structures reflecting the distinct social and sexual specialization in these two phenotypes. Males are extremely short-lived and do not take part in any colony maintenance tasks, as their only function is to find and inseminate a virgin queen. Ant males therefore function as 'simple minded' but extremely targeted sperm vectors. In sharp contrast, workers engage in all the colony tasks except reproduction and need multipurpose brains, consistent with the KCs and OPNs in workers being biased for processing complex information associated with nursing, foraging, colony defence and social communication (Li, 2022).

    Relative to these extremes, gynes and queens had intermediate abundances for almost all brain cell types. This probably reflects that both gynes and queens have maintained functional brain repertoires for a large subset of the social tasks normally done in more advanced ways by workers. Many ants may have retained generalist queen brain functions because they have solitary lives during colony founding, so they need to nurse a first brood and in some species even to forage. However, finding relatively generalist brains in M. pharaonis gynes and queens is remarkable because this species has lost that ancestral independent colony founding behaviour and never needs to operate without worker assistance. However, Monomorium colonies have very many queens, some of which may fail to become inseminated. Such failed queens are known to survive, albeit for less time than inseminated queens, and perform worker-like behaviours, which may have selected for the maintenance of general cognitive abilities in the gyne/queen caste (Li, 2022).

    Overall, the results confirm the concept of complementary divergence in brain function between superorganismal colony members and strongly suggest that fine-tuned brain-level division of labour is an integrated part of sex, caste and reproductive role differentiation, in ways that are not expected to evolve in social systems where a variable number of colony members retain breeder potential even though they may first have helper roles. In many ways, the separate individual brains in colonies of ants such as M. pharaonis combine into a modularly coordinated super-neural organization maintained by advanced communication between colony members. Individual brains are continuously turned over when adult colony members hatch and die, but functional homeostasis and balanced interactions between modules continue, similar to how cells in a metazoan body are turned over without compromising overall body health. The complementary functions of individual brains across the full panel of adult phenotypes are consistent with natural selection maximizing colony-level fitness, as expected for all superorganismal social insects, but not for animals that form societies without irreversible caste differentiation for life among all colony members (Li, 2022).

    Gynes and queens represent two subsequent functional states of the same reproductive female caste, separated by a single insemination event that induces substantial brain transcriptome remodelling resulting in remarkable shifts in behaviour. The gene regulatory network that mediates this reproductive role differentiation is insemination-specific rather than queen-specific, because it has been co-opted by distantly related ant species that secondarily shifted to reproduction via worker-gamergates rather than queens. The present study further explored this convergent evolutionary scenario across castes at the brain cell level. There are parallel cellular shifts across these two caste-specific reproductive role transitions induced by insemination. In particular, a cluster of ensheathing glia with neuroprotective and anti-ageing functions was expanded in both M. pharaonis queens and H. saltator gamergates. It is therefore speculated that ensheathing glia modification might represent one of the proximate mechanisms that ancestrally prolonged queen longevity in ants and whose co-option secondarily extended worker lifespan when they became inseminated as gamergate reproductives. This quantitative reinforcement mechanism of particular neural modules in adulthood effectively decouples queen and worker ageing, so that extremely divergent caste-specific lifespans could evolve (Li, 2022).

    Insemination also induced the expansion of dopamine neurons and a cluster of downstream Dop2R expressing neurons in M. pharaonis queens, and the counterpart cell cluster in H. saltator was found to be convergently expanded in gamergates as well. Experimental confirmation of the gonadotrophic function of dopamine via feeding M. pharaonis gynes with l-dopa suggests that dormant ovary maturation in gynes may be switched into an accelerating trajectory by elevating functionality of dopamine neurons. The downstream Dop2R neurons also preferentially expressed Dh31, a diuretic hormone known to regulate ovulation in flies. The simultaneously expanded dopamine neurons and downstream Dop2R neurons may thus constitute an integral and conserved neural module to realize enhanced reproductive potential in ant queens well beyond the normal fertility levels of solitary insects (Li, 2022).

    Characterization of reproductive dormancy in male Drosophila melanogaster

    Insects are known to respond to seasonal and adverse environmental changes by entering dormancy, also known as diapause. In some insect species, including Drosophila melanogaster, dormancy occurs in the adult organism and postpones reproduction. This adult dormancy has been studied in female flies where it is characterized by arrested development of ovaries, altered nutrient stores, lowered metabolism, increased stress and immune resistance and drastically extended lifespan. Male dormancy, however, has not been investigated in D. melanogaster, and its physiology is poorly known in most insects. This study shows that unmated 3-6 h old male flies placed at low temperature (11 ° C) and short photoperiod (10 Light:14 Dark) enter a state of dormancy with arrested spermatogenesis and development of testes and male accessory glands. Over 3 weeks of diapause a dynamic increase is seen in stored carbohydrates and an initial increase and then a decrease in lipids. An up-regulated expression of genes involved in metabolism, stress responses and innate immunity is also noted. Interestingly, it was found that male flies that entered reproductive dormancy do not attempt to mate females kept under non-diapause conditions (25 ° C, 12L:12D), and conversely non-diapausing males do not mate females in dormancy. In summary, this study shows that male D. melanogaster can enter reproductive dormancy. However, these data suggest that dormant male flies deplete stored nutrients faster than females, studied earlier, and that males take longer to recover reproductive capacity after reintroduction to non-diapause conditions (Kubrak, 2016).

    Behavioral senescence and aging-related changes in motor neurons and brain neuromodulator levels are ameliorated by lifespan-extending reproductive dormancy in Drosophila

    The lifespan of Drosophila can be extended substantially by inducing reproductive dormancy (also known as diapause) by lowered temperature and short days. This increase of longevity is accompanied by lowered metabolism and increased stress tolerance. This study asked whether behavioral senescence is ameliorated during adult dormancy. To study this flies were kept for seven or more weeks in normal rearing conditions or in diapause conditions and compared to 1-week-old flies in different behavioral assays of sleep, negative geotaxis and exploratory walking. The senescence of geotaxis and locomotor behavior seen under normal rearing conditions was negligible in flies kept in dormancy. The normal senescence of rhythmic activity and sleep patterns during the daytime was also reduced by adult dormancy. To monitor age-associated changes in neuronal circuits regulating activity rhythms, sleep and walking behavior antisera were applied to tyrosine hydroxylase (TH), serotonin and several neuropeptides to examine changes in expression levels and neuron morphology. In most neuron types the levels of stored neuromodulators decreased during normal aging, but not in diapause treated flies. No signs of neurodegeneration were seen in either condition. These data suggest that age-related changes in motor neurons could be the cause of part of the behavioral senescence and that this is ameliorated by reproductive diapause. Thus, it is likely that the retained levels of neuromodulators in dormant flies alleviate behavioral senescence (Liao, 2017).

    Selection for reproduction under short photoperiods changes diapause-associated traits and induces widespread genomic divergence

    The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are interrelated is not well understood. This study performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and / or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence between the selection and control line replicates in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled ones detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, this study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity (Kauranen, 2019).

    Effects of Food and Temperature on Drosophila melanogaster Reproductive Dormancy as Revealed by Quantification of a GFP-Tagged Yolk Protein in the Ovary

    When exposed to harsh environmental conditions, such as food scarcity and/or low temperature, Drosophila melanogaster females enter reproductive dormancy, a metabolic state that enhances stress resistance for survival at the expense of reproduction. Although the absence of egg chambers carrying yolk from the ovary has been used to define reproductive dormancy in this species, this definition is susceptible to false judgements of dormancy events: e.g. a trace amount of yolk could escape visual detection; a fly is judged to be in the non-dormancy state if it has a single yolk-containing egg chamber even when other egg chambers are devoid of yolk. In this study, an alternative method is proposed for describing the maturation state of oocytes, in which the amount of yolk in the entire ovary is quantified by the fluorescence intensity derived from GFP, which is expressed as a fusion with the major yolk protein Yp1. Yolk deposition was shown to increase with temperature with a sigmoidal function, and the quality of food substantially alters the maximum accumulation of yolk attainable at a given temperature. The Yp1::GFP reporter will serve as a reliable tool for quantifying the amount of yolk and provides a new means for defining the dormancy state in D. melanogaster (Hara, 2021).

    Seasonal changes in photoperiod and temperature lead to changes in cuticular hydrocarbon profiles and affect mating success in Drosophila suzukii

    Seasonal plasticity in insects is often triggered by temperature and photoperiod changes. When climatic conditions become sub-optimal, insects might undergo reproductive diapause, a form of seasonal plasticity delaying the development of reproductive organs and activities. During the reproductive diapause, the cuticular hydrocarbon (CHC) profile, which covers the insect body surface, might also change to protect insects from desiccation and cold temperature. However, CHCs are often important cues and signals for mate recognition and changes in CHC composition might affect mate recognition. This study investigated the CHC profile composition and the mating success of Drosophila suzukii in 1- and 5-day-old males and females of summer and winter morphs. CHC compositions differed with age and morphs. However, no significant differences were found between the sexes of the same age and morph. The results of the behavioral assays show that summer morph pairs start to mate earlier in their life, have a shorter mating duration, and have more offspring compared to winter morph pairs. It is hypothesized that CHC profiles of winter morphs are adapted to survive winter conditions, potentially at the cost of reduced mate recognition cues (Karpati, 2023).

    Unveiling Subtle Geographical Clines: Phenotypic Effects and Dynamics of Circadian Clock Gene Polymorphisms
    Understanding of the gene regulatory network that constitutes the circadian clock has greatly increased in recent decades, notably due to the use of Drosophila as a model system. In contrast, the analysis of natural genetic variation that enables the robust function of the clock under a broad range of environments has developed more slowly. The current study analyzed comprehensive genome sequencing data from wild European populations of Drosophila, which were densely sampled through time and space. Hundreds of single nucleotide polymorphisms (SNPs) were identified in nine genes associated with the clock, 276 of which exhibited a latitudinal cline in their allele frequencies. While the effect sizes of these clinal patterns were small, indicating subtle adaptations driven by natural selection, they provided important insights into the genetic dynamics of circadian rhythms in natural populations. Nine SNPs in different genes were chosen and their impact on circadian and seasonal phenotypes was assessed by reconstructing outbred populations fixed for either of the SNP alleles, from inbred DGRP strains. The circadian free-running period of the locomotor activity rhythm was affected by an SNP in doubletime (dbt) and eyes absent (Eya). The SNPs in Clock (Clk), Shaggy (Sgg), period (per), and timeless (tim) affected the acrophase. the time period in a cycle during which the cycle crests or peaks. The alleles of the SNP in Eya conferred different levels of diapause and the chill coma recovery response (Khatib, 2023).

    Clinal variation in the temperature and photoperiodic control of reproductive diapause in Drosophila montana females
    Insect adaptation to climatic conditions at different latitudes has required changes in life-history traits linked with survival and reproduction. Several species, including Drosophila montana, show robust latitudinal variation in the critical day length (CDL), below which more than half of the emerging females enter reproductive diapause at a given temperature. This study used a novel approach to find out whether D. montana also shows latitudinal variation in the critical temperature (CTemp), above which the photoperiodic regulation of diapause is disturbed so that the females develop ovaries in daylengths that are far below their CDL. CTemp was estimated for 53 strains from different latitudes on 3 continents after measuring their diapause proportions at a range of temperatures in 12 h daylength (for 29 of the strains also in continuous darkness). In 12 h daylength, CTemp increased towards high latitudes alongside an increase in CDL, and in 3 high-latitude strains diapause proportion exceeded 50 % in all temperatures. In continuous darkness, the diapause proportion was above 50 % in the lowest temperature(s) in only 9 strains, all of which came from high latitudes. In the second part of the study, changes were measured in CTemp and CDL in a selection experiment favouring reproduction in short daylength (photoperiodic selection) and by exercising selection for females that reproduce in LD12:12 at low temperature (photoperiodic and temperature selection). In both experiments selection induced parallel changes in CDL and CTemp, confirming correlations seen between these traits along latitudinal clines. Overall, the findings suggest that selection towards strong photoperiodic diapause and long CDL at high latitudes has decreased the dependency of D. montana diapause on environmental temperature. Accordingly, the prevalence and timing of the diapause of D. montana is likely to be leβ vulnerable to climate warming in high- than low-latitude populations (Lankinen, 2023).

    Female reproductive dormancy in Drosophila is regulated by DH31-producing neurons projecting into the corpus allatum

    Female insects can enter reproductive diapause, a state of suspended egg development, to conserve energy under adverse environments. In many insects, including the fruit fly, Drosophila melanogaster, reproductive diapause, also frequently called reproductive dormancy, is induced under low-temperature and short-day conditions by the downregulation of juvenile hormone (JH) biosynthesis in the corpus allatum (CA). This study demonstrates that neuropeptide Diuretic hormone 31 (DH31) produced by brain neurons that project into the CA plays an essential role in regulating reproductive dormancy by suppressing JH biosynthesis in adult D. melanogaster. The CA expresses the gene encoding the DH31 receptor, which is required for DH31-triggered elevation of intracellular cAMP in the CA. Knocking down Dh31 in these CA-projecting neurons or DH31 receptor in the CA suppresses the decrease of JH titer, normally observed under dormancy-inducing conditions, leading to abnormal yolk accumulation in the ovaries. These findings provide the first molecular genetic evidence demonstrating that CA-projecting peptidergic neurons play an essential role in regulating reproductive dormancy by suppressing JH biosynthesis (Kurogi, 2023).

    Characterization of clock-related proteins and neuropeptides in Drosophila littoralis and their putative role in diapause>

    Insects from high latitudes spend the winter in a state of overwintering diapause, which is characterized by arrested reproduction, reduced food intake and metabolism, and increased life span. The main trigger to enter diapause is the decreasing day length in summer-autumn. It is thus assumed that the circadian clock acts as an internal sensor for measuring photoperiod and orchestrates appropriate seasonal changes in physiology and metabolism through various neurohormones. However, little is known about the neuronal organization of the circadian clock network and the neurosecretory system that controls diapause in high-latitude insects. This was addressed by mapping the expression of clock proteins and neuropeptides/neurohormones in the high-latitude fly Drosophila littoralis. The principal organization of both systems was found to be similar to that in Drosophila melanogaster, but with some striking differences in neuropeptide expression levels and patterns. The small ventrolateral clock neurons that express pigment-dispersing factor (PDF) and short neuropeptide F (sNPF) and are most important for robust circadian rhythmicity in D. melanogaster virtually lack PDF and sNPF expression in D. littoralis. In contrast, dorsolateral clock neurons that express ion transport peptide in D. melanogaster additionally express allatostatin-C and appear suited to transfer day-length information to the neurosecretory system of D. littoralis. The lateral neurosecretory cells of D. littoralis contain more neuropeptides than D. melanogaster. Among them, the cells that coexpress corazonin, PDF, and diuretic hormone 44 appear most suited to control diapause. This work sets the stage to investigate the roles of these diverse neuropeptides in regulating insect diapause (Manoli, 2023).

    Evolution of multiple sensory systems drives novel egg-laying behavior in the fruit pest Drosophila suzukii

    The rise of a pest species represents a unique opportunity to address how species evolve new behaviors and adapt to novel ecological niches. This question was addressed by studying the egg-laying behavior of Drosophila suzukii, an invasive agricultural pest species that has spread from Southeast Asia to Europe and North America in the last decade. While most closely related Drosophila species lay their eggs on decaying plant substrates, D. suzukii oviposits on ripening fruit, thereby causing substantial economic losses to the fruit industry. D. suzukii has evolved an enlarged, serrated ovipositor that presumably plays a key role by enabling females to pierce the skin of ripe fruit. This study explored how D. suzukii selects oviposition sites, and how this behavior differs from that of closely related species. Behavioral experiments were combined in multiple species with neurogenetics and mutant analysis in D. suzukii to show that this species has evolved a specific preference for oviposition on ripe fruit. The results also establish that changes in mechanosensation, olfaction, and presumably gustation have contributed to this ecological shift. These observations support a model in which the emergence of D. suzukii as an agricultural pest is the consequence of the progressive modification of several sensory systems, which collectively underlie a radical change in oviposition behavior (Karageorgi, 2017).

    Upregulation of juvenile hormone titers in female Drosophila melanogaster through mating experiences and host food occupied by eggs and larvae

    Juvenile hormone (JH) plays a crucial role in the determination of developmental timing in insects. In Drosophila melanogaster, reports indicate that JH titers are the highest immediately following eclosion and that the mating experience increases the titers in females. However, the titers have not been successively measured for an extended period of time after eclosion. This study reveals that JH titers are increased after eclosion in virgin females when supplied with food that is occupied by eggs and larvae as well as in mated females. With the presence of eggs and larvae, food induced the virgin females to lay unfertilized eggs. When combined with previous work indicating that females are attracted to such food where they prefer to lay eggs, these results suggest that flies can prepare themselves to lay eggs by changing the titers of JH under the presence of growing larvae, ensuring that the food is an appropriate place to oviposit (Sugime, 2017).

    Physiological responses of insects to microbial fermentation products: insights from the interactions between Drosophila and acetic acid

    Acetic acid is a fermentation product of many microorganisms, including some that inhabit the food and guts of Drosophila. This study investigated the effect of dietary acetic acid on oviposition and larval performance of Drosophila. At all concentrations tested (0.34-3.4%), acetic acid promoted egg deposition by mated females in no-choice assays; and females preferred to oviposit on diet with acetic acid relative to acetic acid-free diet. However, acetic acid depressed larval performance, particularly extending the development time of both larvae colonized with the bacterium Acetobacter pomorum and axenic (microbe-free) larvae. The larvae may incur an energetic cost associated with dissipating the high acid load on acetic acid-supplemented diets. This effect was compounded by suppressed population growth of A. pomorum on the 3.4% acetic acid diet, such that the gnotobiotic Drosophila on this diet displayed traits characteristic of axenic Drosophila, specifically reduced developmental rate and elevated lipid content. It is concluded that acetic acid is deleterious to larval Drosophila, and hypothesized that acetic acid may function as a reliable cue for females to oviposit in substrates bearing microbial communities that promote larval nutrition (Kim, D., 2017).

    Chronic exposure to dim artificial light at night decreases fecundity and adult survival in Drosophila melanogaster
    The presence of artificial light at night (ALAN) is expanding in geographical range and increasing in intensity to such an extent that species living in urban environments may never experience natural darkness. The negative ecological consequences of artificial night lighting have been identified in several key life history traits across multiple taxa (albeit with a strong vertebrate focus); comparable data for invertebrates is lacking. This study explored the effect of chronic exposure to different night-time lighting intensities on growth, reproduction and survival in Drosophila melanogaster. Three generations of flies were reared under identical daytime light conditions (2600lx) and one of four ecologically relevant ALAN treatments (0, 1, 10 or 100lx), then variation was explored in oviposition, number of eggs produced, juvenile growth and survival and adult survival. In the presence of light at night (1, 10 and 100lx treatments), the probability of a female commencing oviposition and the number of eggs laid was significantly reduced. This did not translate into differences at the juvenile phase: juvenile development times and the probability of eclosing as an adult were comparable across all treatments. However, a direct link was demonstrated between chronic exposure to light at night (greater than 1lx) and adult survival. The data highlight that ALAN has the capacity to cause dramatic shifts in multiple life history traits at both the individual and population level. Such shifts are likely to be species-specific, however a more in depth understanding of the broad-scale impact of ALAN and the relevant mechanisms driving biological change is urgently required as research moves into an increasing brightly lit future (McLay, 2017).

    Peptidoglycan sensing by octopaminergic neurons modulates Drosophila oviposition

    As infectious diseases pose a threat to host integrity, eukaryotes have evolved mechanisms to eliminate pathogens. In addition to develop strategies reducing infection, animals can engage in behaviours that lower the impact of the infection. The molecular mechanisms by which microbes impact host behaviour are not well understood. This study demonstrated that bacterial infection of Drosophila females reduces oviposition and that bacterial cell wall peptidoglycan, the component that activates Drosophila antibacterial response, is also the elicitor of this behavioral change. Peptidoglycan regulates egg laying rate by activating PGRP-LC -> NF-κB (Relish) signaling pathway in octopaminergic neurons and that, a dedicated peptidoglycan degrading enzyme acts in these neurons to buffer this behavioural response. This study shows that a unique ligand and signaling cascade are used in immune cells to mount an immune response and in neurons to control fly behavior following infection. This may represent a case of behavioural immunity (Kurz, 2017).

    In addition to activate direct antimicrobial strategies, eukaryotes have developed behavioral mechanisms that facilitate the avoidance of pathogens or lower the impact of the infection. These phenotypes grouped under the term 'behavioral immunity' or 'sickness behavior' refer to a suite of neuronal mechanisms that allow organisms to detect the potential presence of disease-causing agents and to engage in behaviors which prevent contact with the invaders or reduce the consequences of the infection. Although such microbe-induced behavioral changes have been reported in Lepidoptera and Orthoptera, deciphering the molecular mechanisms involved is experimentally challenging in these insects. Indeed, such an analysis requires a model organism with genetic tools allowing the manipulation of actors and regulators of both the immune and neuronal systems. Recent reports, mainly in Drosophila, start to unravel some aspects of these peculiar host-microbe interactions. Stensmyr et al. demonstrated that Drosophila avoid food contaminated by pathogenic bacteria by using an olfactory pathway exquisitely tuned to a single microbial odor, Geosmin (Stensmyr, 2012). Produced by harmful microorganisms, Geosmin is detected by specific Drosophila olfactory sensory neurons which then transfer the message to higher brain centers. Activation of this olfactory circuit ultimately induces an avoidance response, and suppresses egg-laying and feeding behaviors, thereby reducing the infection risk of both the adult flies and their offspring. Drosophila not only modify their behavior to avoid contamination by microbes or parasites, but also once they have been contaminated in order to reduce the impact of infection. For instance, direct exposure to bacteria impacts sleep patterns and induces hygienic grooming. In addition, Drosophila plastically increases the production of recombinant offspring in response to parasite infection. Although certainly involving a neuro-immunological integration, these microbe-induced behavioral changes are rarely understood at the molecular level, namely with no information on the nature of the elicitor and on the cellular and molecular machineries that link bacteria detection to behavioral changes. Moreover, canonical immune signaling pathways were never reported as being involved in those processes (Kurz, 2017).

    The data demonstrate that bacteria derived cell wall peptidoglycan (PGN) entry into the fly body cavity has, at least, two physiological consequences. In addition to activate innate immune response in fat body cells, it also blocks mature egg delivery in oviduct and hence reduces egg laying of infected females. It was further demonstrated that this bacterially induced behavioral change is due to an NF-κB pathway-dependent modulation in octopaminergic neurons. Evidence is presented that both responses, that are potentially detrimental if not down-regulated, are fine-tuned by distinct and specific PGN degrading enzymes. It is proposed that by regulating the level of internal PGN, flies adapt their egg-laying behavior to environmental conditions. In standard environmental conditions, PGRP-LB ensures that low level of PGN does not affect egg laying. However, whenever PGN concentration reaches a certain threshold, which either reflects an infection status or the presence of a highly contaminated food supply, NF-κB pathway activation in neurons is blocking egg release. As PGN of ingested bacteria is capable of reaching the internal fluid and triggering dedicated signaling cascades, one could imagine that such a mechanism prevents flies from laing their eggs in highly contaminated food, in which their development and that of the hatching larvae could be impaired by microbes. In this context, PGRP-LB mediated PGN scavenging is crucial since a non-regulated behavioral immune response would lead to a severe drop in the amount of progeny which may not be in keeping with the real threat. Another possibility could be that a reduced egg production will favor immune effector production. Indeed, it is often considered that the energy cost of an acute innate immune response needs can be balanced by a decreased offspring production. Blocking the energy-consuming egg production in infected flies could be a way for them to mobilize resources required for full activation of innate immune defences. A similar depression of oviposition has recently been documented in females flies exposed to parasitoid wasps who lay their eggs in Drosophila larvae. However, while visual perception of wasps by female flies induces a long-term decline in oviposition associated with an early stage-specific oocyte apoptosis, PGN effects are transient and rather lead to a late stage oocyte accumulation suggesting that although the final outcome is the same, the mechanisms differ (Kurz, 2017).

    The data from this study indicate that PGN sensing acts on egg-laying behavior via neuronal modulation. NF-κB pathway signaling in octopaminergic neurons was identified as the actor of this PGN-dependent oviposition reduction. It would be informative to test whether bacterial infection is also affecting other octopamine-mediated behaviors such as reward in olfactory or visual learning, male-male courtship, male aggressive behavior. This would require to further characterize the nature of the octopamine neurons whose activation is modulated by infection and to consider that the phenotypes defined as being part of the sickness behaviours might be orchestrated directly by the immune system following the perception of microbes. Indeed, a PGRP-LBPD reporter line not only labels cells in the reproductive tract but also in thoraco-abdominal ganglia and in the brain with projections to proboscis, wings and legs. Likewise, octopaminergic neurons have been shown to innervate numerous areas in the brain and in the thoraco-abdominal ganglion and to project to various reproductive structures such as ovaries, oviducts and uterus, further work will be needed to exactly pinpoint the identity of the affected octopaminergic neurons, their targets and their effect on fly behavior. In addition, the question remains as to how NF-κB activation can modulate octopaminergic neurons activity. Among the possibilities is the modulation of octopamine neuron excitability, the regulation of octopamine production or its secretion. Knowing the NF-κB protein itself is required for this behavioral response and that increasing the amount of available octopamine via overexpression of the TβH enzyme rescues the oviposition drop, it is expected that IMD pathway activation in neurons will have transcriptional consequences. However, other hypotheses might be considered since Dorsal, a member of the other Drosophila NF-κB signaling cascade Toll, has been shown to function post-transcriptionally together with IκB and IRAK at the post-synaptic membrane to specify glutamate receptor density. It should also be noticed that PGRP-LC has recently been shown to control presynaptic homeostatic plasticity in mouse (Harris, 2015). One of the future challenges will be to understand how NF-κB activation is reducing octopaminergic signals (Kurz, 2017).

    This study shows that Drosophila uses an unique bacteria associated molecular pattern to activate different processes related to host defence, namely the production of antimicrobial peptides and the modulation of oviposition behavior. Interestingly, it appears that in order to fine-tune these responses, different isoforms of the same PGN scavenging enzyme, PGRP-LB, are required. While the secreted PGRP-LBPC isoform certainly acts non cell-autonomously to dampen immune activation by circulating PGN, a putatively intracellular isoform PGRP-LBPD controls the effect of PGN on oviposition. Even more remarkable, this response is not transmitted via PGRP-LC but rather by the intracytoplasmic PGRP-LE receptor. Previous work has shown that PGRP-LE is also regulating response to bacteria in some part of the gut. Thus, it will be important to understand how PGN is trafficking within and through cells, and how PGRP-LBPD modulates PGRP-LE-dependent IMD pathway activation and whether it is also required to modulate other PGN/PGRP-LE-dependent responses (Kurz, 2017).

    In essence, the results demonstrate that PGN, when ingested or introduced into the body cavity, not only activates antibacterial immune response but also influences neuronally controlled behaviors in flies. Importantly, the sickness behavior deciphered in this study does not appear to be a side effect of an energetically expensive immune response, but rather the result of a specific regulation. An orchestration of different processes required for the immune response was also exemplified by a recent report linking metabolism and immunity. Although not dissected to the molecular level, previous studies in mammals have suggested that similar interactions between PGN and neuronally controlled activities. For instance, PGN derived muropeptide MDP has been shown to display powerful somnogenic effect when injected into rabbit braint. It has also been shown that PGN produced by symbiotic microbiota may 'leak' into the bloodstream and reach organs distant to the gut, such as the bones. Finally, recent findings show that bacterial cell wall peptidoglycan traverses the murine placenta and reach the developing fetal brain where it triggers a TLR2-dependent fetal neuroproliferative response. A future challenge will be to test whether an NF-κB-dependent response to PGN is also taking place in mammalian neurons and directly influences the animal behavior (Kurz, 2017).

    A single pair of neurons modulates egg-laying decisions in Drosophila

    Animals have to judge environmental cues and choose the most suitable option for them from many different options. Female fruit flies selecting an optimum site to deposit their eggs is a biologically important reproductive behavior. When given the direct choice between ovipositing their eggs in a sucrose-containing medium or a caffeine-containing medium, female flies prefer the latter. However, the neural circuits and molecules that regulate this decision-making processes during egg-laying site selection remain poorly understood. The present study found that amnesiac (amn) mutant flies show significant defects in egg-laying decisions, and such defects can be reversed by expressing the wild-type amn transgene in two dorsal paired medial (DPM) neurons in the brain. Silencing neuronal activity with an inward rectifier potassium channel (Kir2.1) in DPM neurons also impaired egg-laying decisions. Finally, the activity in mushroom body αβ neurons was required for the egg-laying behavior, suggesting a possible 'DPM-αβ neurons' brain circuit modulating egg-laying decisions. These results highlight the brain circuits and molecular mechanisms of egg-laying decisions in Drosophila (Wu, 2015).

    amn encodes a preproneuropeptide with limited similarity to pituitary-adenylyl-cyclase-activating peptide (PACAP). It has been reported that AMN plays a critical role in behaviors of Drosophila such as olfactory memory and sleep (Liu, 2008). To examine the role of the amn gene in egg-laying decisions, a collection of amn mutants were analyzed for their egg-laying preference in the behavioral chambers. Interestingly, amn1, amn28A, amnc651, and amnX8 mutants showed significant defects in egg-laying preference compared to wild-type flies. The egg-laying preference was further examined in the chamber containing sucrose or caffeine substrate in one side and a plain substrate in the opposite side. Consistent with the previous findings, wild-type female flies avoided laying eggs on sucrose or caffeine substrates when the other option was a plain substrate. All the amn mutants show significant difference in egg-laying preference in sucrose/plain or caffeine/plain chambers compared to wild-type flies. These results indicate the amn gene is critical for egg-laying decisions in sucrose/caffeine, sucrose/plain, and caffeine/plain mediums (Wu, 2015).

    Although the amn gene is expressed throughout the fly brain, targeting expression of the amn gene in two DPM neurons restores the olfactory memory in amn mutant flies (Waddell, 2000). Tests were performed to see whether the amn gene product in DPM neurons is involved in egg-laying decisions. A GAL4/UAS system was used to target expression of the wild-type amn transgene (amn+) in DPM neuron by applying three independent DPM specific GAL4 drivers, the C316-GAL4, VT6412-GAL4, and VT64246-GAL4. amn1 is an EMS-induced mutation in the allele of the amn gene that causes a significant reduction in the amn transcript. Therefore, amn1 was chosen to perform the following rescue experiment. Flies carrying the amn1/amn1; +/+; C316-GAL4/UAS-amn+, or amn1/amn1; +/+; VT6412-GAL4/UAS-amn+, or amn1/amn1; +/+; VT64246-GAL4/UAS-amn+ showed normal egg-laying preferences compared to wild-type flies, indicating that targeting expression of the amn transgene in DPM neurons restored typical egg-laying preference. In addition, acute silencing of the neuronal activity in DPM neurons by an inward rectifier potassium channel (Kir2.1) disrupts egg-laying preferences, suggesting a role of neurotransmission in DPM neurons for execution of normal egg-laying preference (Wu, 2015).

    The fibers of DPM neurons innervate the mushroom body, and both axons and dendrites are evenly distributed in the lobes and the anterior peduncle of the mushroom body. Therefore, the role of the mushroom body neurons was examined in egg-laying preferences of female flies. The Drosophila mushroom body consists of 2000 neurons in each hemisphere of the brain, and the neurons in the mushroom body can be classified into the γ, α'β', and αβ subsets. The effects were examined of acute inhibition of activity in different subsets of mushroom body neurons by tubP-GAL80ts; UAS-Kir2.1 combined with R16A06-GAL4 (γ neurons) or VT30604-GAL4 (α'β' neurons) or VT49246-GAL4 (αβ neurons. Surprisingly, only inhibiting the neuronal activity in the αβ neurons disrupted the normal female egg-laying preference. These data suggest that the release of the AMN neuropeptide from DPM neurons onto the mushroom body αβ neurons regulates egg-laying preference in female flies (Wu, 2015).

    The egg-laying site selection by female fruit flies provides a suitable system to study the cellular mechanisms of a simple decision-making behavior. When given the direct choice between a sucrose-containing medium and a caffeine-containing medium, flies prefer to lay eggs on the latter. This decision-making process during egg-laying site selection is unchanged in aged animals, suggesting that aging does not dramatically alter the neural activity involved in egg-laying decisions (Wu, 2015).

    amn1 is the first amnesiac mutant isolated from the behavioral screening for olfactory memory mutants. This study has identified the crucial role of the amn gene on egg-laying decisions in female flies. The egg-laying preference is altered in amn1, amn28A, and amnC651, and amnX8 mutants compared to wild-type flies, implying that the amn gene product is important for normal egg-laying decisions. Interestingly, it was observed that the amnX8 showed significant difference in egg-laying preference in sucrose/caffeine or sucrose/plain medium compared to the other amn mutants. The original amn1 is an EMS-induced mutant allele in the amn gene while amn28A and amnc651 are P-element-induced mutations in the amn gene. The amnX8 was made by imprecise excision of the P-element from amn28A, and a significant increase in ethanol-sensitive phenotype was found in amnX8 compared to amn1 and amn28A. It is noteworthy that amnX8 contains possibly other GAL4 insertions elsewhere in the genome left after excision of amn28A, which may cause a significant negative value of egg-laying preference index in sucrose/caffeine medium. Genetic expression of the wild-type amn transgene in DPM neurons of amn1 mutant flies restores the deficiency of egg-laying preference, suggesting that the expression of AMN in DPM neurons is sufficient for normal egg-laying decisions. The AMN neuropeptide is a homologue of the vertebrate PACAP that mediates several physiological functions through stimulation of cAMP production, implying that the cAMP-signaling pathway is important for decision-making processes during egg-laying site selection in Drosophila (Bhattacharya, 2004; Wu, 2015 and references therein).

    Both the axons and dendrites of DPM neurons are evenly distributed in different lobes of the mushroom body, suggesting that DPM neurons receive from and transmit to the mushroom body. It has been reported that the neurotransmissions from DPM or mushroom body α'β' neurons are required for olfactory memory consolidation. In addition, the projections of DPM neurons to the α'β' lobes of the mushroom body are sufficient for stabilizing olfactory memory. These data suggest the possible reciprocal feedback circuits between DPM-mushroom body α'β' neurons for olfactory memory consolidation. The current data indicate that AMN release from DPM neurons is critical for normal egg-laying decisions. Silencing the activity in mushroom body αβ neurons also affects this behavior, suggesting that the neural circuitry downstream of DPM neurons modulates egg-laying decisions. However, the neural activity in mushroom body α'β' neurons is not required for normal egg-laying decisions, which indicates the involvement of separate subsets of mushroom body neuron during olfactory memory consolidation and egg-laying decisions. In addition to the AMN neuropeptide, it has been shown that DPM neurons also release serotonin (5HT) onto the mushroom body αβ neurons via the action of the 5HT1A receptor. Whether 5HT and the 5HT1A receptor are required for egg-laying decisions is still unknown (Wu, 2015).

    Interestingly, a recent study identified that different subsets of dopaminergic neurons play opposing roles in egg-laying preference on ethanol substrate in a concentration-dependent manner (Azanchi, 2013). Neuronal activity in the mushroom body α'β' neurons and the ellipsoid body R2 neurons is also required for normal egg-laying preference for ethanol in female flies (Azanchi, 2013). It is speculated that egg-laying decisions on different substrates (i.e. different concentrations of ethanol-containing foods or sucrose/caffeine containing medium) are mediated by independent subsets of mushroom body neurons. Further study is needed to establish the molecular and neural circuits in the mushroom body involved in decision-making processes during egg-laying site selection in Drosophila (Wu, 2015).

    Ethanol confers differential protection against generalist and specialist parasitoids of Drosophila melanogaster

    As parasites coevolve with their hosts, they can evolve counter-defenses that render host immune responses ineffective. These counter-defenses are more likely to evolve in specialist parasites than generalist parasites; the latter face variable selection pressures between the different hosts they infect. Natural populations of Drosophila are commonly threatened by endoparasitoid wasps in the genus Leptopilina, including the specialist L. boulardi and the generalist L. heterotoma, and both wasp species can incapacitate the cellular immune response of D. melanogaster larvae. Given that ethanol tolerance is high in D. melanogaster and stronger in the specialist wasp than the generalist, whether fly larvae could use ethanol as an anti-parasite defense and whether its effectiveness would differ against the two wasp species was tested. Fly larvae benefited from eating ethanol-containing food during exposure to L. heterotoma; a two-fold decrease in parasitization intensity and a 24-fold increase in fly survival to adulthood were observed. Although host ethanol consumption did not affect L. boulardi parasitization rates or intensities, it led to a modest increase in fly survival. Thus, ethanol conferred stronger protection against the generalist wasp than the specialist. Overall, these results suggest that D. melanogaster larvae obtain protection from certain parasitoid wasp species through their mothers' innate oviposition preferences for ethanol-containing food sources (Lynch, 2017).

    Olfactory neurons and brain centers directing oviposition decisions in Drosophila

    The sense of smell influences many behaviors, yet how odors are represented in the brain remains unclear. A major challenge to studying olfaction is the lack of methods allowing activation of specific types of olfactory neurons in an ethologically relevant setting. To address this, a genetic method was developed in Drosophila called olfactogenetics in which a narrowly tuned odorant receptor, Or56a, is ectopically expressed in different olfactory neuron types. Stimulation with geosmin (the only known Or56a ligand) in an Or56a mutant background leads to specific activation of only target olfactory neuron types. This approach was used to identify olfactory sensory neurons (OSNs) that directly guide oviposition decisions. Five OSN-types (Or71a, Or47b, Or49a, Or67b, and Or7a) were identified that, when activated alone, suppress oviposition. Projection neurons partnering with these OSNs share a region of innervation in the lateral horn, suggesting that oviposition site selection might be encoded in this brain region (Chin, 2018).

    A post-ingestive amino acid sensor promotes food consumption in Drosophila

    Adequate protein intake is crucial for the survival and well-being of animals. How animals assess prospective protein sources and ensure dietary amino acid intake plays a critical role in protein homeostasis. By using a quantitative feeding assay, this study shows that three amino acids, L-glutamate (L-Glu), L-alanine (L-Ala) and L-aspartate (L-Asp), but not their D-enantiomers or the other 17 natural L-amino acids combined, rapidly promote food consumption in the fruit fly Drosophila melanogaster. This feeding-promoting effect of dietary amino acids is independent of mating experience and internal nutritional status. In vivo and ex vivo calcium imagings show that six brain neurons expressing diuretic hormone 44 (DH44) can be rapidly and directly activated by these amino acids, suggesting that these neurons are an amino acid sensor. Genetic inactivation of DH44(+) neurons abolishes the increase in food consumption induced by dietary amino acids, whereas genetic activation of these neurons is sufficient to promote feeding, suggesting that DH44(+) neurons mediate the effect of dietary amino acids to promote food consumption. Single-cell transcriptome analysis and immunostaining reveal that a putative amino acid transporter, CG13248, is enriched in DH44(+) neurons. Knocking down CG13248 expression in DH44(+) neurons blocks the increase in food consumption and eliminates calcium responses induced by dietary amino acids. Therefore, these data identify DH44(+) neuron as a key sensor to detect amino acids and to enhance food intake via a putative transporter CG13248. These results shed critical light on the regulation of protein homeostasis at organismal levels by the nervous system (Yang, 2018).

    Sensory deficiencies affect resource selection and associational effects at two spatial scales

    Many insect species have limited sensory abilities and may not be able to perceive the quality of different resource types while approaching patchily distributed resources. These restrictions may lead to differences in selection rates between separate patches and between different resource types within a patch, which may have consequences for associational effects between resources. This study used an oviposition assay containing different frequencies of apple and banana substrates divided over two patches to compare resource selection rates of wild-type Drosophila melanogaster at the between- and within-patch scales. Next, the wild-type behavior was compared with that of the olfactory-deficient strain Orco (2) and the gustatory-deficient strain Poxn (DeltaM22-B5), and comparable responses were found to patch heterogeneity and similarly strong selection rates for apple at both scales for the wild-type and olfactory-deficient flies. Their oviposition behavior translated into associational susceptibility for apple and associational resistance for banana. The gustatory-deficient flies, on the other hand, no longer had a strong selection rate for apple, strongly differed in between- and within-patch selection rates from the wild-type flies, and caused no associational effects between the resources. This study suggests that differences in sensory capabilities can affect resource selection at different search behavior scales in different ways and in turn underlie associational effects between resources at different spatial scales (Verschut, 2018).

    Correlated decision making across multiple phases of olfactory guided search in Drosophila improves search efficiency

    Nearly all motile organisms must search for food, often requiring multiple phases of exploration across heterogeneous environments. The fruit fly, Drosophila, has emerged as an effective model system for studying this behavior, however, little is known about the extent to which experiences at one point in their search might influence decisions in another. To investigate whether prior experiences impact flies' search behavior after landing, individually labelled fruit flies were tracked as they explored three odor emitting but food-barren objects. Two features of their behavior correlated with the distance they travel on foot. First, flies walked larger distances when they approached the odor source, which they were almost twice as likely to do when landing on the patch farthest downwind. Computational fluid dynamics simulations suggest this patch may have had a stronger baseline odor, but only ∼15% higher than the other two. This small increase, together with flies' high olfactory sensitivity, suggests that perhaps their flight trajectory used to approach the patches plays a role. Second, flies also walked larger distances when the time elapsed since their last visit was longer. However, the correlation is subtle and subject to a large degree of variability. Using agent-based models, it was shown that this small correlation can increase search efficiency by 25-50% across many scenarios. Furthermore, the models developed in this study provide mechanistic hypotheses explaining the variability through either a noisy or straightforward decision-making process. Surprisingly, these stochastic decision-making algorithms enhance search efficiency in challenging but realistic search scenarios compared to deterministic strategies (Breugel, 2021).

    Drosophila re-zero their path integrator at the center of a fictive food patch

    The ability to keep track of one's location in space is a critical behavior for animals navigating to and from a salient location, and its computational basis is now beginning to be unraveled. This study tracked flies in a ring-shaped channel as they executed bouts of search triggered by optogenetic activation of sugar receptors. Unlike experiments in open field arenas, which produce highly tortuous search trajectories, the geometrically constrained paradigm enabled monitoring flies' decisions to move toward or away from the fictive food. The results suggest that flies use path integration to remember the location of a food site even after it has disappeared, and flies can remember the location of a former food site even after walking around the arena one or more times. To determine the behavioral algorithms underlying Drosophila search, multiple state transition models were developed and found that flies likely accomplish path integration by combining odometry and compass navigation to keep track of their position relative to the fictive food. The results indicate that whereas flies re-zero their path integrator at food when only one feeding site is present, they adjust their path integrator to a central location between sites when experiencing food at two or more locations. Together, this work provides a simple experimental paradigm and theoretical framework to advance investigations of the neural basis of path integration (Behbahani, 2021).

    Transcriptional Correlates of Chronic Alcohol Neuroadaptation in Drosophila Larvae

    When presented with the choice, Drosophila melanogaster females will often prefer to lay eggs on food containing a significant amount of alcohol. While, in some cases, this behavioral decision can provide a survival advantage to the developing larvae, it can also lead to developmental and cognitive problems. Alcohol consumption can affect executive functions, episodic memory, and other brain function capacities. However, in the fruit fly, the initial cognitive effects of alcohol consumption have been shown to reverse upon persistent exposure to alcohol. Using an olfactory conditioning assay where an odorant is implemented as a conditioned stimulus and paired with a heat shock as an unconditioned stimulus, a previous study has shown that when exposed to a short acute dose of alcohol, Drosophila larvae can no longer learn this association. Interestingly, upon prolonged chronic alcohol exposure, larvae seem to successfully avoid the conditioned stimulus just as well as control alcohol-naive larvae, suggestive of alcohol-induced neuroadaptations. However, the mechanisms by which Drosophila adapt to the presence of alcohol remains unknown. This study explores the transcriptional correlates of neuroadaptation in Drosophila larvae exposed to chronic alcohol to understand the genetic and cellular components responsible for this adaptation. For this, RNA sequencing technology was employed to evaluate differences in gene expression in the brain of larvae chronically exposed to alcohol. The results suggest that alcohol-induced neuroadaptations are modulated by a diverse array of synaptic genes within the larval brain through a series of epigenetic modulators (Anqueira-Gonzalez, 2021).

    A heuristic underlies the search for relief in Drosophila melanogaster

    Humans rely on multiple types of sensory information to make decisions, and strategies that shorten decision-making time by taking into account fewer but essential elements of information are preferred to strategies that require complex analyses. Such shortcuts to decision making are known as heuristics. The identification of heuristic principles in species phylogenetically distant to humans would shed light on the evolutionary origin of speed-accuracy trade-offs and offer the possibility for investigating the brain representations of such trade-offs, urgency and uncertainty. By performing experiments on spatial learning in the invertebrate Drosophila melanogaster, this study showed that the fly's search strategies conform to a spatial heuristic-the nearest neighbor rule-to avoid bitter taste (a negative stimulation). That is, Drosophila visits a salient location closest to its current position to stop the negative stimulation; only if this strategy proves unsuccessful does the fly use other learned associations to avoid bitter taste. Characterizing a heuristic in D. melanogaster supports the view that invertebrates can, when making choices, operate on economic principles, as well as the conclusion that heuristic decision making dates to at least 600 million years ago (Meda, 2021).

    Wosniack, M. E., Festa, D., Hu, N., Gjorgjieva, J. and Berni, J. (2022). Adaptation of Drosophila larva foraging in response to changes in food resources. Elife 11. PubMed ID: 36458693

    Adaptation of Drosophila larva foraging in response to changes in food resources

    All animals face the challenge of finding nutritious resources in a changing environment. To maximize lifetime fitness, the exploratory behavior has to be flexible, but which behavioral elements adapt and what triggers those changes remain elusive. Using experiments and modeling, this study characterized extensively how Drosophila larvae foraging adapts to different food quality and distribution and how the foraging genetic background influences this adaptation. This work shows that different food properties modulated specific motor programs. Food quality controls the traveled distance by modulating crawling speed and frequency of pauses and turns. Food distribution, and in particular the food-no food interface, controls turning behavior, stimulating turns toward the food when reaching the patch border and increasing the proportion of time spent within patches of food. Finally, the polymorphism in the foraging gene (rover-sitter) of the larvae adjusts the magnitude of the behavioral response to different food conditions. This study defines several levels of control of foraging and provides the basis for the systematic identification of the neuronal circuits and mechanisms controlling each behavioral response (Wosniack, 20232)

    Age of both parents influences reproduction and egg dumping behavior in Drosophila melanogaster

    Trans-generational maternal effects have been shown to influence a broad range of offspring phenotypes. However, very little is known about paternal trans-generational effects. This study tested the trans-generational effects of maternal and paternal age, and their interaction, on daughter and son reproductive fitness in Drosophila melanogaster. Significant effects were found of parent ages on offspring reproductive fitness over 10 days post-fertilization. In daughters, older (45 days old) mothers conferred lower reproductive fitness compared to younger mothers (3 days old). In sons, father's age significantly affected reproductive fitness. The effects of two old parents were additive in both sexes and reproductive fitness was lowest when the focal individual had two old parents. Interestingly, daughter fertility was sensitive to father's age but son fertility was insensitive to mother's age, suggesting a sexual asymmetry in trans-generational effects. The egg-laying dynamics in daughters dramatically shaped this relationship. Daughters with two old parents demonstrated an extreme egg dumping behavior on day one and laid >2.35 X the number of eggs than the other three age class treatments. This study reveals significant trans-generational maternal and paternal age effects on fertility and an association with a novel egg laying behavioral phenotype in Drosophila (Mossman, 2019).

    Reproductive fitness of Drosophila is maximised by optimal developmental temperature

    Whether the character of developmental plasticity is adaptive or non-adaptive has often been a matter of controversy. Although thermal developmental plasticity has been studied in Drosophila for several traits, it is not entirely clear how it affects reproductive fitness. This study, therefore, investigated how developmental temperature affects reproductive performance (early fecundity and egg-to-adult viability) of wild-caught Drosophila melanogaster. Competing hypotheses on the character of developmental thermal plasticity were characterized using a full-factorial design with three developmental and adulthood temperatures within the natural thermal range of this species. To account for potential intraspecific differences, flies were examined from tropical (India) and temperate (Slovakia) climate zones. The results show that flies from both populations raised at an intermediate developmental temperature (25 ° C) have comparable or higher early fecundity and fertility at all tested adulthood temperatures, while lower (17 ° C) or higher developmental temperatures (29 ° C) did not entail any advantage under the tested thermal regimes. Importantly, the superior thermal performance of flies raised at 25 ° C is apparent even after taking two traits positively associated with reproductive output into account: body size and ovariole number. Thus, in D. melanogaster, development at a given temperature does not necessarily provide any advantage in this thermal environment in terms of reproductive fitness. These findings strongly support the optimal developmental temperature hypothesis, which states that in different thermal environments, the highest fitness is achieved when an organism is raised at its optimal developmental temperature (Klepsatel, 2019).

    Sweet neurons inhibit texture discrimination by signaling TMC-expressing mechanosensitive neurons in Drosophila

    Integration of stimuli of different modalities is an important but incompletely understood process during decision making. This study shows that Drosophila are capable of integrating mechanosensory and chemosensory information of choice options when deciding where to deposit their eggs. Specifically, females switch from preferring the softer option for egg-laying when both options are sugar free to being indifferent between them when both contain sucrose. Such sucrose-induced indifference between options of different hardness requires functional sweet neurons, and, curiously, the Transmembrane Channel-like (TMC)-expressing mechanosensitive neurons that have been previously shown to promote discrimination of substrate hardness during feeding. Further, axons of sweet neurons directly contact axons of TMC-expressing neurons in the brain and stimulation of sweet neurons increases Ca(2+) influx into axons of TMC-expressing neurons. These results uncover one mechanism by which Drosophila integrate taste and tactile information when deciding where to deposit their eggs and reveal that TMC-expressing neurons play opposing roles in hardness discrimination in two different decisions (Wu, 2019).

