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

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

The rich variety of behaviours observed in animals arises through the interplay between sensory processing and motor control. To understand these sensorimotor transformations, it is useful to build models that predict not only neural responses to sensory input but also how each neuron causally contributes to behaviour. This study demonstrates a novel modelling approach to identify a one-to-one mapping between internal units in a deep neural network and real neurons by predicting the behavioural changes that arise from systematic perturbations of more than a dozen neuronal cell types. A key ingredient that is introduced is 'knockout training', which involves perturbing the network during training to match the perturbations of the real neurons during behavioural experiments. This approach was applied to model the sensorimotor transformations of Drosophila melanogaster males during a complex, visually guided social behaviour. The visual projection neurons at the interface between the optic lobe and central brain form a set of discrete channels, and prior work indicates that each channel encodes a specific visual feature to drive a particular behaviour. This model reaches a different conclusion: combinations of visual projection neurons, including those involved in non-social behaviours, drive male interactions with the female, forming a rich population code for behaviour. Overall, this framework consolidates behavioural effects elicited from various neural perturbations into a single, unified model, providing a map from stimulus to neuronal cell type to behaviour, and enabling future incorporation of wiring diagrams of the brain into the model (Cowley, 2024).

This study develop knockout training, a novel solution to identify a one-to-one mapping between internal units in a deep neural network (DNN) and real neurons in the brain of a fly. The model makes predictions about how neurons respond to sensory stimuli and drive behaviour. Although silencing each LC neuron type (LC neuron types receive input from the lobula and lobula plate in the optic lobe and send axons to optic glomeruli in the central brain) )on its own may have a small to medium effect on behaviour, the 1-to-1 network infers how the LC types work together as a population to drive the courtship behaviour of the male. The model extends beyond findings from direct recordings of LC neuron, even in behaving flies. The 1-to-1 network provides information on LC visual responses in freely behaving flies (not head-fixed, as is required for recordings) engaging in natural social interactions and can generate LC responses to any arbitrary visual stimulus. In fact, it was demonstrate that the 1-to-1 network predicts actual responses to stimuli that the model had not seen during training. The model also makes testable predictions about which combinations of LC types are both necessary and sufficient for specific courtship behaviours. A major new finding of this work is which and to what extent LC neuron types contribute to song production, an integral part of courtship guided by visual feedback. Given that the same visual stimulus sequence can drive multiple LC types, this neuron-to-behaviour relationship is not readily inferred from LC recordings alone. The 1-to-1 network is the first large-scale hypothesis of how the LC types work together to encode stimuli and contribute to behaviour; this model and code is shared with the community to inspire future experiments and models (Cowley, 2024).

The main conclusion of this study is that the complex courtship behaviour of the male relies on combinations of visual projections neurons-including those also involved in non-social behaviours. However, the extent to which other behaviours beyond those observed during courtship also rely on a population code is not yet known. Knockout training on the LC types could easily be applied to other visuomotor behaviours (for example, escape responses or flight) to make direct comparisons. Given the extent of interconnectivity between LC types and convergence of LC types onto common downstream cell types, it is posited that population coding for behaviour, particularly in natural contexts, might be the norm. By contrast, for behaviours that rely on quick and robust processing, such as escape from a predator, the arrangement of LC types into optic glomeruli may facilitate the fast readout of specific channels. One issue raised by the use of a multiplexed code is how the fly brain produces the correct behaviour at the correct time. For example, LPLC2 neurons synapse onto the giant fibre neuron to drive an escape take-off, but the 1-to-1 network predicts that this same cell type encodes female size and contributes to the forward velocity of the male during courtship; recent work has also found LPLC2 contributes to evasive flight turns. Future experiments are needed to understand how the same LC cell type can contribute to different behaviours in different contexts (Cowley, 2024).

This modelling approach comes with limitations. For example, if silencing an LC type does not lead to a noticeable change in behaviour, the 1-to-1 network cannot infer the tuning of that LC type. In addition, many silenced LC types resulted in stronger-not weaker-courtship, suggesting that these LC neurons may act partially as distractors to prevent relentless pursuit of the female. This approach also found some mismatches between real LC responses and the responses of the 1-to-1 network; although this may be owing to differences in internal state between freely moving males during natural courtship (training data for the model) versus head-fixed males passively viewing stimuli (neural recordings), training on neural data and behavioural data together may help to improve both neural and behavioural prediction. An experimental limitation of using natural behaviour arises because the statistics of the visual experience cannot be matched between LC-silenced and control males (for example, an LC9-silenced male spends much less time near the female); future experiments can use virtual reality or robotic females to present identical stimulus sequences to control and silenced males (Cowley, 2024).

Following recent studies using Deep Neural Networks (DNNs) to predict responses of visual neurons, this study used DNNs in the 1-to-1 network that are highly expressive function approximators but lack biological realism. The model-agnostic knockout training procedure can be used to train more biologically inspired models that incorporate constraints from the FlyWire connectome and emerging male brain wiring diagrams to include recurrent connections, lateral connections between LC types and delays. An intriguing future direction is to apply this framework to other bottlenecks within the Drosophila brain, such as the descending and ascending neurons that link the brain and nerve cord. and in more complex systems for which genetic control over cell types is available. This work shows that constraining models with causal perturbations of neurons during complex behaviour is an important ingredient in revealing the relationships between stimulus, neurons and behaviour (Cowley, 2024).

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).

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

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Temperature fluctuations are challenging for ectotherms which are not able to regulate body temperature by physiological means and thus have to adjust their thermal environment via behavior. However, little is yet known about whether microbial symbionts influence thermal preference (T(p)) in ectotherms by modulating their physiology. Several recent studies have demonstrated substantial effects of Wolbachia infections on host T(p) in different Drosophila species. These data indicate that the direction and strength of thermal preference variation is strongly dependent on host and symbiont genotypes and highly variable among studies. By employing highly controlled experiments, this study investigated the impact of several environmental factors including humidity, food quality, light exposure, and experimental setup that may influence T(p) measurements in adult Drosophila melanogaster flies. Additionally, the effects of Wolbachia infection on T(p) of Drosophila was assessed at different developmental stages, which has not been done before. Only subtle effects were found of Wolbachia on host T(p) which are strongly affected by experimental variation in adult, but not during juvenile life stages. These in-depth analyses show that environmental variation has a substantial influence on T(p) which demonstrates the necessity of careful experimental design and cautious interpretations of T(p) measurements together with a thorough description of the methods and equipment used to conduct behavioral studies (Strunov, 2023).

Thermal tolerance is a fundamental physiological complex trait for survival in many species. This study took a multipronged approach to dissect the genetic architecture that controls thermal tolerance in natural populations using the Drosophila Synthetic Population Resource (DSPR) as a model system. First, quantitative genetics and Quantitative Trait Loci (QTL) mapping were used to identify major effect regions within the genome that influences thermal tolerance, then integrated RNA-sequencing to identify differences in gene expression, and lastly, the RNAi system was used to 1) alter tissue-specific gene expression and 2) functionally validate the findings. This powerful integration of approaches not only allows for the identification of the genetic basis of thermal tolerance but also the physiology of thermal tolerance in a natural population, which ultimately elucidates thermal tolerance through a fitness-associated lens (Williams-Simon, 2023).

Temperature sensation involves thermosensitive TRP (thermoTRP) and non-TRP channels. Drosophila larval Class III (CIII) neurons serve as the primary cold nociceptors and express a suite of thermoTRP channels i mplicated in noxious cold sensation. How CIII neurons code temperature remains unclear.This study combined computational and electrophysiological methods to address this question. In electrophysiological experiments, two basic cold-evoked patterns of CIII neurons were identified: bursting and spiking. In response to a fast temperature drop to noxious cold, CIII neurons distinctly mark different phases of the stimulus. Bursts frequently occurred along with the fast temperature drop, forming a peak in the spiking rate and likely coding the high rate of the temperature change. Single spikes dominated at a steady temperature and exhibited frequency adaptation following the peak. When temperature decreased slowly to the same value, mainly spiking activity was observed, with bursts occurring sporadically throughout the stimulation. The spike and the burst frequencies positively correlated with the rate of the temperature drop. Using a computational model, the distinction is explained in the occurrence of the two CIII cold-evoked patterns bursting and spiking, using the dynamics of a thermoTRP current. A two-parameter activity map (Temperature, constant TRP current conductance) marks parameters that support silent, spiking, and bursting regimes. Projecting on the map the instantaneous TRP conductance, governed by activation and inactivation processes, reflects temperature coding responses as a path across silent, spiking, or bursting domains on the map. The map sheds light on how various parameter sets for TRP kinetics represent various types of cold-evoked responses. Together, these results indicate that bursting detects the high rate of temperature change, whereas tonic spiking could reflect both the rate of change and magnitude of steady cold temperature (Maksymchuk, 2023).

Thermosensation is critical for the survival of animals. However, mechanisms through which nutritional status modulates thermosensation remain unclear. This study shows that hungry Drosophila exhibit a strong hot avoidance behavior (HAB) compared to food-sated flies. Hot stimulus increases the activity of α'β' mushroom body neurons (MBns), with weak activity in the sated state and strong activity in the hungry state. Furthermore, it was shown that α'β' MBn receives the same level of hot input from the mALT projection neurons via cholinergic transmission in sated and hungry states. Differences in α'β' MBn activity between food-sated and hungry flies following heat stimuli are regulated by distinct Drosophila insulin-like peptides (Dilps). Dilp2 is secreted by insulin-producing cells (IPCs) and regulates HAB during satiety, whereas Dilp6 is secreted by the fat body and regulates HAB during the hungry state. Dilp2 induces PI3K/AKT signaling, whereas Dilp6 induces Ras/ERK signaling in α'β' MBn to regulate HAB in different feeding conditions. Finally, it was shown that the 2 α'β'-related MB output neurons (MBONs), MBON-α'3 and MBON-β'1, are necessary for the output of integrated hot avoidance information from α'β' MBn. These results demonstrate the presence of dual insulin modulation pathways in α'β' MBn, which are important for suitable behavioral responses in Drosophila during thermoregulation under different feeding states (Chiang, 2023).

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

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

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

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

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

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

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

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

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

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

The effects of density are key in determining population dynamics, since they can positively or negatively affect the fitness of individuals. These effects have great relevance for polyphagous insects for which immature stages develop within a single site of finite feeding resources. Drosophila suzukii is a crop pest that induces severe economic losses for agricultural production; however, little is known about the effects of density on its life-history traits. This study, (i) investigated the egg distribution resulting from females' egg-laying strategy and (ii) tested the immediate (on immatures) and delayed (on adults) effects of larval density on emergence rate, development time, potential fecundity, and adult size. The density used varied in a range between 1 and 50 larvae. 44.27% of the blueberries used for the oviposition assay contained between 1 and 11 eggs in aggregates. The high experimental density (50 larvae) has no immediate effect in the emergence rate but has effect on larval developmental time. This trait was involved in a trade-off with adult life-history traits: The time of larval development was reduced as larval density increased, but smaller and less fertile females were produced. These results clearly highlight the consequences of larval crowding on the juveniles and adults of this fly (Reyes-Ramirez, 2023).

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

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

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

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

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).

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).

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

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

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

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

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

Fluctuating environments threaten fertility and viability. To better match the immediate, local environment, many organisms adopt alternative phenotypic states, a phenomenon called "phenotypic plasticity." Natural populations that predictably encounter fluctuating environments tend to be more plastic than conspecific populations that encounter a constant environment, suggesting that phenotypic plasticity can be adaptive. Despite pervasive evidence of such "adaptive phenotypic plasticity," gene regulatory mechanisms underlying plasticity remains poorly understood. This study tested the hypothesis that environment-dependent phenotypic plasticity is mediated by epigenetic factors. To test this hypothesis, the adaptive reproductive arrest of Drosophila melanogaster females, called diapause, was exploited. Using an inbred line from a natural population with high diapause plasticity, it was demonstrated that diapause is determined epigenetically: only a subset of genetically identical individuals enter diapause and this diapause plasticity is epigenetically transmitted for at least three generations. Upon screening a suite of epigenetic marks, it was discovered that the active histone marks H3K4me3 and H3K36me1 are depleted in diapausing ovaries. Using ovary-specific knockdown of histone mark writers and erasers, it was demonstrated that H3K4me3 and H3K36me1 depletion promotes diapause. Given that diapause is highly polygenic, that is, distinct suites of alleles mediate diapause plasticity across distinct genotypes, the potential for genetic variation in diapause-determining epigenetic marks was also investigated. Specifically, it was asked if these histone marks were similarly depleted in diapause of a genotypically distinct line. Evidence was found of divergence in both the gene expression program and histone mark abundance. This study reveals chromatin determinants of phenotypic plasticity and suggests that these determinants may be genotype-dependent, offering new insight into how organisms may exploit and evolve epigenetic mechanisms to persist in fluctuating environments (Evans, 2023).

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).

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).

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

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-LB^PD 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-LB^PC isoform certainly acts non cell-autonomously to dampen immune activation by circulating PGN, a putatively intracellular isoform PGRP-LB^PD 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-LB^PD 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).

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, amn^28A, amn^c651, and amn^X8 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-GAL80^ts; 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, amn^28A, and amn^C651, and amn^X8 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 amn^X8 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 amn^28A and amn^c651 are P-element-induced mutations in the amn gene. The amn^X8 was made by imprecise excision of the P-element from amn^28A, and a significant increase in ethanol-sensitive phenotype was found in amn^X8 compared to amn¹ and amn^28A. It is noteworthy that amn^X8 contains possibly other GAL4 insertions elsewhere in the genome left after excision of amn^28A, 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).

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).

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

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

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

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

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

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

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

All animals face the challenge of finding nutritious resources in a changing environment. To maximize lifetime fitness, the exploratory behavior has to be flexible, but which behavioral elements adapt and what triggers those changes remain elusive. Using experiments and modeling, this study characterized extensively how Drosophila larvae foraging adapts to different food quality and distribution and how the foraging genetic background influences this adaptation. This work shows that different food properties modulated specific motor programs. Food quality controls the traveled distance by modulating crawling speed and frequency of pauses and turns. Food distribution, and in particular the food-no food interface, controls turning behavior, stimulating turns toward the food when reaching the patch border and increasing the proportion of time spent within patches of food. Finally, the polymorphism in the foraging gene (rover-sitter) of the larvae adjusts the magnitude of the behavioral response to different food conditions. This study defines several levels of control of foraging and provides the basis for the systematic identification of the neuronal circuits and mechanisms controlling each behavioral response (Wosniack, 2022)

This study employs supervised machine learning algorithms to test whether locomotive features during exploratory activity in open field arenas can serve as predictors for the genotype of fruit flies. Because of the nonlinearity in locomotive trajectories, traditional statistical methods that are used to compare exploratory activity between genotypes of fruit flies may not reveal all insights. 10-minute-long trajectories of four different genotypes of fruit flies in an open-field arena environment were captured. Turn angles and step size features extracted from the trajectories were used for training supervised learning models to predict the genotype of the fruit flies. Using the first five minute locomotive trajectories, an accuracy of 83% was achieved in differentiating wild-type flies from three other mutant genotypes. Using the final 5 min and the entire ten minute duration decreased the performance indicating that the most variations between the genotypes in their exploratory activity are exhibited in the first few minutes. Feature importance analysis revealed that turn angle is a better predictor than step size in predicting fruit fly genotype. Overall, this study demonstrates that features of trajectories can be used to predict the genotype of fruit flies through supervised machine learning methods (Nguyen, 2023).

Detailed descriptions of behavior provide critical insight into the structure and function of nervous systems. In Drosophila larvae and many other systems, short behavioral experiments have been successful in characterizing rapid responses to a range of stimuli at the population level. However, the lack of long-term continuous observation makes it difficult to dissect comprehensive behavioral dynamics of individual animals and how behavior (and therefore the nervous system) develops over time. To allow for long-term continuous observations in individual fly larvae, a robotic instrument was engineered that automatically tracks and transports larvae throughout an arena. The flexibility and reliability of its design enables controlled stimulus delivery and continuous measurement over developmental time scales, yielding an unprecedented level of detailed locomotion data. This study utilized the new system's capabilities to perform continuous observation of exploratory search behavior over a duration of 6 hr with and without a thermal gradient present, and in a single larva for over 30 hr. Long-term free-roaming behavior and analogous short-term experiments show similar dynamics that take place at the beginning of each experiment. Finally, characterization of larval thermotaxis in individuals reveals a bimodal distribution in navigation efficiency, identifying distinct phenotypes that are obfuscated when only analyzing population averages (Yu, 2023)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Behavioural avoidance has obvious benefits for animals facing environmental stressors such as pathogen-contaminated foods. Most current bioinsecticides are based on the environmental and opportunistic bacterium Bacillus thuringiensis (Bt) that kills targeted insect pests upon ingestion. While food and oviposition avoidance of Bt bioinsecticide by targeted insect species was reported, this remained to be addressed in non-target organisms, especially those affected by chronic exposure to Bt bioinsecticide such as Drosophila species. Here, using a two-choice oviposition test, we showed that female flies of three Drosophila species (four strains of D. melanogaster, D. busckii and D. suzukii) avoided laying eggs in the presence of Bt var. kurstaki bioinsecticide, with potential benefits for the offspring and female's fitness. Avoidance occurred rapidly, regardless of the fraction of the bioinsecticide suspension (spores and toxin crystals versus soluble toxins/compounds) and independently of the female motivation for egg laying. These results suggest that, in addition to recent findings of developmental and physiological alterations upon chronic exposure to non-target Drosophila, this bioinsecticide may modify the competitive interactions between Drosophila species in treated areas and the interactions with their associated natural enemies (Babin, 2023).

Taste systems encode chemical cues that drive vital behaviors. This study has elucidated noncanonical features of taste coding using an unconventional kind of electrophysiological analysis. Taste neurons of Drosophila are much more sensitive than previously thought. They have a low spontaneous firing frequency that depends on taste receptors. Taste neurons have a dual function as olfactory neurons: They are activated by most tested odorants, including N,N-diethyl-meta-toluamide (DEET), at a distance. DEET can also inhibit certain taste neurons, revealing that there are two modes of taste response: activation and inhibition. Electrophysiological OFF responses were characterized and it was found that the tastants that elicit them are related in structure. OFF responses link tastant identity to behavior: the magnitude of the OFF response elicited by a tastant correlated with the egg laying behavior it elicited. In summary, the sensitivity and coding capacity of the taste system are much greater than previously known (Dweck, 2023).

This study elucidated a variety of noncanonical features of taste coding. The results show that the taste system is much more sensitive than previously thought and has an unexpectedly rich capacity for encoding taste information (Dweck, 2023).

Bitter, sugar, and water neurons all showed remarkably low levels of spontaneous firing-on the order of 1 or 2 spikes/s. The low firing frequencies may provide a means of increasing their sensitivity: A low concentration of tastant that elevates the firing frequency by a few spikes per second thereby produces a large fractional increase in activity (Dweck, 2023).

The spontaneous firing rate of I-a neurons depends on Gr receptors it expresses. A simple model to explain this finding is that receptors exist in an equilibrium between an open state and a closed state and that the spontaneous firing frequency is governed by this equilibrium: Spontaneous firing is low if the fraction of receptors in the open state is low. When receptors are removed by mutation, the spontaneous rate may be reduced further. In a GRN such as I-a, with only a few Grs, deletion of individual Grs reduces the spontaneous rate appreciably; in GRNs with many receptors, such as S-a or S-b, removal of one receptor does not have a measurable effect on spontaneous firing (Dweck, 2023).

