• 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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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 results in individuals with similar ecologically relevant traits converging to the same patches, and hence it could indirectly cause assortative mating when mating occurs in those patches. White-eyed mutants of Drosophila fruit flies have a series of disadvantages compared to wild type flies, including a poorer performance under bright light. It has been previously reported that, when given a choice, wild type Drosophila simulans preferred a brightly lit habitat while white-eyed mutants occupied a dimly lit one. This spatial segregation allowed the eye color polymorphism to be maintained for several generations, whereas normally it is quickly replaced by the wild type. This study compared the habitat choice decisions of white-eyed and wild type flies in another species, D. melanogaster. Groups of flies were released in a light gradient, and their departure and settlement behavior was recorded. Departure depended on sex and phenotype, but not on the light conditions of the release point. Settlement depended on sex, and on the interaction between phenotype and light conditions of the point of settlement. Nonetheless, simulations showed that this differential habitat use by the phenotypes would only cause a minimal degree of assortative mating in this species (Peralta-Rincon, 2020).

    Common microbehavioral 'footprint' of two distinct classes of conditioned aversion

    Avoiding unfavorable situations is a vital skill and a constant task for any animal. Situations can be unfavorable because they feature something that the animal wants to escape from, or because they do not feature something that it seeks to obtain. This study investigated whether the microbehavioral mechanisms by which these two classes of aversion come about are shared or distinct. Larval Drosophila were found to avoid odors either previously associated with a punishment, or previously associated with the lack of a reward. These two classes of conditioned aversion are found to be strikingly alike at the microbehavioral level. In both cases larvae show more head casts when oriented toward the odor source than when oriented away, and direct fewer of their head casts toward the odor than away when oriented obliquely to it. Thus, conditioned aversion serving two qualitatively different functions-escape from a punishment or search for a reward-is implemented by the modulation of the same microbehavioral features. These features also underlie conditioned approach, albeit with opposite sign. That is, the larvae show conditioned approach toward odors previously associated with a reward, or with the lack of a punishment. In order to accomplish both these classes of conditioned approach the larvae show fewer head casts when oriented toward an odor, and direct more of their head casts toward it when they are headed obliquely. Given that the Drosophila larva is a genetically tractable model organism that is well suited to study simple circuits at the single-cell level, these analyses can guide future research into the neuronal circuits underlying conditioned approach and aversion, and the computational principles of conditioned search and escape (Paisios, 2017).

    Neural circuitry that evokes escape behavior upon activation of nociceptive sensory neurons in Drosophila larvae

    Noxious stimuli trigger a stereotyped escape response in animals. In Drosophila larvae, class IV dendrite arborization (C4 da) sensory neurons in the peripheral nervous system are responsible for perception of multiple nociceptive modalities, including noxious heat and harsh mechanical stimulation, through distinct receptors. Silencing or ablation of C4 da neurons largely eliminates larval responses to noxious stimuli, whereas optogenetic activation of C4 da neurons is sufficient to provoke corkscrew-like rolling behavior similar to what is observed when larvae receive noxious stimuli, such as high temperature or harsh mechanical stimulation. How C4 da activation triggers the escape behavior in the circuit level is still incompletely understood. This study identified segmentally arrayed local interneurons (medial clusters of C4 da second-order interneurons [mCSIs]) in the ventral nerve cord that are necessary and sufficient to trigger rolling behavior. GFP reconstitution across synaptic partners (GRASP) analysis indicates that C4 da axons form synapses with mCSI dendrites. Optogenetic activation of mCSIs induces the rolling behavior, whereas silencing mCSIs reduces the probability of rolling behavior upon C4 da activation. Further anatomical and functional studies suggest that the C4 da-mCSI nociceptive circuit evokes rolling behavior at least in part through segmental nerve a (SNa) motor neurons. These findings thus uncover a local circuit that promotes escape behavior upon noxious stimuli in Drosophila larvae and provide mechanistic insights into how noxious stimuli are transduced into the stereotyped escape behavior in the circuit level (Yoshino, 2017).

    Neural basis for looming size and velocity encoding in the Drosophila giant fiber escape pathway

    Identified neuron classes in vertebrate cortical and subcortical areas and invertebrate peripheral and central brain neuropils encode specific visual features of a panorama. How downstream neurons integrate these features to control vital behaviors, like escape, is unclear. In Drosophila, the timing of a single spike in the giant fiber (GF) descending neuron determines whether a fly uses a short or long takeoff when escaping a looming predator. It has been proposed that GF spike timing results from summation of two visual features whose detection is highly conserved across animals: an object's subtended angular size and its angular velocity. Velocity encoding is attributed to input from lobula columnar type 4 (LC4) visual projection neurons, but the size-encoding source remained unknown. This study shows that lobula plate/lobula columnar, type 2 (LPLC2) visual projection neurons anatomically specialized to detect looming provide the entire GF size component. LPLC2 neurons were found to be necessary for GF-mediated escape, and LPLC2 and LC4 synapse are shown directly onto the GF via reconstruction in a fly brain electron microscopy (EM) volume. LPLC2 silencing eliminates the size component of the GF looming response in patch-clamp recordings, leaving only the velocity component. A model summing a linear function of angular velocity (provided by LC4) and a Gaussian function of angular size (provided by LPLC2) replicates GF looming response dynamics and predicts the peak response time. This study thus presents an identified circuit in which information from looming feature-detecting neurons is combined by a common post-synaptic target to determine behavioral output (Ache, 2019).

    A neuronal pathway that commands deceleration in Drosophila larval light-avoidance

    When facing a sudden danger or aversive condition while engaged in on-going forward motion, animals transiently slow down and make a turn to escape. The neural mechanisms underlying stimulation-induced deceleration in avoidance behavior are largely unknown. This study reports that in Drosophila larvae, light-induced deceleration was commanded by a continuous neural pathway that included prothoracicotropic hormone neurons, eclosion hormone neurons, and tyrosine decarboxylase 2 motor neurons (the PET pathway). Inhibiting neurons in the PET pathway led to defects in light-avoidance due to insufficient deceleration and head casting. On the other hand, activation of PET pathway neurons specifically caused immediate deceleration in larval locomotion. These findings reveal a neural substrate for the emergent deceleration response and provide a new understanding of the relationship between behavioral modules in animal avoidance responses (Gong, 2019).

    Sensory perception of dead conspecifics induces aversive cues and modulates lifespan through serotonin in Drosophila

    Sensory perception modulates health and aging across taxa. Understanding the nature of relevant cues and the mechanisms underlying their action may lead to novel interventions that improve the length and quality of life. This study found that in the vinegar fly, Drosophila melanogaster, exposure to dead conspecifics in the environment induced cues that were aversive to other flies, modulated physiology, and impaired longevity. The effects of exposure to dead conspecifics on aversiveness and lifespan required visual and olfactory function in the exposed flies. Furthermore, the sight of dead flies was sufficient to produce aversive cues and to induce changes in the head metabolome. Genetic and pharmacologic attenuation of serotonergic signaling eliminated the effects of exposure on aversiveness and lifespan. These results indicate that Drosophila have an ability to perceive dead conspecifics in their environment and suggest conserved mechanistic links between neural state, health, and aging; the roots of which might be unearthed using invertebrate model systems (Chakraborty, 2019).

    The behavioral repertoire of Drosophila melanogaster in the presence of two predator species that differ in hunting mode

    The fruit fly, Drosophila melanogaster, has proven to be an excellent model organism for genetic, genomic and neurobiological studies. However, relatively little is known about the natural history of D. melanogaster. In particular, neither the natural predators faced by wild populations of D. melanogaster, nor the anti-predatory behaviors they may employ to escape and avoid their enemies have been documented. This study observed and described the influence of two predators that differ in their mode of hunting: zebra jumping spiders, Salticus scenicus (active hunters) and Chinese praying mantids, Tenodera sinensis (ambush predators) on the behavioral repertoire of Drosophila melanogaster. Three particularly interesting behaviors were documented: abdominal lifting, stopping, and retreat-which were performed at higher frequency by D. melanogaster in the presence of predators. While mantids had only a modest influence on the locomotory activity of D. melanogaster, a significant increase was observed in the overall activity of D. melanogaster in the presence of jumping spiders. Finally, considerable among-individual behavioral variation was observed in response to both predators (Parigi, 2019).

    Multiple phototransduction inputs integrate to mediate UV light-evoked avoidance/attraction behavior in Drosophila

    Short-wavelength light guides many behaviors that are crucial for an insect's survival. In Drosophila melanogaster, short-wavelength light induces both attraction and avoidance behaviors. How light cues evoke two opposite valences of behavioral responses remains unclear. This study comprehensively examined the effects of (1) light intensity, (2) timing of light (duration of exposure, circadian time of day), and (3) phototransduction mechanisms processing light information that determine avoidance versus attraction behavior assayed at high spatiotemporal resolution in Drosophila. External opsin-based photoreceptors signal for attraction behavior in response to low-intensity ultraviolet (UV) light. In contrast, the cell-autonomous neuronal photoreceptors, CRYPTOCHROME (CRY) and RHODOPSIN 7 (RH7), signal avoidance responses to high-intensity UV light. In addition to binary attraction versus avoidance behavioral responses to UV light, flies show distinct clock-dependent spatial preference within a light environment coded by different light input channels (Baik, 2019).

    Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila

    Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. A population of interneurons in the nerve cord of Drosophila, termed Down-and-Back (DnB) neurons, was identified that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior (Burgos, 2018).

    Nocifensive escape behavior in Drosophila larvae consists of C-shaped body bending and rolling, followed by rapid forward crawling. Recent studies have begun to identify circuits that mediate nocifensive behaviors (Kaneko, 2017; Ohyama, 2015; Yoshino, 2017). Prior work identified Basin neurons as multisensory interneurons that drive rolling behavior in response to vibration and noxious stimuli, and identified downstream Goro as command-like neurons for rolling. This study has identified and characterized DnB interneurons that are essential for nocifensive behavior in Drosophila larvae (see Summary model for DnB neurons controlling nocifensive escape). DnB neurons are direct targets of nociceptive cIV neurons and multiple mechanosensory cell types, including cII and cIII gentle touch da neurons and es neurons. Thus, DnBs provide a potential node for multisensory integration of tactile and noxious stimuli. The convergence of input from cIII gentle-touch receptors and cIV nociceptors onto DnB neurons is reminiscent of vertebrate interneurons that receive direct excitatory input from C-fiber/A∂ nociceptors and Aβ mechanoreceptors. Based on these studies nociceptive inputs appear to be integrated with multiple mechanosensory submodalities by Basin and DnB interneurons (Burgos, 2018).

    EM reconstruction of DnB targets supported divergent major downstream circuitry. Output synapses on DnB axons converge on premotor neurons, at least some of which promote peristaltic wave propagation during locomotion. Other downstream neurons receive input from presynaptic sites on the DnB dendrite, and lead to Goro rolling command-like neurons. The spatial segregation of DnB output sites may mirror a functional segregation of downstream circuitry into bending and rolling modules. It is still unclear which muscle groups are recruited and how segments coordinate during body bending and rolling. This study provides evidence that silencing the period-positive median segmental interneuron (PMSI) cohort, which includes direct DnB targets A02g and A02e, reduces rolling behavior. PMSIs are glutamatergic inhibitory premotor neurons that terminate motor neuron bursting to regulate crawling speed (Kohsaka, 2014). Future work to selectively silence groups of premotor neurons will help to elucidate their role in nocifensive escape downstream of DnBs. Although silencing DnB neurons slightly increased the speed of forward locomotion, overall, forward crawling remained intact. Given that peristaltic waves also consist of segmental contractions, links to premotor neurons provide candidate neurons for dual control of crawling and C-shape bending behavior. Notably, DnB neurons target motor neurons innervating LT1 muscles, which have been implicated in larval self-righting behaviors. Self-righting consists of a C-shape type body bend, and 180° turn, so it is possible that LT1 muscles facilitate curved body bends that underlie both self-righting and rolling behavior. It is noted that the impact of DnB neurons on nociceptive circuits is likely to be more broad than indicated by synaptic connections, since EM and marker expression suggest that DnB neurons are peptidergic. Identification of the putative neuropeptide expressed by DnB neurons, and physiological effects, will be an important future direction, particularly given the important role of neuropeptides in vertebrate pain pathways, and recent evidence that mechanical nociception in larvae is under peptidergic control (Burgos, 2018).

    Prior data showed that rolling is directional and is advantageous for dislodging attacking parasitoid wasps. Efficient rolling occurs coincident with deep C-shaped body bends, but the significance of these body bends for escape behavior has not been determined. DnB neural circuitry appears to be critically important for evoking body bend behavior prior to and during nocifensive rolling. Bending may provide the initial, most rapid, form of withdrawal from a noxious stimulus, and may subsequently support rolling locomotion by orienting and focusing the energy of muscle contraction into lateral thrusts. Re-orientation of denticle belts, triangle-shaped extensions of the cuticle, may also aid rapid lateral locomotion by providing substrate traction. Compromised escape rolling upon DnB inactivation may therefore arise both from weakened Goro activation and decreases in body bend angle. Understanding the circuit mechanisms that promote bending downstream of DnB neurons, and the muscle activities and physical mechanisms that underlie rolling behavior are important future aims (Burgos, 2018).

    Analysis of DnB function revealed modular control of nocifensive escape behavior, consistent with EM reconstruction data. When DnB neurons were ectopically activated C-shaped body bending was observed that was often, but not always, associated with rolling. Other, non-rolling, animals bent with minimal crawling, or bent persistently while attempting to crawl forward. These observations provided initial evidence that C-shaped bending and rolling control circuits are separable, and that nocifensive bending could be combined with other behaviors, like pausing or crawling. Loss of function data supported bending as a primary motor output of DnB activity, with probabilistic activation of rolling motor programs. These behaviors could conceivably be linked, such that reduction in bending compromises rolling ability, or could arise from parallel influence of DnB activity on bending and rolling as suggested by EM reconstruction. Consistent with an important role for DnBs in promoting rolling, silencing Goro while activating DnB neurons promoted persistent bending without rolling, and uncoordinated snake-like forward crawling. This result further implicates a separate premotor circuitry in nocifensive body bending. These data further suggest that the bend-roll sequence must be tightly regulated by interactions between the parallel bend-roll premotor circuits, such that bending occurs first to facilitate rolling, which occurs second. However, bending can occur without being followed by rolling, indicating C-shaped bending itself is not sufficient to trigger rolling. Such independent, but sequentially regulated behavioral modules are consistent with hierarchical models of sequence generation as in fly grooming, human speech, roll-crawl sequence, and hunch-bend sequence. It is noted however, that although bending and rolling are sequential, they co-occur for much of the defensive behavior sequence, in contrast to such sequential and non-overlapping behavioral sequences. Elucidating the mechanisms of timing and interaction between the different circuit modules (bend vs roll) identified therefore promises to shed light on the general mechanisms of circuit implementation of sequence generation and co-ordination between different motor modules (Burgos, 2018).

    A neural basis for categorizing sensory stimuli to enhance decision accuracy

    Sensory stimuli with graded intensities often lead to yes-or-no decisions on whether to respond to the stimuli. How this graded-to-binary conversion is implemented in the central nervous system (CNS) remains poorly understood. This study shows that graded encodings of noxious stimuli are categorized in a decision-associated CNS region in the ventral cord of Drosophila larvae, and then decoded by a group of peptidergic neurons for executing binary escape decisions. GABAergic inhibition gates weak nociceptive encodings from being decoded, whereas escalated amplification through the recruitment of second-order neurons boosts nociceptive encodings at intermediate intensities. These two modulations increase the detection accuracy by reducing responses to negligible stimuli whereas enhancing responses to intense stimuli. These findings thus unravel a circuit mechanism that underlies accurate detection of harmful stimuli (Hu, 2020).

    This study identified a neural network that categorizes noxious stimuli of graded intensities to generate binary escape decisions in Drosophila larvae and unraveled a gated amplification mechanism that underlies such binary categorization. In responding to the noxious stimuli, whereas failure in prompt responses may cause harm, excessive escape responses to negligible stimuli would lead to the loss of resources for survival. The gated amplification mechanism could reduce the responses to negligible stimuli whereas enhancing the responses to intense stimuli. In this way, the accuracy in deciding whether to escape from the stimuli is enhanced (Hu, 2020).

