Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the Drosophila larva is simple enough to be tractable, yet complex enough to be interesting. It features about 10,000 neurons and is capable of various taxes, kineses and Pavlovian conditioning. All its neurons are currently being mapped into a light-microscopical atlas, and Gal4 strains are being generated to experimentally access neurons one at a time. In addition, an electron microscopic reconstruction of its nervous system seems within reach. Notably, this electron microscope-based connectome is being drafted for a stage 1 larva. This study undertook a survey the behaviour of stage 1 larvae. In a community-based approach called the Ol1mpiad, stage 1 Drosophila larvae were probed for free locomotion, feeding, responsiveness to substrate vibration, gentle and nociceptive touch, burrowing, olfactory preference and thermotaxis, light avoidance, gustatory choice of various tastants plus odour-taste associative learning, as well as light/dark-electric shock associative learning (see Artificial Intelligence Helps Build Brain Atlas of Fly Behavior). Quantitatively, stage 1 larvae show lower scores in most tasks, arguably because of their smaller size and lower speed. Qualitatively, however, stage 1 larvae perform strikingly similar to stage 3 larvae in almost all cases. These results bolster confidence in mapping brain structure and behaviour across developmental stages (Almeida-Carvalho, 2017).
The mechanism of action selection is a widely shared fundamental process required by animals to interact with the environment and adapt to it. A key step in this process is the filtering of many "distracting" sensory inputs which may disturb action selection. Because it has been suggested that, beyond sharing common mechanisms, action selection may also be processed by shared circuits in vertebrates and invertebrates, it was asked whether invertebrates showed the ability to filter out "distracting" stimuli to maintain a goal-directed action, as seen in vertebrates. In this experiment action selection was studied in wild-type Drosophila melanogaster, by investigating their reaction to the abrupt appearance of a visual distractor during an ongoing locomotor action directed to a specific visual target. Flies tended to shift the original trajectory towards the distractor, thus acknowledging it's presence, but did not appear to commit to it, suggesting that an inhibition process took place in order to continue to carry out the original goal-directed action. To some extent flies appeared to take into account the level of salience of the abrupt distractor appearance as a basis for the ensuing motor program. However, they did not engage in a complete change in their initial motor program in favour of the distractor. These results provide interesting insights into the selection-for-action mechanism, in a context requiring action-centered attention which might have appeared rather early in the course of evolution (Frighetto, 2019).
Illicit use of psychostimulants, such as cocaine and methamphetamine, constitutes a significant public health problem. Whereas neural mechanisms that mediate the effects of these drugs are well-characterized, genetic factors that account for individual variation in susceptibility to substance abuse and addiction remain largely unknown. Drosophila melanogaster can serve as a translational model for studies on substance abuse, since flies have a dopamine transporter that can bind cocaine and methamphetamine, and exposure to these compounds elicits effects similar to those observed in people, suggesting conserved evolutionary mechanisms underlying drug responses. This study used the D. melanogaster Genetic Reference Panel to investigate the genetic basis for variation in psychostimulant drug consumption, to determine whether similar or distinct genetic networks underlie variation in consumption of cocaine and methamphetamine, and to assess the extent of sexual dimorphism and effect of genetic context on variation in voluntary drug consumption. Quantification of natural genetic variation in voluntary consumption, preference, and change in consumption and preference over time for cocaine and methamphetamine uncovered significant genetic variation for all traits, including sex-, exposure- and drug-specific genetic variation. Genome wide association analyses identified both shared and drug-specific candidate genes, which could be integrated in genetic interaction networks. The effects were assessed of ubiquitous RNA interference (RNAi) on consumption behaviors for 34 candidate genes: all affected at least one behavior. Finally, RNAi knockdown in the nervous system was used to implicate dopaminergic neurons and the mushroom bodies as part of the neural circuitry underlying experience-dependent development of drug preference (Highfill, 2019).
Monoamine serotonin (5HT) has been linked to aggression for many years across species. However, elaboration of the neurochemical pathways that govern aggression has proven difficult because monoaminergic neurons also regulate other behaviors. There are approximately 100 serotonergic neurons in the Drosophila nervous system, and they influence sleep, circadian rhythms, memory, and courtship. In the Drosophila model of aggression, the acute shut down of the entire serotonergic system yields flies that fight less, whereas induced activation of 5HT neurons promotes aggression. Using intersectional genetics, the population of 5HT neurons that can be reproducibly manipulated were restricted to identify those that modulate aggression. Although similar approaches were used recently to find aggression-modulating dopaminergic and Fru(M)-positive peptidergic neurons, the downstream anatomical targets of the neurons that make up aggression-controlling circuits remain poorly understood. This study identified a symmetrical pair of serotonergic PLP neurons (5HT-PLP neurons) that are necessary for the proper escalation of aggression. Silencing these neurons reduced aggression in male flies, and activating them increased aggression in male flies. GFP reconstitution across synaptic partners (GRASP) analyses suggested that 5HT-PLP neurons form contacts with 5HT1A receptor-expressing neurons in two distinct anatomical regions of the brain. Activation of these 5HT1A receptor-expressing neurons, in turn, caused reductions in aggression. These studies, therefore, suggest that aggression may be held in check, at least in part, by inhibitory input from 5HT1A receptor-bearing neurons, which can be released by activation of the 5HT-PLP neurons (Alekseyenko, 2014).
Displays of appropriate levels of aggression rely on the ability of an animal to analyze many factors, including the following: the correct identification and evaluation of the abilities of potential competitors; the evaluation of the value of a territory and the likelihood of acquiring it; and the physiological state of the animal. Multiple sensory systems and circuits will be utilized in making such evaluations. The fixed number of neurons and neuronal circuits in nervous systems might limit the abilities of an animal to evaluate such a multiplicity of factors, but great flexibility is introduced into the system by the availability of neuromodulators. These have the capability of rapidly, efficiently, and reversibly reconfiguring the networks of neurons without changing the 'hardwiring.' The current studies illustrate the modulation by 1-2 pairs of serotonergic neurons that enhance aggression. Other modulatory neurons and systems that influence aggression have been identified previously in Drosophila, including dopaminergic neurons, FruM-positive octopamine neurons that influence the behavioral choice between courtship and aggression, FruM-positive tachykinin neurons that enhance aggression, and neuropeptide F circuits that decrease aggression. The arbors of processes of the 5HT-PLP neurons examined in this study densely innervate several integrative centers in the fly brain, but thus far, they do not seem to overlap with the processes of the other reported aggression-influencing neuromodulatory neurons. The 5HT-PLP neurons do not coexpress FruM or Dsx. Thus, the modulatory control of the male-specific higher-level aggression appears to involve both sex-specific regulatory factors and other as-yet-unidentified control elements. The current studies further suggest that going to higher-intensity levels in fights may be held in check by inhibition, which can be released by activation of the 5HT-PLP neurons. Learning more about the neurons and neuronal circuits involved with a suggested downstream aggression-suppressing system and with the sensory systems that trigger aggression in the first place will be essential steps in further unraveling the complex circuitry that controls the release of aggression in Drosophila (Alekseyenko, 2014).
In summary, using a Drosophila model system and an intersectional genetic strategy, this study identified a pair of serotonergic neurons in the PLP cluster that modulate aggressive behavior. These neurons arborize through several neuropil regions in the central brain, where they influence the escalation of aggression, at least in part, via 5HT1A receptor-bearing neurons and also independently influence locomotion and sleep. The single-cell resolution in identification of neuronal connections and explorations of their functions in behaving animals provides an entry point into unraveling the circuitry associated with complex behaviors like aggression (Alekseyenko, 2014).
Fighting between different species is widespread in the animal kingdom, yet this phenomenon has been relatively understudied in the field of aggression research. Particularly lacking are studies that test the effect of genetic distance, or relatedness, on aggressive behaviour between species. This study characterized male-male aggression within and between species of fruit flies across the Drosophila phylogeny. Male Drosophila are shown to discriminate between conspecifics and heterospecifics and show a bias for the target of aggression that depends on the genetic relatedness of opponent males. Specifically, males of closely related species treated conspecifics and heterospecifics equally, whereas males of distantly related species were overwhelmingly aggressive towards conspecifics. This is the first study to quantify aggression between Drosophila species and to establish a behavioural bias for aggression against conspecifics versus heterospecifics. The results suggest that future study of heterospecific aggression behaviour in Drosophila is warranted to investigate the degree to which these trends in aggression among species extend to broader behavioural, ecological and evolutionary contexts (Gupta, 2019).
In the Drosophila model of aggression, males and females fight in same-sex pairings, but a wide disparity exists in the levels of aggression displayed by the 2 sexes. A screen of Drosophila Flylight Gal4 lines by driving expression of the gene coding for the temperature sensitive dTRPA1 channel, yielded a single line (GMR26E01-Gal4) displaying greatly enhanced aggression when thermoactivated. Targeted neurons were widely distributed throughout male and female nervous systems, but the enhanced aggression was seen only in females. No effects were seen on female mating behavior, general arousal, or male aggression. The enhancement was quantified by measuring fight patterns characteristic of female and male aggression and confirmed that the effect was female-specific. To reduce the numbers of neurons involved, an intersectional approach was used with a library of enhancer trap flp-recombinase lines. Several crosses reduced the populations of labeled neurons, but only 1 cross yielded a large reduction while maintaining the phenotype. Of particular interest was a small group (2 to 4 pairs) of neurons in the approximate position of the pC1 cluster important in governing male and female social behavior. Female brains have approximately 20 doublesex (dsx)-expressing neurons within pC1 clusters. Using dsx (FLP) instead of 357 (FLP) for the intersectional studies, it was found that the same 2 to 4 pairs of neurons likely were identified with both. These neurons were cholinergic and showed no immunostaining for other transmitter compounds. Blocking the activation of these neurons blocked the enhancement of aggression (Palavicino-Maggio, 2019).
Traumatic brain injury (TBI), caused by repeated concussive head trauma can induce chronic traumatic encephalopathy (CTE), a neurodegenerative disease featuring behavioral symptoms ranging from cognitive deficits to elevated aggression. In a Drosophila model, this study used a high-impact trauma device to induce TBI-like symptoms and to study post-TBI behavioral outcomes. Following TBI, aggression in banged male flies was significantly elevated as compared with that in unbanged flies. Various forms of dietary therapy, especially the high-fat, low-carbohydrate ketogenic diet (KD), have recently been explored for a wide variety of neuropathies. It is thus hypothesized that putatively neuroprotective dietary interventions might be able to suppress post-traumatic elevations in aggressive behavior in animals subjected to head-trauma-inducing strikes, or "bangs". A normal high-carbohydrate Drosophila diet was supplemented with the KD metabolite beta-hydroxybutyrate (beta-HB)-a ketone body (KB). Banged flies raised on a KB-supplemented diet exhibited a marked reduction in aggression, whereas aggression in unbanged flies was equivalent whether dieted with KB supplements or not. Pharmacological blockade of the ATP-sensitive potassium (KATP) channel abrogated KB effects reducing post-TBI aggression while pharmacological activation mimicked them, suggesting a mechanism by which KBs act in this model. KBs did not significantly extend lifespan in banged flies, but markedly extended lifespan in unbanged flies. This study has developed a functional model for the study of post-TBI elevations of aggression. Further, It is concluded that dietary interventions may be a fruitful avenue for further exploration of treatments for TBI- and CTE-related cognitive-behavioral symptoms (Lee, 2019).
Social isolation strongly modulates behavior across the animal kingdom. This study utilized the fruit fly Drosophila melanogaster to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide Drosulfakinin (Dsk), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors. Dsk knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of Dsk neurons each similarly increased isolation-driven aggression. The results suggest a U-shaped dependence of social isolation-induced aggressive behavior on Dsk signaling, similar to the actions of many neuromodulators in other contexts (Agrawal, 2020).
For decades, numerous researchers have documented the presence of the fruit fly or Drosophila melanogaster on alcohol-containing food sources. Although fruit flies are a common laboratory model organism of choice, there is relatively little understood about the ethological relationship between flies and ethanol. This study finds that when male flies inhabit ethanol-containing food substrates they become more aggressive. A possible mechanism was identified for this behavior. The odor of ethanol potentiates the activity of sensory neurons in response to an aggression-promoting pheromone. Finally, it was observed that the odor of ethanol also promotes attraction to a food-related citrus odor. Understanding how flies interact with the complex natural environment they inhabit can provide valuable insight into how different natural stimuli are integrated to promote fundamental behaviors (Park, 2020).
Traumatic experiences generate stressful neurological effects in the exposed persons and animals. Previous studies have demonstrated that in many species, including Drosophila, the defeated animal has a higher probability of losing subsequent fights. However, the neural basis of this "loser effect" is largely unknown. This study reports that elevated serotonin (5-HT) signaling helps a loser to overcome suppressive neurological states. Coerced activation of 5-HT neurons increases aggression in males and promotes losers to both vigorously re-engage in fights and even defeat the previous winners and regain mating motivation. P1 neurons act upstream and 5-HT1B neurons in the ellipsoid body act downstream of 5-HT neurons to arouse losers. These results demonstrate an ancient neural mechanism of regulating depressive behavioral states after distressing events (Hu, 2020).
Aggressive social interactions are used to compete for limited resources and are regulated by complex sensory cues and the organism's internal state. While both sexes exhibit aggression, its neuronal underpinnings are understudied in females. This study identified a population of sexually dimorphic aIPg neurons in the adult Drosophila melanogaster central brain whose optogenetic activation increased, and genetic inactivation reduced, female aggression. Analysis of GAL4 lines identified in an unbiased screen for increased female chasing behavior revealed the involvement of another sexually dimorphic neuron, pC1d, and implicated aIPg and pC1d neurons as core nodes regulating female aggression. Connectomic analysis demonstrated that aIPg neurons and pC1d are interconnected and suggest that aIPg neurons may exert part of their effect by gating the flow of visual information to descending neurons. This work reveals important regulatory components of the neuronal circuitry that underlies female aggressive social interactions and provides tools for their manipulation (Schretter, 2020).