This study found that tastants can inhibit the spontaneous firing rate of GRNs. DEET particularly inhibited the spontaneous firing of the S-a neuron in mutant backgrounds. Thus, tastants can have two modes of action: activation and inhibition. One model to explain this inhibition is that binding of DEET to one or more receptors shifts the equilibrium from the open to the closed configuration. In summary, DEET is likely to bind to at least one receptor and activate it, explaining the increase in firing it elicits in wild type, but can also bind to another receptor and inactivate it, which is revealed in mutant backgrounds lacking a receptor that DEET activates. It is noted that tastants have previously been found to inhibit the excitatory responses of taste neurons to other tastants, but this is a distinct paradigm, and the mechanism of such inhibition may be different (Dweck, 2023).

This study found that taste neurons respond to the vapor of DEET, coumarin (COU), and most members of a panel comprising 47 structurally diverse odorants. The magnitude of the responses depends on the neuron, the odorant, and the concentration; thus, the GRN repertoire provides a representation of the identity and intensity of the odorant. In summary, GRNs can function as ORNs (Dweck, 2023).

The ecological significance of these olfactory responses is an interesting issue. The labellum is on the order of 200 to 240 μm in length in the antero-posterior direction, and sensilla range from ~15 to ~35 μm in length. This studyfound responses to DEET at distances of 1 to 50 μm, but, in nature, these responses to volatile compounds may be detected at greater distances depending on wind, temperature, and concentration. A taste sensillum of a fly that is exploring a potential food source in a microenvironment may often first encounter a compound via the air and shortly thereafter via contact. When contact is made, the stimulus may be part of a complex mixture that includes a wide variety of nonvolatile molecules, some of which could reduce the salience of the volatile cues. The effect of early airborne activation of taste neurons on decision-making awaits further study; in some cases, it could signal the presence of compounds at sufficiently high levels as to be aversive. In any case, these results suggest that Grs, in addition to binding a remarkable diversity of tastants, are also able to bind a wide range of odorants. Chemical compounds can evidently become solubilized in the lymph of taste sensilla and reach membrane receptors whether delivered to the sensillum via a solution or via the air (Dweck, 2023).

The taste system of Drosophila was found to be much more sensitive to tastants in solution than previously thought. The sensitivity may be increased by the low spontaneous firing frequency, as noted above. High sensitivity may be especially adaptive by allowing detection of bitter compounds that are toxic at low levels. The taste system operates as an early warning system, protecting the fly from feeding or laying eggs on toxic food sources. A more sensitive system may provide more information about the suitability of a potential food source or oviposition substrate in nature (Dweck, 2023).

Some sensory systems have evolved extraordinary sensitivity. Rod photoreceptors can signal the presence of a single photon. Moth pheromone-sensing neurons can detect a small number of pheromone molecules. In this regard, it is noted that tastant space is vast, and the current results suggest the interesting possibility that other tastants of particular biological significance may be detected by the fly with even greater sensitivity than have found in this study (Dweck, 2023).

Electrophysiological OFF responses, at high cellular and temporal resolution, were document to a wide variety of tastants, in multiple taste neurons, and in six Drosophila species. OFF responses are elicited by a subset of bitter tastants in D. melanogaster. Among those tastants that elicit OFF responses, the identity of the tastant is encoded in the pattern of OFF responses it elicits from the different GRNs. The magnitude of the OFF response does not correlate with that of the corresponding ON response. The threshold for OFF responses is at least an order of magnitude higher than for ON responses for all bitter compounds tested (Dweck, 2023).

Surprising relationships were found between OFF responses, tastant structure, and behavior. Tastants that produced OFF responses in this study are structurally related to each other. The tastants that produced OFF responses have higher molecular weights than those that did not, with the exception of aristolochic acid (ARI). OFF responses correlated with behavior, in two different ways (Dweck, 2023).

First, egg-laying preference could be predicted by the OFF response: Compounds that elicited larger OFF responses elicited greater aversion to egg laying. By contrast, there was no correlation between ON responses and egg-laying preference. These results suggest that the OFF response is a particularly salient feature of taste coding. It is noted that in establishing this correlation between OFF responses and egg-laying preference, the arithmetic sum of OFF (or ON) responses of I-a, S-a, and S-b bitter neurons were used as a simple representation of the response of the labellar GRN repertoire. In the future, it will interesting to expand this analysis to include other GRNs of the fly and to examine the correlations that emerge when their inputs are weighted in ways suggested by their connectivity in the central nervous system (Dweck, 2023).

Second, proboscis extension response (PER) behavior was inhibited only at concentrations when an OFF response was observed . This finding suggests a critical role for OFF responses in inhibiting the PER, a behavior that operates over a much shorter time scale than egg laying . In summary, results from these two behavioral paradigms suggest that OFF responses provide an informative representation of both the identity and intensity of a tastant (Dweck, 2023).

It is notable that a correlation was found between egg-laying avoidance behavior and OFF responses, but not ON responses. A priori one might have predicted that the onset of a bitter stimulus, producing an ON response, would elicit inhibition of egg laying, with a stronger ON response eliciting stronger inhibition. Rather, it was found that the offset of a bitter stimulus, producing an OFF response and signaling the appearance of a more suitable egg-laying site, may be a more salient signal. That is, to a fly exploring a site in nature, a large OFF response may reflect the end of a strong bitter stimulus and thereby indicate a large improvement in the suitability of the fly's immediate locale for egg laying. One interpretation of these results is that egg-laying behavior is driven largely by a circuit that is activated by signals from the taste system indicating the appearance of a more favorable egg-laying site (Dweck, 2023).

It is interesting that the same four Gr genes are required for both ON and OFF responses in I-a sensilla. One might have expected ON responses to be mediated by one complex of Grs and OFF responses by another. Rather, the results support a model in which a single Gr heteromultimeric complex has evolved an elegant mechanism of signaling both the onset and offset of taste stimuli. The evolution of dual function for a single complex is economical and may expand the coding capacity of a receptor repertoire of a given size (Dweck, 2023).

It will be of great interest to elucidate the mechanism by which Grs produce ON and OFF responses. Determining the structure of a bitter Gr may be highly informative of mechanism, just as determination of Or structures has been. However, GRN signaling appears to have more degrees of freedom than ORN signaling. In addition to having OFF responses, which have not been reported in Drosophila ORNs, GRNs show different patterns of ON and OFF signaling within an individual neuron in response to different stimuli. For example, depending on the stimulus, an individual S-a neuron produces an ON but not an OFF response, an OFF but not an ON response, or both. This great complexity of signaling, which is unusual among sensory receptor neurons, may reflect the great complexity of receptor expression in GRNs (Dweck, 2023).

Together, the results of this study support the view that the taste system of Drosophila has a much greater coding capacity than previously thought. Taste neurons have greater dynamic ranges than previously appreciated, can be inhibited as well as activated, respond to many compounds before contact, and show OFF responses that link taste quality and quantity to behavioral output. The coding mechanisms described here will almost certainly be essential to an understanding of how chemical information is transformed by taste circuits into behavior (Dweck, 2023).

This study has examined taste coding in a limited number of taste neurons, i.e., those of the labellum that are most accessible to electrophysiological analysis. Coding in other taste organs deserves much future exploration. Likewise, this study has examined a diverse set of tastants and odorants, but chemical space is vast, and testing of other compounds may reveal additional features of taste coding. This study examines monomolecular tastants, but, in nature, flies encounter complex mixtures of compounds. Much remains to be learned about the coding of taste mixtures (Dweck, 2023).

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

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

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

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

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

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

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

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

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).

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). >

Little is known about the molecular underpinning of behavioral pleiotropy. The Drosophila melanogaster foraging gene is highly pleiotropic, affecting many independent larval and adult phenotypes. Included in foraging's multiple phenotypes are larval foraging path length, triglyceride levels, and food intake. foraging has a complex structure with four promoters and 21 transcripts that encode nine protein isoforms of a cGMP dependent protein kinase (PKG). This study examined if foraging's complex molecular structure underlies the behavioral pleiotropy associated with this gene. Using a promotor analysis strategy, DNA fragments upstream of each of foraging's transcription start sites was cloned and four separate for^pr-Gal4s were generated. Supporting the hypothesis of modular function, they had discrete, restricted expression patterns throughout the larva. In the CNS, for^pr1-Gal4 and for^pr4-Gal4 were expressed in neurons while for^pr2-Gal4 and for^pr3-Gal4 were expressed in glia cells. In the gastric system, for^pr1-Gal4 and for^pr3-Gal4 were expressed in enteroendocrine cells of the midgut while for^pr2-Gal4 was expressed in the stem cells of the midgut. for^pr3-Gal4 was expressed in the midgut enterocytes, and midgut and hindgut visceral muscle. for^pr4-Gal4's gastric system expression was restricted to the hindgut. Promoter specific expression was found in the larval fat body, salivary glands, and body muscle. The modularity of foraging's molecular structure was also apparent in the phenotypic rescues. Path length, triglyceride levels (bordered on significance), and food intake were rescued of for^pr0 null larvae using different for^pr-Gal4s to drive UAS-for(cDNA). In a foraging null genetic background, for^pr1-Gal4 was the only promoter driven Gal4 to rescue larval path length, for^pr3-Gal4 altered triglyceride levels, and for^pr4-Gal4 rescued food intake. The results refine the spatial expression responsible for foraging's associated phenotypes, as well as the sub-regions of the locus responsible for their expression. foraging's pleiotropy arises at least in part from the individual contributions of its four promoters (Allen, 2018).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

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).

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A general assumption would be that a tastant would cause a similar response in ingestion and choice preference behavior, in either a positive or negative manner. However, the current findings corroborate that certain tastants elicit divergent ingestion and choice preference behavior. Combining molecular genetic tools, behavioral assays, and genetically encoded calcium sensors to assess neuronal activity, the results provide evidence that relatively independent neural systems appear to regulate the two initial processes of feeding in Drosophila larva: searching for palatable food, i.e., choice preference, and eating the selected food, i.e., ingestion. A subset of gustatory neurons housed in the TO, the external gustatory organ of Drosophila larva, detect denatonium and induce avoidance behavior, and DP1, a specific pair of GRNs in the dorsal pharyngeal organ, plays a major role in regulating both ingestion and avoidance in response to CAF (Choi, 2020).

The TO of Drosophila larva is located at the tip of the cephalic lobes, and is thus anatomically likely to be the first organ to contact external stimuli and subsequently cause a change in movement to regulate the initial step of feeding. Similarly, pharyngeal sensilla are located between the external sense organs and digestive organs, and are thus anatomically likely to act in maintaining the ingestion of appetitive foods while stopping ingestion and causing avoidance of aversive cues such as bitter toxins. It could be advantageous for ingestion to be predominantly controlled by pharyngeal sense organs, rather than by external organs, since animals can try out a potential food source before making their decision, rather than blindly avoiding it. This could be a particularly advantageous strategy for insect larvae whose main purpose is to feed. Also, the difference in behavioral responses elicited by the C1 and C7 neurons in the TO and DP1 in the pharyngeal sense organs is likely linked to the difference in brain projection patterns of GRNs from the TO and pharyngeal GRNs from the larval SEZ, with the distinct projection areas of the brain taste center likely being linked to different circuits, resulting in distinct behavioral outputs (Choi, 2020).

In Drosophila larvae, choice and ingestion have generally been grouped together and studied as a group of reflexive behaviors. Sugar processing provides another intriguing example of divergence between choice and ingestion. Larvae generally show increased preference and feeding when exposed to increasing concentrations of fructose or sucrose. At extremely high concentrations such as 2 M or 4 M, larvae still exhibit preference in terms of choice, but show suppression of feeding (or ingestion, as is denoted in this study). Since this suppression of ingestion could be due to high viscosity and/or osmolarity, a direct comparison to the processing of aversive tastants such as bitter chemicals is difficult. However, this example nonetheless provides evidence that relatively independent circuits exist to determine choice and ingestion. Using bitter tastants, this study found that choice and ingestion can manifest in clearly divergent behaviors to the same compounds and elucidate the cellular basis of these observations. Similarities to the observation that external sense organs and pharyngeal organs appear to be involved in somewhat independent behavioral output can be seen in sugar consumption in the adult fly. The activation of sweet GRNs in the legs and labellum initiates feeding behaviors including the proboscis extension reflex, and pharyngeal sweet GRNs play an important role in directing the sustained consumption of sweet compounds (Choi, 2020).

Most of the 22 putative bitter tastants tested in this study, including CAF, cause negative effects in choice preference and ingestion. Nicotine caused a positive P.I. in the choice preference assay. It cannot be completely rule out that nicotine could act as an attractive chemosensory cue at low concentrations, this study found that nicotine inhibits the movement of larvae in the experimental setup. Larvae strongly avoid denatonium, but once they sample denatonium-containing food, they ingest it. This ingestion likely occurs because denatonium is added to the agarose of the entire plate, whereas larvae probably would not ingest as much if they had the choice. Nevertheless, the results suggest that this larval response to denatonium is due to the existence of a functional receptor complex for denatonium in the TO, which does not exist in the pharyngeal sense organs, or at the very least the DP1 neuron. Consistently, ectopic expression of GR59c in DP1 caused a novel calcium response to denatonium and suppression of ingestion in response to denatonium. Some remaining questions regarding sensing of denatonium merit further study. Avoidance to denatonium is defective when either C1 or C7 is inactivated, indicating that C1 and C7 are not redundant in terms of behavior. It is possible that a certain threshold of neuronal activity is required to elicit behavior, or inactivation of one neuron may cause a change in the functions of other GRNs. Although a numerically simple system, larval GRNs also have a multimodal character, and as such a more complicated mechanism might be involved. Also, in the bitter sensing neurons of the adult labellum, two complexes, GR32a/GR66a/GR59c and GR32a/GR66a/GR22e, are each sufficient to confer a response to denatonium. Based on Gr-GAL4 expression, the larval DP1 neuron expresses Gr22e, but not Gr59c, but is not capable of detecting denatonium. This suggests that the GRNs of the larva and adult fly possess different cellular contexts, which could be interesting to unravel. An interesting remaining question is if Gr59c is solely responsible for denatonium sensing in the larval C1 neuron or if the existing Gr22e can rescue denatonium sensing in Gr59c mutants. This would indicate that Gr22e needs a specific co-receptor repertoire for denatonium detection and could help elucidate coding differences in the larva versus the adult fly (Choi, 2020).

The levels at which distinct bitter compounds are detected might reflect the ecological niche of the animal and the toxicity level of a given tastant. The results suggest that information from the DP1 neuron is processed in a circuit that results in negative and aversive behavior in ingestion and choice preference to CAF. The C1 and C7 neuron in the TO elicit avoidance to denatonium in choice preference behavior. Thus, these results suggest that distinct sensory neurons appear to have distinct sensory roles, likely through the expression of specific receptors or specific groups of receptors. Sensory information detected by these sensory neurons appears to be processed through distinct circuits in the central nervous system to mediate changes in ingestion or choice behavior. It is yet unclear whether the different circuits interact to result in a final behavioral output. Further examination of the potential connections between the external and pharyngeal gustatory neurons and interneurons or motor neurons in the brain may provide insight into the overall neural circuit that regulates feeding and locomotion (Choi, 2020).

The function of the central nervous system to regulate food intake can be disrupted by sustained metabolic challenges such as high-fat diet (HFD), which may contribute to various metabolic disorders. Previous work has shown that a group of octopaminergic (OA) neurons mediated starvation-induced hyperactivity, an important aspect of food-seeking behavior. This study found that HFD specifically enhances this behavior. Mechanistically, HFD increases the excitability of these OA neurons to a hunger hormone named adipokinetic hormone (AKH), via increasing the accumulation of AKH receptor (AKHR) in these neurons. Upon HFD, excess dietary lipids are transported by a lipoprotein LTP to enter these OA(+)AKHR(+) neurons via the cognate receptor LpR1, which in turn suppresses autophagy-dependent degradation of AKHR. Taken together, this study has uncovered a mechanism that links HFD, neuronal autophagy, and starvation-induced hyperactivity, providing insight in the reshaping of neural circuitry under metabolic challenges and the progression of metabolic diseases (Huang, 2020).

Obesity and obesity-associated metabolic disorders such as type 2 diabetes and cardiovascular diseases have become a global epidemic. Chronic over-nutrition, especially excessive intake of dietary lipids, is one of the leading causes of these metabolic disturbances. Accumulating evidence has shown that HFD imposes adverse effects on the physiology and metabolism of liver, skeletal muscle, the adipose tissue, and the nervous system. It is therefore of importance to understand the mechanisms underlying HFD-induced changes in different organs and cell types, which will offer critical insight into the diagnosis and treatment of obesity and other metabolic diseases (Huang, 2020).

The central nervous system plays a critical role in regulating energy intake and expenditure. In rodent models, neurons located in the arcuate nucleus of the hypothalamus, particularly neurons expressing Neuropeptide Y (NPY) and Agouti-Related Neuropeptide (AgRP) or those expressing Pro-opiomelanocortin (POMC), are important behavioral and metabolic regulators. These neurons detect various neural and hormonal cues such as circulating glucose and fatty acids, leptin, and ghrelin, and modulate energy intake and expenditure accordingly. Upon the reduction of the internal energy state, NPY/AgRP neurons are activated and exert a robust orexigenic effect. Genetic ablation of NPY/AgRP neurons in neonatal mice completely abolishes food consumption whereas acute activation of these neurons significantly enhances food consumption. NPY/AgRP neurons also antagonize the function of POMC neurons that plays a suppressive role on food consumption. Taken together, these two groups of neurons, among other neuronal populations, work in synergy to ensure a refined balance between energy intake and expenditure, and hence organismal metabolism (Huang, 2020).

In spite of their critical roles, the function of the nervous system to accurately regulate appetite and metabolism may be disrupted by sustained metabolic stress, resulting in eating disorders and various metabolic diseases such as obesity and type 2 diabetes. Several lines of evidence have begun to reveal the underlying neural mechanisms. For example, HFD increases the intrinsic excitability of orexigenic NPY/AgRP neurons, induces leptin resistance, and enhances their inhibitory innervations with anorexigenic POMC neurons, altogether resulting in hypersensitivity to starvation and increased food consumption. Interestingly, besides HFD, other metabolic challenges, including maternal HFD, alcohol consumption, as well as aging, also disrupt normal food intake via affecting the excitability and/or innervation of NPY/AgRP neurons. All these interventions may contribute to the onset and progression of metabolic disorders (Huang, 2020).

Before the actual food consumption, food-seeking behavior is a critical yet largely overlooked behavioral component for the localization and occupation of desirable food sources. Food-seeking behavior has been characterized in rodent models, primarily by the elevation of locomotor activity and increased food approach of starved animals. It has been reported that NPY/AgRP neurons also play a role in food-seeking behavior. However, to ensure adequate food intake, food seeking and food consumption are temporally and spatially separated and even reciprocally inhibited. It remains largely unclear how the neural circuitry of food seeking and food consumption segregated and independently regulated in rodent models. Furthermore, it remains unknown whether HFD also affects food seeking, and if so whether its effects on both food seeking and food consumption share common mechanisms or not. To fully understand the intervention of energy homeostasis by sustained metabolic stress, it is necessary to dissect the neural circuitry underlying food seeking and examine whether and how it is affected by HFD (Huang, 2020).

Fruit flies Drosophila melanogaster share fundamental analogy to vertebrate counterparts on the regulation of energy homeostasis and organismal metabolism despite that they diverged several hundred million years ago. Therefore, it offers a good model to characterize food-seeking behavior in depth and provides insight into the regulation of energy intake and the pathogenesis of metabolic disorders in more complex organisms such as rodents and human (Huang, 2020).