    Information processing in the nervous system is affected by noise, which may be embedded in external sensory stimuli (e.g., sensory noise) or generated within the nervous system (e.g., electric noise). A recent study in C. elegans shows that activation mediated by electrical synapses and disinhibition mediated by glutamatergic chemical synapses form an AND logic gate to integrate the presentation of the salience of attractive odors (Dobosiewicz, 2019). The AND-gate computation in worm AIA interneurons requires multiple sensory neurons to report the presence of attractive odors and, consequently, filters out the noise embedded in the sensory stimuli. Another study on the olfactory system of adult Drosophila reported a mechanism to address the noise that is produced within the nervous system. A three-layered feedforward network averages the noise to enhance peak detection accuracy and then uses coincidence detection to distinguish real signals arrived synchronously from noise caused by spontaneous neural activities. In the nervous system, the noise can be produced at each stage of the sensori-motor transformation. Compared with the two mechanisms mentioned above, which filter out the existing noise, the graded-to-binary conversion through the gated amplification mechanism reported in this study makes the converted signals less vulnerable to the noise produced at later stages of sensori-motor transformation. This is because after the graded signals become binary, the signals are more separated (either suppressed or amplified) according to stimulus intensities and, consequently, the same level of noise is less likely to cause the binary signals to falsely pass the decision threshold than the graded ones. As a result, the ambiguous encoding range of the stimulus intensity is narrowed and the frequency of false decisions is reduced, as demonstrated by computational modeling (Hu, 2020).

    Thresholding of gradually accumulated sensory evidence has been considered to be fundamental for generating yes-or-no decisions. For example, a recent study in mammals has shown that visual evidence of danger can be gradually accumulated by recurrent circuits to overcome the threshold for escape behaviors. Such a mechanism takes time to build up decision-associated activities for decisions with higher accuracy, which leads to the well-known speed-accuracy trade-off in perceptual decision making. However, the current findings add a new dimension to the processing of sensory evidence for perceptual decision making: different from recurrent networks, the recruitment of a number of second-order neurons (SONs) can instantaneously boost the decision-associated activity to reach the decision threshold, which ensures decision speed. Because the gated amplification mechanism reported in this study also ensures the detection accuracy, such a mechanism might bypass the speed-accuracy trade-off in sensory signal detection (Hu, 2020).

    An electron microscopy connectome study reported 13 types of SONs in the Drosophila larval nociceptive system, each of which has distinct connectivity and functions. For example, Basin-4, DnB, and Wave neurons also receive mechanosensory inputs, whereas A08n does not. Moreover, Wave neurons detect stimulus positions on larval body walls. Furthermore, serotonergic modulation acts on this network during development to adjust the nociceptive responses, providing a mechanism for larvae to adjust the escape threshold according to their developmental environment. However, because at least 5 types of SONs are both required and sufficient for larval escape behaviors, it remains a mystery why there exist so many seemingly redundant neurons at the same level in the network. The nociceptive system is a dedicated protective system that responds to potential tissue-damaging insults, so both speed and accuracy of the perceptual decision-making process are important. This is probably why the nociceptive system uses an amplification network formed by a large number of SONs to dissociate time from accuracy in the perceptual decision-making process and avoid the trade-off between decision speed and accuracy (Hu, 2020).

    This study has developed novel unbiased computational toolsets for automatically analyzing the functional connectivity of all neural structures, including both somas and neurites in the larval VNC. Using these toolsets, a decision-associated CNS region, the PMC, was identified the covers the neuropil structure TP. The TP is concentrated with large amounts of neurites, especially those of peptidergic neurons. Although this anatomical structure was identified previously, its function is unknown. The finding of its important function in sensori-motor transformation suggests that this region is possibly a hub for information exchange and integration. The detailed anatomical and functional connectivity of the TP could be a fascinating direction for future studies (Hu, 2020).

    In summary, a neural basis is postulated for converting graded sensory inputs to yes-or-no behavioral decisions. A previous study showed that neurons in the rat posterior parietal cortex encode a graded value of accumulating evidence whereas those in the prefrontal cortex have a more categorical encoding that indicates the decisions. Thus, the categorization of sensory evidence by making graded encodings binary in perceptual decision making is likely an evolutionarily conserved process. In this study, advantage was taken of the powerful genetic model Drosophila to unravel how such computation might be implemented at the cellular and molecular level. Finally, because whole-CNS functional imaging analysis is a key approach to decipher the neural basis for sensori-motor integration and perceptual decision making, it is anticipated that the computational tools developed in this study will facilitate investigations in these fields (Hu, 2020).

    Competitive disinhibition mediates behavioral choice and sequences in Drosophila

    Even a simple sensory stimulus can elicit distinct innate behaviors and sequences. During sensorimotor decisions, competitive interactions among neurons that promote distinct behaviors must ensure the selection and maintenance of one behavior, while suppressing others. The circuit implementation of these competitive interactions is still an open question. By combining comprehensive electron microscopy reconstruction of inhibitory interneuron networks, modeling, electrophysiology, and behavioral studies, this study determined the circuit mechanisms that contribute to the Drosophila larval sensorimotor decision to startle, explore, or perform a sequence of the two in response to a mechanosensory stimulus. Together, these studies reveal that, early in sensory processing, (1) reciprocally connected feedforward inhibitory interneurons implement behavioral choice, (2) local feedback disinhibition provides positive feedback that consolidates and maintains the chosen behavior, and (3) lateral disinhibition promotes sequence transitions. The combination of these interconnected circuit motifs can implement both behavior selection and the serial organization of behaviors into a sequence (Jovanic, 2016).

    Visually guided behavior and optogenetically induced learning in head-fixed flies exploring a virtual landscape

    Studying the intertwined roles of sensation, experience, and directed action in navigation has been facilitated by the development of virtual reality (VR) environments for head-fixed animals, allowing for quantitative measurements of behavior in well-controlled conditions. VR has long featured in studies of Drosophila melanogaster, but these experiments have typically allowed the fly to change only its heading in a visual scene and not its position. This study explores how flies move in two dimensions (2D) using a visual VR environment that more closely captures an animal's experience during free behavior. Flies' 2D interaction with landmarks cannot be automatically derived from their orienting behavior under simpler one-dimensional (1D) conditions. Using novel paradigms, this study demonstrated that flies in 2D VR adapt their behavior in response to optogenetically delivered appetitive and aversive stimuli. Much like free-walking flies after encounters with food, head-fixed flies exploring a 2D VR respond to optogenetic activation of sugar-sensing neurons by initiating a local search, which appears not to rely on visual landmarks. Visual landmarks can, however, help flies to avoid areas in VR where they experience an aversive, optogenetically generated heat stimulus. By coupling aversive virtual heat to the flies' presence near visual landmarks of specific shapes, selective learned avoidance of those landmarks was elicited. Thus, this study demonstrates that head-fixed flies adaptively navigate in 2D virtual environments, but their reliance on visual landmarks is context dependent. These behavioral paradigms set the stage for interrogation of the fly brain circuitry underlying flexible navigation in complex multisensory environments (Haberkern, 2019).

    Serotoninergic Modulation of Phototactic Variability Underpins a Bet-Hedging Strategy in Drosophila melanogaster

    When organisms' environmental conditions vary unpredictably in time, it can be advantageous for individuals to hedge their phenotypic bets. It has been shown that a bet-hedging strategy possibly underlies the high inter-individual diversity of phototactic choice in Drosophila melanogaster. This study shows that fruit flies from a population living in a boreal and relatively unpredictable climate have more variable phototactic biases than fruit flies from a more stable tropical climate, consistent with bet-hedging theory. This study experimentally showed that phototactic variability of D. melanogaster is regulated by the neurotransmitter serotonin (5-HT), which acts as a suppressor of the variability of phototactic choices. When fed 5-HT precursor, boreal flies exhibited lower variability, and they were insensitive to 5-HT inhibitor. The opposite pattern was seen in the tropical flies. Thus, the reduction of 5-HT in fruit flies' brains may be the mechanistic basis of an adaptive bet-hedging strategy in a less predictable boreal climate (Krams, 2021).

    Behavioral evidence for enhanced processing of the minor component of binary odor mixtures in larval Drosophila

    A fundamental problem in deciding between mutually exclusive options is that the decision needs to be categorical although the properties of the options often differ but in grade. In this study, larval Drosophila were trained such that in one set of animals odor A was rewarded, but odor B was not (A+/B), whereas a second set of animals was trained reciprocally (A/B+). The preference was tested of the larvae, either for A, or for B, or for "morphed" mixtures of A and B, that is for mixtures differing in the ratio of the two components. As expected, the larvae showed higher preference when only the previously rewarded odor was presented than when only the previously unrewarded odor was presented. For mixtures of A and B that differed in the ratio of the two components, the major component dominated preference behavior-but it dominated less than expected from a linear relationship between mixture ratio and preference behavior. This suggests that a minor component can have an enhanced impact in a mixture, relative to such a linear expectation. The current paradigm may prove useful in understanding how nervous systems generate discrete outputs in the face of inputs that differ only gradually (Chen, 2017).

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

    The fruit fly larva stands as a powerful model to study decision-making processes that underlie directed navigation. This study has quantitatively measured phototaxis in response to well-defined sensory inputs. Subsequently, a statistical stochastic model based on biased Markov chains was formulated to characterize the behavioural basis of negative phototaxis. These experiments show that larvae make navigational decisions depending on two independent physical variables: light intensity and its spatial gradient. Furthermore, the statistical model quantifies how larvae balance two potentially-contradictory factors: minimizing exposure to light intensity and at the same time maximizing their distance to the light source. The response to the light field is manifestly non-linear, and saturates above an intensity threshold. The model has been validated against experimental biological data yielding insight into the strategy that larvae use to achieve their goal with respect to the navigational cue of light, an important piece of information for future work to study the role of the different neuronal components in larval phototaxis (de Andres-Bragado, 2018).

    Are Drosophila preferences for yeasts stable or contextual?

    Whether there are general mechanisms, driving interspecific chemical communication is uncertain. Saccharomycetaceae yeast and Drosophila fruit flies, both extensively studied research models, share the same fruit habitat, and it has been suggested their interaction comprises a facultative mutualism that is instigated and maintained by yeast volatiles. Using choice tests, experimental evolution, and volatile analyses, this study investigated the maintenance of this relationship and reveal little consistency between behavioral responses of two isolates of sympatric Drosophila species. While D. melanogaster was attracted to a range of different Saccharomycetaceae yeasts and this was independent of fruit type, D. simulans preference appeared specific to a particular S. cerevisiae genotype isolated from a vineyard fly population. This response, however, was not consistent across fruit types and is therefore context-dependent. In addition, D. simulans attraction to an individual S. cerevisiae isolate was pliable over ecological timescales. Volatile candidates were analyzed to identify a common signal for yeast attraction, and while D. melanogaster generally responded to fermentation profiles, D. simulans preference was more discerning and likely threshold-dependent. Overall, there is no strong evidence to support the idea of bespoke interactions with specific yeasts for either of these Drosophila genotypes. Rather the data support the idea Drosophila are generally adapted to sense and locate fruits infested by a range of fungal microbes and/or that yeast-Drosophila interactions may evolve rapidly (Gunther, 2019).

    Honeybees learn landscape features during exploratory orientation flights

    Exploration is an elementary and fundamental form of learning about the structure of the world. Navigating animals explore the environment for safe return to an important place (e.g., a nest site) and to travel between places. Flying central-place foragers like honeybees (Apis mellifera) extend their exploration into distances from which the features of the nest cannot be directly perceived. Bees perform short-range and long-range orientations flights. Short-range flights are performed in the immediate surroundings of the hive and occur more frequently under unfavorable weather conditions, whereas long-range flights lead the bees into different sectors of the surrounding environment. Applying harmonic radar technology for flight tracking, this study addressed the question of whether bees learn landscape features during their first short-range or long-range orientation flight. The homing flights of single bees were compared after they were displaced to areas explored or not explored during the orientation flight. Bees learn the landscape features during the first orientation flight since they returned faster and along straighter flights from explored areas as compared to unexplored areas. The study excluded a range of possible factors that might have guided bees back to the hive based on egocentric navigation strategies (path integration, beacon orientation, and pattern matching of the skyline). It is concluded that bees localize themselves according to learned ground structures and their spatial relations to the hive (Degen, 2016).

    Drosophila increase exploration after visually detecting predators

    Novel stimuli elicit behaviors that are collectively known as specific exploration. These behaviors allow the animal to become more familiar with the novel objects within its environment. Specific exploration is frequently suppressed by defensive reactions to predator cues. This study examined if this suppression occurs in Drosophila melanogaster by measuring the response of these flies to wild harvested predators. The flies used in these experiments have been cultured and had not lived under predator threat for multiple decades. In a circular arena with centrally-caged predators, wild type Drosophila actively avoided the pantropical jumping spider, Plexippus paykulli, and the Texas unicorn mantis, Phyllovates chlorophaena, indicating an innate defensive reaction to these predators. Interestingly, wild type Drosophila males also avoided a centrally-caged mock spider, and the avoidance of the mock spider became exaggerated when it was made to move within the cage. Visually impaired Drosophila failed to detect and avoid the Plexippus paykulli and the moving mock spider, while the broadly anosmic orco2 mutants were fully capable of detecting and avoiding Plexippus paykulli, indicating that these flies principally relied upon vison to perceive the predator stimuli. During early exploration of the arena, exploratory activity increased in the presence of Plexippus paykulli and the moving mock spider. The elevated activity induced by Plexippus paykulli disappeared after the fly had finished exploring, suggesting the flies were capable of habituating the predator cues. Taken together, these results indicate that despite being isolated from predators for decades Drosophila will visually detect these predators, retain innate defensive behaviors, respond by increasing exploratory activity in the arena rather than suppressing activity, and may habituate to normal predator cues (de la Flor, 2017).

    Exploratory search during directed navigation in C. elegans and Drosophila larva

    Many organisms-from bacteria to nematodes to insect larvae-navigate their environments by biasing random movements. In these organisms, navigation in isotropic environments can be characterized as an essentially diffusive and undirected process. In stimulus gradients, movement decisions are biased to drive directed navigation toward favorable environments. How does directed navigation in a gradient modulate random exploration either parallel or orthogonal to the gradient? This study introduces methods originally used for analyzing protein folding trajectories to study the trajectories of the nematode Caenorhabditis elegans and the Drosophila larva in isotropic environments, as well as in thermal and chemical gradients. The statistics of random exploration in any direction are little affected by directed movement along a stimulus gradient. A key constraint on the behavioral strategies of these organisms appears to be the preservation of their capacity to continuously explore their environments in all directions even while moving toward favorable conditions (Klein, 2017).

    Bi-directional control of walking behavior by horizontal optic flow sensors

    Moving animals experience constant sensory feedback, such as panoramic image shifts on the retina, termed optic flow. Underlying neuronal signals are thought to be important for exploratory behavior by signaling unintended course deviations and by providing spatial information about the environment. Particularly in insects, the encoding of self-motion-related optic flow is well understood. However, a gap remains in understanding how the associated neuronal activity controls locomotor trajectories. In flies, visual projection neurons belonging to two groups encode panoramic horizontal motion: horizontal system (HS) cells respond with depolarization to front-to-back motion and hyperpolarization to the opposite direction, and other neurons have the mirror-symmetrical response profile. With primarily monocular sensitivity, the neurons' responses are ambiguous for different rotational and translational self-movement components. Such ambiguities can be greatly reduced by combining signals from both eyes to determine turning and movement speed. This study explores the underlying functional logic by optogenetic HS cell manipulation in tethered walking Drosophila. De- and hyperpolarization were shown to evoke opposite turning behavior, indicating that both direction-selective signals are transmitted to descending pathways for course control. Further experiments reveal a negative effect of bilaterally symmetric de- and hyperpolarization on walking velocity. The results are therefore consistent with a functional architecture in which the HS cells' membrane potential influences walking behavior bi-directionally via two decelerating pathways (Busch, 1018).