Social interactions pivot on an animal's experiences, internal states and feedback from others. This complexity drives the need for precise descriptions of behavior to dissect the fine detail of its genetic and neural circuit bases. In laboratory assays, male Drosophila melanogaster reliably exhibit aggression, and its extent is generally measured by scoring lunges, a feature of aggression in which one male quickly thrusts onto his opponent. This study introduces an explicit approach to identify both the onset and reversals in hierarchical status between opponents and observe that distinct aggressive acts reproducibly precede, concur or follow the establishment of dominance. Lunges were found to be insufficient for establishing dominance. Rather, lunges appear to reflect the dominant state of a male and help in maintaining his social status. Lastly, this study characterized the recurring and escalating structure of aggression that emerges through subsequent reversals in dominance. Collectively, this work provides a framework for studying the complexity of agonistic interactions in male flies, enabling its neurogenetic basis to be understood with precision (Simon, 2020).
Aggression involves both sexually monomorphic and dimorphic actions. How the brain implements these two types of actions is poorly understood. This study has identified three cell types that regulate aggression in Drosophila: one type is sexually shared, and the other two are sex specific. Shared common aggression-promoting (CAP) neurons mediate aggressive approach in both sexes, whereas functionally downstream dimorphic but homologous cell types, called male-specific aggression-promoting (MAP) neurons in males and fpC1 in females, control dimorphic attack. These symmetric circuits underlie the divergence of male and female aggressive behaviors, from their monomorphic appetitive/motivational to their dimorphic consummatory phases. The strength of the monomorphic → dimorphic functional connection is increased by social isolation in both sexes, suggesting that it may be a locus for isolation-dependent enhancement of aggression. Together, these findings reveal a circuit logic for the neural control of behaviors that include both sexually monomorphic and dimorphic actions, which may generalize to other organisms (Chiu, 2020).
In competition for food, mates and territory, most animal species display aggressive behavior through visual threats and/or physical attacks. Such naturally-complex social behaviors have been shaped by evolution. Environmental pressure, such as the one imposed by dietary regimes, forces animals to adapt to specific conditions and ultimately to develop alternative behavioral strategies. The quality of the food resource during contests influence animals' aggression levels. However, little is known regarding the effects of a long-term dietary restriction-based environmental pressure on the development of alternative fighting strategies. To address this, two lines were employed of the wild-type Drosophila melanogaster Canton-S (CS)which originated from the same population but raised under two distinct diets for years. One diet contained both proteins and sugar, while the second one was sugar-free. Male-male aggression assays were set up using both CS lines; differences were found in aggression levels and the fighting strategies employed to establish dominance relationships. CS males raised on a sugar-containing diet started fights with a physical attack and employed a high number of lunges for establishing dominance but displayed few wing threats throughout the fight. In contrast, the sugar-free-raised males favored wing threats as an initial aggressive demonstration and used fewer lunges to establish dominance, but displayed a higher number of wing threats. This study demonstrates that fruit flies that have been raised under different dietary conditions have adapted their patterns of aggressive behavior and developed distinct fighting strategies: one favoring physical attacks, while the other one favoring visual threats (Legros, 2020).
Many animal species show aggression to gain mating partners and to protect territories and other resources from competitors. Both male and female fruit flies of the species Drosophila melanogaster exhibit aggression in same-sex pairings, but the strategies used are sexually dimorphic. The biological basis for the differing aggression strategies, and the cues promoting one form of aggression over the other, are being explored. This study describes a line of genetically masculinized females that switch between male and female aggression patterns based on the sexual identity of their opponents. When these masculinized females are paired with more aggressive opponents, they increase the amount of male-like aggression they use, but do not alter the level of female aggression. This suggests that male aggression may be more highly responsive to behavioral cues than female aggression. Although the masculinized females of this line show opponent-dependent changes in aggression and courtship behavior, locomotor activity and sleep are unaffected. Thus, the driver line used may specifically masculinize neurons involved in social behavior. A discussion of possible different roles of male and female aggression in fruit flies is included in this paper. These results can serve as precursors to future experiments aimed at elucidating the circuitry and triggering cues underlying sexually dimorphic aggressive behavior (Monyak, 2021).
Aggressive behaviours are among the most striking displayed by animals, and aggression strongly impacts fitness in many species. Aggression varies plastically in response to the social environment, but direct tests of how aggression evolves in response to intra-sexual competition are lacking. This study investigated how aggression in both sexes evolves in response to the competitive environment, using populations of Drosophila melanogaster that were experimentally evolved under female-biased, equal, and male-biased sex ratios. After evolution in a female-biased environment-with less male competition for mates-males fought less often on food patches, although the total frequency and duration of aggressive behaviour did not change. In females, evolution in a female-biased environment-where female competition for resources is higher-resulted in more frequent aggressive interactions among mated females, along with a greater increase in post-mating aggression. These changes in female aggression could not be attributed solely to evolution either in females or in male stimulation of female aggression, suggesting that coevolved interactions between the sexes determine female post-mating aggression. Evidence was found consistent with a positive genetic correlation for aggression between males and females, suggesting a shared genetic basis. This study demonstrates the experimental evolution of a behaviour strongly linked to fitness, and the potential for the social environment to shape the evolution of contest behaviours (Bath, 2021).
Aggression between individuals of the same sex is almost ubiquitous across the animal kingdom. Winners of intrasexual contests often garner considerable fitness benefits, through greater access to mates, food, or social dominance. In females, aggression is often tightly linked to reproduction, with females displaying increases in aggressive behavior when mated, gestating or lactating, or when protecting dependent offspring. In the fruit fly, Drosophila melanogaster, females spend twice as long fighting over food after mating as when they are virgins. However, it is unknown when this increase in aggression begins or whether it is consistent across genotypes. This study shows that aggression in females increases between 2 to 4 hours after mating and remains elevated for at least a week after a single mating. In addition, this increase in aggression 24 hours after mating is consistent across three diverse genotypes, suggesting this may be a universal response to mating in the species. This study also reports the first use of automated tracking and classification software to study female aggression in Drosophila and assess its accuracy for this behavior. Dissecting the genetic diversity and temporal patterns of female aggression assists in better understanding its generality and adaptive function, and will facilitate the identification of its underlying mechanisms (Bath, 2020).
Gut microbiome profoundly affects many aspects of host physiology and behaviors. This study reports that gut microbiome modulates aggressive behaviors in Drosophila. Germ-free males showed substantial decrease in inter-male aggression, which could be rescued by microbial re-colonization. These germ-free males are not as competitive as wild-type males for mating with females, although they displayed regular levels of locomotor and courtship behaviors. it was further found that Drosophila microbiome interacted with diet during a critical developmental period for the proper expression of octopamine and manifestation of aggression in adult males. These findings provide insights into how gut microbiome modulates specific host behaviors through interaction with diet during development (Jia, 2021).
Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. A systematic, unbiased screen identified OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. The results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM(+) neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals (Watanabe, 2017).
A rich behavioral literature has implicated OA in the control of invertebrate aggression, although the direction of its effects differs between species. Classic studies in lobsters have shown that injection of OA into the hemolymph promotes a subordinate-like posture, while injection of serotonin (5HT) produces a dominant-like posture. In contrast, hemolymph injections of OA in crickets restore aggressiveness to subordinated animals, mimicking the arousing effects of episodes of free flight. OA has also been suggested to play a role in aggressive motivation restored to defeated crickets by residency in a shelter. In Drosophila, null mutations of TβH strongly suppressed aggressiveness, suggesting a positive-acting role for OA in flies as in crickets. Interestingly, intra-hypothalamic infusion of NE in mammals can also enhance aggression. However, little is known about the neurons on which these amines act directly to influence aggression, in any organism (Watanabe, 2017).
This study applied a novel, unbiased approach to identify OARNs relevant to aggression in Drosophila. Importantly this screen was based not on mutations in OAR genes, but rather upon genetic silencing of neurons that express GAL4 under the control of different OAR gene cis-regulatory modules (CRMs). This screen was agnostic with respect to which OAR gene is involved, or in which neurons that OAR is expressed. It yielded a small population of male-specific, FruM+ OA-sensitive neurons, called aSP2, the activity of which is required for normal levels of aggressiveness. No significant change in UWEs (male-male courtship) was observed when these neurons were activated or silenced. Nevertheless, neuronal silencing in the parental R47A04-GAL4 line increased male-male courtship, perhaps reflecting an inhibitory role for non-aSP2 neurons in that line. Therefore, while it is not possible to completely exclude a role for aSP2 neurons to suppress male-male courtship, the evidence does not strongly support it (Watanabe, 2017).
Multiple lines of evidence suggest that R47A04aSP2 neurons are indeed OA responsive, likely via OAMB. First, these neurons are labeled by a CRM from the Oamb gene. Second, RNAi-mediated knockdown of Oamb in R47A04 neurons reduced aggression, phenocopying the effects of an Oamb null allele. (However, knockdown using the split-GAL4 R47A04aSP2driver only yielded a trend to reduced aggression that did not reach significance, perhaps reflecting a floor effect in this assay.) Third, overexpression of Oamb cDNAs in these neurons using R47A04-GAL4 rescued the Oamb null mutant and enhanced the effect of OA feeding to promote aggression. Fourth,R47A04aSP2 neurons were activated by bath-applied OA in brain explants, and this effect was also blocked by RNAi-mediated knockdown of Oamb. Taken together, these data strongly suggest that aSP2 neurons respond directly to OA to mediate its effects on aggression, although they do not exclude a role for other OA-responsive non-aSP2 neurons in line R47A04. While it was not possible to definitively establish which of the 27 different classes of OANs in Drosophila provide functional OA input to aSP2 cells, some candidate OA neurons labeled in retrograde PA-GFP experiments (VUM and VPM) have previously been implicated in aggression (Watanabe, 2017).
In Drosophila OA, like NE in vertebrates, is thought to promote arousal. Consistent with such a function, OAergic fibers are broadly distributed across the entire Drosophila CNS, as are NE fibers in vertebrates. Thus OARNs could enhance aggression by increasing arousal, and there is evidence for such a function in crickets. However, manipulations of R47A04aSP2neurons that increased or decreased aggression did not affect locomotion, circadian activity, or sleep. This suggests that these neurons influence aggression directly and specifically, rather than by increasing generalized arousal. Other classes of OARNs not investigated in this study have been implicated in sleep-wake arousal (Watanabe, 2017).
Does OA promote aggression in a permissive or instructive manner? While it is clear that OA synthesis and release are required for aggression in Drosophila, whether increasing OA suffices to promote aggression is less clear. It was reported that NaCh Bac-mediated activation of Tdc2-GAL4 neurons enhanced aggression, but the current study neither this manipulation, nor activation of Tdc2 neurons using dTrpA1 or Chrimson, yielded consistent effects. Thus, while OA is essential for normal levels of aggression, it is not clear whether it plays an instructive role to promote this behavior (Watanabe, 2017).
In principle, OA RNs could act directly in command-like neurons that mediate aggression, or rather in cells that play a modulatory role. It was found that aggression was increased by tonically enhancing the excitability of R47A04aSP2 neurons using NaChBac, but not by phasically activating them optogenetically, arguing against a command-like function. Furthermore, the influence of TK FruM neurons, which do promote aggression when phasically activated, was not dependent on the activity of R47A04aSP2 neurons, indicating that the latter are not functionally downstream of the former. Together, these data argue against a role for R47A04aSP2 cells as command-like neurons, or as direct outputs of command neurons, for aggression. Rather, these cells exert a modulatory influence on agonistic behavior (Watanabe, 2017).
In searching for neurons that may interact with R47A04aSP2 cells in their modulatory capacity, P1 neurons, a FruM+ population of 20 neurons/hemibrain was identified that controls male courtship, but which can also promote aggression when activated. It has been argued that this aggression-promoting effect is due to a subset of FruM neurons in the GAL4 line used in these studies, R71G01-GAL4. However, this study shows that conditional expression of FLP-ON Chrimson in a subset of neurons within the R71G01-GAL4 population using Fru-FLP. Nevertheless, these data do not exclude that the aggression-promoting neurons in the P1 cluster expressed Fru-FLP only transiently during development, nor do they exclude the possibility that different subpopulations of neurons within line R71G01 control courtship versus aggression; further studies will be required to resolve these issues (Watanabe, 2017).
The P1 cluster is known to project to downstream cells that are specific for courtship . The present study provides the first evidence that cells in this cluster also functionally activate (and physically contact) aggression-specific neurons. However, due to limitations of the genetic reagents employed, it is not certain that the behavioral, physiological, and anatomical interactions with aSP2 cells demonstrated in this study are all mediated by the same subset of neurons in the P1 cluster. With this caveat in mind, these data suggest that aSP2 neurons are functionally downstream of both a subset(s) of P1 neurons, as well as of OA neurons (Watanabe, 2017).
Feeding flies OA potentiated the activation of R47A04aSP2
neurons by P1 neuron stimulation, in brain explants. Furthermore, activation of aggression by P1 stimulation was enhanced and suppressed by pharmacologically increasing or decreasing OA signaling, respectively. While some off-target effects of the drugs, or an action on non-aSP2 neurons expressing OARs, cannot be excluded these pharmacologic effects were overridden by opposite-direction genetic manipulations of R47A04aSP2neuronal activity. Whether P1 neurons and OANs normally activate aSP2 neurons in vivo, simultaneously or sequentially, is not yet clear. Nevertheless it is striking that P1 and Tdc2 putative inputs occupy adjacent regions of aSP2 dendrites. Taken together, these findings suggest that aSP2 cells may serve as a node through which OA can bias output from a multifunctional social behavior network involving P1 neurons, in a manner that favors aggression. However, aSP2 neurons themselves do not appearto control directly the choice between mating and fighting (Watanabe, 2017).