Previous work showed that fruit flies exhibited robust starvation-induced hyperactivity that was directed towards the localization and acquisition of food sources, therefore resembling an important aspect of food-seeking behavior upon starvation (Yang, 2015). A small subset of OA neurons in the fly brain were identified that specifically regulated starvation-induced hyperactivity (Yu, 2016). Analogous to mammalian systems, a number of neural and hormonal cues are involved in the systemic control of nutrient metabolism and food intake in fruit flies. Among them, Neuropeptide F (NPF), short NPF (sNPF), Leucokinin, and Allatostatin A (AstA), have been shown to regulate food consumption, all of which have known mammalian homologs that regulate food intake. In particular, starvation-induced hyperactivity is regulated by two classes of neuroendocrine cells (Yu, 2016). One is functionally analogous to pancreatic α cells and produce AKH upon starvation, whereas the other produces Drosophila insulin-like peptides (DILPs), resembling the function of pancreatic β cells. These two classes of Drosophila hormones exert antagonistic functions on starvation-induced hyperactivity via the same group of OA neurons in the fly brain (Huang, 2020).

Based on these findings, this study sought to examine whether HFD disrupted the regulation of starvation-induced hyperactivity in fruit flies and aimed to investigate the underlying mechanism. The present study found that HFD-fed flies became significantly more sensitive to starvation and exhibited starvation-induced hyperactivity earlier and stronger than flies fed with normal diet (ND). Meanwhile, HFD did not alter flies' food consumption, suggesting that starvation-induced hyperactivity and food consumption are independently affected by HFD. Several days of HFD treatment did not alter the production of important hormonal cues like AKH and DILPs, but rather increased the sensitivity of the OA neurons that regulated starvation-induced hyperactivity to the hunger hormone AKH. In these OA neurons, constitutive autophagy maintained the homeostasis of AKHR protein, which determined their sensitivity to AKH and hence starvation. HFD feeding suppressed neuronal autophagy via AMPK-TOR signaling and in turn increased the level of AKHR in these OA neurons. Consistently, eliminating autophagy in these neurons mimicked the effect of HFD on starvation-induced hyperactivity whereas promoting autophagy inhibited the induction of hyperactivity by starvation. Furthermore, this study also showed that a specific lipoprotein LTP and its cognate receptor LpR1 likely mediated the effect of HFD on the neuronal autophagy of OA neurons and hence its effect on starvation-induced hyperactivity. Taken together, this study uncovered a novel mechanism that linked HFD, AMPK-TOR signaling, neuronal autophagy, and starvation-induced hyperactivity, shedding crucial light on the reshaping of neural circuitry under metabolic stress and the progression of metabolic diseases (Huang, 2020).

There is accumulating evidence that notes the effect of HFD on food consumption from insects to human, which results in obesity and obesity-associated metabolic diseases. But the effect of HFD on another critical food intake related behavior, food seeking, remains largely uncharacterized. Conceptually, food-seeking behavior in the fruit fly is composed of two behavioral components, increased sensitivity to food cues, and enhanced exploratory locomotion, which altogether facilitates the localization and acquisition of desirable food sources. Previous work has shown that starvation promotes starvation-induced hyperactivity, the exploratory component of food-seeking behavior, via a small group of OA neurons in the fly brain. These hunger-sensing OA neurons sample the metabolic status by detecting two groups of functionally antagonistic hormones, AKH and DILPs, and promote starvation-induced hyperactivity (Yu, 2016; Huang, 2020).

This study has demonstrated that this behavior is compromised by metabolic challenges. After a few days of HFD feeding, flies became behaviorally hypersensitive to starvation and as a result their starvation-induced hyperactivity was greatly enhanced, despite that their food intake and expenditure were not affected. These results suggest that HFD feeding may specifically modulate the activity of the neural circuitry underlying starvation-induced hyperactivity and offers an opportunity to further elucidate the cellular and circuitry mechanisms underlying behavioral abnormalities upon metabolic challenges (Huang, 2020).

As an insect counterpart of mammalian glucagon, AKH acts as a hunger signal to activate its cognate receptor AKHR expressed in the fat body and subsequently triggers lipid mobilization and energy allocation. In the fly brain, a small number of OA neurons also express AKHR. These neurons have been shown to be responsive to starvation and modulate various behaviors including food seeking and drinking (Jourjine, 2016; Yu, 2016). In that sense, these OA+AKHR+ neurons are functionally analogous to mammalian NPY/AgRP neurons in the hypothalamus, which also senses organismal metabolic states and regulates specific food intake behaviors. This study found that OA+AKHR+ neurons exhibited higher AKHR protein accumulation and became hypersensitive to AKH after HFD feeding. Notably, HFD feeding in mammals also increases the excitability of NPY/AgRP neurons, which contributes to the hypersensitivity to starvation and increased food consumption (Vernia, 2016). Thus, HFD may exert a conserved effect in the regulation of neuronal excitability and food intake related behaviors in both fruit flies and mammals (Huang, 2020).

Autophagy, a lysosomal degradative process that maintains cellular homeostasis, is critical for energy homeostasis. Upon cellular starvation, autophagy generates additional energy supply by breaking down macromolecules and subcellular organelles. At the organismal level, autophagy also contributes to the regulation of food intake and hence organismal energy homeostasis. For example, fasting induces autophagy in NPY/AgRP neurons via fatty acid uptake and promotes AgRP expression, which in turn enhances food intake (Kaushik, 2011). In line with these results, eliminating autophagy in NPY/AgRP neurons reduces food intake and hence body weight and fat deposits (Kaushik, 2011). Conversely, loss of autophagy in POMC neurons displays increased food intake and adiposity (Coupe, 2012). Consistently, in the current study, fruit flies neuronal autophagy was critical for the function of OA+AKHR+ neurons to sense hunger and regulate starvation-induced hyperactivity (Huang, 2020).

Accumulating evidence suggests that HFD suppresses autophagy in different peripheral tissue types such as liver, skeletal muscle, and the adipose tissue. Similarly, HFD suppresses autophagy in the hypothalamus, whereas blocking hypothalamic autophagy, particularly in POMC neurons, exacerbates HFD induced obesity. This study showed that HFD suppressed neuronal autophagy in OA+AKHR+ neurons and enhanced AKHR accumulation in these neurons. As a result, OA+AKHR+ neurons became hypersensitive to starvation and promoted starvation-induced hyperactivity. It will be of interest to examine whether HFD also reduces autophagy and increases the accumulation of specific membrane receptors in mammalian NPY/AgRP neurons (Huang, 2020).

This study also sought to examine the cellular mechanism that linked HFD feeding to the reduction of autophagy. HFD feeding activated TOR signaling. TOR, a highly conserved serine-threonine kinase, controls numerous anabolic cellular processes. Yhis study found that TOR signaling was tightly associated with the activity of AKHR+ neurons and the behavioral responses upon HFD feeding. Genetic enhancement of TOR activity in AKHR+ neurons increased AKHR protein accumulation, the sensitivity of these neurons to AKH, and hence starvation-induced hyperactivity, all of which mimicked the effect of HFD feeding. Inhibiting TOR activity exerted an opposite effect. In addition, the effect of HFD on TOR signaling was found to be mediated by AMPK signaling. These results altogether suggest that AMPK-TOR signaling in AKHR+ neurons plays an important role in maintaining the homeostasis of these neurons and determining the responsiveness to HFD feeding. Similarly, rodent studies have shown that manipulating AMPK-TOR signaling results in the dysfunction of NPY/AgRP neurons as well as POMC neurons, which leads to abnormal food consumption and adiposity. It will be of interest to examine whether HFD also modulates AMPK-TOR signaling in these specific hypothalamic neurons (Huang, 2020).

This study next sought to understand how AKHR+ neurons detected HFD, or more specifically, excess lipid ingested by the flies. As an essential nutrient and important energy reserve, dietary lipids were transported via their carrier proteins, named lipoproteins, in the circulation system and regulated multiple cellular signaling pathways. Proteomic analysis helped identify one lipoprotein LTP that was enriched in flies' hemolymph after HFD feeding. Single-cell RNAseq of AKHR+ neurons identified a number of lipoprotein receptors, especially LpR1, highly expressed in these neurons. Therefore, it is proposed that AKHR+ neurons might sense HFD feeding via LTP-LpR1 signaling. Evidently, it was found that eliminating LpR1 in AKHR+ neurons could protect flies from HFD, reducing AKHR accumulation and abolishing the effect of HFD to enhance starvation-induced hyperactivity. Conversely, eliminating LpR1 in the fat body, the major lipid reservoir of flies, created diet-independent hyperlipidemia and mimicked the effect of HFD feeding on flies' starvation-induced hyperactivity. Taken together, a working model is proposed that upon HFD feeding, excess dietary lipids are transported by LTP in the hemolymph, which interacts with its cognate receptor LpR1 in OA+AKHR+ neurons. As a result, these neurons undergo a number of cellular signaling processes and eventually become hypersensitive to starvation (Huang, 2020).

To summarize, the present study establishes a link between an unhealthy diet and abnormalities of food intake related behaviors in a model organism. The underlying mechanism was also deciphered, involving intracellular AMPK-TOR signaling, reduced neuronal autophagy, accumulation of a specific hormone receptor, and increased excitability of a small group of hunger-sensing neurons. This study will shed crucial light on the pathological changes in the central nervous system upon metabolic challenges. Given that the central control of metabolism and food intake related behaviors are highly conserved across different species, it will be of importance to further examine whether similar mechanisms also mediate the effect of HFD feeding on food intake and metabolic diseases in mammals (Huang, 2020).

Sensing satiety is a crucial survival skill for all animal species including human. Despite the discovery of numerous neuromodulators that regulate food intake in Drosophila, the mechanism of satiety sensing remains largely elusive. This study investigated how neuropeptidergic circuitry conveyed satiety state to influence flies' food consumption. Drosophila tackykinin (DTK) and its receptor TAKR99D were identified in an RNAi screening as feeding suppressors. Two pairs of DTK(+) neurons in the fly brain could be activated by elevated D-glucose in the hemolymph and imposed a suppressive effect on feeding. These DTK(+) neurons formed a two-synapse circuitry targeting insulin-producing cells, a well-known feeding suppressor, via TAKR99D(+) neurons, and this circuitry could be rapidly activated during food ingestion and cease feeding. Taken together, this study identified a novel satiety sensor in the fly brain that could detect specific circulating nutrients and in turn modulate feeding, shedding light on the neural regulation of energy homeostasis (Qu, 2020).

Cannabinoids have an important role in regulating feeding behaviors via cannabinoid receptors in mammals. Cannabinoids also exhibit potential therapeutic functions in Drosophila melanogaster, or fruit fly that lacks cannabinoid receptors. However, it remains unclear whether cannabinoids affect food consumption and metabolism in a cannabinoid receptors-independent manner in flies. This study systematically investigated pharmacological functions of various cannabinoids in modulating food preference and consumption in flies. Flies display preferences for consuming cannabinoids, independent of two important sensory regulators Poxn and Orco. Interestingly, phyto- and endo- cannabinoids exhibit an inhibitory effect on food intake. Unexpectedly, the non-selective CB1 receptor antagonist AM251 attenuates the suppression of food intake by endocannabinoids. Moreover, the endocannabinoid anandamide (AEA) and its metabolite inhibit food intake and promote resistance to starvation, possibly through reduced lipid metabolism. Thus, this study has provided insights into a pharmacological role of cannabinoids in feeding behaviors using an adult Drosophila model (He, 2021).

Many insect species exhibit basal social behaviors such as aggregation, which play important roles in their feeding and mating ecologies. However, the evolutionary, genetic, and physiological mechanisms that regulate insect aggregation remain unknown for most species. This study used natural populations of Drosophila melanogaster to identify the genetic architecture that drives larval aggregation feeding behavior. By using quantitative and reverse genetic approaches, a complex neurogenetic network was identified that plays a role in regulating the decision of larvae to feed in either solitude or as a group. Results from single gene, RNAi-knockdown experiments show that several of the identified genes represent key nodes in the genetic network that determines the level of aggregation while feeding. Furthermore, this study showed that a single non-coding variant in the gene CG14205, a putative acyltransferase, is associated with both decreased mRNA expression and increased aggregate formation, which suggests that it has a specific role in inhibiting aggregation behavior. These results identify, for the first time, the genetic components which interact to regulate naturally occurring levels of aggregation in D. melanogaster larvae (McKinney, 2021).

The integration of two or more distinct sensory cues can help animals make more informed decisions about potential food sources, but little is known about how feeding-related multimodal sensory integration happens at the cellular and molecular levels. This study shows that multimodal sensory integration contributes to a stereotyped feeding behavior in the model organism Drosophila melanogaster Simultaneous olfactory and mechanosensory inputs significantly influence a taste-evoked feeding behavior called the proboscis extension reflex (PER). Olfactory and mechanical information are mediated by antennal Or35a neurons and leg hair plate mechanosensory neurons, respectively. The controlled delivery of three different sensory cues can produce a supra-additive PER via the concurrent stimulation of olfactory, taste, and mechanosensory inputs. It is suggested that the fruit fly is a versatile model system to study multisensory integration related to feeding, which also likely exists in vertebrates (Oh, 2021).

Feeding is an essential part of animal life that is greatly impacted by the sense of taste. Although the characterization of taste-detection at the periphery has been extensive, higher order taste and feeding circuits are still being elucidated. This study used an automated closed-loop optogenetic activation screen to detect novel taste and feeding neurons in Drosophila melanogaster. Out of 122 Janelia FlyLight Project GAL4 lines preselected based on expression pattern, this study identified six lines that acutely promote feeding and 35 lines that inhibit it. As proof of principle, R70C07-GAL4, which labels neurons that strongly inhibit feeding, was analyzed. Using split-GAL4 lines to isolate subsets of the R70C07-GAL4 population, both appetitive and aversive neurons were found. Furthermore, this study shows that R70C07-GAL4 labels putative second-order taste interneurons in the subesophageal zone that contact both sweet and bitter sensory neurons. These results serve as a resource for further functional dissection of fly feeding circuits (Lau, 2021).

Animal behaviours are demonstrably governed by sensory stimulation, previous experience and internal states like hunger. With increasing hunger, priorities shift towards foraging and feeding. During foraging, flies are known to employ efficient path integration strategies. However, general long-term activity patterns for both hungry and satiated flies in conditions of foraging remain to be better understood. Similarly, little is known about how permanent contact chemosensory stimulation affects locomotion. To address these questions, a novel, simplistic fly activity tracking setup, the Panopticon, was developed. Using a 3D-printed Petri dish inset, this assay allows recording of walking behaviour, of several flies in parallel, with all arena surfaces covered by a uniform substrate layer. Two constellations of providing food were tested: (i) in single patches and (ii) omnipresent within the substrate layer. Fly tracking is done with FIJI, further assessment, analysis and presentation is done with a custom-built MATLAB analysis framework. This study found that starvation history leads to a long-lasting reduction in locomotion, as well as a delayed place preference for food patches which seems to be not driven by immediate hunger motivation (Mahishi, 2021).

Reproduction induces increased food intake across females of many animal species, providing a physiologically relevant paradigm for the exploration of appetite regulation. By examining the diversity of enteric neurons in Drosophila melanogaster, this study identified a key role for gut-innervating neurons with sex and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. These findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain (Hadjieconomou, 2020).

Internal state has profound effects on brain function. Despite increasingly recognized roles for the gut-brain axis in maintaining energy balance, links between internal state and gastrointestinal innervation remain poorly characterized. Progress has been hindered by neuroanatomical complexity, which is only beginning to be parsed in mammals. The simpler-yet physiologically complex-intestine of Drosophila provides an alternative entry point into the study of gastrointestinal innervation (Hadjieconomou, 2020).

Innervation of the main digestive portion of the adult fly intestine, which encompasses the anterior midgut and the crop and central neurons of the pars intercerebralis (PI) in the brain. PI neurons directly innervate the anterior midgut and the crop, and include insulin-producing neurons and other peptidergic subtypes. The crop is further populated by processes that emanate from cells of the corpora cardiaca, which produce the glucagon-like adipokinetic hormone and are adjacent to the hypocerebral ganglion (HCG). Also adjacent to both the HCG and the corpora cardiaca are the corpus allatum cells, which produce juvenile hormone and extend short local projections. The thoracico-abdominal ganglion of the central nervous system might not innervate these gut regions (Hadjieconomou, 2020).

The crop-an expandable structure found in the intestines of insects-might be disregarded as a passive food store, but several observations suggest active regulation of its physiology. Refeeding flies after starvation results in enlarged, food-filled crops, pointing to modulation of food ingression into and out of the crop. Live imaging or temporal dissections of flies revealed that food always enters the crop before proceeding to the midgut. Additionally, food transit through the crop is dependent on both its palatability and its nutritional value. Therefore, in adult flies, all food transits through the crop, which is nutrient-sensitive and shows chemically and anatomically diverse innervation (Hadjieconomou, 2020).

The crop and anterior midgut are innervated by myosuppressin (Ms)-positive neurons located in the PI and the HCG. PI Ms neurons are distinct from known neuronal subsets, with the exception of eight Ms neurons that co-express the Taotie-GAL4 marker. Two PI Ms neuron populations can be distinguished by cell size: one comprises 18 large cells and another comprises 12 smaller cells. Single-cell clones of large Ms neurons reveal a single process that bifurcates into a longer, probably axonal projection to the gut-which arborizes in the HCG and extends further to innervate the crop-and a shorter, probably dendritic process that reaches the suboesophageal zone, where the axons of peripheral gustatory sensory neurons terminate. A subset of HCG Ms-expressing neurons also innervates the crop, whereas another subset projects locally. This study confirmed the expression of Ms using an endogenously tagged Ms reporter (Ms-GFP) and in situ hybridization Ms innervation was also observed of the hindgut, the rectal ampulla and the heart, and a subset of peripheral Ms-positive neurons innervating the female reproductive tract (Hadjieconomou, 2020).

This study selectively activated or silenced Ms neurons in adult flies. Activation resulted in greatly enlarged crops in flies that were fed ad libitum, consistent with the relaxant properties of Ms on insect muscles ex vivo. By contrast, silencing of Ms neurons prevented crop enlargement in a starved-refed condition in which the crop normally expands. Genetic downregulation or mutation of Ms (using a new mutant) prevented crop enlargement, albeit to a lesser extent than Ms neuron silencing. This could be due to another Ms-neuron-derived neurotransmitter or neuropeptide contributing to crop enlargement, or to loss of the Ms peptide during development in these experiments, resulting in adaptations that render the crop more active than it would be in response to acute loss of the Ms peptide. A Gal4 insertion into the Ms locus was generated that disrupts Ms production (MsTGEM). In contrast to the crop enlargement resulting from TrpA1-mediated activation from Ms-Gal4, TrpA1 expression from this (Ms mutant) MsTGEM-Gal4 driver failed to induce crop enlargement, further confirming a requirement for Ms. Ms neuron subtype-specific downregulations and activations enabled establishing that the PI Ms neurons (in particular, the Taotie-Gal4-positive subset of large PI Ms neurons) induce, and are indispensable for, crop enlargement through their production of Ms neuropeptide (Hadjieconomou, 2020).

