• 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).

    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 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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    Invasive Drosophila suzukii facilitates Drosophila melanogaster infestation and sour rot outbreaks in the vineyards

    How do invasive pests affect interactions between members of pre-existing agrosystems? The invasive pest Drosophila suzukii is suspected to be involved in the aetiology of sour rot, a grapevine disease that otherwise develops following Drosophila melanogaster infestation of wounded berries. This study combined field observations with laboratory assays to disentangle the relative roles of both Drosophila in disease development. The emergence was observed of numerous D. suzukii, but no D. melanogaster flies, from bunches that started showing mild sour rot symptoms days after field collection. However, bunches that already showed severe rot symptoms in the field mostly contained D. melanogaster. In the laboratory, oviposition by D. suzukii triggered sour rot development. An independent assay showed the disease increased grape attractiveness to ovipositing D. melanogaster females. These results suggest that in invaded vineyards, D. suzukii facilitates D. melanogaster infestation and, consequently, favours sour rot outbreaks. Rather than competing with close species, the invader subsequently permits their reproduction in otherwise non-accessible resources and may cause more frequent, or more extensive, disease outbreaks (Rombaut, 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, 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).

    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).

    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).

    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).

    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, 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, 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 courtship 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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).


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