    Sugar intake elicits intelligent searching behavior in flies and honey bees

    This study presents a comparison of the sugar-elicited search behavior in Drosophila melanogaster and Apis mellifera. In both species, intake of sugar-water elicits a complex of searching responses. The most obvious response was an increase in turning frequency. However, it was also found that flies and honey bees returned to the location of the sugar drop. They even returned to the food location when they were prevented from using visual and chemosensory cues. Analyses of the recorded trajectories indicated that flies and bees use two mechanisms, a locomotor pattern involving an increased turning frequency and path integration to increase the probability to stay close or even return to the sugar drop location. However, evidence for the use of path integration in honey bees was less clear. In general, walking trajectories of honey bees showed a higher degree of curvature and were more spacious; two characters which likely masked evidence for the use of path integration in these experiments. Visual cues, i.e., a black dot, presented underneath the sugar drop made flies and honey bees stay closer to the starting point of the search. In honey bees, vertical black columns close to the sugar drop increased the probability to visit similar cues in the vicinity. An additional one trial learning experiment suggested that the intake of sugar-water likely has the potential to initiate an associative learning process. Together, these experiments indicate that the sugar-elicited local search is more complex than previously assumed. Most importantly, this local search behavior appeared to exhibit major behavioral capabilities of large-scale navigation. Thus, it is proposed that sugar-elicited search behavior has the potential to become a fruitful behavioral paradigm to identify neural and molecular mechanisms involved in general mechanisms of navigation (Brockmann, 2018).

    Olfactory and neuromodulatory signals reverse visual object avoidance to approach in Drosophila

    Behavioral reactions of animals to environmental sensory stimuli are sometimes reflexive and stereotyped but can also vary depending on contextual conditions. Engaging in active foraging or flight provokes a reversal in the valence of carbon dioxide responses from aversion to approach in Drosophila, whereas mosquitoes encountering this same chemical cue show enhanced approach toward a small visual object. Sensory plasticity in insects has been broadly attributed to the action of biogenic amines, which modulate behaviors such as olfactory learning, aggression, feeding, and egg laying. Octopamine acts rapidly upon the onset of flight to modulate the response gain of directionally selective motion-detecting neurons in Drosophila. How the action of biogenic amines might couple sensory modalities to each other or to locomotive states remains poorly understood. This study used a visual flight simulator equipped for odor delivery to confirm that flies avoid a small contrasting visual object in odorless air but that the same animals reverse their preference to approach in the presence of attractive food odor. An aversive odor does not reverse object aversion. Optogenetic activation of either octopaminergic neurons or directionally selective motion-detecting neurons that express octopamine receptors elicits visual valence reversal in the absence of odor. The results suggest a parsimonious model in which odor-activated octopamine release excites the motion detection pathway to increase the saliency of either a small object or a bar, eliciting tracking responses by both visual features (Cheng, 2019).

    Characterizing approach behavior of Drosophila melanogaster in Buridan's paradigm

    The Buridan's paradigm is a behavioral task designed for testing visuomotor responses or phototaxis in fruit fly Drosophila melanogaster. In the task, a wing-shortened fruit fly freely moves on a round platform surrounded by a 360° white screen with two vertical black stripes placed at 0° and 180°. A normal fly will tend to approach the stripes one at a time and move back and forth between them. A variety of tasks developed based on the Buridan's paradigm were designed to test other cognitive functions such as visual spatial memory. Although the movement patterns and the behavioral preferences of the flies in the Buridan's or similar tasks have been extensively studies a few decades ago, the protocol and experimental settings are markedly different from what are used today. This study revisited the Buridan's paradigm and systematically investigated the approach behavior of fruit flies under different stimulus settings. While early studies revealed an edge-fixation behavior for a wide stripe in the initial visuomotor responses, no such tendency was discovered in the Buridan's paradigm when observing a longer-term behavior up to minutes, a memory-task relevant time scale. Instead, robust negative photoaxis was observed in which the flies approached the central part of the dark stripes of all sizes. In addition, it was found that stripes of 20°-30° width yielded the best performance of approach. Further, the luminance of the stripes and the background screen were varied; it was discovered that the performance depended on the luminance ratio between the stripes and the screen. This study provided useful information for designing and optimizing the Buridan's paradigm and other behavioral tasks that utilize the approach behavior (Han, 2021).

    Modulations of microbehaviour by associative memory strength in Drosophila larvae

    Finding food is a vital skill and a constant task for any animal, and associative learning of food-predicting cues gives an advantage in this daily struggle. This study investigated what impact the strength of an associative odour-sugar memory has on the microbehaviour of Drosophila larvae. Larvae were found to form stronger memories with increasing concentrations of sugar or odour, and these stronger memories manifest themselves in stronger modulations of two aspects of larval microbehaviour, the rate and the direction of lateral reorientation manoeuvres (so-called head casts). These two modulations of larval behaviour are found to be correlated to each other in every experiment performed, in line with a model that assumes that both modulations are controlled by a common motor output. These analyses can guide future research into the neuronal circuits underlying the translation of associative memories of different strength into behaviour, and may help to understand how these processes are organised in more complex systems (Thane, 2019).

    Scaling the interactive effects of attractive and repellent odours for insect search behaviour

    Insects searching for resources are exposed to a complexity of mixed odours, often involving both attractant and repellent substances. Understanding how insects respond to this complexity of cues is crucial for understanding consumer-resource interactions, but also to develop novel tools to control harmful pests. To advance understanding of insect responses to combinations of attractive and repellent odours, this study formulated three qualitative hypotheses; the response-ratio hypothesis, the repellent-threshold hypothesis and the odour-modulation hypothesis. The hypotheses were tested by exposing Drosophila melanogaster in a wind tunnel to combinations of vinegar as attractant and four known repellents; benzaldehyde, 1-octen-3-ol, geosmin and phenol. The responses to benzaldehyde, 1-octen-3-ol and geosmin provided support for the response-ratio hypothesis, which assumes that the behavioural response depends on the ratio between attractants and repellents. The response to phenol, rather supported the repellent-threshold hypothesis, where aversion only occurs above a threshold concentration of the repellent due to overshadowing of the attractant. It is hypothesized that the different responses may be connected to the localization of receptors, as receptors detecting phenol are located on the maxillary palps whereas receptors detecting the other odorants are located on the antennae (Verschut, 2019).

    Sun navigation requires compass neurons in Drosophila

    Despite their small brains, insects can navigate over long distances by orienting using visual landmarks, skylight polarization, and sun position. Although Drosophila are not generally renowned for their navigational abilities, mark-and-recapture experiments in Death Valley revealed that they can fly nearly 15 km in a single evening. To accomplish such feats on available energy reserves, flies would have to maintain relatively straight headings, relying on celestial cues. Cues such as sun position and polarized light are likely integrated throughout the sensory-motor pathway, including the highly conserved central complex. Recently, a group of Drosophila central complex cells (E-PG neurons) have been shown to function as an internal compass, similar to mammalian head-direction cells. Using an array of genetic tools, this study set out to test whether flies can navigate using the sun and to identify the role of E-PG cells in this behavior. Using a flight simulator, it was found that Drosophila adopt arbitrary headings with respect to a simulated sun, thus performing menotaxis, and individuals remember their heading preference between successive flights-even over several hours. Imaging experiments performed on flying animals revealed that the E-PG cells track sun stimulus motion. When these neurons are silenced, flies no longer adopt and maintain arbitrary headings relative to the sun stimulus but instead exhibit frontal phototaxis. Thus, without the compass system, flies lose the ability to execute menotaxis and revert to a simpler, reflexive behavior (Giraldo, 2018).

    In the absence of normal E-PG function, flies might directly orient toward the sun, because they lack the ability to compare their instantaneous heading to a stored value of their directional preference. Such a loss of function in the compass network might unmask a simpler reflexive behavior, such as phototaxis, that does not require the elaborate circuitry of the central complex. Consistent with this hypothesis, stripe fixation was not different between control and experimental animals. This interpretation is compatible with a recent model that showed that frontal object fixation could result from a simple circuit involving two asymmetric wide-field motion integrators, without the need for the central complex (Giraldo, 2018).

    The findings are consistent with an emerging model of a navigational circuit involving the central complex. E-PG cells have an excitatory relationship with another cell class in the central complex (protocerebral bridge to ellipsoid body and noduli, or P-EN, neurons), creating an angular velocity integrator that allows a fly to maintain its heading in the absence of visual landmarks. Furthermore, the E-PG neurons are homologous to the CL1 neurons described in locusts, monarchs, dung beetle, and bees and likely serve similar functions across taxa. Extracellular recordings from the central complex in cockroaches revealed neurons that act as head-direction cells relative to, or in the absence of, visual landmarks, although precise cell types were not identified. Inputs to E-PG neurons likely occur via the anterior visual pathway from the medulla to the anterior optic tubercle and on to the bulb. From there, tubercle-bulb neurons, one class of which is responsive to the azimuth and elevation of small bright spots, synapse onto ring neurons that project to the ellipsoid body, thus bringing visual information into the compass network. In a recent model of path integration in bees, CL1 neurons are part of a columnar circuit that provides instantaneous heading information to an array of self-excitatory networks that also receive convergent optic flow information, thereby storing a memory of distance traveled in each direction (Stone, 2017). This information is then retrieved as an animal returns home, by driving appropriate steering commands in another class of central complex neurons. The putative memory cells suggested by this model, CPU4 cells, could be homologous to protocerebral bridge-fan-shaped-body noduli (P-FN) neurons described for Drosophila. Furthermore, cells responsive to progressive optic flow are found throughout the central complex of flies, including neuropil in the fan-shaped body containing the P-FN cells. In addition to their role in path integration, the CPU4 network might also function to store the desired heading during sun navigation. Although the results do not directly test this model, they are consistent with the role of CL1 neurons in providing heading direction to circuits that generate steering commands toward an arbitrary orientation whose memory is stored in the network of CPU4 (P-FN) neurons (Giraldo, 2018).

    Stripe fixation and sun navigation behaviors may represent two different flight modes in Drosophila. Stripe fixation is thought to be a short-range behavioral reflex to orient toward near objects, which, in free flight, is quickly terminated by collision avoidance or landing behaviors. In contrast, navigation using the sun is likely a component of long-distance dispersal behavior that could be used in conjunction with polarization vision either in a hierarchical or integrative manner. Individuals could differ in where they lie on the continuum of long-range dispersal to local search, which could explain the inter-individual variation observed in heading fidelity during sun orientation experiments. In general, dispersal is a condition-dependent behavior that is known to vary with hunger or other internal factors. Given the architectural similarity of the central complex among species, the celestial compass identified in Drosophila is likely one module within a conserved behavioral toolkit, allowing orientation and flight over long distances by integrating skylight polarization, the position of the sun or moon, and other visual cues. An independent study has recently found that the E-PG compass neurons are also necessary in walking flies for maintaining arbitrary headings relative to a small bright object. The expanding array of genetic tools developed for flies and the rapid growth in understanding of the neural circuitry involved in rientation and flight make this a promising system for exploring such essential and highly conserved behaviors (Giraldo, 2018).

    Elementary sensory-motor transformations underlying olfactory navigation in walking fruit-flies

    Odor attraction in walking Drosophila melanogaster is commonly used to relate neural function to behavior, but the algorithms underlying attraction are unclear. In this study, a high-throughput assay was developed to measure olfactory behavior in response to well-controlled sensory stimuli. Odor is shown to evokes two behaviors: an upwind run during odor (ON response), and a local search at odor offset (OFF response). Wind orientation requires antennal mechanoreceptors, but search is driven solely by odor. Using dynamic odor stimuli, the dependence of these two behaviors on odor intensity and history was measured. Based on these data, a navigation model was developed that recapitulates the behavior of flies in the apparatus, and generates realistic trajectories when run in a turbulent boundary layer plume. The ability to parse olfactory navigation into quantifiable elementary sensori-motor transformations provides a foundation for dissecting neural circuits that govern olfactory behavior (Alvarez-Salvado, 2018).

    Neural substrates of Drosophila larval anemotaxis

    Animals use sensory information to move toward more favorable conditions. Drosophila larvae can move up or down gradients of odors (chemotax), light (phototax), and temperature (thermotax) by modulating the probability, direction, and size of turns based on sensory input. Whether larvae can anemotax in gradients of mechanosensory cues is unknown. Further, although many of the sensory neurons that mediate taxis have been described, the central circuits are not well understood. This study used high-throughput, quantitative behavioral assays to demonstrate Drosophila larvae anemotax in gradients of wind speeds and to characterize the behavioral strategies involved. Larvae modulate the probability, direction, and size of turns to move away from higher wind speeds. This suggests that similar central decision-making mechanisms underlie taxis in somatosensory and other sensory modalities. By silencing the activity of single or very few neuron types in a behavioral screen, two sensory (chordotonal and multidendritic class III) and six nerve cord neuron types were found to be involved in anemotaxis. The identified neurons were reconstructed in an electron microscopy volume that spans the entire larval nervous system and it was found they received direct input from the mechanosensory neurons or from each other. In this way, local interneurons and first- and second-order subesophageal zone (SEZ) and brain projection neurons were identified. Finally, silencing a dopaminergic brain neuron type impairs anemotaxis. These findings suggest that anemotaxis involves both nerve cord and brain circuits. The candidate neurons and circuitry identified in this study provide a basis for future detailed mechanistic understanding of the circuit principles of anemotaxis (Jovanic, 2019).

    A decision underlies phototaxis in an insect

    Like a moth into the flame-phototaxis is an iconic example for innate preferences. Such preferences probably reflect evolutionary adaptations to predictable situations and have traditionally been conceptualized as hard-wired stimulus-response links. Perhaps for that reason, the century-old discovery of flexibility in Drosophila phototaxis has received little attention. This study reports that across several different behavioural tests, light/dark preference tested in walking is dependent on various aspects of flight. If flying ability is temporarily compromised, walking photopreference reverses concomitantly. Neuronal activity in circuits expressing dopamine and octopamine, respectively, plays a differential role in photopreference, suggesting a potential involvement of these biogenic amines in this case of behavioural flexibility. It is concluded that flies monitor their ability to fly, and that flying ability exerts a fundamental effect on action selection in Drosophila. This work suggests that even behaviours which appear simple and hard-wired comprise a value-driven decision-making stage, negotiating the external situation with the animal's internal state, before an action is selected (Gorostiza, 2016).

    Interestingly, experiments described by McEwen in 1918 and Benzer in 1967 demonstrated that wing defects affect phototaxis also in walking flies. These early works showed that flies with clipped wings did not display the phototactic response to light, whereas cutting the wings from mutants with deformed wings did not decrease their already low response to light any further. The fact that manipulating an unrelated organ, such as wings, affects phototaxis contradicts the assumed hard-wired organization of this behaviour, suggesting that it may not be a simple matter of stimulus and rigid, innate response, but that it contains at least a certain element of flexibility. This work systematically addressed the factors involved in this behavioural flexibility and begin to explore the neurobiological mechanisms behind it (Gorostiza, 2016).

    McEwen's discovery is of interest because of its implications for the supposed rigidity of simple behaviours. The findings of McEwen and Benzer that wing manipulation leads to a decrease in Drosophila phototaxis were reproduced in this study. Slightly altering the conditions of Benzer's countercurrent paradigm (BCP) and comparing performance between two additional experiments, this study found that the decrease in phototaxis is not due to hypoactivity of wing-manipulated flies, but to a more general change in the flies' assessment of their environment. Evidence was discovered that the BCP is just one of several experiments that can measure a fly's general photopreference. Manipulating the wings modulated this preference in all of the selected experiments such that compromised wing utility yielded a decreased preference for brightness (bright stimuli) and an increased preference for darkness (dark stimuli) across the experiments chosen. However, of these experiments, only the BCP can be argued to test phototaxis proper. In Buridan's paradigm, the flies walk between two unreachable black stripes; and in the T-maze, the flies choose between a dark tube and a bright one where the light is coming from an angle perpendicular to their trajectory. Neither of the two paradigms is testing taxis to nor away from a light source. Interestingly, in pilot experiments, this study tested phototaxis in different variations of the T-maze with various LEDs placed at the end of one of two opaque tubes, and only found a reduction of phototaxis and never negative phototaxis. In fact, in these pilot experiments, every possible difference was observed between flying and manipulated flies. In the end, the experimental design was chosen that yielded positive and negative scores, respectively, in wild-type Berlin (WTB) flies purely for practical reasons. Other wild-type strains, such as some Canton S substrains, do not show a negative photopreference in the T-maze after wing clipping. Taken together, these lines of evidence strongly suggest that photopreference in Drosophila is a strain-specific continuum where experimental design assigns more or less arbitrary values along the spectrum. In some special cases, this photopreference manifests itself as phototaxis. If that were the case, phototaxis would constitute an example of a class of experiments not entailing a class of behaviours (Gorostiza, 2016).