The contributions of myosuppressin receptors 1 and 2 (MsR1 and MsR2) were explored. MsR1 expression was observed in crop muscles, in subsets of neurons including the PI and HCG Ms-positive neurons and neurons innervating the ovary and heart; no MsR1 expression was detected in ovarian or heart muscles. Expression of MsR2 was also detected in crop muscles. To investigate the function of the Ms receptor, MsR1 was downregulated specifically in adult crop muscles using two independent driver lines (vm-Gal4 and MsR1crop-Gal4). Both genetic manipulations led to reduced crop enlargement in a starvation-refeeding assay, comparable to that observed for Ms neuron silencing or Ms mutation. Downregulation of MsR2 did not affect crop enlargement. A role for MsR1 in mediating crop enlargement was confirmed using a MsR1TGEM mutant. MsR1 is therefore identified as the crop muscle receptor through which Ms signals to modulate crop enlargement (Hadjieconomou, 2020).

The physiological regulation of crop enlargement was explored and found that it is dependent on sex and on reproductive status: the crops of mated females fed ad libitum (which were used for all the experiments described above) were consistently more expanded than those of virgin female or mated male flies fed ad libitum. Because post-mating changes were not seen in Ms neuron projections, it was asked whether post-mating crop enlargement might result from the release of Ms preferentially in mated females. Ms peptide levels were lower in the PI neuron cell bodies of females only after mating. In the absence of Ms transcriptional changes this observation is consistent with a post-mating increase in the secretion of Ms peptide in females. This effect of mating on Ms levels was specific to mating: nutrient availability did not affect intracellular Ms levels. It was also observed that the Ms neurons of mated females had higher cumulative calcium levels and a reduced amplitude of calcium oscillations compared to virgin females, as detected both by in vivo GCaMP6 calcium imaging and by the calcium-sensitive reporter CaLexA, in which GFP expression is proportional to cumulative neuronal activity. Physiologically, and in contrast to observations in mated females, a reduction of Ms signalling in males or in virgin female flies failed to impair crop enlargement. Consequently, when Ms signalling to crop muscles was prevented, the size of the crop of mated females no longer differed from that of virgin females. Collectively, these findings support the idea that, in female flies, the activity of PI Ms neurons changes after mating to promote Ms release (Hadjieconomou, 2020).

Levels of the steroid hormone ecdysone, which promotes egg production and intestinal stem-cell proliferation, increase after mating. The ecdysone receptor (EcR) is expressed by all PI Ms neurons, which suggests that they might be sensitive to circulating ecdysone. Expression of a dominant-negative EcR-which targets all EcR isoforms-confined to the Ms neurons of adult flies was found to increase intracellular Ms levels in the Ms PI neuron cell bodies of mated females to the levels observed in virgin females, whereas it had no effect on virgin females. Downregulation of EcR (using RNA interference lines that target all isoforms or the B1 isoform specifically) produced comparable results. In both experiments, the amplitude of in vivo calcium oscillations in Ms neurons was increased to levels seen in virgin females. Compromising EcR signalling in adult Ms neurons significantly reduced crop enlargement preferentially in mated females; this phenotype was also apparent when the PI Ms neurons were targeted using Taotie-Gal4. Ecdysone therefore communicates mating status to Ms neurons through its B1 receptor (Hadjieconomou, 2020).

Previous work showed that the adult intestine is resized and metabolically remodelled after mating (Reiff, 2016), but did not investigate possible effects on its hormone-producing enteroendocrine cells. This study now observe a post-mating increase in the number of enteroendocrine cells, including a subset that expresses the hormone bursicon α (Burs), which is known to signal to adipose tissue through an unidentified neuronal relay. An endogenous protein reporter for the Burs receptor Rickets (Rk, also known as Lgr2) revealed its expression in subsets of neurons including all PI Ms neurons (including the Taotie-Gal4-positive subset) and in projections terminating in the HCG. Expression in a subset of the HCG Ms neurons was observed only sporadically (Hadjieconomou, 2020).

Consistent with the regulation of Ms neurons by the increase in Burs derived from enteroendocrine cells after mating, adult-specific downregulation of the Burs receptor gene rk in Ms neurons reverted intracellular Ms levels in the PI Ms neurons of mated females to levels observed in virgin females; there was no effect in virgin females. Like EcR downregulation, rk downregulation in Ms neurons also increased the amplitude of in vivo calcium oscillations in the Ms neuron cell bodies of mated females to values similar to those observed in virgin females. Functionally, both the downregulation of Burs in intestinal enteroendocrine cells and the adult-specific rk downregulation in Ms neurons-either in all neurons or in the Taotie-Gal4-positive subset in the PI-preferentially reduced crop enlargement in mated females. Conversely, stimulating the intestinal release of enteroendocrine hormones-including Burs-from enteroendocrine cells resulted in reduced Ms levels in the Ms neuron cell bodies of virgin females, similar to those observed in mated females, and greatly enlarged crops (Hadjieconomou, 2020).

Thus, steroid and enteroendocrine hormones communicate mating status to the brain. Acting through their receptors in the PI Ms neurons, these hormones change Ms neuronal activity, promoting the release of Ms after mating (Hadjieconomou, 2020).

To investigate the importance of Ms neuron modulation after mating, post-mating crop enlargement was selectively prevented by downregulating MsR1 in adult crop muscles using two independent strategies. This had no discernible effects in males or virgin females, but specifically prevented the increase in food intake that is normally observed in female flies after mating. Comparable results were obtained by blocking the post-mating ecdysone and Burs inputs into the Ms neurons. Downregulation of MsR2 had no such effect. The post-mating change in crop expandability, mediated by Ms and MsR1 signalling, thus causes the increased food intake observed in females after mating (Hadjieconomou, 2020).

The negative pressures that have been reported in the crops of larger insects suggest that the crop may draw food in by generating suction. The increased crop expandability enabled by Ms release after mating could therefore increase food intake through changes in suction. It was observed that mated females ingest more food per sip than virgin females, which is consistent with mated females needing to generate a higher suction pressure to facilitate bigger sips. Crop enlargement was therefore modeled using the Poiseuille equation for incompressible fluid flow in a pipe and found that the crop would need a suction pressure of the order of -1 kPa to achieve the previously reported intake volume per sip. This is in reasonable agreement with previously reported values measured in cockroach crops of between -0.5 and -1 kPa. The model predicts that mated flies would require a modest increase in suction pressure to -1.3kPa in order to facilitate the increased sip size (Hadjieconomou, 2020).

In the model, the change in crop volume drives food intake through increased suction. A crop that cannot enlarge, or a persistently enlarged crop, should therefore result in a comparable reduction in food intake by preventing the generation of suction. This was tested by persistently preventing crop enlargement (using crop-muscle-specific MsR1 knockdown) or by persistently inducing it (using TrpA1-mediated Ms neuron activation from Ms-Gal4 or Taotie-Gal4), after which the diet of these flies was switched from an undyed to a dye-laced food source to assess food intake. As predicted, both genetic manipulations reduced food intake. Conversely, increasing the rate at which the crop expands should increase food intake. This was tested by activating the Ms neurons as in the previous experiment, but this time the dye-laced food source was provided, and its intake was monitored at the same time as the neurons were activated (that is, as inducing greater crop expansion was being induced) rather than after a persistent activation (when the crop is already maximally expanded). Increased food intake was observed under these conditions in the absence of changes in the number of meals. Although further work will be required to elucidate the full dynamics of crop enlargement, filling and emptying, these experiments support the idea that the Ms-induced enlargement of the crop after mating increases food intake at least partly by increasing the suction power of the crop (Hadjieconomou, 2020).

Finally, given the links between nutrient intake and fecundity, it is proposed that the Ms-driven crop enlargement after mating might be adaptive and support reproduction. Crop enlargement was prevented selectively after mating by downregulating MsR1 from crop muscles, as in previous experiments. This resulted in reduced egg production, and the eggs that were produced had reduced viability. It is therefore conclude that the crop and its Ms innervation sustain the increase in food intake after mating, maximising female fecundity (Hadjieconomou, 2020).

These findings lead to a proposal that the maternal increase in food intake during reproduction is adaptive, that the crop is a key reproductive organ, and that Ms is a major effector of post-mating responses. In support of these ideas, the crop is absent in larvae-the juvenile stage of insects-and other Diptera have co-opted it for reproductive behaviours such as the regurgitation of nuptial gifts or the secretion of male pheromones. Ms receptors are also closely related to the Sex peptide receptor (the 'mating sensor' of female flies), and both diverged after duplication of an ancestral receptor that might have responded to the Myoinhibitory peptide (Mip) in the last common ancestor of protostomes. It will be interesting to explore possible links between Ms and Sex peptide signalling, and whether and how these mating signals affect recently described crop mechanosensing mechanisms that restrain ingestion as the crop expands in order to terminate large meals (Hadjieconomou, 2020).

This study has provided evidence for a gut-to-brain axis in Drosophila by identifying central Ms neurons as targets of the gut-derived hormone Burs. These central neurons innervate the gut, 'closing' a gut-brain-gut loop that connects midgut enteroendocrine signals to the crop, a more anterior gut region. This might allow for the functional coordination of different gut portions, while enabling central modulation by sensory cues (for example, gustatory). This study also identified the Ms neurons as the neural targets of ecdysone, which has been shown to promote food intake. Reproduction has pronounced, and in some cases lasting, effects on the human female brain; Ms neurons provide a tractable and physiologically relevant neural substrate for the investigation of the mechanisms involved (Hadjieconomou, 2020).

The human digestive system might be similarly modulated by reproductive cues to affect food intake. In mammals, enteric neurons express sex and reproductive-hormone receptors, and enteroendocrine hormone levels change during reproduction. It is suggested that pregnancy and lactation represent an attractive and relatively unexplored physiological adaptation for the investigation of nutrient intake regulation, organ remodelling and metabolic plasticity-mechanisms that might eventually be leveraged to curb appetite and/or weight gain (Hadjieconomou, 2020).

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes. This study used a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. These findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis (Redhai, 2020).

To investigate nutrient sensing in enterocytes, 111 putative nutrient sensors in D. melanogaster were selected on the basis of their intestinal expression and their predicted structure or function. Using two enterocyte-specific driver lines, their expression was downregulated in midgut enterocytes throughout development under two dietary conditions, nutrient-rich and nutrient-poor; it was reasoned that dysregulation of nutrient-sensing mechanisms may increase or reduce the normal period of larval growth, and might do so in a diet-dependent manner. Enterocyte-specific knockdown of the gene CG11340, also referred to as pHCl-22, resulted in developmental delay. This delay was exacerbated, and was accompanied by significantly reduced larval viability, under nutrient-poor conditions; these phenotypes were confirmed using a second RNAi transgene and a new CG11340 mutant. In the tradition of naming Drosophila genes according to their loss-of-function phenotype, CG11340 was named 'hodor', an acronym for 'hold on, don't rush', in reference to the developmental delay (Redhai, 2020).

A transcriptional reporter revealed that Hodor was expressed in the intestine. A new antibody revealed that Hodor expression was confined to enterocytes in two midgut portions that are known to store metals: the copper cell region and the iron cell region. Within the copper cell region, Hodor was expressed only in so-called interstitial cells. hodor-Gal4 was also present in the interstitial cells of the copper cell region; however, in the experimental conditions used in this study and in contrast to published results, it was not detected in the iron cell region. Apart from the intestine, Hodor was found only in principal cells of the excretory Malpighian tubules. To identify the cells from which Hodor controls systemic growth, region- or cell-type-specific downregulation and rescue experiments were conducted. Only fly lines in which hodor was downregulated in interstitial cells showed slowed larval development. This developmental delay persisted when hodor knockdown was induced post-embryonically during larval growth, and was rescued only in fly lines in which hodor expression was re-instated in cell types that included interstitial cells. The fat body (analogous to liver and adipose tissue) has long been known to couple nutrient availability with developmental rate; however, recent studies have revealed contributions from the intestine, particularly in nutrient-poor conditions. The current findings confirm a role for the intestine in coupling nutrient availability with larval growth, and further implicate a subpopulation of enterocytes-interstitial cells-as important mediators. Interstitial cells were described decades ago in blowfly, but had remained relatively uncharacterized since; their name refers only to their position, interspersed among the acid-secreting copper cells that control microbiota loads (Redhai, 2020).

This study established that the lethality of hodor mutation or knockdown was apparent only during the larval period. The development of hodor mutants was slower throughout larval life, and surviving mutants attained normal pupal and adult sizes. Consistent with previous findings, hodor mutation or knockdown was found to reduce luminal acidity in the copper cell region, suggesting a role specifically for interstitial cells in this process. hodor mutants also had increased gut bacterial titres, which is consistent with the observed functional defects in the copper cell region. Enlarged volumes of both the lumen of the copper cell region and the interstitial cells were also apparent after 1-3 days of (delayed) larval development; ultrastructurally, this was apparent in interstitial cells as a reduction in the complexity of their characteristic basal infoldings. This study was, however, able to rule out all of these defects as reasons for the developmental delay (Redhai, 2020).

During the course of these experiments, it was observed that hodor mutant larvae were more translucent than control larvae. This was suggestive of peripheral lipid depletion, which was confirmed by quantifying and staining for triacylglycerides. Reduced lipid stores did not result from disrupted enterocyte integrity: the intestinal barrier of mutants was intact, both anatomically and functionally. It was observed that hodor mutants had less food in their intestines and accumulated insulin-like peptide Ilp2 in their brains (nutrient-dependent Ilp2 secretion promotes larval development; its accumulation in the brain is commonly interpreted as peptide retention in the absence of transcriptional changes). Consistent with reduced systemic insulin signalling, hodor mutant larval extracts had reduced levels of phospho-Akt and phospho-S6 kinase. As these are all indicators of starvation, food intake was quantified, and it was observed to be reduced in both hodor mutant larvae and in hodor knockdowns targeting interstitial cells. Reduced food intake was apparent soon after hatching and persisted throughout larval development. Ectopic expression of Ilp2-which rescues developmental delay in larvae that lack insulin-like peptides-in hodor mutants partially rescued their developmental delay, but did not increase their food intake. An 'instructive' link between intestinal Hodor and food intake was further suggested by the overexpression of hodor in otherwise wild-type enterocytes; this resulted in larvae that ate more and developed at a normal rate, but had increased lipid stores. Therefore, Hodor controls larval growth from a subset of enterocytes by promoting food intake and systemic insulin signalling. In its absence, larvae fail to eat sufficiently to proceed through development at the normal rate and are leaner. When present in excess, Hodor causes larvae to eat more and accumulate the energy surplus as fat (Redhai, 2020).

In fly adipose tissue, amino acid availability activates Tor signalling to promote systemic growth. This study therefore combined hodor knockout or knockdown with genetic manipulations to alter Tor signalling. In flies with reduced or absent Hodor function, decreasing or increasing Tor signalling in hodor-expressing cells exacerbated or rescued the developmental delay, respectively. The reduced food intake of hodor mutants was also significantly rescued by activation of Tor signalling in hodor-expressing cells. Genetic targeting of Rag GTPases or the Gator1 complex in these cells failed to affect the developmental delay of hodor mutants, which could suggest non-canonical regulation of Tor signalling in Hodor-expressing cells. The systemic effects of Hodor on food intake and larval growth are therefore modulated by Tor signalling within Hodor-expressing interstitial cells (Redhai, 2020).

Hodor belongs to the (typically neuronal) Cys-loop subfamily of ligand-gated ion channels, and is predicted to be a neurotransmitter-gated anion channel. It is known to show activity in response to alkaline conditions in Xenopus oocytes, but the acidic pH of the copper cell region prompted a search for additional ligands. Although alkaline pH-induced Hodor activity was confirmed in oocyte expression systems, Hodor did not respond to typical Cys-loop receptor ligands such as neurotransmitters or amino acids. Instead, the screen identified zinc as an unanticipated ligand, which elicited a strong dose-dependent response only in Hodor-expressing oocytes; this response to zinc showed peak current amplitude values much greater than those observed in response to pH or to other metals such as iron or copper. Force-field-based structural stability and binding affinity calculations identified the amino acid pair E255 and E296 as a potential binding site for the divalent zinc ion. Mutating these residues did not abrogate the zinc-elicited currents, but did result in currents with faster rise time and deactivation kinetics, which supports the idea that zinc is a relevant Hodor ligand. On the basis of its sequence and conductance properties, Hodor has been proposed to transport chloride (Feingold, 2016; Remnant, 2016), and the zinc-elicited currents that were observed in oocytes had a reversal potential that is consistent with chloride selectivity. In vivo experiments in flies showed that supplementation of a low-yeast diet with zinc led to a reduction of chloride levels in interstitial cells, whereas hodor mutation increased chloride levels. Thus, Hodor is a pH-modulated, zinc-gated chloride channel (Redhai, 2020).

Attempts were made to establish the relevance of zinc binding in vivo. Zinc enrichment is observed in both the copper and iron cell regions of the larval gut, revealing an unrecognized role for these Hodor-expressing regions in zinc handling. Mutation of hodor failed to affect this zinc accumulation, although dietary yeast levels did, which is consistent with a role for Hodor in sensing rather than transporting zinc. (Notably, the white mutation-which is frequently used in the genetic background of Drosophila experiments-results in a small but significant reduction in both intestinal zinc accumulation and larval growth rate, although the status of the w gene neither exacerbated nor masked the more substantial, hodor-induced developmental delay. Furthermore, larvae that were fed a low-yeast diet ate significantly more when the diet was supplemented with zinc; this effect was abrogated in hodor mutants. In a food choice experiment, control larvae developed a preference for zinc-supplemented food over time, which suggests that the preference develops after ingestion. Consistent with this idea, zinc preference was specifically abrogated in hodor mutants (their general ability to discriminate between other diets was confirmed. Thus, zinc sensing by Hodor is physiologically relevant in vivo. Metals such as zinc are primarily provided by yeasts in nature; Hodor may be one of several sensors used to direct larvae to nutrient-rich food sources (Redhai, 2020).

The subcellular localization of Hodor suggests that it may normally maintain low cytoplasmic chloride concentrations by transporting it out of the interstitial cells and/or into their lysosomes. In accordance with this, and consistent with its putative lysosomal localization signals, Hodor was specifically enriched in apical compartments containing late endosome or lysosomal markers, as well as decorating the brush border of interstitial cells. The presence of Hodor in a subpopulation of lysosomes was of interest, because chloride transport across lysosomal membranes often sustains the activity of the proton-pumping vacuolar-type ATPase (V-ATPase) that maintains lysosomal acidity and Tor activation on the lysosome. To explore a role for Hodor in enabling Tor signalling, whether the absence of hodor induced autophagy-a hallmark of reduced Tor signalling, was tested. First, the induction of common autophagy markers in interstitial cells after genetic interference with the V-ATPase complex, which is known to promote autophagy by reducing lysosomal acidity and Tor signalling, was confirmed. Similar to reduced V-ATPase function, loss of hodor increased autophagy in interstitial cells. Expression of the dual autophagosome and autolysosome reporter UAS-GFP-mCherry-Atg8a in the intestinal cells of hodor mutants confirmed the induction of autophagy, and revealed two additional features. First, the acidification of autophagic compartments was defective in hodor mutants. Second, the increased autophagy and defective acidification observed in hodor mutants were particularly prominent in the two Hodor-expressing intestinal regions (the copper cell region and the iron cell region), consistent with cell-intrinsic roles for Hodor in these processes. Additional support for the roles of lysosomal function and Tor signalling in controlling whole-body growth from interstitial cells was provided by the finding that most V-ATPase subunits were transcriptionally enriched in the copper cell region. Functionally, the downregulation of V-ATPase subunits specifically in Hodor-expressing cells-and not in other subsets of enterocytes, such as those targeted by R2R4-Gal4-led to developmental delay and reduced food intake, phenotypes comparable to those observed as a result of hodor downregulation. Hence, although the directionality of zinc sensing and chloride transport in interstitial cells remains to be established, the data are consistent with roles for brush-border Hodor in transporting chloride out of interstitial cells-thus maintaining osmolarity and water balance. Lysosomal Hodor may transport chloride into the lysosome to sustain V-ATPase function, lysosomal acidification and TOR signalling, pointing to new links between lysosomal homeostasis in specialized intestinal cells, food intake and systemic growth. Nutrients such as amino acids are important regulators of Tor signalling. The genetic data are consistent with novel input from metals and/or micronutrients into Tor signalling. The nutrient-dependent zinc accumulation in lysosomal organelles-recently described in mammalian cells and nematode worms-suggests that links between zinc, lysosomes and Tor may be of broader importance. Two attractive cell types in which to explore such links are the Paneth cells of the mammalian intestine, which accumulate zinc and regulate intestinal immunity and stem cell homeostasis, and the 'lysosome-rich enterocytes' that have recently been described in fish and mice, which have roles in protein absorption (Redhai, 2020).