Male-specific cuticular hydrocarbons such as 7-tricosene (7-T) are known to be required for aggression. Interestingly, it has recently been shown that gustatory neurons expressing Gr32a, which encodes a putative 7-T receptor, innervate OANs in the SEZ; these OANs are activated by 7-T in a Gr32a-dependent manner. SEZ-innervating OANs include the VPM/VUM subsets seen in PA-GFP retrograde labeling experiments. These data raise the possibility that R47A04aSP2 neurons might be targets of VPM/VUM OANs activated by 7-T. If so, then they could provide a potential link between the influence of male-specific pheromones, OA, and central aggression circuitry. Studies of NE neurons in vertebrates have led to a prevailing view that this neuromodulator is released in a diffuse, 'sprinkler system'-like manner to control brain-wide states like arousal. Recent studies in Drosophila indicate that the broad, brain-wide distribution of OAergic fibers reflects the superposition of close to 30 anatomically distinct subclasses of OANs). The data presented in this study reveal a high level of circuit specificity for OARNs that mediate the effects of OA on aggression, mirroring the anatomical and functional specificity of OANs reported to control this behavior. If this anatomical logic is conserved, then such circuit specificity may underlie the actions of NE in mammals to a greater extent than is generally assumed (Watanabe, 2017).
Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both 'classical' and 'modulatory' signals that could produce diverse and/or complementary effects in associated circuits. This study establishes that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identifed the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. These findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network (Sherer, 2020).
Addressing the functional complexities of 'one neuron, multiple transmitters' is critical to understanding how neuron communication, circuit computation, and behavior can be regulated by a single neuron. Over many decades, significant progress has been made elucidating the functional properties of neurons co-expressing neuropeptides and small molecule neurotransmitters, where the neuropeptide acts as a co-transmitter and modulates the action of the neurotransmitter. Only recently have studies begun to examine the functional significance of co-transmission by a fast-acting neurotransmitter and a slow-acting monoamine (Sherer, 2020).
This study has demonstrated that OA neurons express dVGLUT and has utilized a new genetic tool to remove dVGLUT in OA-glutamate neurons. Quantifying changes in the complex social behaviors of aggression and courtship revealed that dVGLUT in brain OGNs is required to promote aggressive behavior and a specific behavioral pattern, the lunge. In contrast, males deficient for dVGLUT function do not exhibit an increase in inter-male courtship. These results establish a previously undetermined role for dVGLUT in OA neurons located in the adult brain and reveal glutamate uncouples aggression from inter-male courtship. It has been suggested that classical neurotransmitters and monoamines present in the same neuron modulate each other's packaging into synaptic vesicles or after release via autoreceptors. For example, a reduction of dVGLUT in DA-glutamate neurons resulted in decreased AMPH-stimulated hyperlocomotion in Drosophila and mice suggesting a key function of dVGLUT is the mediation of vesicular DA content. In this study, the independent behavioral changes suggests enhancing the packaging of OA into vesicles is not the sole function of dVGLUT co-expression and suggests differences in signaling by OA from OGNs on courtship-related circuitry (Sherer, 2020).
Co-transmission can generate distinct circuit-level effects via multiple mechanisms. One mechanism includes spatial segregation; the release of two neurotransmitters or a neurotransmitter and monoamine from a single neuron occurring at different axon terminals or presynaptic zones. Recent studies examining this possible mechanism have described (1) the release of GLU and DA from different synaptic vesicles in midbrain dopamine neurons and (2) the presence of VMAT and VGLUT microdomains in a subset of rodent mesoaccumbens DA neurons. This study expressed a new conditionally expressed epitope-tagged version of VMAT in OGNs and visualized endogenous dVGLUT via antibody labeling. Within OGNs, the colocalization of VMAT and dVGLUT puncta was not complete suggesting the observed behavioral phenotype differences may be due to spatial differences in OA signaling (Sherer, 2020).
A second mechanism by which co-transmission may generate unique functional properties relies on activating distinct postsynaptic receptors. In Drosophila, recent work has identified a small population of male-specific neurons that express the alpha-like adrenergic receptor, OAMB, as aggression-promoting circuit-level neuronal targets of OA modulation independent of any effect on arousal and separately knockdown of the Rdl GABAa receptor in a specific doublesex+ population stimulated male aggression (Watanabe, 2017). Future experiments identifying downstream targets that express both glutamate and octopamine receptors would be informative, as well as using additional split-Gal4 lines to determine if segregation of transporters is a hallmark of the majority of OGNs. Finally, a third possible mechanism is Glu may be co-released from OGNs and act on autoreceptors to regulate presynaptic OA release (Sherer, 2020).
Deciphering the signaling complexity that allows neural networks to integrate external stimuli with internal states to generate context-appropriate social behavior is a challenging endeavor. Neuromodulators including monoamines are released to signal changes in an animal's environment and positively or negatively reinforce network output. In invertebrates, a role for OA in responding to external chemosensory cues as well as promoting aggression has been well-established. In terms of identifying specific aggression circuit-components that utilize OA, previous results determined OA neurons directly receive male-specific pheromone information and the aSP2 neurons serve as a hub through which OA can bias output from a multi-functional social behavior network towards aggression. The ability of OA to bias behavioral decisions based on positive and negative reinforcement was also recently described for food odors. In vertebrates, it has been proposed that DA-GLU cotransmission in the NAc medial shell might facilitate behavioral switching. The finding that the majority of OA neurons are glutamatergic, suggests that the complex social behavior of aggression may rely on small subsets of neurons that both signal the rapid temporal coding of critical external stimuli as well as the frequency coding of such stimuli resulting in the enhancement of this behavioral network. One implication of the finding regarding the separable OA-dependent inhibition of inter-male courtship is the possibility of identifying specific synapses or axon terminals that when activated gate two different behavioral outcomes. A second implication is that aggressive behavior in other systems may be modified by targeting GLU function in monoamine neurons (Sherer, 2020).
Finally, monoamine-expressing neurons play key roles in human behavior including aggression and illnesses that have an aggressive component such as depression, addiction, anxiety, and Alzheimer's. While progress is being made in addressing the functional complexities of dual transmission, the possible pathological implications of glutamate co-release by monoamine neurons remains virtually unknown. Analyzing the synaptic vesicle and release properties of monoamine-glutamate neurons could offer new possibilities for therapeutic interventions aimed at controlling out-of-context aggression (Sherer, 2020).
Aggression is known to be regulated by pheromonal information in many species. But how central brain neurons processing this information modulate aggression is poorly understood. Using the fruit fly model of Drosophila melanogaster, this study systematically characterize the role of a group of sexually dimorphic GABAergic central brain neurons, popularly known as mAL, in aggression regulation. The mAL neurons are known to be activated by male and female pheromones. This report shows that mAL activation robustly increases aggression, whereas its inactivation decreases aggression and increases intermale courtship, a behavior considered reciprocal to aggression. GABA neurotransmission from mAL is crucial for this behavior regulation. Exploiting the genetic toolkit of the fruit fly model, a small group of approximately three to five GABA(+) central brain neurons were found with anatomical similarities to mAL. Activation of the mAL resembling group of neurons is necessary for increasing intermale aggression. Overall, these findings demonstrate how changes in activity of GABA(+) central brain neurons processing pheromonal information, such as mAL in Drosophila melanogaster, directly modulate the social behavior of aggression in male-male pairings (Sengupta, 2022).
Aggression is an adaptive set of behaviors that allows animals to compete against one another in an environment of limited resources. Typically, males fight for mates and food, whereas females fight for food and nest sites. Although the study of male aggression has been facilitated by the extravagant nature of the ritualized displays involved and the remarkable armaments sported by males of many species the subtler and rarer instances of inter-female aggression have historically received much less attention. In Drosophila, females display high levels of complex and highly structured aggression on a food patch with conspecific females. Other contexts of female aggression have not been explored. Indeed, whether females compete for mating partners, as males do, has remained unknown so far. In the present work, it is reported that Drosophila melanogaster females reliably display aggression toward mating pairs. This aggressive behavior is regulated by mating status and perception of mating opportunities and relies heavily on olfaction. Furthermore, food odor in combination with OR47b-dependent fly odor sensing is required for proper expression of aggressive behavior. Taken together, this study describes a social context linked to reproduction in which Drosophila females aspiring to mate produce consistent and stereotyped displays of aggression. These findings open the door for further inquiries into the neural mechanisms that govern this behavior (Gaspar, 2002).
Aggression is a behavior common in most species; it is controlled by internal and external drivers, including hormones, environmental cues, and social interactions, and underlying pathways are understood in a broad range of species. To date, though, effects of gut microbiota on aggression in the context of gut-brain communication and social behavior have not been completely elucidated. This study examined how manipulation of Drosophila melanogaster microbiota affects aggression as well as the pathways that underlie the behavior in this species. Male flies treated with antibiotics exhibited significantly more aggressive behaviors. Furthermore, they had higher levels of cVA and (Z)-9 Tricosene, pheromones associated with aggression in flies, as well as higher expression of the relevant pheromone receptors and transporters OR67d, OR83b, GR32a, and LUSH. These findings suggest that aggressive behavior is, at least in part, mediated by bacterial species in flies (Grinberg, 2022)
Male sexual aggression towards females is a form of sexual conflict that can result in increased fitness for males through forced copulations (FCs) or coercive matings at the cost of female lifetime fitness. This study used male fruit flies (Drosophila melanogaster) as a model system to uncover the genomic contributions to variation in FC, both due to standing variation in a wild population, and due to plastic changes associated with variation in social experience. RNAseq was used to analyse whole-transcriptome differential expression (DE) in male head tissue associated with evolved changes in FC from lineages previously selected for high and low FC rate and in male flies with varying FC rates due to social experience. Hundreds of genes were identified associated with evolved and plastic variation in FC, however only a small proportion (27 genes) showed consistent DE due to both modes of variation. This trend of low concordance in gene expression effects across broader sets of genes was confirmed to be significant in either the evolved or plastic analyses using multivariate approaches. The gene ontology terms neuropeptide hormone activity and serotonin receptor activity were significantly enriched in the set of significant genes. Of seven genes chosen for RNAi knockdown validation tests, knockdown of four genes showed the expected effect on FC behaviours. Taken together, these results provide important information about the apparently independent genetic architectures that underlie natural variation in sexual aggression due to evolution and plasticity (Scott, 2022).
Females increase aggression for mating opportunities and for acquiring reproductive resources. Although the close relationship between female aggression and mating status is widely appreciated, whether and how female aggression is regulated by mating-related cues remains poorly understood. This study reports an interesting observation that Drosophila virgin females initiate high-frequency attacks toward mated females. 11-cis-vaccenyl acetate (cVA), a male-derived pheromone transferred to females during mating, was shown to promote virgin female aggression. A cVA-responsive neural circuit was subsequently reveal consisting of four orders of neurons, including Or67d, DA1, aSP-g, and pC1 neurons, that mediate cVA-induced virgin female aggression. It was also determined that aSP-g neurons release acetylcholine (ACh) to excite pC1 neurons via the nicotinic ACh receptor nAChRα7. Together, beyond revealing cVA as a mating-related inducer of virgin female aggression, these results identify a neural circuit linking the chemosensory perception of mating-related cues to aggressive behavior in Drosophila females (Wan, 2023).
Neuropeptides influence animal behaviors through complex molecular and cellular mechanisms, the physiological and behavioral effects of which are difficult to predict solely from synaptic connectivity. Many neuropeptides can activate multiple receptors, whose ligand affinity and downstream signaling cascades are often different from one another. Although it is known that the diverse pharmacological characteristics of neuropeptide receptors form the basis of unique neuromodulatory effects on distinct downstream cells, it remains unclear exactly how different receptors shape the downstream activity patterns triggered by a single neuronal neuropeptide source. This study uncovered two separate downstream targets that are differentially modulated by Tachykinin, an aggression-promoting neuropeptide in Drosophila. Tachykinin from a single male-specific neuronal type recruits two separate downstream groups of neurons. One downstream group, synaptically connected to the tachykinergic neurons, expresses the receptor TkR86C and is necessary for aggression. In this case, tachykinin supports cholinergic excitatory synaptic transmission between the tachykinergic and TkR86C downstream neurons. The other downstream group expresses the TkR99D receptor and is recruited primarily when tachykinin is over-expressed in the source neurons. Differential activity patterns in the two groups of downstream neurons correlate with levels of male aggression triggered by the tachykininergic neurons. These findings highlight how the amount of neuropeptide released from a small number of neurons can reshape the activity patterns of multiple downstream neuronal populations. The results lay the foundation for further investigations into the neurophysiological mechanism by which a neuropeptide controls complex behaviors (Wohl, 2023).
Drosophila melanogaster has long been used to demonstrate the effect of inbreeding, particularly in relation to reproductive fitness and stress tolerance. In comparison, less attention has been given to exploring the influence of inbreeding on the innate behavior of D. melanogaster. In this study, multiple replicates of six different types of crosses were set in pair conformation of the laboratory-maintained wild-type D. melanogaster. This resulted in progeny with six different levels of inbreeding coefficients. Larvae and adult flies of varied inbreeding coefficients were subjected to different behavioral assays. In addition to the expected inbreeding depression in the-egg to-adult viability, noticeable aberrations were observed in the crawling and phototaxis behaviors of larvae. Negative geotactic behavior as well as positive phototactic behavior of the flies were also found to be adversely affected with increasing levels of inbreeding. Interestingly, positively phototactic inbred flies demonstrated improved learning compared to outbred flies, potentially the consequence of purging. Flies with higher levels of inbreeding exhibited a delay in the manifestation of aggression and courtship. In summary, these findings demonstrate that inbreeding influences the innate behaviors in D. melanogaster, which in turn may affect the overall biological fitness of the flies (Amanullah, 2023).