    This insight entails that manipulations of different aspects of flight ought to affect this continuum in different ways. Complete loss of flight ought to have more severe effects than manipulations affecting merely individual aspects of flight behaviour, such as wing beat amplitude/frequency (i.e., lift/thrust), torque, flight initiation, flight maintenance, proprioception or motion/wind-speed sensation. This study found some evidence to support this expectation. For instance, clipping only the tips of the wings does not eliminate flight, but affects torque as well as lift/thrust. Flies with the tips of their wings cut behave indifferently in the T-maze and do not avoid the bright tube. Flies without antennae are reluctant to fly and have lost their main sense of air speed detection, but they are still able to fly. Also these flies do not become light averse in the T-maze after the manipulation, but indifferent. Only clipping the wings in these flies abolishes their flight capabilities completely and yields negative scores. Flies with removed gyroscopic halteres, on the other hand, are severely affected in their detection of rotations and usually do not fly, despite being able to still beat their wings and control flight direction using vision alone in stationary flight. These flies avoid the bright arm of the T-maze. Finally, injuries to flight-unrelated parts of the fly's body did not affect photopreference ruling out the preference of darkness being a direct escape response due to bodily harm. Further research is required to establish a quantitative link between the many different aspects of flight behaviour and their relation to photopreference (Gorostiza, 2016).

    Taken together, these experiments so far demonstrate that: (1) the physical state of the wings with regard to their shape, form or degree of intactness influences photopreference; (2) the capability to not just move the wings, but specifically to move them in a way that would support flight also influences the flies' photopreference; and (3) the state of sensory organs related to flight such as antennae or halteres also exerts such an influence, while non-flight-related sensory deprivation shows no such consequences. This multitude of flight-related aspects extends the concept of flying ability beyond mere wing utility: manipulating seemingly any aspect of the entire sensorimotor complex of flight will affect photopreference, and do so reversibly). As it appears that any aspect of flight, sensory or motor, is acutely linked to photopreference, it is straightforward to subsume all of these aspects under the term 'flying ability', emphasizing that flying ability encompasses several more factors in addition to wing utility. The observation that each fly, when it is freshly eclosed from the pupal case and the wings are not yet expanded, goes through a phase of reduced phototaxis that extends beyond wing expansion until the stage when its wings render it capable of flying lends immediate ethological value to a neuronal mechanism linking flying ability with photopreference (Gorostiza, 2016).

    One possible explanation of how the link between flying ability and photopreference may be established mechanistically is via a process reminiscent of learning: at one time point, the flies register a sensory or motor deficit in their flight system and at a later time point they use this experience when making a decision that does not involve flying. Once flying ability is restored, the same choice situation is solved with a different decision again in the absence of flight behaviour. How the flies accomplish this learning task, if indeed learning is involved, is yet unknown, but it is tentatively concluded that it is unlikely that any of the known learning pathways or areas involved in different forms of learning play more than a contributing role. While the molecular learning mechanism remains unidentified, the process appears to be (near) instantaneous. Even though it is not possible to rule out that an unknown learning mechanism exists which is unaccounted for in the screen, it is concluded that at least none of the known learning mechanisms suffices to explain the complete effect size of the shift in photopreference. These results corroborate the findings above, that the switch is instantaneous and does not require thorough training or learning from repeated attempts to fly, let alone flight bouts. They do not rule out smaller contributions due to these known learning processes or an unknown, fast, episodic learning process. It is also possible that the flies constantly monitor their flying ability and hence do not have to remember their flight status. Despite these ambiguities, this study has been able to elucidate some of the underlying neurobiological mechanisms. Much as in other forms of insect learning and valuation, neurons expressing the biogenic amine neuromodulators OA and DA appear to have opposite functions in the modulation of photopreference (Gorostiza, 2016).

    Although both DA and OA play some role in different aspects of flight behaviour, these cannot explain the results. In general, the biogenic amine neuron manipulated flies escape their vial via flight if granted the opportunity. Thus, flight is not abolished in any of the transgenic lines affecting OA, TA or DA neurons. However, there may be more subtle deficits in less readily perceived aspects of flight. Experiments performed with mutant flies lacking OA demonstrated that OA is necessary for initiation and maintenance of flight. However, in the paradigm used in this study, silencing OA/TA neurons promoted approaching light, the opposite effect of what would be expected for a flightless fly. Activating these OA/TA neurons, however, rendered the flies indifferent in the T-maze. OA/TA appear to be involved in flight initiation and maintenance via opponent processes. Transient activation of OA/TA neurons may lead to a subtle alteration of flight performance and reduce photopreference in these flies. Similarly, it has been shown that altering the development of specific DA neurons results in flight deficits (reduction of flight time or loss of flight, depending on the treatment. the manipulations lasted for approximately 30 min during adulthood, ruling out such developmental defects. Work in the laboratory of Gaiti Hasan has also found that silencing of three identified TH-positive interneurons for several days in the adult animal compromises flight to some extent (wing coordination defects during flight initiation and cessation) (Sadaf, 2015). The much shorter manipulation of the current study does not lead to any readily observable flight defect. However, one need not discuss whether or not the aminergic manipulations may have had subtle effects on some aspects of flight behaviour, as these flies can be compared to the wing-clipped siblings with which they were tested simultaneously (i.e., the flies with the maximum shift in photopreference due to completely abolished flight). Comparing the intact DA-inactivated flies and OA/TA-activated flies with their respective wingless siblings (reveals that the choice indices of the pairs of groups become essentially indistinguishable at the restrictive temperature. In other words, intact flies where DA neurons have been inactivated or OA/TA neurons have been activated behave as if their wings had been clipped and their flight capabilities abolished completely, despite them being capable of at least some aspects of flight. Hence, even if there were some contribution of some aspect of flight behaviour being subtly affected by manipulating these aminergic neurons, there is a contribution of activity in these neurons that goes beyond these hypothetical flight deficits. Therefore, it is concluded that neither the OA/TA nor the DA effects can be explained only by subtle defects in one or the other aspect of flight behaviour in the manipulated flies (Gorostiza, 2016).

    The precise neurobiological consequences of manipulating OA/TA and DA neurons, respectively, are less certain, however. The two driver lines (th-GAL4 and tdc2-GAL4) only imperfectly mimicking the expression patterns of the genes from which they were derived. The effectors, moreover, only manipulated the activity of the labelled neurons. One manipulation (shiTS) prevents vesicle recycling and probably affects different vesicle pools differentially, depending on their respective release probabilities and recycling rates. The other effector (TrpA1) depolarizes neurons. It is commonly not known if the labelled neurons may not be co-releasing several different transmitters and/or modulators in the case of supra-threshold depolarization. Hence, without further research, the involvement can be stated only of the labelled neurons, which as populations are likely to be distinct mainly by containing either DA or OA/TA, respectively. Whether it is indeed the release of these biogenic amines or rather the (co-)release of yet unknown factors in these neuronal populations remains to be discovered. Further research will also elucidate the exact relationship between the activities of these two neuronal populations and whether/how it shifts after manipulations of flying ability (Gorostiza, 2016).

    In conclusion, the current findings provide further evidence that even innate preferences, such as those expressed in classic phototaxis experiments, are not completely hard-wired, but depend on the animal's state and presumably other factors, much like in the more complex behaviours previously studied. This endows the animal with the possibility to decide, for example, when it is better to move towards the light or hide in the shadows. Moreover, the fact that flies adapt their photopreference in accordance with their flying ability raises the tantalizing possibility that flies may have the cognitive tools required to evaluate the capability to perform an action and to let that evaluation impact other actions - an observation reminiscent of meta-cognition (Gorostiza, 2016).

    Age- and wavelength-dependency of Drosophila larval phototaxis and behavioral responses to natural lighting conditions

    Animals use various environmental cues as key determinant for their behavioral decisions. Visual systems are hereby responsible to translate light-dependent stimuli into neuronal encoded information. Even though the larval eyes of the fruit fly Drosophila melanogaster are comparably simple, they comprise two types of photoreceptor neurons (PRs), defined by different Rhodopsin genes expressed. Recent findings support that for light avoidance Rhodopsin5 (Rh5) expressing photoreceptors are crucial, while Rhodopsin6 (Rh6) expressing photoreceptors are dispensable under laboratory conditions. However, it remains debated how animals change light preference during larval life. This study shows that larval negative phototaxis is age-independent as it persists in larvae from foraging to wandering developmental stages. Moreover, whether spectrally different Rhodopsins are employed for the detection of different wavelength of light remains unexplored. This study found that negative phototaxis can be elicit by light with wavelengths ranging from ultraviolet (UV) to green. This behavior is uniquely mediated by Rh5 expressing photoreceptors, and therefore suggest that this photoreceptor-type is able to perceive UV up to green light. In contrast to laboratory tests, field experiments revealed that Drosophila larvae uses both types of photoreceptors under natural lighting conditions. The results demonstrate that Drosophila larval eyes mediate avoidance of light stimuli with a wide, ecological relevant range of quantity (intensities) and quality (wavelengths). Thus, the two photoreceptor-types appear more likely to play a role in different aspects of phototaxis under natural lighting conditions, rather than color discrimination (Humberg, 2017).

    Navigational strategies underlying temporal phototaxis in Drosophila larvae

    Navigating across light gradients is essential for survival for many animals. However, there is still a poor understanding of the algorithms that underlie such behaviors. This study developed a novel closed-loop phototaxis assay for Drosophila larvae in which light intensity is always spatially uniform but updates depending on the location of the animal in the arena. Even though larvae can only rely on temporal cues during runs, this study finds that they are capable of finding preferred areas of low light intensity. Further detailed analysis of their behavior reveals that larvae turn more frequently and that heading angle changes increase when they experience brightness increments over extended periods of time. It is suggested that temporal integration of brightness change during runs is an important - and so far largely unexplored - element of phototaxis (Zhu, 2021).


    Ache, J. M., Polsky, J., Alghailani, S., Parekh, R., Breads, P., Peek, M. Y., Bock, D. D., von Reyn, C. R. and Card, G. M. (2019). Neural basis for looming size and velocity encoding in the Drosophila giant fiber escape pathway. Curr Biol 29(6): 1073-1081. PubMed ID: 30827912

    Agrawal, P., Kao, D., Chung, P. and Looger, L. L. (2020). The neuropeptide Drosulfakinin regulates social isolation-induced aggression in Drosophila. J Exp Biol. PubMed ID: 31900346

    Ahn, J. E., Chen, Y. and Amrein, H. (2017). Molecular basis of fatty acid taste in Drosophila. Elife 6. PubMed ID: 29231818

    Alekseyenko, O. V., Chan, Y. B., Fernandez, M. P., Bulow, T., Pankratz, M. J. and Kravitz, E. A. (2014). Single serotonergic neurons that modulate aggression in Drosophila. Curr Biol 24: 2700-2707. PubMed ID: 25447998

    Alekseyenko, O. V., Chan, Y. B., Okaty, B. W., Chang, Y., Dymecki, S. M. and Kravitz, E. A. (2019). Serotonergic modulation of aggression in Drosophila involves GABAergic and cholinergic opposing pathways. Curr Biol. PubMed ID: 31231050

    Allen, A. M., Anreiter, I., Neville, M. C. and Sokolowski, M. B. (2016). Feeding-related traits are affected by dosage of the foraging gene in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28007892

    Allen, A. M., Anreiter, I., Vesterberg, A., Douglas, S. J. and Sokolowski, M. B. (2018). Pleiotropy of the Drosophila melanogaster foraging gene on larval feeding-related traits. J Neurogenet: 1-11. PubMed ID: 30303018Almeida-Carvalho, M. J., Berh, D., Braun, A., Chen, Y. C., Eichler, K., Eschbach, C., Fritsch, P. M. J., Gerber, B., Hoyer, N., Jiang, X., Kleber, J., Klambt, C., Konig, C., Louis, M., Michels, B., Miroschnikow, A., Mirth, C., Miura, D., Niewalda, T., Otto, N., Paisios, E., Pankratz, M. J., Petersen, M., Ramsperger, N., Randel, N., Risse, B., Saumweber, T., Schlegel, P., Schleyer, M., Soba, P., Sprecher, S. G., Tanimura, T., Thum, A. S., Toshima, N., Truman, J. W., Yarali, A. and Zlatic, M. (2017). The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3 Drosophila larvae. J Exp Biol 220(Pt 13): 2452-2475. PubMed ID: 28679796

    Alvarez-Salvado, E., Licata, A. M., Connor, E. G., McHugh, M. K., King, B. M., Stavropoulos, N., Victor, J. D., Crimaldi, J. P. and Nagel, K. I. (2018). Elementary sensory-motor transformations underlying olfactory navigation in walking fruit-flies. Elife 7. PubMed ID: 30129438

    Alwash, N., Allen, A. M., Sokolowski, M. B.. and Levine, J. D. (2021). The Drosophila melanogaster foraging gene affects social networks. J Neurogenet: 1-13. PubMed ID: 34121597

    Andrew, D. R., Moe, M. E., Chen, D., Tello, J. A., Doser, R. L., Conner, W. E., Ghuman, J. K. and Restifo, L. L. (2020). Spontaneous motor-behavior abnormalities in two Drosophila models of neurodevelopmental disorders. J Neurogenet: 1-22. PubMed ID: 33164597

    Asaoka, T., Almagro, J., Ehrhardt, C., Tsai, I., Schleiffer, A., Deszcz, L., Junttila, S., Ringrose, L., Mechtler, K., Kavirayani, A., Gyenesei, A., Hofmann, K., Duchek, P., Rittinger, K. and Ikeda, F. (2016). Linear ubiquitination by LUBEL has a role in Drosophila heat stress response. EMBO Rep 17: 1624-1640. PubMed ID: 27702987

    Asahina, K., Watanabe, K., Duistermars, B. J., Hoopfer, E., Gonzalez, C. R., Eyjolfsdottir, E. A., Perona, P. and Anderson, D. J. (2014). Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila. Cell 156(1-2): 221-235. PubMed ID: 24439378

    Avalos, A., Fang, M., Pan, H., Ramirez Lluch, A., Lipka, A. E., Zhao, S. D., Giray, T., Robinson, G. E., Zhang, G. and Hudson, M. E. (2020). Genomic regions influencing aggressive behavior in honey bees are defined by colony allele frequencies. Proc Natl Acad Sci U S A 117(29): 17135-17141. PubMed ID: 32631983

    Azanchi, R., Kaun, K. R. and Heberlein, U. (2013). Competing dopamine neurons drive oviposition choice for ethanol in Drosophila. Proc Natl Acad Sci U S A 110(52): 21153-21158. PubMed ID: 24324162

    Bademosi, A. T., Steeves, J., Karunanithi, S., Zalucki, O. H., Gormal, R. S., Liu, S., Lauwers, E., Verstreken, P., Anggono, V., Meunier, F. A. and van Swinderen, B. (2018), Trapping of Syntaxin1a in presynaptic nanoclusters by a clinically relevant general anesthetic. Cell Rep 22(2): 427-440. PubMed ID: 29320738

    Bae, J. E., Bang, S., Min, S., Lee, S. H., Kwon, S. H., Lee, Y., Lee, Y. H., Chung, J. and Chae, K. S. (2016). Positive geotactic behaviors induced by geomagnetic field in Drosophila. Mol Brain 9: 55. PubMed ID: 27192976

    Baik, L. S., Recinos, Y., Chevez, J. A., Au, D. D. and Holmes, T. C. (2019). Multiple phototransduction inputs integrate to mediate UV light-evoked avoidance/attraction behavior in Drosophila. J Biol Rhythms: 748730419847339. PubMed ID: 31140349