An extensive reconstruction of the hodor family tree supported the presence of a single member of the family in the ancestor of insects. Because Hodor-like proteins are present only in insects, they may prove to be highly specific targets for the chemical control of disease vectors, particularly given that mosquito genomes contain a single gene rather than the three paralogues that are found in most flies. To test this idea, CRISPR-Cas9 genome editing was used to generate a mutant that lacks the single hodor-like gene (AGAP009616) in the malaria vector Anopheles gambiae. This gene is also expressed in the digestive tract-specifically in the midgut-and in Malphighian tubules. Three independent deletion alleles revealed that AGAP009616 function is essential for the viability of A. gambiae. A target that is expressed in the intestine, such as Hodor, is particularly attractive for vector control as it may circumvent accessibility issues and could be directly targeted using ingestible drugs such as those applied to larval breeding sites (Redhai, 2020).

Metals have received little attention in the contexts of development or whole-body physiology, and are often regarded as passive 'building blocks'. By revealing the roles of a metal sensor in food intake and growth control, these findings highlight the importance of investigating the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis. These mechanisms could prove to be useful in insect vector control (Redhai, 2020).

Feeding decisions are fundamental to survival, and decision making is often disrupted in disease. This study shows that neural activity in a small population of neurons projecting to the fan-shaped body higher-order central brain region of Drosophila represents food choice during sensory conflict. Food deprived flies made tradeoffs between appetitive and aversive values of food. An upstream neuropeptidergic and dopaminergic network was identified that relays internal state and other decision-relevant information to a specific subset of fan-shaped body neurons. These neurons were strongly inhibited by the taste of the rejected food choice, suggesting that they encode behavioral food choice. These findings reveal that fan-shaped body taste responses to food choices are determined not only by taste quality, but also by previous experience (including choice outcome) and hunger state, which are integrated in the fan-shaped body to encode the decision before relay to downstream motor circuits for behavioral implementation (Sareen, 2021).

Feeding is essential for animal survival and reproduction and is regulated by both internal states and external stimuli. However, little is known about how internal states influence the perception of external sensory cues that regulate feeding behavior. This study investigated the neuronal and molecular mechanisms behind nutritional state-mediated regulation of gustatory perception in control of feeding behavior in the brown planthopper and Drosophila. Feeding was found to increase the expression of the cholecystokinin-like peptide, sulfakinin (SK), and the activity of a set of SK-expressing neurons. Starvation elevates the transcription of the sugar receptor Gr64f and SK negatively regulates the expression of Gr64f in both insects. Interestingly, it was found that one of the two known SK receptors, CCKLR-17D3, is expressed by some of Gr64f-expressing neurons in the proboscis and proleg tarsi. Thus, this study has identified SK as a neuropeptide signal in a neuronal circuitry that responds to food intake, and regulates feeding behavior by diminishing gustatory receptor gene expression and activity of sweet sensing GRNs. The findings demonstrate one nutritional state-dependent pathway that modulates sweet perception and thereby feeding behavior, but the experiments cannot exclude further parallel pathways. Importantly, it was shown that the underlying mechanisms are conserved in the two distantly related insect species (Guo, 2021).

Animals must express the appropriate behavior that meets their most pressing physiological needs and their environmental context. However, it is currently unclear how alternative behavioral options are evaluated and appropriate actions are prioritized. This study describes how fruit flies choose between feeding and courtship; two behaviors necessary for survival and reproduction. Sex- and food-deprived male flies were shown to prioritize feeding over courtship initiation, and manipulation of food quality or the animal's internal state fine-tunes this decision. The tyramine signaling pathway was identified as an essential mediator of this decision. Tyramine biosynthesis is regulated by the fly's nutritional state and acts as a satiety signal, favoring courtship over feeding. Tyramine inhibits a subset of feeding-promoting tyramine receptor (TyrR)-expressing neurons and activates P1 neurons, a known command center for courtship. Conversely, the perception of a nutritious food source activates TyrR neurons and inhibits P1 neurons. Therefore, TyrR and P1 neurons are oppositely modulated by starvation, via tyramine levels, and food availability. It is proposed that antagonistic co-regulation of neurons controlling alternative actions is key to prioritizing competing drives in a context- dependent manner (Cheriyamkunnel, 2021).

There is little information on how macronutrient composition and bacterial communities in natural food sources vary across seasons in nature and on how these factors affect the fitness components of insects. In this study, diet samples from an orchard compost heap, which is a natural habitat for many Drosophila species and other arthropods, were collected over 9 months covering all seasons in a temperate climate. It developed D. melanogaster on diet samples and investigated stress resistance and life-history traits as well as the microbial community of flies and compost. Nutrient and microbial community analysis of the diet samples showed marked differences in macronutrient composition and microbial community across seasons. However, except for the duration of development on these diet samples and Critical Thermal maximum, fly stress resistance and life-history traits were unaffected. The resulting differences in the fly microbial community were also more stable and less diverse than the microbial community of the diet samples. This study suggests that when D. melanogaster are exposed to a vastly varying nutritional environment with a rich, diverse microbial community, the detrimental consequences of an unfavourable macronutrient composition are offset by the complex interactions between microbes and nutrients (Davis, 2021).

Body size is a key life-history trait that influences many aspects of an animal's biology and is shaped by a variety of factors, both genetic and environmental. While locally-adapted populations differ in the extent to which body size responds plastically to environmental conditions like diet, there is a limited understanding of what causes these differences. It was hypothesized that populations could differ in the way body size responds to nutrition either by modulating growth rate, development time, feeding rate, or a combination of the above. Using three locally-adapted populations of Drosophila melanogaster from along the east coast of Australia, body size plasticity was investigated across five different diets. How these populations differed in feeding behaviour and developmental timing on each of the diets was assessed. Population-specific plastic responses to nutrition was observed for body size and feeding rate, but not development time. However, differences in feeding rate did not fully explain the differences in the way body size responded to diet. Thus, it is concluded that body size variation in locally-adapted populations is shaped by a combination of growth rate and feeding behaviour. This paves the way for further studies that explore how differences in the regulation of the genetic pathways that control feeding behaviour and growth rate contribute to population-specific responses of body size to diet (Chakraborty, 2021).

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).

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).

The decline of Drosophila climbing behavior is one of the common phenomena of Drosophila aging. The so-called negative geotaxis refers to the natural upward climbing behavior of Drosophila melanogaster after it oscillates to the bottom of the test tube. The strength of climbing ability is regarded as the index of aging change of D. melanogaster. At present, many laboratories use the percentage of 10 fruit flies climbing a specific height in 5 s as a general indicator of the climbing ability of fruit flies. This group research index ignores the climbing performance of a single fruit fly, and the climbing height belongs to the concept of vertical distance in physics, which cannot truly and effectively reflect the concept of curve distance in the actual climbing process of fruit flies. Therefore, based on the image processing algorithm, an experimental method was added to draw the climbing trajectory of a single fruit fly. By comparing the differences in climbing behavior of fruit flies under different sex, group or single, oscillation condition or rotation inversion condition, it was possible to find that the K-Nearest Neighbor target detection algorithm has good applicability in fruit fly climbing experiment, and the climbing ability of fruit flies decreases with age. Under the same experimental conditions, the climbing ability of female fruit flies was greater than that of male fruit flies. The climbing track length of a single fruit fly can better reflect the climbing process of a fruit fly (Zhong, 2022).

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).

Animals integrate information from different sensory modalities, body parts, and time points to inform behavioral choice, but the relevant sensory comparisons and the underlying neural circuits are still largely unknown. This study used the grooming behavior of Drosophila melanogaster as a model to investigate the sensory comparisons that govern a motor sequence. Flies perform grooming movements spontaneously, but when covered with dust, they clean their bodies following an anterior-to-posterior sequence. After investigating different sensory modalities that could detect dust, focus was placed on mechanosensory bristle neurons, whose optogenetic activation induces a similar sequence. Computational modeling predicts that higher sensory input strength to the head will cause anterior grooming to occur first. This prediction was tested using an optogenetic competition assay whereby two targeted light beams independently activate mechanosensory bristle neurons on different body parts. It was found that the initial choice of grooming movement is determined by the ratio of sensory inputs to different body parts. In dust-covered flies, sensory inputs change as a result of successful cleaning movements. Simulations from this model suggest that this change results in sequence progression. One possibility is that flies perform frequent comparisons between anterior and posterior sensory inputs, and the changing ratios drive different behavior choices. Alternatively, flies may track the temporal change in sensory input to a given body part to measure cleaning effectiveness. The first hypothesis is supported by the optogenetic competition experiments: iterative spatial comparisons of sensory inputs between body parts is essential for organizing grooming movements in sequence (Zhang, 2020).

Spontaneous grooming behavior is a component of insect fitness. We quantified spontaneous grooming behavior in 201 sequenced lines of the Drosophila melanogaster Genetic Reference Panel and observed significant genetic variation in spontaneous grooming, with broad-sense heritabilities of 0.25 and 0.24 in females and males, respectively. Although grooming behavior is highly correlated between males and females, significant sex by genotype interactions were observed, indicating that the genetic basis of spontaneous grooming is partially distinct in the two sexes. Genome-wide association analyses of grooming behavior was performed, and 107 molecular polymorphisms associated with spontaneous grooming behavior were mapped, of which 73 were in or near 70 genes and 34 were over 1 kilobase from the nearest gene. The candidate genes were associated with a wide variety of gene ontology terms, and several of the candidate genes were significantly enriched in a genetic interaction network. Functional assessments were performed of 29 candidate genes using RNA interference, and 11 were found to affecte spontaneous grooming behavior. The genes associated with natural variation in Drosophila grooming are involved with glutamate metabolism (Gdh) and transport (Eaat); interact genetically with (CCKLR-17D1) or are in the same gene family as (PGRP-LA) genes previously implicated in grooming behavior; are involved in the development of the nervous system and other tissues; or regulate the Notch and Epidermal growth factor receptor signaling pathways. Several DGRP lines exhibited extreme grooming behavior. Excessive grooming behavior can serve as a model for repetitive behaviors diagnostic of several human neuropsychiatric diseases (Yanagawa, 2020).

Boys with fragile X syndrome (FXS), a leading monogenic cause of intellectual disability, often display repetitive behaviors, a core feature of autism. This study characterized spontaneous-motor-behavior phenotypes of Drosophila dfmr1 mutants, an established model for FXS. Individual 1-day-old adult flies, with mature nervous systems, were recorded in small arenas. Young dfmr1 mutants spent excessive time grooming, with increased bout number and duration; both were rescued by transgenic wild-type dfmr1(+). By two grooming-pattern measures, dfmr1-mutant flies showed elevated repetitions consistent with perseveration, which is common in FXS. In addition, the mutant flies display a preference for grooming posterior body structures, and an increased rate of grooming transitions from one site to another. The possibility was raised that courtship and circadian rhythm defects, previously reported for dfmr1 mutants, are complicated by excessive grooming. Significantly increased grooming was also observed in CASK mutants, despite their dramatically decreased walking phenotype. The mutant flies, a model for human CASK-related neurodevelopmental disorders, displayed consistently elevated grooming indices throughout the assay, but transient locomotory activation immediately after placement in the arena. Based on published data identifying FMRP-target transcripts and functional analyses of mutations causing human genetic neurodevelopmental disorders, the following proteins are proposed as candidate mediators of excessive repetitive behaviors in FXS: CaMKIIα, NMDA receptor subunits 2A and 2B, NLGN3, and SHANK3. Together, these fly-mutant phenotypes and mechanistic insights provide starting points for drug discovery to identify compounds that reduce dysfunctional repetitive behaviors (Andrew, 2020).

Central pattern generators (CPGs) are neurons or neural circuits that produce periodic output without requiring patterned input. More complex behaviors can be assembled from simpler subroutines, and nested CPGs have been proposed to coordinate their repetitive elements, organizing control over different time scales. This study used behavioral experiments to establish that Drosophila grooming may be controlled by nested CPGs. On a short time scale (5-7 Hz, ~ 200 ms/movement), flies clean with periodic leg sweeps and rubs. More surprisingly, transitions between bouts of head sweeping and leg rubbing are also periodic on a longer time scale (0.3-0.6 Hz, ~2 s/bout). This study examined grooming at a range of temperatures to show that the frequencies of both oscillations increase-a hallmark of CPG control-and also that rhythms at the two time scales increase at the same rate, indicating that the nested CPGs may be linked. This relationship holds when sensory drive is held constant using optogenetic activation, but oscillations can decouple in spontaneously grooming flies, showing that alternative control modes are possible. Loss of sensory feedback does not disrupt periodicity but slow down the longer time scale alternation. Nested CPGs simplify the generation of complex but repetitive behaviors, and identifying them in Drosophila grooming presents an opportunity to map the neural circuits that constitute them (Ravbar, 2021).

These behavior and anatomy data provide strong evidence that wPN1 plays an essential, command-like role in coordinating left and right back legs, but higher-resolution behavioral analysis and complete neuronal circuit mapping downstream of wPN1 are necessary to confirm this function (Zhang, 2022).

Behavioral differences can be observed between species or populations (variation) or between individuals in a genetically similar population (variability). This study investigated genetic differences as a possible source of variation and variability in Drosophila grooming. Grooming confers survival and social benefits. Grooming features of five Drosophila species exposed to a dust irritant were analyzed. Aspects of grooming behavior, such as anterior to posterior progression, were conserved between and within species. However, significant differences in activity levels, proportion of time spent in different cleaning movements, and grooming syntax were identified between species. All species tested showed individual variability in the order and duration of action sequences. Genetic diversity was not found to correlate with grooming variability within a species: melanogaster flies bred to increase or decrease genetic heterogeneity exhibited similar variability in grooming syntax. Individual flies observed on consecutive days also showed grooming sequence variability. Standardization of sensory input using optogenetics reduced but did not eliminate this variability. In aggregate, these data suggest that sequence variability may be a conserved feature of grooming behavior itself. These results also demonstrate that large genetic differences result in distinguishable grooming phenotypes (variation), but that genetic heterogeneity within a population does not necessarily correspond to an increase in the range of grooming behavior (variability) (Mueller, 2021).

Mechanosensory neurons located across the body surface respond to tactile stimuli and elicit diverse behavioral responses, from relatively simple stimulus location-aimed movements to complex movement sequences. How mechanosensory neurons and their postsynaptic circuits influence such diverse behaviors remains unclear. Previous work has shown that Drosophila perform a body location-prioritized grooming sequence when mechanosensory neurons at different locations on the head and body are simultaneously stimulated by dust. This study identified nearly all mechanosensory neurons on the Drosophila head that individually elicit aimed grooming of specific head locations, while collectively eliciting a whole head grooming sequence. Different tracing methods were used to reconstruct the projections of these neurons from different locations on the head to their distinct arborizations in the brain. This provides the first synaptic resolution somatotopic map of a head, and defines the parallel-projecting mechanosensory pathways that elicit head grooming (Eichler, 2023).

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

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).

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).

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

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

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

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

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

Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, this study uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. Acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. This work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses (Imambocus, 2021).

When threatened by dangerous or harmful stimuli, animals engage in diverse forms of rapid escape behavior. In Drosophila larvae, escape behavior is characterized by C-shaped bending and lateral rolling, followed by rapid forward crawling. The sensory circuitry that promotes escape has been extensively characterized, but the motor programs underlying escape are unknown. This study characterized the neuromuscular basis of escape. High-speed, volumetric, Swept Confocally-Aligned Planar Excitation (SCAPE) microscopy was used to image muscle activity during larval rolling. Unlike the sequential peristaltic muscle contractions from segment to segment that underlie forward and backward crawling, muscle activity progresses in a circumferential sequence during bending and rolling. Certain muscle subgroups show functional antagonism during bending and rolling. EM connectome data was used to identify premotor to motor connectivity patterns that could drive rolling behavior, and the necessity of specific groups of motor neurons in rolling using neural silencing approaches was tested. The data reveal the body-wide muscle activity patterns and putative premotor circuit organization for escape (Cooney, 2023).

Appropriate modulation of escape behaviors in response to potentially damaging stimuli is essential for survival. Although nociceptive circuitry has been studied, it is poorly understood how genetic contexts affect relevant escape responses. Using an unbiased genome-wide association analysis, this study identified an Ly6/α-neurotoxin family protein, Belly roll (Bero), which negatively regulates Drosophila nociceptive escape behavior. Bero is expressed in abdominal leucokinin-producing neurons (ABLK neurons) and bero knockdown in ABLK neurons resulted in enhanced escape behavior. Furthermore, it was demonstrated that ABLK neurons responded to activation of nociceptors and initiated the behavior. Notably, bero knockdown reduced persistent neuronal activity and increased evoked nociceptive responses in ABLK neurons. These findings reveal that Bero modulates an escape response by regulating distinct neuronal activities in ABLK neurons (Li, 2023).

Acute avoidance of dangerous temperatures is critical for animals to prevent or minimize injury. Therefore, surface receptors have evolved to endow neurons with the capacity to detect noxious heat so that animals can initiate escape behaviors. Animals including humans have evolved intrinsic pain-suppressing systems to attenuate nociception under some circumstances. Using Drosophila melanogaster, this study uncovered a new mechanism through which thermal nociception is suppressed. A single descending neuron was identified in each brain hemisphere, which is the center for suppression of thermal nociception. These Epi neurons, for Epione-the goddess of soothing of pain-express a nociception-suppressing neuropeptide Allatostatin C (AstC), which is related to a mammalian anti-nociceptive peptide, somatostatin. Epi neurons are direct sensors for noxious heat, and when activated they release AstC, which diminishes nociception. Epi neurons also express the heat-activated TRP channel, Painless (Pain), and thermal activation of Epi neurons and the subsequent suppression of thermal nociception depend on Pain. Thus, while TRP channels are well known to sense noxious temperatures to promote avoidance behavior, this work reveals the first role for a TRP channel for detecting noxious temperatures for the purpose of suppressing rather than enhancing nociception behavior in response to hot thermal stimuli (Liu, 2023).

Endogenous pain inhibitory systems can temporarily provide relief. Millions of people suffer from chronic and debilitating pain, some of which might be induced by abnormalities in the descending pain modulatory system. In mammals, neurotransmitters and neuromodulators, including endogenous opioids (β-endorphin, encephalin, and dynorphin) and endogenous cannabinoids, play important roles in nociception inhibition. Brain imaging and electrophysiological studies indicate that the pain-suppressing descending modulatory circuit receives input from multiple brain regions including the rostral anterior cingulate cortex, the periaqueductal gray region, and the rostral ventromedial medulla. However, the key neurons that are activated in the inhibitory pathway, and the target neurons that are silenced, have not been clearly delineated (Liu, 2023).