Inhibitors of enzymes that inactivate amine neurotransmitters (dopamine, serotonin), such as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), are thought to increase neurotransmitter levels and are widely used to treat Parkinson's disease and psychiatric disorders, yet the role of these enzymes in regulating behavior remains unclear. This study investigated the genetic loss of a similar enzyme in the model organism Drosophila melanogaster. Because the enzyme Ebony modifies and inactivates amine neurotransmitters, its loss is assumed to increase neurotransmitter levels, increasing behaviors such as aggression and courtship and decreasing sleep. This study quantitatively confirmed that ebony mutants exhibited increased aggressive behaviors such as boxing but also decreased courtship behaviors and increased sleep. Through tissue-specific knockdown, the role of ebony in these behaviors was found to be specific to glia. Unexpectedly, direct measurement of amine neurotransmitters in ebony brains revealed that their levels were not increased but reduced. Thus, increased aggression is the anomalous behavior for this neurotransmitter profile. It was further found that ebony mutants exhibited increased aggression only when fighting each other, not when fighting wild-type controls. Moreover, fights between ebony mutants were less likely to end with a clear winner than fights between controls or fights between ebony mutants and controls. In ebony vs. control fights, ebony mutants were more likely to win. Together, these results suggest that ebony mutants exhibit prolonged aggressive behavior only in a specific context, with an equally dominant opponent (Pantalia, 2023).
For social animals, the genotypes of group members affect the social environment, and thus individual behavior, often indirectly. This study used genome-wide association studies (GWAS) to determine the influence of individual vs. group genotypes on aggression in honey bees. Aggression in honey bees arises from the coordinated actions of colony members, primarily nonreproductive "soldier" bees, and thus, experiences evolutionary selection at the colony level. This study shows that individual behavior is influenced by colony environment, which in turn, is shaped by allele frequency within colonies. Using a population with a range of aggression, individual whole genomes were sequenced and for genotype-behavior associations were looked for within colonies in a common environment. There were no significant correlations between individual aggression and specific alleles. By contrast, strong correlations were found between colony aggression and the frequencies of specific alleles within colonies, despite a small number of colonies. Associations at the colony level were highly significant and were very similar among both soldiers and foragers, but they covaried with one another. One strongly significant association peak, containing an ortholog of the Drosophila sensory gene dpr4 (see Dips and Dprs) on linkage group (chromosome) 7, showed strong signals of both selection and admixture during the evolution of gentleness in a honey bee population. Links were thus found between colony genetics and group behavior and also, molecular evidence was found for group-level selection, acting at the colony level. It is concluded that group genetics dominates individual genetics in determining the fatal decision of honey bees to sting (Avalos, 2020).
Aggressive behavior is regulated by various neuromodulators such as neuropeptides and biogenic amines. This study found that the neuropeptide Drosulfakinin (Dsk) modulates aggression in Drosophila melanogaster. Knock-out of Dsk or Dsk receptor CCKLR-17D1 reduced aggression. Activation and inactivation of Dsk-expressing neurons increased and decreased male aggressive behavior, respectively. Moreover, data from transsynaptic tracing, electrophysiology and behavioral epistasis reveal that Dsk-expressing neurons function downstream of a subset of P1 neurons (P1(a)-splitGAL4) to control fighting behavior. In addition, winners show increased calcium activity in Dsk-expressing neurons. Conditional overexpression of Dsk promotes social dominance, suggesting a positive correlation between Dsk signaling and winning effects. The mammalian ortholog CCK has been implicated in mammal aggression, thus this work suggests a conserved neuromodulatory system for the modulation of aggressive behavior (Wu, 2020).
Aggression is a common innate behavior in most vertebrate and invertebrate species and a major driving force for natural and sexual selections. It is a critical behavior for defense against conspecifics to obtain food resources and mating partners (Wu, 2020).
Aggressive behavior of fruit flies was first reported by Alfred Sturtevant. Since then, a number of ethological and behavioral studies in flies pave the way for using Drosophila as a genetic system to study aggression. Drosophila provides an excellent system to manipulate genes and genetically defined populations of neurons, leading to the identification of multiple genes and neural circuits that control aggression. The neural circuits of aggression involve the peripheral sensory systems that detect male-specific pheromones and auditory cues necessary for aggression, a subset of P1 neurons, pCd in the central brain controlling aggressive arousal, and AIP neurons controlling threat displays. Aggression is modulated by various monoamines and neuropeptides. Octopamine, serotonin and dopamine are important neuromodulators for fly aggression and the specific aminergic neurons that control aggression have been identified. Neuropeptides such as tachykinin and neuropeptide F are required for normal male aggression. Cholecystokinin (CCK) is a neuropeptide that is linked to a number of psychiatric disorders and involved in various emotional behaviors in humans and other mammals. Infusion of CCK induces panic attack in humans. Enhanced CCK level is detected in a rat model of social defeat. CCK is implicated to act in the periaqueductal gray to potentiate defensive rage behavior in cats. In addition, CCK is a satiety signal in a number of species. Silencing CCK-like peptide Drosulfakinin could decrease satiety signaling and increase intake of food in flies. Co-injection of nesfatin-1 and CCK8 decreased food intake in Siberian sturgeon (Acipenser baerii) (Wu, 2020).
This study investigated the roles of cholecysokinin-like peptide Drosulfakinin (Dsk) in Drosophila aggression. Knock-outs and GAL4 knock-ins were generated for Dsk and candidate Dsk receptors. Loss-of-function in either Dsk or Dsk receptor CCKLR-17D1 reduces aggression. Thermogenetic activation of DskGAL4 neurons promotes aggression, while silencing these neurons suppresses aggression. Transsynaptic tracing, electrophysiology and behavioral epistasis experiments were performed to illustrate that Dsk-expressing neurons are functionally connected with a subset of P1 neurons (P1a-splitGAL4, 8 ~ 10 pairs of P1 Neurons) and act downstream of a subset of P1 neurons to control fighting behavior. Furthermore, this study found that winners show increased calcium activity in Dsk-expressing neurons and that conditional overexpression of Dsk promotes winning effects, implicating an important role of the Dsk system in the establishment of social hierarchy during fly fighting. Previously the mamalian ortholog CCK has been implicated in aggression, thus this work suggests a potentially conserved neural pathway for the modulation of aggressive behavior (Wu, 2020).
This study has systematically dissected the neuromodulatory roles of the Dsk system in fly aggression. At the molecular level, Dsk neuropeptide and its receptor CCKLR-17D1 are important for fly aggression. At the circuit level, Dsk-expressing neurons function downstream of a subset of P1 neurons (P1a-splitGAL4, 8 ~ 10 pairs of P1 Neurons) to control aggression. Furthermore, winners show increased calcium activity of Dsk-expressing neurons. Conditional overexpression of Dsk promotes winner effects, suggesting that Dsk is closely linked to the establishment of dominance. Taken together, these results elucidate the molecular and circuit mechanism underlying male aggression and suggest that cholecystokinin-like neuropeptide is likely to be evolutionarily conserved for the neuromodulation of aggression (Wu, 2020).
A neural circuitry controlling aggression should be composed of multiple modules that extend from sensory inputs to motor outputs. A variety of peptidergic and aminergic neurons are implicated in fly aggression, but it is not clear how these modulatory neurons integrate input signals from other neural circuits to signal specific physiological states. The current data from circuit tracing, functional connectivity and behavioral epistasis suggest that Dsk-expressing neurons function downstream of a subset of P1 neurons and likely summate inputs from a subset of P1 neurons to signal an internal state of aggression. Activation of a subset of P1 neurons triggers both aggression and courtship. Interestingly, while the aggression-promoting effect of activating a subset of P1 neurons is dramatically suppressed by the loss of the Dsk gene, the courtship-promoting effect remains intact in the ΔDsk mutant background. On the other hand, recent study suggested that Dsk neurons might function to antagonize P1 neurons on regulating male courtship. This dissociation suggests that while a subset of P1 neurons signal an arousal state facilitating both aggression and courtship, the Dsk system acts downstream of a subset of P1 neurons specifically required for aggression. It worth mentioning that the P1a-splitGAL4 used in those studies not only labeled a small subset of Fru+ neurons but also several Fru- neurons, and previous study on pC1 neurons suggested that Fru+ pC1 neurons promote courtship and Fru- pC1 neurons promote aggression, so further studies are needed to characterize whether different subset of P1a-splitGAL4 labeled neurons are function differently on aggression and how Dsk system are involved. In addition, it remains unknown whether the Dsk system is responsible for integrating the sensory inputs and arousal state related to aggression, and how it connects to other components of the aggression circuitry, such as Tk neurons and AIP neurons (Wu, 2020).
As a caveat, it has been reported that Dsk is involved in feeding behavior. The current experiment also reproduced the result that ΔDsk mutants show increased food consumption in the Capillary Feeder (CAFE) essay. Previous studies reported a positive correlation between the body size of flies and the aggression level, suggesting that the modulational effects of DSK neurons on aggression and feeding can be separated. Further research is required to disentangle the relationship between DSK neurons modulating aggression and those regulating feeding (Wu, 2020).
This study classified the eight DSK neurons into three subtypes (Type I, II and III) based on the morphology of the neurites or two subtypes (DSK-M and DSK-L) based on the location of the cell bodies. Interestingly, these subtypes also show functional difference in modulating aggression and differential connectivity with the a subset of P1 neurons. Note that Type I and II neurons correspond to DSK-M and Type III neurons correspond to DSK-L. The finding that DSK-M neurons showed stronger responses to a subset of P1 neurons activation is consistent with the behavioral results of the flip-out experiment, in which Type I and II neurons, but not Type III, are critical to aggression. In future research, it would be interesting to use intersect method to more specifically label and manipulate the DSK neuron subtypes (Wu, 2020).
Previous study implicated that the cholecystokinin system is closely linked with various human psychiatric disorders, such as bipolar disorder and panic attacks. Interestingly, verbal aggression is promoted by the administration of cholecystokinin tetrapeptide in human subjects. In cats, cholecystokinin agonists potentiate the defensive rage behavior while the cholecystokinin antagonists suppress it. These results reveal that cholecystokinin-like peptide Dsk and Dsk receptor CCKLR-17D1 are important for Drosophila aggression. In addition, increased calcium activity in Dsk-expressing neurons coincides with winner states. Thus, the cholecystokinin system is linked to aggressive behavior in a variety of species and is likely to be an evolutionarily conserved pathway for modulating aggressiveness (Wu, 2020).
It has long been noticed that hierarchical relationships could be established during fly fights, with winners remaining highly aggressive and winning the subsequent encounters, and losers retreating and losing second fights. The winner state is perceived as a reward signal while losing experience is aversive (Kim, 2018). The establishment of social hierarchy is only observed in males, and this male-specific feature of fly aggression is specified by fruitless. However, neural correlates of dominance have not been reported. In this study, Using a transcriptional reporter of intracellular calcium (TRIC), it was found that winners display increased calcium activity in the median Dsk-expressing neurons. Moreover, conditional overexpression of Dsk specifically in the adult stage increases the flies' aggressiveness and makes them more likely to win against opponents without Dsk overexpression. Thus, both the enhanced Dsk signaling in the brain and the winning-promoting effect of conditional overexpression supported that the Dsk system may be involved in the establishment of social hierarchy during fly aggression (Wu, 2020).
Social isolation strongly modulates behavior across the animal kingdom. This study utilized the fruit fly Drosophila melanogaster to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide Drosulfakinin (Dsk), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors. Dsk knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of Dsk neurons each similarly increased isolation-driven aggression. The results suggest a U-shaped dependence of social isolation-induced aggressive behavior on Dsk signaling, similar to the actions of many neuromodulators in other contexts (Agrawal, 2020).
This study has shown that knockdown of the neuropeptide Dsk or its receptor CCKLR-17D1 in the pars intercerebralis (PI) increases social isolation-driven aggression of male flies. Moreover, Dsk appears to act in a U-shaped fashion, with both knockdown (the current results) and overexpression (Williams, 2014) increasing aggression. Dsk neuronal activity follows a similar trend, with both activation and silencing increasing aggression. Williams (2014) overexpressed the Dsk transcript in the PI region, which resulted in increased aggression; furthermore, activation of PI neurons was also shown to increase aggression in a separate study. Taken together, this suggests that the primary role of these neurons in this context is indeed production and secretion of Dsk. Transcription factors in the fly PI neurons regulating aggression were recently identified, and it was shown that activation of PI neurons increases aggression. However, the downstream neuropeptides were not known. The current findings identify Dsk as a key neuropeptide expressed in the PI region that regulates aggression. Further work will be required to delineate the aggression-modulating functions, if any, of other neuropeptides also secreted from the PI region (Agrawal, 2020).
A recent neural activation screen (Asahina, 2014) explored the role of neuropeptides in aggression in Drosophila, but investigated only group-housed flies. Intriguingly, Asahina (2014) identified tachykinin signaling in the lateral protocerebrum and did not find increased aggression in group-housed (GH) flies upon activation of Dsk neurons. Thus, male-male aggression in GH and solitary-housed (SH) flies appears to be controlled by different neuropeptides in different brain regions. The absence of Dsk neurons from the screen results in GH flies (Asahina, 2014), combined with the results showing suppressed aggression in GH flies regardless of Dsk transcription or neural activity, suggests a mechanism that overrides Dsk function (Agrawal, 2020).
Downregulation of the Dsk receptor CCKLR-17D1 in Dsk/Dilp2 neurons also increased aggression, consistent with the observation that some neuropeptidergic neurons, e.g. those for neuropeptide F, neuropeptide Y and FMRFamide, have receptors to modulate their signaling in an autocrine manner. However, pan-neuronal downregulation of CCKLR-17D1 receptor did not affect aggression, suggesting potential antagonistic effects outside Dsk/Dilp2 neurons (Agrawal, 2020).