    Bath, E., Edmunds, D., Norman, J., Atkins, C., Harper, L., Rostant, W. G., Chapman, T., Wigby, S. and Perry, J. C. (2021). Sex ratio and the evolution of aggression in fruit flies. Proc Biol Sci 288(1947): 20203053. PubMed ID: 33726599

    Bath, E., Biscocho, E. R., Easton-Calabria, A. and Wigby, S. (2020). Temporal and genetic variation in female aggression after mating. PLoS One 15(4): e0229633. PubMed ID: 32348317

    Bentzur, A., Ben-Shaanan, S., Benichou, J. I. C., Costi, E., Levi, M., Ilany, A. and Shohat-Ophir, G. (2020). Early Life Experience Shapes Male Behavior and Social Networks in Drosophila. Curr Biol. PubMed ID: 33186552

    Bezerra Da Silva, C. S., Park, K. R., Blood, R. A. and Walton, V. M. (2019). Intraspecific competition affects the pupation behavior of spotted-wing Drosophila (Drosophila suzukii). Sci Rep 9(1): 7775. PubMed ID: 31123337

    Bhattacharya, A., Lakhman, S. S. and Singh, S. (2004). Modulation of L-type calcium channels in Drosophila via a pituitary adenylyl cyclase-activating polypeptide (PACAP)-mediated pathway. J Biol Chem 279(36): 37291-37297. PubMed ID: 15201281

    Breugel, F. V. (2021). Correlated decision making across multiple phases of olfactory guided search in Drosophila improves search efficiency. J Exp Biol. PubMed ID: 34286337

    Burgos, A., Honjo, K., Ohyama, T., Qian, C. S., Shin, G. J., Gohl, D. M., Silies, M., Tracey, W. D., Zlatic, M., Cardona, A. and Grueber, W. B. (2018). Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila. Elife 7. PubMed ID: 29528286

    Bracker, L. B., Schmid, C. A., Bolini, V. A., Holz, C. A., Prud'homme, B., Sirota, A. and Gompel, N. (2019). Quantitative and discrete evolutionary changes in the egg-laying behavior of single Drosophila females. Front Behav Neurosci 13: 118. PubMed ID: 31191270

    Brankatschk, M., Gutmann, T., Knittelfelder, O., Palladini, A., Prince, E., Grzybek, M., Brankatschk, B., Shevchenko, A., Coskun, U. and Eaton, S. (2018). A temperature-dependent switch in feeding preference improves Drosophila development and survival in the cold. Dev Cell 46(6): 781-793.e784. PubMed ID: 30253170

    Brockmann, A., Basu, P., Shakeel, M., Murata, S., Murashima, N., Boyapati, R. K., Prabhu, N. G., Herman, J. J. and Tanimura, T. (2018). Sugar intake elicits intelligent searching behavior in flies and honey bees. Front Behav Neurosci 12: 280. PubMed ID: 30546299

    Brown, E. B., Slocumb, M. E., Szuperak, M., Kerbs, A., Gibbs, A. G., Kayser, M. S. and Keene, A. C. (2019). Starvation resistance is associated with developmentally specified changes in sleep, feeding and metabolic rate. J Exp Biol. PubMed ID: 30606795

    Brunner, B., Saumweber, J., Samur, M., Weber, D., Schumann, I., Mahishi, D., Rohwedder, A. and Thum, A. S. (2020). Food restriction reconfigures naive and learned choice behavior in Drosophila larvae. J Neurogenet: 1-10. PubMed ID: 31975653

    Burgos, A., Honjo, K., Ohyama, T., Qian, C. S., Shin, G. J., Gohl, D. M., Silies, M., Tracey, W. D., Zlatic, M., Cardona, A. and Grueber, W. B. (2018). Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila. Elife 7. PubMed ID: 29528286

    Busch, C., Borst, A. and Mauss, A. S. (2018). Bi-directional control of walking behavior by horizontal optic flow sensors. Curr Biol. PubMed ID: 30528583

    Cande, J., Namiki, S., Qiu, J., Korff, W., Card, G. M., Shaevitz, J. W., Stern, D. L. and Berman, G. J. (2018). Optogenetic dissection of descending behavioral control in Drosophila. Elife 7. PubMed ID: 29943729

    Cao, W., Song, L., Cheng, J., Yi, N., Cai, L., Huang, F. D. and Ho, M. (2017). An automated rapid iterative negative geotaxis assay for analyzing adult climbing behavior in a Drosophila model of neurodegeneration. J Vis Exp(127). PubMed ID: 28931001

    Carreira-Rosario, A., Zarin, A. A., Clark, M. Q., Manning, L., Fetter, R. D., Cardona, A. and Doe, C. Q. (2018). MDN brain descending neurons coordinately activate backward and inhibit forward locomotion. Elife 7. PubMed ID: 30070205

    Cazale-Debat, L., Houot, B., Farine, J. P., Everaerts, C. and Ferveur, J. F. (2019). Flying Drosophila show sex-specific attraction to fly-labelled food. Sci Rep 9(1): 14947. PubMed ID: 31628403

    Chakraborty, T. S., Gendron, C. M., Lyu, Y., Munneke, A. S., DeMarco, M. N., Hoisington, Z. W. and Pletcher, S. D. (2019). Sensory perception of dead conspecifics induces aversive cues and modulates lifespan through serotonin in Drosophila. Nat Commun 10(1): 2365. PubMed ID: 31147540

    Chatzigeorgiou, M., Bang, S., Hwang, S. W. and Schafer, W. R. (2013). tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans. Nature 494(7435): 95-99. PubMed ID: 23364694

    Chen, J., Reiher, W., Hermann-Luibl, C., Sellami, A., Cognigni, P., Kondo, S., Helfrich-Förster, C., Veenstra, J.A. and Wegener, C. (2016). Allatostatin A signalling in Drosophila regulates feeding and sleep and is modulated by PDF. PLoS Genet 12: e1006346. PubMed ID: 27689358

    Chen, Y. C., Mishra, D., Glass, S. and Gerber, B. (2017). Behavioral evidence for enhanced processing of the minor component of binary odor mixtures in larval Drosophila. Front Psychol 8: 1923. PubMed ID: 29163299

    Chen, Y. D. and Dahanukar, A. (2017). Molecular and cellular organization of taste neurons in adult Drosophila pharynx. Cell Rep 21(10): 2978-2991. PubMed ID: 29212040

    Cheng, K. Y., Colbath, R. A. and Frye, M. A. (2019). Olfactory and neuromodulatory signals reverse visual object avoidance to approach in Drosophila. Curr Biol 29(12): 2058-2065. PubMed ID: 31155354

    Cheung, S. K. and Scott, K. (2017). GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster. PLoS One 12(3): e0175177. PubMed ID: 28362856

    Chiu, H., Hoopfer, E. D., Coughlan, M. L. and Anderson, D. J. (2020). A circuit logic for sexually shared and dimorphic aggressive behaviors in Drosophila. Cell. PubMed ID: 33382967

    Chin, S. G., Maguire, S. E., Huoviala, P., Jefferis, G. and Potter, C. J. (2018). Olfactory neurons and brain centers directing oviposition decisions in Drosophila. Cell Rep 24(6): 1667-1678. PubMed ID: 30089274

    Choi, J., Yu, S., Choi, M. S., Jang, S., Han, I. J., Maier, G. L., Sprecher, S. G. and Kwon, J. Y. (2020). Cellular Basis of Bitter-Driven Aversive Behaviors in Drosophila Larva. eNeuro 7(2). PubMed ID: 32220859

    Chouhan, N. S., Mohan, K. and Ghose, A. (2017). cAMP signaling mediates behavioral flexibility and consolidation of social status in Drosophila aggression. J Exp Biol 220(Pt 23): 4502-4514. PubMed ID: 28993465

    Chowdhury, B., Chan, Y.B. and Kravitz, E.A. (2017). Putative transmembrane transporter modulates higher-level aggression in Drosophila. Proc Natl Acad Sci U S A 114: 2373-2378. PubMed ID: 28193893

    Chwen, Y., Lee, G., Yang, Q., Chi, W., Turkson, S. A., Du, W. A., Kemkemer, C., Zeng, Z. B., Long, M. and Zhuang, X. (2017). Genetic architecture of natural variation underlying adult foraging behavior that is essential for survival of Drosophila melanogaster. Genome Biol Evol 9(5):1357-1369. PubMed ID: 28472322

    Cohen, D., van Swinderen, B. and Tsuchiya, N. (2018). Isoflurane impairs low-frequency feedback but leaves high-frequency feedforward connectivity intact in the fly brain. eNeuro 5(1). PubMed ID: 29541686

    Colinet, H., Pineau, C. and Com, E. (2017). Large scale phosphoprotein profiling to explore Drosophila cold acclimation regulatory mechanisms. Sci Rep 7(1): 1713. PubMed ID: 28490779

    Corfas, R. A., Sharma, T. and Dickinson, M. H. (2019). Diverse food-sensing neurons trigger idiothetic local search in Drosophila. Curr Biol 29(10): 1660-1668. PubMed ID: 31056390

    Coupe, B., Ishii, Y., Dietrich, M. O., Komatsu, M., Horvath, T. L. and Bouret, S. G. (2012). Loss of autophagy in pro-opiomelanocortin neurons perturbs axon growth and causes metabolic dysregulation. Cell Metab 15(2): 247-255. PubMed ID: 22285542

    Crowley-Gall, A., Shaw, M. and Rollmann, S. M. (2018). Host Preference and Olfaction in Drosophila mojavensis. J Hered. PubMed ID: 30299456

    Davies, L. R., Schou, M. F., Kristensen, T. N. and Loeschcke, V. (2018). Linking developmental diet to adult foraging choice in Drosophila melanogaster. J Exp Biol [Epub ahead of print]. PubMed ID: 29666197

    Davis, S. M., Thomas, A. L., Liu, L., Campbell, I. M. and Dierick, H. A. (2017). Isolation of aggressive behavior mutants in Drosophila using a screen for wing damage. Genetics [Epub ahead of print]. PubMed ID: 29109180

    Dawson, E. H., Bailly, T. P. M., Dos Santos, J., Moreno, C., Devilliers, M., Maroni, B., Sueur, C., Casali, A., Ujvari, B., Thomas, F., Montagne, J. and Mery, F. (2018). Social environment mediates cancer progression in Drosophila. Nat Commun 9(1): 3574. PubMed ID: 30177703

    de Andres-Bragado, L., Mazza, C., Senn, W. and Sprecher, S. G. (2018). Statistical modelling of navigational decisions based on intensity versus directionality in Drosophila larval phototaxis. Sci Rep 8(1): 11272. PubMed ID: 30050066

    Degen, J., et al. (2016). Honeybees learn landscape features during exploratory orientation flights. Curr Biol. PubMed ID: 27693138

    de la Flor, M., Chen, L., Manson-Bishop, C., Chu, T. C., Zamora, K., Robbins, D., Gunaratne, G. and Roman, G. (2017). Drosophila increase exploration after visually detecting predators. PLoS One 12(7): e0180749. PubMed ID: 28746346

    Deutsch, D., Pacheco, D., Encarnacion-Rivera, L., Pereira, T., Fathy, R., Clemens, J., Girardin, C., Calhoun, A., Ireland, E., Burke, A., Dorkenwald, S., McKellar, C., Macrina, T., Lu, R., Lee, K., Kemnitz, N., Ih, D., Castro, M., Halageri, A., Jordan, C., Silversmith, W., Wu, J., Seung, H. S. and Murthy, M. (2020). The neural basis for a persistent internal state in Drosophila females. Elife 9. PubMed ID: 33225998

    Dobosiewicz, M., Liu, Q. and Bargmann, C. I. (2019). Reliability of an interneuron response depends on an integrated sensory state. Elife 8. PubMed ID: 31718773

    Dombrovski, M., Poussard, L., Moalem, K., Kmecova, L., Hogan, N., Schott, E., Vaccari, A., Acton, S. and Condron, B. (2017). Cooperative behavior emerges among Drosophila larvae. Curr Biol 27(18): 2821-2826.e2822. PubMed ID: 28918946

    Dombrovski, M., Kim, A., Poussard, L., Vaccari, A., Acton, S., Spillman, E., Condron, B. and Yuan, Q. (2019). A plastic visual pathway regulates cooperative behavior in Drosophila larvae. Curr Biol 29(11): 1866-1876. PubMed ID: 31130457

    Dombrovski, M., Kuhar, R., Mitchell, A., Shelton, H. and Condron, B. (2020). Cooperative foraging during larval stage affects fitness in Drosophila. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 206(5): 743-755. PubMed ID: 32623493

    Duistermars, B. J., Pfeiffer, B. D., Hoopfer, E. D. and Anderson, D. J. (2018). A brain module for scalable control of complex, multi-motor threat displays. Neuron. PubMed ID: 30415997

    Edmunds, D., Wigby, S. and Perry, J. C. (2021). 'Hangry' Drosophila: food deprivation increases male aggression. Anim Behav 177: 183-190. PubMed ID: 34290451

    Elya, C., Lok, T. C., Spencer, Q. E., McCausland, H., Martinez, C. C. and Eisen, M. (2018). Robust manipulation of the behavior of Drosophila melanogaster by a fungal pathogen in the laboratory. Elife 7. PubMed ID: 30047862

    Enriquez, T. and Colinet, H. (2019). Cold acclimation triggers major transcriptional changes in Drosophila suzukii. BMC Genomics 20(1): 413. PubMed ID: 31117947

    Frighetto, G., Zordan, M. A., Castiello, U. and Megighian, A. (2019). Action-based attention in Drosophila melanogaster. J Neurophysiol. PubMed ID: 31042449

    Eriksson, A., Anand, P., Gorson, J., Grijuc, C., Hadelia, E., Stewart, J. C., Holford, M. and Claridge-Chang, A. (2018). Using Drosophila behavioral assays to characterize terebrid venom-peptide bioactivity. Sci Rep 8(1): 15276. PubMed ID: 30323294

    Feingold, D., Knogler, L., Starc, T., Drapeau, P., O'Donnell, M. J., Nilson, L. A. and Dent, J. A. (2019). secCl is a cys-loop ion channel necessary for the chloride conductance that mediates hormone-induced fluid secretion in Drosophila. Sci Rep 9(1): 7464. PubMed ID: 31097722

    Ferdenache, M., Bezzar-Bendjazia, R., Marion-Poll, F. and Kilani-Morakchi, S. (2019). Transgenerational effects from single larval exposure to azadirachtin on life history and behavior traits of Drosophila melanogaster. Sci Rep 9(1): 17015. PubMed ID: 31745147

    Fernandez, R. W., Akinleye, A. A., Nurilov, M., Feliciano, O., Lollar, M., Aijuri, R. R., O'Donnell, J. M. and Simon, A. F. (2017). Modulation of social space by dopamine in Drosophila melanogaster, but no effect on the avoidance of the Drosophila stress odorant. Biol Lett 13(8). PubMed ID: 28794277

    Filice, D. C. S., Bhargava, R. and Dukas, R. (2020). Plasticity in male mating behavior modulates female life history in fruit flies. Evolution 74(2): 365-376. PubMed ID: 31925958

    Galikova, M., Dircksen, H. and Nassel, D. R. (2018). The thirsty fly: Ion transport peptide (ITP) is a novel endocrine regulator of water homeostasis in Drosophila. PLoS Genet 14(8): e1007618. PubMed ID: 30138334

    Giraldo, Y. M., Leitch, K. J., Ros, I. G., Warren, T. L., Weir, P. T. and Dickinson, M. H. (2018). Sun navigation requires compass neurons in Drosophila. Curr Biol 28(17):2845-2852. PubMed ID: 30174187

    Giraldo, D., Adden, A., Kuhlemann, I., Gras, H. and Geurten, B. R. H. (2019). Correcting locomotion dependent observation biases in thermal preference of Drosophila. Sci Rep 9(1): 3974. PubMed ID: 30850647

    Gong, C., Ouyang, Z., Zhao, W., Wang, J., Li, K., Zhou, P., Zhao, T., Zheng, N. and Gong, Z. (2019). A neuronal pathway that commands deceleration in Drosophila larval light-avoidance. Neurosci Bull. PubMed ID: 30810958