    This work showed that activation of sweet neurons by sucrose can promote Drosophila females to become indifferent between two substrates of different hardness during egg-laying, and that such sucrose-induced indifference required input from the TMC-expressing mechanosensitive neurons on the labellum. Specifically, Drosophila females were shown to generally preferred the softer substrate for egg-laying in a two-choice assay when both options were sugar free, but their preference for the softer substrate reduced significantly when both options contained 100 mM sucrose. Such sugar-induced indifference between substrates of different hardness depended on functional molecular sugar receptors and sweet neurons as well as, interestingly, functional TMC channel and TMC-expressing mechanosensitive neurons. Further, anatomical-labeling and Ca2+-imaging results showed that axons of sweet neurons directly contacted those of TMC-expressing neurons in the brain and that depolarizing the sweet neurons increased Ca2+ influx into axon termini of TMC neurons. Thus, such axon-axon contacts provide an anatomical basis for sweet neurons to directly modulate the output of TMC neurons in the brain. Together, these findings suggest that, during egg-laying site selection, activation of sweet neurons can act to inhibit discrimination of substrates of different hardness by enhancing the output of TMC neurons directly. The results thus demonstrate a novel means by which Drosophila integrate specific chemosensory and mechanosensory properties of two competing substrates when evaluating them during a simple decision-making task. However, it is worth pointing out that the mechanism described in this study may not be the only path by which sweet neurons can act to modify discrimination of substrate hardness during egg-laying site selection. First, input from tarsi and antennae played a role, too. While no tmc transcripts were detected on them, it is unclear whether tmc-expressing neurons on these structures (that were missed by the tmc-GAL4) have the same interaction with sweet neurons as the ones on the labellum. Second, while the function of tmc-GAL4-expressing neurons was required for sucrose to dampen hardness discrimination, it was not possible to ascertain that direct artificial activation of these neurons was sufficient to do so in the absence of sucrose as such activation severely reduced females' egg-laying rate. Thus, one important next task is to identify the relevant mechanosensitive input from tarsi and antennae and assess how information they relay might be modulated by activation of sweet neurons during egg-laying site selection (Wu, 2019).

    A second point that is worth discussing is whether the conclusions are compatible with findings from previous reports. While the results suggest that sweet neurons can act to potentiate the output of TMC neurons via axon-axon interaction, two recent studies have shown that activation of mechanosensitive neurons can inhibit the output of sweet neurons. Specifically, Zhang (2016) has shown that activation of TMC neurons can inhibit PER, a motor response triggered by activation of sweet neurons. Further, Jeong (2016) has shown that Nanchung-expressing neurons can inhibit PER and that axons of Nanchung-expressing neurons form inhibitory synapses with axons of sweet neurons. It is proposed that the current conclusions are not incompatible with these earlier reports. First, it is conceivable that axons of mechanosensitive neurons and sweet neurons can have two distinct types of interactions: presynaptic inhibition from mechanosensitive neurons to sweet neurons as well as presynaptic facilitation from sweet neurons to TMC neurons. Second, while 100 mM sucrose may facilitate TMC neurons less when flies were sampling 1.5% agarose than on 0.5% agarose (taking into account that sweet neurons should be suppressed more on 1.5% agarose than on 0.5% agarose), this should reduce the difference in perceived hardness of 0.5% and 1.5% agarose substrates, thus not inconsistent with what was seen. Moreover, it is unclear whether 0.5% and 1.5% agarose exerted very different levels of suppression on output of sweet neurons in this task. For example, Jeong (2016) showed that 0.2% vs. 2% agarose had significantly different impacts on feeding preference for 0.5 mM vs. 1 mM sucrose, however, the concentration of sucrose used in this study was 100 mM. For these reasons, the idea is favored that the conclusions expand the view of the relationship between sweet neurons and mechanosensitive neurons provided by the previous studies (Wu, 2019).

    Another point worth discussing after comparing this work with previous reports is that flies appeared to use two different sensory mechanisms to discriminate substrates of different hardness during feeding and egg-laying, even though they generally preferred the softer substrate in both tasks. Previous studies have shown that flies rely on TMC, Nan, and NompC channels and two specific groups of labellum sensory neurons that express these channels to discriminate substrates of different hardness during feeding (Jeong, 2016; Zhang, 2016; Sanchez-Alcaniz, 2017). In contrast, the current results showed that neither these channels nor these neurons were essential for flies to discriminate substrates of different hardness during egg-laying. More curiously, the results suggest input from mechanosensitive neurons on the labellum (as well as possibly ones on antennae and tarsi) can act to inhibit discrimination of substrates of different hardness during egg-laying. This conclusion is supported in part by the observations that animals without intact labellum or functional TMC-expressing neurons on the labellum showed enhanced discrimination in the presence of sucrose during egg-laying. In contrast, tmc mutants did not discriminate substrates of different hardness well for feeding when given the exact same choices. The striking difference in the requirement of labellum and TMC on substrate hardness discrimination during feeding and egg-laying raises the question of what are the identities of the specific sensory neurons that promote discrimination of substrate hardness during egg-laying. The totality of the current results are consistent with a very tentative model that Drosophila likely use some as-yet-unidentified mechanosensitive neurons on their ovipositor to sense and discriminate substrates of different hardness. This tentative model is based on the following reasons. First, ovipositor is known to possess mechanosensitive neurons; second, flies have been shown to actively probe the substrates with their ovipositor prior to depositing each egg; third and most important, animals that lacked the a significant portion of virtually all other appendages (e.g., labellum, tarsi, wings) but had intact ovipositor were still capable of discriminating substrates of different hardness. Thus, another important next task is to identify the mechanosensitive neurons on the ovipositor -- or possibly on other body parts -- that are critical for discriminating substrate hardness during egg-laying and the central targets of these neurons. Identities of these neurons will provide a much-needed molecular and anatomical basis to start elucidating how texture discrimination and substrate selection during egg-laying site selection is enabled and modulated (Wu, 2019).

    Lastly, what is the potential advantage in allowing sugar detection to inhibit discrimination of egg-laying substrates of different hardness? Strong selectiveness likely costs effort and delays emergence of progenies. Thus, when deciding between two competing substrates that do not differ significantly in values, it might be more advantageous for flies to deposit their eggs on both. In the experiments carried out in this study, difference in values between the plain 0.5% agarose and the plain 1.5% agarose maybe relatively small because while flies preferred the 0.5% agarose over the 1.5% agarose in the two-choice assay, they laid comparable numbers of eggs on them when each was presented in single-choice assays. Thus, the presence of high concentration of sucrose in both substrates may further reduce their differences in values, thereby largely eliminating flies' soft preference. (However, it is worth noting that the idea is favored that adding sucrose to the 0.5% and 1.5% agarose substrates may equalize their values by dampening them, at least in the context of regular assays performed in this study. This is because in regular assays, adding sucrose to an agarose substrate reduces as opposed to increases its value: while flies readily accepted the sucrose-containing substrate for egg-laying, they consistently preferred the plain one when given a choice between a plain one and a sucrose-containing one to choose from. Finally, from an evolutionary point of view, it is proposed that allowing sweet neurons to directly enhance the output of mechanosensitive neurons that can inhibit hardness discrimination during egg-laying may provide a neural substrate for different species to adopt different texture selectivity. For example, in contrast to Drosophila melanogaster, the fruit pest Drosophila suzukii is more receptive to lay eggs on harder substrates and attack both ripe (harder) and rotten (softer) fruits. It may be interesting to test whether modifications of the structure and function of sweet and TMC neurons, and/or the connection between them, contribute to Drosophila suzukii's acceptance of harder substrate during egg-laying (Wu, 2019).

    Parallel mechanosensory pathways direct oviposition decision-making in Drosophila

    Female Drosophila choose their sites for oviposition with deliberation. Female flies employ sensitive chemosensory systems to evaluate chemical cues for egg-laying substrates, but how they determine the physical quality of an oviposition patch remains largely unexplored. This study reports that flies evaluate the stiffness of the substrate surface using sensory structures on their appendages. The TRPV family channel Nanchung is required for the detection of all stiffness ranges tested, whereas two other proteins, Inactive and DmPiezo, interact with Nanchung to sense certain spectral ranges of substrate stiffness differences. Furthermore, Tmc is critical for sensing subtle differences in substrate stiffness. The Tmc channel is expressed in distinct patterns on the labellum and legs and the mechanosensory inputs coordinate to direct the final decision making for egg laying. This study thus reveals the machinery for deliberate egg-laying decision making in fruit flies to ensure optimal survival for their offspring (Zhang, 2020).

    This study revealed an unexpected complexity of stiffness assessment when female flies select their egg-laying site. Multiple peripheral appendages and mechanosensory channels are employed to determine the stiffness difference between adjacent egg-laying substrates, and the parallel information from different mechanosensory pathways is integrated to make the final decision for softer substrate. At the moderate stiffness range (0.25%-0.5%), a group of nan+ mechanosensory neurons in the leg tarsal bristles are activated. Similarly, a lower stiffness difference (0.25%-0.4%) activates a group of nan+/Dmpiezo+ tarsal bristle mechanosensory neurons. The detection of subtle stiffness differences is small, as 0.05% agarose relies on sd-L and md-L neurons. Activation of each pathway imparts an inhibitory tone on egg laying and thus guides the flies to softer substrate. Although it remains to be tested whether nan+/Dmpiezo+ tarsal mechanosensory neurons can be activated by moderate stiffness or sd-L/md-L neurons can be activated by moderate and mild stiffness, behavioral data argue that there is functional redundancy among the sensory pathways (Zhang, 2020).

    Together with previous findings that flies choose egg-laying sites based on internal and external cues, this study demonstrates that the decision-making process for egg-laying sites in female Drosophila is a highly deliberative process that employs multiple sensory modalities and multiple sensory structures within each modality. This deliberateness is essential because choosing the best egg-laying site is the most critical parental behavior among female flies to maximize their offspring's survival. Female flies in the wild certainly face a more difficult task in making such decisions for a far more complicated environment than is available in a lab experiment. Further investigation will be needed to understand how flies make decisions when evaluating complex or conflicting cues from multiple sensory pathways (Zhang, 2020).

    This study has revealed the exquisite ability of female flies to discriminate a texture difference as small as 0.05% in agarose. To do this, flies employ both external sensory structures and proprioceptive sensors to assess the stiffness of the surface. Upon touching the substrate with the legs, tarsal bristles are the first structures to be deformed, leading to the activation of mechanosensory neurons underneath the bristles. In the later probing step, as the proboscis pushes against the substrate, the cuticle of the distal labellum starts to be compressed against the substrate. With innervation to most of the labellum bristles, the Tmc+ md-L neurons are well positioned to detect this information. Proboscis extension will also cause a change of the angle between the labellum and haustrum, and consequently activates the proprioceptive Iav+ sd-L neurons. Loss of either md-L or sd-L neurons on the labellum results in a complete disability to identify a subtle stiffness difference, suggesting that the two structures cooperate functionally to detect weak mechanical stimuli. It remains to be explored how these two sensors coordinate to represent stiffness values in the brain to make the final, accurate selection of softer substrate (Zhang, 2020).

    Under the experimental conditions used in this study, the labellum and legs are the predominant appendages that detect substrate stiffness during egg laying. Nevertheless, the role of the ovipositor structure that executes the oviposition maneuver cannot be overlooked. This notion is supported by a previous study, but the exact neurons or genes remain elusive due to the structural complexity of the ovipositor. Moreover, a female fly pushes her lower abdomen against the substrate in order to insert the eggs into the substrate, and this abdominal bending action may require proprioceptive feedback to represent her body position and strength, although this notion requires further experimental evidence. Although it is possible to build a cumulative picture of mechanosensory regulation of decision making, a comprehensive understanding cannot be achieved before the roles of ovipositor and abdominal proprioception are elucidated (Zhang, 2020).

    So far, a bona fide center in the fly brain for the integration of mechanosensory inputs has not been established. Unlike visual or olfactory pathways, each of which are encoded and represented by discrete brain regions, mechanosensory inputs appear sparsely distributed throughout the brain and neural transduction from the peripheral to the central nervous system (CNS) seems to be largely parallel. In the egg-laying neuronal circuit, the labellum mechanosensory neurons for detecting subtle stiffness differences project extensive arborizations over the SEZ, a brain region critical for gustatory perception. By contrast, leg bristle neurons that sense greater stiffness send their axons to the ventral nerve cord (VNC) and the projections are then relayed to the higher brain regions including the SEZ, ventrolateral protocerebrum (VLP), superior lateral protocerebrum (SLP), and others. This segregation complicates the identification of brain circuitry that integrates parallel mechanosensory inputs from different appendages to direct egg-laying decision making. Previous studies have raised working models for this interaction, most of which are supported by the fact that mechanosensory and gustatory pathways antagonize or facilitate each other in the local SEZ circuits. Based on the results that leg mechanosensory neurons project to multiple brain regions, however, it would seem more likely that integration may also occur at higher brain areas outside the SEZ (Zhang, 2020).

    Furthermore, mechanosensory and gustatory information unambiguously influence one another during decision making for egg laying or feeding. Wu (2019) found that Tmc neurons were required for the loss of softness preference when sugar was provided. This study more symmetrically deciphered the mechanosensory pathways involved in the stiffness detection. Both studies agree that the tarsus and labellum are essential for the flies to choose egg-laying substrates of the optimal stiffness. Wu focused on the discrimination between 0.5% and 1.5% agarose whereas this study focuses on substrates from 0.25% to 0.5% agarose. A major difference in the two experimental setups for these two studies is that the stiffness difference ranges in this study were smaller (0.25%-0.5%), which allowed uncovering of additional mechanosensory mechanisms underlying egg-laying site choice. Nevertheless, the two studies are mutually complementary in deciphering how female flies recognize and integrate substrate texture and chemical cues into final decision making for egg-deposition sites (Zhang, 2020).

    A significant question in the field asks how multiple mechanotransduction channels function in overlapping or parallel pathways to coordinate behavioral responses, as more than one channel type is typically expressed in the same type of mechanosensory neurons. This study found that the mechanosensory channels Nanchung and DmPiezo are required for the discrimination of a mild stiffness difference. However, how the combination of these two channels drives the function of the same neurons remains elusive. Two possibilities are suggested: first, multiple mechanosensitive channels co-express and function in the same neurons in a parallel manner. For example, DmPiezo and PPK function in larval class VI da neurons to mediate mechanical nociceptive response. Another case comes from larval class I da neurons, in which both NompC and Tmc are required for proprioceptive feedback to control larval locomotion. In this scenario, Nanchung and DmPiezo channels may function in parallel signaling pathways required for normal preference to 0.25% over 0.4%. When either pathway is disrupted, females would show a decreased ability to distinguish stiffness differences. Second, the two channels may function in series in the same pathway, with one acting as a sensor and the other as an amplifier. For instance, in fly Cho organ neurons, three TRP channels, Nanchung, NompC, and Inactive, are all required for sound transduction. Nanchung is expressed in most mechanosensory neurons for hearing and proprioception. It is plausible that Nanchung maintains basal neuronal activity and DmPiezo functions as a specific receptor for mechanical force. The current data support this view, as a nanGal4 mutant lost nearly all spike firing whereas DmpiezoKO still maintained a reduced firing activity. Behaviorally, the nanGal4 mutant showed much more severe defects in selecting softer substrate than DmpiezoKO in the mild range. The data also implicate other mechanosensors such as NompC as working in concert with Nanchung in bristle mechanosensory neurons (Zhang, 2020).

    Quantitative and discrete evolutionary changes in the egg-laying behavior of single Drosophila females

    This study focused on oviposition, the act of laying an egg, in flies of the genus Drosophila to describe the elementary behavioral steps or microbehaviors that a single female fly undertakes prior to and during egg laying. The hierarchy and relationships in time of these microbehaviors were analyzed in three closely related Drosophila species with divergent egg-laying preferences and uncovered cryptic differences in their behavioral patterns. Using high-speed imaging, the oviposition behavior of single females of Drosophila suzukii, Drosophila biarmipes and Drosophila melanogaster was quantified in depth in a novel behavioral assay. By computing transitions between microbehaviors, a common ethogram structure was identified underlying oviposition of all three species. Quantifying parameters such as relative time spent on a microbehavior and its average duration, however, revealed clear differences between species. In addition, the temporal dynamics and probability of transitions to different microbehaviors were analyzed relative to a central event of oviposition, ovipositor contact. Although the quantitative analysis highlights behavioral variability across flies, it reveals some interesting trends for each species in the mode of substrate sampling, as well as possible evolutionary differences. Larger datasets derived from automated video annotation will overcome this paucity of data in the future, and use the same framework to reappraise these observed differences. This study reveals a common architecture to the oviposition ethogram of three Drosophila species, indicating its ancestral state. It also indicates that Drosophila suzukii's behavior departs quantitatively and qualitatively from that of the outgroup species, in line with its known divergent ethology. Together, these results illustrate how a global shift in ethology breaks down in the quantitative reorganization of the elementary steps underlying a complex behavior (Bracker, 2019).

    Evolution of ovipositor length in Drosophila suzukii is driven by enhanced cell size expansion and anisotropic tissue reorganization

    Morphological diversity is dominated by variation in body proportion, which can be described with scaling relationships and mathematical equations, following the pioneering work of D'Arcy Thompson and Julian Huxley. Yet, the cellular processes underlying divergence in size and shape of morphological traits between species remain largely unknown. This study compared the ovipositors of two related species, Drosophila melanogaster and D. suzukii. D. suzukii has switched its egg-laying niche from rotting to ripe fruit. Along with this shift, the D. suzukii ovipositor has undergone a significant change in size and shape. Using an allometric approach, this study finds that, while adult ovipositor width has hardly changed between the species, D. suzukii ovipositor length is almost double that of D. melanogaster. This difference mostly arises in a 6-h time window during pupal development. It was observed that the developing ovipositors of the two species comprise an almost identical number of cells, with a similar profile of cell shapes and orientations. After cell division stops, the ovipositor area continues to grow in both species through the isotropic expansion of cell apical area and the anisotropic cellular reorganization of the tissue. Remarkably, it was found that the lengthening of the D. suzukii ovipositor compared to that of D. melanogaster results from the combination of the accelerated expansion of apical cell size and the enhanced anisotropic rearrangement of cells in the tissue. Therefore, the quantitative fine-tuning of morphogenetic processes can drive evolutionary changes in organ size and shape (Green, 2019).

    Transgenerational effects from single larval exposure to azadirachtin on life history and behavior traits of Drosophila melanogaster

    Azadirachtin is one of the successful botanical pesticides in agricultural use with a broad-spectrum insecticide activity, but its possible transgenerational effects have not been under much scrutiny. The effects of sublethal doses of azadirachtin on life-table traits and oviposition behaviour of a model organism in toxicological studies, D. melanogaster, were evaluated. The fecundity and oviposition preference of flies surviving to single azadirachtin-treated larvae of parental generation was adversely affected and resulted in the reduction of the number of eggs laid and increased aversion to this compound over two successive generations. In parental generation, early exposure to azadirachtin affects adult's development by reducing the number of organisms, delay larval and pupal development; male biased sex ratio and induced morphological alterations. Moreover, adult's survival of the two generations was significantly decreased as compared to the control. Therefore, Single preimaginal azadirachtin treatment can affect flies population dynamics via transgenerational reductions in survival and reproduction capacity as well as reinforcement of oviposition avoidance which can contribute as repellent strategies in integrated pest management programs. The transgenerational effects observed suggest a possible reduction both in application frequency and total amount of pesticide used, would help in reducing both control costs and possible ecotoxicological risks (Ferdenache, 2019).

    Geosmin attracts Aedes aegypti mosquitoes to oviposition sites

    Geosmin is one of the most recognizable microbial smells. Some insects, like mosquitoes, require microbial-rich environments for their progeny, whereas for other insects such microbes may prove dangerous. In Drosophila, geosmin is decoded in a precise fashion and induces aversion. This study investigated the effect of geosmin on the behavior of the yellow fever mosquito Aedes aegypti. In contrast to flies, geosmin is not aversive but mediates egg-laying site selection. Female mosquitoes likely associate geosmin with microbes, including cyanobacteria consumed by larvae, who also find geosmin-as well as geosmin-producing cyanobacteria-attractive. Using in vivo multiphoton calcium imaging from transgenic PUb-GCaMP6s mosquitoes, this study shows that Ae. aegypti code geosmin in a qualitatively similar fashion to flies, i.e., through a single olfactory channel with a high degree of sensitivity for this volatile. It was further demonstrated that geosmin can be used as bait under field conditions, and geosmin, which is both expensive and difficult to obtain, can be substituted by beetroot peel extract, providing a cheap and viable potential means for mosquito control and surveillance in developing countries (Melo, 2019).

    Neural circuitry linking mating and egg laying in Drosophila females

    Mating and egg laying are tightly cooordinated events in the reproductive life of all oviparous females. Oviposition is typically rare in virgin females but is initiated after copulation. This study identified the neural circuitry that links egg laying to mating status in Drosophila melanogaster. Activation of female-specific oviposition descending neurons (oviDNs) is necessary and sufficient for egg laying, and is equally potent in virgin and mated females. After mating, sex peptide-a protein from the male seminal fluid-triggers many behavioural and physiological changes in the female, including the onset of egg laying. Sex peptide is detected by sensory neurons in the uterus, and silences these neurons and their postsynaptic ascending neurons in the abdominal ganglion. This study shows that these abdominal ganglion neurons directly activate the female-specific pC1 neurons. GABAergic (gamma-aminobutyric-acid-releasing) oviposition inhibitory neurons (oviINs) mediate feed-forward inhibition from pC1 neurons to both oviDNs and their major excitatory input, the oviposition excitatory neurons (oviENs). By attenuating the abdominal ganglion inputs to pC1 neurons and oviINs, sex peptide disinhibits oviDNs to enable egg laying after mating. This circuitry thus coordinates the two key events in female reproduction: mating and egg laying (Wang, 2020).

    It was reasoned that egg laying is likely to depend on cell types that are female-specific and hence express one or both of the sex-determination genes fruitless (fru) and doublesex (dsx). In particular, egg laying is blocked by either silencing or masculinizing all fru+ neurons. Some of these fru+ neurons are descending interneurons, which project from the brain to the ventral nerve cord and are thought to convey high-level motor commands. This study therefore focused on female-specific fru+ descending neurons and used the split-GAL4 technique to obtain two driver lines that label two female-specific fru+dsx- cholinergic descending neurons per brain hemisphere. In optogenetic activation experiments using Chrimson, both split-GAL4 driver lines reliably induced oviposition behaviour in mated females, with most but not all females also depositing an egg (it is presumed that not all females had an egg in the uterus at the time of neuronal activation). Accordingly, these neurons are referred to as oviposition descending neurons (oviDNs), and to the two split-GAL4 driver lines that label them as oviDN-SS1 and oviDN-SS2 (in which SS denotes stable split-GAL4). Stochastic labelling of single neurons resolved two morphologically distinct types of oviDN, which are refered to as oviDNa and oviDNb cells. In an electron microscopy volume of a full adult female brain (FAFB15), two oviDNa-like cells and one oviDNb-like cell were identified in each hemisphere (Wang, 2020).

    Egg laying by mated females was completely blocked by genetic ablation of oviDNs, and markedly reduced by their chronic silencing. Virgin females in which oviDNs were ablated were as receptive to mating as control females. Several days after mating, the ovaries of oviDN-ablated females contained many mature eggs, and most carried either a fertilized egg or a first-instar larva in the uterus. It is concluded that oviDNs are essential for oviposition, but dispensable for mating, ovulation and fertilization (Wang, 2020).

    It was not possible to generate driver lines that specifically target oviDNa or oviDNb cells. To determine which oviDN subtype is involved in oviposition, a stochastic 'unsilencing' experiment was performed, in which a tdTomato-tagged silencing transgene was targeted to all oviDNs, but stochastically replaced in some of these cells with GFP. Individual females were assayed for egg laying over five days after mating, then dissected and stained to determine their complement of red (tdTomato; silenced) and green (GFP; unsilenced) oviDNs. Females with no unsilenced cells laid no or very few eggs, whereas those with just a single functional oviDN cell generally laid large numbers of eggs. The number of eggs laid per female was variable in these cases, but there was no appreciable difference between females in which an oviDNa cell was unsilenced and those in which an oviDNb cell was unsilenced, nor between females in which either one or two cells of either type were functional. Although the oviDNa and oviDNb subtypes differ in their morphology-and probably their connectivity and physiology-these data suggest that they nonetheless have similar functions in oviposition (Wang, 2020).

    Oviposition involves a coordinated and highly stereotyped sequence of motor actions that progresses from abdomen bending to ovipositor extrusion and egg deposition. Abdomen bending, ovipositor extrusion and egg deposition were all eliminated in females in which oviDNs were ablated. Conversely, abdomen bending and ovipositor extrusion were reliably triggered by strong photoactivation of oviDNs in either virgin or mated females. Egg deposition was also induced, but only in mated females (presumably because mating is required to stimulate ovulation). In all of these oviDN activation experiments, the sequence of motor actions was the same as that in natural egg laying. By varying the stimulus intensity, it was found that egg deposition has a higher activation threshold than abdomen bending and ovipositor extrusion, and that action latencies were shorter at higher stimulus intensities. Moreover, at low stimulus intensities, the oviposition sequence was often truncated, but an action was never skipped, and only once was a single action occurring out of order observed (in a total of 38 flies at each of 3 intensities). These data suggest that oviDNs may use a ramp-to-threshold mechanism to elicit the successive motor actions of oviposition. Notably, the activation thresholds and action latencies were indistinguishable between virgins and mated females, indicating that mating status regulates egg laying through the brain circuits upstream of oviDNs rather than through downstream motor circuits (Wang, 2020).

    The onset of egg laying after mating is induced by sex peptide, a protein of the male seminal fluid that is detected by sex-peptide sensory neurons (SPSNs) of the uterus. Sex peptide silences both SPSNs and their postsynaptic targets in the abdominal ganglion, the SP abdominal ganglion (SAG) neurons. Artificially activating either SPSNs or SAG neurons suppressed egg laying in mated females. Conversely, ablating or silencing these cells increased the number of eggs laid by virgin females. Virgin egg laying as a result of SPSN or SAG ablation depended on oviDNs, as egg laying was prevented if these cells were co-ablated. SPSN and SAG activity is thus critical in keeping oviDNs inactive until after mating. This inhibition is most likely to be indirect, because the SAGs are cholinergic and hence probably excitatory. This study identified and extensively traced the ascending projections of the two SAG neurons in the FAFB volume and found just a single synapse from SAG neurons to oviDNs (Wang, 2020).

    The targets of SAG neurons in the brain have not been identified. Because SAG neurons regulate female receptivity as well as egg laying, it is speculated that their targets could include the female-specific fru-dsx+ pC1 neurons in the protocerebrum, which are known to regulate receptivity. Within the FAFB volume five morphologically distinct pC1 cells were identified in each hemisphere, which are referred to as pC1a-pC1e. Extensive tracing of single pC1a, pC1c and pC1e cells, as well as more limited tracing of pC1b and pC1d cells, suggests that the SAG neurons provide numerous synaptic inputs to the pC1a, pC1b and pC1c cells, with fewer if any direct inputs to pC1d and pC1e cells. Whole-cell recordings were performed from individual pC1 neurons while photoactivating the SAGs; pC1a cells were strongly depolarized, pC1b cells were weakly depolarized and pC1c, pC1d and pC1e cells showed little or no response upon SAG activation. There were numerous synaptic connections amongst all five pC1 subtypes, however, suggesting that any information on mating status that is obtained from SAG neurons by pC1a and pC1b cells is potentially shared across the entire set of pC1 cells (Wang, 2020).

    Two split-GAL4 driver lines were obtained for pC1 neurons: pC1-SS1, which labels pC1a, pC1c and pC1e, and pC1-SS2, which labels all five pC1 cells. Ablation of pC1 cells using either driver resulted in an increase in egg laying in virgin females that was dependent on oviDN function, whereas mated females in which pC1 neurons were chronically activated laid fewer eggs. Brief optogenetic silencing of pC1 neurons in virgins did not acutely trigger egg laying, as would be expected if pC1-inactivated virgins (like pC1-intact mated females) rely on additional substrate-borne cues for the induction of egg laying (Wang, 2020).

    These behavioural data indicate that-similar to SPSNs and SAG neurons-pC1 neurons suppress the function of oviDNs and therefore suppress egg laying in virgin females. Consistent with this interpretation, it was found by in vivo imaging that basal calcium levels in pC1 neurons, although variable, are generally higher in virgin than mated females. Moreover, whole-cell recordings from oviDNs revealed that both oviDNa and oviDNb cells are hyperpolarized after photoactivation of pC1 neurons, and that this effect is sensitive to picrotoxin, a chloride channel blocker. This inhibition is probably indirect, because pC1 neurons are cholinergic and have very few synapses onto the oviDNs (Wang, 2020).

    To look for inhibitory intermediates from pC1 to oviDN cells-as well as excitatory inputs that might stimulate egg laying upon detection of a preferred substrate- the synaptic inputs to oviDNa and oviDNb cells were reconstructed in the Full Adult Fly Brain (FAFB) volume. Sparse split-GAL4 driver lines were obtained for the two cell types with the largest numbers of oviDN input synapses. Whole-cell recordings reliably showed changes in membrane potential in oviDNs after photoactivation of either of these two cell types. The cell type with the most oviDN input synapses is cholinergic, and activation of these cells depolarized oviDNs. These cells were therefore named oviposition excitatory neurons (oviENs). The cell type with the second-highest number of oviDN input synapses is GABAergic, and activation of these cells hyperpolarized oviDNs. Accordingly, these cells were named oviposition inhibitory neurons (oviINs). There is a single oviEN and a single oviIN per hemisphere, and they are reciprocally connected. The oviINs are also reciprocally connected with pC1 cells, and calcium-imaging experiments showed that photoactivation of pC1 cells elicits an excitatory response in oviINs. The pC1 cells have few direct synaptic connections with oviENs, and no connections were detected between SAG neurons and either oviINs or oviENs (Wang, 2020).

    Silencing oviENs in mated females strongly suppressed egg laying, similarly to the effect observed when oviDNs were silenced. By contrast, potentiating oviENs in virgin females caused them to lay significantly more eggs than control virgins, albeit not as many as mated females (presumably because ovulation remains infrequent). Manipulating oviIN activity had the opposite effects: silencing oviINs caused virgins to lay significantly more eggs, whereas depolarizing oviINs reduced the number of eggs laid by mated females. Thus, as expected from the sign of their inputs to oviDNs (that is, excitatory for oviENs; inhibitory for oviINs), oviENs promote egg laying, whereas oviINs inhibit it (Wang, 2020).

    It was hypothesized that oviENs could mediate the external sensory signals that trigger egg laying in mated females, which are likely to include both gustatory and mechanosensory cues from the substrate. When provided with a choice of substrates, females lay more eggs on agarose medium than on a hard surface or a substrate of agarose and sucrose. Therefore in vivo calcium imaging was performed to determine the responses of oviDNs, oviENs and oviINs to the presentation of each of these substrates to the legs. In oviDNs, an increase was observed in calcium levels only upon contact with the agarose substrate. This response was stronger in mated females than in virgins. The agarose-and-sucrose substrate elicited a small reduction in calcium levels, which was more pronounced in virgin females. The oviENs showed a positive calcium response to agarose but to neither of the other two substrates, and this response was indistinguishable between virgins and mated females. The oviINs responded to all three substrates, but more strongly to agarose and sucrose than to agarose alone, and only weakly to the hard surface. Regardless of substrate, oviIN responses were stronger in virgins than in mated females (Wang, 2020).

    In conclusion, these findings support the following model for the neural coordination of mating and egg laying in Drosophila. The oviDNs control the entire oviposition motor programme. They receive excitatory input from oviENs, which respond to stimulatory cues from the substrate, and inhibitory input from oviINs, which convey information about mating status from pC1 cells. In virgins, increased activity of pC1 neurons potentiates oviIN-mediated inhibition of both oviDNs and oviENs, which suppresses egg laying. After mating, sex peptide silences SAG inputs onto pC1 neurons, thereby decreasing the activity of pC1 neurons and oviINs to facilitate egg laying when a preferred substrate is encountered. Reciprocal connections between oviINs and oviENs might ensure that oviDNs respond to oviEN activation with the appropriate temporal pattern and dynamic range, through feed-forward and feedback inhibition, respectively. The oviDNs, oviENs and oviINs all have numerous synaptic inputs in addition to those that have been described in this stduy-all of which remain functionally uncharacterized. These inputs may mediate other controls on the egg-laying process, such as the presence of an egg in the uterus and the nutritional state of the female. The pC1 neurons might also regulate other female behaviours that switch after mating, perhaps through different sets of output neurons. Notably, the male counterparts of pC1 neurons are thought to encode an analogous state of courtship arousal that modulates command pathways for specific motor actions such as courtship song and 'licking'. Thus, functionally analogous but anatomically divergent circuits-shaped during development by fru and dsx-could account for the distinct reproductive behaviours of Drosophila males and females (Wang, 2020).

    Symbiotic bacteria attenuate Drosophila oviposition repellence to alkaline through acidification

    Metazoans harbor a wealth of symbionts that are ever-changing the environment by taking up resources and/or excreting metabolites. One such common environmental modification is a change in pH. Conventional wisdom holds that symbionts facilitate the survival and production of their hosts in the wild, but this notion lacks empirical evidence. This study reports that symbiotic bacteria in the genus Enterococcus attenuate the oviposition avoidance of alkaline environments in Drosophila. The effects of alkalinity on oviposition preference was studied for the first time, and it was found that flies are robustly disinclined to oviposit on alkali-containing substrates. This innate repulsion to alkaline environments is explained, in part, by the fact that alkalinity compromises the health and lifespan of both offspring and parent Drosophila. Enterococcus dramatically diminished or even completely reversed the ovipositional avoidance of alkalinity in Drosophila. Mechanistically, Enterococcus generate abundant lactate during fermentation, which neutralizes the residual alkali in an egg-laying substrate. In conclusion, Enterococcus protects Drosophila from alkali stress by acidifying the ovipositional substrate, and ultimately improves the fitness of the Drosophila population. These results demonstrate that symbionts are profound factors in the Drosophila ovipositional decision, and extend understanding of the intimate interactions between Drosophila and their symbionts (Liu, 2020).

    Neuropeptide F signaling regulates parasitoid-specific germline development and egg-laying in Drosophila

    Drosophila larvae and pupae are at high risk of parasitoid infection in nature. To circumvent parasitic stress, fruit flies have developed various survival strategies, including cellular and behavioral defenses. This study shows that adult Drosophila females exposed to the parasitic wasps, Leptopilina boulardi, decrease their total egg-lay by deploying at least two strategies: Retention of fully developed follicles reduces the number of eggs laid, while induction of caspase-mediated apoptosis eliminates the vitellogenic follicles. These reproductive defense strategies require both visual and olfactory cues, but not the MB247-positive mushroom body neuronal function, suggesting a novel mode of sensory integration mediates reduced egg-laying in the presence of a parasitoid. It was further shown that neuropeptide F (NPF) signaling is necessary for both retaining matured follicles and activating apoptosis in vitellogenic follicles. Whereas previous studies have found that gut-derived NPF controls germ stem cell proliferation, this study shows that sensory-induced changes in germ cell development specifically require brain-derived NPF signaling, which recruits a subset of NPFR-expressing cell-types that control follicle development and retention. Importantly, it was found that reduced egg-lay behavior is specific to parasitic wasps that infect the developing Drosophila larvae, but not the pupae. These findings demonstrate that female fruit flies use multimodal sensory integration and neuroendocrine signaling via NPF to engage in parasite-specific cellular and behavioral survival strategies (Sadanandappa, 2021).

    A sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior

    Although males and females largely share the same genome and nervous system, they differ profoundly in reproductive investments and require distinct behavioral, morphological, and physiological adaptations. How can the nervous system, while bound by both developmental and biophysical constraints, produce these sex differences in behavior? This study uncovered a novel dimorphism in Drosophila melanogaster that allows deployment of completely different behavioral repertoires in males and females with minimum changes to circuit architecture. Sexual differentiation of only a small number of higher order neurons in the brain leads to a change in connectivity related to the primary reproductive needs of both sexes-courtship pursuit in males and communal oviposition in females. This study explains how an apparently similar brain generates distinct behavioral repertoires in the two sexes and presents a fundamental principle of neural circuit organization that may be extended to other species (Nojima, 2021).

    Sexually reproducing species exhibit sex differences in social interactions to boost reproductive success and survival of progeny. Comparing and contrasting the anatomy, activity, and function of sexually dimorphic neurons in the brain of males and females across taxa are starting to reveal the fundamental principles of neural circuit organization underlying these sex differences in behavior. A variety of alternative neuronal circuit configurations have been proposed to generate sexually dimorphic behaviors. Many studies have identified sex differences in sensory inputs in various species; however, such differences in higher order brain circuits that organize species- and sex-specific instinctive behaviors in response to sensory cues are still poorly characterized (Nojima, 2021).

    Sex is determined early in an animal's development and initiates many irreversible sexual differentiation events that influence how the genome and the environment interact to give rise to sex-specific morphology and behavior. In Drosophila, selective expression of two sex determination transcription factors (TFs), Doublesex (Dsx) and Fruitless (Fru), define cell-type-specific developmental programs that govern functional connectivity and lay the foundations through which innate sexual behaviors are genetically predetermined. Because both fru- and dsx-expressing neurons are essential for male and female reproductive behaviors, studies in the adult have focused on neurons that express these TFs to identify anatomical or molecular sex differences in neuronal populations. This allows entry to the neural circuits underlying sex-typical behaviors and identification of the neuronal nodes that control component behaviors and behavioral sequencing (Nojima, 2021).

    Dsx proteins, which are part of the structurally and functionally conserved Doublesex and Male-abnormal-3 Related Transcription factors (DMRT) protein family, are critical for sex-specific differentiation throughout the animal kingdom. In the insect phylum, Dsx proteins act at the interface between sex determination and sexual differentiation, regulating a myriad of somatic sexual differences both inside and outside the nervous system. The dsx gene has functions in both sexes: its transcripts undergo sex-specific alternative splicing to encode either a male- or female-specific isoform. dsx expression is highly regulated in both male and female flies, as shown by its temporally and spatially restricted expression patterns through development, with only a select group of neurons expressing dsx. The dsx gene is expressed in some 150 and 30-40 neurons per hemisphere in the male and female brains, which reside in 10 and 7 to 8 discrete anatomical clusters, respectively. This restricted expression of dsx in higher order neurons in the brain suggests these neurons may act as key sex-specific processing nodes of sensory information (Nojima, 2021).

    To study the fundamental principles of neural circuit organization underlying sex differences in behavior, this study identified and mapped dsx+ sexual dimorphisms in the CNS. This analyses revealed that all dsx+ clusters are either sexually dimorphic or sex specific; none are sexually monomorphic. To examine higher order processing differences between the sexes, this study focused on the dsx+ anterior dorsal neuron (aDN) cluster, as it is present in both sexes yet has sexually dimorphic dendritic arborizations associated with sensory perception. These anatomical differences lead to sex-specific connectivity, with male aDN inputs being exclusively visual, while female inputs are primarily olfactory. Finally, this study shows that this unique sexually dimorphic neuronal hub that reroutes distinct sensory pathways gives rise to functionally distinct social behaviors between the sexes: visual tracking during courtship in males and communal egg-laying site selection in females (Nojima, 2021).

    This study identified a small cluster of two neurons per hemisphere in the central brain, which reconfigures circuit logic in a sex-specific manner. Perhaps most surprising is the seemingly unrelated behaviors these equivalent neurons control in each sex-visual tracking during courtship in males and communal egg laying in females. Ultimately, these circuit reconfigurations lead to the same end result-an increase in reproductive success. These findings highlight a flexible strategy used to structure the nervous system, where relatively minor modifications in neuronal networks allow each sex to respond to their social environment in a sex-appropriate manner (Nojima, 2021).

    The behavioral function of the male aDN cluster appears to be related to visual aspects of courtship behavior. A set of visual projection neurons, LC10a, was previously identified as involved in tracking and following behaviors in the male during courtship; however, no apparent sex differences in their anatomy or their physiological responses to visual stimuli were detected. It would seem these sex differences in behavior arise from the sex-specific downstream connectivity of LC10a neurons in the central brain. This study identified aDNs connecting downstream to LC10a in males only. aDN inactivation mirrors visual tracking defects displayed upon LC10a inactivation; therefore, the male aDN cluster confers sex specificity to visually guided tracking of females during courtship (Nojima, 2021).

    This study also identified AL5a neurons to be downstream of LC10a in both sexes. Interestingly, it has been reported that AL5a is likely upstream of the fru+ cluster Lv2/pIP-b/pIP8 thought to exchange and integrate visual information from the right and left hemispheres of the brain. This male-specific connectivity is compatible with a potential role for AL5a in mediating visual information necessary for wing choice during courtship, a behavior these neurons have been shown to elicit when activated (Nojima, 2021).

    The two LC10a downstream clusters that this study identified, aDN and AL5a, also show differences in their anatomical connectivity and physiological responses. Whereas AL5a is downstream of LC10a in both sexes, aDN is only connected to LC10a in the male. Despite direct anatomical connectivity between LC10a and aDN in males, functional connectivity was only uncovered under conditions of pharmacological disinhibition. This observation might hint at inhibitory modulation of aDN that depends on the male's internal state, e.g., his mating drive, or additional cues that influence his courtship arousal. A previous study found that, in sexually satiated males, calcium responses in courtship 'decision-making' P1 neurons were absent when stimulating upstream neurons but could be restored to the levels observed in naive males by application of PTX. It is tempting to speculate that inhibition in the LC10a -> aDN pathway is similarly linked to sexual arousal. In contrast, AL5a responses to LC10a stimulation occurred in the absence of PTX and were markedly larger in AL5a than in aDN. The variation in calcium signals could be due to the considerable difference in cell numbers comprising each cluster (2 aDN versus 24 AL5a) or due to inputs from different AOTu regions. aDNs sample from the whole glomerulus region, whereas the AL5a cluster is restricted to the dorsal part of the AOTu, suggesting they extract information from broad versus specific parts of the visual field, respectively. Future investigation will be aimed at linking the clusters' anatomical differences with their differential processing of visual information to facilitate distinct behavioral roles (Nojima, 2021).

    In females, the aDN cluster does not receive visual information but appears to sample from a range of sensory modalities, with information received via the antennal lobe dominating its inputs, suggesting its involvement in a complex behavior requiring multisensory integration. One such behavior is female egg-laying site selection, which is critical to the success of offspring. For Drosophila, offspring survival rates depend on the selection of oviposition sites that are shared with conspecifics, a process known to rely on olfaction (Nojima, 2021).

    This study has shown that aDNs are highly integrated into circuitry known to regulate oviposition. The excitatory oviEN, which is anatomically similar to the aDNs, responds to information about substrate suitability via gustatory and mechanosensory cues in the legs and directly influences aDN output. Silencing oviEN function suppresses egg laying itself, whereas silencing aDN does not affect the overall number of eggs laid. Instead, aDN-silenced females are no longer able to show a preference to lay eggs communally, losing a female-specific social behavior essential for offspring survival. While both oviEN and aDN output directly onto the oviposition motor program (through oviDNs), oviENs are the largest contributors to oviDN dendritic budgets, with aDN being relatively minor contributors. Thus, the aDN cluster acts as a modulator of egg laying choice, whereas the oviEN more generally affects the mechanics of egg laying (Nojima, 2021).

    As the oviposition of fertilized eggs is a female behavior that can only be displayed after mating, the behavioral programs required are likely inhibited in virgin females. The activity of the inhibitory neuron oviIN depends on female mating status and thus appears to act as a general inhibitor of egg-laying circuitry in virgin females. oviINs form axo-axonic synapses with both the aDN and oviEN, suggesting they gate their outputs by presynaptic inhibition in a state-dependent manner. Intriguingly, as both oviEN and oviIN form axo-axonic synapses with aDN, this suggests a potential gating mechanism by which their relative strengths inhibit or facilitate output from aDN onto downstream targets (Nojima, 2021).

    Consistent with aDNs' behavioral function in egg-laying site selection, a female post-mating behavior, this study found differences in the aDN physiological responses in mated versus virgin females. Stimulation of OSNs resulted in significantly stronger aDN calcium responses in mated females compared to virgins. This finding might hint at a state-dependent inhibition of olfactory inputs into aDN in females, potentially analogous to the inhibition of visual inputs to aDN observed in males. The difference in physiological responses between mated and virgin females was not observed when stimulating PNs, which are downstream of OSNs but upstream of aDN. There are different possible explanations for this discrepancy, including differences in the populations of neurons targeted by the driver lines used to target PNs versus OSNs or inhibition in virgin females occurring at the level of OSN to PN connectivity; therefore, activating PNs directly bypasses the state-dependent inhibition. In addition to state-dependent effects, there also seemed to be differences in the calcium responses in different neuronal compartments. This finding could be explained by the position of the input synapses of different upstream neurons into the aDN (e.g., dendritic versus axonic). The exact mechanism of how aDN integrates these different inputs and transforms them into an output that guides egg-laying site selection remains to be examined (Nojima, 2021).

    The principal output of the female aDN is the previously undescribed SMP156 neuron, which itself outputs primarily in the IB, where its axons show cross-hemisphere connectivity, suggesting it acts as integrators of sensory information from different directions. The major SMP156 output neuron type (IB011) projects to the lobula in the opposite hemisphere, potentially integrating olfactory and visual information as observed in other flying insects during pheromone orientation. Olfactory navigation requires comparisons of left and right inputs, e.g., when male moths orient themselves toward conspecific females in response to sex pheromones. Determination of position and direction applies to males pursuing females and females following pheromonal cues to locate a communal egg-laying site. It is proposed that the aDN cluster in females selectively integrates sensory information, relaying it to SMP156, which confers directionality and processes information relevant to locating an appropriate egg-laying site. In the absence of a male connectome for comparison, it can only be speculated about potential shared downstream connectivity. As the male aDN output sites are mainly overlapping with female sites in the SMP, it is possible that the male visual pathway also inputs into SMP156, or a similar neuron associated with the IB, potentially feeding back onto visual pathways, supporting appropriate tracking of the female. A male connectome and more genetic tools will help reveal the full extent of downstream functional connectivity and convergence between the sexes (Nojima, 2021).

    As fundamental features of most animal species, sexual dimorphisms and sex differences have particular importance for the function of the nervous system. These innate sex-specific adaptations are built during development and orchestrate interactions between sensory information and specific brain regions to shape the phenotype, including the emergent properties of the sex-specific neural circuitry. Evolutionary forces acting on these neural systems have generated adaptive sex differences in behavior. In Drosophila, males compete for a mate through courtship displays, while a female's investment is focused on the success of their offspring. These sex-specific behaviors are guided by the perception and processing of sensory cues, ensuring responses lead to reproductive success. This study has shown how a sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior and provides insight on how similar mechanisms may be implemented in the brains of other sexually dimorphic species (Nojima, 2021).

    Sleep correlates with behavioral decision making critical for reproductive output in Drosophila melanogaster

    Balance between sleep, wakefulness and arousal is important for survival of organisms and species as a whole. While, the benefits of sleep both in terms of quantity and quality is widely recognized across species, sleep has a cost for organismal survival and reproduction. here the study focuses on how sleep duration, sleep depth and sleep pressure affect the ability of animals to engage in courtship and egg-laying behaviors critical for reproductive success. Using isogenic lines from the Drosophila Genetic Reference Panel with variable sleep phenotypes this study investigated the relationship between sleep and reproductive behaviors, courtship and oviposition. This study found that three out of five lines with decreased sleep and increased arousal phenotypes, showed increased courtship and decreased latency to court as compared to normal and long sleeping lines. However, the male courtship phenotype is dependent on context and genotype as some but not all long sleeping-low courting lines elevate their courtship in the presence of short sleeping-high courting flies. Sleep phenotypes were less variable and minimally susceptible to social experience. In addition to male courtship, Oviposition was found to be less sensitive to sleep length. Taken together this extensive behavioral analysis shows complex bidirectional interactions between genotype and environment and add to the growing evidence linking sleep duration and sleep-wake switch parameters to behavioral decision making critical to reproductive output (Buchert, 2021).

    A functional division of Drosophila sweet taste neurons that is value-based and task-specific

    Sucrose is an attractive feeding substance and a positive reinforcer for Drosophila. But Drosophila females have been shown to robustly reject a sucrose-containing option for egg-laying when given a choice between a plain and a sucrose-containing option in specific contexts. How the sweet taste system of Drosophila promotes context-dependent devaluation of an egg-laying option that contains sucrose, an otherwise highly appetitive tastant, is unknown. This study reports that devaluation of sweetness/sucrose for egg-laying is executed by a sensory pathway recruited specifically by the sweet neurons on the legs of Drosophila First, silencing just the leg sweet neurons caused acceptance of the sucrose option in a sucrose versus plain decision, whereas expressing the channelrhodopsin CsChrimson in them caused rejection of a plain option that was "baited" with light over another that was not. Analogous bidirectional manipulations of other sweet neurons did not produce these effects. Second, circuit tracing revealed that the leg sweet neurons receive different presynaptic neuromodulations compared to some other sweet neurons and were the only ones with postsynaptic partners that projected prominently to the superior lateral protocerebrum (SLP) in the brain. Third, silencing one specific SLP-projecting postsynaptic partner of the leg sweet neurons reduced sucrose rejection, whereas expressing CsChrimson in it promoted rejection of a light-baited option during egg-laying. These results uncover that the Drosophila sweet taste system exhibits a functional division that is value-based and task-specific, challenging the conventional view that the system adheres to a simple labeled-line coding scheme (Chen, 2022).

    Social and physical environment independently affect oviposition decisions in Drosophila

    In response to environmental stimuli, including variation in the presence of conspecifics, genotypes show highly plastic responses in behavioral and physiological traits influencing reproduction. Although extensively documented in males, such female responses are rather less studied. It is expected that females would be highly responsive to environmental variation and to differentially allocate resources to increase offspring fitness, given the major contribution of mothers to offspring number, size, and developmental conditions. Using Drosophila melanogaster, this study (1) manipulated exposure to conspecific females, which mothers could use to anticipate the number of potential mates and larval density, and; (2) tested how this interacts with the spatial distribution of potential oviposition sites, with females from higher densities expected to prefer clustered resources that can support a larger number of larvae. It was found that high density females were slower to start copulating and reduced their copulation duration, the opposite effect to that observed in males. There was a parallel, perhaps related, effect on egg production: females previously housed in groups laid fewer eggs than those housed in solitude. Resource patchiness also influenced oviposition behavior: females preferred aggregated substrate, which attracted more females to lay eggs. However, no interaction was found between prior housing conditions and resource patchiness, indicating that females did not perceive the value of different resource distributions differently when exposed to environments that could signal expected levels of larval competition. This study shows that, although exposure to consexual competition changes copulatory behaviors of females, the distribution of oviposition resources has a greater effect on oviposition decisions (Churchill, 2021).