A pain inhibitory system has also been documented in worms. In C. elegans avoidance responses that are mediated through the polymodal ASH neurons are suppressed by complex signaling pathways initiated by octopamine and neuropeptides. Drosophila has also been employed to study the inhibition of nociception in addition to the far more extensive studies focusing on the mechanisms for detecting noxious stimuli, such as excessive heat, has been shown to to initiate escape responses. A Drosophila channel, Painless (Pain), which is related to the TRP channel in the fly's compound eye, is critical for sensing noxious heat. This work, which followed the seminal discovery of TRPV1 as a heat sensor in mammals and the finding that a related TRPV channel (Osm-9) contributes to several other sensory modalities in C. elegans, contributed significantly to the notion that TRP channels are evolutionarily conserved polymodal sensors (Liu, 2023).

In addition to Pain, two other Drosophila TRP channels also function in sensing high temperatures to promote escape behavior: Pyrexia (Pyx) and TRPA1. However, it is unclear whether any TRP channel serves to detect noxious heat for the purpose of alleviating thermally induced nociception (Liu, 2023).

This work used the fruit fly, Drosophila melanogaster, to investigate an intrinsic system for suppression of thermal nociception. A pair of bilaterally symmetrical neurons in the brain was identified that is required for decreasing the nociceptive response to hot temperatures. These Epi neurons respond directly to heat and release a neuropeptide, Allatostatin C (AstC), which is required for suppression of nociception. The ability of Epi neurons to sense noxious heat depends on Pain, demonstrating a role for a thermo-TRP in suppressing rather than enhancing the nociceptive response to high temperatures (Liu, 2023).

This study found that a single pair of bilaterally symmetrical Epi neurons in the fly brain is critical for suppressing thermal nociception. The importance of Epi neurons is underscored by the observation that artificial activation of these neurons is sufficient to suppress the aversive jump response to hot temperatures and that inhibition of signaling from these neurons increases the jump responses to moderate heat. The profound effect of a single pair of neurons in reducing thermal nociception is surprising given that multiple brain regions appear to function in pain suppression in mammals (Liu, 2023).

The dendrites of Epi neurons arborize to multiple regions of the brain, such as the optic lobes (OLs), the lateral horn (LH), and a region near the mushroom bodies, indicating that Epi neurons receive multiple signal inputs. The LH is a higher-order processing center that receives input from the antennal (olfactory) lobes and then sends relays to other brain regions such as the mushroom bodies. Therefore, it is intriguing to speculate that the Epi neurons may be activated by noxious odorants and aversive visual cues, which attenuate the avoidance behavioral responses to these stimuli. Epi neurons might also receive input from attractive olfactory and visual cues, which in turn diminish the escape responses to noxious stimuli such as high temperatures. In addition, the axons of Epi neurons project to the VNC, consistent with a role in descending control of motor output (Liu, 2023).

A key question is the mechanism through which Epi neurons respond to hot temperatures and alleviate thermal nociception. Epi neurons were found to be directly activated by hot temperatures and do so through activation of the thermo-TRP channel, Pain, which is expressed in Epi neurons. The Pain channel is critical for suppressing nociception since mutation of the pain gene causes an increase in thermal pain sensitivity (hyperalgesia). While Epi neurons respond directly to heat and are anti-nociceptors, other neurons in the fly brain, the so-called anterior cell neurons, respond directly to suboptimal warm temperatures. In contrast to the anti-nociceptive Epi neurons, the AC neurons function in thermal avoidance, which is mediated through thermal activation of TRPA1 (Liu, 2023).

The next question is the mechanism through which activation of Epi neurons suppresses thermal pain. Epi neurons express a neuropeptide, AstC, which binds to receptors that have sequence homology (39.0% identity for AstC-R1; 38.5% identity for AstC-R2) to human opioid receptors, which function in the suppression of nociception in mammals. Moreover, mutation of AstC or knockdown of AstC in Epi neurons causes thermal hyperalgesia, and mutation of AstC-R1 elicits a similar phenotype. Heat stimulation diminishes the level of AstC in Epi neurons, indicating that activation of these neurons promotes release of AstC. It is concluded that Epi neurons alleviate thermal nociception through a mechanism that depends on heat sensing by the Pain channel, leading to release of AstC (Liu, 2023).

Surprisingly, mutation of pain also reduced expression of AstC in Epi neurons below the level of detection. This effect was not due to elimination of Epi neurons since pain mutant brains express UAS-GCaMP6f under control of the Epi-Gal4. Expression of neuropeptides has been linked to neuronal activity. Moreover, there is an example in which a thermosensory TRPV channel affects expression of a neuropeptide receptor. Pain is activated by thermal heat, with the most pronounced activation in the noxious heat range. However, even at temperatures significantly below the flex point in which a given temperature rapidly opens the gate of a thermosensory TRP, such as Pain, there is some channel activity. It is suggested that low levels of Pain and Epi neuron activities are necessary for expression of AstC, while high levels of activities that are induced by noxious heat are required for release of the AstC (Liu, 2023).

A feature of activation of Epi neurons is that the pain suppression due to an acute 30-second activation of Epi neurons is sustained for several minutes. It is suggested that the slow termination of the pain suppression following stimulation of these neurons is mediated by release of the neuromodulator AstC, which persists for several minutes. Epi neurons appear to be non-adapting, as chronic activation of these neurons with the NaChBac channel leads to similar levels of pain suppression as acute stimulation with channelrhodopsin. This non-adapting feature of Epi neurons may be beneficial because it allows for pain suppression under conditions in which the aversive response to heat needs to be suppressed sufficiently long enough to allow activities that promote survival. Given that fruit flies are poikilothermic, and their body temperature equilibrates with the environment, direct activation of Epi neurons would allow the flies to suppress nociception and enter excessively warm environments to feed or avoid predators (Liu, 2023).

In conclusion, this study unveils a molecular and cellular basis for pain suppression in Drosophila. The observation that Pain is essential for suppressing nociception is surprising given that all other thermal-TRP channels function in avoidance of suboptimal or noxious temperatures. Mutation of pain in fly larvae eliminates the sensitivity to hot temperatures (hypoalgesia). Thus, it is remarkable that the same TRP channel has opposite functions in nociception and anti-nociception in larvae and adults (Liu, 2023).

Evolution has generated an enormous variety of morphological, physiological, and behavioral traits in animals. How do behaviors evolve in different directions in species equipped with similar neurons and molecular components? This study adopted a comparative approach to investigate the similarities and differences of escape behaviors in response to noxious stimuli and their underlying neural circuits between closely related drosophilid species. Drosophilids show a wide range of escape behaviors in response to noxious cues, including escape crawling, stopping, head casting, and rolling. This study found that D. santomea, compared with its close relative D. melanogaster, shows a higher probability of rolling in response to noxious stimulation. To assess whether this behavioral difference could be attributed to differences in neural circuitry, focused ion beam-scanning electron microscope volumes of the ventral nerve cord of D. santomea were generated to reconstruct the downstream partners of mdIV, a nociceptive sensory neuron in D. melanogaster. Along with partner interneurons of mdVI (including Basin-2, a multisensory integration neuron necessary for rolling) previously identified in D. melanogaster, two additional partners of mdVI were identified in D. santomea. Finally, this study showed that joint activation of one of the partners (Basin-1) and a common partner (Basin-2) in D. melanogaster increased rolling probability, suggesting that the high rolling probability in D. santomea is mediated by the additional activation of Basin-1 by mdIV. These results provide a plausible mechanistic explanation for how closely related species exhibit quantitative differences in the likelihood of expressing the same behavior (Zhu, 2023).

Animals rapidly acquire surrounding information to perform the appropriate behavior. Although social learning is more efficient and accessible than self-learning for animals, the detailed regulatory mechanism of social learning remains unknown, mainly because of the complicated information transfer between animals, especially for aversive conditioning information transmission. The current study revealed that, during social learning, the neural circuit in observer flies used to process acquired aversive conditioning information from demonstrator flies differs from the circuit used for self-learned classic aversive conditioning. This aversive information transfer is species dependent. Solitary flies cannot learn this information through social learning, suggesting that this ability is not an innate behavior. Neurons used to process and execute avoidance behavior to escape from electrically shocked flies are all in the same brain region, indicating that the fly brain has a common center for integrating external stimuli with internal states to generate flight behavior (Wu, 2023).

Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking. Although dysfunction of the dopaminergic valuation system of the brain is known to contribute towards unconstrained reward seeking, the underlying reasons for this behaviour are unclear. This study describes dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. It is proposed that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals (Jovanoski, 2023).

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

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

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

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

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

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

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

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

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

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

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

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).

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

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

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).

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

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

Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. This study shows that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. These findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies (Cai, 2021).

Olfactory perception influences nutrition and promotes physiological and mental well-being. In flies, a dedicated olfactory circuit elicits avoidance behaviours towards geosmin-a volatile compound that is released by mould and some bacteria. Olfactory receptors also mediate an initial attraction to food that contains certain enteropathogens. After infection with these pathogens, however, an avoidance behaviour is triggered by immune receptors in the brain, gustatory bitter neurons, and the neuropeptide leukokinin. Whether changes in olfactory perception contribute to this behavioural switch from attraction to avoidance remains unclear (Cai, 2021).

In Drosophila, odorants are sensed by olfactory receptor neurons in the head, the antenna and the maxillary palp. Olfactory receptor neurons synapse into projection neurons at the antennal lobe (AL), where the signal is converted into a spatiotemporal code in 50 glomerular compartments. Projection neurons axons project to higher olfactory centres to instruct innate and learned behaviour. In this system, glia and neurons operate as a tightly coupled unit to maintain olfactory sensitivity (Cai, 2021).

In ageing flies, olfactory perception of both aversive and attractive odours declines, but the mechanism(s) of this decline remain unclear. Olfactory perception and other neurological processes also decline in ageing mammals, often influenced by gastrointestinal signals (Cai, 2021).

This study investigated the communication between the gut and the brain, and how it influences infection-induced avoidance behaviour, infection tolerance, and olfactory decline during ageing (Cai, 2021).

A modified capillary feeder (CAFE) assay was used to measure choice between food that did or did not contain Erwinia carotovora carotovora 15 (Ecc15), a non-lethal enteropathogen that causes intestinal inflammation. Consistent with recent reports, naive flies consumed more Ecc15-containing food than normal food. However, when orally infected with Ecc15 for 24 h before the feeding assay, flies developed a distinct aversion to food that contained Ecc15. To assess whether this involved changes in olfactory perception, the 'preference index' for attractive (such as putrescine) or aversive (such as 3-octanol) odours was determined in T-maze assays. Preference or aversion for attractive or aversive odours, respectively, declined after infection, which indicates that infection causes a non-selective decline in olfactory discrimination. This was transient, as olfactory discrimination recovered 5 days after infection, coincident with the clearance of bacteria and epithelial regeneration in the intestine. Olfactory discrimination was not influenced by starvation or exposure to heat-killed Ecc15. Consistent with their reported role in sensing pathogenic bacteria, the CO2 receptor Gr63a or the odorant receptor co-receptor Orco was required for the attraction to Ecc15 food: Orco1 and Gr63a1 mutants ingested less Ecc15 food under naive conditions, and when infected, failed to further reduce ingestion of Ecc15-containing food. Together, these observations suggest that after an initial odorant-mediated attraction, flies develop aversion to enteropathogens, through a concerted activation of gustatory and immune receptors and suppression of olfaction (Cai, 2021).

After oral infection with Ecc15, damaged intestinal enterocytes produce the inflammatory IL-6-like cytokines Unpaired 2 and 3 (Upd2 and Upd3) to stimulate intestinal stem-cell proliferation and epithelial regeneration. Proteins of the Upd family activate the JAK-STAT signalling pathway through the receptor Domeless (Dome) and the JAK homologue Hopscotch (Hop). Using the 2xSTAT::GFP reporter for JAK-STAT pathway activity, this study found upregulated GFP expression in the brain 4 h after oral Ecc15 infection, as well as after oral infection with the more lethal enteropathogen Pseudomonas entomophila (PE) that damages the gut epithelium. JAK-STAT activity was observed in a sparse population of cells of the brain that stained positive for the glial marker Repo. Subtype-specific Gal4 drivers revealed that among the five subtypes of Drosophila glia (astrocytes, ensheathing, perineural, subperineural and cortex glia), ensheathing glia (EG) were the main population that upregulate STAT activity in response to Ecc15 infection. This was confirmed using four different Gal4 drivers to label EG and by flow cytometry. Infection did not influence numbers and membranous processes (labelled using UAS::mCD4GFP) of EG at the AL, and glomerular compartmentalization in the AL and lobe size remained unaffected. JAK-STAT activation in EG was sufficient and required for infection-induced changes in olfactory discrimination, as overexpression of constitutively active Hop (hop^tuml) in EG reduced olfactory discrimination, whereas loss of Dome or STAT in all glia (repo::Gal4), or specifically in EG (GMR56F03::Gal4), rescued the decline of olfactory discrimination caused by Ecc15 infection. Overexpression of hop^tuml in EG also reduced ingestion of Ecc15-containing food and promoted survival of flies fed PE-containing food, whereas knockdown of Dome or STAT in EG increased ingestion of Ecc15-containing food in infected flies and increased mortality on PE-containing food. It is proposed that the corresponding changes in ingestion of PE-laced food contribute to reduced mortality, but it is possible that additional genetic background conditions influence mortality, as seen, for example, in Orco-mutant flies, which ingest less bacteria but show increased susceptibility to PE (Cai, 2021).

To test whether gut-derived Upd proteins directly contribute to the infection-induced activation of JAK-STAT signalling in EGs, intestinal enterocyte-specific perturbations were performed using Mex1::Gal4, an enterocyte driver with no expression in the brain. Indeed, JAK-STAT activation in glia at the AL could be triggered in naive flies or prevented in infected flies by overexpression or knockdown, respectively, of Upd2 and Upd3 in enterocytes. Consistently, enterocyte-derived Upd2 and Upd3 were sufficient and required for the modulation of olfactory discrimination caused by infection. Knockdown of Upd2 or Upd3 did not affect olfaction in naive flies, and perturbing these ligands in fatbody (cg::Gal4) or haemocytes (hml::Gal4), tissues that are sources for Upd proteins in other contexts, did not significantly affect STAT activity in glia at the AL (Cai, 2021).

Loss of olfactory sensitivity is an early sign of normal ageing and neurodegeneration. In ageing Drosophila, olfactory perception has been reported to deteriorate before vision, a decline that was possible to recapitulate in T-maze assays. Glomerular compartments in the AL became less organized and less distinct in geriatric (60-70-day-old) flies and AL size increased with age. This correlates with a reduction in the number of EG and of glial membranous processes, changes that are expected to affect AL structure, and thus probably contribute to the age-related decline in olfaction (Cai, 2021).

Ageing in Drosophila is accompanied by the development of intestinal inflammation, and is associated with the constitutive expression and release of Upd cytokines. Consistently, JAK-STAT activity in the AL of EG was increased in old flies, and knockdown of Dome or STAT by RNA interference (RNAi) in EG specifically or in all glia rescued the decline of olfactory discrimination in old flies. The loss of Dome in EG also rescued the age-related decline of EGs and restored the size of the AL. JAK-STAT activation in the EG of old flies is a consequence of intestinal Upd release, as knocking down Upd2 and Upd3 in enterocytes alleviated STAT activation in the AL, and prevented the age-related decline of olfactory discrimination (Cai, 2021).

This age-related decline of olfactory discrimination was independent of the microbiota, as germ-free old flies still exhibited reduced olfaction sensitivity, increased JAK-STAT signalling in the AL, decreased numbers of EG, loss of glial cellular processes, and an enlarged AL. These results are consistent with the observation that the age-related increase in Upd released from the gut is also independent of the microbiota (Cai, 2021).

To understand why EG but not other glia selectively respond to Upd ligands and activate JAK-STAT signalling during ageing or infection, single-cell RNA sequencing (scRNA-seq) was performed on purified glia from young and old flies. Either all glia (labelled using repo::Gal4) or EG selectively (labelled using GMR56F03::Gal4) were profiled using Smart-seq2. The expression of dome was significantly higher in EG than in other glia, consistent with the specific upregulation of socs36E, a known target of JAK-STAT signalling, in EG but not in other glia during ageing. These results are supported by a similar upregulation of Socs36E in EG of old flies observed in a previous scRNA-seq dataset (Cai, 2021).

Bulk RNA sequencing analysis on glia (repo::Gal4, UAS::tdTomato) purified from central brains of flies expressing a 10xSTAT::GFP reporter revealed that the transcriptomes of STAT::GFP⁺ glia from Ecc15 infected and uninfected flies were more similar to each other than to STAT::GFP^- glia of either condition, which indicates that JAK-STAT induction has a stronger influence on glial transcriptomes than other infection-related changes. Differentially expressed genes (866 genes using a cut-off of twofold change, P < 0.001, false discovery rate (FDR) < 0.01 and reads > 0.5) were significantly enriched in genes that encode proteins involved in lipid metabolism and carbohydrate transmembrane transport. These included the lipid binding protein Glial lazarillo (GLaz, a homologue of apolipoprotein D in mammals), which facilitates lipid transport from neurons to glia in flies; the lipid droplet surface binding proteins Lsd-1 and Lsd-2; the diacylglycerol O-acyltransferase Midway, which is a central regulator of triacylglycerol biosynthesis, and Coatomer, which is responsible for protein delivery to lipid droplets (LDs). This induction of lipid storage genes was coupled with induction of the monocarboxylate transporter (MCT) Outsiders (Out), and the MCT accessory protein Basigin (Bsg), sugar transporters (Tret1-1 and Tret1-2), and 17 enzymes involved in β-oxidation (Cai, 2021).

Glial MCTs promote lipid production in neurons and LD accumulation in glia by establishing a neuron and glia 'lactate shuttle'. To test a potential role for STAT signalling in influencing this shuttle at the AL, the accumulation of LDs was assessed at the AL in infected young flies using a combination of a neutral lipid probe (LipidTox, deep red) and a lipid peroxidation probe (C11-Bodipy, 581/591). A transient accumulation of LDs was observed 24 h after infection that decreased 4 days after infection, possibly owing to increased levels of β-oxidation. Overexpression of hop^tuml in the EG of young flies also promoted LD accumulation, whereas knocking down Dome or STAT rescued infection-induced accumulation. GLaz and Out were required for LD accumulation after infection, and overexpression of Upd2 and Upd3 in the gut induced LD accumulation at the AL, whereas knockdown of Upd2 or Upd3 alleviated LD accumulation in infected flies. Infection or JAK-STAT perturbation did not influence lipid peroxidation in LDs in young flies (Cai, 2021).

During neuronal stress, neurons can preferentially transfer fatty acids to glia, causing lipid accumulation and increasing fatty acid β-oxidation in glia. This study observed a significant induction of LDs specifically in EG at the AL in old flies, phenocopying hop^tuml overexpression. As fatty acid β-oxidation is a source of reactive oxygen species (ROS) that can result in lipid peroxidation, and lipid peroxidation in pigment cells (glia of the retina) promotes the demise of photoreceptors in the retina (whereas oxidative stress contributes to age-related dysfunction of cholinergic projection neurons within the olfactory circuit) it was reasoned that overall levels of ROS might increase in glia with age. Various genetically encoded ROS sensors were expressed in all glia (repo::Gal4) or in EG only (GMR56F03::Gal4) to measure levels of hydrogen peroxide (H2O2; measured by RoGFP2_Orp1) or the glutathione redox potential (measured by RoGFP2_Grx1) within the mitochondria or cytosol, respectively. Cytosolic levels of H2O2 were increased in the EG of old flies (both cytosolic and mitochondrial H2O2 levels were increased in all glia), whereas the cytosolic glutathione redox potential remained unchanged. In contrast to acute intestinal infection in young flies, lipids were peroxidated in LDs of old flies. Knocking down STAT specifically in EG, or knocking down Upd2 and Upd3 in gut enterocytes, inhibited LD accumulation and alleviated lipid peroxidation in old flies (Cai, 2021).