    Gorostiza, E. A., Colomb, J. and Brembs, B. (2016). A decision underlies phototaxis in an insect.Open Biol 6(12). PubMed ID: 28003472

    Grangeteau, C., Yahou, F., Everaerts, C., Dupont, S., Farine, J. P., Beney, L. and Ferveur, J. F. (2018). Yeast quality in juvenile diet affects Drosophila melanogaster adult life traits. Sci Rep 8(1): 13070. PubMed ID: 30166573

    Green, J. E., Cavey, M., Medina Caturegli, E., Aigouy, B., Gompel, N. and Prud'homme, B. (2019). Evolution of ovipositor length in Drosophila suzukii is driven by enhanced cell size expansion and anisotropic tissue reorganization. Curr Biol 29(12): 2075-2082. PubMed ID: 31178315

    Gunther, C. S., Knight, S. J., Jones, R. and Goddard, M. R. (2019). Are Drosophila preferences for yeasts stable or contextual? Ecol Evol 9(14): 8075-8086. PubMed ID: 31380072

    Gupta, T., Howe, S. E., Zorman, M. L. and Lockwood, B. L. (2019). Aggression and discrimination among closely versus distantly related species of Drosophila. R Soc Open Sci 6(6): 190069. PubMed ID: 31312482

    Haberkern, H., Basnak, M. A., Ahanonu, B., Schauder, D., Cohen, J. D., Bolstad, M., Bruns, C. and Jayaraman, V. (2019). Visually guided behavior and optogenetically induced learning in head-fixed flies exploring a virtual landscape. Curr Biol 29(10): 1647-1659. PubMed ID: 31056392

    Hadjieconomou, D., King, G., Gaspar, P., Mineo, A., Blackie, L., Ameku, T., Studd, C., de Mendoza, A., Diao, F., White, B. H., Brown, A. E. X., Placais, P. Y., Preat, T. and Miguel-Aliaga, I. (2020). Enteric neurons increase maternal food intake during reproduction. Nature 587(7834): 455-459. PubMed ID: 33116314

    Han, R., Wei, T. M., Tseng, S. C. and Lo, C. C. (2021). Characterizing approach behavior of Drosophila melanogaster in Buridan's paradigm. PLoS One 16(1): e0245990. PubMed ID: 33507934

    Harris, N., Braiser, D. J., Dickman, D. K., Fetter, R. D., Tong, A. and Davis, G. W. (2015). The innate immune receptor PGRP-LC controls presynaptic homeostatic plasticity. Neuron 88(6): 1157-1164. PubMed ID: 26687223

    He, J., Hommen, F., Lauer, N., Balmert, S. and Scholz, H. (2020). Serotonin transporter dependent modulation of food-seeking behavior. PLoS One 15(1): e0227554. PubMed ID: 31978073

    He, J., Tan, A. M. X., Ng, S. Y., Rui, M. and Yu, F. (2021). Cannabinoids modulate food preference and consumption in Drosophila melanogaster. Sci Rep 11(1): 4709. PubMed ID: 33633260

    Hergarden, A. C., Tayler, T. D. and Anderson, D. J. (2012). Allatostatin-A neurons inhibit feeding behavior in adult Drosophila. Proc Natl Acad Sci U S A 109(10): 3967-3972. PubMed ID: 22345563

    Highfill, C. A., Baker, B. M., Stevens, S. D., Anholt, R. R. H. and Mackay, T. F. C. (2019). Genetics of cocaine and methamphetamine consumption and preference in Drosophila melanogaster. PLoS Genet 15(5): e1007834. PubMed ID: 31107875

    Himmel, N. J., Letcher, J. M., Sakurai, A., Gray, T. R., Benson, M. N., Donaldson, K. J. and Cox, D. N. (2021). Identification of a neural basis for cold acclimation in Drosophila larvae. iScience 24(6): 102657. PubMed ID: 34151240

    Hoopfer, E.D., Jung, Y., Inagaki, H.K., Rubin, G.M. and Anderson, D.J. (2015). P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila. Elife [Epub ahead of print]. PubMed ID: 26714106

    Hopkins, D., Envall, T., Poikela, N., Pentikainen, O. T. and Kankare, M. (2018). Effects of cold acclimation and dsRNA injections on Gs1l gene splicing in Drosophila montana. Sci Rep 8(1): 7577. PubMed ID: 29765071

    Hu, S. W., Yang, Y. T., Sun, Y., Zhan, Y. P. and Zhu, Y. (2020). Serotonin Signals Overcome Loser Mentality in Drosophila. iScience 23(11): 101651. PubMed ID: 33117967

    Hu, Y., Wang, C., Yang, L., Pan, G., Liu, H., Yu, G. and Ye, B. (2020). A neural basis for categorizing sensory stimuli to enhance decision accuracy. Curr Biol. 30(24):4896-4909. PubMed ID: 33065003

    Huang, R., Song, T., Su, H., Lai, Z., Qin, W., Tian, Y., Dong, X. and Wang, L. (2020). High-fat diet enhances starvation-induced hyperactivity via sensitizing hunger-sensing neurons in Drosophila. Elife 9. PubMed ID: 32324135

    Humberg, T. H. and Sprecher, S. G. (2017). Age- and wavelength-dependency of Drosophila larval phototaxis and behavioral responses to natural lighting conditions. Front Behav Neurosci 11: 66. PubMed ID: 28473759

    Jezovit, J. A., Rooke, R., Schneider, J. and Levine, J. D. (2020). Behavioral and environmental contributions to drosophilid social networks. Proc Natl Acad Sci U S A 117(21): 11573-11583. PubMed ID: 32404421

    Jia, Y., Jin, S., Hu, K., Geng, L., Han, C., Kang, R., Pang, Y., Ling, E., Tan, E. K., Pan, Y. and Liu, W. (2021). Gut microbiome modulates Drosophila aggression through octopamine signaling. Nat Commun 12(1): 2698. PubMed ID: 33976215

    Jiang, L., Cheng, Y., Gao, S., Zhong, Y., Ma, C., Wang, T. and Zhu, Y. (2020). Emergence of social cluster by collective pairwise encounters in Drosophila. Elife 9. PubMed ID: 31959283

    Jourjine, N., Mullaney, B. C., Mann, K. and Scott, K. (2016). Coupled sensing of hunger and thirst signals balances sugar and water consumption. Cell 166(4): 855-866. PubMed ID: 27477513

    Joseph, R. M., Sun, J. S., Tam, E. and Carlson, J. R. (2017). A receptor and neuron that activate a circuit limiting sucrose consumption. Elife 6. PubMed ID: 28332980

    Jourjine, N., Mullaney, B.C., Mann, K. and Scott, K. (2016). Coupled sensing of hunger and thirst signals balances sugar and water consumption. Cell [Epub ahead of print]. PubMed ID: 27477513

    Jovanic, T., Schneider-Mizell, C.M., Shao, M., Masson, J.B., Denisov, G., Fetter, R.D., Mensh, B.D., Truman, J.W., Cardona, A. and Zlatic, M. (2016). Competitive disinhibition mediates behavioral choice and sequences in Drosophila. Cell [Epub ahead of print]. PubMed ID: 27720450

    Jovanic, T., Winding, M., Cardona, A., Truman, J. W., Gershow, M. and Zlatic, M. (2019). Neural substrates of Drosophila larval anemotaxis. Curr Biol. PubMed ID: 30744969

    Kacsoh, B. Z., Bozler, J. and Bosco, G. (2018). Drosophila species learn dialects through communal living. PLoS Genet 14(7): e1007430. PubMed ID: 30024883

    Kanellopoulos, A. K., Mariano, V., Spinazzi, M., Woo, Y. J., McLean, C., Pech, U., Li, K. W., Armstrong, J. D., Giangrande, A., Callaerts, P., Smit, A. B., Abrahams, B. S., Fiala, A., Achsel, T. and Bagni, C. (2020). Aralar Sequesters GABA into Hyperactive Mitochondria, Causing Social Behavior Deficits. Cell 180(6): 1178-1197.e1120. PubMed ID: 32200800

    Kacsoh, B. Z., Bozler, J., Hodge, S. and Bosco, G. (2019). Neural circuitry of social learning in Drosophila requires multiple inputs to facilitate inter-species communication. Commun Biol 2: 309. PubMed ID: 31428697

    Karageorgi, M., Bracker, L. B., Lebreton, S., Minervino, C., Cavey, M., Siju, K. P., Grunwald Kadow, I. C., Gompel, N. and Prud'homme, B. (2017). Evolution of multiple sensory systems drives novel egg-laying behavior in the fruit pest Drosophila suzukii. Curr Biol [Epub ahead of print]. PubMed ID: 28285999

    Kauranen, H., Kinnunen, J., Hiillos, A. L., Lankinen, P., Hopkins, D., Wiberg, R. A. W., Ritchie, M. G. and Hoikkala, A. (2019). Selection for reproduction under short photoperiods changes diapause-associated traits and induces widespread genomic divergence. J Exp Biol. PubMed ID: 31511345

    Kaushik, S., Rodriguez-Navarro, J. A., Arias, E., Kiffin, R., Sahu, S., Schwartz, G. J., Cuervo, A. M. and Singh, R. (2011). Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance. Cell Metab 14(2): 173-183. PubMed ID: 21803288

    Kawashima, Y., Geleoc, G. S., Kurima, K., Labay, V., Lelli, A., Asai, Y., Makishima, T., Wu, D. K., Della Santina, C. C., Holt, J. R. and Griffith, A. J. (2011). Mechanotransduction in mouse inner ear hair cells requires transmembrane channel-like genes. J Clin Invest 121(12): 4796-4809. PubMed ID: 22105175

    Keesey, I. W., Koerte, S., Khallaf, M. A., Retzke, T., Guillou, A., Grosse-Wilde, E., Buchon, N., Knaden, M. and Hansson, B. S. (2017). Pathogenic bacteria enhance dispersal through alteration of Drosophila social communication. Nat Commun 8(1): 265. PubMed ID: 28814724

    Khodaei, L. and Long, T. A. F. (2019). Kin recognition and co-operative foraging in Drosophila melanogaster larvae. J Evol Biol. PubMed ID: 31454451

    Kim, D. H., Shin, M., Jung, S. H., Kim, Y. J. and Jones, W. D. (2017). A fat-derived metabolite regulates a peptidergic feeding circuit in Drosophila. PLoS Biol 15(3): e2000532. PubMed ID: 28350856

    Kim, G., Huang, J. H., McMullen, J. G., Newell, P. D. and Douglas, A. E. (2017). Physiological responses of insects to microbial fermentation products: insights from the interactions between Drosophila and acetic acid. J Insect Physiol [Epub ahead of print]. PubMed ID: 28522417

    Kim, D., Alvarez, M., Lechuga, L. M. and Louis, M. (2017). Species-specific modulation of food-search behavior by respiration and chemosensation in Drosophila larvae. Elife 6. PubMed ID: 28871963

    Kim, H., Jeong, Y. T., Choi, M. S., Choi, J., Moon, S. J. and Kwon, J. Y. (2017). Involvement of a Gr2a-expressing Drosophila pharyngeal gustatory receptor neuron in regulation of aversion to high-salt foods. Mol Cells [Epub ahead of print]. PubMed ID: 28535667

    Kim, I. S. and Dickinson, M. H. (2017). Idiothetic path Integration in the fruit fly Drosophila melanogaster. Curr Biol 27(15): 2227-2238.e2223. PubMed ID: 28736164

    Kim, H., Kirkhart, C. and Scott, K. (2017). Long-range projection neurons in the taste circuit of Drosophila. Elife 6. PubMed ID: 28164781

    Kim, Y. K., Saver, M., Simon, J., Kent, C. F., Shao, L., Eddison, M., Agrawal, P., Texada, M., Truman, J. W. and Heberlein, U. (2018). Repetitive aggressive encounters generate a long-lasting internal state in Drosophila melanogaster males. Proc Natl Acad Sci U S A 115(5): 1099-1104. PubMed ID: 29339481

    Klein, M., Krivov, S. V., Ferrer, A. J., Luo, L., Samuel, A. D. and Karplus, M. (2017). Exploratory search during directed navigation in C. elegans and Drosophila larva. Elife 6. PubMed ID: 29083306

    Klepsatel, P., Girish, T. N., Dircksen, H. and Galikova, M. (2019). Reproductive fitness of Drosophila is maximised by optimal developmental temperature. J Exp Biol 222(Pt 10). PubMed ID: 31064855

    Kohsaka, H., Takasu, E., Morimoto, T. and Nose, A. (2014). A group of segmental premotor interneurons regulates the speed of axial locomotion in Drosophila larvae. Curr Biol 24(22): 2632-2642. PubMed ID: 25438948

    Koniger, A., Arif, S. and Grath, S. (2019). Three quantitative trait loci explain more than 60% of variation for chill coma recovery time in a natural population of Drosophila ananassae. G3 (Bethesda) 9(11): 3715-3725. PubMed ID: 31690597

    Krams, I. A., Krama, T., Krams, R., Trakimas, G., Popovs, S., Joers, P., Munkevics, M., Elferts, D., Rantala, M. J., Makņa, J. and de Bivort, B. L. (2021). Serotoninergic Modulation of Phototactic Variability Underpins a Bet-Hedging Strategy in Drosophila melanogaster. Front Behav Neurosci 15: 659331. PubMed ID: 33935664

    Kubrak, O. I., Kucerova, L., Theopold, U., Nylin, S. and Nassel, D. R. (2016). Characterization of reproductive dormancy in male Drosophila melanogaster. Front Physiol 7: 572. PubMed ID: 27932997

    Kudow, N., Kamikouchi, A. and Tanimura, T. (2019). Softness sensing and learning in Drosophila larvae. J Exp Biol. PubMed ID: 30833462

    Kudo, A., Shigenobu, S., Kadota, K., Nozawa, M., Shibata, T. F., Ishikawa, Y. and Matsuo, T. (2017). Comparative analysis of the brain transcriptome in a hyper-aggressive fruit fly, Drosophila prolongata. Insect Biochem Mol Biol 82: 11-20. PubMed ID: 28115271

    Kurz, C. L., Charroux, B., Chaduli, D., Viallat-Lieutaud, A. and Royet, J. (2017). Peptidoglycan sensing by octopaminergic neurons modulates Drosophila oviposition. Elife 6. PubMed ID: 28264763

    Landayan, D., Feldman, D. S. and Wolf, F. W. (2018). Satiation state-dependent dopaminergic control of foraging in Drosophila. Sci Rep 8(1): 5777. PubMed ID: 29636522

    Lau, C. K. S., Jelen, M. and Gordon, M. D. (2021). A closed-loop optogenetic screen for neurons controlling feeding in Drosophila. G3 (Bethesda) 11(5). PubMed ID: 33714999

    Lee, D. C., et al. (2019). Dietary supplementation with the ketogenic diet metabolite beta-hydroxybutyrate ameliorates post-TBI aggression in young-adult male Drosophila. Front Neurosci 13: 1140. PubMed ID: 31736687

    Legros, J., Tang, G., Gautrais, J., Fernandez, M. P. and Trannoy, S. (2020). Long-Term Dietary Restriction Leads to Development of Alternative Fighting Strategies. Front Behav Neurosci 14: 599676. PubMed ID: 33519392

    Leitão-Gonçalves, R., Carvalho-Santos, Z., Francisco, A.P., Fioreze, G.T., Anjos, M., Baltazar, C., Elias, A.P., Itskov, P.M., Piper, M.D.W. and Ribeiro, C. (2017). Commensal bacteria and essential amino acids control food choice behavior and reproduction. PLoS Biol 15: e2000862. PubMed ID: 28441450

    Leung, A., Cohen, D., van Swinderen, B. and Tsuchiya, N. (2021). Integrated information structure collapses with anesthetic loss of conscious arousal in Drosophila melanogaster. PLoS Comput Biol 17(2): e1008722. PubMed ID: 33635858

    Li, N., Stanewsky, R., Popay, T., Warman, G. and Cheeseman, J. (2020). The Effect of General Anaesthesia on Circadian Rhythms in Behaviour and Clock Gene Expression of Drosophila melanogaster. Clocks Sleep 2(4): 434-441. PubMed ID: 33113932