To address potential developmental effects of Dsk signaling, it would be useful to temporally restrict neural perturbation. However,efforts to conditionally silence Dsk+ neurons only in the adult using temperature-sensitive UAS-Kir2.1-GAL80ts were inconclusive, because prolonged exposure of flies (including controls) to the permissive temperature (30°C) affected their basal locomotion and aggression. To address potential off-target targets of the TRiP Dsk RNAi line, another RNAi line against Dsk (VDRC 14201) was tested but no significant reduction in Dsk levels were observed. It would be useful to test other Dsk loss-of-function alleles in future. However, the current conclusions about the involvement of Dsk in isolation-mediated aggression are supported by the similar effects from knockdown of its receptor CCKLR-17D1, as well as silencing and activation of Dsk-secreting neurons (Agrawal, 2020).
The U-shaped ('hormetic') response of the aggression phenotype to both Dsk levels and Dsk+ neuronal activity is similar to such responses seen for NPF and dopamine neurons in Drosophila aggression. Such effects are not unexpected, given the ubiquity of such hormetic responses in neuromodulator signaling pathways and receptors in general. At the level of individual G-protein coupled receptors, such U-shaped responses (low-dose agonism, high-dose antagonism) arise directly from equations considering receptor expression level and the effects of receptor activation on downstream signaling pathways. At the circuit level, it is thought that such U-shaped responses help to maintain neuronal activity patterns, and the resulting behaviors, near homeostatic optima, with deviations resulting in negative feedback (Agrawal, 2020).
There have been a number of prior studies on the genetic basis of aggression in Drosophila, many of them performed with DNA microarrays rather than with RNA-seq, that record counts for specific transcripts of interest. These studies counted all transcripts within cells. Four such studies have been performed in recent years, each identifying a large number of putative aggression-related genes. Given that the involvement of Dsk in aggression is quite context-specific. Asahina (2014) explicitly ruled out involvement of Dsk in aggression of group-housed flies. Therefore, it is perhaps unsurprising that it was not found in several of the screens. In fact, the only one of these four studies to uncover Dsk was the one that utilized socially isolated flies, strengthening the notion that Dsk specifically links social isolation to aggression. It was this link with social behavior that drew attention to Dsk, and indeed the current experiments bear out that this function is mediated through activity in the brain. The PI region has been shown to be the seat of regulation of many other social and sexually dimorphic behaviors (Agrawal, 2020).
In mammals, the Dsk homologue cholecystokinin (CCK) and its receptors regulate aggression, anxiety and social-defeat responses. For instance, intravenous injection of the smallest isoform, CCK-4, in humans reliably induces panic attack and is often used to screen anxiolytic drug candidates. However, in other contexts, such as in mating and juvenile play, CCK encodes strong positive valence. CCK colocalizes with dopamine in the ventral striatum, and microinjection of CCK into the rat nucleus accumbens phenocopies the effects of dopamine agonists, increasing attention and reward-related behaviors, further supporting its role in positive valence encoding. CCK actions differ across brain regions, in a context-dependent manner. For instance, time pinned (negative valence) during rough-and-tumble play correlated with increased CCK levels in the posterior cortex and decreased levels in hypothalamus. However, lower hypothalamic CCK also correlated with positive-valence play aspects including dorsal contacts and 50 kHz ultrasonic vocalizations. Thus, CCK can encode both positive- and negative-valence aspects of complex behaviors differentially across the brain. As with many neuromodulators, CCK appears to act in a U-shaped fashion, with increases and decreases of signaling from baseline levels often producing similar phenotypes (Agrawal, 2020).
Taken together, the results suggest an evolutionarily conserved role for neuropeptide signaling through the Drosulfakinin pathway (homologue of cholecystokinin) in promoting aggression. Intriguingly, this pathway only seems active in socially isolated flies; in socially enriched flies, aggression is controlled by tachykinin (a.k.a. Substance P) signaling. The PI region, in which the Dsk/Dilp2 neurons reside, has considerable similarities with the hypothalamus, a brain region crucial for regulating aggression in mammals, with the most relevant activity localized to the ventrolateral subdivision of the ventromedial hypothalamus, where CCK neurons reside. Thus, the predominant aggression-regulating mechanism in rodents bears strong homology to the fly pathway regulating aggression of socially deprived, but not socially enriched, individuals (Agrawal, 2020).
Animal aggressiveness is controlled by genetic and environmental factors. Among environmental factors, social experience plays an important role in modulating aggression in vertebrates and invertebrates. In Drosophila, pheromonal activation of olfactory neurons contributes to social suppression of aggression. While it was reported that impairment in vision decreases the level of aggression in Drosophila, it remains unknown if visual perception also contributes to the modulation of aggression by social experience. This study investigated the role of visual perception in the control of aggression in Drosophila. Several genetic approaches were taken to examine the effects of blocking visual circuit activity on fly aggressive behaviors. In wild type, group housing greatly suppresses aggressiveness. Loss of vision by mutating the ninaB gene does not affect social suppression of fly aggression. Similar suppression of aggressiveness by group housing is observed in fly mutants carrying a mutation in the eya gene leading to complete loss of eyes. Chronic visual loss does not affect the level of aggressiveness of single-housed flies that lack social experience prior to behavioral tests. When visual circuit activity is acutely blocked during behavioral test, however, single-housed flies display higher levels of aggressiveness than that of control flies. It is concluded that visual perception does not play a major role in social suppression of aggression in Drosophila. For single-housed individuals lacking social experience prior to behavioral tests, visual perception decreases the level of aggressiveness (Ramin, 2014).
How brains are hardwired to produce aggressive behavior, and how aggression circuits are related to those that mediate courtship, is not well understood. This large-scale screen for aggression-promoting neurons in Drosophila identifies several independent hits that enhance both inter-male aggression and courtship. Genetic intersections reveal that P1 interneurons, previously thought to exclusively control male courtship, are responsible for both phenotypes. The aggression phenotype is fly-intrinsic, and requires male-specific chemosensory cues on the opponent. Optogenetic experiments indicate that P1 activation promotes aggression vs. wing extension at low vs. high thresholds, respectively. High frequency photostimulation promotes wing extension and aggression in an inverse manner, during light ON and OFF, respectively. P1 activation enhances aggression by promoting a persistent internal state, which could endure for minutes prior to social contact. Thus P1 neurons promote an internal state that facilitates both aggression and courtship, and can control these social behaviors in a threshold-dependent manner (Hoopfer, 2015).
This study describes the first large-scale neuronal activation screen for aggression neurons in Drosophila. Using the thermosensitive ion channel dTrpA1, a collection of over 3,000 GAL4 lines was screened for flies that exhibited increased fighting following thermogenetic neuronal activation.
Among ~20 hits obtained, three exhibited both increased aggression and male-male courtship behavior. Intersectional refinement of expression patterns using split-GAL4 indicated that both social behaviors are controlled, in all three hits, by a subpopulation of ~8-10 P1 neurons per hemibrain. P1 cells are male-specific, FruM+ interneurons that integrate pheromonal and visual cues to promote male courtship behavior. The results indicate, surprisingly, that at least a subset of P1 neurons, previously thought to control exclusively courtship, can promote male aggression as well. Moreover, it was shown that they exert this influence by inducing a persistent fly-intrinsic state, lasting for minutes, that enhances these behaviors. These data define a sexually dimorphic neural circuit node that may link internal states to the control of mating and fighting, and identify a potentially conserved circuit 'motif' for the control of social behaviors (Hoopfer, 2015).
Individuals are not merely subject to their social environments; they choose and create them, through a process called social environment (or social niche) construction. This study identified multiple mechanisms of social environment construction that differ among natural genotypes of Drosophila melanogaster and investigated their consequences for the development of aggressive behavior. Male genotypes differed in the group sizes that they preferred and in their aggressive behavior; both of these behaviors influenced social experience, demonstrating that these behaviors function as social environment-constructing traits. Further, the effects of social experience-as determined in part by social environment construction-carried over to affect focal male aggression at a later time and with a new opponent. These results provide manipulative experimental support for longstanding hypotheses in psychology, that genetic variation in social environment construction has a causal role in behavioral development. More broadly, these results imply that studies of the genetic basis of complex traits should be expanded to include mechanisms by which genetic variation shapes the environments that individuals experience (Saltz, 2016).
Aggressive behavior is observed in many animals, but its intensity differs between species. In a model animal of genetics, Drosophila melanogaster, genetic basis of aggressive behavior has been studied intensively, including transcriptome analyses to identify genes whose expression level was associated with intra-species variation in aggressiveness. However, whether these genes are also involved in the evolution of aggressiveness among different species has not been examined. De novo transcriptome analysis was performed in this study in the brain of Drosophila prolongata to identify genes associated with the evolution of aggressiveness. Males of D. prolongata were hyper-aggressive compared with closely related species. Comparison of the brain transcriptomes identified 21 differentially expressed genes in males of D. prolongata. They did not overlap with the list of aggression-related genes identified in D. melanogaster, suggesting that genes involved in the evolution of aggressiveness were independent of those associated with the intra-species variation in aggressiveness in Drosophila. Although females of D. prolongata were not aggressive as the males, expression levels of the 21 genes identified in this study were more similar between sexes than between species (Kudo, 2017).
By selection of winners of dyadic fights for 35 generations, this study
generated a hyperaggressive Bully
line of flies that almost always win fights against the parental wild-type
Canton-S stock. Maintenance of the Bully phenotype is temperature
dependent during development: the phenotype is lost when flies are reared
at 19 °C. No similar effect is seen with the parent line. This difference
served as the basis for RNA-seq experiments which identify a limited
number of genes that are differentially expressed by twofold or greater in
the Bullies; one of these is a putative transmembrane transporter, CG13646,
which shows consistent and reproducible twofold down-regulation in
Bullies. The causal effect of this gene on the phenotype was examined with
a mutant line for CG13646, and with an RNAi approach. In all
cases, reduction in expression of CG13646 by approximately half
leads to a hyperaggressive phenotype partially resembling that seen in the
Bully flies. This gene is a member of a very interesting family of solute
carrier proteins (SLCs), some of which have been suggested as being
involved in glutamine/glutamate and GABA cycles of metabolism in
excitatory and inhibitory nerve terminals in mammalian systems (Chowdhury, 2017).
Mutations in proline dehydrogenase (PRODH) are linked to behavioral alterations in schizophrenia and as part of DiGeorge and velo-cardio-facial syndromes, but the role of PRODH in their etiology remains unclear. This study established a Drosophila model to study the role of PRODH in behavioral disorders. The distribution was determined of the Drosophila PRODH homolog slgA in the brain, and knock-down and overexpression of human PRODH and slgA in the lateral neurons ventral (LNv) were shown to lead to altered aggressive behavior. SlgA acts in an isoform-specific manner and is regulated by casein kinase II (CkII). The data suggest that these effects are, at least partially, due to effects on mitochondrial function. It is thus shown that precise regulation of proline metabolism is essential to drive normal behavior and Drosophila aggression is a model behavior relevant for the study of mechanisms impaired in neuropsychiatric disorders (Zwarts, 2017).
Human psychiatric disorders such as schizophrenia, bipolar disorder and attention-deficit/hyper-activity disorder often include adverse behaviors including increased aggressiveness. Individuals with psychiatric disorders often exhibit social withdrawal, which can further increase the probability of conducting a violent act. This study used the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP) to investigate the genetic basis of variation in male aggressive behavior for flies reared in a socialized and socially isolated environment. Genetic variation was identified for aggressive behavior, as well as significant genotype by social environmental interaction (GSEI); i.e., variation among DGRP genotypes in the degree to which social isolation affected aggression. Genome-wide association (GWA) analyses was performed to identify genetic variants associated with aggression within each environment. Genomic prediction was used to partition genetic variants into gene ontology (GO) terms and constituent genes, and GO terms and genes were identified with high prediction accuracies in both social environments and for GSEI. The top predictive GO terms significantly increased the proportion of variance explained, compared to prediction models based on all segregating variants. Genomic prediction was performed across environments, and genes in common were identified between the social environments which turned to be enriched for genome-wide associated variants. A large proportion of the associated genes have previously been associated with aggressive behavior in Drosophila and mice. Further, many of these genes have human orthologs that have been associated with neurological disorders, indicating partially shared genetic mechanisms underlying aggression in animal models and human psychiatric disorders (Rohde, 2017).
Understanding how social experiences are represented in the brain and shape future responses is a major challenge in the study of behavior. This problem was addressed by studying behavioral, transcriptomic and epigenetic responses to intrusion in honey bees. Previous research showed that initial exposure to an intruder provokes an immediate attack; this study now shows that this also leads to longer-term changes in behavior in the response to a second intruder, with increases in the probability of responding aggressively and the intensity of aggression lasting 2 and 1 h, respectively. Previous research also documented the whole-brain transcriptomic response; it is now shown that in the mushroom bodies (MBs) there are 2 waves of gene expression, the first highlighted by genes related to cytoskeleton remodeling, and the second highlighted by genes related to hormones, stress response and transcription factors (TFs). Overall, 16 of 37 (43%) of the TFs whose cis-motifs were enriched in the promoters of the differentially expressed genes (DEGs) were also predicted from transcriptional regulatory network analysis to regulate the MB transcriptional response, highlighting the strong role played by a relatively small subset of TFs in the MB's transcriptomic response to social challenge. Whole brain histone profiling showed few changes in chromatin accessibility in response to social challenge; most DEGs were 'ready' to be activated. These results show how biological embedding of a social challenge involves temporally dynamic changes in the neurogenomic state of a prominent region of the insect brain that are likely to influence future behavior (Shpigler, 2017).
Aggression is a complex social behavior that is widespread in nature. To date only a limited number of genes that affect aggression have been identified, in large part because the complexity of the phenotype makes screening difficult and time consuming regardless of the species that is studied. Aggressive group-housed Drosophila melanogaster males inflict damage on each other's wings; wing damage negatively affects their ability to fly and mate. Using this wing-damage phenotype, males from ~1,400 chemically mutagenized strains were screened and ~40 mutant strains were found with substantial wing damage. Five of these mutants also had increased aggressive behavior. To identify the causal mutation in one of the top aggressive strains, whole genome sequencing and genomic duplications rescue strategies were used. A novel mutation was identified in the voltage-gated potassium channel Shaker (Sh) and a nearby previously identified Sh mutation was also shown to exhibit increased aggression. This simple screen can be used to dissect the molecular mechanisms underlying aggression (Davis, 2017).