    Sugar sensation and mechanosensation in the egg-laying preference shift of Drosophila suzukii

    The agricultural pest Drosophila suzukii differs from most other Drosophila species in that it lays eggs in ripe, rather than overripe, fruit. Previously, it was shown that changes in bitter taste sensation accompanied this adaptation. This study shows that D. suzukii has also undergone a variety of changes in sweet taste sensation. D. suzukii has a weaker preference than Drosophila melanogaster for laying eggs on substrates containing all three primary fruit sugars: sucrose, fructose, and glucose. Major subsets of D. suzukii taste sensilla have lost electrophysiological responses to sugars. Expression of several key sugar receptor genes is reduced in the taste organs of D. suzukii. By contrast, certain mechanosensory channel genes, including no mechanoreceptor potential C, are expressed at higher levels in the taste organs of D. suzukii, which has a higher preference for stiff substrates. Finally, it was found that D. suzukii responds differently from D. melanogaster to combinations of sweet and mechanosensory cues. Thus, the two species differ in sweet sensation, mechanosensation, and their integration, which are all likely to contribute to the differences in their egg-laying preferences in nature (Wang, 2022).

    Oviposition behaviour in Drosophila melanogaster: Genetic and behavioural decoupling between oviposition acceptance and preference for natural fruits

    In phytophagous insects, oviposition behaviour is an important component of habitat selection and, given the multiplicity of genetic and environmental factors affecting its expression, is defined as a complex character resulting from the sum of interdependent traits. This study examined two components of egg-laying behaviour: oviposition acceptance (OA) and oviposition preference (OP) in Drosophila melanogaster using three natural fruits as resources (grape, tomato and orange) by means of no-choice and two-choice experiments, respectively. This experimental design showed that the results obtained in two-choice assays (OP) cannot be accounted for by those resulting from no-choice assays (OA). Since the genomes of all lines used are completely sequenced, a genome-wide association study was performed to identify and characterize the genetic underpinnings of these oviposition behaviour traits. The analyses revealed different candidate genes affecting natural genetic variation of both OA and OP traits. Moreover, the results suggest behavioural and genetic decoupling between OA and OP and that egg-laying behaviour is plastic and context-dependent. Such independence in the genetic architectures of OA and OP variation may influence different aspects of oviposition behaviour, including plasticity, canalization, host shift and maintenance of genetic variability, which contributes to the adoption of adaptive strategies during habitat selection (Fanara, 2022).

    An internal expectation guides Drosophila egg-laying decisions

    To better understand how animals make ethologically relevant decisions, egg-laying substrate choice was studied in Drosophila. Flies were found to dynamically increase or decrease their egg-laying rates while exploring substrates so as to target eggs to the best, recently visited option. Visiting the best option typically yielded inhibition of egg laying on other substrates for many minutes. The data support a model in which flies compare the current substrate's value with an internally constructed expectation on the value of available options to regulate the likelihood of laying an egg. Dopamine neuron activity is critical for learning and/or expressing this expectation, similar to its role in certain tasks in vertebrates. Integrating sensory experiences over minutes to generate an estimate of the quality of available options allows flies to use a dynamic reference point for judging the current substrate and might be a general way in which decisions are made (Vijayan, 2022).

    Flexible neural control of transition points within the egg-laying behavioral sequence in Drosophila

    Innate behaviors are frequently comprised of ordered sequences of component actions that progress to satisfy essential drives. Progression is governed by specialized sensory cues that induce transitions between components within the appropriate context. This study has characterized the structure of the egg-laying behavioral sequence in Drosophila and found significant variability in the transitions between component actions that affords the organism an adaptive flexibility. Distinct classes of interoceptive and exteroceptive sensory neurons were identified that control the timing and direction of transitions between the terminal components of the sequence. A pair of motor neurons was identified that enact the final transition to egg expulsion. These results provide a logic for the organization of innate behavior in which sensory information processed at critical junctures allows for flexible adjustments in component actions to satisfy drives across varied internal and external environments (Cury, 2023).

    Natural genetic variation in a dopamine receptor is associated with variation in female fertility in Drosophila melanogaster

    Fertility is a major component of fitness but its genetic architecture remains poorly understood. Using a full diallel cross of 50 Drosophila Genetic Reference Panel inbred lines with whole genome sequences, this study found substantial genetic variation in fertility largely attributable to females. Genes associated with variation in female fertility were mapped by genome-wide association analysis of common variants in the fly genome. Validation of candidate genes by RNAi knockdown confirmed the role of the dopamine 2-like receptor (Dop2R) in promoting egg laying.The Dop2R effect was replicated in an independently collected productivity dataset and showed that the effect of the Dop2R variant was mediated in part by regulatory gene expression variation. This study demonstrates the strong potential of genome-wide association analysis in this diverse panel of inbred strains and subsequent functional analyses for understanding the genetic architecture of fitness traits (Lyman, 2023).

    Aggregation pheromones have a non-linear effect on oviposition behavior in Drosophila melanogaster

    Female fruit flies (Drosophila melanogaster) oviposit at communal sites where the larvae may cooperate or compete for resources depending on group size. This offers a model system to determine how females assess quantitative social information. The concentration of pheromones found on a substrate was found to increase linearly with the number of adult flies that have visited that site. Females prefer oviposition sites with pheromone concentrations corresponding to an intermediate number of previous visitors, whereas sites with low or high concentrations are unattractive. This dose-dependent decision is based on a blend of 11-cis-Vaccenyl Acetate (cVA) indicating the number of previous visitors and heptanal (a novel pheromone deriving from the oxidation of 7-Tricosene), which acts as a dose-independent co-factor. This response is mediated by detection of cVA by odorant receptor neurons Or67d and Or65a, and at least five different odorant receptor neurons for heptanal. These results identify a mechanism allowing individuals to transform a linear increase of pheromones into a non-linear behavioral response (Verschut, 2023).

    Effect of acetic acid bacteria colonization on oviposition and feeding site choice in Drosophila suzukii and its related species

    Oviposition site choice has a large impact on offspring performance. Unlike other vinegar flies that colonize decaying fruits, Drosophila suzukii lay eggs into hard ripening fruits by using their enlarged and serrated ovipositors (oviscapts). This behavior has an advantage over other species by providing access to the host fruit earlier and avoiding competition. However, the larvae are not fully adapted to a low-protein diet, and the availability of intact healthy fruits is seasonally restricted. Thus, to investigate oviposition site preference for microbial growth in this species, an oviposition assay was conducted using single species of commensal Drosophila acetic acid bacteria, Acetobacter and Gluconobacter. The oviposition site preferences for media with or without bacterial growth were quantified in multiple strains of D. suzukii and its closely related species, D. subpulchrella and D. biarmipes, and a typical fermenting-fruit consumer, D. melanogaster. These comparisons demonstrated a continuous degree of preference for sites with Acetobacter growth both within and across species, suggesting that the niche separation is notable but not complete. The preference for Gluconobacter showed large variations among replicates and no clear differences between the strains. In addition, the lack of interspecific differences in feeding site preference for Acetobacter -containing media implies that the interspecific divergence in oviposition site preference occurred independently from the feeding site preference (Sato, 2023).

    Two odorant receptors regulate 1-octen-3-ol induced oviposition behavior in the oriental fruit fly

    The oriental fruit fly Bactrocera dorsalis (Hendel) is a notorious pest of fruit crops. Gravid females locate suitable oviposition sites by detecting host plant volatiles. This study demonstrates that 1-octen-3-ol, a volatile from mango, guides the oviposition behavior of female flies. Two odorant receptors (BdorOR7a-6 and BdorOR13a) are identified as key receptors for 1-octen-3-ol perception by qPCR analysis, heterologous expression in Xenopus laevis oocytes and HEK 293 cells followed by in vitro binding assays, as well as CRISPR/Cas9 genome editing in B. dorsalis. Molecular docking and site-directed mutagenesis are used to determine major binding sites for 1-octen-3-ol. The results demonstrate the potential of 1-octen-3-ol to attract gravid females and molecular mechanism of its perception in B. dorsalis. BdorOR7a-6 and BdorOR13a can therefore be used as molecular targets for the development of female attractants. Furthermore, site-directed mutagenesis data will facilitate the chemical engineering of 1-octen-3-ol to generate more efficient attractants (Xu, 2023).

    Transmission of beneficial yeasts accompanies offspring production in Drosophila-An initial evolutionary stage of insect maternal care through manipulation of microbial load

    Parent-to-offspring transmission of beneficial microorganisms is intimately interwoven with the evolution of social behaviors. Ancestral stages of complex sociality-microbe vectoring interrelationships may be characterized by high costs of intensive parental care and hence only a weak link between the transmission of microbial symbionts and offspring production. This study investigate the relationship between yeast symbiont transmission and egg-laying, as well as some general factors thought to drive the "farming" of microscopic fungi by the fruit fly Drosophila melanogaster, an insect with no obvious parental care but which is highly dependent on dietary microbes during offspring development. The process of transmitting microbes involves flies ingesting microbes from their previous environment, storing and vectoring them, and finally depositing them to a new environment. This study revealed that fecal materials of adult flies play a significant role in this process, as they contain viable yeast cells that support larval development. During single patch visits, egg-laying female flies transmitted more yeast cells than non-egg-laying females, suggesting that dietary symbiont transmission is not random, but linked to offspring production. The crop, an extension of the foregut, was identified as an organ capable of storing viable yeast cells during travel between egg-laying sites. However, the amount of yeast in the crop reduced rapidly during periods of starvation. Although females starved for 24 h deposited a smaller amount of yeast than those starved for 6 h, the yeast inoculum produced still promoted the development of larval offspring. The results of these experiments suggest that female Drosophila fruit flies have the ability to store and regulate the transfer of microorganisms beneficial to their offspring via the shedding of fecal material. It is argue that this observation may represent an initial evolutionary stage of maternal care through the manipulation of microbial load, from which more specialized feedbacks of sociality and microbe management may evolve (Cho, 2023).

    A rise-to-threshold process for a relative-value decision

    Whereas progress has been made in the identification of neural signals related to rapid, cued decisions, less is known about how brains guide and terminate more ethologically relevant decisions in which an animal's own behaviour governs the options experienced over minutes. Drosophila search for many seconds to minutes for egg-laying sites with high relative value and have neurons, called oviDNs, whose activity fulfills necessity and sufficiency criteria for initiating the egg-deposition motor programme This study shows that oviDNs express a calcium signal that (1) dips when an egg is internally prepared (ovulated), (2) drifts up and down over seconds to minutes-in a manner influenced by the relative value of substrates-as a fly determines whether to lay an egg and (3) reaches a consistent peak level just before the abdomen bend for egg deposition. This signal is apparent in the cell bodies of oviDNs in the brain and it probably reflects a behaviourally relevant rise-to-threshold process in the ventral nerve cord, where the synaptic terminals of oviDNs are located and where their output can influence behaviour. Perturbational evidence is provided that the egg-deposition motor programme is initiated once this process hits a threshold and that subthreshold variation in this process regulates the time spent considering options and, ultimately, the choice taken. Finally, a small recurrent circuit was found that feeds into oviDNs, and that activity in each of its constituent cell types was shown to be required for laying an egg. These results argue that a rise-to-threshold process regulates a relative-value, self-paced decision and provide initial insight into the underlying circuit mechanism for building this process (Vijayan, 2023).

    Genetic architecture of natural variation underlying adult foraging behavior that is essential for survival of Drosophila melanogaster

    Foraging behavior is critical for the fitness of individuals. However, the genetic basis of variation in foraging behavior and the evolutionary forces underlying such natural variation have rarely been investigated. A systematic approach was developed to assay the variation in survival rate in a foraging environment for adult flies derived from a wild Drosophila melanogaster population. Despite being such an essential trait, there is substantial variation of foraging behavior among D. melanogaster strains. Importantly, this study provided the first evaluation of the potential caveats of using inbred Drosophila strains to perform genome-wide association studies on life-history traits, and concluded that inbreeding depression is unlikely a major contributor for the observed large variation in adult foraging behavior. Adult foraging behavior has a strong genetic component and, unlike larval foraging behavior, depends on multiple loci. Identified candidate genes are enriched in those with high expression in adult heads and, demonstrated by expression knock down assay, are involved in maintaining normal functions of the nervous system. This study not only identified candidate genes for foraging behavior that is relevant to individual fitness, but also shed light on the initial stage underlying the evolution of the behavior (Chwen, 2017).

    Coupled sensing of hunger and thirst signals balances sugar and water consumption

    Hunger and thirst are ancient homeostatic drives for food and water consumption. Although molecular and neural mechanisms underlying these drives are currently being uncovered, less is known about how hunger and thirst interact. This study used molecular genetic, behavioral, and anatomical studies in Drosophila to identify four neurons that modulate food and water consumption. Activation of these neurons promotes sugar consumption and restricts water consumption, whereas inactivation promotes water consumption and restricts sugar consumption. By calcium imaging studies, it was shown that these neurons are directly regulated by a hormone signal of nutrient levels and by osmolality. Finally, a hormone receptor and an osmolality-sensitive ion channel that underlie this regulation were identified. Thus, a small population of neurons senses internal signals of nutrient and water availability to balance sugar and water consumption. These results suggest an elegant mechanism by which interoceptive neurons oppositely regulate homeostatic drives to eat and drink (Jourjine, 2016).

    This study has uncovered a neural mechanism that coordinates two essential homeostatic behaviors: sugar and water consumption. This coordination is achieved by two neurons per SEZ hemisphere of the Drosophila brain, the ISNs, which are sensitive to internal signals for both hunger and thirst and whose activity oppositely regulates sugar and water consumption. The antagonistic manner in which ISNs couple these behaviors suggests a regulatory principle by which animal nervous systems might promote internal osmotic and metabolic homeostasis (Jourjine, 2016).

    Low internal osmolality and high AKH are signals of water satiety and hunger, respectively. ISN activity increases both in the presence of low extracellular osmolality and AKH. Increasing ISN activity promotes sugar consumption and reduces water consumption. Conversely, high internal osmolality and low AKH are signals of thirst and food satiety. ISN activity decreases and AKH responses are reduced in the presence of high extracellular osmolality or insulin. Decreasing ISN activity increases water consumption and reduces sugar consumption (Jourjine, 2016).

    How do ISNs achieve opposite regulation of a single behavior, consumption, in a manner that depends on the substance being consumed? One possibility is that the downstream targets of ISNs include interneurons involved in the behavioral response to water and sugar taste. This model predicts that increased ISN activity promotes the ability of sugar taste interneurons to drive consumption while inhibiting the ability of water taste interneurons to do so. It may be possible to test hypotheses about the neural circuits in which ISNs participate through the use of large-scale calcium imaging (Jourjine, 2016).

    ISNs regulate sugar and water consumption in a manner that appropriately reflects internal hunger and thirst states. This study shows that two genes, AKHR and nanchung, are expressed in ISNs and function to confer sensitivity to these states (Jourjine, 2016).

    AKHR is a G protein coupled receptor expressed in the fat body and the brain that has been well characterized in the context of insect metabolic regulation. The ligand for this receptor, AKH, is secreted into the hemolymph by specialized neurosecretory cells in the corpus cardiacum, where it acts under conditions of food deprivation. This study identified a role for AKH in regulating the activity of four interneurons in the SEZ, the ISNs, and this activity is shown to promote sugar consumption. AKH abundance in the hemolymph therefore promotes feeding via the ISNs. Manipulating AKHR exclusively in the ISNs provided a means to separate the metabolic and neural effects of AKH, uncovering a role for AKH in the nervous system (Jourjine, 2016).

    Sensors for internal hemolymph osmolality have not previously been described. This study finds that the non-selective cation channel Nanchung is expressed in ISNs and is required for their responses to low osmolality. Although it is not known if Nan is the direct osmosensor in ISNs, previous studies found that Nan confers low osmolality responses when expressed in heterologous cells, consistent with this notion. Nan family members of the TRPV4 family have been shown to participate in osmosensation in Caenorhabditis elegans and mammals, suggesting an ancient and conserved function. Nanchung participates in sensory detection of mechanical stimuli in Drosophila, including proprioception, audition, and low humidity sensing. It is interesting that the same molecule that is involved in external sensory detection of mechanical stimuli also participates in internal detection of osmolality, a mechanical stimulus. Similar molecular re-tooling has recently been described for the GR43a gustatory receptor, which acts as a sensory receptor to monitor fructose in the environment and as an internal sensor monitoring circulating fructose levels in brain hemolymph (Jourjine, 2016).

    In the mammalian brain, osmosensitive neurons are generally found in areas that lack a blood-brain barrier. The blood-brain barrier of Drosophila expresses multiple aquaporins and may potentially regulate hemolymph osmolality. Whether changes in hemolymph osmolality are regulated by the blood-brain barrier to impact ISN activity is an interesting question for future study (Jourjine, 2016).

    ISNs oppositely regulate the behavioral responses to hunger and thirst states. How might this type of coordination be adaptive? One possibility is suggested by the fact that sugar and water consumption perturb internal osmotic homeostasis in opposite directions. In Drosophila and mammals, sugar consumption leads to increased blood-sugar levels and increased blood osmolality. Conversely, water consumption leads to lowered blood osmolality. The current studies show that ISNs are sensitive to extracellular osmolality and that they oppositely regulate sugar and water consumption. Under high osmotic conditions, decreased ISN activity promotes water consumption, reducing internal osmolality. Under low osmotic conditions, increased ISN activity promotes sucrose consumption, increasing internal osmolality. Thus, ISNs may monitor internal osmolality to reciprocally regulate sugar and water consumption to restore homeostasis (Jourjine, 2016).

    Reciprocal regulation of food and water consumption has been reported in both classical and recent rodent studies. For example, increasing blood osmolality promotes water consumption and inhibits food consumption in rats, whereas decreasing osmolality has the opposite effect. In addition, ghrelin, a key internal signal for hunger in mammals, is sufficient not only to promote feeding but also to inhibit water consumption in rats. Thus, vertebrates and invertebrates may share mechanisms for coupling water and sugar consumption in a manner that promotes homeostasis. In Drosophila, the convergence of internal signals onto the ISNs provides a mechanism to weigh homeostatic deviations and drive consumption to restore balance (Jourjine, 2016).

    Other neurons in the Drosophila brain process homeostatic needs for water and sugar separately. For example, water reward and sugar reward are processed by different subsets of mushroom body input neurons, likely independent of gustatory sensory activation. Neuropeptide F, small Neuropeptide F, and dopamine are all signals of nutrient deprivation that promote nutrient intake. Circulating glucose and fructose in the hemolymph also report the nutritional state and alter feeding behavior by direct activation of a few central neurons. The ISNs are unique in that they detect multiple internal state signals and use this information to weigh competing needs. In addition to parallel, independent pathways for eating and drinking, this study demonstrates the existence of a pathway that couples these drives (Jourjine, 2016).

    The thirsty fly: Ion transport peptide (ITP) is a novel endocrine regulator of water homeostasis in Drosophila

    Animals need to continuously adjust their water metabolism to the internal and external conditions. Homeostasis of body fluids thus requires tight regulation of water intake and excretion, and a balance between ingestion of water and solid food. This study investigated how these processes are coordinated in Drosophila melanogaster. The first thirst-promoting and anti-diuretic hormone of Drosophila was identified, encoded by the gene Ion transport peptide (ITP). This endocrine regulator belongs to the CHH (crustacean hyperglycemic hormone) family of peptide hormones. Using genetic gain- and loss-of-function experiments, this study showed that ITP signaling acts analogous to the human vasopressin and renin-angiotensin systems; expression of ITP is elevated by dehydration of the fly, and the peptide increases thirst while repressing excretion, promoting thus conservation of water resources. ITP responds to both osmotic and desiccation stress, and dysregulation of ITP signaling compromises the fly's ability to cope with these stressors. In addition to the regulation of thirst and excretion, ITP also suppresses food intake. Altogether, this work identifies ITP as an important endocrine regulator of thirst and excretion, which integrates water homeostasis with feeding of Drosophila (Galikova, 2018).

    Maintenance of homeostasis is based on ingestion and metabolism of water and nutrients in a manner that reflects the internal needs of the animal, but the precise regulatory mechanisms are incompletely understood. Despite the strong evolutionary conservation of the main pathways underlying energy homeostasis, there is a considerable diversity in the strategies involved in the maintenance of water balance. In insects, this variability arises mainly from the diversity of their habitats and life history strategies. For example, some blood-sucking insects are able to ingest a blood meal that exceeds their body volume up to twelve-fold; their feeding is hence coupled to massive post-prandial diuresis of the excessive water and ions. However, in most of the non-blood sucking terrestrial insects, water conservation is more important than water secretion. Studies on water balance in insects have historically focused mainly on the hormonal regulation of water excretion. These studies investigated the correlations between the hormone titers and diuresis, and analyzed the effects of injections or in vitro applications of the tested compounds. These works contributed to a better understanding of water regulation at the level of fluid secretion by the Malpighian tubules and water reabsorption in the hindgut. Development of genetic tools for Drosophila has allowed analysis of diuretic hormones by direct genetic manipulations. However, no anti-diuretic hormone has been identified in Drosophila until now (Galikova, 2018).

    Drosophila is under laboratory conditions raised on media that provide both nutrients and water, and flies therefore do not regulate food and water intake independently. Nevertheless, insects, including Drosophila, can sense water and exhibit hygrotactic behavior. If given the opportunity, flies differentiate between food and water sources, and are able to seek and drink free water, or ingest media rich in water but devoid of nutrients. Recently, a small group of neurons were identified in the Drosophila brain that antagonistically regulate thirst and hunger. These neurons sense osmolarity cell-autonomously with the cation channel Nanchung, and internal nutrients indirectly via Adipokinetic hormone signaling. Although several hormones have been shown to regulate feeding and satiety, no endocrine regulator of thirst has been identified in Drosophila so far (Galikova, 2018).

    The mechanisms that orchestrate water sensing, water-seeking behavior and conservation of water remain unclear. It is hypothesized that these processes are likely coordinated by endocrine signaling. Physiological roles of Drosophila hormones are mostly well characterized; one of the few exceptions is Ion transport peptide (ITP), which belongs to the family of crustacean hyperglycemic hormones (CHH). CHHs promote water reuptake and hence, act as an anti-diuretic hormones in crustaceans. The locust homolog of ITP promotes water reabsorption by acting on chloride channels in the hindgut. Drosophila has a single ITP gene that gives rise to an amidated ITP hormone and to two longer forms called ITP-like peptides. The functions of Drosophila ITP have not been investigated so far, except for a study that has shown a role of ITP in modulation of evening activity by the circadian clock circuitry. The findings from the crustacean and locust members of the CHH family suggest that Drosophila ITP might be involved in the regulation of water balance as well. This study tested this hypothesis by investigating the effects of gain- and loss-of-function of ITP on key aspects of water homeostasis, such as body water content, desiccation and osmotic stress resistance, food and water intake, and excretion. This work identified master regulatory roles of ITP in water homeostasis of Drosophila; ITP levels increase under desiccation stress and protect the fly from water loss by increasing thirst, reducing excretion rate, and promoting ingestion of water instead of food. Altogether, this work identifies the first anti-diuretic and drinking-promoting hormone in Drosophila, which also coordinates water balance with feeding behavior (Galikova, 2018).

    With the colonization of dry land and evolution of terrestrial life, conservation, rather than elimination of water became the main challenge for the maintenance of water homeostasis. Despite the differences in the organization of the endocrine systems, the main principles of fluid homeostasis are the same in vertebrates and invertebrates; these include thirst, compensation for the feeding-induced increase in osmolarity by water intake, and water re-absorption by the excretory systems. In humans, water homeostasis is regulated primarily by an osmostat located in the hypothalamus. This osmostat increases water levels by triggering thirst, and reduces the water loss by inducing release of the anti-diuretic hormone vasopressin. In addition to the regulation by osmolarity, thirst is also induced by the changes in the blood volume both via vasopressin and the renin-angiotensin system. Even though thirst and water retention are physiologically coupled, their regulation occurs independently. This study shows that these regulations are simplified in Drosophila, where the same hormone promotes thirst, reduces appetite, and increases water storage. Thus, ITP acts as a functional analog of both vasopressin and renin-angiotensin. Interestingly, like the vasopressin and renin-angiotensin system, also ITP is regulated by body water content (Galikova, 2018).

    Over-expression of ITP increases water content by 4.5%, whereas RNAi dehydrates the fly by 3.3%. The physiological consequences of such mild changes of water levels are not known in Drosophila, but for comparison, in human patients, loss of as little as 2% water significantly impairs cognitive abilities, and liquid overload and hypervolemia represent harmful conditions as well (Galikova, 2018).

    The current findings show that knockdown of ITP leads to increased water excretion similar to human disorders caused by defective water re-absorbance in kidney, such as diabetes insipidus. Conversely, ITP over-expression results in increased water retention reminiscent of the human syndrome of inappropriate anti-diuretic hormone secretion (SIADH). ITP manipulations may thus become useful tools to induce and study pathologies associated with these human disorders in Drosophila (Galikova, 2018).

    ITP is the first identified hormone that regulates drinking in Drosophila. Thus, it acts as a functional analog of the renin-angiotensin system of mammals. Similar to the renin-angiotensin system, ITP is most likely activated by hypovolemia. The neural circuits that control drinking and are regulated by ITP, however, remain to be investigated. Neurons that repress drinking in Drosophila have already been identified in the suboesophageal zone. These neurons are regulated cell autonomously by an ion channel that senses osmolarity. ITP-knockdown flies do not have the drive to drink despite their state of dehydration, whereas ITP over-expressing flies drink despite their excessive water content. Thus, unlike the Nanchung-expressing repressors of drinking (Jourjine, 2016), the ITP-regulated neurons are not regulated by the volume of body water, but rather by ITP itself (Galikova, 2018).

    In insects, primary urine is produced by the Malpighian tubules that are functional analogs of mammalian kidneys. Water enters the lumen of these tubules by passive diffusion along the ionic gradient maintained by the vacuolar V-H+-ATPase. The function of the Malpighian tubules is hormonally regulated by diuretic hormones, which in Drosophila include products of the genes capa, DH31, DH44 and leucokinin. Urine then enters the hindgut, where it mixes with the gut contents. Importantly, considerable parts of the water and ions are subsequently re-absorbed in the ileum and rectum. This study shows that ITP reduces excretion of water by reducing the defection rate. Thus, it is likely that Drosophila ITP promotes water reabsorption in the hindgut similar to its homologs in the desert locust Schistocerca gregaria or in the European green crab Carcinus maenas. It is noteworthy that ITP-expressing neurons in the abdominal ganglia innervate Drosophila hindgut, suggesting that in addition to the hormonal regulation, the hindgut may also be regulated by ITP in a paracrine fashion. In crabs and in the red flour beetle Tribolium castaneum¸ CHH- or ITP-producing endocrine cells, respectively, have even been detected in gut epithelia. Thus, whether produced in the neurosecretory cells or in the endocrine cells of the gut, the actions of CHHs and ITPs on the hindgut appear to be evolutionarily conserved (Galikova, 2018).

    In mammals, an increase in osmolarity due to food intake results in postprandial thirst, and conversely, dehydration inhibits feeding when water is not available and this is likely also the case in Drosophila. The current findings of the ITP-driven positive regulation of water intake, concomitant with a negative regulation of feeding likely represents another level of regulation of thirst and hunger, acting in parallel to that of the four drink-repressing neurons in the suboesophageal zone (Galikova, 2018).

    Whereas many terrestrial arthropods frequently experience arid conditions, salt stress is not very common in non-blood feeding terrestrial insects. Nevertheless, desiccation and salt stress resistance have been traditional tests in the studies of Drosophila diuretic hormones. RNAi against diuretic hormones increases desiccation resistance, as shown for capa, DH44 [14] and leucokinin genes. However, it remains unclear whether these hormones contribute to the natural response to the desiccation and osmotic stress. For example, desiccation does not change expression of diuretic hormones DH44 and leucokinin. In contrast, ITP seems to be a natural component of the desiccation and osmotic stress responses, since both stressors trigger an increase in ITP expression. The role of ITP in thirst, hunger and excretion suggest that the ITP-regulated changes in behavior and physiology represent natural responses to cope with the reduction of body water. Consistently, knockdown of ITP reduces survival under desiccation and osmotic stress. However, it is unclear why over-expression of ITP reduces resistance to desiccation and osmotic stress. The UAS-GAL4 based manipulations may increase ITP levels far beyond the physiological range, which (although not lethal under standard feeding) might reduce survival under stressful conditions. Given the role of ITP in the ion transport across the hindgut epithelia of locusts, it is tempting to speculate that a similar mechanism exists in Drosophila. In such a scenario, the non-physiological doses of ITP might considerably increase osmolarity of hemolymph. This would be toxic when feeding on a food medium with a high salt content, as well as under desiccation conditions (which further increase osmolarity) (Galikova, 2018).

    Although ITP has been known for a long time, its function has remained enigmatic in Drosophila. Pioneering work on its roles in Drosophila physiology suggests that ITP codes for a master regulator of water balance, which also integrates the water homeostasis with energy metabolism. Thus, this study not only shows that this member of the CHH family has an evolutionarily conserved anti-diuretic role in Drosophila as it has in other arthropods, but also reveals novel functions of this peptide family in food and water intake. It remains to be investigated to what extent these roles are conserved in other insect species or even in crustaceans, but the strong evolutionary conservation of the gene structure suggests that this might be the case. It is possible that the fly ITP regulates, in addition to its role in water balance, other processes that are known to be CHH-regulated in crustaceans. For example, the high developmental lethality of ITP RNAi, together with the previously described lethality of ITP mutants imply that Drosophila ITP plays a critical role during development, perhaps analogous to the role of CHHs in crustacean molting (Galikova, 2018).

    Although identification of the cellular sources of ITP that are responsible for the functions of this hormone was beyond the scope of this manuscript, the expression pattern of the gene already provides some tempting hints. Previous in situ-hybridizations and immunohistochemistry experiments based on a locust anti-ITP antibody showed that Drosophila ITP is expressed in several neuronal types. Using an antibody specific to Drosophila ITP, this study confirmed that these cells include ipc-1 and ipc-2a neurosecretory neurons in the brain, ipc-3 and ipc-4 interneurons, three pairs of iag cells in the abdominal ganglia, and the LBD neurons in abdominal segments A7 and A8. Although ITP is expressed in several interneurons, the most prominent cells of the brain that express ITP are the neurosecretory protocerebral ipc-1 and the ipc-2a neurons, which send axons towards neurohemal release sites in the corpora cardiaca, corpora allata, and aorta. Experiments based on the Impl2 driver showed that a proper response to desiccation and osmotic stress requires production of ITP in the ipc-1 neurons, ipc-2a neurons, or LBD neurons, or in their combination. The ITP production in these cells becomes nevertheless critical only under desiccation and osmotic stress. In contrast to the global manipulations, ITPi targeted to these neurons is not sufficient to impair water balance under standard conditions. Thus, water content is regulated either via ITP produced by cells outside of the Impl2 expression pattern, or the ITP-producing neurons are redundant in their ability to produce sufficient ITP to maintain water homeostasis under standard conditions. Altogether, additional cell type-specific manipulations are required to differentiate whether thirst, excretion and food intake are regulated by specific neurons, or whether different ITP-producing neurosecretory cells act redundantly to produce sufficient amount of the hormone to regulate physiology of the fly (Galikova, 2018).

    Another key step towards understanding the ITP actions is the identification of the hitherto unknown Drosophila ITP receptor. This will facilitate cell- and tissue-specific manipulations to unravel the neural circuit(s) responsible for the roles of ITP in the control of thirst and hunger, and allow more detailed studies of the peripheral roles of ITP in defecation and water excretion (Galikova, 2018).

    The basis of food texture sensation in Drosophila

    Food texture has enormous effects on food preferences. However, the mechanosensory cells and key molecules responsible for sensing the physical properties of food are unknown. This study shows that akin to mammals, the fruit fly, Drosophila melanogaster, prefers food with a specific hardness or viscosity. This food texture discrimination depends upon a previously unknown multidendritic (md-L) neuron, which extends elaborate dendritic arbors innervating the bases of taste hairs. The md-L neurons exhibit directional selectivity in response to mechanical stimuli. Moreover, these neurons orchestrate different feeding behaviors depending on the magnitude of the stimulus. It was demonstrated that the single Drosophila transmembrane channel-like (TMC) protein is expressed in md-L neurons, where it is required for sensing two key textural features of food-hardness and viscosity. The study proposes that md-L neurons are long sought after mechanoreceptor cells through which food mechanics are perceived and encoded by a taste organ, and that this sensation depends on TMC (Zhang, 2016).

    Food preferences are affected greatly by the qualities of food, including nutrient value, texture, and the taste valence of sweet, bitter, salty, and sour qualities. During the last 15 years, many of the gustatory receptor proteins that participate in the discrimination of the chemical composition of food have been defined. In sharp contrast, the basis through which food texture is detected is enigmatic, despite the universal appreciation that the physical properties of food greatly influence decisions to consume a prospective offering. There are specific tactile features associated with liquid or solid food. Viscosity and creaminess are typical textural features of liquid food, whereas hardness, crispiness, and softness are the main physical characteristics of solid food. Similar to food tastes, food texture provides important information concerning food quality, including freshness and wholesomeness. For instance, people prefer freshly baked bread with relatively soft texture, and tend to reject older bread with a harder texture, even though the chemical composition has not changed significantly over the course of a couple of days. Furthermore, while exploring the food landscape, an animal must make assessments of food hardness and viscosity in order to exert the appropriate force to chew or ingest. Insufficient chewing force results in poor food processing, while excessive force can cause injury to the tongue or teeth (Zhang, 2016).

    Food texture in mammals is predominantly detected through poorly understood mechanisms in taste organs. In rodents and humans, a subset of trigeminal nerves such as the lingual nerve provides somatosensitive afferents to the tongue. Due to the intrinsic mechanical properties of food, mastication produces compression and shearing forces, which in turn activate mechanosensory neuronsin taste organs. However, the molecular identities of mechanosensory neurons and signaling proteins that enable animals to detect food texture are unknown. To address the fundamental issue concerning the cellular and molecular mechanisms that function in the sensation of food texture, this study turned to the fruit fly, Drosophila melanogaster, as an animal model. In flies, food quality is evaluated largely through external sensory hairs (sensilla), which decorate the fly tongue (the labellum) and several other body parts. These sensilla, which house several sensory neurons, allow the chemical composition of foods, such as sugars and bitter compounds, to be detected prior to entering the mouthparts (Zhang, 2016).

    This study found that Drosophila can discriminate between foods on the basis of hardness and viscosity. A previously unknown type of mechanosensory neuron was identified in the fly tongue that is dedicated to detecting food mechanics. These multidendritic neurons in the labellum (md-L) extend their projections into the bases of most of the external sensilla and are activated by deflections induced by hard and viscous food. The ability of md-L neurons to sense food mechanics is virtually lost due to elimination of the only Drosophila member of the transmembrane channel-like (TMC) family. Mice and humans each encode eight TMC proteins, and mutations in the founding member of this family, TMC1, cause deafness in mammals. This study found that tmc is broadly tuned to detect both soft and hard food textures. Remarkably, optogenetic stimulation of the md-L neurons with different light intensities yields opposing behavioral outcomes-weak light promotes feeding, while strong light represses feeding. It is concluded that md-L neurons and TMC are critical cellular and molecular components that enable external sensory bristles on the fly tongue to communicate textural features to the brain, and do so through a pre-ingestive mechanism (Zhang, 2016).

    This study demonstrates that the attraction of wild-type flies to the same concentration of sucrose is altered by the viscosity or hardness of the food. If the sucrose-containing substrate is too sticky, soft, or hard, the appeal of the food declines. These observations establish the Drosophila taste system as a model to explore the cellular and molecular underpinnings that allow an animal to sense food texture. Moreover, similar to the chemosensory evaluation of food by external sensilla decorating the labellum, the textural assessment of foods is pre-ingestive in flies (Zhang, 2016).

    This study has identified md-L, a previously undefined neuron in each of the two bilateral symmetrical labella, which extend a complex array of dendrites to the bases of many sensilla. Several observations demonstrate that md-L neurons play an indispensable role in food texture sensation. First, selective abolition of neurotransmission from md-L caused significant impairments in food texture discrimination. Second, laser ablation of md-L resulted in severe defects in perceiving the viscosity or hardness of foods. Third, low or moderate artificial activation of md-L neurons was sufficient to trigger proboscis extension. Thus, the loss-of-function and gain-of-function analyses of md-L neurons has lead to a conclusion that md-L neurons are key mechanoreceptor cells controlling sensation of food mechanics (Zhang, 2016).

    Unexpectedly, while low-intensity optogenetic stimulation of md-L provoked proboscis extension, high-intensity light induced contraction of the proboscis. Thus, md-L neurons are tuned to different levels of mechanical stimuli that give rise to drastically different feeding behaviors. it is proposed that weak or moderate light mimics the response to softer foods that simulates feeding, while strong light induces a higher level of activity that mimics hard foods and discourages feeding. When a fly is offered sucrose in combination with optogenetic stimulation of md-L neurons with strong light, this caused the animal to reject the otherwise appetitive food. It is proposed that this rejection occurred because the animal perceived the texture of the sucrose as too hard. Thus, it is suggested that texture sensation is mediated by md-L neurons through an intensity-dependent rather than a labeled-line mechanism. While md-L are required, it is not excluded that other neurons in the labella contribute to food texture sensation. Ultrastructural studies of taste sensilla led to the proposal that a neuron positioned at the base of each taste sensillum is a mechanosensory neuron. However, it currently remains unclear as to whether these neurons contribute to some aspect of food texture detection (Zhang, 2016).

    In Drosophila, most taste sensilla point toward the ventral direction. The md-L neuron produced much stronger neuronal activity in response to forces applied to taste hairs that were deflected dorsally than those deflected in other directions. Thus, taste sensilla are most sensitive to force applied opposite to the direction in which they point. Notably, this direction-dependent feature of taste sensilla is reminiscent of the directional sensitivity of hair in mammals, suggesting that it is a widely used neural coding strategy for sensation in the animal kingdom (Zhang, 2016).

    The directional sensitivity of taste sensilla differs from the macrochaete bristles in the thorax, since these latter bristles are most sensitive to force applied in the same direction in which they point. The profound differences inmforce-directional sensitivity reflect the functional divergence between these two types of mechanosensory bristles. The direction-tuning feature of md-L neurons might be an evolutionary adaptation to help fruit flies sample food. While exploring the food landscape, a fruit fly normally extends its proboscis in the ventral direction. As a consequence, the forces arising from the food will bend taste sensilla in the opposite dorsal direction (Zhang, 2016).

    Thus, it is suggested that md-L neurons evolved to become most sensitive to forces emanating from the dorsal direction It is concluded that Drosophila TMC is required for detecting food hardness. TMC is expressed and required in md-L neurons. Furthermore, loss of tmc greatly reduced the ability to behaviorally discriminate the preferred softness (1% agarose) or smoothness (sucrose solution only) from harder or stickier food options, respectively. However, the responses to tastants, such as sucrose, salt, or caffeine, were unaffected in tmc1, indicating that TMC was specifically required for sensing food texture rather than the chemical composition of food (Zhang, 2016).

    An important question concerns the mechanism through which TMC enables md-L neurons to sense food hardness. It is proposed that deflection of gustatory sensilla by food hardness imposes mechanical force on these neurons. The harder the food, the greater the stimulation of md-L neurons, which sense force through the dendrites innervating the bases of many sensilla. Given the expression of TMC in dendrites, an appealing possibility is that TMC is a key component of a mechanically activated channel that endows the fly tongue with the ability to sense food hardness. A TMC protein (TMC-1) is expressed in worms and is proposed to be required for salt sensation (Chatzigeorgiou, 2013). Furthermore, TMC-1 plays a critical role in alkali sensation in vivo (Wang, 2016). As such, it appears that the worm TMC-1 controls multiple aspects of chemosensation. Mammalian TMC1 and TMC2 are required for hearing and expressed in the inner ear (Kawashima, 2011; Pan, 2013). Currently, it is not known if mammalian TMCs are subunits of a channel, or whether they are mechanically activated, since problems with cell-surface expression of these proteins in heterologous expression systems have precluded biophysical characterizations. It is possible that TMCs may depend on additional subunits for trafficking or to form functional ion channels. Drosophila TMC may also be one subunit of a mechanically activated channel, and it is proposed that this feature might allow md-L neurons to be stimulated in response to bending of taste sensilla by hard foods (Zhang, 2016).

    In conclusion, this study has elucidated a cellular mechanism through which food mechanics influence the taste preference of an animal. The md-L neurons define a novel class of mechanosensory neurons that enable flies to detect food hardness and viscosity. A future question concerns the mapping of the brain region where mechanical and chemosensory pathways converge to dictate gustatory decisions. An appealing possibility is that md-L and GRN axons coordinately signal to a pair of command interneurons (Fdg neurons) that have extensive arborizations in the SEZ and control feeding behavior. Finally, the results demonstrate that TMC is essential for food texture sensation. These results raise the possibility that homologs of fly TMC may be dedicated to the gustatory discrimination of texture in many other animals, including mammals (Zhang, 2016).

    Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila

    Starvation induces sustained increase in locomotion, which facilitates food localization and acquisition and hence composes an important aspect of food-seeking behavior. This study investigated how nutritional states modulate starvation-induced hyperactivity in adult Drosophila. The receptor of adipokinetic hormone (AKHR), the insect analog of glucagon, is required for starvation-induced hyperactivity. AKHR is expressed in a small group of octopaminergic neurons in the brain. Silencing AKHR+ neurons and blocking octopamine signaling in these neurons eliminates starvation-induced hyperactivity, whereas activation of these neurons accelerates the onset of hyperactivity upon starvation. Neither AKHR nor AKHR+ neurons are involved in increased food consumption upon starvation, suggesting that starvation-induced hyperactivity and food consumption are independently regulated. Single cell analysis of AKHR+ neurons identified the co-expression of Drosophila insulin-like receptor (dInR), which imposes suppressive effect on starvation-induced hyperactivity. Therefore, insulin and glucagon signaling exert opposite effects on starvation-induced hyperactivity via a common neural target in Drosophila (Yu, 2016).

    Food seeking and food consumption are essential for the acquisition of food sources, and hence survival, growth, and reproduction of animal species. Starvation influences food-seeking behavior via both modulating the perception of food cues as well as enhancing flies' locomotor activity. Accumulated evidence has suggested that starvation modulates the activity of ORNs via multiple neural and hormonal cues, which in turn facilitates odor driven food search and food consumption. Similarly, starvation also modulates the perception of food taste via the relative sensitivity of appetitive sweet-sensing and aversive bitter-sensing GRNs,which may in turn increase the attractiveness of food taste. However, how starvation increases the locomotor activity of flies remains largely uncharacterized (Yu, 2016).

    Consistent with previous reports, this study has shown that starved fruit flies exhibit sustained increase in their locomotor activity, which can be suppressed by food consumption induced by both nutritive and non-nutritive food cues. The present study has shown that a small group of neurons located in the subesophageal zone (SEZ) region of the fly brain are both necessary and sufficient for starvation induced hyperactivity. These neurons sense the changes in flies' internal nutritional states by directly responding to two sets of hormones, AKH and DILPs, and modulate locomotor activity in response. Single cell analysis has identified that these AKHR+dInR+ neurons are octopaminergic, which offers an entry point to trace the downstream neural circuitry that regulates starvation-induced hyperactivity. For example, there are seven candidate octopamine receptors in fruit flies and it would be of interest to investigate whether any of these receptors and the receptor-expressing neurons are involved in locomotor regulation upon starvation (Yu, 2016).

    AKH and DILPs are two sets of functionally counteracting hormones in fruit flies. As its mammalian analog glucagon, the reduction in circulating sugars induces the release of AKH, which in turn mobilizes fat storage and provides energy supply for flies. In contrast, DILPs, the insect analog of mammalian insulin, function as satiety hormones. Dietary nutrient induces the release of DILPs into the hemolymph, which in turn promotes protein synthesis, body growth, and other anabolic processes. This study has shown that these two hormonal signaling systems exert opposite effects on starvation-induced hyperactivity via a small group of AKHR+InR+ octopaminergic neurons. These results suggest that these AKHR+dInR+ neurons can integrate the inputs from the two hormonal signaling systems representing hunger and satiety at the same time, and modulate flies' locomotor activity. This elegant yet concise design allows these neurons to be responsive to rapid changes in the internal nutritional states as well as food availability. Furthermore, it is possible that besides hunger and satiety, other physiological states such as wakefulness, stress, and emotions also influence flies' locomotor activity. Notably, single cell analysis has shown that these AKHR+dInR+ neurons also sparsely express other neuropeptide receptors, suggesting that at least small portions of these neurons may also receive input from other neuropeptidergic systems (Yu, 2016).

    Starved animals exhibited increased locomotion and food consumption, the transition of which relies on the detection of food cues. But whether these two behaviors are interdependently or independently regulated remains unclear. This study has shown that these two behaviors are dissociable from each other in fruit flies. On the one hand, although AKHR+ neurons exert robust modulatory effect on starvation-induced hyperactivity, these neurons are neither necessary nor sufficient for starvation-induced food consumption. On the other hand, the regulation of food consumption is independent of starvation-induced hyperactivity as well. Previous studies have shown that a small subset of GABAergic neurons in the fly brain regulates food consumption but exerts no effect on 10 starvation-induced hyperactivity (Pool, 2014). In addition, several neuropeptides are known to regulate food consumption, such as Hugin, NPF, sNPF, Leucokinin, and AstA. However this study found in an RNAi screen that the receptors of these neuropeptides were not involved in the regulation of starvation-induced hyperactivity. Taken together, it is likely that starvation-induced hyperactivity and food consumption are independently regulated by different sets of hormonal cues, and that AKHR+ neurons are only involved in the former but not the latter. These results may shed light on the regulation of food intake in mammals, especially whether starvation-induced hyperactivity and food consumption are also independently regulated by different sets of hormones and distinct neural circuitry in mammals (Yu, 2016).

    Allatostatin A signalling in Drosophila regulates feeding and sleep and is modulated by PDF

    Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. This study shows that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing posterior lateral protocerebrum (PLP) neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. The PLP neurons are identified to be a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, these results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF (Chen, 2016).

    Neuropeptides and peptide hormones transfer a wide variety of neuronal or physiological information from one cell to the other by activating specific receptors on their target cells. Most if not all peptides are pleiotropic and can orchestrate diverse physiological, neuronal or behavioural processes. In vertebrates, such a pleiotropic effect is especially prominent in the regulation of feeding and sleep. Many different peptides (e.g. orexin/hypocretin, ghrelin, obestatin) modulate different aspects of both behaviours, which reciprocally influence each other. The temporal pattern of neuroendocrine activity and neuropeptide release is shaped by sleep homeostasis and the circadian clock which, in turn, reciprocally affects feeding and sleep-wake cycles. Significant progress has been made in this field during recent years. Still little characterised, however, is the neuronal architecture that enables the relevant peptidergic neurons to integrate energy status, circadian time and sleep-wake status in order to coordinate the timing of sleep, locomotor activity and feeding. Information about the output signals by which endogenous clocks provide time- and non-circadian information to relevant peptidergic cells is still limited (Chen, 2016).

    During the last years, the fruit fly Drosophila has become an important model for research into the regulation of feeding and sleep. Drosophila offers advanced genetic tools, a small brain with only about 100.000 neurons and a quantifiable sleep- and feeding behaviour that shows characteristics very similar to that of mammals. These features greatly facilitate the analysis of the neuronal and endocrine underpinnings of feeding and sleep. Like in most animals, feeding and sleep follow a circadian pattern in the fruit fly with little characterised neuronal and hormonal pathways downstream of the central clock. Like in mammals, a number of neuropeptides have been shown to be involved in the regulation of feeding or sleep in Drosophila. Yet, so far, only sNPF and likely also NPF are implicated in the regulation of both feeding and sleep. Also Insulin-like peptide (DILP)-expressing neurons (IPCs) in the pars intercerebralis affect feeding and sleep, yet only feeding seems to be directly dependent on DILP signalling (Chen, 2016).

    Recent work by Hergarden (2012) demonstrated that neurons expressing neuropeptides of the allatostatin A (AstA) family regulate feeding behaviour of the fruit fly. Constitutive activation of AstA cells contained in the AstA1-Gal4 expression pattern by ectopic expression of the bacterial low threshold voltage-gated NaChBac channel potently inhibited starvation-induced feeding. In contrast, constitutive inactivation of AstA1 cells by expression of the inwardly rectifying Kir2.1 potassium channel increased feeding under restricted food availability. NaChBac activation of AstA1 cells also inhibited the starvation-induced increase of the proboscis extension reflex (PER), a behavioural indicator for glucose responsiveness (Hergarden, 2012). The AstA1 expression pattern includes a large number of brain neurons plus gut-innervating thoracico-abdominal ganglion (TAG) neurons and enteroendocrine cells (EECs) in the posterior midgut (Hergarden, 2012). This broad expression pattern is consistent with earlier described patterns of AstA-like immunoreactivity and suggests multiple functions for AstA. Earlier work had demonstrated an effect of AstA on gut motility. Two AstA receptors, DAR-1 (= AlstR) and DAR-2 are characterised for Drosophila. Different genome-based phylogenetic GPCR analyses independently demonstrated their homology with the galanin receptor family of vertebrates (Chen, 2016).

    Using anatomical subdivision and genetic manipulation of neuronal activity, this study aimed to identify AstA functions and assign them to subsets of AstA expressing cells. The results revealed new interconnected AstA functions that link feeding and sleep and identify AstA-expressing PLP neurons and EECs as a target of the central clock output factor PDF. Pleiotropic AstA signalling seems capable of coordinating multiple aspects of physiology and behaviour in a coherent manner to adapt the fly to a digestive energy-saving state. The functional range of AstA signalling in the fly is thus reminiscent of the pleiotropy found in mammalian galanin signalling (Chen, 2016).