    Liao, S., Broughton, S. and Nassel, D. R. (2017). Behavioral senescence and aging-related changes in motor neurons and brain neuromodulator levels are ameliorated by lifespan-extending reproductive dormancy in Drosophila. Front Cell Neurosci 11: 111. PubMed ID: 28503133

    Lirakis, M., Dolezal, M. and Schlotterer, C. (2018). Redefining reproductive dormancy in Drosophila as a general stress response to cold temperatures. J Insect Physiol 107: 175-185. PubMed ID: 29649483

    Liu, G., Nath, T., Linneweber, G. A., Claeys, A., Guo, Z., Li, J., Bengochea, M., De Backer, S., Weyn, B., Sneyders, M., Nicasy, H., Yu, P., Scheunders, P. and Hassan, B. A. (2018). A simple computer vision pipeline reveals the effects of isolation on social interaction dynamics in Drosophila. PLoS Comput Biol 14(8): e1006410. PubMed ID: 30161262

    Liu, Q., Tabuchi, M., Liu, S., Kodama, L., Horiuchi, W., Daniels, J., Chiu, L., Baldoni, D. and Wu, M. N. (2017). Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger. Science 356(6337): 534-539. PubMed ID: 28473588

    Liu, W., Guo, F., Lu, B. and Guo, A. (2008). amnesiac regulates sleep onset and maintenance in Drosophila melanogaster. Biochem Biophys Res Commun 372(4): 798-803. PubMed ID: 18514063

    Liu, W., Wang, J., Zhang, H. Y., Yang, Y. C., Kang, R. X., Bai, P., Fu, H., Chen, L. R., Gao, Y. P. and Tan, E. K. (2020). Symbiotic bacteria attenuate Drosophila oviposition repellence to alkaline through acidification. Insect Sci. PubMed ID: 32725723

    Lynch, Z. R., Schlenke, T. A., Morran, L. T. and de Roode, J. C. (2017). Ethanol confers differential protection against generalist and specialist parasitoids of Drosophila melanogaster. PLoS One 12(7): e0180182. PubMed ID: 28700600

    MacMillan, H. A., Nazal, B., Wali, S., Yerushalmi, G. Y., Misyura, L., Donini, A. and Paluzzi, J. P. (2018). Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance. J Exp Biol. PubMed ID: 30104306

    Mahishi, D., Triphan, T., Hesse, R. and Huetteroth, W. (2021). The Panopticon-Assessing the Effect of Starvation on Prolonged Fly Activity and Place Preference. Front Behav Neurosci 15: 640146. PubMed ID: 33841109

    Mansourian, S., Enjin, A., Jirle, E. V., Ramesh, V., Rehermann, G., Becher, P. G., Pool, J. E. and Stensmyr, M. C. (2018). Wild African Drosophila melanogaster are seasonal specialists on marula fruit. Curr Biol 28(24):3960-3968. PubMed ID: 30528579

    Martelli, C., Pech, U., Kobbenbring, S., Pauls, D., Bahl, B., Sommer, M. V., Pooryasin, A., Barth, J., Arias, C. W. P., Vassiliou, C., Luna, A. J. F., Poppinga, H., Richter, F. G., Wegener, C., Fiala, A. and Riemensperger, T. (2017). SIFamide translates hunger signals into appetitive and feeding behavior in Drosophila. Cell Rep 20(2): 464-478. PubMed ID: 28700946

    McKinney, R. M., Valdez, R. and Ben-Shahar, Y. (2021). The genetic architecture of larval aggregation behavior in Drosophila. J Neurogenet: 1-16. PubMed ID: 33629904

    McLay, L. K., Green, M. P. and Jones, T. M. (2017). Chronic exposure to dim artificial light at night decreases fecundity and adult survival in Drosophila melanogaster. J Insect Physiol 100: 15-20. PubMed ID: 28499591

    Meichtry, L. B., Poetini, M. R., Dahleh, M. M. M., Araujo, S. M., Musachio, E. A. S., Bortolotto, V. C., de Freitas Couto, S., Somacal, S., Emanuelli, T., Gayer, M. C., Roehrs, R., Guerra, G. P. and Prigol, M. (2020). Addition of saturated and trans-fatty acids to the diet induces depressive and anxiety-like behaviors in Drosophila melanogaster. Neuroscience. PubMed ID: 32738432

    Melo, N., Wolff, G. H., Costa-da-Silva, A. L., Arribas, R., Triana, M. F., Gugger, M., Riffell, J. A., DeGennaro, M. and Stensmyr, M. C. (2019). Geosmin attracts Aedes aegypti mosquitoes to oviposition sites. Curr Biol. PubMed ID: 31839454

    Miroschnikow, A., Schlegel, P., Schoofs, A., Hueckesfeld, S., Li, F., Schneider-Mizell, C. M., Fetter, R. D., Truman, J. W., Cardona, A. and Pankratz, M. J. (2018). Convergence of monosynaptic and polysynaptic sensory paths onto common motor outputs in a Drosophila feeding connectome. Elife 7. PubMed ID: 30526854

    Monyak, R. E., Golbari, N. M., Chan, Y. B., Pranevicius, A., Tang, G., Fernandez, M. P. and Kravitz, E. A. (2021). Masculinized Drosophila females adapt their fighting strategies to their opponent. J Exp Biol. PubMed ID: 33568440

    Mossman, J. A., Mabeza, R. M. S., Blake, E., Mehta, N. and Rand, D. M. (2019). Age of both parents influences reproduction and egg dumping behavior in Drosophila melanogaster. J Hered. PubMed ID: 30753690

    Musso, P. Y., Junca, P., Jelen, M., Feldman-Kiss, D., Zhang, H., Chan, R. C. and Gordon, M. D. (2019). Closed-loop optogenetic activation of peripheral or central neurons modulates feeding in freely moving Drosophila. Elife 8. PubMed ID: 31322499

    Murata, S., Brockmann, A. and Tanimura, T. (2017). Pharyngeal stimulation with sugar triggers local searching behavior in Drosophila. J Exp Biol [Epub ahead of print]. PubMed ID: 28684466

    Murgier, J., Everaerts, C., Farine, J. P. and Ferveur, J. F. (2019). Live yeast in juvenile diet induces species-specific effects on Drosophila adult behaviour and fitness. Sci Rep 9(1): 8873. PubMed ID: 31222019

    Nojima, T., Rings, A., Allen, A. M., Otto, N., Verschut, T. A., Billeter, J. C., Neville, M. C. and Goodwin, S. F. (2021). A sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior. Curr Biol. PubMed ID: 33508219

    Oh, S. M., Jeong, K., Seo, J. T. and Moon, S. J. (2021). Multisensory interactions regulate feeding behavior in Drosophila. Proc Natl Acad Sci U S A 118(7). PubMed ID: 33558226

    Ohyama, T., Schneider-Mizell, C. M., Fetter, R. D., Aleman, J. V., Franconville, R., Rivera-Alba, M., Mensh, B. D., Branson, K. M., Simpson, J. H., Truman, J. W., Cardona, A. and Zlatic, M. (2015). A multilevel multimodal circuit enhances action selection in Drosophila. Nature 520(7549): 633-639. PubMed ID: 25896325

    Olufs, Z. P. G., Loewen, C. A., Ganetzky, B., Wassarman, D. A. and Perouansky, M. (2018). Genetic variability affects absolute and relative potencies and kinetics of the anesthetics isoflurane and sevoflurane in Drosophila melanogaster. Sci Rep 8(1): 2348. PubMed ID: 29402974

    Paisios, E., Rjosk, A., Pamir, E. and Schleyer, M. (2017). Common microbehavioral 'footprint' of two distinct classes of conditioned aversion. Learn Mem 24(5): 191-198. PubMed ID: 28416630

    Palavicino-Maggio, C. B., Chan, Y. B., McKellar, C. and Kravitz, E. A. (2019). A small number of cholinergic neurons mediate hyperaggression in female Drosophila. Proc Natl Acad Sci U S A 116(34): 17029-17038. PubMed ID: 31391301

    Pan, B., Geleoc, G. S., Asai, Y., Horwitz, G. C., Kurima, K., Ishikawa, K., Kawashima, Y., Griffith, A. J. and Holt, J. R. (2013). TMC1 and TMC2 are components of the mechanotransduction channel in hair cells of the mammalian inner ear. Neuron 79(3): 504-515. PubMed ID: 23871232

    Parigi, A., Porter, C., Cermak, M., Pitchers, W. R. and Dworkin, I. (2019). The behavioral repertoire of Drosophila melanogaster in the presence of two predator species that differ in hunting mode. PLoS One 14(5): e0216860. PubMed ID: 31150415

    Park, A., Tran, T. and Atkinson, N. S. (2018). Monitoring food preference in Drosophila by oligonucleotide tagging. Proc Natl Acad Sci U S A 115(36): 9020-9025. PubMed ID: 30127010

    Park, A., Tran, T., Scheuermann, E. A., Smith, D. P. and Atkinson, N. S. (2020). Alcohol potentiates a pheromone signal in flies. Elife 9. PubMed ID: 33141025

    Peralta-Rincon, J. R., Aoulad, F. Z., Prado, A. and Edelaar, P. (2020). Phenotype-dependent habitat choice is too weak to cause assortative mating between Drosophila melanogaster strains differing in light sensitivity. PLoS One 15(10): e0234223. PubMed ID: 33057335

    Pool, A. H., Kvello, P., Mann, K., Cheung, S. K., Gordon, M. D., Wang, L. and Scott, K. (2014). Four GABAergic interneurons impose feeding restraint in Drosophila. Neuron 83: 164-177. PubMed ID: 24991960

    Prasad, N. and Hens, K. (2018). Sugar promotes feeding in flies via the serine protease homolog scarface. Cell Rep 24(12): 3194-3206. PubMed ID: 30232002

    Pu, Y., Zhang, Y., Zhang, Y. and Shen, P. (2018). Two Drosophila Neuropeptide Y-like neurons define a reward module for transforming appetitive odor representations to motivation. Sci Rep 8(1): 11658. PubMed ID: 30076343

    Porcelli, D., Gaston, K.J., Butlin, R.K. and Snook, R.R. (2016). Local adaptation of reproductive performance during thermal stress. J Evol Biol [Epub ahead of print]. PubMed ID: 27862539

    Qi, W., Wang, G. and Wang, L. (2020). A novel satiety sensor detects circulating glucose and suppresses food consumption via insulin-producing cells in Drosophila. Cell Res. PubMed ID: 33273704

    Qiao, B., Li, C., Allen, V. W., Shirasu-Hiza, M. and Syed, S. (2018). Automated analysis of long-term grooming behavior in Drosophila using a k-nearest neighbors classifier. Elife 7. PubMed ID: 29485401

    Ramin, M., Domocos, C., Slawaska-Eng, D. and Rao, Y. (2014). Aggression and social experience: genetic analysis of visual circuit activity in the control of aggressiveness in Drosophila. Mol Brain 7: 55. PubMed ID: 25116850

    Ramin, M., Li, Y., Chang, W. T., Shaw, H. and Rao, Y. (2019). The peacefulness gene promotes aggression in Drosophila. Mol Brain 12(1): 1. PubMed ID: 30606245

    Redhai, S., Pilgrim, C., Gaspar, P., Giesen, L. V., Lopes, T., Riabinina, O., Grenier, T., Milona, A., Chanana, B., Swadling, J. B., Wang, Y. F., Dahalan, F., Yuan, M., Wilsch-Brauninger, M., Lin, W. H., Dennison, N., Capriotti, P., Lawniczak, M. K. N., Baines, R. A., Warnecke, T., Windbichler, N., Leulier, F., Bellono, N. W. and Miguel-Aliaga, I. (2020). An intestinal zinc sensor regulates food intake and developmental growth. Nature 580(7802): 263-268. PubMed ID: 32269334

    Reisenman, C. E. and Scott, K. (2019). Food-derived volatiles enhance consumption in Drosophila melanogaster. J Exp Biol. PubMed ID: 31085598

    Ryvkin, J., Bentzur, A., Shmueli, A., Tannenbaum, M., Shallom, O., Dokarker, S., Benichou, J. I. C., Levi, M. and Shohat-Ophir, G. (2021). Transcriptome Analysis of NPFR Neurons Reveals a Connection Between Proteome Diversity and Social Behavior. Front Behav Neurosci 15: 628662. PubMed ID: 33867948

    Robles-Murguia, M., Rao, D., Finkelstein, D., Xu, B., Fan, Y. and Demontis, F. (2020). Muscle-derived Dpp regulates feeding initiation via endocrine modulation of brain dopamine biosynthesis. Genes Dev 34(1-2): 37-52. PubMed ID: 31831628

    Rohde, P. D., Gaertner, B., Wards, K., Sorensen, P. and Mackay, T. F. C. (2017). Genomic analysis of genotype by social environment interaction for Drosophila aggressive behavior. Genetics [Epub ahead of print]. PubMed ID: 28550016

    Rombaut, A., Guilhot, R., Xuereb, A., Benoit, L., Chapuis, M. P., Gibert, P. and Fellous, S. (2017). Invasive Drosophila suzukii facilitates Drosophila melanogaster infestation and sour rot outbreaks in the vineyards. R Soc Open Sci 4(3): 170117. PubMed ID: 28405407

    Rooke, R., Rasool, A., Schneider, J. and Levine, J. D. (2020). Drosophila melanogaster behaviour changes in different social environments based on group size and density. Commun Biol 3(1): 304. PubMed ID: 32533063

    Rouse, J., McDowall, L., Mitchell, Z., Duncan, E. J. and Bretman, A. (2020). Social competition stimulates cognitive performance in a sex-specific manner. Proc Biol Sci 287(1935): 20201424. PubMed ID: 32933446

    Sadaf, S., Reddy, O. V., Sane, S. P. and Hasan, G. (2015). Neural control of wing coordination in flies. Curr Biol 25(1): 80-86. PubMed ID: 25496964

    Saltz, J. B. (2016). Genetic variation in social environment construction influences the development of aggressive behavior in Drosophila melanogaster. Heredity (Edinb) [Epub ahead of print]. PubMed ID: 27848947

    Sadanandappa, M. K., Sathyanarayana, S. H., Kondo, S. and Bosco, G. (2021). Neuropeptide F signaling regulates parasitoid-specific germline development and egg-laying in Drosophila. PLoS Genet 17(3): e1009456. PubMed ID: 33770070

    Sareen, P. F., McCurdy, L. Y. and Nitabach, M. N. (2021). A neuronal ensemble encoding adaptive choice during sensory conflict in Drosophila. Nat Commun. 12(1):4131. PubMed ID: 34226544

    Saxena, N., Natesan, D. and Sane, S. P. (2017). Odor source localization in complex visual environments by fruit flies. J Exp Biol. PubMed ID: 29146771

    Sayin, S., De Backer, J. F., Siju, K. P., Wosniack, M. E., Lewis, L. P., Frisch, L. M., Gansen, B., Schlegel, P., Edmondson-Stait, A., Sharifi, N., Fisher, C. B., Calle-Schuler, S. A., Lauritzen, J. S., Bock, D. D., Costa, M., Jefferis, G., Gjorgjieva, J. and Grunwald Kadow, I. C. (2019). A neural circuit arbitrates between persistence and withdrawal in hungry Drosophila. Neuron 104(3):544-558. PubMed ID: 31471123

    Sherer, L. M., Catudio Garrett, E., Morgan, H. R., Brewer, E. D., Sirrs, L. A., Shearin, H. K., Williams, J. L., McCabe, B. D., Stowers, R. S. and Certel, S. J. (2020). Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons. PLoS Genet 16(2): e1008609. PubMed ID: 32097408

    Schlegel, P., Texada, M. J., Miroschnikow, A., Schoofs, A., Huckesfeld, S., Peters, M., Schneider-Mizell, C. M., Lacin, H., Li, F., Fetter, R. D., Truman, J. W., Cardona, A. and Pankratz, M. J. (2016). Synaptic transmission parallels neuromodulation in a central food-intake circuit. Elife 5: e16799. PubMed ID: 27845623