Carbon dioxide is produced by many organic processes and is a convenient volatile cue for insects that are searching for blood hosts, flowers, communal nests, fruit and wildfires. Although Drosophila melanogaster feed on yeast that produce CO2 and ethanol during fermentation, laboratory experiments suggest that walking flies avoid CO2. This study resolved this paradox by showing that both flying and walking Drosophila find CO2 attractive, but only when they are in an active state associated with foraging. Their aversion to CO2 at low-activity levels may be an adaptation to avoid parasites that seek CO2, or to avoid succumbing to respiratory acidosis in the presence of high concentrations of CO2 that exist in nature. In contrast to CO2, flies are attracted to ethanol in all behavioural states, and invest twice the time searching near ethanol compared to CO2. These behavioural differences reflect the fact that ethanol is a unique signature of yeast fermentation, whereas CO2 is generated by many natural processes. Using genetic tools, it was determined that the evolutionarily conserved ionotropic co-receptor IR25a is required for CO2 attraction, and that the receptors necessary for CO2 avoidance are not involved in this attraction. This study lays the foundation for future research to determine the neural circuits that underlie both state- and odorant-dependent decision-making in Drosophila (van Breugel, 2018).
D. melanogaster feed, mate and deposit eggs on rotting fruit. Between 10 and 14 days later, the next generation of flies must locate a fresh ferment. Because of the high volatility of CO2, the emission of CO2 is greatest near the start of fermentation, whereas ethanol emission increases more slowly. Other odours associated with fermentation (for example, acetic acid and ethyl acetate) form later, when bacteria break down ethanol. In trap assays, Drosophila show a preference for two-day-old apple juice ferments compared to older solutions, which suggests that they might be attracted to CO2. Although it is difficult to estimate concentrations of CO2 in wild ferments, the CO2 concentration in bottles commonly used to rear flies has been determined to be 0.5-1% (van Breugel, 2018).
This evidence that CO2 might attract Drosophila contradicts previous studies conducted using small chambers. To study how flies respond to odours under more-ethological conditions, the flight trajectories was recorded of flies in a wind tunnel that contained a landing platform, which was programmed to periodically release plumes of CO2 or ethanol. Both odours elicited approaches, landings and explorations of a conspicuous visual feature, which is consistent with previous experiments with flies and mosquitoes. Flies were more likely to approach the platform or dark spot in the presence of ethanol compared to CO2, but were equally likely to land in response to either odour (van Breugel, 2018).
To quantify the behaviour of flies after they land, a platform was designed that is suitable for automated tracking. At a flow rate of 60 ml min-1 CO2, the CO2 concentration near the surface of the platform was approximately 3%. After landing near a source of CO2, ethanol or apple cider vinegar, flies exhibited a local search behaviour that was similar to so-called 'dances'. Flies spent twice the amount of time exploring platforms that emitted ethanol compared to CO2 or vinegar. Flies approached a source that emitted both ethanol and CO2 more frequently than they approached vinegar, or either odour alone. Vinegar elicited smaller local searches and slightly fewer approaches compared to CO2, consistent with the hypothesis that vinegar might indicate a less favourable, late-stage ferment. Flies spent significantly less time standing still on the platform in the presence of CO2 compared to any other odour, with a mean walking speed > 2 mm s-1 (van Breugel, 2018).
One previous study showed that Drosophila are attracted to CO2 while flying on a tether. The current results confirm this observation in freely flying flies; however, it was also found that flies remain attracted to CO2 after they land, which contradicts previous studies. One potential explanation is that flies in constrained walking chambers might behave differently to those that arrived on the open wind tunnel platform after tracking the odour plume and landing. To test this hypothesis, an enclosed arena was built in which flies were unable to fly, and they were presented with pulses of 5% CO2. Groups of 10 starved flies presented with CO2 after acclimating to the arena for 10 min exhibited aversion, as previously reported. However, if allowed to acclimate in the chamber for two hours, the flies exhibited attraction to CO2 (van Breugel, 2018).
To study the response of these flies in more detail, the behaviour of flies was recorded for 20 h, while providing 10-min presentations of CO2 from alternating sides of the arena every 40 min. To control for humidity, 20 ml min-1 of H2O-saturated air was continuously pumped through the odour ports on both sides of the chamber. The flies exhibited a clear circadian rhythm within the chamber, as indicated by their mean walking speed. At times of peak activity-near dusk and dawn-flies showed a strong initial attraction to CO2, which decayed stereotypically during the 10-min presentation. At times of low activity-at mid-day and during the night-flies exhibited a mild aversion to CO2. Starving flies for 24 h before the experiment changed their activity profile, resulting in a slightly elevated attraction during the night. Ethanol, by contrast, elicited sustained attraction regardless of baseline activity (van Breugel, 2018).
To probe this relationship between activity and CO2 attraction, the temperature was increased and the wind speed-manipulations that are known to elevate and depress activity were elevated, respectively. When wthe bulk-flow rate was increased to 100 ml min-1, flies exhibited a peak walking speed of about 1.5 mm s-1 at dusk-nearly half the speed measured at a flow rate of 20 ml min-1. Instead of showing attraction, these flies exhibited aversion to 5% CO2, although they were still attracted to ethanol. This result helps to explain why previous studies that used higher flows (100-1,000 ml min-1) to present CO2 observed aversion. To further explore the effect of wind, the aristae of the flies, which destroys their primary means of detecting airflow but does not interfere with the detection of odours, were clipped. The flies without aristae exhibited the same walking speed and attraction to CO2 at the high flow rate as was exhibited by normal flies at the low flow rate. Warming flies with intact aristae to 32°C also increased their baseline activity and recovered their attraction to CO2 at the higher flow rate. Pooling data across all experimental conditions, it was found that flies were attracted to CO2 when they had a baseline walking speed that was above about 2.4 mm s-1. This value is similar to the walking speed that was observed in the wind tunnel assay, which was higher for CO2 than the other odours. To confirm that activity-dependent attraction to CO2 is not a function of social interactions, 29 single flies, which behaved similarly to the cohorts of 10, were tested. Three concentrations of CO2 (1.7%, 5% and 15%) were also tested and found that the 5% concentration elicited the strongest response, consistent with wind tunnel experiments (van Breugel, 2018).
Although the responses of flies to ethanol and CO2 were similar at stimulus onset, attraction to ethanol was more sustained. The time course of behaviour was notably similar in the walking arena and wind tunnel, which suggests that the behavioural dynamics of olfactory attraction are robust to the stimulus environment and may represent an adaptation for using information that broad (CO2) and more specific (ethanol) odorants provide (van Breugel, 2018).
Previous research shows that CO2 aversion is mediated by Gr63a and Gr21a receptors; high concentrations of CO2 are also detected by an acid-sensitive ionotropic receptor, IR64a10. In the current assay, mutant flies that lack the IR64a receptor showed no significant change in their behaviour compared to wild type. Consistent with previous work, mutants that lack the Gr63a receptor exhibited no aversion to CO2; however, they were still attracted to CO2 when active. Mutant flies that are homozygous for both Gr63a and IR64a behaved similarly to the Gr63a mutants. It is noteworthy that the characteristic decaying time course of attraction was unaffected in Gr63a mutants, even though these flies showed no aversion. Thus, the decay in attraction to CO2 is not caused by an increase in aversion over time (van Breugel, 2018).
Given that CO2 attraction is not mediated by Gr63a, Gr21a or IR64a, it was of interest to confirm that the attraction is indeed a chemosensory response. To determine whether CO2 attraction is mediated by either an olfactory or ionotropic receptor, a mutant was tested that lacks the olfactory and ionotropic co-receptors (Orco, IR25a and IR8a) as well as Gr63a. These near-anosmic mutants exhibited no detectable behavioural response to CO2. Flies in which the third antennal segment was surgically removed showed no response to CO2, despite normal levels of activity. Together with the arista ablations, these experiments show that CO2 attraction is mediated by receptors on the third antennal segment. To further confirm this, each co-receptor mutant was tested individually, and it was found that mutants that lack IR25a did not exhibit wild-type CO2 attraction, whereas Orco and IR8a mutants did. Mutant flies that lack Orco, IR8a and Gr63a also exhibit wild-type attraction to CO2, confirming that the only required co-receptor is IR25a. IR25a has previously been implicated in a wide range of behaviours, including temperature and humidity sensation. The temperature in the arena near the CO2 port was measured, and no change was found in temperature as a result of the stimulus. To eliminate the possibility of a humidity artifact, an IR40a mutant, which still exhibited attraction to CO2, was tested. In summary, these experiments show that CO2 attraction is mediated by a separate chemosensory pathway from that which governs aversion, and that CO2 attraction requires the IR25a co-receptor. IR25a is the most highly conserved olfactory receptor among insects. It is possible that other insect species that lack Gr63a26 but that still respond to CO2 use the same IR25a-dependent pathway. Unfortunately, the GAL4 driver for the IR25a promoter is expressed only in about half of the endogenous IR25a-expressing neurons, which makes imaging experiments that aim to identify which glomerulus is involved difficult at this time (van Breugel, 2018).
The finding that active flies are attracted to CO2 makes ethological sense, given that CO2 is generated by yeast-the preferred food of these flies. Why it might be that Drosophila avoid CO2 when in a low-activity state was considered. Flies do not exhibit this state-dependent reaction to ethanol and vinegar; perhaps the aversion to CO2 at low activity is an adaptation that minimizes encounters with parasites that seek CO2. Alternatively, the behaviour may help flies to avoid respiratory acidosis when near high concentrations of CO2 within the environment. Previous studies have suggested that CO2 serves as an aversive pheromone by which stressed flies signal others to flee a local environment. However, an alternative explanation is that agitated flies release CO2 not as a social signal but simply because it is present in their tracheal system owing to their process of discontinuous respiration. Further work on this state-dependent reaction to CO2 will require experiments that carefully consider the natural ethology of the flies (van Breugel, 2018).
Foraging is a goal-directed behavior that balances the need to explore the environment for resources with the need to exploit those resources. In Drosophila melanogaster, distinct phenotypes have been observed in relation to the foraging (for) gene, labeled the rover and sitter. Adult rovers explore their environs more extensively than do adult sitters. This study explored whether this distinction would be conserved in humans. A distinction was used from regulatory mode theory between those who "get on with it," so-called locomotors, and those who prefer to ensure they "do the right thing," so-called assessors. In this logic, rovers and locomotors share similarities in goal pursuit, as do sitters and assessors. Genetic variation in PRKG1, the human ortholog of for, is associated with preferential adoption of a specific regulatory mode. Next, participants performed a foraging task to see whether genetic differences associated with distinct regulatory modes would be associated with distinct goal pursuit patterns. Assessors tended to hug the boundary of the foraging environment, much like behaviors seen in Drosophila adult sitters. In a patchy foraging environment, assessors adopted more cautious search strategies maximizing exploitation. These results show that distinct patterns of goal pursuit are associated with particular genotypes of PRKG1, the human ortholog of for (Struk, 2019).
Mechanosensation provides animals with important sensory information in addition to olfaction and gustation during feeding behavior. This study used Drosophila larvae to investigate the role of softness sensing in behavior and learning. In the natural environment, Drosophila larvae need to dig into soft foods for feeding. Finding foods that are soft enough to dig into is likely to be essential for their survival. This study reports that Drosophila larvae can discriminate between different agar concentrations and prefer softer agar. Interestingly, it was shown that larvae on a harder place search for a softer place using memory associated with an odor and that they evaluate foods by balancing softness and sweetness. These findings suggest that Drosophila larvae integrate mechanosensory information with chemosensory input while foraging. Moreover, it was found that the larval preference for softness is affected by genetic background (Kudow, 2019).
Foraging animals may benefit from remembering the location of a newly discovered food patch while continuing to explore nearby. For example, after encountering a drop of yeast or sugar, hungry flies often perform a local search. That is, rather than remaining on the food or simply walking away, flies execute a series of exploratory excursions during which they repeatedly depart and return to the resource. Fruit flies, Drosophila melanogaster, can perform this food-centered search behavior in the absence of external landmarks, instead relying on internal (idiothetic) cues. This path-integration behavior may represent a deeply conserved navigational capacity in insects, but its underlying neural basis remains unknown. This study used optogenetic activation to screen candidate cell classes and found that local searches can be initiated by diverse sensory neurons. Optogenetically induced searches resemble those triggered by actual food, are modulated by starvation state, and exhibit key features of path integration. Flies perform tightly centered searches around the fictive food site, even within a constrained maze, and they can return to the fictive food site after long excursions. Together, these results suggest that flies enact local searches in response to a wide variety of food-associated cues and that these sensory pathways may converge upon a common neural system for navigation. Using a virtual reality system, this study demonstrated that local searches can be optogenetically induced in tethered flies walking on a spherical treadmill, laying the groundwork for future studies to image the brain during path integration (Corfas, 2019).
To discover sensory pathways triggering local search, the behavior of individual female flies was tracked as they explored a circular arena with a featureless optogenetic activation zone at its center. The assay consists of an initial 10-min baseline control period followed by a 30-min period during which animals receive a 1-s pulse of red light (628 nm) whenever they enter the activation zone. For flies expressing the light-sensitive channel CsChrimson in food-sensing neurons, the activation zone should act as a patch of fictive food, potentially able to elicit a local search. Using this setup, gustatory, olfactory, and reward-signaling neurons were screened to identify cell classes that trigger local search. Aside from the light pulses used for optogenetic activation, the animals are in complete darkness and must rely on internal cues to navigate the open-field portion of the arena. To examine whether flies were conducting local search, trajectories were analyzed, beginning at the activation zone and ending at the arena edge. Prior to testing, flies were subjected to 33-42 h of starvation, during which they had access to water only (Corfas, 2019).