    This study shows that AstA cells via AstA signalling subserve an anorexigenic and sleep-promoting function in Drosophila. In mammals, a variety of neuropeptides and peptide hormones affect both sleep and feeding, and the results provide evidence that also further such peptides exist in the fly besides sNPF and possibly NPF. More specifically, the results with a new AstA34-Gal4 driver line show that activation of AstA-expressing PLP brain neurons or numerous EECs in the midgut strongly reduces food intake and promotes sleep. These behavioural effects are congruent with the anatomy of these cells. PLP interneurons are well positioned to modulate sleep as they widely arborise in the posterior superior protocerebrum, a projection area of sleep-relevant dopaminergic neurons, superior (dorsal) fan-shaped body neurons and neurons of the pars intercerebralis. AstA EECs in Drosophila are 'open type' EECs, possessing apical extensions that reach the gut lumen and likely express gustatory receptors. AstA-expressing EECs are thus potentially able to humorally signal nutritional information from the gut to brain centres regulating feeding and possibly also sleep and locomotor activity. If AstA is involved in inhibiting feeding and promoting sleep, one could expect AstA mutants to display decreased sleep and increased feeding in the absence of any other manipulation of AstA cells. It was observed, however, that a functional loss of the AstA gene did neither affect feeding nor locomotor activity under the experimental conditions with unrestricted access to a food source. This may suggest that AstA signalling is not part of a core feeding network, but represents an extrinsic modulator which becomes activated under specific yet so far uncharacterised conditions. Alternatively, as suggested by the observed difference in effect of constitutive vs. conditional electrical silencing of AstA cells, flies may be able to genetically or neuronally compensate for a constitutive loss of AstA signalling during development (Chen, 2016).

    In larval Drosophila, AstA inhibits midgut peristalsis and affects K+ transport in order to concentrate ingested food. Together with the finding of a sleep-promoting and feeding-inhibiting effect of AstA, it is proposed that pleiotropic AstA signalling serves to coordinate behaviour and gut physiology to allow for efficient digestion. After food intake, AstA from the PLP neurons or EECs cause inhibition of further feeding, and -as the need for food search behaviour is relieved and nutrients need to be taken up- promotes sleep and inhibits gut peristalsis. Based on the gut content, enteroendocrine AstA is released and hormonally activates DAR-2 on key metabolic centers to tune adipokinetic hormone and insulin signalling, and -at least in other insects- stimulates digestive enzyme activity in the midgut (Chen, 2016 and references therein).

    The AstA receptors are homologues of the vertebrate galanin receptors that have pleiotropic functions. When activated in specific brain areas, galanin signalling has a strong orexigenic effect and has also been implicated in the control of arousal and sleep in mammals. In zebrafish, transgenic heat-shock induced expression of galanin decreased swimming activity, the latency to rest at night and decreased the responsiveness to various stimuli. Furthermore, the allatostatin/galanin-like receptor NPR-9 inhibits local search behaviour on food in the nematode C. elegans. Similar to AstA in Drosophila, galanin modulates intestinal motility and ion transport. Thus, in broad terms, the involvement of DARs/galanin receptors in modulating feeding, gut physiology and arousal/sleep appears to be evolutionarily conserved (Chen, 2016 and references therein).

    The neuronal clock network in Drosophila is intrinsically and extrinsically modulated by a variety of peptides (sNPF, NPF, calcitonin-gene related peptide/DH31, ion transport peptide, myoinhibiting peptides and PDF), which all affect sleep and locomotor activity and in part also act as clock output factors. Imaging results and constitutive activation of the PDF signalling pathway by t-PDF now suggest that the PLP neurons are modulated by PDF originating from the sLNv clock neurons. Unlike the peptides above, AstA from PLP neurons is outside and downstream of the central clock and seems not to modulate the clock network. Due to their anatomy and position, PLP neurons thus appear well-suited candidate cells by which clock neurons could modulate the complex cross-regulatory network regulating sleep, locomotor activity and perhaps also feeding. The rather mild effects on sleep and feeding of either t-PDF expression in AstA cells or thermogenetic activation of the sLNvs implies that this pathway is not the major output target of the central clock (if there is any) to modulate feeding and locomotor activity/sleep. This study found no shift in the circadian period or phase of feeding and locomotory activity/sleep upon AstA cell activation, suggesting that the main function of PDF-to-AstA cell signalling is not to time the respective behaviours but to modulate their amplitude. Similar non-timing functions of PDF have been demonstrated for other behaviours, including geotaxis and rival-induced mating duration (Chen, 2016).

    At first sight, the current data suggesting that PDF activates PLP neurons to promote sleep seem to contradict earlier findings. Since pdf01 mutants show increased sleep during the photophase, the arousal effect appears to be the dominant effect of PDF which is due to signalling between ventral lateral clock neurons (LNvs), with a major contribution of the PDF-expressing large LNvs. The PLP neurons are only contacted by the sLNvs, which upon activation induced a time-specific increase in sleep, but did not increase arousal. Thus, the sLNv-PLP pathway likely represents a sleep-promoting clock output branch. Besides PDF, the sLNvs but not the lLNvs also co-localise the sleep-promoting peptide sNPF. A recent report shows that hormonal PDF released from abdominal PDF neurons serves to couple the central clock with a peripheral clock in the oenocytes. Furthermore, the posterior midgut is innervated by the abdominal PDF neurons, and PDFR is expressed in the midgut. It is thus possible that the AstA-expressing EECs represent additional PDF targets and may contribute to the PDF-related effects of AstA cells (Chen, 2016).

    In conclusion, the lack of effect on feeding upon AstA cell silencing under non-restricted food availability and an unaltered circadian locomotor rhythmicity after AstA cell silencing suggests that AstA signalling is neither a primary signal in feeding regulation nor in the clock output pathway timing rhythmic behaviour. Rather-like mammalian galanin signalling - it seems to be one out of several modulatory pathways that allow to adapt the intensity of feeding and locomotor activity/sleep to specific physiological or environmental conditions. For example, decreased locomotor activity to save energy and increased digestion efficiency to maximise energy uptake may be most important during restricted food conditions, at which AstA cell silencing leads to increased feeding (Hergarden, 2012). While our results allow now to raise such speculations, it is clear that more research is needed to reveal the conditions at which AstA signalling is functional and the modulatory PDF input is strongest (Chen, 2016).

    Feeding-related traits are affected by dosage of the foraging gene in Drosophila melanogaster

    Nutrient acquisition and energy storage are critical parts of achieving metabolic homeostasis. The foraging gene in Drosophila melanogaster has previously been implicated in multiple feeding-related and metabolic traits. Before foraging's functions can be further dissected, a precise genetic null mutant is needed to definitively map its amorphic phenotypes. This study used homologous recombination to precisely delete foraging, generating the for0 null allele, and used recombineering to re-integrate a full copy of the gene, generating the {forBAC} rescue allele. Total loss of foraging expression in larvae results in reduced larval path length and food intake behavior, while conversely showing an increase in triglyceride levels. Furthermore, varying foraging gene dosage demonstrates a linear dose-response on these phenotypes in relation to foraging gene expression levels. These experiments have unequivocally proven a causal, dose-dependent relationship between the foraging gene and its pleiotropic influence on these feeding-related traits. In that regard, this analysis of foraging's transcription start sites, termination sites, and splicing patterns using RACE and full length cDNA sequencing, revealed 4 independent promoters, pr1-4, that produce 21 transcripts with 9 distinct ORFs. The use of alternative promoters and alternative splicing at the foraging locus creates diversity and flexibility in the regulation of gene expression, and ultimately function. Future studies will exploit these genetic tools to precisely dissect the isoform- and tissue-specific requirements of foraging's functions and shed light on the genetic control of feeding-related traits involved in energy homeostasis (Allen, 2016). >

    Pleiotropy of the Drosophila melanogaster foraging gene on larval feeding-related traits

    Little is known about the molecular underpinning of behavioral pleiotropy. The Drosophila melanogaster foraging gene is highly pleiotropic, affecting many independent larval and adult phenotypes. Included in foraging's multiple phenotypes are larval foraging path length, triglyceride levels, and food intake. foraging has a complex structure with four promoters and 21 transcripts that encode nine protein isoforms of a cGMP dependent protein kinase (PKG). This study examined if foraging's complex molecular structure underlies the behavioral pleiotropy associated with this gene. Using a promotor analysis strategy, DNA fragments upstream of each of foraging's transcription start sites was cloned and four separate forpr-Gal4s were generated. Supporting the hypothesis of modular function, they had discrete, restricted expression patterns throughout the larva. In the CNS, forpr1-Gal4 and forpr4-Gal4 were expressed in neurons while forpr2-Gal4 and forpr3-Gal4 were expressed in glia cells. In the gastric system, forpr1-Gal4 and forpr3-Gal4 were expressed in enteroendocrine cells of the midgut while forpr2-Gal4 was expressed in the stem cells of the midgut. forpr3-Gal4 was expressed in the midgut enterocytes, and midgut and hindgut visceral muscle. forpr4-Gal4's gastric system expression was restricted to the hindgut. Promoter specific expression was found in the larval fat body, salivary glands, and body muscle. The modularity of foraging's molecular structure was also apparent in the phenotypic rescues. Path length, triglyceride levels (bordered on significance), and food intake were rescued of forpr0 null larvae using different forpr-Gal4s to drive UAS-for(cDNA). In a foraging null genetic background, forpr1-Gal4 was the only promoter driven Gal4 to rescue larval path length, forpr3-Gal4 altered triglyceride levels, and forpr4-Gal4 rescued food intake. The results refine the spatial expression responsible for foraging's associated phenotypes, as well as the sub-regions of the locus responsible for their expression. foraging's pleiotropy arises at least in part from the individual contributions of its four promoters (Allen, 2018).

    Odor source localization in complex visual environments by fruit flies

    Flying insects routinely forage in complex and cluttered sensory environments. Their search for a food or a pheromone source typically begins with a whiff of odor, which triggers a flight response, eventually bringing the insect near the odor source. However, pinpointing the precise location of an odor source requires use of both visual and olfactory modalities, aided by odor plumes. This study investigated odor-tracking behavior in fruit flies (Drosophila melanogaster) presented with low- or high-contrast visual landmarks, either paired with or separate from an attractive odor cue. These experiments were conducted either in a gentle air stream which generated laminar odor plumes, or in still air in which odor dissipates uniformly in all directions. Trajectories of flies revealed several novel features of their odor-tracking behavior in addition to those previously documented. First, in both moving and still air, odor-seeking flies rely on co-occurrence of visual landmarks with olfactory cues to guide them to odorant objects. Second, flies abruptly decelerate upon encountering an odor plume, thereafter steering towards nearest visual objects that had no inherent salience in the absence of odor. Thus, interception of an attractive odor increases their salience to nearby high-contrast visual landmarks. Third, flies adopt distinct odor tracking strategies during flight in moving vs. still air. Whereas they weave in and out of plumes towards an odor source in airflow, their approach is more incremental in still air. Both strategies are robust and flexible, and enable flies to reliably find odor sources under diverse visual and airflow environments (Saxena, 2017).

    Satiation state-dependent dopaminergic control of foraging in Drosophila

    Hunger evokes stereotypic behaviors that favor the discovery of nutrients. The neural pathways that coordinate internal and external cues to motivate foraging behaviors are only partly known. Drosophila that are food deprived increase locomotor activity, are more efficient in locating a discrete source of nutrition, and are willing to overcome adversity to obtain food. A simple open field assay was developed that allows flies to freely perform multiple steps of the foraging sequence, and it was shown that two distinct dopaminergic neural circuits regulate measures of foraging behaviors. One group, the PAM neurons, functions in food deprived flies while the other functions in well fed flies, and both promote foraging. These satiation state-dependent circuits converge on dopamine D1 receptor-expressing Kenyon cells of the mushroom body, where neural activity promotes foraging independent of satiation state. These findings provide evidence for active foraging in well-fed flies that is separable from hunger-driven foraging (Landayan, 2018).

    Linking developmental diet to adult foraging choice in Drosophila melanogaster

    Rather than maximizing intake of available macronutrients, insects increase intake of some nutrients and restrict intake of others. This selective consumption influences, and potentially optimizes developmental time, reproduction and lifespan of the organism. Studies so far have focused on discriminating between protein and carbohydrate and the consequences on fitness components at different life stages. However, it is largely unknown if and how the developmental diets, which may entail habitat specific nutrient restrictions, affect the selective consumption of adults. Adult female D. melanogaster were shown to opt for the same protein to carbohydrate (P:C) ratio regardless of their developmental diet (P:C ratio of 1:1, 1:4 or 1:8). Males choose a diet that makes up for deficiencies; when protein is low during development, males increase protein consumption despite this being detrimental to starvation resistance. The sexual dimorphism in foragingchoice could be due to the different energetic requirements of males and females. To investigate the effect of developmental diet on lifespan once an adult nutritional environment had been established, a no choice experiment was conducted. Here adult lifespan increased as P:C ratio decreased irrespective of developmental diet, thus demonstrating a 'cancelling out' effect of nutritional environment experienced during early life stages. This study provides novel insights into how developmental diet is linked to adult diet by presenting evidence for sexual dimorphism in foraging choice as well as life stage dependency of diet on lifespan (Davies, 2018).

    Taotie neurons regulate appetite in Drosophila

    The brain has an essential role in maintaining a balance between energy intake and expenditure of the body. Deciphering the processes underlying the decision-making for timely feeding of appropriate amounts may improve understanding of physiological and psychological disorders related to feeding control. This study identified a group of appetite-enhancing neurons in a behavioural screen for flies with increased appetite. Manipulating the activity of these neurons, which were name Taotie neurons, induces bidirectional changes in feeding motivation. Long-term stimulation of Taotie neurons results in flies with highly obese phenotypes. Furthermore, it was shown that the in vivo activity of Taotie neurons in the neuroendocrine region reflects the hunger/satiety states of un-manipulated animals, and that appetitive-enhancing Taotie neurons control the secretion of insulin, a known regulator of feeding behaviour. Thus, this study reveals a new set of neurons regulating feeding behaviour in the high brain regions that represents physiological hunger states and control feeding behaviour in Drosophila (Zhan, 2016).

    Motor control of Drosophila feeding behavior

    The precise coordination of body parts is essential for survival and behavior of higher organisms. While progress has been made towards the identification of central mechanisms coordinating limb movement, only limited knowledge exists regarding the generation and execution of sequential motor action patterns at the level of individual motoneurons. This study used Drosophila proboscis extension as a model system for a reaching-like behavior. A neuroanatomical description is provided of the motoneurons and muscles contributing to proboscis motion. Using genetic targeting in combination with artificial activation and silencing assays, the individual motoneurons controlling the five major sequential steps of proboscis extension and retraction were identified. Activity-manipulations during naturally evoked proboscis extension show that orchestration of serial motoneuron activation does not rely on feed-forward mechanisms. The data support a model in which central command circuits recruit individual motoneurons to generate task-specific proboscis extension sequences (Schwarz, 2017).

    Pathogen induced food evasion behavior in Drosophila larvae

    Recognizing a deadly pathogen and generating an appropriate immune reaction is essential for any organism to survive in its natural habitat. Unlike vertebrates and higher primates, invertebrates depend solely on the innate immune system to defend themselves from an attacking pathogen. This paper reports a behavioral defense strategy observed in Drosophila larvae that help them escape and limit an otherwise lethal infection. A bacterial infection in the gut is sensed by the larval central nervous system which generates an alteration in its food preference, leading them to stop feeding and move away from the infectious food source. This behavioral response is dependent on the internal nutritive state of the larvae. Using this novel behavioral assay as a read-out, hugin neuropeptide was found to be involved in evasion response and detection of bacterial signals (Surendran, 2017).

    Drosophila divalent metal ion transporter Malvolio is required in dopaminergic neurons for feeding decisions

    Members of the natural resistance-associated macrophage protein (NRAMP) family are evolutionarily conserved metal ion transporters that play an essential role in regulating intracellular divalent cation homeostasis in both prokaryotes and eukaryotes. Malvolio (Mvl), the sole NRAMP family member in insects, plays a role in food choice behaviors in Drosophila and other species. However, the specific physiological and cellular processes that require the action of Mvl for appropriate feeding decisions remain elusive. This study shows that normal food choice requires Mvl function specifically in the dopaminergic system, and can be rescued by supplementing food with manganese. Collectively, data indicate that the action of the Mvl transporter affects food choice behavior via the regulation of dopaminergic innervation of the mushroom bodies, a principle brain region associated with decision-making in insects. These data suggest that the homeostatic regulation of the intraneuronal levels of divalent cations plays an important role in the development and function of the dopaminergic system and associated behaviors (LaMora, 2017).

    Synaptic transmission parallels neuromodulation in a central food-intake circuit

    NeuromedinU is a potent regulator of food intake and activity in mammals. In Drosophila, neurons producing the homologous neuropeptide hugin regulate feeding and locomotion in a similar manner. This study used EM-based reconstruction to generate the entire connectome of hugin-producing neurons in the Drosophila larval CNS (see EM reconstruction of hugin neurons and their synaptic sites). Hugin neurons were shown to use synaptic transmission in addition to peptidergic neuromodulation, and acetylcholine was identified as a key transmitter. Hugin neuropeptide and acetylcholine are both necessary for the regulatory effect on feeding. Subtypes of hugin neurons connect chemosensory to endocrine system by combinations of synaptic and peptide-receptor connections. Targets include endocrine neurons producing DH44, a CRH-like peptide, and insulin-like peptides. Homologs of these peptides are likewise downstream of neuromedinU, revealing striking parallels in flies and mammals. It is proposed that hugin neurons are part of an ancient physiological control system that has been conserved at functional and molecular level (Schlegel, 2016).

    Almost all neurons in Drosophila are uniquely identifiable and stereotyped. This enabled identification and reconstruction of a set of 20 peptidergic neurons in an ssTEM volume spanning an entire larval CNS. These neurons produce the neuropeptide hugin and have previously been grouped into four classes based on their projection targets. Neurons of the same morphological class (a) were very similar with respect to the distribution of synaptic sites, (b) shared a large fraction of their pre- and postsynaptic partners and (c) in case of the interneuron classes (hugin-PC and hugin-VNC), neurons were reciprocally connected along their axons with other neurons of the same class. This raises the question why the CNS sustains multiple copies of morphologically very similar neurons. Comparable features have been described for a population of neurons which produce crustacean cardioactive peptide (CCAP) in Drosophila. The reciprocal connections as well as the overlap in synaptic partners suggest that the activity of neurons within each interneuron class is likely coordinately regulated and could help sustain persistent activity within the population. In the mammalian pyramidal network of the medial prefrontal cortex, reciprocal connectivity between neurons is thought to contribute to the network's robustness by synchronizing activity within subpopulations and to support persistent activity. Similar interconnectivity and shared synaptic inputs have also been demonstrated for peptidergic neurons producing gonadotropin-releasing hormone (GnRH) and oxytocin in the hypothalamus. Likewise, this is thought to synchronize neuronal activity and allow periodic bursting (Schlegel, 2016).

    Previous studies showed that specific phenotypes and functions can be assigned to certain classes of hugin neurons: hugin-VNC neurons increase locomotion motor rhythms but do not affect food intake, whereas hugin-PC neurons decrease food intake and are necessary for processing of aversive gustatory cues. For hugin-RG or hugin-PH such specific functional effects have not yet been described. One conceivable scenario would be that each hugin class mediates specific aspects of an overarching 'hugin phenotype'. This would require that under physiological conditions all hugin classes are coordinately active. However, no evidence of such coordination was found on the level of synaptic connectivity. Instead, each hugin class forms an independent microcircuit with its own unique set of pre- and postsynaptic partners. It is thus predicted that each class of hugin-producing neurons has a distinct context and function in which it is relevant for the organism (Schlegel, 2016).

    Data presented in this study provide the neural substrate for previous observation as well as open new avenues for future studies. One of the key features in hugin connectivity is the sensory input to hugin-PC, hugin-VNC and, to a lesser extent, hugin-RG. While hugin-PC neurons are known to play a role in gustatory processing, there is no detailed study of this aspect for hugin-VNC or hugin-RG neurons. Sensory inputs to hugin neurons are very heterogeneous, which suggests that they have an integrative/processing rather than a simple relay function (Schlegel, 2016).

    Hugin neurons also have profound effects on specific motor systems: hugin-PC neurons decelerate motor patterns for pharyngeal pumping whereas hugin-VNC neurons accelerate locomotion motor patterns. For hugin-PC, this study has demonstrated that this effect is mediated by both synaptic and hugin peptide transmissions. For hugin-VNC, this effect is independent of the hugin neuropeptide, suggesting synaptic transmission to play a key role. Suprisingly, no direct synaptic connections to the relevant motor neurons were found. However, the kinetics of the effects of hugin neurons on motor systems have not yet been studied at a high enough temporal resolution (i.e., by intracellular recordings) to assume monosynaptic connections. It is thus well conceivable that connections to the respective motor systems are polysynaptic and occur further downstream. Alternatively, this may involve an additional non-synaptic (peptidergic) step. A strong candidate for this is the neuroendocrine system which this study has identified as the major downstream target of hugin-PC neurons. Among the endocrine targets of hugin, the insulin-producing cells (IPCs) have long been known to centrally regulate feeding behavior. It is not known if insulin-signaling directly affects motor patterns in Drosophila. Nevertheless, increased insulin signaling has strong inhibitory effects on food-related sensory processing and feeding behavior. Whether the neuroendocrine system is a mediator of the suppressive effects of hugin-PC neurons on food intake remains to be determined (Schlegel, 2016).

    The first functional description of hugin in Drosophila was done in larval and adult, while more recent publications have focused entirely on the larva. One of the main reasons for this is the smaller behavioral repertoire of the larva: the lack of all but the most fundamental behaviors makes it well suited to address basic questions. Nevertheless, it stands to reason that elementary circuits should be conserved between larval and adult flies. To date, there is no systematic comparison of hugin across the life cycle of Drosophila. However, there is indication that hugin neurons retain their functionality from larva to the adult fly. First, morphology of hugin neurons remains virtually the same between larva and adults. Second, hugin neurons seem to serve similar purposes in both stages: they acts as a brake on feeding behavior - likely as response to aversive sensory cues. In larvae, artificial activation of this brake shuts down feeding. In adults, removal of this break by silencing of hugin neurons leads to a facilitation (earlier onset) of feeding. Such conservation of neuropeptidergic function between larval and adult Drosophila has been observed only in a few cases. Prominent examples are short and long neuropeptide F, both of which show strong similarities with mammalian NPY. The lack of additional examples is not necessarily due to actual divergence of peptide function but rather due to the lack of data across both larva and adult. Given the wealth of existing data on hugin in larvae, it would be of great interest to investigate whether and to what extent the known features (connectivity, function, etc.) of this system are maintained throughout Drosophila's life history (Schlegel, 2016).

    A neural network is a highly dynamic structure and is subject to constant change, yet it is constrained by its connectivity and operates within the framework defined by the connections made between its neurons. On one hand, this connectivity is based on anatomical connections formed between members of the network, namely synapses and gap junctions. On the other hand, there are non-anatomical connections that do not require physical contact due to the signaling molecules, such as neuropeptides/-hormones, being able to travel considerable distances before binding their receptors. The integrated analysis in this study of the operational framework for a set of neurons genetically defined by the expression of a common neuropeptide, positions hugin-producing neurons as a novel component in the regulation of neuroendocrine activity and the integration of sensory inputs. Most hugin neurons receive chemosensory input in the subesophageal zone, the brainstem analog of Drosophila. Of these, one class is embedded into a network whose downstream targets are median neurosecretory cells (mNSCs) of the pars intercerebralis, a region homologous to the mammalian hypothalamus. Hugin neurons target mNSCs by two mechanisms. First, by classic synaptic transmission as the current data strongly suggest that acetylcholine (ACh) acts as transmitter at these synapses. Accordingly, subsets of mNSCs have been shown to express a muscarinic ACh receptor. Whether additional ACh receptors are expressed is unknown. Second, by non-anatomical, neuromodulatory transmission using a peptide-receptor connection, as demonstrated by the expression of hugin G-protein-coupled receptor PK2-R1 (CG8784) in mNSCs. Strikingly, while PK2-R1 is expressed in all mNSCs, the hugin neurons have many synaptic contacts onto insulin-producing cells but few to DMS and DH44 neurons. This mismatch in synaptic vs. peptide targets among the mNSCs suggests an intricate influence of hugin-producing neurons on this neuroendocrine center. In favor of a complex regulation is that those mNSCs that are synaptically connected to hugin neurons additionally express a pyrokinin-1 receptor (PK1-R, CG9918) which, like PK2-R1, is related to mammalian neuromedinU receptors. There is some evidence that PK1-R might also be activated by the hugin neuropeptide, which would add another regulatory layer (Schlegel, 2016).

    The concept of multiple messenger molecules within a single neuron is well established and appears to be widespread among many organisms and neuron types. For example, cholinergic transmission plays an important role in mediating the effect of Neuromedin U (NMU) in mammals. This has been demonstrated in the context of anxiety but not yet for feeding behavior. There are, however, only few examples of simultaneous employment of neuromodulation and fast synaptic transmission in which specific targets of both messengers have been investigated at single-cell level. In many cases, targets and effects of classic and peptide co-transmitters seem to diverge. In contrast, AgRP neurons in the mammalian hypothalamus employ neuropeptide Y, the eponymous agouty-related protein (AgRP) and the small molecule transmitter GABA to target pro-opiomelanocortin (POMC) neurons in order to control energy homeostasis. Also, reminiscent of the current observations is the situation in the frog sympathetic ganglia, where preganglionic neurons use both ACh and a neuropeptide to target so-called C cells but only the neuropeptide additionally targets B cells. In both targets, the neuropeptide elicits late, slow excitatory postsynaptic potentials (EPSPs). It is conceivable that hugin-producing neurons act in a similar manner by exerting a slow, lasting neuromodulatory effect on all mNSCs and a fast, transient effect exclusively on synaptically connected mNSCs. Alternatively, the hugin neuropeptide could facilitate the postsynaptic effect of acetylcholine. Such is the case in Aplysia where a command-like neuron for feeding employs acetylcholine and two neuropeptides, feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP2). Both peptides work cooperatively on a postsynaptically connected motor neuron to enhance EPSPs in response to cholinergic transmission (Schlegel, 2016).

    In addition to the different timescales that neuropeptides and small molecule transmitters operate on, they can also be employed under different circumstances. It is commonly thought that low-frequency neuronal activity is sufficient to trigger fast transmission using small molecule transmitters, whereas slow transmission employing neuropeptides requires higher frequency activity. Hugin-producing neurons could employ peptidergic transmission only as a result of strong excitatory (e.g. sensory) input. There are, however, cases in which base activity of neurons is already sufficient for graded neuropeptide release: Aplysia ARC motor neurons employ ACh as well as neuropeptides and ACh is generally released at lower firing rates than the neuropeptide. This allows the motor neuron to function as purely cholinergic when firing slowly and as cholinergic/peptidergic when firing rapidly. However, peptide release already occurs at the lower end of the physiological activity of those neurons. It remains to be seen how synaptic and peptidergic transmission in hugin neurons relate to each other (Schlegel, 2016).

    The present study is one of very few detailed descriptions of differential targets of co-transmission and the first of its kind in Drosophila. These finding should provide a basis for elucidating some of the intriguing modes of action of peptidergic neurons (Schlegel, 2016).

    The mammalian homolog of hugin, neuromedinU (NMU), is found in the CNS as well as in the gastrointestinal tract. Its two receptors, NMUR1 and NMUR2, show differential expression. NMUR2 is abundant in the brain and the spinal cord, whereas NMUR1 is expressed in peripheral tissues, in particular in the gastrointestinal tract. Both receptors mediate different effects of NMU. The peripheral NMUR1 is expressed in pancreatic islet β cells in humans and allows NMU to potently suppress glucose-induced insulin secretion. The same study also showed that Limostatin (Lst) is a functional homolog of this peripheral NMU in Drosophila: Lst is expressed by glucose-sensing, gut-associated endocrine cells and suppresses the secretion of insulin-like peptides. The second, centrally expressed NMU receptor, NMUR2, is necessary for the effect of NMU on food intake and physical activity. In this context, NMU is well established as a factor in regulation of the hypothalamo-pituitary axis and has a range of effects in the hypothalamus, the most important being the release of corticotropin-releasing hormone (CRH). This study shows that a subset of hugin-producing neurons targets the pars intercerebralis, the Drosophila homolog of the hypothalamus, in a similar fashion: neuroendocrine target cells in the pars intercerebralis produce a range of peptides, including diuretic hormone 44 which belongs to the insect CRH-like peptide family. Given these similarities, it is proposed that hugin is homologous to central NMU just as Lst is a homologous to peripheral NMU. Demonstration that central NMU and hugin circuits share similar features beyond targeting neuroendocrine centers, e.g. the integration of chemosensory inputs, will require further studies on NMU regulation and connectivity (Schlegel, 2016).

    Previous work on vertebrate and invertebrate neuroendocrine centers suggests that they evolved from a simple brain consisting of cells with dual sensory/neurosecretory properties, which later diversified into optimized single-function cells. There is evidence that despite the increase in neuronal specialization and complexity, connections between sensory and endocrine centers have been conserved throughout evolution. It is proposed that the connection between endocrine and chemosensory centers provided by hugin neurons represents such a conserved circuit that controls basic functions like feeding, locomotion, energy homeostasis and sex (Schlegel, 2016).

    Indisputably, the NMU system in mammals is much more complex as NMU is found more widespread within the CNS and almost certainly involves a larger number of different neuron types. This complexity, however, only underlines the use of numerically smaller nervous systems such as Drosophila's to generate a foundation to build upon. Moreover, NMU/NMU-like systems may have similar functions not just in mammals and Drosophila but also other vertebrates such as fish and other invertebrates such as C. elegans. In summary, these findings should encourage research in other organisms, such as the involvement of NMU and NMU homologs in relaying chemosensory information onto endocrine systems, and more ambitiously, to elucidate their connectomes in order to allow comparative analyses of the underlying network architecture (Schlegel, 2016).

    GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster

    Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. This study tested this hypothesis by examining GABA receptors and neurons that promote consumption. Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation (Cheung, 2017).

    The dissection of neural circuits that underlie consumption remains an important challenge toward understanding the regulation of feeding behavior. This study identifies neurons that regulate the consumption of non-appetitive and appetitive substances, and depend on the expression of RDL receptor for proper regulation of consumption. These RDL receptor-expressing neurons are able to orchestrate consumption regardless of taste quality, as knockdown of Rdl expression within these neurons not only causes overconsumption of sugar, bitter, and water substances, but tasteless substances as well. Acute activation of these neurons also caused overconsumption of sweet, bitter and water substances, whereas blocking neurotransmission of these neurons results in decreased sucrose consumption in starved flies. These studies reveal a subset of neurons that play a critical role in promoting consumption (Cheung, 2017).

    Previous studies have identified two different classes of interneurons that trigger sucrose consumption. FDG neurons are located in the SEZ and respond to sugar stimulation on the proboscis and the cholinergic IN1 neurons respond to sugar stimulation of the internal mouthparts. These two classes of neurons respond selectively to sucrose, suggesting that there is a pathway selective for regulating sucrose consumption. Similarly, ectopic activation of these neurons increased consumption of sucrose but not water or bitter. These studies indicate that consumption of sucrose is regulated independently of consumption of water or bitter and argue for distinct circuits mediating consumption for each class of tastant. The RDL-expressing neurons differ from previously identified consumption neurons because either knockdown of Rdl or optogenetic activation of these neurons elicited consumption not only of appetitive substances, but also of non-appetitive substances. One model suggested by these studies that bears testing is that there may be distinct circuits for sweet, water, and bitter food sources that all converge on the RDL-expressing neurons (Cheung, 2017).

    Knockdown of Rdl results in increased consumption of water, sucrose and bitter substances. These RDL neurons may be inhibited by GABAergic neurons such as DSOG1. Previous studies indicate that DSOG1 neurons act as a tonic inhibitor of consumption. Flies with silenced DSOG1 neurons overconsume all taste substances independent of taste quality and nutritional state, very similar to the phenotype observed when activating the RDL neurons in this study. An attractive model is that GABA release from DSOG1 inhibits the RDL neurons, restricting consumption. Indeed, 'studies show that RDL neuronal silencing is able to suppress the DSOG1-silencing phenotype. Although the data are consistent with the model that DSOG1 acts on the RDL neurons, it remains possible that the RDL neurons and DSOG1 influence parallel pathways. Further characterization of the RDL neurons that promote consumption and the DSOG1 neurons that inhibit consumption will enable distinguishing of these models (Cheung, 2017).

    This study demonstrates that RDL function in a subset of neurons is critical for the regulation of consumption of all substances, regardless of taste modality. Further studies characterizing these neurons and their interactions with the different neurons that regulate feeding will provide insight into the temporal dynamics and plasticity in feeding decisions (Cheung, 2017).

    A receptor and neuron that activate a circuit limiting sucrose consumption

    The neural control of sugar consumption is critical for normal metabolism. In contrast to sugar-sensing taste neurons that promote consumption, this study identified a taste neuron that limits sucrose consumption in Drosophila. Silencing of the neuron increases sucrose feeding; optogenetic activation decreases it. The feeding inhibition depends on the IR60b receptor, as shown by behavioral analysis and Ca2+ imaging of an IR60b mutant. The IR60b phenotype shows a high degree of chemical specificity when tested with a broad panel of tastants. An automated analysis of feeding behavior in freely moving flies shows that IR60b limits the duration of individual feeding bouts. This receptor and neuron provide the molecular and cellular underpinnings of a new element in the circuit logic of feeding regulation. A dynamic model is proposed in which sucrose acts via IR60b to activate a circuit that inhibits feeding and prevents overconsumption (Joseph, 2017).

    A fat-derived metabolite regulates a peptidergic feeding circuit in Drosophila

    This study shows that the enzymatic cofactor tetrahydrobiopterin (BH4) inhibits feeding in Drosophila. BH4 biosynthesis requires the sequential action of the conserved enzymes Punch, Purple, and Sepiapterin Reductase (Sptr). Although increased feeding is observed upon loss of Punch and Purple in the adult fat body, loss of Sptr must occur in the brain. Sptr expression is required in four adult neurons that express neuropeptide F (NPF), the fly homologue of the vertebrate appetite regulator neuropeptide Y (NPY). As expected, feeding flies BH4 rescues the loss of Punch and Purple in the fat body and the loss of Sptr in NPF neurons. Mechanistically, it was found BH4 deficiency reduces NPF staining, likely by promoting its release, while excess BH4 increases NPF accumulation without altering its expression. This study thus shows that, because of its physically distributed biosynthesis, BH4 acts as a fat-derived signal that induces satiety by inhibiting the activity of the NPF neurons (Kim, 2017).

    Commensal bacteria and essential amino acids control food choice behavior and reproduction

    Choosing the right nutrients to consume is essential to health and wellbeing across species. However, the factors that influence these decisions are poorly understood. This is particularly true for dietary proteins, which are important determinants of lifespan and reproduction. This study shows that in Drosophila melanogaster, essential amino acids (eAAs) and the concerted action of the commensal bacteria Acetobacter pomorum and Lactobacilli are critical modulators of food choice. Using a chemically defined diet, it was shown that the absence of any single eAA from the diet is sufficient to elicit specific appetites for amino acid (AA)-rich food. Furthermore, commensal bacteria buffer the animal from the lack of dietary eAAs: both increased yeast appetite and decreased reproduction induced by eAA deprivation are rescued by the presence of commensals. Surprisingly, these effects do not seem to be due to changes in AA titers, suggesting that gut bacteria act through a different mechanism to change behavior and reproduction. Thus, eAAs and commensal bacteria are potent modulators of feeding decisions and reproductive output. This demonstrates how the interaction of specific nutrients with the microbiome can shape behavioral decisions and life history traits (Leitão-Gonçalves, 2017).

    Involvement of a Gr2a-expressing Drosophila pharyngeal gustatory receptor neuron in regulation of aversion to high-salt foods

    Regulation of feeding is essential for animal survival. The pharyngeal sense organs can act as a second checkpoint of food quality, due to their position between external taste organs such as the labellum which initially assess food quality, and the digestive tract. Growing evidence provides support that the pharyngeal sensory neurons regulate feeding, but much is still unknown. This study found that a pair of gustatory receptor neurons in the LSO, a Drosophila adult pharyngeal organ which expresses four gustatory receptors, is involved in feeding inhibition in response to high concentrations of sodium ions. RNAi experiments and mutant analysis showed that the gustatory receptor Gr2a is necessary for this process. This feeding preference determined by whether a food source is perceived as appetizing or not is influenced by nutritional conditions, such that when the animal is hungry, the need for energy dominates over how appealing the food source is. These results provide experimental evidence that factors involved in feeding function in a context-dependent manner (Kim, 2017).

    Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger

    Free-living animals must not only regulate the amount of food they consume but also choose which types of food to ingest. The shifting of food preference driven by nutrient-specific hunger can be essential for survival, yet little is known about the underlying mechanisms. This study identified a dopamine circuit that encodes protein-specific hunger in Drosophila. The activity of these neurons increased after substantial protein deprivation. Activation of this circuit simultaneously promoted protein intake and restricted sugar consumption, via signaling to distinct downstream neurons. Protein starvation triggered branch-specific plastic changes in these dopaminergic neurons, thus enabling sustained protein consumption. These studies reveal a crucial circuit mechanism by which animals adjust their dietary strategy to maintain protein homeostasis (Liu, 2017).

    Pharyngeal stimulation with sugar triggers local searching behavior in Drosophila

    Foraging behavior is essential for all organisms to find food containing nutritional chemicals. A hungry fly of Drosophila melanogaster performs local searching behavior after drinking a small amount of sugar solution. Using video tracking this study examined how the searching behavior is regulated in D. melanogaster. A small amount of highly concentrated sugar solution was found to induce a long-lasting searching behavior. After the intake of sugar solution, a fly moved around in circles and repeatedly returned to the position where the sugar droplet had been placed. The non-nutritious sugar, D-arabinose, but not the non-sweet nutritious sugar, D-sorbitol, was effective in inducing the behavior, indicating that sweet sensation is essential. Furthermore, pox-neuro mutant flies with no external taste bristles showed local searching behavior, suggesting the involvement of the pharyngeal taste organ. Experimental activation of pharyngeal sugar-sensitive gustatory receptor neurons by capsaicin using the Gal4/UAS system induced local searching behavior. In contrast, inhibition of pharyngeal sugar-responsive gustatory receptor neurons abolished the searching behavior. Together these results indicate that in Drosophila the pharyngeal taste-receptor neurons trigger searching behavior immediately after ingestion (Murata, 2017).

    Idiothetic path Integration in the fruit fly Drosophila melanogaster

    After discovering a small drop of food, hungry flies exhibit a peculiar behavior in which they repeatedly stray from, but then return to, the newly discovered resource. To study this behavior in more detail, hungry Drosophila were tracked as they explored a large arena, focusing on the question of how flies remain near the food. To determine whether flies use external stimuli, visual, olfactory, and pheromonal cues were individually eliminated. In all cases, flies still exhibited a centralized search behavior, suggesting that none of these cues are absolutely required for navigation back to the food. To simultaneously eliminate visual and olfactory cues associated with the position of the food, an apparatus was constructed in which the food could be rapidly translated from the center of the arena. Flies continued to search around the original location, even after the food was moved to a new position. A random search model based on measured locomotor statistics could not reproduce the centered nature of the animal's trajectory. It is concluded that this behavior is best explained by a form of path integration in which the flies use idiothetic cues to search near the location of the food. It is argued that the use of path integration to perform a centered local search is not a specialization of Drosophila but rather represents an ancient behavioral mode that is homologous to the more elaborate foraging strategies of central place foragers such as ants (Kim, I. S., 2017).

    Species-specific modulation of food-search behavior by respiration and chemosensation in Drosophila larvae

    Animals explore their environment to encounter suitable food resources. Despite its vital importance, this behavior puts individuals at risk by consuming limited internal energy during locomotion. A novel assay has been developed to investigate how food-search behavior is organized in Drosophila melanogaster larvae dwelling in hydrogels mimicking their natural habitat. Three main behavioral modes are defined: resting at the gel's surface, digging while feeding near the surface, and apneic dives. In unstimulated conditions, larvae spend most of their time digging. By contrast, deep and long exploratory dives are promoted by olfactory stimulations. Hypoxia and chemical repellents impair diving. Remarkable differences are reported in the dig-and-dive behavior of D. melanogaster and the fruit-pest D. suzukii. The present paradigm offers an opportunity to study how sensory and physiological cues are integrated to balance the limitations of dwelling in imperfect environmental conditions and the risks associated with searching for potentially more favorable conditions (Kim, D., 2017).

    SIFamide translates hunger signals into appetitive and feeding behavior in Drosophila

    Animal behavior is, on the one hand, controlled by neuronal circuits that integrate external sensory stimuli and induce appropriate motor responses. On the other hand, stimulus-evoked or internally generated behavior can be influenced by motivational conditions, e.g., the metabolic state. Motivational states are determined by physiological parameters whose homeostatic imbalances are signaled to and processed within the brain, often mediated by modulatory peptides. This study investigate the regulation of appetitive and feeding behavior in the fruit fly, Drosophila melanogaster. Four neurons in the fly brain that release SIFamide were found to be integral elements of a complex neuropeptide network that regulates feeding. SIFamidergic cells integrate feeding stimulating (orexigenic) and feeding suppressant (anorexigenic) signals to appropriately sensitize sensory circuits, promote appetitive behavior, and enhance food intake. This study advances the cellular dissection of evolutionarily conserved signaling pathways that convert peripheral metabolic signals into feeding-related behavior (Martelli, 2017).

    Animals have interlaced neuronal and endocrine systems to control feeding behavior by integrating internal information about metabolic needs and external stimuli signaling the availability and quality of nutrition. In mammals, various internal sensors monitor the metabolic state and convey endocrine and neuronal signals to peripheral organs and the brain, e.g., through the release of peptides, such as leptin, ghrelin, insulin, and peptide YY, or through the neuronal activity of the sensory vagus nerve afferents. The hypothalamus (HT) represents a main integrator of these signals and contains neuronal circuits regulating energy homeostasis. Antagonistically acting populations of neurons in the arcuate nucleus that express neuropeptide Y (NPY), agouti-related peptide (AgRP), peptides derived from the precursors pro-opiomelanocortin (POMC), or cocaine- and amphetamine-regulated transcript (CART), respectively, integrate these peripheral signals. Activating NPY/AgRP-releasing and orexin-releasing neurons, or injection of these peptides, enhances food intake, whereas activating POMC- and CART-expressing neurons or injection of these peptides decreases it. How exactly these peptides modulate neuronal circuits that control feeding-related behavior remains unclear (Martelli, 2017).

    The brain of the fruit fly, Drosophila melanogaster, is much simpler in terms of cell numbers when compared to the mammalian brain. Its often individually identifiable neurons can be genetically targeted and manipulated or monitored using DNA-encoded Ca2+ sensors. Feeding-related behavior ranging from odor-guided foraging to food uptake has been exceedingly well described in Drosophila and other flies. Neural circuits controlling distinct aspects of feeding, e.g., the detection of gustatory and olfactory food stimuli, internal sensing of hemolymph sugar concentration, motor control of proboscis extension, food intake, and feeding-induced suppression of alternative behaviors like locomotion, have been characterized. Also in flies, peptidergic neurons modulate feeding behavior. The release of short neuropeptide F (sNPF) increases appetitive odor-guided behavior and food uptake. Conversely, drosulfakinin, a cholecystokinin homolog, allatostatin A (AstA), and myosin inhibitory peptide (MIP) reduce food intake. However, a function for the neuropeptide SIFamide in feeding-related behavior remains unclear. The SIFamide amino acid sequence is largely conserved across the arthropod lineage and has been implicated in behavior and sleep in Drosophila, aggression in a freshwater prawn, as well as in various feeding-related physiological processes, e.g., the modulation of the stomatogastric ganglion in lobsters or the control of salivary glands in blood-sucking ticks. The SIFamide receptor (SIFaR) is a homolog of the vertebrate gonadotropin inhibitory hormone receptor (GnIHR), although their respective ligands, SIFamide and GnIH, are not sequence related. GnIHR regulates food intake and reproductive behavior in opposite directions, thereby promoting feeding behavior over alternative behavioral tasks in periods of metabolic needs. However, it remains unclear whether the functions of the SIFamide- and GnIH-signaling pathways, respectively, are conserved across phyla (Martelli, 2017).

    This study used Drosophila to study the role of SIFamide in feeding behavior. Thermogenetic activation of SIFamidergic neurons was shown to enhance appetitive behavior evoked by gustatory and olfactory stimuli, as well as food intake. Second, it was shown that release of SIFamide sensitizes olfactory signaling in the antennal lobe (AL). Third, it was demonstrated that orexigenic as well as anorexigenic peptidergic neurons interact anatomically and functionally with SIFamidergic cells in the brain. These findings together identify SIFamide neurons as an interface between intrinsic metabolic signals and sensory neuronal circuits mediating appetitive behavior and food intake (Martelli, 2017).

    A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste

    Fat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. The highly palatable taste of free fatty acids (FAs), one of the building blocks of fat, promotes food consumption, activates reward circuitry. A broad population of sugar-sensing taste neurons expressing Gustatory Receptor 64f (Gr64f) is required for reflexive feeding responses to both FAs and sugars. This study reports a genetic silencing screen to identify specific populations of taste neurons that mediate fatty acid (FA) taste. Neurons identified by expression of Ionotropic Receptor 56d (IR56d) were found to be necessary and sufficient for reflexive feeding response to FAs. Functional imaging reveals that IR56d-expressing neurons are responsive to short- and medium-chain FAs. Silencing IR56d neurons selectively abolishes FA taste, and their activation is sufficient to drive feeding responses. Analysis of co-expression with Gr64f identifies two subpopulations of IR56d-expressing neurons. While physiological imaging reveals that both populations are responsive to FAs, IR56d/Gr64f neurons are activated by medium-chain FAs and are sufficient for reflexive feeding response to FAs. Moreover, flies can discriminate between sugar and FAs in an aversive taste memory assay, indicating that FA taste is a unique modality in Drosophila. Taken together, these findings localize FA taste within the Drosophila gustatory center and provide an opportunity to investigate discrimination between different categories of appetitive tastants (Tauber, 2017).

    Sweet-sensing neurons in Drosophila have been broadly classified as those responding to sugars and other attractive tastants such as glycerol and amino acids. The findings presented in this study further localize the reflexive feeding response to hexanoic and octanoic acids, both medium-chain FAs, to a small population of FA-responsive taste neurons that partially overlap with sweet-sensing neurons. Previous work has shown that genetic silencing of most sweet-sensing neurons using Gr64f-GAL4 abolished FA response, suggesting that these neurons detect sugars and FAs. In flies, some subpopulations of Gr64f neurons are selectively sensitive to certain tastants including a Gr64e population that is sensitive to glycerol and a Gr5a subset that is sensitive to trehalose. To localize the Gr64f neurons responsible for FA taste, a targeted screen was conducted and neurons were silenced that are believed to overlap with Gr64f neurons, which led to a study of the IR56d population of neurons. Silencing IR56d neurons appears to selectively disrupt HxA response without affecting response to sucrose, supporting the notion that independent mechanisms within the Gr64f population mediate responses to sugars and FAs (Tauber, 2017).

    It is possible that FA-sensitive neurons are broadly tuned to FAs or selectively respond to distinct classes of FAs. Ca2+ imaging experiments indicate that IR56d neurons are responsive to medium-chain HxA (C6, saturated) and octanoic acid (C8, saturated) in both anterior and posterior regions if the SEZ, and to short-chain pentanoic acid (C5, saturated), but only in the anterior projections. No IR56d neurons were found that were responsive to long-chain oleic acid (C18, mono-unsaturated). These findings are supported by behavioral data revealing that flies exhibit PER in response to pentanoic acid, HxA, and octanoic acid, but not oleic acid. Therefore, it seems likely that flies are strongly responsive to short/medium-chain FAs, but are less responsive to long-chain and/or unsaturated FAs. The finding that PER elicited by pentanoic acid occurs even when Ir56d neurons are genetically silenced suggests independent populations of taste neurons drive PER in response to short-chain and medium-chain FAs. Further, IR56d neurons may be activated by long-chain FAs that were not tested, and these could modulate feeding response and induce PER. Nevertheless, the findings presented in this study reveal specificity for medium-chain FAs within a defined population of taste neurons (Tauber, 2017).

    Many of the neurons identified by IR56d expression express multiple taste receptors including IR56d, Gr64f and Gr5a. These neurons likely express many additional candidate taste receptors, and future studies are needed to identify the receptor(s) that are responsive to FAs. IRs are related to ionotropic Glutamate receptors and respond to diverse tastants and odorants, making them excellent candidates for detecting FAs. While IR56d remains an excellent candidate, it will be necessary to examine potential IR co-receptors that may be critical for IR-dependent sensation. For example, IR25a is relatively broadly expressed and likely functions as a co-receptor for numerous IR-dependent sensory processes including temperature sensing and hygrosensation. It is possible that multiple IRs are required for FA taste, with some acting as co-receptors and others detecting specific FAs. While future work is required to identify the molecular bases for FA taste, the identification of FA sensitivity in IR56d neurons provides a system to interrogate the cellular mechanisms of FA taste (Tauber, 2017).

    The PER response induced by two different medium-chain FAs, hexanoic and octanoic acids, suggests they may be part of Drosophila melanogaster diet. Typical dietary fats, including many plant based oils, such as coconut oil, are rich in longer-chain FAs including palmitic acid, oleic acid and linoleic acid. However, medium-chain FAs are present in fermenting fruits such as guava and also in pollen. Moreover, the medium-chain FAs (mostly C6-C10) are excreted by yeast during fermentation, possibly helping with finding yeast-rich feeding substrates, raising the possibility that flies have developed a response to FAs in order to locate suitable fermented food sources. Further, previous work has shown that a diet of HxA alone is sufficient for survival. Therefore, it is possible that FA attraction evolved to promote consumption of calorically rich fermenting fruits consumed by Drosophila (Tauber, 2017).