    Schretter, C. E., Aso, Y., Robie, A. A., Dreher, M., Dolan, M. J., Chen, N., Ito, M., Yang, T., Parekh, R., Branson, K. M. and Rubin, G. M. (2020). Cell types and neuronal circuitry underlying female aggression in Drosophila. Elife 9. PubMed ID: 33141021

    Schwarz, O., Bohra, A. A., Liu, X., Reichert, H., VijayRaghavan, K. and Pielage, J. (2017). Motor control of Drosophila feeding behavior. Elife 6 [Epub ahead of print]. PubMed ID: 28211791

    Sehdev, A., Mohammed, Y. G., Tafrali, C. and Szyszka, P. (2019). Social foraging extends associative odor-food memory expression in an automated learning assay for Drosophila melanogaster. J Exp Biol 222(Pt 19). PubMed ID: 31527181

    Shpigler, H. Y., Saul, M. C., Murdoch, E. E., Cash-Ahmed, A. C., Seward, C. H., Sloofman, L., Chandrasekaran, S., Sinha, S., Stubbs, L. J. and Robinson, G. E. (2017). Behavioral, transcriptomic and epigenetic responses to social challenge in honey bees. Genes Brain Behav 16(6): 579-591. PubMed ID: 28328153

    Simoes, J. M., Levy, J. I., Zaharieva, E. E., Vinson, L. T., Zhao, P., Alpert, M. H., Kath, W. L., Para, A. and Gallio, M. (2021). Robustness and plasticity in Drosophila heat avoidance. Nat Commun 12(1): 2044. PubMed ID: 33824330

    Sokabe, T., Chen, H.C., Luo, J. and Montell, C. (2016). A switch in thermal preference in Drosophila larvae depends on multiple rhodopsins. Cell Rep 17: 336-344. PubMed ID: 27705783

    Sørensen, J.G., Schou, M.F., Kristensen, T.N. and Loeschcke, V. (2016). Thermal fluctuations affect the transcriptome through mechanisms independent of average temperature. Sci Rep 6: 30975. PubMed ID: 27487917

    Søvik, E., LaMora, A., Seehra, G., Barron, A.B., Duncan, J.G. and Ben-Shahar, Y. (2017). Drosophila divalent metal ion transporter Malvolio is required in dopaminergic neurons for feeding decisions. Genes Brain Behav [Epub ahead of print]. PubMed ID: 28220999

    Stensmyr, M. C., Dweck, H. K., Farhan, A., Ibba, I., Strutz, A., Mukunda, L., Linz, J., Grabe, V., Steck, K., Lavista-Llanos, S., Wicher, D., Sachse, S., Knaden, M., Becher, P. G., Seki, Y. and Hansson, B. S. (2012). A conserved dedicated olfactory circuit for detecting harmful microbes in Drosophila. Cell 151(6): 1345-1357. PubMed ID: 23217715

    Stern, U., Srivastava, H., Chen, H. L., Mohammad, F., Claridge-Chang, A. and Yang, C. H. (2019). Learning a spatial task by trial and error in Drosophila. Curr Biol. PubMed ID: 31327716

    Stone, T., Webb, B., Adden, A., Weddig, N. B., Honkanen, A., Templin, R., Wcislo, W., Scimeca, L., Warrant, E. and Heinze, S. (2017). An anatomically constrained model for path integration in the bee brain. Curr Biol 27(20): 3069-3085 e3011. PubMed ID: 28988858

    Struk, A. A., Mugon, J., Huston, A., Scholer, A. A., Stadler, G., Higgins, E. T., Sokolowski, M. B. and Danckert, J. (2019). Self-regulation and the foraging gene (PRKG1) in humans. Proc Natl Acad Sci U S A. PubMed ID: 30782798

    Sugime, Y., Watanabe, D., Yasuno, Y., Shinada, T., Miura, T. and Tanaka, N. K. (2017). Upregulation of juvenile hormone titers in female Drosophila melanogaster through mating experiences and host food occupied by eggs and larvae. Zoolog Sci 34(1): 52-57. PubMed ID: 28148219

    Sun, Y., Qiu, R., Li, X., Cheng, Y., Gao, S., Kong, F., Liu, L. and Zhu, Y. (2020). Social attraction in Drosophila is regulated by the mushroom body and serotonergic system. Nat Commun 11(1): 5350. PubMed ID: 33093442

    Surendran, S., Huckesfeld, S., Waschle, B. and Pankratz, M. J. (2017). Pathogen induced food evasion behavior in Drosophila larvae. J Exp Biol [Epub ahead of print]. PubMed ID: 28254879

    Takagi, S., Cocanougher, B. T., Niki, S., Miyamoto, D., Kohsaka, H., Kazama, H., Fetter, R. D., Truman, J. W., Zlatic, M., Cardona, A. and Nose, A. (2017). Divergent connectivity of homologous command-like neurons mediates segment-specific touch responses in Drosophila. Neuron 96(6): 1373-1387 e1376. PubMed ID: 29198754

    Tang, X., Roessingh, S., Hayley, S. E., Chu, M. L., Tanaka, N. K., Wolfgang, W., Song, S., Stanewsky, R. and Hamada, F. N. (2017). The role of PDF neurons in setting preferred temperature before dawn in Drosophila. Elife 6. PubMed ID: 28463109

    Tastekin, I., Riedl, J., Schilling-Kurz, V., Gomez-Marin, A., Truman, J. W. and Louis, M. (2015). Role of the subesophageal zone in sensorimotor control of orientation in Drosophila larva. Curr Biol 25(11): 1448-1460. PubMed ID: 25959970

    Tastekin, I., Khandelwal, A., Tadres, D., Fessner, N. D., Truman, J. W., Zlatic, M., Cardona, A. and Louis, M. (2018). Sensorimotor pathway controlling stopping behavior during chemotaxis in the Drosophila melanogaster larva. Elife 7. pii: e38740. PubMed ID: 30465650

    Tauber, J. M., Brown, E. B., Li, Y., Yurgel, M. E., Masek, P. and Keene, A. C. (2017). A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste. PLoS Genet 13(11): e1007059. PubMed ID: 29121639

    Thane, M., Viswanathan, V., Meyer, T. C., Paisios, E. and Schleyer, M. (2019). Modulations of microbehaviour by associative memory strength in Drosophila larvae. PLoS One 14(10): e0224154. PubMed ID: 31634372

    Ton, H. T., Phan, T. X., Abramyan, A. M., Shi, L. and Ahern, G. P. (2017). Identification of a putative binding site critical for general anesthetic activation of TRPA1. Proc Natl Acad Sci U S A 114(14): 3762-3767. PubMed ID: 28320952

    Kottler, B., Bao, H., Zalucki, O., Imlach, W., Troup, M., van Alphen, B., Paulk, A., Zhang, B. and van Swinderen, B. (2013). A sleep/wake circuit controls isoflurane sensitivity in Drosophila. Curr Biol 23(7): 594-598. PubMed ID: 23499534

    Troup, M., Zalucki, O. H., Kottler, B. D., Karunanithi, S., Anggono, V. and van Swinderen, B. (2019). Syntaxin1A neomorphic mutations promote rapid recovery from isoflurane anesthesia in Drosophila melanogaster. Anesthesiology. PubMed ID: 31356232

    Tsao, C. H., Chen, C. C., Lin, C. H., Yang, H. Y. and Lin, S. (2018). Drosophila mushroom bodies integrate hunger and satiety signals to control innate food-seeking behavior. Elife 7. PubMed ID: 29547121

    Umezaki, Y., Hayley, S. E., Chu, M. L., Seo, H. W., Shah, P. and Hamada, F. N. (2018). Feeding-state-dependent modulation of temperature preference requires insulin signaling in Drosophila warm-sensing neurons. Curr Biol 28(5): 779-787.e773. PubMed ID: 29478858

    van Breugel, F., Huda, A. and Dickinson, M. H. (2018). Distinct activity-gated pathways mediate attraction and aversion to CO2 in Drosophila. Nature. PubMed ID: 30464346

    Vernia, S., Edwards, Y. J., Han, M. S., Cavanagh-Kyros, J., Barrett, T., Kim, J. K. and Davis, R. J. (2016). An alternative splicing program promotes adipose tissue thermogenesis. Elife 5. PubMed ID: 27635635

    Verschut, T. A., Carlsson, M. A. and Hamback, P. A. (2019). Scaling the interactive effects of attractive and repellent odours for insect search behaviour. Sci Rep 9(1): 15309. PubMed ID: 31653955

    Vigoder, F. M., Parker, D. J., Cook, N., Tourniere, O., Sneddon, T. and Ritchie, M. G. (2016). Inducing Cold-Sensitivity in the Frigophilic Fly Drosophila montana by RNAi. PLoS One 11: e0165724. PubMed ID: 27832122

    von Heckel, K., Stephan, W. and Hutter, S. (2016). Canalization of gene expression is a major signature of regulatory cold adaptation in temperate Drosophila melanogaster. BMC Genomics 17: 574. PubMed ID: 27502401

    Waddell, S., Armstrong, J. D., Kitamoto, T., Kaiser, K. and Quinn, W. G. (2000). The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory. Cell 103(5): 805-813. PubMed ID: 11114336

    Wang, F., Wang, K., Forknall, N., Patrick, C., Yang, T., Parekh, R., Bock, D. and Dickson, B. J. (2020). Neural circuitry linking mating and egg laying in Drosophila females. Nature 579(7797): 101-105. PubMed ID: 32103180

    Wang, X., Li, G., Liu, J., Liu, J. and Xu, X. Z. (2016). TMC-1 mediates alkaline sensation in C. elegans through nociceptive neurons. Neuron 91(1): 146-154. PubMed ID: 27321925

    Watanabe, K., Chiu, H., Pfeiffer, B. D., Wong, A. M., Hoopfer, E. D., Rubin, G. M. and Anderson, D. J. (2017). A circuit node that integrates convergent input from neuromodulatory and social behavior-promoting neurons to control aggression in Drosophila. Neuron 95(5): 1112-1128 e1117. PubMed ID: 28858617

    Wilinski, D., Winzeler, J., Duren, W., Persons, J. L., Holme, K. J., Mosquera, J., Khabiri, M., Kinchen, J. M., Freddolino, P. L., Karnovsky, A. and Dus, M. (2019). Rapid metabolic shifts occur during the transition between hunger and satiety in Drosophila melanogaster. Nat Commun 10(1): 4052. PubMed ID: 31492856

    Williams, M. J., Akram, M., Barkauskaite, D., Patil, S., Kotsidou, E., Kheder, S., Vitale, G., Filaferro, M., Blemings, S. W., Maestri, G., Hazim, N., Vergoni, A. V. and Schioth, H. B. (2020). CCAP regulates feeding behavior via the NPF pathway in Drosophila adults. Proc Natl Acad Sci U S A. PubMed ID: 32179671

    Wu, C.L., Fu, T.F., Chou, Y.Y. and Yeh, S.R. (2015). A single pair of neurons modulates egg-laying decisions in Drosophila. PLoS One 10: e0121335. PubMed ID: 25781933

    Wu, F., Deng, B., Xiao, N., Wang, T., Li, Y., Wang, R., Shi, K., Luo, D. G., Rao, Y. and Zhou, C. (2020). A neuropeptide regulates fighting behavior in Drosophila melanogaster. Elife 9. PubMed ID: 32314736

    Wu, S. F., Ja, Y. L., Zhang, Y. J. and Yang, C. H. (2019). Sweet neurons inhibit texture discrimination by signaling TMC-expressing mechanosensitive neurons in Drosophila. Elife 8. PubMed ID: 31184585

    Yang, Z., Yu, Y., Zhang, V., Tian, Y., Qi, W. and Wang, L. (2015). Octopamine mediates starvation-induced hyperactivity in adult Drosophila. Proc Natl Acad Sci U S A 112(16): 5219-5224. PubMed ID: 25848004

    Yang, Z., Huang, R., Fu, X., Wang, G., Qi, W., Mao, D., Shi, Z., Shen, W. L. and Wang, L. (2018). A post-ingestive amino acid sensor promotes food consumption in Drosophila. Cell Res. PubMed ID: 30209352

    Yen, H. H., Han, R. and Lo, C. C. (2019). Quantification of visual fixation behavior and spatial orientation memory in Drosophila melanogaster. Front Behav Neurosci 13: 215. PubMed ID: 31572145

    Yu, Y., Huang, R., Ye, J., Zhang, V., Wu, C., Cheng, G., Jia, J. and Wang, L. (2016). Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila. Elife 5. PubMed ID: 27612383

    Versace, E., Caffini, M., Werkhoven, Z. and de Bivort, B. L. (2020). Individual, but not population asymmetries, are modulated by social environment and genotype in Drosophila melanogaster. Sci Rep 10(1): 4480. PubMed ID: 32161330

    Verschut, T. A., Inouye, B. D. and Hamback, P. A. (2018). Sensory deficiencies affect resource selection and associational effects at two spatial scales. Ecol Evol 8(21): 10569-10577. PubMed ID: 30464828

    Williams, M. J., Goergen, P., Rajendran, J., Zheleznyakova, G., Hagglund, M. G., Perland, E., Bagchi, S., Kalogeropoulou, A., Khan, Z., Fredriksson, R. and Schioth, H. B. (2014). Obesity-linked homologues TfAP-2 and Twz establish meal Frequency in Drosophila melanogaster. PLoS Genet 10: e1004499. PubMed ID: 25187989

    Yanagawa, A., Huang, W., Yamamoto, A., Wada-Katsumata, A., Schal, C. and Mackay, T. F. C. (2020). Genetic Basis of Natural Variation in Spontaneous Grooming in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32727922

    Yerushalmi, G. Y., Misyura, L., Donini, A. and MacMillan, H. A. (2016). Chronic dietary salt stress mitigates hyperkalemia and facilitates chill coma recovery in Drosophila melanogaster. J Insect Physiol 95: 89-97. PubMed ID: 27642001

    Yoshino, J., Morikawa, R. K., Hasegawa, E. and Emoto, K. (2017). Neural circuitry that evokes escape behavior upon activation of nociceptive sensory neurons in Drosophila larvae. Curr Biol 27(16):2499-2504. PubMed ID: 28803873

    Yu, Y., Huang, R., Ye, J., Zhang, V., Wu, C., Cheng, G., Jia, J. and Wang, L. (2016). Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila. Elife 5: e15693. PubMed ID: 27612383

    Zhan, Y. P., Liu, L. and Zhu, Y. (2016). Taotie neurons regulate appetite in Drosophila. Nat Commun 7: 13633. PubMed ID: 27924813

    Zhang, L., Yu, J., Guo, X., Wei, J., Liu, T. and Zhang, W. (2020). Parallel mechanosensory pathways direct oviposition decision-making in Drosophila. Curr Biol. PubMed ID: 32649914

    Zhang, N., Guo, L. and Simpson, J. H. (2020). Spatial comparisons of mechanosensory information govern the grooming sequence in Drosophila. Curr Biol. PubMed ID: 32142695

    Zhang, Y.V., Aikin, T.J., Li, Z. and Montell, C. (2016). The basis of food texture sensation in Drosophila. Neuron 91(4):863-877. PubMed ID: 27478019

    Zhao, W., Zhou, P., Gong, C., Ouyang, Z., Wang, J., Zheng, N. and Gong, Z. (2019). A disinhibitory mechanism biases Drosophila innate light preference. Nat Commun 10(1): 124. PubMed ID: 30631066

    Zhou, Y., Cao, L. H., Sui, X. W., Guo, X. Q. and Luo, D. G. (2019). Mechanosensory circuits coordinate two opposing motor actions in Drosophila feeding. Sci Adv 5(5): eaaw5141. PubMed ID: 31131327

    Zhu, M. L., Herrera, K. J., Vogt, K. and Bahl, A. (2021). Navigational strategies underlying temporal phototaxis in Drosophila larvae. J Exp Biol. PubMed ID: 33954778

    Zwarts, L., Vulsteke, V., Buhl, E., Hodge, J. J. and Callaerts, P. (2017). SlgA, the homologue of the human schizophrenia associated PRODH gene, acts in clock neurons to regulate Drosophila aggression. Dis Model Mech. PubMed ID: 28331058

    Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

    The Interactive Fly resides on the
    Society for Developmental Biology's Web server.