It is known that flies perform local searches after discovering a drop of sucrose, suggesting that sweet-sensing neurons may be sufficient to initiate this behavior. To test this, Gr43a-GAL4 >UAS-CsChrimson flies were used to activate fructose-sensing neurons whenever the flies entered the activation zone. Activation of these gustatory neurons triggered local searches remarkably similar to those previously observed in response to actual food, consisting of a series of excursions from and returns to the fictive food site. Unlike parental controls, Gr43a-GAL4 >UAS-CsChrimson flies extensively searched the area surrounding the activation zone (~30 cm2) after receiving a light pulse. These search trajectories were highly centered at the activation zone and consisted of numerous revisits to the activation zone-both features of local searches shown to require path integration. During local search, flies cumulatively walked ~30-300 cm (approximately 100-1,000 body lengths) before eventually straying to the arena edge. Prior studies, using another Gr43a-GAL4 line, suggest that Gr43a is expressed in neurons of the pharynx and brain that measure post-ingestive sugar levels as well as in sugar-sensing neurons in the periphery. However, this study found that nearly identical local searches are triggered by activation using the Gr5a-GAL4 driver, which only labels peripheral sugar-sensing neurons. Therefore, non-pharyngeal sugar sensors are capable of eliciting local search. This result is in disagreement with recent experiments suggesting that only pharyngeal sugar sensors can trigger local search; however, that study did not examine the effect of activating specific subsets of sugar-sensing neurons. The use of fictive food in the current experiments provides further evidence that flies are in fact using idiothetic path integration during local search rather than relying on external (allothetic) cues coming from an actual drop of food, such as visual appearance, odor or humidity gradients, or tracks of food residue deposited during prior search excursions (Corfas, 2019).
Previous work has shown that, compared to sucrose-triggered searches, searches triggered by a drop of 5% yeast solution elicits search trajectories that are even longer and include more revisits to the food, suggesting that proteinaceous food components may also initiate this behavior. Amino acids present in yeast are a coveted source of nutrition for mated females, which require a protein source to produce eggs. The ionotropic receptor Ir76b has been implicated in the detection of the taste of yeast, amino acids, carbonation, and other important nutrients, such as salt, polyamines, and fatty acids. Ir76b-GAL4>UAS-CsChrimson flies were tested; activation of these amino-acid sensors resulted in a modest increase in residence near the activation zone, due largely to the animals ceasing locomotor activity. However, the activation did not trigger a local search-the trajectories covered little distance and rarely included a revisit to the activation site. The failure to elicit local searches via activation of Ir76b-GAL4 may be due to the fact that this line labels a large population of neurons associated with diverse sensory functions. Indeed, whereas silencing of these neurons disrupts preference for feeding on yeast, direct activation of Ir76b-GAL4 neurons has never been shown to trigger feeding behavior (Corfas, 2019).
Food odorants also trigger search behavior in insects. In flight, for example, encounters with an odor plume elicit the stereotyped cast and surge maneuvers that enable insects to localize the source of an advected odor. Recent studies have demonstrated that this also occurs during walking-flies increase their turn rate when they exit a plume of apple cider vinegar (ACV) odor. Attraction to the smell of ACV in Drosophila is mediated primarily by neurons expressing the olfactory receptor Or42b. Optogenetic activation of Or42b-GAL4 neurons produces attraction behavior in flies, as does activation of Or59b-GAL4 neurons, which respond to acetate esters found in food odors. Simultaneous optogenetic activation of nearly all the olfactory receptor neurons via Orco-GAL4 also produces attraction in flies. This study tested whether these three classes of olfactory neurons could trigger a local search and found that activation of Orco- and Or59b-GAL4 neurons did not elicit searches. Activation of ACV-odor-sensing Or42b-GAL4 neurons resulted in increased residence near the activation zone, much like the results from searches triggered by activation of Ir76b-GAL4, but it did not produce local searches, according to the metrics used in this study (Corfas, 2019).
The water content of food drops might be enough to evoke local search. In Drosophila, water sensation is mediated by the osmosensitive ion channel ppk28, a member of the degenerin/epithelial sodium channel family. Activation of water-sensing ppk28-GAL4 neurons in food-deprived flies did not result in local search. This result is in agreement with previous behavioral experiments showing that Drosophila do not produce local search bouts after encountering a drop of pure water (Corfas, 2019).
It was hypothesized that reward-signaling neurons of the central nervous system might also trigger local searches. Neuropeptide F (NPF) is a highly conserved hunger-signaling neuropeptide that stimulates a variety of Drosophila behaviors, including feeding. NPF-GAL4 labels neurons in the posterior region of the Drosophila brain, and activation of these cells is rewarding in the context of olfactory conditioning. Much like Ir76b-GAL4 and Or42b-GAL4, activation of NPF-GAL4 neurons was shown to result in a modest increase in residence near the activation zone but not statistically significant local searches. Another set of reward-signaling neurons are the dopaminergic protocerebral anterior medial (PAM) neurons, which are activated by sugar ingestion and innervate the mushroom body, a structure critical for forming associative memories. Activation of PAM neurons via R58E02-GAL4 is known to mediate reward during olfactory conditioning, and silencing PAM neurons inhibits food occupancy during foraging. However,activation of R58E02-GAL4 neurons did not produce search behavior (Corfas, 2019).
Because local searches are initiated by activation of sugar receptors, it was hypothesized that starvation state may influence the extent of optogenetically induced searches. The influence of starvation has been observed for sucrose-induced searches in Drosophila as well as for protein- and water-induced searches in the blowfly (Phormia regina). Until this point, all of the experiments were conducted with animals allowed access only to water for 33-42 h preceding the trial. To examine the importance of starvation in promoting local search, activation of sugar-sensing Gr43a-GAL4 neurons was tested in flies that were reared continuously on food or starved for only 9-18 h. As expected, longer starvation times result in more extensive searches, with longer trajectories and more revisits to the activation zone (Corfas, 2019).
Tests were performed to see whether additional food deprivation could produce searches triggered by sensory pathways that had weak behavioral effects in the original screen. For these experiments, flies were starved for 7 days: 5 days with access to sucrose solution followed by 2 days with access to only water. Even in 7-day-starved animals, activation of amino-acid-sensing Ir76b-GAL4 neurons did not elicit substantial local search, despite the fact that protein-deprived mated females are known to develop a strong preference for amino-acid-containing food. However, activation of ACV-odor-sensing Or42b-GAL4neurons in 7-day-starved animals resulted in extensive and centralized local searches, comparable to those triggered by sugar-sensing neurons. This finding is consistent with work showing that starvation promotes food search behavior in Drosophila and that this effect is mediated by neuropeptidergic modulation of Or42b-GAL4 neuron activity. Local search was found to be triggered by activation of NPF-GAL4 neurons in 7-day-starved animals. This effect may be related to previous work showing that NPF-GAL4 neurons are activated by food odors in a starvation-dependent manner. Finally, it was found that nutritional deprivation can even produce searches triggered by water sensation-activation of ppk28-GAL4 neurons elicited robust local searches in animals subjected to a desiccating environment without food or water (Corfas, 2019).
Collectively, these results show that optogenetic activation of a variety of food-associated sensors can trigger searches and that this behavior is influenced by the internal nutritional state, much like searches triggered by actual food. Previous work has shown that flies regulate their consumption of sugar and yeast depending on whether they are deficient in that specific nutrient. When a fly finds a patch of yeast, for example, its decision to stay or leave depends strongly on whether it is deficient in amino acids. Thus, it may be that flies only perform local search when they experience a food cue associated with a nutrient they currently need. To test this hypothesis further, animals were starved on a synthetic food medium that allowed deprivation of flies specifically of either sugar or protein while supplying them with an otherwise complete and balanced diet. Activation of Gr43a-GAL4 sugar sensors produced robust local searches in animals subjected to sugar deprivation but not in animals subjected to long-term protein deprivation, suggesting that sugar-sensation-triggered searches are a response to a specific nutritional need. However, activation of ACV-odor-sensing Or42b-GAL4 neurons did not elicit searches in protein-deprived flies and elicited modest searches in sugar-deprived flies. However, activation of Or42b-GAL4 neurons does elicit robust searches in 7-day-starved animals subjected to a combination of sugar and protein deprivation. One possible functional explanation for these differing results is that substances sensed via contact, such as sugar or water, produce local search conditional on the specific internal state of that nutrient. In contrast, because the detection of volatile compounds is a less reliable indicator of the nutritional content of nearby food, the potency with which an odor can elicit local search may depend on the general nutritional state of the animal. Determining the ethological connection between food-triggered search and nutrient homeostasis will require further investigation (Corfas, 2019).
In his initial description of food-induced local search, it has been demonstrated that when a hungry blowfly discovers a drop of food, it performs a proboscis extension response (PER)-a reflex associated with appetitive cues. To explore the role of proboscis extension in optogenetically induced local search, tests were performed to see whether activation of each of these neuron classes elicits PER. As has been previously reported, activation of sugar-sensing Gr5a-GAL4 neurons elicits PER. This study found that activation of Gr43a-GAL4 neurons also elicits PER in a starvation-dependent manner, indicating that fructose triggers a feeding reflex similar to that triggered by other sugars. Activation of water sensors via ppk28-GAL4 neurons also resulted in PER, even in animals that had not been subjected to dry starvation. Activation of hunger-signaling neurons via NPF-GAL4 was found to elicit strong PER , demonstrating a novel function for these neurons. However, none of the other neuron classes in the screen consistently triggered PER, including Or42b-GAL4 neurons, indicating that local search can be initiated by receptors that do not by themselves elicit PER (Corfas, 2019).
Together, these results suggest that local searches are triggered by both contact chemosensory cues that signal that the fly is on food (e.g., water or sugar) as well as volatile cues that indicate food is nearby (e.g., the odor of ACV). It appears that flies even initiate local searches around a location associated with a rewarding stimulus (i.e., activation of NPF-GAL4 neurons) without accompanying activation of peripheral chemosensors. Although searches triggered by sugar, water, odor, and reward signaling appear broadly similar in these experiments, it is likely that their underlying behavioral structure differs. For example, it was found that whereas activation of Gr43a-, Gr5a-, Ir76b-, ppk28-, or NPF-GAL4 results in decreased locomotion or complete stopping, activation of ACV-odor-sensing Or42b-GAL4 neurons only elicits a brief startle response, similar to controls. The absence of slowing at the initiation of searches triggered by Or42b-GAL4 neurons is consistent with the interpretation that these searches are related to the casting behaviors elicited by loss of an odor plume. Future studies using the current paradigm may determine whether local searches triggered by distinct food-associated stimuli are stereotyped or are instead accomplished through diverse behavioral strategies (Corfas, 2019).
The results show that optogenetically induced local searches resemble those evoked by actual food, suggesting that flies are using idiothetic path integration to keep track of their position relative to the activation zone. Unlike in previous studies using real food, it was possible to monitor every occasion at which the fly senses the fictive food and can easily reinforce the memory of its location. Using the data from Gr43a-GAL4>UAS-CsChrimson animals in the original screen, the search trajectories occurring after each optogenetic stimulation were examined. Many of these trajectories lasted only a few seconds and covered only a few centimeters before the fly returned to the activation zone, thus receiving another optogenetic pulse. In many cases, however, flies performed a centered local search lasting minutes and covering hundreds of body lengths without an intervening optogenetic stimulation. This implies that a persistent internal representation of space underlies this behavior-without sustaining a centered search, flies would quickly stray to the arena edge. It was also observed that flies can update the center of their search upon discovering another activation zone, as has been found with searches around real food. Moreover, flies repeatedly shift the center of their search between activation zones, resembling experiments in which flies foraged among an array of food patches. In those experiments, flies were found to execute local searches around food sites but also discovered new food sites by exploring further-a behavior dependent on the internal nutrient state of the fly (Corfas, 2019).
The ability to execute a sustained search centered around a fictive food site in complete darkness, and moreover to carry this out in an environment with arbitrary geometric constraints, strongly suggests that flies can keep track of their location relative to the activation zone. This feat of idiothetic path integration has previously been compared to other insect behaviors, such as the foraging excursions of desert ants (Cataglyphis fortis), which routinely embark on long and winding runs through featureless terrain and yet are able to return to their nest in a direct path. To accomplish this, these ants keep track of both the distance and the direction of their travel, enabling them to integrate their position relative to a point of origin. During food-triggered searches, Drosophila may be using the same computational strategies as Cataglyphis and thus may be relying on the same highly conserved brain structures. In particular, studies point to the importance of the central complex-a sensorimotor hub of the insect brain that processes numerous aspects of locomotion, navigation, and decision making. Wedge neurons of the ellipsoid body encode azimuthal heading, potentially serving as a compass for path integration, celestial navigation, and other behaviors. Whereas less is known about how insects monitor odometry, it is thought that step counting can be achieved by using proprioceptive feedback or efferent copies of motor commands to integrate the distance traveled (Corfas, 2019).
It is proposed that optogenetic activation of Gr43a- and Gr5a-GAL4 sugar sensors may be a potent tool in future experiments seeking to characterize the neural implementation of path integration. Among the sensory pathways studied, these sweet-sensing neurons are the most reliable triggers of local search. However, the comparatively weaker searches elicited by activation of other neural pathways in this study may be a consequence of differences in the levels or anatomical depth of transgene expression rather than a reflection of their contribution to search behavior. Regardless of this experimental limitation, the fact that so many sensory modalities can trigger local searches suggests a convergence of these pathways onto the set of brain structures underlying navigation. This is consistent with anatomical studies of the central complex showing that it receives a variety of indirect sensory inputs as well as direct innervation by a large subset of NPF-GAL4 neurons (Corfas, 2019).