    The use of sucrose and hexanoic acid (HxA) in an aversive taste memory paradigm reveals flies can discriminate between these attractive tastants. Sugars induce broad activation of Gr64f neurons, while the activation induced by HxA appears more restricted, and therefore it is possible that differences in activation allow for differentiation. Alternatively, it was found that HxA also activates anterior-projecting IR56d neurons that emanate from the taste pegs and do not co-express Gr64f, raising the possibility that differential response of these neurons to sucrose and FAs allows discrimination. Considering the different biochemical pathways involved in sugar and FA sensing, their identity may also be coded by unique temporal and spatial dynamics of sensory neuron activation. Differences in activation are suggested to provide a mechanism for olfactory discrimination within defined neural populations, and it is possible that similar mechanisms are utilized for attractive tastants. In Drosophila, attractive tastants have been found to induce a wide range of excitatory responses ranging from acute to prolonged firing, providing a potential mechanism for discrimination. While the sensillar response to FAs has not been reported, the differences in Ca2+ response to sugar or HxA presentation within the SEZ suggest differences in temporal activation (Tauber, 2017).

    The findings of this study reveal the population of IR56d neurons that innervate the anterior SEZ, which emanate from the taste pegs, are dispensable for PER in response to FAs. However, it is possible these neurons are still involved in discrimination between FAs and sugars. These neurons are not responsive to sucrose, therefore distinct anatomical activation may account for the gustatory discrimination between attractive substances. The taste pegs have previously been implicated in sensing non-sugar attractive tastants including polyamines and carbonation, raising the possibility that these neurons are responsive to multiple taste modalities (Hassain 2016; Fischler, 2007). Selectively silencing the IR56d taste peg neurons and measuring discrimination between FAs and sugars may determine whether distinct classes of IR56d neurons mediate taste feeding response and taste discrimination (Tauber, 2017).

    This study found that flies can discriminate between sugars and FAs, but it is not known whether they can discriminate qualitatively between different classes of FAs. A previous study examining discrimination between different sugars found that flies are unable to discriminate based on quality, but could discriminate based on perceived palatability. This study found that pentanoic acid elicits a PER response that is independent of IR56d neurons. The findings, coupled with evidence that distinct populations of neurons respond to FAs from different classes, raise the possibility that flies may discriminate between FAs based on the identity of neurons activated by each FA, or classes of FAs (Tauber, 2017).

    We previously reported that PLC signaling in sweet-sensing Gr64f neurons is required for FA taste (Masek, 201). Flies with mutation or knockdown of the PLC-β ortholog norpA do not respond to HxA or octanoic acid but respond normally to sugars, revealing independent intercellular signaling mechanisms likely underlie response to FAs and sugars. This study found that knockdown of norpA in IR56d neurons abolishes FA taste without disrupting the taste of sucrose. These findings phenocopy norpA mutants and broad knockdown of norpA in Gr64f neurons, fortifying the notion that PLC signaling is selectively required for FA taste. Testing the response of norpA deficient flies to FAs that are sensed independently of IR56d will inform whether PLC is more generally required for FA taste, or is only specific to medium-chain FAs detected by IR56d neurons (Tauber, 2017).

    While taste coding within the SEZ has been extensively investigated, less is known about the higher order circuits governing taste. Sweet-sensing neurons connect to the antennal mechanosensory and motor center (AMMC) and downstream PAM dopamine neurons that are activated by sugar. In addition, a separate population of dopamine neurons, the PPL1 cluster, is required for olfactory appetitive memory and taste aversive conditioning. To date, higher order neurons responsive to FA taste have not been identified. It is possible that sugar and FA taste signal through shared higher order dopamine neurons or, alternatively, each taste modality may activate distinct populations of higher order neurons that convey valence to the mushroom bodies, the memory and sensory integration center in insects (Tauber, 2017).

    While both sugars and FAs activate shared neurons, the ability to discriminate between these tastants provides a model for investigating sensory discrimination. There is growing evidence of multimodal coding within Drosophila sensory neurons, and in downstream targets. Flies harboring only a single functional type of olfactory receptor neurons are able to discriminate between odorants, presumably due to differences in temporal activation between the odorants. Further, in the larval taste system, a single gustatory receptor neuron is responsive to both attractive and aversive compounds, and mediates the integration of these competing stimuli. In addition to integration of distinct cues by the sensory system, the Drosophila mushroom bodies, and courtship circuitry integrate complex sensory cues within the brain. Future studies on how the central brain processes sugar and FA taste will help elucidate mechanisms of discrimination between sugars and FAs (Tauber, 2017).

    Despite the role of FAs in promoting feeding, surprisingly little is known about how FAs promote taste in any model system. Fats contain many sensory cues and separating the taste of fat per se, from other cues such as texture, viscosity and smell is a particular challenge in mammals. A number of studies have implicated the lipid binding protein CD36 as contributing to FA taste. CD36 is expressed in gustatory oral tissue, and appears to be selectively involved in FA taste. CD36 knockout animals show no preference for FAs but retain preference for sugars. The Drosophila homolog of CD36, Sensory neuron membrane protein 1, is expressed in the olfactory system and required for sensation of the pheromone cis-vaccenyl acetate, and therefore is unlikely to mediate FA taste. Additionally, a number of FA-binding GPCRs are expressed in taste cells, but their role in FA taste has not been identified. The ability to selectively manipulate and ablate defined classes of sensory neurons in Drosophila allows for the disambiguation of taste from other sensory processes. Identifying FA receptors and neural circuitry mediating FA taste and discrimination will provide a framework for investigating similar processes in mammalian systems (Tauber, 2017).

    Taken together, this study provides insight into the coding of FAs within the fly gustatory system. The results reveal a population of sweet-sensing neurons that are tuned for medium-chain FAs, but not short- or long-chain FAs. Further, the finding that flies are capable of discriminating between FAs and sugars suggests coding differences, either spatial or temporal neuronal activation, and provides a mechanism to distinguish between tastants of the same valence. Understanding how FAs are coded within the fly brain provides a model for understanding taste in more complex systems and will offer insight into generalizable mechanisms for taste discrimination (Tauber, 2017).

    Molecular basis of fatty acid taste in Drosophila

    Behavioral studies have established that Drosophila appetitive taste responses towards fatty acids are mediated by sweet sensing Gustatory Receptor Neurons (GRNs). This study shows that sweet GRN activation requires the function of the Ionotropic Receptor genes IR25a, IR76b and IR56d. The former two IR genes are expressed in several neurons per sensillum, while IR56d expression is restricted to sweet GRNs. Importantly, loss of appetitive behavioral responses to fatty acids in IR25a and IR76b mutant flies can be completely rescued by expression of respective transgenes in sweet GRNs. Interestingly, appetitive behavioral responses of wild type flies to hexanoic acid reach a plateau at ~1%, but decrease with higher concentration, a property mediated through IR25a/IR76b independent activation of bitter GRNs. With previous report on sour taste, these studies suggest that IR-based receptors mediate different taste qualities through cell-type specific IR subunits (Ahn, 2017).

    IR genes have emerged as a second large gene family encoding chemoreceptors in insects. In the Drosophila olfactory system, IRs function as multimeric receptors in coeloconic olfactory sensory neurons (OSN) and are thought to sense volatile carboxylic acids, amines and aldehydes. Expression analyses have shown that each coeloconic OSN expresses up to four IR genes, including high levels of either IR8a or IR25a. IR25a and IR8a are distinct from other IRs in that they are more conserved to each other and iGluRs, and they share a long amino terminal domain absent in all other IRs. These observations, along with functional analyses of basiconic olfactory neurons that express combinations of IR genes, led to a model in which IR based olfactory receptors are tetrameric complexes thought to consist of up to three different subunits that contain at least one core unit (IR8a or IR25a) and two additional IRs that determine ligand binding specificity. The findings presented in this paper expand this concept to taste receptors that sense fatty acids through the sweet GRNs found in tarsal taste sensilla (Ahn, 2017).

    This analysis extends the multimodal role of IR25a and IR76b to the taste systems. Consistent with gene expression arrays, this paper shows that up to three GRNs, including many sweet and bitter GRNs, co-express IR25a and IR76b. Functional studies have established a novel role for these two IR proteins in fatty acid taste, which revealed that these two subunits are not only critically important to elicit Proboscis Extension Reflex (PER) responses in flies when challenged with fatty acids, but are also necessary for fatty acid induced Ca2+ increases in tarsal sweet GRNs. Based on these findings and with consideration of their established role in other sensory systems, it is proposed that IR25a and IR76b play central roles in sweet GRNs in a multimeric receptor complex for initiating appetitive taste behavior to these chemicals. Intriguingly, both genes are also co-expressed in two other GRNs of most tarsal taste sensilla, strongly arguing for additional taste functions. While the subset of tarsal bitter GRNs activated by hexanoic acid does not require either gene, the third GRN (the sour GRN) is narrowly tuned to acids in an IR25a/IR76b dependent manner. These observations suggest that modality specific IRs are likely expressed in a cell-type specific fashion whereby they complement IR25a/IR76b to function as either a fatty acid or a sour taste receptor. Indeed, the screen identified IR56d, a gene that is expressed in sweet GRNs of tarsal taste sensilla, as a likely candidate encoding an IR subunit specific for a fatty acid taste receptor. It remains to be seen whether IR25a, IR76b and IR56d comprise all subunits that constitute this receptor or whether yet additional IRs are necessary to mediate responses to these chemicals (Ahn, 2017).

    The fact that different food chemicals can activate a single class of neurons raises the question how flies discriminate between sugars and fatty acids. First, the difference is noted in sensitivity of appetitive GRNs to sugars and fatty acids, respectively: The most responsive GRN for sugars is the one associated with the 5v sensilla, followed by that with the 5s and finally the 5b sensilla, while the responsiveness for fatty acids is the reverse (5b > 5s > 5v). Second, fatty acids induces weaker PER responses from stimulation of the labial palps as opposed to tarsi, while sugars induce equally strong PER responses from stimulation of either taste organ. Third, at least some fatty acids activate bitter GRNs, and hence, generate more complex activation patterns in the brain than sugars, which are not known to activate neurons other than sweet GRNs. These properties may provide a rationale for differential coding of these two classes of chemicals in the brain. Finally, sugars but not fatty acids are soluble in water, and hence, the specific solvents in which these chemicals are presented provides different textural quality, which was recently shown to play a role in taste perception (Ahn, 2017).

    NorpA, which encodes a phospholipase C (PLC), plays a critical role in sweet GRNs for appetitive feeding responses to fatty acids, but it is dispensable for behavioral responses to sugars. This study found that its absence also selectively abolishes Ca2+ responses to fatty acids, but not sugars, in sweet cells. NorpA is known for its role as downstream effector of G-protein coupled receptors in the fly's visual system, but interestingly it is also required for olfactory responses in neurons of the maxillary palps, which express ORs that are thought to function as ligand-gated ion channels. It is noted that fatty acid taste in mammals is in part mediated by two G-protein coupled receptors, GPR40 and GPR120, and that one of these (GPR120) was found to signal through a phospholipase C. Thus, future studies will be necessary to gain insights for how PLC mediates chemosensory responses through ORs and the phylogenetically unrelated IRs (Ahn, 2017).

    Multimeric IR based receptors were recently shown to be required in non-chemosensory processes. Specifically, Dorsal Organ Cool Cells (DOCCs) located in the larval brain, express and require the function of three IRs (IR21a IR25a and IR93a), thereby allowing larvae to avoid temperatures below ~20°C. Similarly, two sets of cells in the antennal sacculus of adult flies, requiring the functions of IR25a and IR93a and either IR40a or IR68a, were shown to mediate a fly's preferred humidity environment, which is generally in the dry range, but is also dependent on the fly's hydration state. Intriguingly, these non-chemosensory IR complexes share a common theme with the fatty acid and carboxylic acid taste receptors in that they all require a core unit (IR25a) and two additional IRs that mediate specificity for a particular stimulus type (i.e., temperature, humidity, fat, acid) (Ahn, 2017).

    An IR76b based sodium channel and an IR76b based amino acid receptor appear to lack an obligate core unit (IR25a or IR8a) found in olfactory receptors or fatty acid and carboxylic acid taste receptors. The IR76b sodium channel mediates salt responses in a heterologous systems independently of any other IRs, while a proposed multimeric IR76b containing receptor mediates amino acids taste in wild type and IR25a mutant flies (IR8a is not expressed in taste neurons). It will be interesting to elucidate the compositions of complete IR based amino acid and sour taste receptors, and (with regard of amino acid receptors) to identify the neurons that mediate this taste modality (Ahn, 2017).

    Molecular and cellular organization of taste neurons in adult Drosophila pharynx

    The Drosophila pharyngeal taste organs are poorly characterized despite their location at important sites for monitoring food quality. Functional analysis of pharyngeal neurons has been hindered by the paucity of molecular tools to manipulate them, as well as their relative inaccessibility for neurophysiological investigations. This study generated receptor-to-neuron maps of all three pharyngeal taste organs by performing a comprehensive chemoreceptor-GAL4/LexA expression analysis. The organization of pharyngeal neurons reveals similarities and distinctions in receptor repertoires and neuronal groupings compared to external taste neurons. The mapping results were validated by pinpointing a single pharyngeal neuron required for feeding avoidance of L-canavanine. Inducible activation of pharyngeal taste neurons reveals functional differences between external and internal taste neurons and functional subdivision within pharyngeal sweet neurons. These results provide roadmaps of pharyngeal taste organs in an insect model system for probing the role of these understudied neurons in controlling feeding behaviors (Chen, 2017).

    In Drosophila, taste neurons located in sensilla in several body regions sense and distinguish nutritive substances such as sugars, amino acids, and low salt, and potentially harmful ones such as high salt, acids, and a diverse variety of bitter compounds. Hair-like sensilla on the labellum, distal segments of the legs (tarsi), anterior wing margins, and ovipositor have access to chemicals in external substrates. Pit-like sensilla (taste pegs) on the oral surface have access only once the fly extends its proboscis and opens the labellar palps; similar sensilla in the pharynx have access only when food intake is initiated. Based on its anatomical position, the pharynx is considered to act as a gatekeeper to control ingestion, promoting the intake of appetitive foods and blocking that of toxins (Chen, 2017).

    Three distinct internal taste organs are present in the adult fly pharynx: the labral sense organ (LSO), the ventral cibarial sense organ (VCSO), and dorsal cibarial sense organ (DCSO). The VCSO and DCSO are paired on opposite sides of the rostrum, whereas the LSO is located in the haustellum. The organization and neuronal composition of all three organs, based on both light and electron microscopy data, have been described in detail. Nine separate sensilla are present in the LSO, of which 1-6 are innervated by a single mechanosensory neuron each. The remaining three, named 7-9, are uniporous sensilla, a feature that ascribes chemosensory function to them. Sensillum 7 is the largest one, with eight chemosensory neurons. Sensilla 8 and 9 have two neurons each (one mechanosensory and one chemosensory). Although one study reported two sensilla in the VCSO, this and other studies have observed three sensilla in the VCSO, innervated by a total of eight chemosensory neurons. The DCSO has two sensilla, each containing three chemosensory neurons. Notwithstanding the availability of detailed anatomical descriptions of pharyngeal taste organs, little is known about their function. The internal location of these organs poses challenges for electrophysiological analysis of taste neurons located within them. Additionally, few molecular tools are currently described to manipulate the function of selected pharyngeal taste neurons (Chen, 2017).

    The expression and function of members of several chemosensory receptor gene families such as gustatory receptors (Grs), ionotropic receptors (Irs), Pickpocket (Ppk) channels, and transient receptor potential channels (Trps) have been found in external gustatory receptor neurons (GRNs) of the labellum and the tarsal segments. A number of Gr- and Ir-GAL4 drivers are also shown to label pharyngeal organs, but only a few, including Gr43a and members of sweet Gr clade, Gr2a, Ir60b, and TrpA1, have been mapped to specific taste neurons (Chen, 2017).

    This study generated receptor-to-neuron maps for three pharyngeal taste organs by a systematic expression analysis of chemoreceptor reporter lines that represent Gr, Ir, and Ppk receptor families. The maps reveal a large and diverse chemoreceptor repertoire in the pharynx. Some receptors are expressed in combinations that are predictive of neuronal sweet or bitter taste function based on analysis of external GRNs. By contrast, some pharyngeal taste neurons express receptor combinations that are distinct from any that have been reported in other organs, leaving open questions about their functional roles. This study validated he receptor-to-neuron maps derived from reporter gene expression by assessing roles of pharyngeal GRNs predicted to detect L-canavanine, a bitter tastant for which a complete receptor repertoire has been reported. Interestingly, a systematic activation analysis of different classes of pharyngeal taste neurons reveals functional differences between external and internal taste neurons for bitter avoidance and functional subdivision within pharyngeal sweet neurons for sweet acceptance. Together, this study provides a molecular map of pharyngeal taste organs, which will serve as a resource for future studies of the roles of pharyngeal taste neurons in food evaluation (Chen, 2017).

    Internal pharyngeal taste organs are the least explored taste organs, despite their obvious importance in insect feeding behaviors, which are crucial drivers for damaging crops and vectoring disease. The receptor-to-neuron maps of pharyngeal taste organs suggest a high degree of molecular complexity, with co-expression of different chemoreceptor family members in many pharyngeal GRNs. In particular, none of the pharyngeal GRNs were found to express Gr genes alone; rather, one or more Ir genes were always expressed in the same neurons. Gr and Ir genes are also co-expressed in some external sweet and bitter-sensing GRNs. Thus, both classes of receptors are likely to contribute to responses of Gr/Ir-expressing neurons in the LSO and VCSO, but whether they interact functionally or act independently remains to be determined. In the LSO, expression of sweet Grs and Ir76b overlaps in pharyngeal sweet GRNs, as observed in tarsi as well. In the pharynx, this study also found co-expression of ppk28 with Ir genes, which has not been described for external GRNs. These observations invite explorations of possible crosstalk, and its functional significance, between the two classes of receptors (Chen, 2017).

    Pharyngeal GRNs also exhibit distinctive functional groupings. All external bitter GRNs have always been found grouped with sweet GRNs in taste hairs. By contrast, canonical sweet and bitter GRNs appear to segregate in different sensilla in the LSO, which is most well characterized for this perspective. L8 and L9 may be functionally identical and house only one Gr66a-expressing bitter GRN each, whereas L7 contains two sweet GRNs (L7-1 and L7-2). Moreover, external hairs typically have two to four GRNs, each of which has a distinct functional profile. In the LSO duplications are found (L7-1 and L7-2 are identical, as are L7-4 and L7-5), although differences between these pairs of GRNs may emerge as additional chemoreceptors are mapped in the pharynx. Finally, it is difficult to ascribe putative functions to most pharyngeal GRNs based on existing knowledge of receptor function in external counterparts. The L7-3 Gr-expressing neuron, for example, does not express members of the sweet clade, but neither does it express any of the common bitter Grs (Gr32a, Gr66a, and Gr89a) that would corroborate its role as a bitter GRN. Similarly, with the exception of salt neurons that may express Ir76b alone, there are few known functions for GRNs that solely express Ir genes. One possibility is that some of these GRNs possess novel chemoreceptor family ligand interactions. For example, L7-7 is involved in sensing sucrose but limiting sugar ingestion, representing an Ir neuron that operates in a negative circuit module for sugar intake. In addition, another recent study suggests that TRPA1 expression in L8 and L9 of the LSO is involved in feeding avoidance to bacterial endotoxins lipopolysaccharides (LPS). Alternatively, some pharyngeal GRNs may evaluate characteristics other than palatability, such as temperature or viscosity. Ir25a, which is broadly expressed in all 24 pharyngeal GRNs, is required for cool sensing and thermosensing. It will be worth investigating whether one or more pharyngeal GRNs act to integrate information about temperature and chemical quality of food substrates (Chen, 2017).

    Expression analyses also hint at some functional subdivisions between pharyngeal taste organs. The LSO contains a smaller proportion of Gr-expressing neurons than the VCSO, which also expresses a larger number of Gr genes that are co-expressed with Gr66a. Thus, broader bitter taste function might be expected in the VCSO. By contrast, sweet taste function appears to be more dominant in the LSO; its sweet GRNs express more sweet Gr-GAL4 drivers than the ones in the VCSO, and their activation is sufficient to drive feeding preference. VCSO sweet GRNs fail to promote ingestion by themselves but may contribute to an increase in feeding preference when activated simultaneously with those in the LSO. Thus, there may be synergistic or hierarchical interactions between LSO and VCSO sweet taste circuits, with the latter coming into play only once the former is activated. The finding that Gr and Ir genes are expressed in the LSO and VCSO but only Ir genes in the DCSO is also striking and raises the possibility that the DCSO, which is present at the most internal location relative to the others, may serve a unique role in controlling ingestion (Chen, 2017).

    Based on its molecular signature, the V5 neuron was identified as an L-canavanine-sensing neuron in the pharynx. As predicted, feeding avoidance of L-canavanine is dependent on V5. It was thus unexpected that capsaicin-mediated activation of bitter pharyngeal GRNs, which include V5, did not induce strong feeding avoidance either in the absence or presence of sugar. Because the strength and pattern of pharyngeal neuronal activation by bitter tastants or capsaicin is unknown, it is possible that capsaicin response may be weaker than that of canonical bitter tastants. Alternatively, sweet and bitter inputs from internal and external neurons may be summed differently. It is known that activation of one or few external sweet neurons can lead to proboscis extension, for example, but a larger number of bitter neurons may need to be activated for avoidance (Chen, 2017).

    The afferents of pharyngeal GRNs target regions of the SEZ that are distinct from areas in which afferents from labellar and tarsal GRNs terminate. Interestingly, pharyngeal GRN projections between molecularly different classes of neurons, as well as between GRNs of the LSO and VCSO, are also distinct. Projections of sugar-sensing GRNs were found in separate ipsilateral regions, whereas those of neurons predicted to detect aversive tastants were found at the midline, suggesting the presence of contralateral termini. These observations may inform future functional studies of pharyngeal GRNs. L7-6 neurons, for example, would be predicted to sense aversive compounds based on the presence of their termini at the midline. Analysis of pharyngeal GRN projections also suggests distinct connectivity to higher order neuronal circuits. With the molecular tools described here, future investigations of pharyngeal GRNs and pharyngeal taste circuits will provide insight into how internal taste is integrated with external taste to control various aspects of feeding behavior (Chen, 2017).

    Drosophila mushroom bodies integrate hunger and satiety signals to control innate food-seeking behavior

    The fruit fly can evaluate its energy state and decide whether to pursue food-related cues. This study reveals that the mushroom body (MB) integrates hunger and satiety signals to control food-seeking behavior. Five pathways in the MB were found to be essential for hungry flies to locate and approach food. Blocking the MB-intrinsic Kenyon cells (KCs) and the MB output neurons (MBONs) in these pathways impairs food-seeking behavior. Starvation bi-directionally modulates MBON responses to a food odor, suggesting that hunger and satiety controls occur at the KC-to-MBON synapses. These controls are mediated by six types of dopaminergic neurons (DANs). By manipulating these DANs, it was possible to inhibit food-seeking behavior in hungry flies or promote food seeking in fed flies. Finally, this study showed that the DANs potentially receive multiple inputs of hunger and satiety signals. This work demonstrates an information-rich central circuit in the fly brain that controls hunger-driven food-seeking behavior (Tsao, 2018).

    Yeast quality in juvenile diet affects Drosophila melanogaster adult life traits

    .Diet quality is critical for animal development and survival. Fungi can provide nutrients that are essential to organisms that are unable to synthetize them, such as ergosterol in Drosophila melanogaster. Drosophila studies examining the influence of yeast quality in the diet have generally either provided the diet over the whole life span (larva to adult) or during the adult stage and have rarely focussed on the juvenile diet. This study tested the effect of yeast quality in the larval diet on pre-adult development and adult weight, survival, reproduction and food preference. The yeast Saccharomyces cerevisiae was added in three forms in three treatments-live, heated or dried-to food used as the juvenile diet or was not added (empty treatment). Adults resulting from the larvae raised on these four juvenile diets were all maintained on a similar standard laboratory food diet. The data indicate that yeast quality in the juvenile diet affects larva-to-pupa-but not pupa-to-adult-development. Importantly, adult survival, food preference, mating behaviour and cuticular pheromones strongly varied with the juvenile diet. Therefore, the variation of yeast quality in the pre-adult Drosophila diet affects key adult life traits involved in food search, reproduction and survival (Grangeteau, 2018).

    Host Preference and Olfaction in Drosophila mojavensis

    Many organisms live in complex environments that vary geographically in resource availability. This environmental heterogeneity can lead to changes within species in their phenotypic traits. For example, in many herbivorous insects, variation in host plant availability has been shown to influence insect host preference behavior. This behavior can be mediated in part through the insect olfactory system and the odor-evoked responses of olfactory receptor neurons (ORNs), which are in turn mediated by their corresponding odorant receptor genes. The desert dwelling fly Drosophila mojavensis is a model species for understanding the mechanisms underlying host preference in a heterogeneous environment. Depending on geographic region, one to multiple host plant species are available. Electrophysiological studies were conducted and variation was found in responses of ORNs to host plant volatiles both within and between 2 populations-particularly to the odorant 4-methylphenol. Flies from select localities within each population were found to lack a response to 4-methylphenol. Experiments then assessed the extent to which these electrophysiological differences were associated with difference in several odor-mediated behavioral responses. No association between the presence/absence of these odor-evoked responses and short range olfactory behavior or oviposition behavior was observed. However, differences in odor-induced feeding behavior in response to 4-methylphenol were found. Localities that exhibit an odor-evoked response to the odorant had increased feeding behavior in the presence of the odorant. This study sets the stage for future work examining the functional genetics underlying variation in odor perception (Crowley-Gall, 2018).

    Monitoring food preference in Drosophila by oligonucleotide tagging

    Drosophila melanogaster is a powerful model organism for dissecting the neurogenetic basis of appetitive and aversive behaviors. However, some methods used to assay food preference require or cause starvation. This can be problematic for fly ethanol research because it can be difficult to dissociate caloric preference for ethanol from pharmacological preference for the drug. BARCODE, a starvation-independent assay that uses trace levels of oligonucleotide tags was designed to differentially mark food types. In BARCODE, flies feed ad libitum, and relative food preference is monitored by qPCR of the oligonucleotides. Persistence of the ingested oligomers within the fly records the feeding history of the fly over several days. Using BARCODE, this study identified a sexually dimorphic preference for ethanol. Females are attracted to ethanol-laden foods, whereas males avoid consuming it. Furthermore, genetically feminizing male mushroom body lobes induces preference for ethanol. In addition, it was demonstrated that BARCODE can be used for multiplex diet measurements when animals are presented with more than two food choices (Park, 2018).

    A temperature-dependent switch in feeding preference improves Drosophila development and survival in the cold

    How cold-blooded animals acclimate to temperature and what determines the limits of their viable temperature range are not understood. This study shows that Drosophila alter their dietary preference from yeast to plants when temperatures drop below 15 degrees C and that the different lipids present in plants improve survival at low temperatures. Drosophila require dietary unsaturated fatty acids present in plants to adjust membrane fluidity and maintain motor coordination. Feeding on plants extends lifespan and survival for many months at temperatures consistent with overwintering in temperate climates. Thus, physiological alterations caused by a temperature-dependent dietary shift could help Drosophila survive seasonal temperature changes (Brankatschkm 2018).

    Two Drosophila Neuropeptide Y-like neurons define a reward module for transforming appetitive odor representations to motivation.

    Neuropeptides, many of which are conserved among vertebrate and invertebrate animals, are implicated in the regulation of motivational states that selectively facilitate goal-directed behaviors. After a brief presentation of appetitive odors, Drosophila larvae display an impulsive-like feeding activity in readily accessible palatable food. This innate appetitive response may require coordinated signaling activities of dopamine (DA) and neuropeptide F (NPF; a fly homolog of neuropeptide Y). This study provides anatomical and functional evidence, at single-cell resolution, that two NPF neurons define a reward module in the highest-order brain region for cognitive processing of food-related olfactory representations. First, laser lesioning of these NPF neurons abolished odor induction of appetitive arousal, while their genetic activation mimicked the behavioral effect of appetitive odors. Further, a circuit analysis shows that each of the two NPF neurons relays its signals to a subset of target neurons in the larval hindbrain-like region. Finally, the NPF neurons discriminatively responded to appetitive odor stimuli, and their odor responses were blocked by targeted lesioning of a pair of dopaminergic third-order olfactory neurons that appear to be presynaptic to the NPF neurons. Therefore, the two NPF neurons contribute to appetitive odor induction of impulsive-like feeding by selectively decoding DA-encoded ascending olfactory inputs and relaying NPF-encoded descending motivational outputs for behavioral execution (Pu, 2018).

    This study has taken a multifaceted approach to functionally dissect the role of the NPF system in appetitive odor-aroused feeding motivation of Drosophila larvae. The anatomical and functional evidence suggest that two DM-NPF neurons, one in each brain hemisphere, define two parallel neuronal pathways that function in a largely autonomous manner. In each pathway, the DM-NPF neuron defines the highest-order circuit module for food odor processing. In the lateral horn, the DM-NPF neuron receives ascending DA signals from four upstream DL2 neurons. Subsequently, it selectively converts such inputs to descending NPF-encoded motivational outputs, which are relayed to a subset of NPFR1 neurons in larval hindbrain-like region (SEZ) for organizing feeding-related peripheral activities. Through combined use of targeted laser microsurgery and dTrpA1-mediated neuronal activation, this study also showed that remote activation of two DM-NPF neurons in behaving fed larvae appear to be sufficient to mimic the appetizing effect of food odor stimulation. Therefore, these findings suggest that the DM-NPF neurons define a module of the highest order in a food reward circuit that prepares larvae for reward-driven feeding of palatable food (Pu, 2018).

    One of the key features of the appetitive odor-aroused feeding response by fed larvae is its requirement of optimal levels of odor stimulation; Odor stimuli that are either too strong or too weak are not effective. In heterozygous Dop1R1 fed larvae, the effective doses of odor vapors required to induce appetitive arousal were much higher, as evidenced by the right-shift in its dose-response curve. Further, fed larvae with a reduced Dop1R1 activity in NPF neurons also phenocopied heterozygous Dop1R1 fed larvae. This work has provided cellular evidence that DM-NPF neurons display excitatory responses only to odor stimuli at appetitive doses. Together, these findings point to the presence of a Dop1R1-mediated gating mechanism that tunes the NPF neuronal response to odor-evoked DA signals, and thereby selectively assigns appetitive significance to DA signals that are otherwise meaningless behaviorally (Pu, 2018).

    This study has provided several lines of evidence suggesting that in each brain hemisphere, the DM-NPF neuron receives ascending DA signals from an assembly of four dopaminergic DL2 neurons in the lateral horn. First, using an npf-lexA driver that predominantly labels the two DM-NPF neurons, it was found the dendrites of these neurons were highly enriched in the lateral horn, which is known to mediate innate olfactory behaviors. Second, the four DL2 neurons, which function as third-order olfactory neurons, were previously shown to project their axons exclusively to the lateral horn. Third, the split GFP assay also points to the presence of synaptic connections between the DM-NPF and the upstream DL2 neurons in the lateral horn. Finally, the functional imaging analysis shows that the DM-NPF neuron acts downstream from four DL2 neurons. When stimulated by a stream of appetitive odor vapor, the DL2 neurons responded more rapidly than the NPF neuron, and lesions in the DM-NPF neuron had no effect on the odor response of the DL2 neurons. In contrast, the NPF neuronal response to the same odor stimulus required the presence of the DL2 neurons. In combination, these findings have revealed a previously uncharacterized brain center where a DA/NPF-mediated circuit mechanism underlies cognitive processing of food odors for appetitive motivation (Pu, 2018).

    In summary, evidence is provided that the activity of a pair of NPY-like neurons defines a reward system in fly larval brain responsible for cognitive processing of food-related olfactory representations. However, when the two DM-NPF neurons were selectively lesioned, the rapid responses of DL2 neurons to a PA stimulus (within 2-5 seconds) remained intact. In a previous study, functional knockdown of the NPFR1 activity in the DL2 neurons attenuated their rapid excitation by a transient odor stimulus (e.g., a puff of PA vapor). Therefore, these findings have raised the possibility that the two DL-NPF neurons, which project their axons ipsilaterally within the brain lobe, may define a separate neural mechanism, and this NPF mechanism may set the basal level of NPF activity in un-stimulated larval brains to facilitate the sensitive detection of distant food sources by foraging larvae (Pu, 2018).

    Sugar promotes feeding in flies via the serine protease homolog scarface

    A balanced diet of macronutrients is critical for animal health. A lack of specific elements can have profound effects on behavior, reproduction, and lifespan. This study used Drosophila to understand how the brain responds to carbohydrate deprivation. Serine protease homologs (SPHs) were enriched among genes that are transcriptionally regulated in flies deprived of carbohydrates. Stimulation of neurons expressing one of these SPHs, Scarface (Scaf), or overexpression of scaf positively regulates feeding on nutritious sugars, whereas inhibition of these neurons or knockdown of scaf reduces feeding. This modulation of food intake occurs only in sated flies while hunger-induced feeding is unaffected. Furthermore, scaf expression correlates with the presence of sugar in the food. As Scaf and Scaf neurons promote feeding independent of the hunger state, and the levels of scaf are positively regulated by the presence of sugar, it is concluded that scaf mediates the hedonic control of feeding (Prasad, 2018).

    Recent studies have shown that nutrient balance is a major determinant of behavior. A study in orb-weaving spiders has shown that the nutrient balance of a predator can alter foraging behavior, while in Drosophila, intake of macronutrients (particularly carbohydrates) can influence male pre- and post-copulatory reproductive traits. Furthermore, the dietary yeast and sucrose content of the diet has sex-dependent effects on the sleep architecture of the fly. This study has determined on a systems level the transcriptional response of the brain to deprivation of a macronutrient, namely carbohydrates. The data demonstrate that the brain mounts a distinct transcriptional response under these conditions. This distinct response can start to explain the changes in behavior observed upon alterations of individual macronutrients in the diet. Thr data also provide a repertoire of genes that change expression upon carbohydrate deprivation. This valuable resource can be mined to understand and link molecular mechanisms with specific responses of the brain to carbohydrate deficiency (Prasad, 2018).

    The findings suggest that SPs and SPHs play an important role in modulating fly behavior when the fly is deprived of sugar. The SPH scaf positively regulates feeding, depending on the presence of sugar in the food. However, the mechanism of action of scaf is not clear. It is possible that Scaf is cleaved into smaller peptides that play a role in neuronal communication or that Scaf competes with an active SP for specific substrates. In embryos, scaf expression is upregulated by activation of the JNK pathway and acts as an antagonist of JNK signaling. Hence, Scaf regulates its own expression levels. This negative-feedback loop may provide an interesting mechanism to control ad libitum feeding in flies. As sugar positively regulates the expression levels of scaf, sugar-rich food would induce constitutively high levels of scaf expression, which in turn would cause continuous feeding. The autoregulatory capacity of scaf may explain the fact that this does not happen in natural conditions, as Scaf downregulates its own expression. Interestingly, pharmacological inhibition of JNK signaling reduces food intake and protects against obesity in diet-induced obese mice (Prasad, 2018).

    Several studies have demonstrated that the brain can detect differences in the caloric content of the available food. The current data show that scaf expression increases when flies are fed on sugar-rich food. Therefore, Scaf neurons must receive information about the sugar content of the food and respond by regulating the levels of scaf. Scaf neurons are located in the SEZ of the adult brain and the VNC, and it cannot be currently determine if the effect on feeding is caused only by SEZ neurons. Scaf neurons appear to be second-order neurons and their polarity suggests that they can convey information to higher brain centers. Gustatory neurons from external mouthparts and the pharynx project into the SEZ, and the SEZ plays an important role in processing gustatory information. The dendritic projections of SEZ Scaf neurons around the foramen and in the SEZ therefore indicate that these neurons may be a part of the neuronal circuitry that relays gustatory information to higher brain centers. Similar neurons that transmit information about sugar have been reported earlier (Kim, 2017). Scaf neurons could be a parallel set of neurons that transmit information about the sugar content of the food when the fly eats. Scaf neuron activity would motivate the fly to continue feeding on food that is rich in sugars rather than feeding on sugar-deficient food sources. This may be important for survival, as it prevents the fly from feeding on nonnutritious food and encourages the fly to build up energy reserves even when it is no longer hungry (Prasad, 2018).

    Regulating food intake is an important process toward the maintenance of energy homeostasis. Neuronal and hormonal mechanisms regulate the feeding drive, depending on the internal state of the body and the quality of the available food. The drive to consume palatable, energy-dense food may ensure survival in times of scarcity but when dysregulated may result in overfeeding and obesity. Studies in mice suggest that the neural circuits responsible for the homeostatic control of feeding are dispensable when feeding is assessed on a high-fat, high-sugar diet, thus demonstrating independent homeostatic and hedonic control of feeding. This study has shown that scaf and Scaf neurons promote feeding on nutritious sugars independent of the hunger state of the fly. Scaf responds to the presence of nutritive sugars in food, and Scaf neurons do not evaluate the quality of food. The enhanced feeding motivation that was noticed upon activation of Scaf neurons and upon scaf overexpression may be due to its effect on downstream neurons. Manipulation of scaf or Scaf neuron activity results in a change in feeding only in sated state due to the fine balance between the internal state of the body and the quality of the food in regulating feeding drive. In the sated state, when the feeding drive due to the internal state is low or absent, increased activity of Scaf neurons or overexpression of scaf can easily enhance the feeding drive on nutritive sugars, while silencing Scaf neurons or downregulating the levels of scaf reduces the feeding drive. These effects may be due to enhanced or decreased activation of the downstream feeding machinery to which Scaf neurons convey the information about the nutrient content of the food. In starved state, the drive to feed is already high. As pointed out earlier, other circuits also transmit information about sugar content to higher brain centers. The enhanced feeding drive in the starved state coupled with information about the food from other neurons is likely sufficient to drive feeding to an extent that would render the feeding enhancement caused by manipulation of scaf or Scaf neurons unobservable (Prasad, 2018).

    Starvation resistance is associated with developmentally specified changes in sleep, feeding and metabolic rate

    Food shortage represents a primary challenge to survival, and animals have adapted diverse developmental, physiological, and behavioral strategies to survive when food becomes unavailable. Starvation resistance is strongly influenced by ecological and evolutionary history, yet the genetic basis for the evolution of starvation resistance remains poorly understood. The fruit fly, Drosophila melanogaster, provides a powerful model for leveraging experimental evolution to investigate traits associated with starvation resistance. While control populations only live a few days without food, selection for starvation resistance results in populations that can survive weeks. Previous work has shown that selection for starvation resistance results in increased sleep and reduced feeding in adult flies. This study investigated the ontogeny of starvation resistance-associated behavioral and metabolic phenotypes in these experimentally selected flies. Selection for starvation resistance was found to result in delayed development and a reduction in metabolic rate in larvae that persists into adulthood, suggesting that these traits may allow for the accumulation of energy stores and an increase in body size within these selected populations. In addition, larval sleep was found to be largely unaffected by starvation selection and feeding increases during the late larval stages, suggesting that experimental evolution for starvation resistance produces developmentally specified changes in behavioral regulation. Together, these findings reveal a critical role for development in the evolution of starvation resistance and indicate that selection can selectively influence behavior during defined developmental timepoints (Brown, 2019).

    Wild african Drosophila melanogaster are seasonal specialists on marula fruit

    Although the vinegar fly Drosophila melanogaster is arguably the most studied organism on the planet, fundamental aspects of this species' natural ecology have remained enigmatic. This study has investigated a wild population of D. melanogaster from a mopane forest in Zimbabwe. These flies are closely associated with marula fruit (Sclerocarya birrea), and it is proposed that this seasonally abundant and predominantly Southern African fruit is a key ancestral host of D. melanogaster. Moreover, when fruiting, marula is nearly exclusively used by D. melanogaster, suggesting that these forest-dwelling D. melanogaster are seasonal specialists, in a similar manner to, e.g., Drosophila erecta on screw pine cones. It was further demonstrated that the main chemicals released by marula activate odorant receptors that mediate species-specific host choice (Or22a) and oviposition site selection (Or19a). The Or22a-expressing neurons-ab3A-respond strongly to the marula ester ethyl isovalerate, a volatile rarely encountered in high amounts in other fruit. Or22a differs among African populations sampled from a wide range of habitats, in line with a function associated with host fruit usage. Flies from Southern Africa, most of which carry a distinct allele at the Or22a/Or22b locus, have ab3A neurons that are more sensitive to ethyl isovalerate than, e.g., European flies. Finally, the possibility is discussed that marula, which is also a culturally and nutritionally important resource to humans, may have helped the transition to commensalism in D. melanogaster (Mansourian, 2018).

    The vinegar fly Drosophila melanogaster displays preference toward certain fruit and strongly favors citrus for egg laying. The presence of a distinct host partiality is intriguing and implies that D. melanogaster during its evolutionary history likely has had a close association with a specific fruit, or group of fruit, with characteristics akin to citrus. This ancestral host is, however, likely not found among members of the Asian genus Citrus, but rather among fruit found within the Miombo and Mopane forests of the fly's predicted Urheimat in Southern Africa, more precisely in present day Zimbabwe and Zambia, and displays physical and chemical properties that fit with the known preference of D. melanogaster. In brief, marula has a thick rind similar to that of citrus, which encloses a sugary (and highly fermentable) juicy pulp, with a pH similar to that of orange, features all favored by D. melanogaster. Marula emits terpenes and esters, which in terms of total emission contribution, as well as in numbers, are the primary chemical components, as determined via gas chromatography-mass spectroscopy analysis of headspace collections. The two main chemicals, ethyl isovalerate (an ester) and β-caryophyllene (a sesquiterpene), together make up ~55% of the headspace. Both terpenes and esters are known to be important and ecologically relevant olfactory cues for D. melanogaster. In short, marula fulfills the criteria on essentially all counts and is accordingly a good candidate ancestral host (Mansourian, 2018).

    Do flies from native habitats then use marula? To answer this question, an expedition was mounted to Southern Africa in search of forest-dwelling D. melanogaster and marula. Specifically, mopane woodlands of the Matopos national park in Southwestern Zimbabwe, a site situated within the predicted ancestral range, was searched. The Matopos covers 424 km2, hosts no permanent human habitation, and is covered in Mopane and kopje woodlands (Mansourian, 2018).

    Once in the Matopos, marula trees, as well as fruiting trees with fermenting fruit below, were localized. among which fly traps baited with marula were placed. Over the next days, these traps caught numerous D. melanogaster. Traps placed under an additional 5 marula trees yielded another 67 D. melanogaster specimens. At all examined sites, though, D. simulans outnumbered D. melanogaster. These flies will be referred to as 'wild,' in line with their presence in undisturbed wilderness, with the caveat that their ultimate origin remains unknown (Mansourian, 2018).

    The forest flies were provided with a choice of marula versus orange, the favorite breeding substrate of domestic D. melanogaster. Paired traps, containing either marula or orange, were placed under a fruiting marula tree. Similar to the laboratory strain, the wild D. melanogaster showed a strong preference for marula. Interestingly, though, D. simulans displayed no such preference, indicating that the marula preference is exclusive to D. melanogaster and, moreover, that marula is not simply overall a more suitable fruit resource to Drosophila spp. Marula was dissected in search of fly eggs and larvae, and in all fruit examined, drosophilid larvae were located, from which D. melanogaster adults later emerged. In short, wild African D. melanogaster are drawn to the odor of marula, prefer marula to orange, and use marula as breeding substrate (Mansourian, 2018).

    To investigate the general distribution of D. melanogaster in the Matopos, traps (baited with fermenting marula) were placed at five locations with no fruiting marula trees nearby, but with otherwise similar vegetation (including other fruiting trees). Strikingly, D. melanogaster was absent, or very sparse, in traps at these locations. On the other hand, D. simulans was as abundant at sites with marula as it was in sites without. The distribution pattern of D. melanogaster in the Matopos hence indicates niche confinement and, in turn, a specialized lifestyle. D. melanogaster as a seasonal fruit specialist would actually not be surprising given. (2) the observed presence of a distinct egg-laying preference, and (3) the fact that host specialization is a prevalent feature in the melanogaster subgroup. Drosophila sechellia exclusively breeds in noni fruit, whereas Drosophila erecta and Drosophila orena are seasonal specialists on Pandanus cones and Syzygium waterberries respectively. Drosophila teissieri is closely associated with Parinari fruit, which limits its geographic range. A nonrandom subset of olfactory genes is associated with host preference in the fruit fly Drosophila orena, whereas Drosophila santomea is found with figs from Ficus clamydocarpa trees. Thus, seasonal host specialization in D. melanogaster would fall into the pattern displayed by most (if not all) of its close relatives. Outside of marula season, these forest flies may go into diapause, much like they do in temperate regions, or switch to opportunism, utilizing alternate breeding substrates. One such alternative could be figs, which are present year-round in the Matoposand in terms of biomass are even more abundant than marula. D. melanogaster has moreover been reared from figs in Africa, which are also an alternate host for the seasonal specialist D. erecta outside of Pandanus season (Mansourian, 2018).

    Wild African D. melanogaster hence not only utilize marula for parts of the year, marula appears to be exclusively utilized. It was asked how domestic flies react to this fruit. To this end, a two-choice assay to examine egg-laying preference in Canton-Special (Canton-S) wild-type flies. The Canton-S strain was established sometime before 1916 from a population in Canton, Ohio, well outside the sub-Saharan range of marula. The citrus preference of these flies was verified in the oviposition assay. Given a choice between orange and banana, the flies clearly preferred citrus as oviposition substrate. Having confirmed the assay, orange versus marula was tested, and indeed, flies provided this choice strongly preferred marula, similar to Wild African D. melanogaster. The ancestral marula preference is accordingly conserved in non-African flies (Mansourian, 2018).

    Which chemicals then mediate the marula preference? The same two-choice assay was used and the major chemical components of the headspace were tested individually. Previous work has shown that fly food spiked with terpenes confers positive egg-laying site selection, and thus the main terpene (β-caryophyllene), which as expected generated preferential oviposition, was tested. The main ester component, ethyl isovalerate, also conferred oviposition preference, as well as attraction in a T-maze assay. The preference of marula over orange may hence be mediated by the high presence of esters in the former. In line with this reasoning, flies provided with a choice of orange spiked with ethyl isovalerate against marula failed to make a choice (Mansourian, 2018).

    In D. sechellia and D. erecta, host specialization is linked to the Or22a circuit, which in both species is activated by distinct esters from the respective hosts. It was thus asked whether the primary marula ester ethyl isovalerate also activates Or22a-expressing olfactory sensory neurons (OSNs) in D. melanogaster. To investigate this issue, functional imaging of the antennal lobe was performed in flies expressing the calcium reporter GCaMP6m. Stimulation with ethyl isovalerate yielded strong calcium signals in the DM2 glomerulus (the target of the Or22a-expressing OSNs) already at 10-7 dilution. In line with its chemistry, marula odor also triggered strong Ca2+ signals from DM2, whereas orange odor triggered weak to no activity from the same glomerulus. Thus, similar to its specialized siblings, the main ester from the preferred host activates Or22a. Silencing of the Or22a pathway via Or22a-Gal4>UAS-TNT did not, however, abolish the marula oviposition preference, suggesting that additional pathways are involved in this behavior. Rather than mediating egg-laying preference, the primary function of Or22a may instead be locating the host over distance. Hence, up-wind flight navigation toward marula of flies with Or22a silenced (via Or22a-Gal4>UAS-TNT) was examined in a wind tunnel assay. Flies with non-functional Or22a input showed a reduced ability to localize marula compared to control flies, suggesting that these neurons' predominant function is to assist the fly in locating its host over distance. The importance of these neurons in this context is also evident from D. sechellia, which has a numerical increase of Or22a-expressing OSNs, which likely affords an improved ability to find noni fruit over distance (Mansourian, 2018).

    Since marula is restricted to sub-Saharan Africa, most D. melanogaster have to make do with alternative hosts. If Or22a indeed is linked to the specific chemistry of the host, local adaptation of the Or22a locus would be expected between D. melanogaster populations from diverse environments that may utilize disparate hosts. Thus, local genetic differentiation (as indexed by FST was estimated within the OR family between genomes from 10 African populations, plus one European. For each window centered on an olfactory receptor gene, the FST quantile was evaluated for each pairwise population comparison (the proportion of all windows on the same chromosome arm that showed stronger allele frequency differences [higher FST]) between these same two populations. The Or22a locus, and the adjacent tandem paralog Or22b, shows striking genetic differentiation between almost all population pairs, in stark contrast to most of the other ORs, for which little or no sign of local adaptation can be discerned (Mansourian, 2018).

    In cases where other ORs did show strong FST outliers (quantiles < 0.0001), differentiation in one or a few populations was often most apparent. These genes included Or33a, Or65b, and Or67a. Interestingly, these receptors also appear to have important functions. Or33a has unknown function, but like Or22a, it shows variable expression across species and has undergone serial duplication in Drosophila suzukii and Drosophila biarmipes. Or65b is expressed in pheromone-sensing neurons, but its function has not been established. In short, unlike most members of the OR family in D. melanogaster, Or22a (and its closely linked paralog, Or22b) shows strong signs of local adaptation, in line with a function associated with host-specific chemistry (Mansourian, 2018).

    At the molecular level, Or22a (and Or22b) thus differs between populations, but does this local differentiation also translate into functional changes in the ab3A neurons where these genes are expressed? The most conspicuous alteration among the investigated populations in the Or22a/Or22b locus is a deletion allele, whereby a segment stretching from the second exon of Or22a to the start of the second exon of Or22b has been deleted, generating a chimeric receptor, Or22ab. In light of the chimeric appearance of Or22ab, this variant appears to be a derived deletion (following a more ancient duplication to create these paralogs), rather than a representation of the ancestral state of the Or22 locus (Mansourian, 2018).

    The data support a prior suggestion that the Or22ab fusion variant is quite ancient. This variant is at a very high frequency within the ancestral range (e.g., 88% in Zambia). Nucleotide diversity of flanking sequences, which should accrue on the order of 4 Ne ~ 10 million generations in this species, is at or above typical levels among Zambia haplotypes carrying this deletion. Hence, it is likely that the fusion variant existed well before the species expanded beyond its ancestral range on the order of 150,000 generations ago, or ~10,000 years ago. In contrast, putatively ancestral full-length Or22a/Or22b haplotypes from Zambia show strongly reduced diversity across the deletion region. This pattern could reflect a low long-term population size of the full-length allele, in accordance with its current rarity in the ancestral range. In some populations, such as in Europe or the Ethiopian highlands, the full-length allele has become predominant. Many of these haplotypes show identical or nearly identical sequences, in line with prior evidence for positive selection linked to the Or22a/Or22b haplotype in Europe. It is noted that some populations with similarly high frequencies of the fusion variant are strongly differentiated from each other at the Or22a/b locus, which could imply either parallel increases of the fusion variant on distinct haplotypes or additional variants under spatially varying selection at this locus (Mansourian, 2018).