Olfactory discrimination was partially rescued in old and young infected flies after knockdown of GLaz and Out in EG. Knockdown of GLaz and Out also led to more Ecc15 food consumption, increased mortality after PE exposure, and reduced LD accumulation in the glia of old flies (Cai, 2021).

To confirm that infection or ageing-induced metabolic changes in EG affect neuron or glia metabolic coupling at the AL, the consequences of perturbing projection neurons directly were assessed using GH146::Gal4. Knocking down Out but not lactate dehydrogenase (Ldh) in projection neurons rescued olfactory discrimination of infected or aged flies, whereas food preference or mortality was not influenced. Overexpression of lipase 4 (Lip-4) in projection neurons, or knockdown of the neuronal lipid binding protein Neural lazarillo (NLaz), significantly improved olfactory discrimination in infected or old flies, and overexpression of Lip-4 increased Ecc15 food consumption and increased mortality after PE exposure (Cai, 2021).

This work suggests that gut-derived inflammatory cytokines modulate the metabolic coupling of glia and neurons in the brain of Drosophila to induce an adaptive temporary halt of olfactory discrimination after intestinal infection, but also contribute to age-related olfactory decline. It is proposed that gut-derived Upd2 and Upd3 reprogram lipid metabolism in EG, increasing lactate and lipid transport between glia and olfactory neurons, resulting in LD accumulation and upregulation of mitochondrial β-oxidation, potentially a source of increased ROS production. Chronic activation of this metabolic shift in old flies results in the accumulation of peroxidated lipids in EG, promoting their decay and contributing to the previously described functional decline of olfactory neurons. Detailed characterization of this metabolic reprogramming, and further exploration of the role of lipid synthesis in projection neurons for glial lipid accumulation and for olfactory discrimination are important avenues for further study (Cai, 2021).

These findings further determine the regulation of avoidance behaviour against enteropathogens in insects. In addition to gustatory bitter neurons and immune receptors in octopaminergic neurons, Upd proteins constitute a direct endocrine signal from the damaged intestinal epithelium in this complex but essential behaviour. It is proposed that Upd-mediated suppression of olfactory discrimination is required to prevent olfaction-mediated attraction to a food source after pathogenicity has been established and aversion is induced by gustatory neurons. It remains unclear, however, whether gustatory neurons are also affected by JAK-STAT signalling in EG. Whether similar mechanisms are conserved and control infection-induced loss of sensory perception in vertebrates including humans will be interesting to explore (Cai, 2021).

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).

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).

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).

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

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

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

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

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

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

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

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

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

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

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

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

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).

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).

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

Human's unique character traits make their behavior consistent and define their individuality. Yet, this consistency does not entail that people behave repetitively like machines. Like humans, animals also combine personality traits with spontaneity to produce adaptive behavior: consistent, but not fully predictable. This study examined an iconically rigid behavioral trait, insect phototaxis, that nevertheless also contains both components of individuality and spontaneity. In a light/dark T-maze, approximately 70% of a group of Drosophila fruit flies choose the bright arm of the T-Maze, while the remaining 30% walk into the dark. Taking the photopositive and the photonegative subgroups and re-testing them reveals the spontaneous component: a similar 70-30 distribution emerges in each of the two subgroups. Increasing the number of choices to ten choices, reveals the individuality component: flies with an extremely negative series of first choices were more likely to show photonegative behavior in subsequent choices and vice versa. General behavioral traits, independent of light/dark preference, contributed to the development of this individuality. The interaction of individuality and spontaneity together explains why group averages, even for such seemingly stereotypical behaviors, are poor predictors of individual choices (Steymans, 2021).

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

Arican, C., Schmitt, F. J., Rossler, W., Strube-Bloss, M. F., Nawrot, M. P. (2023). The mushroom body output encodes behavioral decision during sensory-motor transformation. Curr Biol, 33(19):4217-4224. PubMed ID: 37657449

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

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

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

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

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

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

Alpert, M. H., Gil, H., Para, A. and Gallio, M. (2022). A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat. Curr Biol. PubMed ID: 35981537

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

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

Amanullah, A., Arzoo, S., Aslam, A., Qureshi, I. W. and Hussain, M. (2023). Inbreeding-Driven Innate Behavioral Changes in Drosophila melanogaster. Biology (Basel) 12(7). PubMed ID: 37508357

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

Anqueira-Gonzalez, A., Acevedo-Gonzalez, J. P., Montes-Mercado, A., Irizarry-Hernandez, C., Fuenzalida-Uribe, N. L. and Ghezzi, A. (2021). Transcriptional Correlates of Chronic Alcohol Neuroadaptation in Drosophila Larvae. Front Behav Neurosci 15: 768694. PubMed ID: 34803626

Arican, C., Schmitt, F. J., Rossler, W., Strube-Bloss, M. F., Nawrot, M. P. (2023). The mushroom body output encodes behavioral decision during sensory-motor transformation. Curr Biol, 33(19):4217-4224. PubMed ID: 37657449

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

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

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

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

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

Babin, A., Gatti, J. L., Poirie, M. (2023). Bacillus thuringiensis bioinsecticide influences Drosophila oviposition decision. R Soc Open Sci, 10(8):230565 PubMed ID: 37650056

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

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

Banu, A., Gowda, S. B. M., Salim, S. and Mohammad, F. (2022). Serotonergic control of feeding microstructure in Drosophila. Front Behav Neurosci 16: 1105579. PubMed ID: 36733453

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

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

Behbahani, A. H., Palmer, E. H., Corfas, R. A. and Dickinson, M. H. (2021). Drosophila re-zero their path integrator at the center of a fictive food patch. Curr Biol. PubMed ID: 34450090

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

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

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

Borchardt, L. A., Scharenbrock, A. R., Olufs, Z. P. G., Wassarman, D. A. and Perouansky, M. (2023). Mutations in Complex I of the Mitochondrial Electron-Transport Chain Sensitize the Fruit Fly (Drosophila melanogaster) to Ether and Non-Ether Volatile Anesthetics. Int J Mol Sci 24(3). PubMed ID: 36768163

Brand, J. A., Yee, W. K. W., Aitkenhead, I. J., Martin, J. M., Polverino, G., Chown, S. L., Wong, B. B. M. and Dowling, D. K. (2023). Temperature change exerts sex-specific effects on behavioural variation. Proc Biol Sci 290(2002): 20230110. PubMed ID: 37403505

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

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

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

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

Bressan, G. N., Cardoso, P. M., Reckziegel, J. and Fachinetto, R. (2023). Reserpine and PCPA reduce heat tolerance in Drosophila melanogaster. Life Sci 318: 121497. PubMed ID: 36780938

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

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

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

Buchert, S. N., Murakami, P., Kalavadia, A. H., Reyes, M. T. and Sitaraman, D. (2021). Sleep correlates with behavioral decision making critical for reproductive output in Drosophila melanogaster. Comp Biochem Physiol A Mol Integr Physiol: 111114. PubMed ID: 34785379

Buhr, A., Schiemann, R. and Meyer, H. (2023). Neprilysin 4: an essential peptidase with multifaceted physiological relevance. Biol Chem. PubMed ID: 36653344

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

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

Cai, X. T., Li, H., Borch Jensen, M., Maksoud, E., Borneo, J., Liang, Y., Quake, S. R., Luo, L., Haghighi, P. and Jasper, H. (2021). Gut cytokines modulate olfaction through metabolic reprogramming of glia. Nature. PubMed ID: 34290404

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

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

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

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

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

Chakraborty, A., Sgro, C. M. and Mirth, C. K. (2021). The proximate sources of genetic variation in body size plasticity: the relative contributions of feeding behaviour and development in Drosophila melanogaster. J Insect Physiol: 104321. PubMed ID: 34653505

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

Chechi, T. S., Narasimhan, A., Biswas, B. and Prasad, N. G. (2022). Male mating success evolves in response to increased levels of male-male competition. Evolution. PubMed ID: 35598115

Chen, H. L., Motevalli, D., Stern, U. and Yang, C. H. (2022). A functional division of Drosophila sweet taste neurons that is value-based and task-specific. Proc Natl Acad Sci U S A 119(3). PubMed ID: 35031566

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

Chen, S. C., Tang, X., Goda, T., Umezaki, Y., Riley, A. C., Sekiguchi, M., Yoshii, T. and Hamada, F. N. (2022). Dorsal clock networks drive temperature preference rhythms in Drosophila. Cell Rep 39(2): 110668. PubMed ID: 35417715

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

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

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

Cheriyamkunnel, S. J., Rose, S., Jacob, P. F., Blackburn, L. A., Glasgow, S., Moorse, J., Winstanley, M., Moynihan, P. J., Waddell, S. and Rezaval, C. (2021). A neuronal mechanism controlling the choice between feeding and sexual behaviors in Drosophila. Curr Biol. PubMed ID: 34358444

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

Chiang, M. H., Lin, Y. C., Chen, S. F., Lee, P. S., Fu, T. F., Wu, T., Wu, C. L. (2023). Independent insulin signaling modulators govern hot avoidance under different feeding states. PLoS Biol, 21(10):e3002332 PubMed ID: 37847673

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

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

Cho, H. and Rohlfs, M. (2023). Transmission of beneficial yeasts accompanies offspring production in Drosophila-An initial evolutionary stage of insect maternal care through manipulation of microbial load? Ecol Evol 13(6): e10184. PubMed ID: 37332518

Cho, L. C., Yu, C. C. and Kao, C. F. (2021). Social perception of young adults prolongs the lifespan of aged Drosophila. NPJ Aging Mech Dis 7(1): 21. PubMed ID: 34471134

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

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

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

Churchill, E. R., Dytham, C., Bridle, J. R. and Thom, M. D. F. (2021). Social and physical environment independently affect oviposition decisions in Drosophila. Behav Ecol 32(6): 1391-1399. PubMed ID: 34949961

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

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

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

Cooney, P. C., Li, W., Huang, Y., Hormigo, R., Tabachnik, T., Hillman, E. M. C., Grueber, W. B. and Zarin, A. A. (2023). Neuromuscular Basis of Drosophila Larval Escape Behavior. bioRxiv. PubMed ID: 36778508

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

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

Cowley, B. R., Calhoun, A. J., Rangarajan, N., Ireland, E., Turner, M. H., Pillow, J. W., Murthy, M. (2024). Mapping model units to visual neurons reveals population code for social behaviour. Nature, 629(8014):1100-1108 PubMed ID: 38778103

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

Cury, K. M. and Axel, R. (2023). Flexible neural control of transition points within the egg-laying behavioral sequence in Drosophila. Nat Neurosci 26(6): 1054-1067. PubMed ID: 37217726

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

Davies, L. R., Loeschcke, V., Schou, M. F., Schramm, A. and Kristensen, T. N. (2021). The importance of environmental microbes for Drosophila melanogaster during seasonal macronutrient variability. Sci Rep 11(1): 18850. PubMed ID: 34552121

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

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

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

Deere, J. U. and Devineni, A. V. (2022). Taste cues elicit prolonged modulation of feeding behavior in Drosophila. iScience 25(10): 105159. PubMed ID: 36204264

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

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

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

Dhakal, S., Ren, Q., Liu, J., Akitake, B., Tekin, I., Montell, C. and Lee, Y. (2022). Drosophila TRPgamma is required in neuroendocrine cells for post-ingestive food selection. Elife 11. PubMed ID: 35416769

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

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

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

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

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

Dweck, H. K. M., Carlson, J. R. (2023). Diverse mechanisms of taste coding in Drosophila. Sci Adv, 9(46):eadj7032 PubMed ID: 37976361

Eddison, M. (2021). A genetic screen for Drosophila social isolation mutants and analysis of sex pistol. Sci Rep 11(1): 17395. PubMed ID: 34462500

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

Eichler, K., Hampel, S., Alejandro-Garcia, A., Calle-Schuler, S. A., Santana-Cruz, A., Kmecova, L., Blagburn, J. M., Hoopfer, E. D. and Seeds, A. M. (2023). Somatotopic organization among parallel sensory pathways that promote a grooming sequence in Drosophila. bioRxiv. PubMed ID: 36798384

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

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

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

Evans, A. D., Fairbanks, R. A., Schmidt, P., Levine, M. T. (2023). Histone methylation regulates reproductive diapause in Drosophila melanogaster. PLoS Genet, 19(9):e1010906 PubMed ID: 37703303

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

Fanara, J. J., Beti, M. I. L., Gandini, L. and Hasson, E. (2022). Oviposition behaviour in Drosophila melanogaster: Genetic and behavioural decoupling between oviposition acceptance and preference for natural fruits. J Evol Biol. PubMed ID: 36357966

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

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

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

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

Fulgham, C. V., Dreyer, A. P., Nasseri, A., Miller, A. N., Love, J., Martin, M. M., Jabr, D. A., Saurabh, S. and Cavanaugh, D. J. (2021). Central and Peripheral Clock Control of Circadian Feeding Rhythms. J Biol Rhythms: 7487304211045835. PubMed ID: 34547954

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

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

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

Girardeau, A. R., Foley, B. R. and Saltz, J. B. (2021). Comparing single- and mixed-species groups in fruit flies: differences in group dynamics, but not group formation. J Hered. PubMed ID: 34453172

Gaspar, M., Dias, S. and Vasconcelos, M. L. (2022). Mating pair drives aggressive behavior in female Drosophila. Curr Biol 32(21): 4734-4742. PubMed ID: 36167074

Gordon, J. and Masek, P. (2021). Excessive energy expenditure due to acute physical restraint disrupts Drosophila motivational feeding response. Sci Rep 11(1): 24208. PubMed ID: 34921197

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

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

Grinberg, M., Levin, R., Neuman, H., Ziv, O., Turjeman, S., Gamliel, G., Nosenko, R. and Koren, O. (2022). Antibiotics increase aggression behavior and aggression-related pheromones and receptors in Drosophila melanogaster. iScience 25(6): 104371. PubMed ID: 35620429

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

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

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

Guo, D., Zhang, Y. J., Zhang, S., Li, J., Guo, C., Pan, Y. F., Zhang, N., Liu, C. X., Jia, Y. L., Li, C. Y., Ma, J. Y., Nassel, D. R., Gao, C. F. and Wu, S. F. (2021). Cholecystokinin-like peptide mediates satiety by inhibiting sugar attraction. PLoS Genet 17(8): e1009724. PubMed ID: 34398892

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

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

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

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

Hara, Y. and Yamamoto, D. (2021). Effects of Food and Temperature on Drosophila melanogaster Reproductive Dormancy as Revealed by Quantification of a GFP-Tagged Yolk Protein in the Ovary. Front Physiol 12: 803144. PubMed ID: 35046840

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

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

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

He, L., Wu, B., Shi, J., Du, J. and Zhao, Z. (2023). Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila. Cell Rep 42(8): 112912. PubMed ID: 37531254

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

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

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

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

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

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

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

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

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

Imambocus, B. N., Zhou, F., Formozov, A., Wittich, A., Tenedini, F. M., Hu, C., Sauter, K., Macarenhas Varela, E., Heredia, F., Casimiro, A. P., Macedo, A., Schlegel, P., Yang, C. H., Miguel-Aliaga, I., Wiegert, J. S., Pankratz, M. J., Gontijo, A. M., Cardona, A. and Soba, P. (2021). A neuropeptidergic circuit gates selective escape behavior of Drosophila larvae. Curr Biol. PubMed ID: 34798050

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

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

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

Jorgensen, L. B., Malte, H., Orsted, M., Klahn, N. A. and Overgaard, J. (2021). A unifying model to estimate thermal tolerance limits in ectotherms across static, dynamic and fluctuating exposures to thermal stress. Sci Rep 11(1): 12840. PubMed ID: 34145337

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

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

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

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

Jovanoski, K. D., Duquenoy, L., Mitchell, J., Kapoor, I., Treiber, C. D., Croset, V., Dempsey, G., Parepalli, S., Cognigni, P., Otto, N., Felsenberg, J., Waddell, S. (2023). Dopaminergic systems create reward seeking despite adverse consequences. Nature, 623(7986):356-365 PubMed ID: 37880370

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

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

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

Kapila, R., Kashyap, M., Gulati, A., Narasimhan, A., Poddar, S., Mukhopadhaya, A. and Prasad, N. G. (2021). Evolution of sex-specific heat stress tolerance and larval Hsp70 expression in populations of Drosophila melanogaster adapted to larval crowding. J Evol Biol. PubMed ID: 34197669

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

Karpati, Z., Deutsch, F., Kiss, B. and Schmitt, T. (2023). Seasonal changes in photoperiod and temperature lead to changes in cuticular hydrocarbon profiles and affect mating success in Drosophila suzukii. Sci Rep 13(1): 5649. PubMed ID: 37024537

Kasturacharya, N., Dhall, J. K. and Hasan, G. (2023). A STIM dependent dopamine-neuropeptide axis maintains the larval drive to feed and grow in Drosophila. PLoS Genet 19(6): e1010435. PubMed ID: 37363909

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

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

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

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

Kelly, K. P., Alassaf, M., Sullivan, C. E., Brent, A. E., Goldberg, Z. H., Poling, M. E., Dubrulle, J. and Rajan, A. (2022). Fat body phospholipid state dictates hunger-driven feeding behavior. Elife 11. PubMed ID: 36201241

Khatib, L., Subasi, B. S., Fishman, B., Kapun, M. and Tauber, E. (2023). Unveiling Subtle Geographical Clines: Phenotypic Effects and Dynamics of Circadian Clock Gene Polymorphisms. Biology (Basel) 12(6). PubMed ID: 37372143

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

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

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

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

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

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

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

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

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

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

Klepsatel, P. and Galikova, M. (2022). Developmental temperature affects thermal dependence of locomotor activity in Drosophila. J Therm Biol 103: 103153. PubMed ID: 35027204

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

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

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

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

Krill, J. L. and Dawson-Scully, K. (2021). Characterization of a novel stimulus-induced glial calcium wave in Drosophila larval peripheral segmental nerves and its role in PKG-modulated thermoprotection. J Neurogenet: 1-15. PubMed ID: 34309496

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

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

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

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

Kurogi, Y., Imura, E., Mizuno, Y., Hoshino, R., Nouzova, M., Matsuyama, S., Mizoguchi, A., Kondo, S., Tanimoto, H., Noriega, F. G. and Niwa, R. (2023). Female reproductive dormancy in Drosophila is regulated by DH31-producing neurons projecting into the corpus allatum. Development 150(10). PubMed ID: 37218457

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

Lankinen, P., Kastally, C. and Hoikkala, A. (2023). Clinal variation in the temperature and photoperiodic control of reproductive diapause in Drosophila montana females. J Insect Physiol: 104556. PubMed ID: 37598869

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

Layh, S., Nagarajan-Radha, V., Lemos, B. and Dowling, D. K. (2023). Y chromosome-linked variation affects locomotor activity in male Drosophila melanogaster and is robust to differences in thermal environment. Heredity (Edinb). PubMed ID: 36914794

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

Leech, T., McDowall, L., Hopkins, K. P., Sait, S. M., Harrison, X. A. and Bretman, A. (2021). Social environment drives sex and age-specific variation in Drosophila melanogaster microbiome composition and predicted function. Mol Ecol. PubMed ID: 34494339