    Consequently, most D. melanogaster in Southern Africa will likely carry the Or22ab allele, which prompts the question: do their ab3A neurons respond to the marula ester? A strain in which Or22ab is fixed (RG18N) was selected, and single-sensillum recordings (SSRs) were performed. Measurements from ab3A neurons revealed strong responses to stimulation with ethyl isovalerate. The ab3A neurons in RG18N actually responded more strongly to ethyl isovalerate than to ethyl hexanoate-the primary ligand of Or22a, where ethyl hexanoate yielded a stronger response than ethyl isovalerate. In short, African D. melanogaster not only detect ethyl isovalerate, but also are even more sensitive to this marula compound than flies from outside Africa. It is noted that the distribution of populations with a high frequency of Or22ab overlaps with the distribution of marula. However, whether the Or22ab allele is an adaptation toward marula remains to be shown. Heterologous expression and detailed functional characterization of this interesting receptor variant will be a topic for future studies (Mansourian, 2018).

    The Matopos is best known for its elaborately painted caves-made by now-vanished San tribes during Late Pleistocene to Early Holocene. For these tribes, marula played a pivotal role, and archeological excavations of their cave homes have uncovered enormous quantities of marula stones. From the Pomongwe cave alone, remains of at least 24 million marula stones were recovered, which only represents the carbonized remains, and hence but a fraction of the marula that must have once been brought into this cave. The San evidently spent considerable time collecting and processing marula, which would have been the staple food item during many months of the year. Thus, just like D. melanogaster, these San tribes appear to have been seasonal specialists on marula as well (Mansourian, 2018).

    The marula-San link offers a plausible scenario by which D. melanogaster became a human commensal. The smell of the stored marula emanating from the caves would have attracted flies from far and wide. Flies would have found a steady supply of marula and fermenting leftovers inside the caves, long after the fruit's presence in the surrounding woodlands had diminished. In other words, the time frame for using the optimal breeding substrate would have been increased considerably. Inside the caves, the flies would also have benefitted from a reduced risk of predation, as well as protection from adverse weather conditions. Over time, the cave flies would have accumulated adaptations helpful for human commensalism. Relevant traits may have included a willingness to enter darker enclosures and an increased tolerance of ethanol, both of which differentiate D. melanogaster from its closest relatives. Thus, it was asked whether D. melanogaster actually enter these caves. To this end, four traps baited with fermenting marula wkere placed along the far wall of the Nswatugi cave. Over three days, these traps caught a number of D. melanogaster specimens, but no D. simulans, in contrast to the closest traps (n = 3) placed under fruiting marula trees outside the cave, where D. simulans greatly outnumbered D. melanogaster (Mansourian, 2018).

    The archeological record indicates that systematic and intensive marula use began ~12,000 years ago. At ~9,500 years ago, marula harvesting reached massive proportions, finally ebbing out ~8,000 years ago . These dates coincide with demographic data from D. melanogaster, which point to a within-Africa expansion starting ~10,000 years ago, an expansion presumably representing the dispersal of the commensal population throughout its new niche. In short, archeological and demographic data would support the notion that marula use by the San may have been a factor in turning the woodland species D. melanogaster into the cosmopolitan species of today (Mansourian, 2018).

    This study has demonstrated that D. melanogaster from a mopane forest within the predicted ancestral range are seasonal specialists on marula fruit. The odor of this seasonally abundant and widely distributed fruit activates select key odorant receptors previously implicated as having particular importance to D. melanogaster, and it is argued that marula is the ancestral primary host of the fly. Flies from sub-Saharan Africa were shown to carry a specific allele of one of these odorant receptors and are also more responsive to a key marula chemical. Finally, it is speculate that the marula specialization might have been important in driving commensalism (Mansourian, 2018).

    The finding of a woodland population of D. melanogaster within the ancestral habitat opens up a range of interesting questions to be addressed. For example, how do these flies differ from their commensal relatives, i.e., which genetic factors underlie this shift in lifestyle? The finding that D. melanogaster appears to have a close association with a single host fruit will furthermore facilitate studies relating to host specific chemosensory adaptations, which so far have had to be conducted in other insects in which the wealth of tools available in D. melanogaster are unavailable (Mansourian, 2018).

    Sensorimotor pathway controlling stopping behavior during chemotaxis in the Drosophila melanogaster larva

    Sensory navigation results from coordinated transitions between distinct behavioral programs. During chemotaxis in the Drosophila melanogaster larva, the detection of positive odor gradients extends runs while negative gradients promote stops and turns. This algorithm represents a foundation for the control of sensory navigation across phyla. The present work identified an olfactory descending neuron, PDM-DN, which plays a pivotal role in the organization of stops and turns in response to the detection of graded changes in odor concentrations. Artificial activation of this descending neuron induces deterministic stops followed by the initiation of turning maneuvers through head casts. Using electron microscopy, the main pathway was reconstructed that connects the PDM-DN neuron to the peripheral olfactory system and to the pre-motor circuit responsible for the actuation of forward peristalsis. The results set the stage for a detailed mechanistic analysis of the sensorimotor conversion of graded olfactory inputs into action selection to perform goal-oriented navigation (Tastekin, 2018).

    The Drosophila melanogaster larva has a numerically simple nervous system that comprises ~10,000 neurons directing a rich repertoire of behaviors that includes navigation in chemical, light and thermal gradients. The larva displays stereotyped behavioral programs that can be decomposed into forward motion ('run'), locomotor pauses ('stops') followed by exploratory lateral-head movements ('head casts') and turns. Although the decomposition of the behavioral continuum displayed by the larva into discrete 'actions' represents an approximation, this approximation has proved valuable in various model organisms, and it permitted the identification and functional characterization of neural circuits in Drosophila. The sensorimotor algorithm directing innate navigation in the larva is shared across sensory modalities. Movements toward favorable directions elongate runs, whereas movements toward unfavorable directions promote turning. The goal of the present study was to identify the neural circuits that implement the sensorimotor conversion of the OSN activity into the probability of switching from a run to a stop-turn (Tastekin, 2018).

    Neural circuits in the brain are connected to the premotor system in the VNC by descending nerve fibers. In adult flies, descending neurons represent a relatively small population of ~1100 cells, accounting for less than 1% of the total number of neurons in the nervous system. By establishing the main connections between the centers carrying out sensory processing in the brain and the central-pattern-generating (CPG) circuits in the VNC, descending neurons are thought to play a key role in the control of sensorimotor behaviors. In adult flies, activation of the 'moonwalking' descending neuron induces backward locomotion. In larvae, activation of the recently-identified 'mooncrawler' neuron triggers backward locomotion and blocks forward locomotion (Carreira-Rosario, 2018). Complex sequences of actions can be elicited by the activation of a single descending neuron, such as courtship song production and flight escapes. Using a collection of Split-Gal4 driver lines that labels relatively sparse sets of descending neurons, a majority of descending neurons was found to elicit only one stereotyped behavior (Cande, 2017), but the same behavior could be elicited by distinct descending neurons. The behavioral effects of gain-of-function manipulations showed dependence on the ongoing motor state of the animal. By contrast, very little is known about the number and the organization of descending neurons in the larva. To identify descending neurons participating in the control of innate larval chemotaxis, a behavioral screen was carried on a collection of sparse driver lines (Tastekin, 2018).

    The behavioral screen was designed based on two assumptions. First, larvae display two types of navigational behaviors: attraction - the most common response elicited by volatile odors - and repulsion, a behavior elicited for chemical alarm cues such as the pheromone emitted by a natural predator of the Drosophila larva, the parasitoid wasp. Based on the selectivity of the behavioral responses induced by individual descending neurons in adult flies, it was reasoned that attractive and aversive responses might be controlled by different descending pathways. To focus on positive (attractive) chemotaxis, an assay was devised that elicited purely attractive behavior. Second, the functional deconstruction of the peripheral olfactory system of the larva has shown that single olfactory sensory neurons (OSNs) are sufficient to direct robust chemotaxis. It was assumed that the activity of a single OSN, that expressing Or42a, was more likely to feed into a single descending neuron than the activity of an ensemble of OSNs, which might activate multiple descending pathways. For this reason, the screen was conducted with ethyl butyrate, an odor that primarily binds to the Or42a odorant receptors (Tastekin, 2018).

    The loss-of-function screen led to the identification of two main classes of neuronal subsets with a phenotypic defect in innate chemotaxis. The first class labeled different sets of mushroom body (MB) neurons (i.e. Kenyon cells, mushroom body input neurons and mushroom body output neurons). Although the MB is not traditionally associated with the control of innate orientation behavior, recent work has uncovered that the MB participates in the control of chemotaxis in adult flies. Considering the effects of MB impairment on learned olfactory behaviors, it is possible that a loss-of-function of particular subsets of MB neurons unbalances the net MB output, thereby producing a dysfunction in innate chemotaxis. The second class of neurons that the loss-of-function screen pointed out included descending neurons. Given that descending neurons form a bottleneck in sensorimotor pathways, concentration was placed on this neuron class in the rest of the work. Among the descending neurons identified in the behavioral screen, the anatomical features of the PDM-DN stood out as promising: the dendritic arborizations of this descending neuron cover regions of the lateral horn (LH) and the MB peduncle. On the output side, PDM-DN has large axonal varicosities in the subesophageal zone (SEZ) -- a region previously implicated in the control of run-to-turn transitions during larval chemotaxis (Tastekin, 2015). The axon terminals of PDM-DN extend dorsally to the 4th abdominal segment, suggesting that this neuron might directly act on the premotor system. Altogether, PDM-DN emerged as a strong descending-neuron candidate that transforms information about the larva's sensory experience collected from the LH and the MB into a modulation of the larva's motor output (Tastekin, 2018).

    Using a set of complementary manipulations to test the effects of silencing or activating PDM-DN, the role was examined of this neuron on specific aspects of the sensorimotor control of innate chemotaxis. First, it was demonstrated that PDM-DN activity contributes to the proper timing of run-to-turn transitions during chemotaxis. During down-gradient runs, the detection of negative changes in odor intensity leads to a graded increase in the probability of switching from a run to a turn. Abrupt termination of the Or42a-OSN activity triggers near deterministic stops. Upon constitutive loss-of-function of PDM-DN, larvae had a significantly lower probability of turning. As a result of the inaccurate timing of their turns, larvae with impaired function of PDM-DN were unable to accumulate in the vicinity of the odor source with the same precision as their controls. Remarkably, manipulations inducing a loss of function of PDM-DN did not affect the ability to turn toward the gradient, arguing that distinct sensorimotor pathways control the timing and the direction of turning maneuvers. By expressing CsChrimson in the PDM-DN neuron, it was established that acute optogenetic activation of PDM-DN elicits near deterministic stops. Upon prolonged gain-of-function stimulations, the release of stopping behavior was accompanied by lateral head casts. Together these results indicate that PDM-DN acts as a command-like element in the sensorimotor pathway that converts changes in the activity of the Or42a and Or42b OSNs into the probabilistic release of reorientation maneuvers. The primary effect of the activity of PDM-DN is to promote switching between run and stop-turn behaviors. Its secondary effect is to induce exploratory scans of the local odor gradient in preparation of a turn (Tastekin, 2018).

    If PDM-DN is part of the sensorimotor pathway controlling chemotaxis, its activation must be dependent on the present -and potentially past- activity of the peripheral OSNs. This hypothesis was tested in functional perturbation experiments. It was reported that the PDM-DN silencing phenotype depends on the olfactory sensory information: in odor gradients, silencing PDM-DN activity affected the release of turns during down-gradient runs, but not during up-gradient runs. By contrast, silencing PDM-DN did not affect the basal turn rate in the absence of odor gradients. The effects were tested of the ongoing activity of peripheral OSNs on the release of stops upon medium-intensity optogenetic activation of PDM-DN. Larvae carrying a PDM-DN>Chrimson transgene were optogenetically stimulated during up-gradient and down-gradient runs. Interestingly, no significant difference was found in the probability of releasing a stop-turn maneuver. This trend was further confirmed by comparing the time course of the tail speed -a proxy for stopping behavior- before and during optogenetic stimulation for up-gradient and down-gradient runs. No difference was found between up-gradient and down-gradient runs. A detailed inspection of the behavior associated with individual trials led to the same conclusion. This result suggests that PDM-DN itself does not integrate the history of the activity of Or42a and Or42b OSNs, otherwise the gain-of-function perturbations should have produced a higher probability of triggering stops during down-gradient runs compared to up-gradient runs. It is also possible that the light stimulation used in these experiments of Figure 3J was still too high to reveal differences due to the integration of distinct sensory experiences between up-gradient and down-gradient runs. By comparison, it was observed that the secondary effect of optogenetic activation of PDM-DN - the promotion of head casting- was strongly dependent on the ongoing olfactory experience of the larva: during down-gradient runs, PDM-DN activation led to vigorous and wide-amplitude head casts, whereas PDM-DN activation led to milder casting behavior during up-gradient runs. Based on this result and the observation that the PDM-DN loss-of-function does not affect the accuracy of individual turns, it is proposed that the head-casting component of reorientation maneuvers is gated by the activity of PDM-DN, but that it is also controlled by other descending pathway(s) that integrate the ongoing activity of the olfactory system (Tastekin, 2018).

    The larval nervous system is amenable to a detailed reconstruction of neural circuits through electron microscopy (EM). While the EM reconstruction is achieved in the nervous systems of younger larvae (first-instar L1 developmental stage) than those tested behaviorally (third instar, L3), it has beee shown that the sensorimotor circuit involving the control of innate chemotaxis is fully functional at the L1 stage (Almeida-Carvalho, 2017). By comparing the anatomy of the PDM-DN neuron between light-scanning and EM microscopy, the PDM-DN neuron was pinpointed in the EM stack. The main pre-synaptic partners of PDM-DN were reconstructed all the way to the peripheral olfactory system. This reconstruction relied on earlier work in which the circuit diagram of the larval antennal lobe was fully mapped at the resolution of single synapses. PDM-DN receives olfactory inputs in the lateral horn region via two lateral-horn interneurons that form a feedforward circuit. Consistent with the fact that the loss-of-function screen involved an odor (ethyl butyrate) that only activates a small number of OSNs, PDM-DN was found to receive olfactory inputs from a subset of OSNs activated by this odor: Or42a and Or42b OSNs. Given the incompleteness of the loss-of-function phenotype of PDM-DN, it is speculated that redundant descending pathways controlled by the same set of OSNs might trigger different behavioral modules in a context-dependent manner (Tastekin, 2018).

    What is the logic underlying the transformation of the activity patterns of OSNs into the all-or-none activation of the PDM-DN neuron? On the one hand, positive gradients promote sustained activity of the Or42a and Or42b OSNs. The Or42a OSN encodes the time derivative (slope) of ramps of odor concentrations. On the other hand, the suppression of stop-turn during up-gradient runs implies that the activity of PDM-DN must be negatively correlated with the activity of the Or42a and Or42b OSNs. Strong activation of these two OSNs must suppress the activity of the PDM-DN neuron while inhibition of these OSNs must trigger the firing of PDM-DN. Although the activity of the uniglomerular Or42a and Or42b PNs is expected to be roughly proportional to the activity of their cognate OSNs, it is possible that these PNs extract higher-order features from dynamic patterns of odor concentrations, such as the acceleration of the stimulus. This implies that the circuitry connecting the Or42a and Or42b OSNs to PDM-DN must produce an inversion of the sign of the incoming olfactory stimulations to gate PDM-DN activity only when the OSN activity is low (Tastekin, 2018).

    The two main upstream partners of PDM-DN are located in the lateral-horn (LH) region: LH-LN1 and LH-LN2. These neurons form a feedforward motif where LH-LN1 outputs on LH-LN2 and PDM-DN whereas as LH-LN2 output on PDM-DN. Feedforward motifs (or feedforward loops) fulfill important regulatory functions in biological networks. Depending on the signs of the interactions between the LH-LN1, LH-LN2 and PDM-DN, this motif could act as pulse generator or a filter dampening off-responses of a sensory unit. Given that the inputs of the Or42a and Or42b uPNs into this circuit will be correlated with changes in stimulus intensity, it is concluded that either the synapses between LH-LN1 and PDM-DN or those between LH-LN2 and PDM-DN must be inhibitory. The absence of driver lines specific to the LH-LN1 and LH-LN2 neurons prevented resolving of the sign of each interaction. In light of the ability of PDM-DN to deterministically trigger stops, it is speculated that the 3-element feedforward circuit in the LH must represent the neural correlate of the action selection underpinning the sensorimotor control of the onset of reorientation maneuvers. Future work will be necessary to clarify how dynamic trains of sensory inputs are converted into the transient activity of PDM-DN. In unpublished experiments, attempts were made to characterize the response of PDM-DN to optogenetically-controlled activation of peripheral OSNs in brain explants. In spite of multiple attempts, these experiments were unsuccessful at producing reliable patterns of PDM-DN activity - a negative result that suggests that the absence of proprioceptive feedback in brain explants precludes the proper function of PDM-DN. Imaging the activity of PDM-DN in freely behaving animals might overcome this limitation in the future (Tastekin, 2018).

    Optogenetically-controlled activation of PDM-DN produces two distinct motor responses: (1) a cessation of forward peristalsis inducing a switch from running to stopping and (2) exploratory head movements followed by a turn. The release of these two actions appears to be part of a hierarchy. The termination of peristalsis is near immediate and largely stereotypical across trials. By contrast, the release of head casting takes a couple of seconds. Significant inter-trial variability is observed for the head-casting behavior. Part of the variability in the head-casting behavior is reflected in the idiosyncratic nature of asymmetrical contractions in the thoracic and anterior abdominal segments, which might be influenced by experience-dependent factors. In agreement with a recent study in adult flies, our results argue that a single descending neuron can contribute to the sensorimotor control of different actions. While stopping behavior limits overshoots of the odor source, sensory-dependent release of patterns of head casts enable the larva to scan the local odor gradients to reorient toward the direction of higher concentrations. By taking advantage of the EM reconstruction, a circuit diagram was built of the main partners downstream from PDM-DN and attempts were made to delineate the neural pathway actuating stops in forward locomotion and head-casting behaviors (Tastekin, 2018).

    Forward locomotion through peristalsis rely on the coordinated inter-segmental propagation of waves of muscle contractions from the posterior (tail) to the anterior end (head) of the body segments. This cyclic behavior emerges from the activity of a network of pre-motor neurons that spans the entire set of abdominal segments of the VNC. The cessation of forward peristalsis (stopping behavior) can be accomplished in at least three different ways: (1) by preventing the initiation of forward peristaltic waves; (2) by inhibiting forward wave propagation or (3) by the combination of both mechanisms. The following observations support a model in which PDM-DN mediates stopping behavior by inhibiting the initiation of forward peristaltic waves in the posterior abdominal segments of the VNC (mechanism 1). First, the analysis of peristaltic wave propagation in freely behaving larvae responding to PDM-DN activation suggested that optogenetic activation of PDM-DN is insufficient to inhibit wave propagation once the wave has already been initiated. Second, a detailed tracking of the segmental contractions in restrained larvae showed that PDM-DN activation can inhibit wave initiation in the posterior segments, but not in the anterior segments. The 4th abdominal segment (A4) appears to be the 'hinge' region beyond which PDM-DN fails to inhibit the wave propagation. Third, similar observations were made by using calcium imaging to monitor fictive patterns of locomotion in isolated CNS preparations in response to PDM-DN activation. In agreement with published results related to the sequential ablation of abdominal segments in CNS explants, this study found that PDM-DN activation blocks forward wave propagation most effectively between the 5th and the 7th abdominal segments (A5-A7). It is concluded that PDM-DN might specifically target the pre-motor circuit responsible for the initiation of forward locomotion in the most posterior segments, while enabling asymmetrical contractions of the thoracic and anterior abdominal segments to scan the local odor gradient through head casts and to implement turning maneuvers (Tastekin, 2018).

    What are the neural mechanisms underlying the inhibitory action of PDM-DN on the pre-motor system of the larva? The EM reconstruction of the downstream partners of PDM-DN revealed that that PDM-DN synapses onto a set of local and descending interneurons in the SEZ region. Previous work has shown that the SEZ comprises a subset of neurons that are necessary and sufficient to trigger reorientation maneuvers in response to multi-sensory stimuli (Tastekin, 2015). In agreement with this finding, the activity of the SEZ region correlates with the initiation and execution of forward peristaltic waves. The SEZ of the larva also participates in the control of switches between feeding and crawling behaviors. More generally, the SEZ acts as a pre-motor hub that integrates dynamic sensory inputs and coordinate the release of specific motor programs. This study found that the PDM-DN relays its 'command' through a small set of larval descending neurons that have their dendritic harbors in the SEZ. The main downstream partner of PDM-DN is a descending neuron SEZ-DN1 also known as Pair-1, which projects to the posterior abdominal segments. SEZ-DN1 synapses on a circuit of segmentally repeated excitatory premotor neurons (A27h) that is involved in the propagation of forward peristaltic waves. The synapses between SEZ-DN1 and the A27h circuit are mainly restricted to the posterior abdominal segments A5-A7. It is proposed that PDM-DN inhibits the initiation of forward peristaltic waves via SEZ-DN1. Neurotransmitter profiling of PDM-DN demonstrated that this neuron is excitatory. Given the inhibitory effect of PDM-DN activation on peristalsis, it was hypothesized that SEZ-DN1 must inhibit the activation of the A27h neurons. In agreement with a companion study (Carreira-Rosario, 2018), co-labeling of SEZ-DN1 with GABA antibody corroborated the inhibitory nature of this neuron. Like PDM-DN, optogenetic activation of SEZ-DN1 is sufficient to evoke stopping patterns of forward locomotion. In imaging experiments where fictive motor waves were visualized with GCaMP6f, this study demonstrated that acute optogenetic activation of SEZ-DN1 interrupted the propagation of peristaltic waves. The connectivity between PDM-DN and SEZ-DN1 was further established by eliciting robust patterns of SEZ-DN1 activity upon optogenetic activation of PDM-DN. Although the possibility that parallel pathways downstream from PDM-DN contribute to the induction of stopping behavior, it is proposed that SEZ-DN1 is a descending neuron that can trigger stopping behavior by inhibiting forward wave initiation in the most posterior abdominal segments of the VNC - a conclusion supported by recent work (Carreira-Rosario, 2018). The bilateral projection of PDM-DN on the left and right SEZ-DN1 neurons explains the ability of unilateral optogenetic activation of PDM-DN to produce symmetrical block in peristalsis (Tastekin, 2018).

    Having identified SEZ-DN1 as the main actuator of pauses upon PDM-DN gain-of-function, the second phenotype triggered by PDM-DN activation was analyzed: the release of head casting behavior in preparation of turning maneuvers. By reviewing the downstream partners of PDM-DN, it was discovered that at least two pathways might be implicated in the release of head-casting behavior: SEZ-LN1 and SEZ-DN2. The SEZ-LN1 neuron lies upstream from an uncharacterized premotor neuron, which gives asymmetrical input into anterior RP neurons that control either dorsal or ventral muscles on the body walls. It is speculated that asymmetrical contractions of dorsal and ventral muscles in the anterior segments facilitate head casting and turning behaviors. Likewise, SEZ-DN2 gives input into pre-motor neurons upstream of prothoracic accessory nerve (PaN) motor neurons that are thought to mediate head tilting - a behavior frequently observed during reorientation maneuvers. Due to the absence of sparse driver lines that specifically label SEZ-LN1 and SEZ-DN2, the function of these neurons could not be examined (Tastekin, 2018).

    In summary, the present study describes the reconstruction of a sensorimotor pathway from the peripheral sensory system down to the motor system. descending neuron, PDM-DN, was identified and characterized that plays a central role in controlling the release of reorientation maneuvers based on the integration of sensory experience. This command-like neuron illustrates the versatility of the behavioral control descending neurons are capable of. While stopping behavior is deterministically triggered by PDM-DN activation, the release of head casting and turning behaviors was context-dependent. The results argue that these two behavioral programs - stopping and head-casting - are partly controlled by independent pathways under the control of different descending neurons. EM reconstruction and functional analysis revealed that PDM-DN employs distinct SEZ and abdominal interneurons to differentially regulate stopping and head casting/turning behaviors. Considering the striking similarity between the navigation algorithms that control orientation to different sensory modalities (thermotaxis, phototaxis and chemotaxis), it is plausible that PDM-DN contributes to the control of stopping behavior elicited by visual and thermal signals too. Alternatively, multiple parallel descending pathways might contribute to the sensory control of switches between running and stopping. To produce a coherent motor outcome, 'commands' arising from these different pathways would have to be integrated downstream from PDM-DN. Where and how this integration takes place remains a mystery that has now become experimentally tractable (Tastekin, 2018).

    Convergence of monosynaptic and polysynaptic sensory paths onto common motor outputs in a Drosophila feeding connectome

    This study reconstructed, from a whole CNS EM volume, the synaptic map of input and output neurons that underlie food intake behavior of Drosophila larvae. Input neurons originate from enteric, pharyngeal and external sensory organs and converge onto seven distinct sensory synaptic compartments within the CNS. Output neurons consist of feeding motor, serotonergic modulatory and neuroendocrine neurons. Monosynaptic connections from a set of sensory synaptic compartments cover the motor, modulatory and neuroendocrine targets in overlapping domains. Polysynaptic routes are superimposed on top of monosynaptic connections, resulting in divergent sensory paths that converge on common outputs. A completely different set of sensory compartments is connected to the mushroom body calyx. The mushroom body output neurons are connected to interneurons that directly target the feeding output neurons. These results illustrate a circuit architecture in which monosynaptic and multisynaptic connections from sensory inputs traverse onto output neurons via a series of converging paths (Miroschnikow, 2018).

    Motor outputs of a nervous system can be broadly defined into those carried out by the muscles to produce movements and by the glands for secretion. Both of these behavioral and physiological events are regulated by a network of output neurons, interneurons and sensory neurons, and a major open question is how one neural path is selected from multiple possible paths to produce a desired output. Nervous system complexity and tool availability have strongly dictated the type of experimental system and analysis that can be used to address this issue, such as a focus on a particular organism, behavior or type of neuron. In this context, the detailed illustrations of different parts of nervous systems at neuronal level as pioneered by Cajal, to the first complete description of a nervous system wiring diagram at synaptic level for C. elegans, demonstrate the power of systematic neuroanatomical analysis in providing a foundation and guide for studying nervous system function. However, the technical challenges posed by such analysis have limited the type of organisms for which synaptic resolution mapping can be performed at the scale of an entire nervous system (Miroschnikow, 2018).

    Analysis of the neural circuits that mediate food intake in the Drosophila larvae offers numerous advantages in meeting the challenge of neuroanatomical mapping at a whole brain level, and combining it with the ability to perform behavioral and physiological experiments. The muscle system that generates the different movements necessary for transporting food from the pharynx to the esophagus, as well as the endocrine system responsible for secreting various hormones for metabolism and growth, have both been well described. These are also complemented by the analysis of feeding behavior in adult flies. Although there is broad knowledge at the morphological level on the organs underlying larval feeding behavior and physiology, as well as on the nerves innervating them in the periphery, the central connectivity of the afferent and efferent neurons within these nerves are largely unknown. At the same time, advances in the EM reconstruction of an entire CNS of a first instar larva offers an opportunity to elucidate an animals' feeding system on a brain-wide scale and at synaptic resolution. As part of this community effort, we recently performed an integrated analysis of fast synaptic and neuropeptide receptor connections for an identified cluster of 20 interneurons that express the neuropeptide hugin, a homolog of the mammalian neuropeptide neuromedin U, and which regulates food intake behavior. This analysis showed that the class of hugin neurons modulating food intake receives direct synaptic inputs from a specific group of sensory neurons, and in turn, makes mono-synaptic contacts to output neuroendocrine cells. The study not only provided a starting point for a combined approach to studying synaptic and neuropeptidergic circuits, but a basis for a comprehensive mapping of the sensory and output neurons that innervate the major feeding and endocrine organs. (Miroschnikow, 2018).

    Feeding is one of the most universal and important activities that animals engage in. Despite large differences in the morphology of the external feeding organs, the internal gut structures are quite similar across different animals; indeed, even within closely related species, there can be large differences in the external organs that detect and gather food, whereas the internal organs that transport food through the alimentary canal are much more similar. Recent studies have also pointed out the functional similarities between the subesophageal zone in insects and the brainstem in vertebrates for regulating feeding behavior. In mammals, the different cranial nerves from the medulla innervate distinct muscles and glands of the foregut. For example, the VIIth cranial nerve (facial nerve) carries taste sensory information from anterior 2/3 of the tongue, and innervates the salivary glands, and lip and facial muscles. The IXth cranial nerve (glossopharyngeal nerve) receives taste inputs from the posterior 1/3 of the tongue, and innervates the salivary glands and pharynx muscles. The Xth cranial nerve (vagus nerve) receives majority of the sensory inputs from the enteric nervous system of the gut, and innervates pharynx and esophagus muscles. The XIth cranial nerve (spinal accessory nerve) and the XIIth cranial nerve (hypoglossal nerve) are thought to carry strictly motor information which innervate the pharynx and neck muscles, and the tongue muscles. The distinct cranial nerves project onto topographically distinct areas in the medulla of the brainstem. It is also noted that olfactory information is carried by cranial nerve I, a strictly sensory nerve that projects to the olfactory bulb (OB), an area topographically distinct from the brainstem. In addition, there are direct neuronal connections between the brainstem and the hypothalamus, the key neuroendocrine center of vertebrates (Miroschnikow, 2018).

    Analogously, distinct pharyngeal nerves of the Drosophila larva are connected to the subesophageal zone (SEZ), and also carry sensory and motor information that regulate different parts of the body. The AN (antennal nerve) carries sensory information from the olfactory, pharyngeal and internal organs, and innervates the pharyngeal muscles for pumping in food. The serotonergic neurons that innervate the major endocrine center and the enteric nervous system also project through the AN. Note also that the olfactory sensory organs project to the antennal lobe (AL), which abuts the SEZ yet is topographically separate. The MxN (maxillary nerve) carries external and pharyngeal sensory information, and innervates the mouth hooks, whose movements are involved in both feeding and locomotion. The PaN (prothoracic accessory nerve) carries external sensory information from the upper head region, and innervates the muscles involved in head tilting. Furthermore, the SEZ has direct connections to median neurosecretory cells (mNSCs) and the ring gland. In sum, although a large body of knowledge exists on the gross anatomy of the nerves that target the feeding organs in vertebrates and invertebrates, the synaptic pathways within the brain that interconnect the sensory inputs and output neurons of the individual nerves remain to be elucidated (Miroschnikow, 2018).

    This paper has reconstructed all sensory, serotonergic modulatory (Se0) and motor neurons of the three pharyngeal nerves that underlie the feeding motor program of Drosophila larvae. The activity of these nerves has previously been shown to be sufficient for generating the feeding motor pattern in isolated nervous system preparations, and that the central pattern generators (CPGs) for food intake lie in the SEZ. This study then identified all monosynaptic connections between the sensory inputs and the motor, Se0 and previously described median neurosecretory ouput neurons, thus providing a full monosynaptic reflex circuit for food intake. Polysynaptic pathways were also mapped that are integrated onto the monosynaptic reflex circuits. In addition, the multisynaptic non-olfactory neuron connections from the sensory neurons to the mushroom body memory circuit were mapped, and these were shown to be different from those involved in monosynaptic reflex circuits. Finally, a set of mushroom body output neurons were traced onto the neurosecretory and other feeding output neurons. Reflex circuits can be seen to represent the simplest synaptic architecture in the nervous system, as formulated by Charles Sherrington. Anatomical reconstructions of monosynaptic and polysynaptic reflex circuits can also be seen in the works of Cajal. A model is proposed of how different mono- and polysynaptic pathways can be traversed from a set of sensory neurons to specific output neurons, which has relevance for understanding the mechanisms of action selection (Miroschnikow, 2018).

    This study provides a comprehensive synaptic map of the sensory and output neurons that underlie food intake and metabolic homeostasis in Drosophila larva. Seven topographically distinct sensory compartments, based on modality and peripheral origin, subdivide the SEZ, a region with functional similarities to the vertebrate brainstem. Sensory neurons that form monosynaptic connections are mostly of enteric origin, and are distinct from those that form multisynaptic connections to the mushroom body (MB) memory circuit. Different polysynaptic connections are superimposed on the monosynaptic input-ouput pairs that comprise the reflex arc. Such circuit architecture may be used for controlling feeding reflexes and other instinctive behaviors (Miroschnikow, 2018).

    Reflex circuits represent a basic circuit architecture of the nervous system, whose anatomical and physiological foundations were laid down by Cajal and Sherrington. The Drosophila larval feeding reflex circuit comprises the motor neurons that innervate the muscles involved in pharyngeal pumping, as well as the neurosecretory neurons that target the endocrine organs. They also include a cluster of serotonergic neurons that innervate the entire enteric nervous system, and which may have neuromodulatory effects on the feeding system in a global manner. The vast majority of output neurons are targeted monosynaptically from a set of topographically distinct sensory synaptic compartments in the CNS. These compartments target the output neurons in overlapping domains: the first, ACa, targets all neuroendocrine cells as well as the serotonergic neurons; the second, AVa, targets a subset of neuroendocrine cells, the serotonergic neurons and most of the pharyngeal motor neurons, while the third, AVp, targets the serotonergic neurons and a different set of pharyngeal motor neurons. With these outputs, one can in principle fulfill the most basic physiological and behavioral needs for feeding: neurosecretory cells for metabolic regulation and pharyngeal motor neurons for food intake. This set of monosynaptic connections can thus be seen to represent an elemental circuit for feeding, since the connections between the input and output neurons cannot be broken down any further (Miroschnikow, 2018).

    Vast majority of the sensory inputs comprising this 'elemental feeding circuit'derive from the enteric nervous system to target the pharyngeal muscles involved in food intake and neuroendocrine output organs. However, there is a small number of monosynaptic reflex connections that originate from the somatosensory compartment. The output neurons targeted by these somatosensory neurons are motor neurons that control mouth hook movements and head tilting, movements which are involved in both feeding and locomotion. In this context, it is noteworthy that monosynaptic reflex connections are found to a much lesser degree in the larval ventral nerve cord, which generates locomotion. An analogous situation exists in C. elegans, where majority of the monosynaptic reflex circuits are found in the head motor neurons and not in the body. One reason could be due to the relative complexity in the response necessary for food intake as compared to locomotion. For example, a decision to finally not to swallow a harmful substance, once in the mouth, may require a more local response, for example muscles limited to a very specific region of the pharynx and esophagus, where monosynaptic arc might suffice. By contrast, initiating escape behaviors requires a more global response with respect to the range and coordination of body movements involved, although it also employs multimodal sensory integration via a multilayered circuit (Miroschnikow, 2018).

    The inter-sensory connections show a combination of hierarchical and reciprocal connections, which may increase the regulatory capability and could be especially important for monosynaptic circuits. By contrast, very few monosynaptic connections exist between the larval olfactory, chordotonal or nociceptive class IV sensory neurons in the body. Interestingly, there is also a much higher percentage of intersensory connections between olfactory receptor neurons in the adult as compared to the larva, which could function in gain modulation at low signal intensities. This might be attributable to adults requiring faster processing of olfactory information during flight navigation (or mating), and/or to minimize metabolic cost. Whether such explanation also applies to the differences in intersensory connection between the different types of sensory neurons in the larvae remains to be determined (Miroschnikow, 2018).

    Very few cases were found where a monosynaptic path between any sensory-output pair is not additionally connected via a polysynaptic path. An interesting question in the context of action selection mechanism is which path a sensory signal uses to reach a specific target neuron. For example, a very strong sensory signal may result in a monosynaptic reflex path being used. However, a weaker sensory signal may result in using a different path, such as one with less threshold for activation. This would also enable the integration of different types of sensory signals through the usage of multiple interneurons, since the interneurons may receive sensory inputs that are not present in monosynaptic connections. For example, sensory neurons can target the neuroendocrine cells directly (monosynaptically), or through a hugin interneuron (di-synaptically). The sensory compartments that directly target the neuroendocrine cells are of enteric origin; however, when hugin neurons are utilized as interneurons, not only is the number of sensory neurons from the same sensory compartment increased, but sensory neurons are added from a completely new peripheral origin. Thus, the hugin interneurons enable sensory inputs from different peripheral origins, for example to integrate enteric inputs with pharyngeal gustatory inputs, to influence an output response, which, in this case, is to stop feeding (Miroschnikow, 2018).

    The coexistence of polysynaptic and monosynaptic paths could also be relevant for circuit variability and compensation: destruction of any given path would still enable the circuit to function, but with more restrictions on the precise types of sensory information it can respond to. In certain cases, this may even lead to strengthening of alternate paths as a form of synaptic plasticity (Miroschnikow, 2018).

    An open issue is how the sensory synaptic compartments might be connected to the feeding central pattern generators (CPGs) which have been demonstrated to exist in the SEZ, especially since CPGs are defined as neural circuits that can generate rhythmic motor patterns in the absence of sensory input. However, the modulation of CPG rhythmic activity can be brought about by sensory and neuromodulatory inputs. A complete circuit reconstruction of the larval SEZ circuit may shed some light on the circuit structure of feeding CPGs (Miroschnikow, 2018).

    A more complex circuit architecture is represented by the MB, the site of associative learning and memory in insects: a completely different set of sensory synaptic compartments is used to connect the various projection neurons to the MB calyx. Thus, the MB module is not superimposed onto the monosynaptic reflex circuits but rather forms a separate unit. The classical studies by Pavlov demonstrated conditioned reflex based on an external signal and an autonomic secretory response in response to food. Although a comparable autonomic response has not been analyzed in the larvae, analogous associative behavior based on odor choice response has been well studied. It is also noteworthy that in the Aplysia, classical conditioning of the gill withdrawal reflex involves monosynaptic connections between a sensory neuron (mechanosensory) and a motor neuron, and neuromodulation by serotonin. This constellation has similarities with the elemental feeding circuit consisting of sensory, motor and serotonergic modulatory neurons. For more complex circuits of feeding behavior in the mouse, a memory device for physiological state, such as hunger, has been reported involving synaptic and neuropeptide hormone circuits. Functional studies on MB output neurons such as the MBON-f1, which may be part of a 'psychomotor' pathway and which targets a number of interneurons that connect to the neurosecretory, serotonergic and pharyngeal motor neurons, may help address how memory circuits interact with feeding circuits (Miroschnikow, 2018).

    Feeding behavior manifests itself from the most primitive instincts of lower animals, to deep psychological and social aspects in humans. It encompasses cogitating on the finest aspects of food taste and the memories evoked by the experience, to sudden reflex reactions upon unexpectedly biting down on a hard seed or shell. Both of these extremes are mediated, to a large degree, by a common set of feeding organs, but the way these organs become utilized can vary greatly. The architecture of the feeding circuit described in this study allows the various types of sensory inputs to converge on a limited number of output responses. The monosynaptic pathways would be used when fastest response is needed. The presence of polysynaptic paths would enable slower and finer control of these reflex events by allowing different sensory inputs, strengths or modalities to act on the monosynaptic circuit. This can be placed in the context in the control of emotions and survival circuits, or by cortex regulation of basic physiological or autonomic processes. In a striking example, pupil dilation, a reflex response, has been used as an indicator of cognitive activity. Here, a major function of having more complex circuit modules on top of monosynaptic circuits may be to allow a finer regulation of feeding reflexes, and perhaps of other reflexes or instinctive behaviors (Miroschnikow, 2018).

    As an outlook, this analysis provides an architectural framework of how a feeding circuit is organized in the CNS. The circuit is divided into two main axes that connect the input to the output systems: the sensory-neurosecretory cell axis and the sensory-motor neuron axis. The sensory system targets overlapping domains of the output neurons; for example, a set of sensory neurons targets exclusively the neuroendocrine cells, other targets both neuroendocrine and pharyngeal motor neurons, and another just the pharyngeal motor neurons. The inputs derive mostly from the internal organs. These connections form the monosynaptic reflex circuits. With these circuits, one can perform the major requirements of feeding regulation, from food intake and ingestion to metabolic homeostasis. Additional multisynaptic circuits, such as the CPGs, those involving sensory signaling from the somatosensory system (external inputs), or those comprising the memory circuits, are integrated or added to expand the behavioral repertoire of the animal (see Input-output synaptic organization of the larval feeding system and its connectivity architecture in the brain). Although circuit construction may proceed from internal to the external, the sequence is reversed in a feeding animal: the first sensory cues are external (olfactory), resulting in locomotion (somatic muscles) that can be influenced by memory of previous experience; this is followed by external taste cues, resulting in food intake into the mouth; the final action is the swallowing of food, involving pharyngeal and enteric signals and reflex circuits. However, regardless of the types of sensory inputs, and whether these are transmitted through a reflex arc, a memory circuit or some other multisynaptic circuits in the brain, they will likely converge onto a certain set of output neurons, what Sherrington referred to as the 'final common path'. The current work is a first step towards finding the common paths (Miroschnikow, 2018)

    secCl is a cys-loop ion channel necessary for the chloride conductance that mediates hormone-induced fluid secretion in Drosophila

    Organisms use circulating diuretic hormones to control water balance (osmolarity), thereby avoiding dehydration and managing excretion of waste products. The hormones act through G-protein-coupled receptors to activate second messenger systems that in turn control the permeability of secretory epithelia to ions like chloride. In insects, the chloride channel mediating the effects of diuretic hormones was unknown. Surprisingly, this study found a pentameric, cys-loop chloride channel, secCl (CG7589), a type of channel normally associated with neurotransmission, mediating hormone-induced transepithelial chloride conductance. This discovery is important because: 1) it describes an unexpected role for pentameric receptors in the membrane permeability of secretory epithelial cells, and 2) it suggests that neurotransmitter-gated ion channels may have evolved from channels involved in secretion (Feingold, 2019).

    Food-derived volatiles enhance consumption in Drosophila melanogaster

    Insects use multiple sensory modalities when searching for and accepting a food source, in particular odor and taste cues. Food-derived odorants are generally involved in mediating long-and short-range attraction. Taste cues, on the other hand, act directly by contact with the food source, promoting the ingestion of nutritious food and the avoidance of toxic substances. It is possible, however, that insects integrate information from these sensory modalities during the process of feeding itself. Using a simple feeding assay, this study investigated whether odors modulate food consumption in the fruit fly Drosophila melanogaster. The presence of both single food-derived odorants and complex odor mixtures enhanced consumption of an appetitive food. Feeding enhancement depended on the concentration and the chemical identity of the odorant. Volatile cues alone were sufficient to mediate this effect, as feeding was also increased when animals were prevented from contacting the odor source. Both males and females, including virgin females, increased ingestion in the presence of food-derived volatiles. Moreover, the presence of food-derived odorants significantly increased the consumption of food mixtures containing aversive bitter compounds, suggesting that flies integrate diverse olfactory and gustatory cues to guide feeding decisions, including in situations in which animals are confronted with stimuli of opposite valence. Overall, these results show that food-derived olfactory cues directly modulate feeding in D. melanogaster, enhancing ingestion (Reisenman, 2019).

    Live yeast in juvenile diet induces species-specific effects on Drosophila adult behaviour and fitness

    The presence and the amount of specific yeasts in the diet of saprophagous insects such as Drosophila can affect their development and fitness. However, the impact of different yeast species in the juvenile diet has rarely been investigated. This study measured the behavioural and fitness effects of three live yeasts (Saccharomyces cerevisiae = SC; Hanseniaspora uvarum = HU; Metschnikowia pulcherrima = MP) added to the diet of Drosophila melanogaster larvae. Beside these live yeast species naturally found in natural Drosophila populations or in their food sources, the inactivated "drySC" yeast widely used in Drosophila research laboratories was tested. All flies were transferred to drySC medium immediately after adult emergence, and several life traits and behaviours were measured. These four yeast diets had different effects on pre-imaginal development: HU-rich diet tended to shorten the "egg-to-pupa" period of development while MP-rich diet induced higher larval lethality compared to other diets. Pre- and postzygotic reproduction-related characters (copulatory ability, fecundity, cuticular pheromones) varied according to juvenile diet and sex. Juvenile diet also changed adult food choice preference and longevity. These results indicate that specific yeast species present in natural food sources and ingested by larvae can affect their adult characters crucial for fitness (Murgier, 2019).

    Mechanosensory circuits coordinate two opposing motor actions in Drosophila feeding

    Mechanoreception detects physical forces in the senses of hearing, touch, and proprioception. This study shows that labellar mechanoreception wires two motor circuits to facilitate and terminate Drosophila feeding. Using patch-clamp recordings, Mechanosensory neurons (MSNs) in taste pegs of the inner labella and taste bristles of the outer labella were identified, both of which rely on the same mechanoreceptor, NOMPC (no mechanoreceptor potential C), to transduce mechanical deflection. Connecting with distinct brain motor circuits, bristle MSNs drive labellar spread to facilitate feeding and peg MSNs elicit proboscis retraction to terminate feeding. Bitter sense modulates these two mechanosensory circuits in opposing manners, preventing labellar spread by bristle MSNs and promoting proboscis retraction by peg MSNs. Together, these labeled-line circuits enable labellar peg and bristle MSNs to use the same mechanoreceptors to direct opposing feeding actions and differentially integrate gustatory information in shaping feeding decisions (Zhou, 2019).

    Closed-loop optogenetic activation of peripheral or central neurons modulates feeding in freely moving Drosophila

    Manipulating feeding circuits in freely moving animals is challenging, in part because the timing of sensory inputs is affected by the animal's behavior. To address this challenge in Drosophila, the Sip-Triggered Optogenetic Behavior Enclosure ('STROBE') was developed. The STROBE is a closed-looped system for real-time optogenetic activation of feeding flies, designed to evoke neural excitation coincident with food contact. Previous work has demonstrated the STROBE's utility in probing the valence of fly sensory neurons. This study provides a thorough characterization of the STROBE system, demonstrates that STROBE-driven behavior is modified by hunger and the presence of taste ligands, and found that mushroom body dopaminergic input neurons and their respective post-synaptic partners drive opposing feeding behaviors following activation. Together, these results establish the STROBE as a new tool for dissecting fly feeding circuits and suggest a role for mushroom body circuits in processing naive taste responses (Musso, 2019).

    Rapid metabolic shifts occur during the transition between hunger and satiety in Drosophila melanogaster

    Metabolites are active controllers of cellular physiology, but their role in complex behaviors is less clear. This study reports metabolic changes that occur during the transition between hunger and satiety in Drosophila melanogaster. To analyze these data in the context of fruit fly metabolic networks, this study developed Flyscape, an open-access tool. In response to eating, metabolic profiles change in quick, but distinct ways in the heads and bodies. Consumption of a high sugar diet dulls the metabolic and behavioral differences between the fasted and fed state and reshapes the way nutrients are utilized upon eating. Specifically, high dietary sugar was found to increase TCA cycle activity, alter neurochemicals, and deplete 1-carbon metabolism and brain health metabolites N-acetyl-aspartate and kynurenine. Together, this work identifies the metabolic transitions that occur during hunger and satiation, and provides a platform to study the role of metabolites and diet in complex behavior (Wilinski, 2019).

    Jacob and Monod's work on the lac operon showed that metabolites can actively control cellular physiology. Yet, for most of the last century, understanding of metabolism has been confined to its energetic function. Nutrients and their metabolic by-products have energetic value because they provide animals with fuel and biomass to support cellular functions. However, metabolites also have informational value: they function both as messengers by carrying data about the nutrient environment and as transducers by directly controlling gene expression, proteostasis, and signal transduction. In the last decade, the shift in understanding of metabolites from fuel and passive by-products, to dynamic entities that control cellular activities have highlighted the potential implications of metabolic regulation in biology. While the role of metabolic signaling and reprograming has been studied in the fields of development, immunology, and cancer, less is known about how these processes impact the brain, especially in the context of complex behaviors (Wilinski, 2019).

    This study began tackling this question by quantifying the changes in metabolite levels during the transition between hunger and satiety in Drosophila melanogaster fruit fly heads and bodies. While the neuroendocrine pathways involved in hunger and satiety have been studied, the exact metabolite changes that occur during the transition to satiation are unknown. Thus, mapping them is the first step to begin studying the role of metabolic signaling in a complex behavior such as feeding. To ask how diet composition influences metabolite levels,the metabolic profiles of fasted and refed fruit flies fed a high sugar diet were measured for different days. As with many omics studies, understanding how metabolites fit into different cellular pathways and vary across conditions is a major challenge. To this end, Flyscape, an open-access application for Cytoscape that visualizes metabolomics data in the context of D. melanogaster metabolic networks and integrates them with other omics data, such as transcriptomics and proteomics, was created (Wilinski, 2019).

    Using a combination of behavioral, metabolomics, and transcriptional studies and by employing Flyscape, this study shows that fly heads and bodies have largely non-overlapping changes in metabolic profiles between the two feeding states (fasted and refed), and that compared to bodies, heads seem tuned to rapid changes in glucose availability at both the metabolite and transcriptional levels. Consumption of a 30% high sugar diet rapidly dulls differences in metabolic profiles between fasted and refed flies and reprograms the way nutrients are assigned to pathways. Together this work provides a starting point to study the role of metabolism in complex behavior by allowing researchers to exploit a genetically tractable organism in studies of specific diet-linked disorders (Wilinski, 2019).

    Metabolites are biologically active compounds that are more than just fuel for the body: they modulate cellular physiology and play a central role in health and disease. Since their influence on complex behaviors is unclear, this question was studied by first mapping the metabolic changes that underlie the transition between hunger and satiety, which is easy to quantify with behavioral assays. To do this, a feeding protocol was developed that resulted in rapid changes in the foraging and feeding behaviors of Drosophila melanogaster flies and was then used to measure metabolites that change in the heads and bodies during the shift between hunger and satiety. It was also asked how short- and long-term consumption of a high sugar diet, which is known to promote obesity and alter feeding patterns in flies, changes acute behavioral and metabolic responses to fasting and refeeding. To aid the analysis of D. melanogaster metabolomics data, Flyscape, an open-access application for Cytoscape where users can visualize and understand metabolomics data in the context of D. melanogaster networks, was developed (Wilinski, 2019).

    It was found that while metabolites change rapidly upon refeeding in both the head and body tissues, the responses of each to fasting and eating are distinct. Overall, heads show large fold increases in glycolytic, pentose-phosphate, 1-carbon metabolism, and hexosamine biosynthesis pathways intermediates that were largely absent in bodies, pointing to a faster response to nutrient availability. Consistent with this observation, it was found that the RNA abundance of several nutrient transporters and metabolic enzymes also changes rapidly in the brains of fasted and refed flies. Of note, the levels of biosynthetic precursors to neurotransmitters responded to feeding state only in head tissue even when present in both samples: choline, N-acetyl serotonin, glutamate, and GABA increased in refed flies, while aspartate and N-acetylaspartate (NAA) were higher in fasted animals. Since these molecules have known or emerging roles in modulating cellular behavior, it is tempting to speculate that the changes that were observed could have informational value and, thus, affect brain function. However, it is also possible that the behaviors with the fasted and refed states have little to do with metabolite levels, and are instead mediated by circuit and synaptic mechanisms that are modulated by hormone levels, neuropeptide signaling, and inter-organ communication. Nonetheless, considering that the internal energy state of animals influences many behaviors, including feeding, learning and memory, and sleep, this work on the identification of the metabolic signatures characterizing these states, opens the road to functionally test the role of metabolic signaling in behavior (Wilinski, 2019).

    Consumption of a high sugar diet profoundly altered the metabolic profiles of fly bodies. Importantly, many of the metabolic hallmarks that characterize humans with obesity occurred in flies fed a high sugar diet, such as elevations in branched-chain amino acids, advanced-glycan products, glutamate, and α-ketoglutarate, are consistent with the findings that fruit flies fed a high sugar diet develop obesity-related illnesses. Bodies also showed signs of TCA cycle dysfunction and a depletion in nucleotide metabolism, as previously observed in tissues of obese mammals and humans. These findings extend previous studies on the effect of a high sugar diet on the metabolism of fruit fly larvae and provide an inroad to study the contributions of these metabolic changes contribute to obesity and metabolic disease in a genetically tractable model organism (Wilinski, 2019).

    In both heads and bodies, a high sugar diet also led to a flattening of the difference in metabolite levels between the fasted and refed state. Given that fluctuations in metabolites critically control cell physiology through gene regulation, protein modification, and second messenger signaling, an enticing question is if and how this metabolic dulling impacts cell function and physiology. For example, it was observed that in heads nearly all the fluctuations in the levels of neurotransmitters and their precursors between feeding states disappeared with both acute and long-term consumption of the high sugar diet, but whether these or other changes in metabolites levels with fasting and sugar diet exposure influence neuronal circuit function and behavior remains to be seen (Wilinski, 2019).

    Metabolic remodeling has been widely studied in both stem cells and cancer and found to play a crucial role in cell physiology and disease progression. To see if consumption of a high sugar diet also leads to a shift in head metabolic state, the metabolic profiles of the heads of flies fed a high sugar diet was examined for 2, 5 and 7 days. It was found that longer exposure to this diet led to higher TCA cycle and hexosamine biosynthesis and lower glycolytic activity. The hexosamine biosynthesis pathway is considered a sensor of cellular sugar levels and is involved in modulating feeding behavior, especially when animals eat a high sugar diet. Neurons have high demand for cellular ATP for action potential generation and the restoration of membrane potential, but glycolysis for fuel is dispensable and inefficient in neurons, and instead used for cellular signaling events, such as those occurring in glucosensing neurons or synaptic plasticity. The high need for energy is met by glia, which metabolize glucose and trehalose into TCA cycle intermediates such as lactate and pyruvate, which are then transported into neurons to fuel cellular processes. The decrease in glycolytic and the increase in TCA intermediates that was found in the heads of flies on a high sugar diet, raises the question of how these changes impact the proper functioning of neurons, especially in the contexts of cellular bioenergetics, neurodegenerative diseases, and information exchange. Exposure to a high sugar diet also depleted metabolites involved in 1-carbon metabolism. Given the relationship between 1-carbon metabolism, histone/DNA/RNA methylation levels, and gene expression, an exciting question is how changes in brain function and behavior with diet-induced obesity may be related to alterations in gene expression due to this reprogramming of metabolism. Finally, the metabolites NAA and kynurenine also showed depletion in the heads of flies on a high sugar diet. NAA, the second most abundant human brain metabolite, is lower in the brains of people with a variety of neuronal diseases and conditions, including depression, schizophrenia, dementia, Alzheimer's, stroke, and traumatic brain injury. Like human brains, fly heads contain high levels of NAA; it was found that these change with internal energy state and diet. In flies, high internal energy conditions, either due to the refed state or consumption of a high sugar diet, had lower NAA, while fasting increased it. Interestingly, NAA levels are also lower in the brains of humans with obesity. Thus, these data suggest the possibility that NAA is not only a sentinel of brain health, but also a marker of the overall brain energy state and point to a potential role for this metabolite in fueling cellular energetics in a stress or nutrient-deprived state. Experiments that functionally address the effects of different NAA levels on brain physiology, will help elucidate the function of this metabolite. The changes in kynurenine levels with fasting and a high sugar diet are also worth noting. Kynurenine and kynurenic acid, the by-products of tryptophan degradation, are increased by exercise, decreased by chronic-stress and trauma, and were recently linked to the physiology of depression. Together, the link between obesity and depression, and the current data showing that kynurenine is correlated with brain energy state and feeding behavior, warrant a deeper investigation on the role of this metabolite in the fine-grained control of food intake, energy balance, and mood (Wilinski, 2019).

    Overall, the data suggest that the internal energy state of the animal, whether it is fasting, satiety, or a high sugar diet alters the way in which nutrients are assigned to metabolic pathways. Metabolic adaptations to environmental and nutritional challenges vary depending on the tissue metabolic needs. While a few of these changes occur gradually and dull metabolite fluctuations between the fasted and fed states, the data show that most transitions are new and may reflect a passage to a new state, reminiscent of some sort of metabolic reprogramming. Mapping these metabolic transitions is the first step towards understanding their potential effects on the physiology of the brain and the role of metabolic signaling in the development of brain conditions associated with diet, such as neurodegeneration, depression, and seizures. In particular, by pinpointing the metabolites that characterize different internal energy states, this work has shed light on the types of metabolic information available to potentially modulate complex behaviors that change with internal energy, such as feeding, sleep, mood, and cognition. While this manuscript does not draw any causal connections between metabolites levels and complex behavior, this analysis provides a springboard to study the function of metabolites as messengers and transducers of environmental information in neuroscience (Wilinski, 2019).

    A neural circuit arbitrates between persistence and withdrawal in hungry Drosophila

    In pursuit of food, hungry animals mobilize significant energy resources and overcome exhaustion and fear. How need and motivation control the decision to continue or change behavior is not understood. Using a single fly treadmill, this study shows that hungry flies persistently track a food odor and increase their effort over repeated trials in the absence of reward suggesting that need dominates negative experience. It was further shown that odor tracking is regulated by two mushroom body output neurons (MBONs) connecting the MB to the lateral horn. These MBONs, together with dopaminergic neurons and Dop1R2 signaling, control behavioral persistence. Conversely, an octopaminergic neuron, VPM4, which directly innervates one of the MBONs, acts as a brake on odor tracking by connecting feeding and olfaction. Together, these data suggest a function for the MB in internal state-dependent expression of behavior that can be suppressed by external inputs conveying a competing behavioral drive (Sayin, 2019).

    Flexibility is an important factor in an ever in-flux environment, where scarcity and competition are the norm. Without persistence to achieve its goals, however, an animal's strive to secure food, protect its offspring, or maintain its social status is in jeopardy. Therefore, sensory cues related to food or danger often elicit strong impulses. However, these impulses must be strictly controlled to allow for coherent goal-directed behavior and to permit behavioral transitions when sensible. Inhibition of antagonistic behavioral drives at the cognitive and physiological level has been proposed as a major task of a nervous system. Which sensory cues and ultimately which behaviors are prioritized and win depends on the animal's metabolic state, internal motivation, and current behavioral context. How this is implemented at the level of individual neurons, circuit motifs, and mechanisms remains an important open question (Sayin, 2019).

    Like most animals, energy-deprived flies prioritize food seeking and feeding behavior. To find food, flies can follow olfactory or visual cues over long distances. External gustatory cues provide information about the type and quality of the eventually encountered food. However, only internal nutrient levels will provide reliable feedback about the quality and quantity of a food source and ultimately suppress food-seeking behaviors. Therefore, food odor, the taste of food, and post-ingestive internal feedback signals induce sequential and partly antagonistic behaviors. Interestingly, chemosensory and internal feedback systems typically mediated by distinct neuromodulators appear to converge in the mushroom body (MB). How neurons and neural circuits signal and combine external and internal cues to maintain or suppress competing behavioral drives is not well understood (Sayin, 2019).

    In mammals, norepinephrine (NE) released by a brain stem nucleus, the locus coeruleus, has been implicated in controlling the balance between persistence and action selection. The potential functional counterpart of NE in insects could be octopamine (OA). Flies lacking OA indeed show reduced arousal, for instance upon starvation. Additionally, OA neurons (OANs) gate appetitive memory formation of odors and also modulate taste neurons and feeding behavior. OANs are organized in distinct clusters and project axons to diverse higher brain regions in a cell type-specific manner. The precise roles and important types of OA and NE neurons in state-dependent action selection remain to be elucidated (Sayin, 2019).

    Similar to NE and OA, dopamine (DA) is being studied in many aspects of behavioral adaptation and flexibility. Different classes of DA neurons (DANs) innervating primarily the MB signal negative or positive context, or even wrong predictions (Sayin, 2019 and references therein).

    This study took advantage of the small number and discrete organization of neuromodulatory neurons in the fly brain to analyze the mechanistic relationship between motivation-dependent persistence in one behavior and the decision to disengage and change to another behavior. Using a single fly spherical treadmill assay, this study found that hungry flies increase their effort to track a food odor with every unrewarded trial. MB output through two identified MBONs (MBON-γ1pedc>α/β and MBON-α2sc) is required for persistent odor tracking. MBON-α2sc provides a MB connection to the lateral horn (LH), where it can modify innate food odor attraction. Furthermore, this study pinpoints a specific type of OAN, VPM4 (ventral paired medial), which connects feeding centers directly to MBON-γ1pedc>α/β and disrupts food odor tracking. Finally, the experimental data suggest that persistent tracking depends on DANs, including PPL1-γ1pedc, and signaling through dopamine receptor Dop1R2 in αβ-type KCs. Based on these results, it is proposed that MB output and a direct external input, depending on internal state and motivation, gradually promote or interrupt ongoing behavior (Sayin, 2019).

    What drives gradually increasing persistence in behavior? For the fly, a model is proposed by which a circuit module of KCs, MBONs, and DANs drive gradually increasing odor tracking, which can be efficiently suppressed by extrinsic MBON-innervating feeding-related OANs. Behavioral persistence has been previously analyzed in flies in a different context. For instance, courtship of fly males and copulation with a female are maintained by dopaminergic neurons in the ventral nerve cord, where they counteract GABAergic neurons. In that scenario, DANs in the ventral nerve cord maintain an ongoing behavior and prevent male premature disengagement before successful insemination (Sayin, 2019).

    The experimental data also implicate DANs, primarily from within the PPL1 (e.g., PPL1-γ1pedc) and PPL2ab clusters, and Dop1R2 signaling. In particular, inactivation of synaptic output of DANs positive for TH-Gal4 as well as loss of Dop1R2 in αβ-type KCs reduced the increase in odor tracking from trial to trial, while not affecting the speed at first odor stimulation. These data suggest that TH+ DANs promote goal-directed movement, i.e., odor tracking, through a Dop1R2-dependent mechanism in KCs (Sayin, 2019).

    MBON-γ1pedc>αβ, which receives dopaminergic input by PPL1-γ1pedc, is required for odor tracking. Moreover, this study also observed a trial-to-trial decrease in odor response of this MBON, matching the dopamine-induced synaptic depression previously observed in MBONs upon learning. Notably, PPL1-γ1pedc activates Dop1R2 in MBON-γ1pedc>αβ, a signal recently found to be critical for appetitive long-term memory. Nevertheless, it appears that, in addition to PPL1-γ1pedc, other DANs regulate behavioral persistence by modulating in particular αβ-KCs. It is intriguing to speculate about a common function of Dop1R2 in the formation of long-lasting aversive memory induced by repeatedly pairing odor with an aversive experience and the behavior examined in this study: increased and persistent expression of a behavior induced by the experience of repeated failure to reach a goal (Sayin, 2019).

    The experimental data further implicated MBON-α2sc, which is connected to MBON-γ1pedc>αβ. Calcium imaging data are consistent with an inhibitory interaction between the two MBONs. However, some of the behavioral data and prior imaging data do not support an inhibitory connection. Furthermore, MBON-γ1pedc>αβ projects to other brain regions and downstream targets, and similarly MBON-α2sc receives additional inputs—all of which could be equally or more important for persistent behavior than a direct connection between these two MBONs. Finally, some DANs respond to movement, including PPL1-γ2α'1/MV1. Although no essential role of this particular neuron was found in odor tracking persistence, movement might contribute to the activity of MBONs responding the odorant (Sayin, 2019).

    Remarkably, MBON-α2sc connects the MB to neurons within the LH. Thus, it is speculated that the LH might assign an odor to its corresponding behavioral category, such as 'food-related' for vinegar, while the MB acts as a top-down control to gauge the expression of an innate behavior (i.e., tracking an appetitive odor) according to state and experience (Sayin, 2019).

    The behavioral data led to the proposal of a circuit model. Using computational modeling, this study tested whether the MB network including DANs and MBONs could, in theory, produce the observed behavior. Indeed, it was found that a simplified recurrent circuit of KCs, DANs, and MBONs can account for the observed behavioral persistence and also the measured MBON-γ1pedc>αβ odor responses. While this model cannot replace experimental evidence, it forms a useful theoretical framework for future studies on the role of the MB in behavioral persistence (Sayin, 2019).

    Based on the present data and computational predictions, a model is proposed by which the recurrent circuit architecture of the MB, in addition to storing information for future behavior, is ideally suited to maintain and gradually change ongoing behavior, for instance by modulating output of the LH, according to the animal's internal state and needs (Sayin, 2019).

    The use of an olfactory treadmill has allowed dissection of the different aspects of a food search. In particular, how does food and feeding suppress food search if the sensory cue, the odor, is still present? OA-VPM4 connects feeding centers (i.e., SEZ) directly with odor tracking-promoting MBON-γ1pedc>αβ and inhibits its activity suggesting an inhibitory connection between VPM4 and the MBON. Nevertheless, it cannot be excluded that OA-VPM4 signals through multiple mechanisms including OA and possibly other neurotransmitters. In addition, a recent study showed that activation of VPM4 promotes proboscis extension to sugar. Although a direct role in taste detection through pharynx or labellum appears unlikely, it is possible that feeding behavior itself (e.g., lymphatic sugar, food texture, activity of feeding muscles) are detected and/or promoted by these neurons and then brought to the MB. It is proposef that VPM4 is a direct mediator between olfactory-guided food search and the rewarding experience of feeding and related behavior (Sayin, 2019).

    The data provide a neural circuit mechanism empowering flies to express and prioritize behavior in a need- and state-dependent manner. It is exciting to speculate that fundamentally similar circuit motifs might exist in NE and DA neuron-containing circuits in the mammalian brain, governing the organization of behavior in a flexible and context-dependent manner by integrating internal and external context. For instance, noradrenergic neurons of the brainstem nucleus of the solitary tract (NST) receive taste information, and input from the gastrointestinal tracts, lungs, and heart. Neurons in the NST project to multiple brain regions including the amygdala, hypothalamus, and insular cortex, all of which receive internal state as well as other sensory information (Sayin, 2019).

    The data in the fly provide an experimental and theoretical framework for a better understanding of the fundamental circuit mechanisms underpinning neuromodulation of context-dependent behavioral persistence and withdrawal (Sayin, 2019).

    Flying Drosophila show sex-specific attraction to fly-labelled food

    Animals searching for food and sexual partners often use odourant mixtures combining food-derived molecules and pheromones. For orientation, the vinegar fly Drosophila melanogaster uses three types of chemical cues: (i) the male volatile pheromone 11-cis-vaccenyl acetate (cVA), (ii) sex-specific cuticular hydrocarbons (CHs; and CH-derived compounds), and (iii) food-derived molecules resulting from microbiota activity. To evaluate the effects of these chemicals on odour-tracking behaviour, Drosophila individuals were tested in a wind tunnel. Upwind flight and food preference were measured in individual control males and females presented with a choice of two food sources labelled by fly lines producing varying amounts of CHs and/or cVA. The flies originated from different species or strains, or their microbiota was manipulated. The following was found: (i) fly-labelled food could attract-but never repel-flies; (ii) the landing frequency on fly-labelled food was positively correlated with an increased flight duration; (iii) male-but not female or non-sex-specific-CHs tended to increase the landing frequency on fly-labelled food; (iv) cVA increased female-but not male-preference for cVA-rich food; and (v) microbiota-derived compounds only affected male upwind flight latency. Therefore, sex pheromones interact with food volatile chemicals to induce sex-specific flight responses in Drosophila (Cazale-Debat, 2019).

    Muscle-derived Dpp regulates feeding initiation via endocrine modulation of brain dopamine biosynthesis

    In animals, the brain regulates feeding behavior in response to local energy demands of peripheral tissues, which secrete orexigenic and anorexigenic hormones. Although skeletal muscle is a key peripheral tissue, it remains unknown whether muscle-secreted hormones regulate feeding. In Drosophila, this study found that decapentaplegic (dpp), the homolog of human bone morphogenetic proteins BMP2 and BMP4, is a muscle-secreted factor (a myokine) that is induced by nutrient sensing and that circulates and signals to the brain. Muscle-restricted dpp RNAi promotes foraging and feeding initiation, whereas dpp overexpression reduces it. This regulation of feeding by muscle-derived Dpp stems from modulation of brain tyrosine hydroxylase (TH) expression and dopamine biosynthesis. Consistently, Dpp receptor signaling in dopaminergic neurons regulates TH expression and feeding initiation via the downstream transcriptional repressor Schnurri. Moreover, pharmacologic modulation of TH activity rescues the changes in feeding initiation due to modulation of dpp expression in muscle. These findings indicate that muscle-to-brain endocrine signaling mediated by the myokine Dpp regulates feeding behavior (Robles-Murguia, 2020).

    Food restriction reconfigures naive and learned choice behavior in Drosophila larvae

    In many animals, the establishment and expression of food-related memory is limited by the presence of food and promoted by its absence, implying that this behavior is driven by motivation. In the past, this has already been demonstrated in various insects including honeybees and adult Drosophila. For Drosophila larvae, which are characterized by an immense growth and the resulting need for constant food intake, however, knowledge is rather limited. Accordingly, this study has analyzed whether starvation modulates larval memory formation or expression after appetitive classical olfactory conditioning, in which an odor is associated with a sugar reward. Odor-sugar memory of starved larvae was shown to last longer than in fed larvae, although the initial performance is comparable. 80 minutes after odor fructose conditioning, only starved but not fed larvae show a reliable odor-fructose memory. This is likely due to a specific increase in the stability of anesthesia-resistant memory (ARM). Furthermore, it was observe that starved larvae, in contrast to fed ones, prefer sugars that offer a nutritional benefit in addition to their sweetness. Taken together this work shows that Drosophila larvae adjust the expression of learned and naive choice behaviors in the absence of food. These effects are only short-lasting probably due to their lifestyle and their higher internal motivation to feed. In the future, the extensive use of established genetic tools will allow identification of development-specific differences arising at the neuronal and molecular level (Brunner, 2020).

    Serotonin transporter dependent modulation of food-seeking behavior

    The olfactory pathway integrates the odor information required to generate correct behavioral responses. To address how changes of serotonin signaling in two contralaterally projecting, serotonin-immunoreactive deutocerebral neurons impacts key odorant attraction in Drosophila melanogaster, this study selectively altered serotonin signaling using the serotonin transporter with mutated serotonin binding sites in these neurons, and the consequence on odorant-guided food seeking was analyzed. The expression of the mutated serotonin transporter selectively changed the odorant attraction in an odorant-specific manner. The shift in attraction was not influenced by more up-stream serotonergic mechanisms mediating behavioral inhibition. The expression of the mutated serotonin transporter in CSD neurons did not influence other behaviors associated with food seeking such as olfactory learning and memory or food consumption. Evidence is provided that the change in the attraction by serotonin transporter function might be achieved by increased serotonin signaling and by different serotonin receptors. The 5-HT1B receptor positively regulated the attraction to low and negatively regulated the attraction to high concentrations of acetic acid. In contrast, 5-HT1A and 5-HT2A receptors negatively regulated the attraction in projection neurons to high acetic acid concentrations. These results provide insights into how serotonin signaling in two serotonergic neurons selectively regulates the behavioral response to key odorants during food seeking (He, 2020).

    CCAP regulates feeding behavior via the NPF pathway in Drosophila adults

    The intake of macronutrients is crucial for the fitness of any animal and is mainly regulated by peripheral signals to the brain. How the brain receives and translates these peripheral signals or how these interactions lead to changes in feeding behavior is not well-understood. This study discovered that 2 crustacean cardioactive peptide (CCAP)-expressing neurons in Drosophila adults regulate feeding behavior and metabolism. Notably, loss of CCAP, or knocking down the CCAP receptor (CCAP-R) in 2 dorsal median neurons, inhibits the release of neuropeptide F (NPF), which regulates feeding behavior. Furthermore, under starvation conditions, flies normally have an increased sensitivity to sugar; however, loss of CCAP, or CCAP-R in 2 dorsal median NPF neurons, inhibited sugar sensitivity in satiated and starved flies. Separate from its regulation of NPF signaling, the CCAP peptide also regulates triglyceride levels. Additionally, genetic and optogenetic studies demonstrate that CCAP signaling is necessary and sufficient to stimulate a reflexive feeding behavior, the proboscis extension reflex (PER), elicited when external food cues are interpreted as palatable. Dopaminergic signaling was also sufficient to induce a PER. On the other hand, although necessary, NPF neurons were not able to induce a PER. These data illustrate that the CCAP peptide is a central regulator of feeding behavior and metabolism in adult flies, and that NPF neurons have an important regulatory role within this system (Williams, 2020).

    This study demonstrates that 2 residual CCAP neurons not only signal for food intake but also have a role in regulating metabolism, where they are important for maintaining triglyceride levels. CCAP signaling activates an NPF pathway for proper sensing of sugars for food intake. Of note, flies lacking CCAP, or CCAP-R in 2 dorsal median P1 NPF neurons, are not able to distinguish nutritive from nonnutritive sugar. However, CCAP signaling to these P1 NPF neurons is not sufficient for the NPF feeding phenotypes, as knocking down CCAP-R specifically in these neurons or inhibiting these 2 neurons using the inward-rectifying channel Kir2.1 was not able to recapitulate the phenotypes. This hints at other CCAP- and NPF-regulated neurons being involved in the control of feeding behavior. One possibility is the peripheral L1-I NPF neurons, as they did react to starvation by increasing NPF protein levels and exhibited increased NPF protein levels when CCAP neurons were activated. When flies sense palatable food, a reflexive behavior known as the proboscis extension reflex is initiated. This study shows that CCAP is both necessary and sufficient to induce this reflex. Moreover, dopaminergic neurons are also sufficient to induce this response, but not NPF neurons. Thus, this study has identified CCAP as a possible key node in regulating feeding behavior (Williams, 2020).

    Previously it was shown that under acute starvation one of the Drosophila NPY homologs, NPF, initiates a response that activates dopaminergic signaling, leading to the sensitization of gustatory neurons (Gr5a) toward sugar taste. This study shows that Drosophila CCAP regulates the activity of the 2 dorsal median P1 NPF neurons and that this is sufficient to control food intake. First, CCAP neurons project toward the 2 dorsal median P1 NPF neurons. Second, knocking down CCAP expression, or CCAP-R expression in the dorsal median P1 NPF neurons, increases NPF expression under ad libitum conditions (CCAP-R knockdown), as well as in response to starvation (both CCAP and CCAP-R). It is interesting that loss of CCAP-R already significantly influences NPF expression under conditions where flies are fed ad libitum. Possibly, NPF is being released at low levels even under fed conditions. It is known that NPF also regulates sleep and the reward system. CCAP may signal upstream of NPF to regulate sleep and reward as well. This possibility should be tested in the future. Furthermore, activating CCAP neurons using thermogenetics was sufficient to reduce NPF expression in the dorsal median P1 NPF neurons, indicating the neurons were more active. On the other hand, activating CCAP neurons increased NPF expression in the dorsal lateral L1-I neurons, indicating that activation of CCAP neurons inhibited these NPF-expressing neurons. This increase of NPF in the peripheral L1-I neurons was also observed when flies were starved. From these data, it is concluded that activating CCAP neurons in turn activates 2 dorsal median NPF neurons, leading to sugar sensitization. Tge inability to recapitulate the CCAPexc7 feeding phenotypes by knocking down CCAP-R expression specifically in the dorsal median P1 NPF neurons, or inhibiting these 2 neurons using the inward-rectifying channel Kir2.1, may indicate that the peripheral L1-I NPF neurons also play an important role in regulating food intake. More work is needed to understand CCAP's possible regulation of the peripheral neurons, as loss of CCAP had no influence on NPF protein levels in these neurons (Williams, 2020).

    This study found that CCAP neurons were not only necessary, but also sufficient, to induce the PER. Previously, by the use of optogenetics, it was determined that Gr5a neurons were sufficient to induce the PER. On the other hand, although NPF neurons and dopaminergic neurons were determined to be necessary for a proper PER when flies were presented with varying concentrations of sugar, it was not established whether they were sufficient. Using optogenetics, this study determined that while dopaminergic neurons (ple-GAL4) were able to induce a PER, NPF neurons (NPF-GAL4 or R64F05-GAL4) were not sufficient (Williams, 2020).

    Interestingly, adult flies lacking the CCAP peptide had significantly lower triglyceride levels. This was not observed when CCAP-R was specifically knocked down in NPF neurons. In order to maintain homeostasis, the brain must process extrinsic and intrinsic information. In Drosophila, different peptides have been shown to regulate these signals, such as diuretic hormone 44 (Dh44), corazonin (Crz), allatostatin A (AstA), and SIFamide (SIFa). Furthermore, similar to mammals, insulin-like peptides and a glucagon-like hormone (AKH) are also involved in regulating feeding behavior. Interestingly, Dh44 was shown to be necessary for the fly to sense postprandial nutritional information, and this study showed that flies lacking either CCAP or CCAP-R in NPF neurons were unable to determine between nutritional and nonnutritional sugars. That said, it must be mentioned this experiment only lasted 1 h and longer times may be necessary to truly understand if CCAP is involved in regulating postprandial nutritional signals. Another possibility is that CCAP neurons regulate Crz signaling. Crz is a Drosophila peptide related to mammalian gonadotropin-releasing hormone. Activation of Crz-expressing neurons was reported to reduce triglyceride levels, while loss of Crz regulation of insulin-producing cells leads to increased triglyceride storage, suggesting that Crz signals to decrease energy reserves. It is possible that CCAP signals to inhibit Crz in order to control the decrease in energy reserves under starvation conditions. Furthermore, loss or activation of SIFa neurons in adult flies produced feeding phenotypes very similar to when CCAP signaling is inhibited or activated, meaning there could be an interaction between SIFa and CCAP as well (Williams, 2020).

    In summary, these experiments identify 2 CCAP peptidergic neurons as being required to induce feeding behavior via the NPF pathway in adult Drosophila. It is suggested that CCAP-expressing neurons regulate feeding behavior and are necessary for the proper sensing of sugars, while also regulating triglyceride levels. Continued studies of these 2 CCAP neurons, their neuronal network, as well as how they regulate feeding behavior and metabolism may help in understanding of satiety control and how peripheral physiological signals are translated into behavioral changes by the brain (Williams, 2020).

    Cellular Basis of Bitter-Driven Aversive Behaviors in Drosophila Larva

    Feeding, a critical behavior for survival, consists of a complex series of behavioral steps. In Drosophila larvae, the initial steps of feeding are food choice, during which the quality of a potential food source is judged, and ingestion, during which the selected food source is ingested into the digestive tract. It remains unclear whether these steps employ different mechanisms of neural perception. This study provides insight into the two initial steps of feeding in Drosophila larva. Substrate choice and ingestion were found to be determined by independent circuits at the cellular level. First, 22 candidate bitter compounds were taken, and their influence on choice preference and ingestion behavior was examined. Interestingly, certain bitter tastants caused different responses in choice and ingestion, suggesting distinct mechanisms of perception. Evidence is further provided that certain gustatory receptor neurons (GRNs) in the external terminal organ (TO) are involved in determining choice preference, and a pair of larval pharyngeal GRNs is involved in mediating both avoidance and suppression of ingestion. These results show that feeding behavior is coordinated by a multistep regulatory process employing relatively independent neural elements. These findings are consistent with a model in which distinct sensory pathways act as modulatory circuits controlling distinct subprograms during feeding (Choi, 2020).

    Feeding, a critical behavior for survival, consists of a complex series of behavioral steps. In Drosophila larvae, the initial steps of feeding are food choice, during which the quality of a potential food source is judged, and ingestion, during which the selected food source is ingested into the digestive tract. It remains unclear whether these steps employ different mechanisms of neural perception. This study provides insight into the two initial steps of feeding in Drosophila larva. Substrate choice and ingestion were found to be determined by independent circuits at the cellular level. First, 22 candidate bitter compounds were taken, and their influence on choice preference and ingestion behavior was examined. Interestingly, certain bitter tastants caused different responses in choice and ingestion, suggesting distinct mechanisms of perception. Evidence is provided that certain gustatory receptor neurons (GRNs) in the external terminal organ (TO) are involved in determining choice preference, and a pair of larval pharyngeal GRNs is involved in mediating both avoidance and suppression of ingestion. These results show that feeding behavior is coordinated by a multistep regulatory process employing relatively independent neural elements. These findings are consistent with a model in which distinct sensory pathways act as modulatory circuits controlling distinct subprograms during feeding (Choi, 2020).

    A general assumption would be that a tastant would cause a similar response in ingestion and choice preference behavior, in either a positive or negative manner. However, the current findings corroborate that certain tastants elicit divergent ingestion and choice preference behavior. Combining molecular genetic tools, behavioral assays, and genetically encoded calcium sensors to assess neuronal activity, the results provide evidence that relatively independent neural systems appear to regulate the two initial processes of feeding in Drosophila larva: searching for palatable food, i.e., choice preference, and eating the selected food, i.e., ingestion. A subset of gustatory neurons housed in the TO, the external gustatory organ of Drosophila larva, detect denatonium and induce avoidance behavior, and DP1, a specific pair of GRNs in the dorsal pharyngeal organ, plays a major role in regulating both ingestion and avoidance in response to CAF (Choi, 2020).

    The TO of Drosophila larva is located at the tip of the cephalic lobes, and is thus anatomically likely to be the first organ to contact external stimuli and subsequently cause a change in movement to regulate the initial step of feeding. Similarly, pharyngeal sensilla are located between the external sense organs and digestive organs, and are thus anatomically likely to act in maintaining the ingestion of appetitive foods while stopping ingestion and causing avoidance of aversive cues such as bitter toxins. It could be advantageous for ingestion to be predominantly controlled by pharyngeal sense organs, rather than by external organs, since animals can try out a potential food source before making their decision, rather than blindly avoiding it. This could be a particularly advantageous strategy for insect larvae whose main purpose is to feed. Also, the difference in behavioral responses elicited by the C1 and C7 neurons in the TO and DP1 in the pharyngeal sense organs is likely linked to the difference in brain projection patterns of GRNs from the TO and pharyngeal GRNs from the larval SEZ, with the distinct projection areas of the brain taste center likely being linked to different circuits, resulting in distinct behavioral outputs (Choi, 2020).

    In Drosophila larvae, choice and ingestion have generally been grouped together and studied as a group of reflexive behaviors. Sugar processing provides another intriguing example of divergence between choice and ingestion. Larvae generally show increased preference and feeding when exposed to increasing concentrations of fructose or sucrose. At extremely high concentrations such as 2 M or 4 M, larvae still exhibit preference in terms of choice, but show suppression of feeding (or ingestion, as is denoted in this study). Since this suppression of ingestion could be due to high viscosity and/or osmolarity, a direct comparison to the processing of aversive tastants such as bitter chemicals is difficult. However, this example nonetheless provides evidence that relatively independent circuits exist to determine choice and ingestion. Using bitter tastants, this study found that choice and ingestion can manifest in clearly divergent behaviors to the same compounds and elucidate the cellular basis of these observations. Similarities to the observation that external sense organs and pharyngeal organs appear to be involved in somewhat independent behavioral output can be seen in sugar consumption in the adult fly. The activation of sweet GRNs in the legs and labellum initiates feeding behaviors including the proboscis extension reflex, and pharyngeal sweet GRNs play an important role in directing the sustained consumption of sweet compounds (Choi, 2020).

    Most of the 22 putative bitter tastants tested in this study, including CAF, cause negative effects in choice preference and ingestion. Nicotine caused a positive P.I. in the choice preference assay. It cannot be completely rule out that nicotine could act as an attractive chemosensory cue at low concentrations, this study found that nicotine inhibits the movement of larvae in the experimental setup. Larvae strongly avoid denatonium, but once they sample denatonium-containing food, they ingest it. This ingestion likely occurs because denatonium is added to the agarose of the entire plate, whereas larvae probably would not ingest as much if they had the choice. Nevertheless, the results suggest that this larval response to denatonium is due to the existence of a functional receptor complex for denatonium in the TO, which does not exist in the pharyngeal sense organs, or at the very least the DP1 neuron. Consistently, ectopic expression of GR59c in DP1 caused a novel calcium response to denatonium and suppression of ingestion in response to denatonium. Some remaining questions regarding sensing of denatonium merit further study. Avoidance to denatonium is defective when either C1 or C7 is inactivated, indicating that C1 and C7 are not redundant in terms of behavior. It is possible that a certain threshold of neuronal activity is required to elicit behavior, or inactivation of one neuron may cause a change in the functions of other GRNs. Although a numerically simple system, larval GRNs also have a multimodal character, and as such a more complicated mechanism might be involved. Also, in the bitter sensing neurons of the adult labellum, two complexes, GR32a/GR66a/GR59c and GR32a/GR66a/GR22e, are each sufficient to confer a response to denatonium. Based on Gr-GAL4 expression, the larval DP1 neuron expresses Gr22e, but not Gr59c, but is not capable of detecting denatonium. This suggests that the GRNs of the larva and adult fly possess different cellular contexts, which could be interesting to unravel. An interesting remaining question is if Gr59c is solely responsible for denatonium sensing in the larval C1 neuron or if the existing Gr22e can rescue denatonium sensing in Gr59c mutants. This would indicate that Gr22e needs a specific co-receptor repertoire for denatonium detection and could help elucidate coding differences in the larva versus the adult fly (Choi, 2020).

    The levels at which distinct bitter compounds are detected might reflect the ecological niche of the animal and the toxicity level of a given tastant. The results suggest that information from the DP1 neuron is processed in a circuit that results in negative and aversive behavior in ingestion and choice preference to CAF. The C1 and C7 neuron in the TO elicit avoidance to denatonium in choice preference behavior. Thus, these results suggest that distinct sensory neurons appear to have distinct sensory roles, likely through the expression of specific receptors or specific groups of receptors. Sensory information detected by these sensory neurons appears to be processed through distinct circuits in the central nervous system to mediate changes in ingestion or choice behavior. It is yet unclear whether the different circuits interact to result in a final behavioral output. Further examination of the potential connections between the external and pharyngeal gustatory neurons and interneurons or motor neurons in the brain may provide insight into the overall neural circuit that regulates feeding and locomotion (Choi, 2020).

    High-fat diet enhances starvation-induced hyperactivity via sensitizing hunger-sensing neurons in Drosophila

    The function of the central nervous system to regulate food intake can be disrupted by sustained metabolic challenges such as high-fat diet (HFD), which may contribute to various metabolic disorders. Previous work has shown that a group of octopaminergic (OA) neurons mediated starvation-induced hyperactivity, an important aspect of food-seeking behavior. This study found that HFD specifically enhances this behavior. Mechanistically, HFD increases the excitability of these OA neurons to a hunger hormone named adipokinetic hormone (AKH), via increasing the accumulation of AKH receptor (AKHR) in these neurons. Upon HFD, excess dietary lipids are transported by a lipoprotein LTP to enter these OA(+)AKHR(+) neurons via the cognate receptor LpR1, which in turn suppresses autophagy-dependent degradation of AKHR. Taken together, this study has uncovered a mechanism that links HFD, neuronal autophagy, and starvation-induced hyperactivity, providing insight in the reshaping of neural circuitry under metabolic challenges and the progression of metabolic diseases (Huang, 2020).

    Obesity and obesity-associated metabolic disorders such as type 2 diabetes and cardiovascular diseases have become a global epidemic. Chronic over-nutrition, especially excessive intake of dietary lipids, is one of the leading causes of these metabolic disturbances. Accumulating evidence has shown that HFD imposes adverse effects on the physiology and metabolism of liver, skeletal muscle, the adipose tissue, and the nervous system. It is therefore of importance to understand the mechanisms underlying HFD-induced changes in different organs and cell types, which will offer critical insight into the diagnosis and treatment of obesity and other metabolic diseases (Huang, 2020).

    The central nervous system plays a critical role in regulating energy intake and expenditure. In rodent models, neurons located in the arcuate nucleus of the hypothalamus, particularly neurons expressing Neuropeptide Y (NPY) and Agouti-Related Neuropeptide (AgRP) or those expressing Pro-opiomelanocortin (POMC), are important behavioral and metabolic regulators. These neurons detect various neural and hormonal cues such as circulating glucose and fatty acids, leptin, and ghrelin, and modulate energy intake and expenditure accordingly. Upon the reduction of the internal energy state, NPY/AgRP neurons are activated and exert a robust orexigenic effect. Genetic ablation of NPY/AgRP neurons in neonatal mice completely abolishes food consumption whereas acute activation of these neurons significantly enhances food consumption. NPY/AgRP neurons also antagonize the function of POMC neurons that plays a suppressive role on food consumption. Taken together, these two groups of neurons, among other neuronal populations, work in synergy to ensure a refined balance between energy intake and expenditure, and hence organismal metabolism (Huang, 2020).

    In spite of their critical roles, the function of the nervous system to accurately regulate appetite and metabolism may be disrupted by sustained metabolic stress, resulting in eating disorders and various metabolic diseases such as obesity and type 2 diabetes. Several lines of evidence have begun to reveal the underlying neural mechanisms. For example, HFD increases the intrinsic excitability of orexigenic NPY/AgRP neurons, induces leptin resistance, and enhances their inhibitory innervations with anorexigenic POMC neurons, altogether resulting in hypersensitivity to starvation and increased food consumption. Interestingly, besides HFD, other metabolic challenges, including maternal HFD, alcohol consumption, as well as aging, also disrupt normal food intake via affecting the excitability and/or innervation of NPY/AgRP neurons. All these interventions may contribute to the onset and progression of metabolic disorders (Huang, 2020).

    Before the actual food consumption, food-seeking behavior is a critical yet largely overlooked behavioral component for the localization and occupation of desirable food sources. Food-seeking behavior has been characterized in rodent models, primarily by the elevation of locomotor activity and increased food approach of starved animals. It has been reported that NPY/AgRP neurons also play a role in food-seeking behavior. However, to ensure adequate food intake, food seeking and food consumption are temporally and spatially separated and even reciprocally inhibited. It remains largely unclear how the neural circuitry of food seeking and food consumption segregated and independently regulated in rodent models. Furthermore, it remains unknown whether HFD also affects food seeking, and if so whether its effects on both food seeking and food consumption share common mechanisms or not. To fully understand the intervention of energy homeostasis by sustained metabolic stress, it is necessary to dissect the neural circuitry underlying food seeking and examine whether and how it is affected by HFD (Huang, 2020).

    Fruit flies Drosophila melanogaster share fundamental analogy to vertebrate counterparts on the regulation of energy homeostasis and organismal metabolism despite that they diverged several hundred million years ago. Therefore, it offers a good model to characterize food-seeking behavior in depth and provides insight into the regulation of energy intake and the pathogenesis of metabolic disorders in more complex organisms such as rodents and human (Huang, 2020).

    Previous work showed that fruit flies exhibited robust starvation-induced hyperactivity that was directed towards the localization and acquisition of food sources, therefore resembling an important aspect of food-seeking behavior upon starvation (Yang, 2015). A small subset of OA neurons in the fly brain were identified that specifically regulated starvation-induced hyperactivity (Yu, 2016). Analogous to mammalian systems, a number of neural and hormonal cues are involved in the systemic control of nutrient metabolism and food intake in fruit flies. Among them, Neuropeptide F (NPF), short NPF (sNPF), Leucokinin, and Allatostatin A (AstA), have been shown to regulate food consumption, all of which have known mammalian homologs that regulate food intake. In particular, starvation-induced hyperactivity is regulated by two classes of neuroendocrine cells (Yu, 2016). One is functionally analogous to pancreatic α cells and produce AKH upon starvation, whereas the other produces Drosophila insulin-like peptides (DILPs), resembling the function of pancreatic β cells. These two classes of Drosophila hormones exert antagonistic functions on starvation-induced hyperactivity via the same group of OA neurons in the fly brain (Huang, 2020).

    Based on these findings, this study sought to examine whether HFD disrupted the regulation of starvation-induced hyperactivity in fruit flies and aimed to investigate the underlying mechanism. The present study found that HFD-fed flies became significantly more sensitive to starvation and exhibited starvation-induced hyperactivity earlier and stronger than flies fed with normal diet (ND). Meanwhile, HFD did not alter flies' food consumption, suggesting that starvation-induced hyperactivity and food consumption are independently affected by HFD. Several days of HFD treatment did not alter the production of important hormonal cues like AKH and DILPs, but rather increased the sensitivity of the OA neurons that regulated starvation-induced hyperactivity to the hunger hormone AKH. In these OA neurons, constitutive autophagy maintained the homeostasis of AKHR protein, which determined their sensitivity to AKH and hence starvation. HFD feeding suppressed neuronal autophagy via AMPK-TOR signaling and in turn increased the level of AKHR in these OA neurons. Consistently, eliminating autophagy in these neurons mimicked the effect of HFD on starvation-induced hyperactivity whereas promoting autophagy inhibited the induction of hyperactivity by starvation. Furthermore, this study also showed that a specific lipoprotein LTP and its cognate receptor LpR1 likely mediated the effect of HFD on the neuronal autophagy of OA neurons and hence its effect on starvation-induced hyperactivity. Taken together, this study uncovered a novel mechanism that linked HFD, AMPK-TOR signaling, neuronal autophagy, and starvation-induced hyperactivity, shedding crucial light on the reshaping of neural circuitry under metabolic stress and the progression of metabolic diseases (Huang, 2020).

    There is accumulating evidence that notes the effect of HFD on food consumption from insects to human, which results in obesity and obesity-associated metabolic diseases. But the effect of HFD on another critical food intake related behavior, food seeking, remains largely uncharacterized. Conceptually, food-seeking behavior in the fruit fly is composed of two behavioral components, increased sensitivity to food cues, and enhanced exploratory locomotion, which altogether facilitates the localization and acquisition of desirable food sources. Previous work has shown that starvation promotes starvation-induced hyperactivity, the exploratory component of food-seeking behavior, via a small group of OA neurons in the fly brain. These hunger-sensing OA neurons sample the metabolic status by detecting two groups of functionally antagonistic hormones, AKH and DILPs, and promote starvation-induced hyperactivity (Yu, 2016; Huang, 2020).

    This study has demonstrated that this behavior is compromised by metabolic challenges. After a few days of HFD feeding, flies became behaviorally hypersensitive to starvation and as a result their starvation-induced hyperactivity was greatly enhanced, despite that their food intake and expenditure were not affected. These results suggest that HFD feeding may specifically modulate the activity of the neural circuitry underlying starvation-induced hyperactivity and offers an opportunity to further elucidate the cellular and circuitry mechanisms underlying behavioral abnormalities upon metabolic challenges (Huang, 2020).

    As an insect counterpart of mammalian glucagon, AKH acts as a hunger signal to activate its cognate receptor AKHR expressed in the fat body and subsequently triggers lipid mobilization and energy allocation. In the fly brain, a small number of OA neurons also express AKHR. These neurons have been shown to be responsive to starvation and modulate various behaviors including food seeking and drinking (Jourjine, 2016; Yu, 2016). In that sense, these OA+AKHR+ neurons are functionally analogous to mammalian NPY/AgRP neurons in the hypothalamus, which also senses organismal metabolic states and regulates specific food intake behaviors. This study found that OA+AKHR+ neurons exhibited higher AKHR protein accumulation and became hypersensitive to AKH after HFD feeding. Notably, HFD feeding in mammals also increases the excitability of NPY/AgRP neurons, which contributes to the hypersensitivity to starvation and increased food consumption (Vernia, 2016). Thus, HFD may exert a conserved effect in the regulation of neuronal excitability and food intake related behaviors in both fruit flies and mammals (Huang, 2020).

    Autophagy, a lysosomal degradative process that maintains cellular homeostasis, is critical for energy homeostasis. Upon cellular starvation, autophagy generates additional energy supply by breaking down macromolecules and subcellular organelles. At the organismal level, autophagy also contributes to the regulation of food intake and hence organismal energy homeostasis. For example, fasting induces autophagy in NPY/AgRP neurons via fatty acid uptake and promotes AgRP expression, which in turn enhances food intake (Kaushik, 2011). In line with these results, eliminating autophagy in NPY/AgRP neurons reduces food intake and hence body weight and fat deposits (Kaushik, 2011). Conversely, loss of autophagy in POMC neurons displays increased food intake and adiposity (Coupe, 2012). Consistently, in the current study, fruit flies neuronal autophagy was critical for the function of OA+AKHR+ neurons to sense hunger and regulate starvation-induced hyperactivity (Huang, 2020).

    Accumulating evidence suggests that HFD suppresses autophagy in different peripheral tissue types such as liver, skeletal muscle, and the adipose tissue. Similarly, HFD suppresses autophagy in the hypothalamus, whereas blocking hypothalamic autophagy, particularly in POMC neurons, exacerbates HFD induced obesity. This study showed that HFD suppressed neuronal autophagy in OA+AKHR+ neurons and enhanced AKHR accumulation in these neurons. As a result, OA+AKHR+ neurons became hypersensitive to starvation and promoted starvation-induced hyperactivity. It will be of interest to examine whether HFD also reduces autophagy and increases the accumulation of specific membrane receptors in mammalian NPY/AgRP neurons (Huang, 2020).

    This study also sought to examine the cellular mechanism that linked HFD feeding to the reduction of autophagy. HFD feeding activated TOR signaling. TOR, a highly conserved serine-threonine kinase, controls numerous anabolic cellular processes. Yhis study found that TOR signaling was tightly associated with the activity of AKHR+ neurons and the behavioral responses upon HFD feeding. Genetic enhancement of TOR activity in AKHR+ neurons increased AKHR protein accumulation, the sensitivity of these neurons to AKH, and hence starvation-induced hyperactivity, all of which mimicked the effect of HFD feeding. Inhibiting TOR activity exerted an opposite effect. In addition, the effect of HFD on TOR signaling was found to be mediated by AMPK signaling. These results altogether suggest that AMPK-TOR signaling in AKHR+ neurons plays an important role in maintaining the homeostasis of these neurons and determining the responsiveness to HFD feeding. Similarly, rodent studies have shown that manipulating AMPK-TOR signaling results in the dysfunction of NPY/AgRP neurons as well as POMC neurons, which leads to abnormal food consumption and adiposity. It will be of interest to examine whether HFD also modulates AMPK-TOR signaling in these specific hypothalamic neurons (Huang, 2020).

    This study next sought to understand how AKHR+ neurons detected HFD, or more specifically, excess lipid ingested by the flies. As an essential nutrient and important energy reserve, dietary lipids were transported via their carrier proteins, named lipoproteins, in the circulation system and regulated multiple cellular signaling pathways. Proteomic analysis helped identify one lipoprotein LTP that was enriched in flies' hemolymph after HFD feeding. Single-cell RNAseq of AKHR+ neurons identified a number of lipoprotein receptors, especially LpR1, highly expressed in these neurons. Therefore, it is proposed that AKHR+ neurons might sense HFD feeding via LTP-LpR1 signaling. Evidently, it was found that eliminating LpR1 in AKHR+ neurons could protect flies from HFD, reducing AKHR accumulation and abolishing the effect of HFD to enhance starvation-induced hyperactivity. Conversely, eliminating LpR1 in the fat body, the major lipid reservoir of flies, created diet-independent hyperlipidemia and mimicked the effect of HFD feeding on flies' starvation-induced hyperactivity. Taken together, a working model is proposed that upon HFD feeding, excess dietary lipids are transported by LTP in the hemolymph, which interacts with its cognate receptor LpR1 in OA+AKHR+ neurons. As a result, these neurons undergo a number of cellular signaling processes and eventually become hypersensitive to starvation (Huang, 2020).

    To summarize, the present study establishes a link between an unhealthy diet and abnormalities of food intake related behaviors in a model organism. The underlying mechanism was also deciphered, involving intracellular AMPK-TOR signaling, reduced neuronal autophagy, accumulation of a specific hormone receptor, and increased excitability of a small group of hunger-sensing neurons. This study will shed crucial light on the pathological changes in the central nervous system upon metabolic challenges. Given that the central control of metabolism and food intake related behaviors are highly conserved across different species, it will be of importance to further examine whether similar mechanisms also mediate the effect of HFD feeding on food intake and metabolic diseases in mammals (Huang, 2020).

    A novel satiety sensor detects circulating glucose and suppresses food consumption via insulin-producing cells in Drosophila

    Sensing satiety is a crucial survival skill for all animal species including human. Despite the discovery of numerous neuromodulators that regulate food intake in Drosophila, the mechanism of satiety sensing remains largely elusive. This study investigated how neuropeptidergic circuitry conveyed satiety state to influence flies' food consumption. Drosophila tackykinin (DTK) and its receptor TAKR99D were identified in an RNAi screening as feeding suppressors. Two pairs of DTK(+) neurons in the fly brain could be activated by elevated D-glucose in the hemolymph and imposed a suppressive effect on feeding. These DTK(+) neurons formed a two-synapse circuitry targeting insulin-producing cells, a well-known feeding suppressor, via TAKR99D(+) neurons, and this circuitry could be rapidly activated during food ingestion and cease feeding. Taken together, this study identified a novel satiety sensor in the fly brain that could detect specific circulating nutrients and in turn modulate feeding, shedding light on the neural regulation of energy homeostasis (Qu, 2020).