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

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

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

Lez-Segarra, A. J. G. Pontes, G., Jourjine, N., Toro, A. D. and Scott, K. (2023). Hunger- and thirst-sensing neurons modulate a neuroendocrine network to coordinate sugar and water ingestion. bioRxiv. PubMed ID: 37066363

Li, K., Tsukasa, Y., Kurio, M., Maeta, K., Tsumadori, A., Baba, S., Nishimura, R., Murakami, A., Onodera, K., Morimoto, T., Uemura, T. and Usui, T. (2023). Belly roll, a GPI-anchored Ly6 protein, regulates Drosophila melanogaster escape behaviors by modulating the excitability of nociceptive peptidergic interneurons. Elife 12. PubMed ID: 37309249

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

Li, Q., Wang, M., Zhang, P., Liu, Y., Guo, Q., Zhu, Y., Wen, T., Dai, X., Zhang, X., Nagel, M., Dethlefsen, B. H., Xie, N., Zhao, J., Jiang, W., Han, L., Wu, L., Zhong, W., Wang, Z., Wei, X., Dai, W., Liu, L., Xu, X., Lu, H., Yang, H., Wang, J., Boomsma, J. J., Liu, C., Zhang, G. and Liu, W. (2022). A single-cell transcriptomic atlas tracking the neural basis of division of labour in an ant superorganism. Nat Ecol Evol 6(8): 1191-1204. PubMed ID: 35711063

Li, W., Wang, Z., Syed, S., Lyu, C., Lincoln, S., O'Neil, J., Nguyen, A. D., Feng, I. and Young, M. W. (2021). Chronic social isolation signals starvation and reduces sleep in Drosophila. Nature 597(7875): 239-244. PubMed ID: 34408325

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

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

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

Liu, J., Liu, W., Thakur, D., Mack, J., Spina, A. and Montell, C. (2023). Alleviation of thermal nociception depends on heat-sensitive neurons and a TRP channel in the brain. Curr Biol 33(12): 2397-2406. PubMed ID: 37201520

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

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

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

Liu, Z., Pan, X., Guo, J., Li, L., Tang, Y., Wu, G., Li, M. and Wang, H. (2023). Long-term sevoflurane exposure resulted in temporary rather than lasting cognitive impairment in Drosophila. Behav Brain Res 442: 114327. PubMed ID: 36738841

Lyman, R. F., Lyman, R. A., Yamamoto, A., Huang, W., Harbison, S. T., Zhou, S., Anholt, R. R. H. and Mackay, T. F. C. (2023). Natural genetic variation in a dopamine receptor is associated with variation in female fertility in Drosophila melanogaster. Proc Biol Sci 290(1996): 20230375. PubMed ID: 37040806

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

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

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

Maksymchuk, N., Sakurai, A., Cox, D. N., Cymbalyuk, G. S. (2023). Cold-Temperature Coding with Bursting and Spiking Based on TRP Channel Dynamics in Drosophila Larva Sensory Neurons. Int J Mol Sci, 24(19) PubMed ID: 37834085

Manoli, G., Zandawala, M., Yoshii, T. and Helfrich-Forster, C. (2023). Characterization of clock-related proteins and neuropeptides in Drosophila littoralis and their putative role in diapause. J Comp Neurol 531(15): 1525-1549. PubMed ID: 37493077

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

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

Maruko, A., Iijima, K. M., Ando, K. (2023). Dissecting the daily feeding pattern: Peripheral CLOCK/CYCLE generate the feeding/fasting episodes and neuronal molecular clocks synchronize them. iScience, 26(11):108164 PubMed ID: 37915609

McDonough-Goldstein, C. E., Pitnick, S. and Dorus, S. (2022). Drosophila female reproductive glands contribute to mating plug composition and the timing of sperm ejection. Proc Biol Sci 289(1968): 20212213. PubMed ID: 35105240

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

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

Meda, N., Menti, G. M., Megighian, A. and Zordan, M. A. (2021). A heuristic underlies the search for relief in Drosophila melanogaster. Ann N Y Acad Sci. PubMed ID: 34928521

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

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

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

Mishra, A., Tung, S., Sruti, V. R. S., Shreenidhi, P. M. and Dey, S. (2022). Desiccation Stress Acts as Cause as well as Cost of Dispersal in Drosophila melanogaster. Am Nat 199(4): E111-e123. PubMed ID: 35324379

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

Morimoto, J., Wenzel, M., Derous, D., Henry, Y. and Colinet, H. (2022). The transcriptomic signature of responses to larval crowding in Drosophila melanogaster. Insect Sci. PubMed ID: 36115064

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

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

Mueller, J. M., Zhang, N., Carlson, J. M. and Simpson, J. H. (2021). Variation and Variability in Drosophila Grooming Behavior. Front Behav Neurosci 15: 769372. PubMed ID: 35087385

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

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

Muria, A., Musso, P. Y., Durrieu, M., Portugal, F. R., Ronsin, B., Gordon, M. D., Jeanson, R. and Isabel, G. (2021). Social facilitation of long-lasting memory is mediated by CO(2) in Drosophila. Curr Biol. PubMed ID: 33740428

Nakamizo-Dojo, M., Ishii, K., Yoshino, J., Tsuji, M., Emoto, K. (2023). Descending GABAergic pathway links brain sugar-sensing to peripheral nociceptive gating in Drosophila. Nat Commun, 14(1):6515 PubMed ID: 37845214

Navas, C. A., Agudelo-Cantero, G. A. and Loeschcke, V. (2022). Thermal boldness: Volunteer exploration of extreme temperatures in fruit flies. J Insect Physiol 136: 104330. PubMed ID: 34848182

Nguyen, M., Roman, G. W., Soibam, B. (2023). Drosophila genotypes can be predicted from their exploration locomotive trajectories using supervised machine learning. Behavioural processes, 212:104944 PubMed ID: 37717930

Niu, M., Zhang, X., Li, W., Wang, J. and Li, Y. (2021). dFRAME: A Video Recording-Based Analytical Method for Studying Feeding Rhythm in Drosophila. Front Genet 12: 763200. PubMed ID: 34721548

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

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

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

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

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

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

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

Pantalia, M., Lin, Z., Tener, S. J., Qiao, B., Tang, G., Ulgherait, M., O'Connor, R., Delventhal, R., Volpi, J., Syed, S., Itzhak, N., Canman, J. C., Fernandez, M. P. and Shirasu-Hiza, M. (2023). Drosophila mutants lacking the glial neurotransmitter-modifying enzyme Ebony exhibit low neurotransmitter levels and altered behavior. Sci Rep 13(1): 10411. PubMed ID: 37369755

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

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

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

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

Poe, A. R., Zhu, L., Tang, S. H., Valencia, E., Kayser, M. S. (2023). Energetic Demands Regulate Sleep-Wake Rhythm Circuit Development. bioRxiv, PubMed ID: 37786713

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

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

Prince, E., Kretzschmar, J., Trautenberg, L. C., Broschk, S. and Brankatschk, M. (2021). DIlp7-Producing Neurons Regulate Insulin-Producing Cells in Drosophila. Front Physiol 12: 630390. PubMed ID: 34385929

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

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

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

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

Ramakrishnan, P., Joshi, A., Tulasi, M. and Yadav, P. (2022). Monochromatic visible lights modulate the timing of pre-adult developmental traits in Drosophila melanogaster. Photochem Photobiol Sci. PubMed ID: 36583814

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

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

Ravbar, P., Zhang, N. and Simpson, J. H. (2021). Behavioral evidence for nested central pattern generator control of Drosophila grooming. Elife 10. PubMed ID: 34936550

Reyes-Ramirez, A., Belgaidi, Z., Gibert, P., Pommier, T., Siberchicot, A., Mouton, L., Desouhant, E. (2023). Larval density in the invasive Drosophila suzukii: Immediate and delayed effects on life-history traits. Ecology and evolution, 13(8):e10433 PubMed ID: 37636864

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

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

Rodriguez, M., Pagola, L., Norry, F. M. and Ferrero, P. (2021). Cardiac performance in heat-stressed flies of heat-susceptible and heat-resistant Drosophila melanogaster. J Insect Physiol 133: 104268. PubMed ID: 34171365

Rodrigues, L. R., Zwoinska, M. K., Axel, W. W. R. and Snook, R. R. (2022). The genetic basis and adult reproductive consequences of developmental thermal plasticity. J Anim Ecol. PubMed ID: 35060127

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

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

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

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

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

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

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

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

Sakurai, A., Littleton, J. T., Kojima, H. and Yoshihara, M. (2021). Alteration in information flow through a pair of feeding command neurons underlies a form of Pavlovian conditioning in the Drosophila brain. Curr Biol. PubMed ID: 34352215

Salachan, P. V. and Sorensen, J. G. (2022). Molecular mechanisms underlying plasticity in a thermally varying environment. Mol Ecol. PubMed ID: 35397190

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

Santos, M. A., Carromeu-Santos, A., Quina, A. S., Santos, M., Matos, M. and Simoes, P. (2021). No evidence for short-term evolutionary response to a warming environment in Drosophila. Evolution. PubMed ID: 34617283

Santos, M. A., Antunes, M. A., Grandela, A., Quina, A. S., Santos, M., Matos, M. and Simoes, P. (2023). Slow and population specific evolutionary response to a warming environment. Sci Rep 13(1): 9700. PubMed ID: 37322066

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

Sato, A., Yew, J. Y. and Takahashi, A. (2023). Effect of acetic acid bacteria colonization on oviposition and feeding site choice in Drosophila suzukii and its related species. bioRxiv. PubMed ID: 36993389

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

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

Scott, A. M., Dworkin, I. and Dukas, R. (2021). Evolution of sociability by artificial selection. Evolution. PubMed ID: 34605553

Scott, A. M., Yan, J. L., Baxter, C. M., Dworkin, I. and Dukas, R. (2022). The genetic basis of variation in sexual aggression: Evolution versus social plasticity. Mol Ecol 31(10): 2865-2881. PubMed ID: 35313034

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

Shiu, P. K., Sterne, G. R., Spiller, N., Franconville, R., Sandoval, A., Zhou, J., Simha, N., Kang, C. H., Yu, S., Kim, J. S., Dorkenwald, S., Matsliah, A., Schlegel, P., Yu, S. C., McKellar, C. E., Sterling, A., Costa, M., Eichler, K., Jefferis, G., Murthy, M., Bates, A. S., Eckstein, N., Funke, J., Bidaye, S. S., Hampel, S., Seeds, A. M. and Scott, K. (2023). A leaky integrate-and-fire computational model based on the connectome of the entire adult Drosophila brain reveals insights into sensorimotor processing. bioRxiv. PubMed ID: 37205514

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

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

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

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

Sengupta, S., Chan, Y. B., Palavicino-Maggio, C. B. and Kravitz, E. A. (2022). GABA transmission from mAL interneurons regulates aggression in Drosophila males. Proc Natl Acad Sci U S A 119(5) PubMed ID: 35082150

Shell, B. C., Luo, Y., Pletcher, S. and Grotewiel, M. (2021). Expansion and application of dye tracers for measuring solid food intake and food preference in Drosophila. Sci Rep 11(1): 20044. PubMed ID: 34625601

Shin, M. and Venton, B. J. (2022). Fast-Scan Cyclic Voltammetry (FSCV) Reveals Behaviorally Evoked Dopamine Release by Sugar Feeding in the Adult Drosophila Mushroom Body. Angew Chem Int Ed Engl 61(44): e202207399. PubMed ID: 35989453

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

Silva-Lopez, J., Godoy, P., Jara, L. and Godoy-Herrera, R. (2023). Interaction and integration among behaviors of adult Drosophila in nature. PLoS One 18(7): e0278427. PubMed ID: 37440503

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

Singh, V. J., Potdar, S. and Sheeba, V. (2022). Effects of Food Availability Cycles on Phase and Period of Activity-rest Rhythm in Drosophila melanogaster. J Biol Rhythms 37(5): 528-544. PubMed ID: 35983646

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

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

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

Steymans, I., Pujol-Lereis, L. M., Brembs, B. and Gorostiza, E. A. (2021). Collective action or individual choice: Spontaneity and individuality contribute to decision-making in Drosophila. PLoS One 16(8): e0256560. PubMed ID: 34437617

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

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

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

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

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

Sultanova, Z., Ivimey-Cook, E. R., Chapman, T. and Maklakov, A. A. (2021). Fitness benefits of dietary restriction. Proc Biol Sci 288(1963): 20211787. PubMed ID: 34814748

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

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

Suzuki, M., Kuromi, H., Shindo, M., Sakata, N., Niimi, N., Fukui, K., Saitoe, M. and Sango, K. (2023). A Drosophila model of diabetic neuropathy reveals a role of proteasome activity in the glia. iScience 26(6): 106997. PubMed ID: 37378316

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

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

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

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

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

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

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

Strunov, A., Schoenherr, C. and Kapun, M. (2023). Wolbachia has subtle effects on thermal preference in highly inbred Drosophila melanogaster which vary with life stage and environmental conditions. Sci Rep 13(1): 13792. PubMed ID: 37612420

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

Troup, M., Tainton-Heap, L. A. L. and van Swinderen, B. (2023). Neural Ensemble Fragmentation in the Anesthetized Drosophila Brain. J Neurosci 43(14): 2537-2551. PubMed ID: 36868857

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

Ueno, T. and Takahashi, Y. (2021). Mitochondrial polymorphism shapes intrapopulation behavioural variation in wild Drosophila. Biol Lett 17(7): 20210194. PubMed ID: 34314641.

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

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

van Breugel, F. (2021). Correlated decision making across multiple phases of olfactory-guided search in Drosophila improves search efficiency. J Exp Biol 224(16). PubMed ID: 34415028

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

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

Verschut, T. A., Ng, R., Doubovetzky, N. P., Le Calvez, G., Sneep, J. L., Minnaard, A. J., Su, C. Y., Carlsson, M. A., Wertheim, B. and Billeter, J. C. (2023). Aggregation pheromones have a non-linear effect on oviposition behavior in Drosophila melanogaster. Nat Commun 14(1): 1544. PubMed ID: 36941252

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

Vijayan, V., Wang, F., Wang, K., Chakravorty, A., Adachi, A., Akhlaghpour, H., Dickson, B. J. and Maimon, G. (2023). A rise-to-threshold process for a relative-value decision. Nature 619(7970): 563-571. PubMed ID: 37407812

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

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

Walsh, B. S., Parratt, S. R., Snook, R. R., Bretman, A., Atkinson, D. and Price, T. A. R. (2022). Female fruit flies cannot protect stored sperm from high temperature damage. J Therm Biol 105: 103209. PubMed ID: 35393050

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

Wang, W., Dweck, H. K. M., Talross, G. J. S., Zaidi, A., Gendron, J. M. and Carlson, J. R. (2022). Sugar sensation and mechanosensation in the egg-laying preference shift of Drosophila suzukii. Elife 11. PubMed ID: 36398882

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

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

Wice, E. W. and Saltz, J. B. (2023). Indirect genetic effects for social network structure in Drosophila melanogaster. Philos Trans R Soc Lond B Biol Sci 378(1874): 20220075. PubMed ID: 36802774

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

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

Williamson, M., Mitchell, A. and Condron, B. (2021). Birth temperature followed by a visual critical period determines cooperative group membership. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. PubMed ID: 34611741.

Williams-Simon, P. A., Oster, C., Moaton, J. A., Ghidey, R., Ng'oma, E., Middleton, K. M., Zars, T. and King, E. G. (2023). Naturally segregating genetic variants contribute to thermal tolerance in a D. melanogaster model system. bioRxiv. PubMed ID: 37461510

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

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

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

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

Xu, L., Jiang, H. B., Yu, J. L., Pan, D., Tao, Y., Lei, Q., Chen, Y., Liu, Z. and Wang, J. J. (2023). Two odorant receptors regulate 1-octen-3-ol induced oviposition behavior in the oriental fruit fly. Commun Biol 6(1): 176. PubMed ID: 36792777

Yang, T., Yuan, Z., Liu, C., Liu, T. and Zhang, W. (2021). A neural circuit integrates pharyngeal sensation to control feeding. Cell Rep 37(6): 109983. PubMed ID: 34758309

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

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

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

Yu, J., Dancausse, S., Paz, M., Faderin, T., Gaviria, M., Shomar, J. W., Zucker, D., Venkatachalam, V. and Klein, M. (2023). Continuous, long-term crawling behavior characterized by a robotic transport system. Elife 12. PubMed ID: 37535068

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

Venkitachalam, S., Das, S., Deep, A. and Joshi, A. (2022). Density-dependent selection in Drosophila: evolution of egg size and hatching time. J Genet 101. PubMed ID: 35129132

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

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

Vijayan, V., Wang, Z., Chandra, V., Chakravorty, A., Li, R., Sarbanes, S. L., Akhlaghpour, H. and Maimon, G. (2022). An internal expectation guides Drosophila egg-laying decisions. Sci Adv 8(43): eabn3852. PubMed ID: 36306348

Wan, X., Shen, P., Shi, K., Li, J., Wu, F. and Zhou, C. (2023). A Neural Circuit Controlling Virgin Female Aggression Induced by Mating-related Cues in Drosophila. Neurosci Bull. PubMed ID: 36941515

Werkhoven, Z., Bravin, A., Skutt-Kakaria, K., Reimers, P., Pallares, L. F., Ayroles, J. and de Bivort, B. L. (2021). The structure of behavioral variation within a genotype. Elife 10. PubMed ID: 34664550.

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

Wohl, M. P., Liu, J. and Asahina, K. (2023). Drosophila tachykininergic neurons modulate the activity of two groups of receptor-expressing neurons to regulate aggressive tone. J Neurosci. PubMed ID: 36977580

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

Wu, M. S., Liao, T. W., Wu, C. Y., Hsieh, T. H., Kuo, P. C., Li, Y. C., Cheng, K. C., Chiang, H. C. (2023). Aversive conditioning information transmission in Drosophila. Cell Rep, 42(10):113207 PubMed ID: 37782557

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

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

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

Yost, R. T., Liang, E., Stewart, M. P., Chui, S., Greco, A. F., Long, S. Q., McDonald, I. S., McDowell, T., McNeil, J. N. and Simon, A. F. (2021). Drosophila melanogaster Stress Odorant (dSO) Displays the Characteristics of an Interspecific Alarm Cue. J Chem Ecol. PubMed ID: 34402994

Yu, J., Guo, X., Zheng, S. and Zhang, W. (2023). A dedicate sensorimotor circuit enables fine texture discrimination by active touch. PLoS Genet 19(1): e1010562. PubMed ID: 36649336

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

Yu, Z., Shi, J., Jiang, X., Song, Y., Du, J. and Zhao, Z. (2022). Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae. Pest Manag Sci. PubMed ID: 36396604

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

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

Zhang, N. and Simpson, J. H. (2022). A pair of commissural command neurons induces Drosophila wing grooming. iScience 25(2): 103792. PubMed ID: 35243214

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

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

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

Zhong, L., Yang, Z., Tang, H., Xu, Y., Liu, X. and Shen, J. (2022). Differential analysis of negative geotaxis climbing trajectories in Drosophila under different conditions. Arch Insect Biochem Physiol: e21922. PubMed ID: 35666567

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

Zhu, J., Boivin, J. C., Pang, S., Xu, C. S., Lu, Z., Saalfeld, S., Hess, H. F. and Ohyama, T. (2023). Comparative connectomics and escape behavior in larvae of closely related Drosophila species. Curr Biol 33(12): 2491-2503. PubMed ID: 37285846

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

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

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