    Cannabinoids modulate food preference and consumption in Drosophila melanogaster

    Cannabinoids have an important role in regulating feeding behaviors via cannabinoid receptors in mammals. Cannabinoids also exhibit potential therapeutic functions in Drosophila melanogaster, or fruit fly that lacks cannabinoid receptors. However, it remains unclear whether cannabinoids affect food consumption and metabolism in a cannabinoid receptors-independent manner in flies. This study systematically investigated pharmacological functions of various cannabinoids in modulating food preference and consumption in flies. Flies display preferences for consuming cannabinoids, independent of two important sensory regulators Poxn and Orco. Interestingly, phyto- and endo- cannabinoids exhibit an inhibitory effect on food intake. Unexpectedly, the non-selective CB1 receptor antagonist AM251 attenuates the suppression of food intake by endocannabinoids. Moreover, the endocannabinoid anandamide (AEA) and its metabolite inhibit food intake and promote resistance to starvation, possibly through reduced lipid metabolism. Thus, this study has provided insights into a pharmacological role of cannabinoids in feeding behaviors using an adult Drosophila model (He, 2021).

    The genetic architecture of larval aggregation behavior in Drosophila

    Many insect species exhibit basal social behaviors such as aggregation, which play important roles in their feeding and mating ecologies. However, the evolutionary, genetic, and physiological mechanisms that regulate insect aggregation remain unknown for most species. This study used natural populations of Drosophila melanogaster to identify the genetic architecture that drives larval aggregation feeding behavior. By using quantitative and reverse genetic approaches, a complex neurogenetic network was identified that plays a role in regulating the decision of larvae to feed in either solitude or as a group. Results from single gene, RNAi-knockdown experiments show that several of the identified genes represent key nodes in the genetic network that determines the level of aggregation while feeding. Furthermore, this study showed that a single non-coding variant in the gene CG14205, a putative acyltransferase, is associated with both decreased mRNA expression and increased aggregate formation, which suggests that it has a specific role in inhibiting aggregation behavior. These results identify, for the first time, the genetic components which interact to regulate naturally occurring levels of aggregation in D. melanogaster larvae (McKinney, 2021).

    Multisensory interactions regulate feeding behavior in Drosophila

    The integration of two or more distinct sensory cues can help animals make more informed decisions about potential food sources, but little is known about how feeding-related multimodal sensory integration happens at the cellular and molecular levels. This study shows that multimodal sensory integration contributes to a stereotyped feeding behavior in the model organism Drosophila melanogaster Simultaneous olfactory and mechanosensory inputs significantly influence a taste-evoked feeding behavior called the proboscis extension reflex (PER). Olfactory and mechanical information are mediated by antennal Or35a neurons and leg hair plate mechanosensory neurons, respectively. The controlled delivery of three different sensory cues can produce a supra-additive PER via the concurrent stimulation of olfactory, taste, and mechanosensory inputs. It is suggested that the fruit fly is a versatile model system to study multisensory integration related to feeding, which also likely exists in vertebrates (Oh, 2021).

    A closed-loop optogenetic screen for neurons controlling feeding in Drosophila

    Feeding is an essential part of animal life that is greatly impacted by the sense of taste. Although the characterization of taste-detection at the periphery has been extensive, higher order taste and feeding circuits are still being elucidated. This study used an automated closed-loop optogenetic activation screen to detect novel taste and feeding neurons in Drosophila melanogaster. Out of 122 Janelia FlyLight Project GAL4 lines preselected based on expression pattern, this study identified six lines that acutely promote feeding and 35 lines that inhibit it. As proof of principle, R70C07-GAL4, which labels neurons that strongly inhibit feeding, was analyzed. Using split-GAL4 lines to isolate subsets of the R70C07-GAL4 population, both appetitive and aversive neurons were found. Furthermore, this study shows that R70C07-GAL4 labels putative second-order taste interneurons in the subesophageal zone that contact both sweet and bitter sensory neurons. These results serve as a resource for further functional dissection of fly feeding circuits (Lau, 2021).

    The Panopticon-Assessing the Effect of Starvation on Prolonged Fly Activity and Place Preference

    Animal behaviours are demonstrably governed by sensory stimulation, previous experience and internal states like hunger. With increasing hunger, priorities shift towards foraging and feeding. During foraging, flies are known to employ efficient path integration strategies. However, general long-term activity patterns for both hungry and satiated flies in conditions of foraging remain to be better understood. Similarly, little is known about how permanent contact chemosensory stimulation affects locomotion. To address these questions, a novel, simplistic fly activity tracking setup, the Panopticon, was developed. Using a 3D-printed Petri dish inset, this assay allows recording of walking behaviour, of several flies in parallel, with all arena surfaces covered by a uniform substrate layer. Two constellations of providing food were tested: (i) in single patches and (ii) omnipresent within the substrate layer. Fly tracking is done with FIJI, further assessment, analysis and presentation is done with a custom-built MATLAB analysis framework. This study found that starvation history leads to a long-lasting reduction in locomotion, as well as a delayed place preference for food patches which seems to be not driven by immediate hunger motivation (Mahishi, 2021).

    Enteric neurons increase maternal food intake during reproduction

    Reproduction induces increased food intake across females of many animal species, providing a physiologically relevant paradigm for the exploration of appetite regulation. By examining the diversity of enteric neurons in Drosophila melanogaster, this study identified a key role for gut-innervating neurons with sex and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. These findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain (Hadjieconomou, 2020).

    Internal state has profound effects on brain function. Despite increasingly recognized roles for the gut-brain axis in maintaining energy balance, links between internal state and gastrointestinal innervation remain poorly characterized. Progress has been hindered by neuroanatomical complexity, which is only beginning to be parsed in mammals. The simpler-yet physiologically complex-intestine of Drosophila provides an alternative entry point into the study of gastrointestinal innervation (Hadjieconomou, 2020).

    Innervation of the main digestive portion of the adult fly intestine, which encompasses the anterior midgut and the crop and central neurons of the pars intercerebralis (PI) in the brain. PI neurons directly innervate the anterior midgut and the crop, and include insulin-producing neurons and other peptidergic subtypes. The crop is further populated by processes that emanate from cells of the corpora cardiaca, which produce the glucagon-like adipokinetic hormone and are adjacent to the hypocerebral ganglion (HCG). Also adjacent to both the HCG and the corpora cardiaca are the corpus allatum cells, which produce juvenile hormone and extend short local projections. The thoracico-abdominal ganglion of the central nervous system might not innervate these gut regions (Hadjieconomou, 2020).

    The crop-an expandable structure found in the intestines of insects-might be disregarded as a passive food store, but several observations suggest active regulation of its physiology. Refeeding flies after starvation results in enlarged, food-filled crops, pointing to modulation of food ingression into and out of the crop. Live imaging or temporal dissections of flies revealed that food always enters the crop before proceeding to the midgut. Additionally, food transit through the crop is dependent on both its palatability and its nutritional value. Therefore, in adult flies, all food transits through the crop, which is nutrient-sensitive and shows chemically and anatomically diverse innervation (Hadjieconomou, 2020).

    The crop and anterior midgut are innervated by myosuppressin (Ms)-positive neurons located in the PI and the HCG. PI Ms neurons are distinct from known neuronal subsets, with the exception of eight Ms neurons that co-express the Taotie-GAL4 marker. Two PI Ms neuron populations can be distinguished by cell size: one comprises 18 large cells and another comprises 12 smaller cells. Single-cell clones of large Ms neurons reveal a single process that bifurcates into a longer, probably axonal projection to the gut-which arborizes in the HCG and extends further to innervate the crop-and a shorter, probably dendritic process that reaches the suboesophageal zone, where the axons of peripheral gustatory sensory neurons terminate. A subset of HCG Ms-expressing neurons also innervates the crop, whereas another subset projects locally. This study confirmed the expression of Ms using an endogenously tagged Ms reporter (Ms-GFP) and in situ hybridization Ms innervation was also observed of the hindgut, the rectal ampulla and the heart, and a subset of peripheral Ms-positive neurons innervating the female reproductive tract (Hadjieconomou, 2020).

    This study selectively activated or silenced Ms neurons in adult flies. Activation resulted in greatly enlarged crops in flies that were fed ad libitum, consistent with the relaxant properties of Ms on insect muscles ex vivo. By contrast, silencing of Ms neurons prevented crop enlargement in a starved-refed condition in which the crop normally expands. Genetic downregulation or mutation of Ms (using a new mutant) prevented crop enlargement, albeit to a lesser extent than Ms neuron silencing. This could be due to another Ms-neuron-derived neurotransmitter or neuropeptide contributing to crop enlargement, or to loss of the Ms peptide during development in these experiments, resulting in adaptations that render the crop more active than it would be in response to acute loss of the Ms peptide. A Gal4 insertion into the Ms locus was generated that disrupts Ms production (MsTGEM). In contrast to the crop enlargement resulting from TrpA1-mediated activation from Ms-Gal4, TrpA1 expression from this (Ms mutant) MsTGEM-Gal4 driver failed to induce crop enlargement, further confirming a requirement for Ms. Ms neuron subtype-specific downregulations and activations enabled establishing that the PI Ms neurons (in particular, the Taotie-Gal4-positive subset of large PI Ms neurons) induce, and are indispensable for, crop enlargement through their production of Ms neuropeptide (Hadjieconomou, 2020).

    The contributions of myosuppressin receptors 1 and 2 (MsR1 and MsR2) were explored. MsR1 expression was observed in crop muscles, in subsets of neurons including the PI and HCG Ms-positive neurons and neurons innervating the ovary and heart; no MsR1 expression was detected in ovarian or heart muscles. Expression of MsR2 was also detected in crop muscles. To investigate the function of the Ms receptor, MsR1 was downregulated specifically in adult crop muscles using two independent driver lines (vm-Gal4 and MsR1crop-Gal4). Both genetic manipulations led to reduced crop enlargement in a starvation-refeeding assay, comparable to that observed for Ms neuron silencing or Ms mutation. Downregulation of MsR2 did not affect crop enlargement. A role for MsR1 in mediating crop enlargement was confirmed using a MsR1TGEM mutant. MsR1 is therefore identified as the crop muscle receptor through which Ms signals to modulate crop enlargement (Hadjieconomou, 2020).

    The physiological regulation of crop enlargement was explored and found that it is dependent on sex and on reproductive status: the crops of mated females fed ad libitum (which were used for all the experiments described above) were consistently more expanded than those of virgin female or mated male flies fed ad libitum. Because post-mating changes were not seen in Ms neuron projections, it was asked whether post-mating crop enlargement might result from the release of Ms preferentially in mated females. Ms peptide levels were lower in the PI neuron cell bodies of females only after mating. In the absence of Ms transcriptional changes this observation is consistent with a post-mating increase in the secretion of Ms peptide in females. This effect of mating on Ms levels was specific to mating: nutrient availability did not affect intracellular Ms levels. It was also observed that the Ms neurons of mated females had higher cumulative calcium levels and a reduced amplitude of calcium oscillations compared to virgin females, as detected both by in vivo GCaMP6 calcium imaging and by the calcium-sensitive reporter CaLexA, in which GFP expression is proportional to cumulative neuronal activity. Physiologically, and in contrast to observations in mated females, a reduction of Ms signalling in males or in virgin female flies failed to impair crop enlargement. Consequently, when Ms signalling to crop muscles was prevented, the size of the crop of mated females no longer differed from that of virgin females. Collectively, these findings support the idea that, in female flies, the activity of PI Ms neurons changes after mating to promote Ms release (Hadjieconomou, 2020).

    Levels of the steroid hormone ecdysone, which promotes egg production and intestinal stem-cell proliferation, increase after mating. The ecdysone receptor (EcR) is expressed by all PI Ms neurons, which suggests that they might be sensitive to circulating ecdysone. Expression of a dominant-negative EcR-which targets all EcR isoforms-confined to the Ms neurons of adult flies was found to increase intracellular Ms levels in the Ms PI neuron cell bodies of mated females to the levels observed in virgin females, whereas it had no effect on virgin females. Downregulation of EcR (using RNA interference lines that target all isoforms or the B1 isoform specifically) produced comparable results. In both experiments, the amplitude of in vivo calcium oscillations in Ms neurons was increased to levels seen in virgin females. Compromising EcR signalling in adult Ms neurons significantly reduced crop enlargement preferentially in mated females; this phenotype was also apparent when the PI Ms neurons were targeted using Taotie-Gal4. Ecdysone therefore communicates mating status to Ms neurons through its B1 receptor (Hadjieconomou, 2020).

    Previous work showed that the adult intestine is resized and metabolically remodelled after mating (Reiff, 2016), but did not investigate possible effects on its hormone-producing enteroendocrine cells. This study now observe a post-mating increase in the number of enteroendocrine cells, including a subset that expresses the hormone bursicon α (Burs), which is known to signal to adipose tissue through an unidentified neuronal relay. An endogenous protein reporter for the Burs receptor Rickets (Rk, also known as Lgr2) revealed its expression in subsets of neurons including all PI Ms neurons (including the Taotie-Gal4-positive subset) and in projections terminating in the HCG. Expression in a subset of the HCG Ms neurons was observed only sporadically (Hadjieconomou, 2020).

    Consistent with the regulation of Ms neurons by the increase in Burs derived from enteroendocrine cells after mating, adult-specific downregulation of the Burs receptor gene rk in Ms neurons reverted intracellular Ms levels in the PI Ms neurons of mated females to levels observed in virgin females; there was no effect in virgin females. Like EcR downregulation, rk downregulation in Ms neurons also increased the amplitude of in vivo calcium oscillations in the Ms neuron cell bodies of mated females to values similar to those observed in virgin females. Functionally, both the downregulation of Burs in intestinal enteroendocrine cells and the adult-specific rk downregulation in Ms neurons-either in all neurons or in the Taotie-Gal4-positive subset in the PI-preferentially reduced crop enlargement in mated females. Conversely, stimulating the intestinal release of enteroendocrine hormones-including Burs-from enteroendocrine cells resulted in reduced Ms levels in the Ms neuron cell bodies of virgin females, similar to those observed in mated females, and greatly enlarged crops (Hadjieconomou, 2020).

    Thus, steroid and enteroendocrine hormones communicate mating status to the brain. Acting through their receptors in the PI Ms neurons, these hormones change Ms neuronal activity, promoting the release of Ms after mating (Hadjieconomou, 2020).

    To investigate the importance of Ms neuron modulation after mating, post-mating crop enlargement was selectively prevented by downregulating MsR1 in adult crop muscles using two independent strategies. This had no discernible effects in males or virgin females, but specifically prevented the increase in food intake that is normally observed in female flies after mating. Comparable results were obtained by blocking the post-mating ecdysone and Burs inputs into the Ms neurons. Downregulation of MsR2 had no such effect. The post-mating change in crop expandability, mediated by Ms and MsR1 signalling, thus causes the increased food intake observed in females after mating (Hadjieconomou, 2020).

    The negative pressures that have been reported in the crops of larger insects suggest that the crop may draw food in by generating suction. The increased crop expandability enabled by Ms release after mating could therefore increase food intake through changes in suction. It was observed that mated females ingest more food per sip than virgin females, which is consistent with mated females needing to generate a higher suction pressure to facilitate bigger sips. Crop enlargement was therefore modeled using the Poiseuille equation for incompressible fluid flow in a pipe and found that the crop would need a suction pressure of the order of -1 kPa to achieve the previously reported intake volume per sip. This is in reasonable agreement with previously reported values measured in cockroach crops of between -0.5 and -1 kPa. The model predicts that mated flies would require a modest increase in suction pressure to -1.3kPa in order to facilitate the increased sip size (Hadjieconomou, 2020).

    In the model, the change in crop volume drives food intake through increased suction. A crop that cannot enlarge, or a persistently enlarged crop, should therefore result in a comparable reduction in food intake by preventing the generation of suction. This was tested by persistently preventing crop enlargement (using crop-muscle-specific MsR1 knockdown) or by persistently inducing it (using TrpA1-mediated Ms neuron activation from Ms-Gal4 or Taotie-Gal4), after which the diet of these flies was switched from an undyed to a dye-laced food source to assess food intake. As predicted, both genetic manipulations reduced food intake. Conversely, increasing the rate at which the crop expands should increase food intake. This was tested by activating the Ms neurons as in the previous experiment, but this time the dye-laced food source was provided, and its intake was monitored at the same time as the neurons were activated (that is, as inducing greater crop expansion was being induced) rather than after a persistent activation (when the crop is already maximally expanded). Increased food intake was observed under these conditions in the absence of changes in the number of meals. Although further work will be required to elucidate the full dynamics of crop enlargement, filling and emptying, these experiments support the idea that the Ms-induced enlargement of the crop after mating increases food intake at least partly by increasing the suction power of the crop (Hadjieconomou, 2020).

    Finally, given the links between nutrient intake and fecundity, it is proposed that the Ms-driven crop enlargement after mating might be adaptive and support reproduction. Crop enlargement was prevented selectively after mating by downregulating MsR1 from crop muscles, as in previous experiments. This resulted in reduced egg production, and the eggs that were produced had reduced viability. It is therefore conclude that the crop and its Ms innervation sustain the increase in food intake after mating, maximising female fecundity (Hadjieconomou, 2020).

    These findings lead to a proposal that the maternal increase in food intake during reproduction is adaptive, that the crop is a key reproductive organ, and that Ms is a major effector of post-mating responses. In support of these ideas, the crop is absent in larvae-the juvenile stage of insects-and other Diptera have co-opted it for reproductive behaviours such as the regurgitation of nuptial gifts or the secretion of male pheromones. Ms receptors are also closely related to the Sex peptide receptor (the 'mating sensor' of female flies), and both diverged after duplication of an ancestral receptor that might have responded to the Myoinhibitory peptide (Mip) in the last common ancestor of protostomes. It will be interesting to explore possible links between Ms and Sex peptide signalling, and whether and how these mating signals affect recently described crop mechanosensing mechanisms that restrain ingestion as the crop expands in order to terminate large meals (Hadjieconomou, 2020).

    This study has provided evidence for a gut-to-brain axis in Drosophila by identifying central Ms neurons as targets of the gut-derived hormone Burs. These central neurons innervate the gut, 'closing' a gut-brain-gut loop that connects midgut enteroendocrine signals to the crop, a more anterior gut region. This might allow for the functional coordination of different gut portions, while enabling central modulation by sensory cues (for example, gustatory). This study also identified the Ms neurons as the neural targets of ecdysone, which has been shown to promote food intake. Reproduction has pronounced, and in some cases lasting, effects on the human female brain; Ms neurons provide a tractable and physiologically relevant neural substrate for the investigation of the mechanisms involved (Hadjieconomou, 2020).

    The human digestive system might be similarly modulated by reproductive cues to affect food intake. In mammals, enteric neurons express sex and reproductive-hormone receptors, and enteroendocrine hormone levels change during reproduction. It is suggested that pregnancy and lactation represent an attractive and relatively unexplored physiological adaptation for the investigation of nutrient intake regulation, organ remodelling and metabolic plasticity-mechanisms that might eventually be leveraged to curb appetite and/or weight gain (Hadjieconomou, 2020).

    An intestinal zinc sensor regulates food intake and developmental growth

    In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes. This study used a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. These findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis (Redhai, 2020).

    To investigate nutrient sensing in enterocytes, 111 putative nutrient sensors in D. melanogaster were selected on the basis of their intestinal expression and their predicted structure or function. Using two enterocyte-specific driver lines, their expression was downregulated in midgut enterocytes throughout development under two dietary conditions, nutrient-rich and nutrient-poor; it was reasoned that dysregulation of nutrient-sensing mechanisms may increase or reduce the normal period of larval growth, and might do so in a diet-dependent manner. Enterocyte-specific knockdown of the gene CG11340, also referred to as pHCl-22, resulted in developmental delay. This delay was exacerbated, and was accompanied by significantly reduced larval viability, under nutrient-poor conditions; these phenotypes were confirmed using a second RNAi transgene and a new CG11340 mutant. In the tradition of naming Drosophila genes according to their loss-of-function phenotype, CG11340 was named 'hodor', an acronym for 'hold on, don't rush', in reference to the developmental delay (Redhai, 2020).

    A transcriptional reporter revealed that Hodor was expressed in the intestine. A new antibody revealed that Hodor expression was confined to enterocytes in two midgut portions that are known to store metals: the copper cell region and the iron cell region. Within the copper cell region, Hodor was expressed only in so-called interstitial cells. hodor-Gal4 was also present in the interstitial cells of the copper cell region; however, in the experimental conditions used in this study and in contrast to published results, it was not detected in the iron cell region. Apart from the intestine, Hodor was found only in principal cells of the excretory Malpighian tubules. To identify the cells from which Hodor controls systemic growth, region- or cell-type-specific downregulation and rescue experiments were conducted. Only fly lines in which hodor was downregulated in interstitial cells showed slowed larval development. This developmental delay persisted when hodor knockdown was induced post-embryonically during larval growth, and was rescued only in fly lines in which hodor expression was re-instated in cell types that included interstitial cells. The fat body (analogous to liver and adipose tissue) has long been known to couple nutrient availability with developmental rate; however, recent studies have revealed contributions from the intestine, particularly in nutrient-poor conditions. The current findings confirm a role for the intestine in coupling nutrient availability with larval growth, and further implicate a subpopulation of enterocytes-interstitial cells-as important mediators. Interstitial cells were described decades ago in blowfly, but had remained relatively uncharacterized since; their name refers only to their position, interspersed among the acid-secreting copper cells that control microbiota loads (Redhai, 2020).

    This study established that the lethality of hodor mutation or knockdown was apparent only during the larval period. The development of hodor mutants was slower throughout larval life, and surviving mutants attained normal pupal and adult sizes. Consistent with previous findings, hodor mutation or knockdown was found to reduce luminal acidity in the copper cell region, suggesting a role specifically for interstitial cells in this process. hodor mutants also had increased gut bacterial titres, which is consistent with the observed functional defects in the copper cell region. Enlarged volumes of both the lumen of the copper cell region and the interstitial cells were also apparent after 1-3 days of (delayed) larval development; ultrastructurally, this was apparent in interstitial cells as a reduction in the complexity of their characteristic basal infoldings. This study was, however, able to rule out all of these defects as reasons for the developmental delay (Redhai, 2020).

    During the course of these experiments, it was observed that hodor mutant larvae were more translucent than control larvae. This was suggestive of peripheral lipid depletion, which was confirmed by quantifying and staining for triacylglycerides. Reduced lipid stores did not result from disrupted enterocyte integrity: the intestinal barrier of mutants was intact, both anatomically and functionally. It was observed that hodor mutants had less food in their intestines and accumulated insulin-like peptide Ilp2 in their brains (nutrient-dependent Ilp2 secretion promotes larval development; its accumulation in the brain is commonly interpreted as peptide retention in the absence of transcriptional changes). Consistent with reduced systemic insulin signalling, hodor mutant larval extracts had reduced levels of phospho-Akt and phospho-S6 kinase. As these are all indicators of starvation, food intake was quantified, and it was observed to be reduced in both hodor mutant larvae and in hodor knockdowns targeting interstitial cells. Reduced food intake was apparent soon after hatching and persisted throughout larval development. Ectopic expression of Ilp2-which rescues developmental delay in larvae that lack insulin-like peptides-in hodor mutants partially rescued their developmental delay, but did not increase their food intake. An 'instructive' link between intestinal Hodor and food intake was further suggested by the overexpression of hodor in otherwise wild-type enterocytes; this resulted in larvae that ate more and developed at a normal rate, but had increased lipid stores. Therefore, Hodor controls larval growth from a subset of enterocytes by promoting food intake and systemic insulin signalling. In its absence, larvae fail to eat sufficiently to proceed through development at the normal rate and are leaner. When present in excess, Hodor causes larvae to eat more and accumulate the energy surplus as fat (Redhai, 2020).

    In fly adipose tissue, amino acid availability activates Tor signalling to promote systemic growth. This study therefore combined hodor knockout or knockdown with genetic manipulations to alter Tor signalling. In flies with reduced or absent Hodor function, decreasing or increasing Tor signalling in hodor-expressing cells exacerbated or rescued the developmental delay, respectively. The reduced food intake of hodor mutants was also significantly rescued by activation of Tor signalling in hodor-expressing cells. Genetic targeting of Rag GTPases or the Gator1 complex in these cells failed to affect the developmental delay of hodor mutants, which could suggest non-canonical regulation of Tor signalling in Hodor-expressing cells. The systemic effects of Hodor on food intake and larval growth are therefore modulated by Tor signalling within Hodor-expressing interstitial cells (Redhai, 2020).

    Hodor belongs to the (typically neuronal) Cys-loop subfamily of ligand-gated ion channels, and is predicted to be a neurotransmitter-gated anion channel. It is known to show activity in response to alkaline conditions in Xenopus oocytes, but the acidic pH of the copper cell region prompted a search for additional ligands. Although alkaline pH-induced Hodor activity was confirmed in oocyte expression systems, Hodor did not respond to typical Cys-loop receptor ligands such as neurotransmitters or amino acids. Instead, the screen identified zinc as an unanticipated ligand, which elicited a strong dose-dependent response only in Hodor-expressing oocytes; this response to zinc showed peak current amplitude values much greater than those observed in response to pH or to other metals such as iron or copper. Force-field-based structural stability and binding affinity calculations identified the amino acid pair E255 and E296 as a potential binding site for the divalent zinc ion. Mutating these residues did not abrogate the zinc-elicited currents, but did result in currents with faster rise time and deactivation kinetics, which supports the idea that zinc is a relevant Hodor ligand. On the basis of its sequence and conductance properties, Hodor has been proposed to transport chloride (Feingold, 2016; Remnant, 2016), and the zinc-elicited currents that were observed in oocytes had a reversal potential that is consistent with chloride selectivity. In vivo experiments in flies showed that supplementation of a low-yeast diet with zinc led to a reduction of chloride levels in interstitial cells, whereas hodor mutation increased chloride levels. Thus, Hodor is a pH-modulated, zinc-gated chloride channel (Redhai, 2020).

    Attempts were made to establish the relevance of zinc binding in vivo. Zinc enrichment is observed in both the copper and iron cell regions of the larval gut, revealing an unrecognized role for these Hodor-expressing regions in zinc handling. Mutation of hodor failed to affect this zinc accumulation, although dietary yeast levels did, which is consistent with a role for Hodor in sensing rather than transporting zinc. (Notably, the white mutation-which is frequently used in the genetic background of Drosophila experiments-results in a small but significant reduction in both intestinal zinc accumulation and larval growth rate, although the status of the w gene neither exacerbated nor masked the more substantial, hodor-induced developmental delay. Furthermore, larvae that were fed a low-yeast diet ate significantly more when the diet was supplemented with zinc; this effect was abrogated in hodor mutants. In a food choice experiment, control larvae developed a preference for zinc-supplemented food over time, which suggests that the preference develops after ingestion. Consistent with this idea, zinc preference was specifically abrogated in hodor mutants (their general ability to discriminate between other diets was confirmed. Thus, zinc sensing by Hodor is physiologically relevant in vivo. Metals such as zinc are primarily provided by yeasts in nature; Hodor may be one of several sensors used to direct larvae to nutrient-rich food sources (Redhai, 2020).

    The subcellular localization of Hodor suggests that it may normally maintain low cytoplasmic chloride concentrations by transporting it out of the interstitial cells and/or into their lysosomes. In accordance with this, and consistent with its putative lysosomal localization signals, Hodor was specifically enriched in apical compartments containing late endosome or lysosomal markers, as well as decorating the brush border of interstitial cells. The presence of Hodor in a subpopulation of lysosomes was of interest, because chloride transport across lysosomal membranes often sustains the activity of the proton-pumping vacuolar-type ATPase (V-ATPase) that maintains lysosomal acidity and Tor activation on the lysosome. To explore a role for Hodor in enabling Tor signalling, whether the absence of hodor induced autophagy-a hallmark of reduced Tor signalling, was tested. First, the induction of common autophagy markers in interstitial cells after genetic interference with the V-ATPase complex, which is known to promote autophagy by reducing lysosomal acidity and Tor signalling, was confirmed. Similar to reduced V-ATPase function, loss of hodor increased autophagy in interstitial cells. Expression of the dual autophagosome and autolysosome reporter UAS-GFP-mCherry-Atg8a in the intestinal cells of hodor mutants confirmed the induction of autophagy, and revealed two additional features. First, the acidification of autophagic compartments was defective in hodor mutants. Second, the increased autophagy and defective acidification observed in hodor mutants were particularly prominent in the two Hodor-expressing intestinal regions (the copper cell region and the iron cell region), consistent with cell-intrinsic roles for Hodor in these processes. Additional support for the roles of lysosomal function and Tor signalling in controlling whole-body growth from interstitial cells was provided by the finding that most V-ATPase subunits were transcriptionally enriched in the copper cell region. Functionally, the downregulation of V-ATPase subunits specifically in Hodor-expressing cells-and not in other subsets of enterocytes, such as those targeted by R2R4-Gal4-led to developmental delay and reduced food intake, phenotypes comparable to those observed as a result of hodor downregulation. Hence, although the directionality of zinc sensing and chloride transport in interstitial cells remains to be established, the data are consistent with roles for brush-border Hodor in transporting chloride out of interstitial cells-thus maintaining osmolarity and water balance. Lysosomal Hodor may transport chloride into the lysosome to sustain V-ATPase function, lysosomal acidification and TOR signalling, pointing to new links between lysosomal homeostasis in specialized intestinal cells, food intake and systemic growth. Nutrients such as amino acids are important regulators of Tor signalling. The genetic data are consistent with novel input from metals and/or micronutrients into Tor signalling. The nutrient-dependent zinc accumulation in lysosomal organelles-recently described in mammalian cells and nematode worms-suggests that links between zinc, lysosomes and Tor may be of broader importance. Two attractive cell types in which to explore such links are the Paneth cells of the mammalian intestine, which accumulate zinc and regulate intestinal immunity and stem cell homeostasis, and the 'lysosome-rich enterocytes' that have recently been described in fish and mice, which have roles in protein absorption (Redhai, 2020).

    An extensive reconstruction of the hodor family tree supported the presence of a single member of the family in the ancestor of insects. Because Hodor-like proteins are present only in insects, they may prove to be highly specific targets for the chemical control of disease vectors, particularly given that mosquito genomes contain a single gene rather than the three paralogues that are found in most flies. To test this idea, CRISPR-Cas9 genome editing was used to generate a mutant that lacks the single hodor-like gene (AGAP009616) in the malaria vector Anopheles gambiae. This gene is also expressed in the digestive tract-specifically in the midgut-and in Malphighian tubules. Three independent deletion alleles revealed that AGAP009616 function is essential for the viability of A. gambiae. A target that is expressed in the intestine, such as Hodor, is particularly attractive for vector control as it may circumvent accessibility issues and could be directly targeted using ingestible drugs such as those applied to larval breeding sites (Redhai, 2020).

    Metals have received little attention in the contexts of development or whole-body physiology, and are often regarded as passive 'building blocks'. By revealing the roles of a metal sensor in food intake and growth control, these findings highlight the importance of investigating the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis. These mechanisms could prove to be useful in insect vector control (Redhai, 2020).

    A neuronal ensemble encoding adaptive choice during sensory conflict in Drosophila

    Feeding decisions are fundamental to survival, and decision making is often disrupted in disease. This study shows that neural activity in a small population of neurons projecting to the fan-shaped body higher-order central brain region of Drosophila represents food choice during sensory conflict. Food deprived flies made tradeoffs between appetitive and aversive values of food. An upstream neuropeptidergic and dopaminergic network was identified that relays internal state and other decision-relevant information to a specific subset of fan-shaped body neurons. These neurons were strongly inhibited by the taste of the rejected food choice, suggesting that they encode behavioral food choice. These findings reveal that fan-shaped body taste responses to food choices are determined not only by taste quality, but also by previous experience (including choice outcome) and hunger state, which are integrated in the fan-shaped body to encode the decision before relay to downstream motor circuits for behavioral implementation (Sareen, 2021).

    Cholecystokinin-like peptide mediates satiety by inhibiting sugar attraction

    Feeding is essential for animal survival and reproduction and is regulated by both internal states and external stimuli. However, little is known about how internal states influence the perception of external sensory cues that regulate feeding behavior. This study investigated the neuronal and molecular mechanisms behind nutritional state-mediated regulation of gustatory perception in control of feeding behavior in the brown planthopper and Drosophila. Feeding was found to increase the expression of the cholecystokinin-like peptide, sulfakinin (SK), and the activity of a set of SK-expressing neurons. Starvation elevates the transcription of the sugar receptor Gr64f and SK negatively regulates the expression of Gr64f in both insects. Interestingly, it was found that one of the two known SK receptors, CCKLR-17D3, is expressed by some of Gr64f-expressing neurons in the proboscis and proleg tarsi. Thus, this study has identified SK as a neuropeptide signal in a neuronal circuitry that responds to food intake, and regulates feeding behavior by diminishing gustatory receptor gene expression and activity of sweet sensing GRNs. The findings demonstrate one nutritional state-dependent pathway that modulates sweet perception and thereby feeding behavior, but the experiments cannot exclude further parallel pathways. Importantly, it was shown that the underlying mechanisms are conserved in the two distantly related insect species (Guo, 2021).

    A neuronal mechanism controlling the choice between feeding and sexual behaviors in Drosophila

    Animals must express the appropriate behavior that meets their most pressing physiological needs and their environmental context. However, it is currently unclear how alternative behavioral options are evaluated and appropriate actions are prioritized. This study describes how fruit flies choose between feeding and courtship; two behaviors necessary for survival and reproduction. Sex- and food-deprived male flies were shown to prioritize feeding over courtship initiation, and manipulation of food quality or the animal's internal state fine-tunes this decision. The tyramine signaling pathway was identified as an essential mediator of this decision. Tyramine biosynthesis is regulated by the fly's nutritional state and acts as a satiety signal, favoring courtship over feeding. Tyramine inhibits a subset of feeding-promoting tyramine receptor (TyrR)-expressing neurons and activates P1 neurons, a known command center for courtship. Conversely, the perception of a nutritious food source activates TyrR neurons and inhibits P1 neurons. Therefore, TyrR and P1 neurons are oppositely modulated by starvation, via tyramine levels, and food availability. It is proposed that antagonistic co-regulation of neurons controlling alternative actions is key to prioritizing competing drives in a context- dependent manner (Cheriyamkunnel, 2021).

    The importance of environmental microbes for Drosophila melanogaster during seasonal macronutrient variability

    There is little information on how macronutrient composition and bacterial communities in natural food sources vary across seasons in nature and on how these factors affect the fitness components of insects. In this study, diet samples from an orchard compost heap, which is a natural habitat for many Drosophila species and other arthropods, were collected over 9 months covering all seasons in a temperate climate. It developed D. melanogaster on diet samples and investigated stress resistance and life-history traits as well as the microbial community of flies and compost. Nutrient and microbial community analysis of the diet samples showed marked differences in macronutrient composition and microbial community across seasons. However, except for the duration of development on these diet samples and Critical Thermal maximum, fly stress resistance and life-history traits were unaffected. The resulting differences in the fly microbial community were also more stable and less diverse than the microbial community of the diet samples. This study suggests that when D. melanogaster are exposed to a vastly varying nutritional environment with a rich, diverse microbial community, the detrimental consequences of an unfavourable macronutrient composition are offset by the complex interactions between microbes and nutrients (Davis, 2021).

    The proximate sources of genetic variation in body size plasticity: the relative contributions of feeding behaviour and development in Drosophila melanogaster

    Body size is a key life-history trait that influences many aspects of an animal's biology and is shaped by a variety of factors, both genetic and environmental. While locally-adapted populations differ in the extent to which body size responds plastically to environmental conditions like diet, there is a limited understanding of what causes these differences. It was hypothesized that populations could differ in the way body size responds to nutrition either by modulating growth rate, development time, feeding rate, or a combination of the above. Using three locally-adapted populations of Drosophila melanogaster from along the east coast of Australia, body size plasticity was investigated across five different diets. How these populations differed in feeding behaviour and developmental timing on each of the diets was assessed. Population-specific plastic responses to nutrition was observed for body size and feeding rate, but not development time. However, differences in feeding rate did not fully explain the differences in the way body size responded to diet. Thus, it is concluded that body size variation in locally-adapted populations is shaped by a combination of growth rate and feeding behaviour. This paves the way for further studies that explore how differences in the regulation of the genetic pathways that control feeding behaviour and growth rate contribute to population-specific responses of body size to diet (Chakraborty, 2021).

    Positive geotactic behaviors induced by geomagnetic field in Drosophila

    Appropriate vertical movement is critical for the survival of flying animals. Although negative geotaxis (moving away from Earth) driven by gravity has been extensively studied, much less is understood concerning a static regulatory mechanism for inducing positive geotaxis (moving toward Earth). Using Drosophila melanogaster as a model organism, this study showed that geomagnetic field (GMF) induces positive geotaxis and antagonizes negative gravitaxis. Remarkably, GMF acts as a sensory cue for an appetite-driven associative learning behavior through the GMF-induced positive geotaxis. This GMF-induced positive geotaxis requires the three geotaxis genes, such as cry, the cation channel pyx and pdf, and the corresponding neurons residing in Johnston's organ of the fly's antennae. These findings provide a novel concept with the neurogenetic basis on the regulation of vertical movement by GMF in the flying animals (Bae, 2016).

    An automated rapid iterative negative geotaxis assay for analyzing adult climbing behavior in a Drosophila model of neurodegeneration

    Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. This study describes a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of seconds to minutes. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single flies, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration (Cao, 2017).

    Differential analysis of negative geotaxis climbing trajectories in Drosophila under different conditions

    The decline of Drosophila climbing behavior is one of the common phenomena of Drosophila aging. The so-called negative geotaxis refers to the natural upward climbing behavior of Drosophila melanogaster after it oscillates to the bottom of the test tube. The strength of climbing ability is regarded as the index of aging change of D. melanogaster. At present, many laboratories use the percentage of 10 fruit flies climbing a specific height in 5 s as a general indicator of the climbing ability of fruit flies. This group research index ignores the climbing performance of a single fruit fly, and the climbing height belongs to the concept of vertical distance in physics, which cannot truly and effectively reflect the concept of curve distance in the actual climbing process of fruit flies. Therefore, based on the image processing algorithm, an experimental method was added to draw the climbing trajectory of a single fruit fly. By comparing the differences in climbing behavior of fruit flies under different sex, group or single, oscillation condition or rotation inversion condition, it was possible to find that the K-Nearest Neighbor target detection algorithm has good applicability in fruit fly climbing experiment, and the climbing ability of fruit flies decreases with age. Under the same experimental conditions, the climbing ability of female fruit flies was greater than that of male fruit flies. The climbing track length of a single fruit fly can better reflect the climbing process of a fruit fly (Zhong, 2022).

    Automated analysis of long-term grooming behavior in Drosophila using a k-nearest neighbors classifier

    Despite being pervasive, the control of programmed grooming is poorly understood. This study addressed this gap by developing a high-throughput platform that allows long-term detection of grooming in Drosophila melanogaster. In this method, a k-nearest neighbors algorithm automatically classifies fly behavior and finds grooming events with over 90% accuracy in diverse genotypes. The data show that flies spend ~13% of their waking time grooming, driven largely by two major internal programs. One of these programs regulates the timing of grooming and involves the core circadian clock components cycle, clock, and period. The second program regulates the duration of grooming and, while dependent on cycle and clock, appears to be independent of period. This emerging dual control model in which one program controls timing and another controls duration, resembles the two-process regulatory model of sleep. Together, this quantitative approach presents the opportunity for further dissection of mechanisms controlling long-term grooming in Drosophila (Qiao, 2018).

    Spatial comparisons of mechanosensory information govern the grooming sequence in Drosophila

    Animals integrate information from different sensory modalities, body parts, and time points to inform behavioral choice, but the relevant sensory comparisons and the underlying neural circuits are still largely unknown. This study used the grooming behavior of Drosophila melanogaster as a model to investigate the sensory comparisons that govern a motor sequence. Flies perform grooming movements spontaneously, but when covered with dust, they clean their bodies following an anterior-to-posterior sequence. After investigating different sensory modalities that could detect dust, focus was placed on mechanosensory bristle neurons, whose optogenetic activation induces a similar sequence. Computational modeling predicts that higher sensory input strength to the head will cause anterior grooming to occur first. This prediction was tested using an optogenetic competition assay whereby two targeted light beams independently activate mechanosensory bristle neurons on different body parts. It was found that the initial choice of grooming movement is determined by the ratio of sensory inputs to different body parts. In dust-covered flies, sensory inputs change as a result of successful cleaning movements. Simulations from this model suggest that this change results in sequence progression. One possibility is that flies perform frequent comparisons between anterior and posterior sensory inputs, and the changing ratios drive different behavior choices. Alternatively, flies may track the temporal change in sensory input to a given body part to measure cleaning effectiveness. The first hypothesis is supported by the optogenetic competition experiments: iterative spatial comparisons of sensory inputs between body parts is essential for organizing grooming movements in sequence (Zhang, 2020).

    Genetic Basis of Natural Variation in Spontaneous Grooming in Drosophila melanogaster

    Spontaneous grooming behavior is a component of insect fitness. We quantified spontaneous grooming behavior in 201 sequenced lines of the Drosophila melanogaster Genetic Reference Panel and observed significant genetic variation in spontaneous grooming, with broad-sense heritabilities of 0.25 and 0.24 in females and males, respectively. Although grooming behavior is highly correlated between males and females, significant sex by genotype interactions were observed, indicating that the genetic basis of spontaneous grooming is partially distinct in the two sexes. Genome-wide association analyses of grooming behavior was performed, and 107 molecular polymorphisms associated with spontaneous grooming behavior were mapped, of which 73 were in or near 70 genes and 34 were over 1 kilobase from the nearest gene. The candidate genes were associated with a wide variety of gene ontology terms, and several of the candidate genes were significantly enriched in a genetic interaction network. Functional assessments were performed of 29 candidate genes using RNA interference, and 11 were found to affecte spontaneous grooming behavior. The genes associated with natural variation in Drosophila grooming are involved with glutamate metabolism (Gdh) and transport (Eaat); interact genetically with (CCKLR-17D1) or are in the same gene family as (PGRP-LA) genes previously implicated in grooming behavior; are involved in the development of the nervous system and other tissues; or regulate the Notch and Epidermal growth factor receptor signaling pathways. Several DGRP lines exhibited extreme grooming behavior. Excessive grooming behavior can serve as a model for repetitive behaviors diagnostic of several human neuropsychiatric diseases (Yanagawa, 2020).

    Spontaneous motor-behavior abnormalities in two Drosophila models of neurodevelopmental disorders

    Boys with fragile X syndrome (FXS), a leading monogenic cause of intellectual disability, often display repetitive behaviors, a core feature of autism. This study characterized spontaneous-motor-behavior phenotypes of Drosophila dfmr1 mutants, an established model for FXS. Individual 1-day-old adult flies, with mature nervous systems, were recorded in small arenas. Young dfmr1 mutants spent excessive time grooming, with increased bout number and duration; both were rescued by transgenic wild-type dfmr1(+). By two grooming-pattern measures, dfmr1-mutant flies showed elevated repetitions consistent with perseveration, which is common in FXS. In addition, the mutant flies display a preference for grooming posterior body structures, and an increased rate of grooming transitions from one site to another. The possibility was raised that courtship and circadian rhythm defects, previously reported for dfmr1 mutants, are complicated by excessive grooming. Significantly increased grooming was also observed in CASK mutants, despite their dramatically decreased walking phenotype. The mutant flies, a model for human CASK-related neurodevelopmental disorders, displayed consistently elevated grooming indices throughout the assay, but transient locomotory activation immediately after placement in the arena. Based on published data identifying FMRP-target transcripts and functional analyses of mutations causing human genetic neurodevelopmental disorders, the following proteins are proposed as candidate mediators of excessive repetitive behaviors in FXS: CaMKIIα, NMDA receptor subunits 2A and 2B, NLGN3, and SHANK3. Together, these fly-mutant phenotypes and mechanistic insights provide starting points for drug discovery to identify compounds that reduce dysfunctional repetitive behaviors (Andrew, 2020).

    Behavioral evidence for nested central pattern generator control of Drosophila grooming

    Central pattern generators (CPGs) are neurons or neural circuits that produce periodic output without requiring patterned input. More complex behaviors can be assembled from simpler subroutines, and nested CPGs have been proposed to coordinate their repetitive elements, organizing control over different time scales. This study used behavioral experiments to establish that Drosophila grooming may be controlled by nested CPGs. On a short time scale (5-7 Hz, ~ 200 ms/movement), flies clean with periodic leg sweeps and rubs. More surprisingly, transitions between bouts of head sweeping and leg rubbing are also periodic on a longer time scale (0.3-0.6 Hz, ~2 s/bout). This study examined grooming at a range of temperatures to show that the frequencies of both oscillations increase-a hallmark of CPG control-and also that rhythms at the two time scales increase at the same rate, indicating that the nested CPGs may be linked. This relationship holds when sensory drive is held constant using optogenetic activation, but oscillations can decouple in spontaneously grooming flies, showing that alternative control modes are possible. Loss of sensory feedback does not disrupt periodicity but slow down the longer time scale alternation. Nested CPGs simplify the generation of complex but repetitive behaviors, and identifying them in Drosophila grooming presents an opportunity to map the neural circuits that constitute them (Ravbar, 2021).

    A pair of commissural command neurons induces Drosophila wing grooming

    In many behaviors such walking and swimming, animals need to coordinate their left and right limbs. In Drosophila, wing grooming can be induced by activation of sensory organs called campaniform sensilla. Flies usually clean one wing at a time, coordinating their left and right hind legs to sweep the dorsal and ventral surfaces of the wing. This study has identified a pair of interneurons located in the ventral nerve cord that was named wing projection neurons 1 (wPN1) whose optogenetic activation induces wing grooming. Inhibition of wPN1 activity reduces wing grooming. They receive synaptic input from ipsilateral wing campaniform sensilla and wing mechanosensory bristle neurons, and they extend axonal arbors to the hind leg neuropils. Although they project contralaterally, their activation induces ipsilateral wing grooming. Anatomical and behavioral data support a role for wPN1 as command neurons coordinating both hind legs to work together to clean the stimulated wing (Zhang, 2022).

    This study has identified wPN1 as potential command neurons for wing grooming. Although the criteria for command neurons is debated, neurons whose activation can induce a coherent motor program and whose function is required for that behavior to occur normally, such as wPN1, provide important entry points to map the neural circuits governing that behavior. The characterization of wPN1 reveals additional complexities in the control of grooming. Wing scratch remains unaffected, indicating additional sensory-to-motor pathways between wings and legs. Other left-right coordination circuits must also exist, since inhibition of wPN1 disrupts wing grooming but leaves hind-leg rubbing largely normal. wPN1 provides an entry point to identify downstream commissural and pre-motor neurons that coordinate left and right leg movements (Zhang, 2022).

    Wing cleaning normally occurs late in the hierarchical sequence of grooming subroutines, and wPN1 may provide insights into when it is selected. Perhaps sensory information from wing campaniform sensilla and mechanosensory bristles integrate or accumulate to a threshold sufficient to activate wPN1, or perhaps wPN1 is transiently inhibited by circuits driving other grooming behaviors. Identification of wPN1 makes these testable hypotheses with the potential to reveal a general control mechanism for sequential motor behaviors (Zhang, 2022).

    These behavior and anatomy data provide strong evidence that wPN1 plays an essential, command-like role in coordinating left and right back legs, but higher-resolution behavioral analysis and complete neuronal circuit mapping downstream of wPN1 are necessary to confirm this function (Zhang, 2022).

    Variation and Variability in Drosophila Grooming Behavior

    Behavioral differences can be observed between species or populations (variation) or between individuals in a genetically similar population (variability). This study investigated genetic differences as a possible source of variation and variability in Drosophila grooming. Grooming confers survival and social benefits. Grooming features of five Drosophila species exposed to a dust irritant were analyzed. Aspects of grooming behavior, such as anterior to posterior progression, were conserved between and within species. However, significant differences in activity levels, proportion of time spent in different cleaning movements, and grooming syntax were identified between species. All species tested showed individual variability in the order and duration of action sequences. Genetic diversity was not found to correlate with grooming variability within a species: melanogaster flies bred to increase or decrease genetic heterogeneity exhibited similar variability in grooming syntax. Individual flies observed on consecutive days also showed grooming sequence variability. Standardization of sensory input using optogenetics reduced but did not eliminate this variability. In aggregate, these data suggest that sequence variability may be a conserved feature of grooming behavior itself. These results also demonstrate that large genetic differences result in distinguishable grooming phenotypes (variation), but that genetic heterogeneity within a population does not necessarily correspond to an increase in the range of grooming behavior (variability) (Mueller, 2021).

    Somatotopic organization among parallel sensory pathways that promote a grooming sequence in Drosophila

    Mechanosensory neurons located across the body surface respond to tactile stimuli and elicit diverse behavioral responses, from relatively simple stimulus location-aimed movements to complex movement sequences. How mechanosensory neurons and their postsynaptic circuits influence such diverse behaviors remains unclear. Previous work has shown that Drosophila perform a body location-prioritized grooming sequence when mechanosensory neurons at different locations on the head and body are simultaneously stimulated by dust. This study identified nearly all mechanosensory neurons on the Drosophila head that individually elicit aimed grooming of specific head locations, while collectively eliciting a whole head grooming sequence. Different tracing methods were used to reconstruct the projections of these neurons from different locations on the head to their distinct arborizations in the brain. This provides the first synaptic resolution somatotopic map of a head, and defines the parallel-projecting mechanosensory pathways that elicit head grooming (Eichler, 2023).

    Phenotype-dependent habitat choice is too weak to cause assortative mating between Drosophila melanogaster strains differing in light sensitivity

    Matching habitat choice is gaining attention as a mechanism for maintaining biodiversity and driving speciation. It revolves around the idea that individuals select the habitat in which they perceive to obtain greater fitness based on a prior evaluation of their local performance across heterogeneous environments. This re