Elucidating the function of these circuits in path integration would require the ability to record neural activity in the Drosophila brain during local search. To this end, a preparation was developed to elicit local searches in a tethered fly walking on an air-suspended spherical treadmill. Similar setups have been successfully used to examine the path integration behavior of Cataglyphis ants. The fly's fictive path was reconstructed in real time using the FicTrac machine vision system, and a closed-loop program controlled optogenetic stimulation to present fictive food sites in the virtual 2D environment. As the fly walked, it would at certain points receive optogenetic stimulation; this virtual location became a fictive food site with an activation zone, thus mimicking the free-walking experiments. If the fly strayed far away from the activation zone, the fictive food site was abolished, and a new fictive food site was spawned soon after at the fly's new position. In this manner, flies experienced numerous virtual fictive food sites, and this study later examined whether they performed local searches in each case. Trials included an initial baseline period and a final post-experimental period during which mock fictive food sites were created, but the fly received no activation (Corfas, 2019).
Activation of Gr43a-GAL4 sugar sensors triggered local searches in the virtual environment that resembled searches in free-walking flies. The spatial scale of the searches was smaller than that of free-walking flies, perhaps due to increased error accumulation in idiothetic path integration caused by a mismatch between the fly's intended locomotion and the machine-reconstructed fictive path. Nevertheless, compared to the baseline and post conditions, and unlike parental controls, these searches covered greater distances, consisted of numerous revisits to the activation zone, and were highly centered at the fictive food site, suggesting that flies walking on the treadmill apparatus are capable of performing idiothetic path integration. As in free-walking flies, activation of Gr43a-GAL4 neurons in tethered flies elicited a reduction in walking speed and proboscis extension, accompanied by a strong startle response. These results are consistent with a recent report, using a similar setup in which flies explore a virtual environment with visual features. That study found that activation of sugar receptors triggers local search in a virtual landscape and that visual landmarks do not contribute to this behavior, supporting the hypothesis that flies are performing idiothetic path integration. Adapting these setups for use with a 2-photon microscope may permit future studies to examine how sensory stimuli, reward signals, spatial information, and memory are encoded and integrated to produce path integration (Corfas, 2019).
In summary, this study found that hungry flies initiate a sustained local search when they experience a fictive food stimulus. This search behavior appears to constitute a generalized foraging response, as it can be triggered by multiple types of food-associated neurons, including water-, sugar-, and vinegar-odor-sensing neurons, as well as by hunger-signaling neurons of the central nervous system. Like local searches triggered by real food, optogenetically induced local searches are modulated by internal nutritional state and show key features of idiothetic path integration. The results suggest that flies are able to keep track of their spatial position relative to a fictive food stimulus, even within a constrained maze. Long-lasting local search bouts can be initiated repeatedly by the brief activation of specific sets of neurons, and a system was developed to reconstitute this behavior in a tethered fly, thus establishing a promising entry point to tracing the neural pathways underlying path integration in insects (Corfas, 2019).
The ability to use memory to return to specific locations for foraging is advantageous for survival. Although recent reports have demonstrated that the fruit flies Drosophila melanogaster are capable of visual cue-driven place learning and idiothetic path integration, the depth and flexibility of Drosophila's ability to solve spatial tasks and the underlying neural substrate and genetic basis have not been extensively explored. This study shows that Drosophila can remember a reward-baited location through reinforcement learning and do so quickly and without requiring vision. This study found that both sighted and blind flies can learn-by trial and error-to repeatedly return to an unmarked location where a brief stimulation of the 0273-GAL4 neurons was available for each visit. Optogenetic stimulation of these neurons enabled learning by employing both a cholinergic pathway that promoted self-stimulation and a dopaminergic pathway that likely promoted association of relevant cues with reward. Lastly, inhibiting activities of specific neurons time-locked with stimulation of 0273-GAL4 neurons showed that mushroom bodies (MB) and central complex (CX) both play a role in promoting learning of the task. This work uncovered new depth in flies' ability to learn a spatial task and established an assay with a level of throughput that permits a systematic genetic interrogation of flies' ability to learn spatial tasks (Stern, 2019).
Animals socially interact during foraging and share information about the quality and location of food sources. The mechanisms of social information transfer during foraging have been mostly studied at the behavioral level, and its underlying neural mechanisms are largely unknown. Fruit flies have become a model for studying the neural bases of social information transfer, because they provide a large genetic toolbox to monitor and manipulate neuronal activity, and they show a rich repertoire of social behaviors. Fruit flies aggregate, they use social information for choosing a suitable mating partner and oviposition site, and they show better aversive learning when in groups. However, the effects of social interactions on associative odor-food learning have not yet been investigated. This paper presents an automated learning and memory assay for walking flies that allows the study of the effect of group size on social interactions and on the formation and expression of associative odor-food memories. Both inter-fly attraction and the duration of odor-food memory expression were found to increase with group size. This study opens up opportunities to investigate how social interactions during foraging are relayed in the neural circuitry of learning and memory expression (Sehdev, 2019).
Cooperative behavior can confer advantages to animals. This is especially true for cooperative foraging which provides fitness benefits through more efficient acquisition and consumption of food. This study has taken advantage of an experimental model system featuring cooperative foraging behavior in Drosophila. Under crowded conditions, fly larvae form coordinated digging groups (clusters). where individuals are linked together by sensory cues and group membership requires prior experience. However, fitness benefits of Drosophila larval clustering remain unknown. This study demonstrates that animals raised in crowded conditions on food partially processed by other larvae experience a developmental delay presumably due to the decreased nutritional value of the substrate. Intriguingly, same conditions promote the formation of cooperative foraging clusters which further extends larval stage compared to non-clustering animals. Remarkably, this developmental retardation also results in a relative increase in wing size, serving an indicator of adult fitness. Thus, this study finds that the clustering-induced developmental delay is accompanied by fitness benefits. Therefore, cooperative foraging, while delaying development, may have evolved to give Drosophila larvae benefits when presented with competition for limited food resources (Dombrovski, 2020).
Environmental stress is one of the important causes of biological dispersal. At the same time, the process of dispersal itself can incur and/or increase susceptibility to stress for the dispersing individuals. Therefore, in principle, stress can serve as both a cause and a cost of dispersal. These potentially contrasting roles of a key environmental stress (desiccation) were studied using Drosophila melanogaster. By modulating water and rest availability, it was asked whether (a) dispersers are individuals that are more susceptible to desiccation stress, (b) dispersers pay a cost in terms of reduced resistance to desiccation stress, (c) dispersal evolution alters the desiccation cost of dispersal, and (d) females pay a reproductive cost of dispersal. Desiccation was was found to be a clear cause of dispersal in both sexes, as both male and female dispersal propensity increased with increasing duration of desiccation. However, the desiccation cost of dispersal was male biased, a trend unaffected by dispersal evolution. Instead, females paid a fecundity cost of dispersal. The complex relationship between desiccation and dispersal, which can lead to both positive and negative associations, were discussed. Furthermore, the sex differences highlighted here may translate into differences in movement patterns, thereby giving rise to sex-biased dispersal patterns (Mishra, 2022).
Social rituals, such as male-male aggression in Drosophila, are often stereotyped and the component behavioral patterns modular. The likelihood of transition from one behavioral pattern to another is malleable by experience and confers flexibility to the behavioral repertoire. Experience-dependent modification of innate aggressive behavior in flies alters fighting strategies during fights and establishes dominant-subordinate relationships. Dominance hierarchies resulting from agonistic encounters are consolidated to longer-lasting, social-status-dependent behavioral modifications, resulting in a robust loser effect. This study shows that cAMP dynamics regulated by the calcium-calmodulin-dependent adenylyl cyclase, Rut, and the cAMP phosphodiesterase, Dnc, but not the Amn gene product, in specific neuronal groups of the mushroom body and central complex, mediate behavioral plasticity necessary to establish dominant-subordinate relationships. rut and dnc mutant flies were unable to alter fighting strategies and establish dominance relationships during agonistic interactions. This real-time flexibility during a fight was independent of changes in aggression levels. Longer-term consolidation of social status in the form of a loser effect, however, required additional Amn-dependent inputs to cAMP signaling and involved a circuit-level association between the alpha/beta and gamma neurons of the mushroom body. These findings implicate cAMP signaling in mediating the plasticity of behavioral patterns in aggressive behavior and in the generation of a temporally stable memory trace that manifests as a loser effect (Chouhan, 2017).
Multiple studies have investigated the mechanisms of aggressive behavior in Drosophila; however, little is known about the effects of chronic fighting experience. This study investigated if repeated fighting encounters would induce an internal state that could affect the expression of subsequent behavior. Wild-type males were trained to become winners or losers by repeatedly pairing them with hypoaggressive or hyperaggressive opponents, respectively. As described previously, it was observed that chronic losers tend to lose subsequent fights, while chronic winners tend to win them. Olfactory conditioning experiments showed that winning is perceived as rewarding, while losing is perceived as aversive. Moreover, the effect of chronic fighting experience generalized to other behaviors, such as gap-crossing and courtship. It is proposed that in response to repeatedly winning or losing aggressive encounters, male flies form an internal state that displays persistence and generalization; fight outcomes can also have positive or negative valence. Furthermore, it was shown that the activities of the PPL1-gamma1pedc dopaminergic neuron and the MBON-gamma1pedc>α/β mushroom body output neuron are required for aversion to an olfactory cue associated with losing fights (Kim, 2018).
Threat displays are a universal feature of agonistic interactions. Whether threats are part of a continuum of aggressive behaviors or separately controlled remains unclear. Threats were analyzed in Drosophila; they are triggered by male cues and visual motion, and comprised of multiple motor elements that can be flexibly combined. A cluster of approximately 3 neurons was isolated whose activity is necessary for threat displays but not for other aggressive behaviors, and whose artificial activation suffices to evoke naturalistic threats in solitary flies, suggesting that the neural control of threats is modular with respect to other aggressive behaviors. Artificially evoked threats suffice to repel opponents from a resource in the absence of contact aggression. Depending on its level of artificial activation, this neural threat module can evoke different motor elements in a threshold-dependent manner. Such scalable modules may represent fundamental "building blocks" of neural circuits that mediate complex multi-motor behaviors (Duistermars, 2018).
Natural aggressiveness is commonly observed in all animal species, and is displayed frequently when animals compete for food, territory and mating. Aggression is an innate behaviour, and is influenced by both environmental and genetic factors. However, the genetics of aggression remains largely unclear. This study identified the peacefulness (pfs) gene as a novel player in the control of male-male aggression in Drosophila. Mutations in pfs decreased intermale aggressiveness, but did not affect locomotor activity, olfactory avoidance response and sexual behaviours. pfs encodes for the evolutionarily conserved molybdenum cofactor (MoCo) synthesis 1 protein (Mocs1), which catalyzes the first step in the MoCo biosynthesis pathway. Neuronal-specific knockdown of pfs decreased aggressiveness. By contrast, overexpression of pfs greatly increased aggressiveness. Knocking down Cinnamon (Cin) catalyzing the final step in the MoCo synthesis pathway, caused a pfs-like aggression phenotype. In humans, inhibition of MoCo-dependent enzymes displays anti-aggressive effects. Thus, the control of aggression by Pfs-dependent MoCo pathways may be conserved throughout evolution (Ramin, 2019).
Pathological aggression is commonly associated with psychiatric and neurological disorders and can impose a substantial burden and cost on human society. Serotonin (5HT) has long been implicated in the regulation of aggression in a wide variety of animal species. In Drosophila, a small group of serotonergic neurons selectively modulates the escalation of aggression. This study has identified downstream targets of serotonergic input-two types of neurons with opposing roles in aggression control. The dendritic fields of both neurons converge on a single optic glomerulus LC12, suggesting a key pathway linking visual input to the aggression circuitry. The first type is an inhibitory GABAergic neuron: its activation leads to a decrease in aggression. The second neuron type is excitatory: its silencing reduces and its activation increases aggression. RNA sequencing (RNA-seq) profiling of this neuron type identified that it uses acetylcholine as a neurotransmitter and likely expresses 5HT1A, short neuropeptide F receptor (sNPFR), and the resistant to dieldrin (RDL) category of GABA receptors. Knockdown of RDL receptors in these neurons increases aggression, suggesting the possibility of a direct crosstalk between the inhibitory GABAergic and the excitatory cholinergic neurons. These data show further that neurons utilizing serotonin, GABA, ACh, and short neuropeptide F interact in the LC12 optic glomerulus. Parallel cholinergic and GABAergic pathways descending from this sensory integration area may be key elements in fine-tuning the regulation of aggression (Alekseyenko, 2019).
General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors (see Drosophila Rdl). In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. This study exploited the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, this study showed that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, the combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels (Ton, 2017).
Hierarchically organized brains communicate through feedforward (FF) and feedback (FB) pathways. In mammals, FF and FB are mediated by higher and lower frequencies during wakefulness. FB is preferentially impaired by general anesthetics in multiple mammalian species. This suggests FB serves critical functions in waking brains. The brain of Drosophila melanogaster is also hierarchically organized, but the presence of FB in these brains is not established. This study examined FB in the fly brain, by simultaneously recording local field potentials (LFPs) from low-order peripheral structures and higher-order central structures. The data was analyzed using Granger causality (GC), the first application of this analysis technique to recordings from the insect brain. The analysis revealed that low frequencies (0.1-5 Hz) mediated FB from the center to the periphery, while higher frequencies (10-45 Hz) mediated FF in the opposite direction. Further, isoflurane anesthesia preferentially reduced FB. The results imply that the spectral characteristics of FF and FB may be a signature of hierarchically organized brains that is conserved from insects to mammals. It is speculated that general anesthetics may induce unresponsiveness across species by targeting the mechanisms that support FB (Cohen, 2018).
Genetic variability affects the response to numerous xenobiotics but its role in the clinically-observed irregular responses to general anesthetics remains uncertain. To investigate the pharmacogenetics of volatile general anesthetics (VGAs), a Serial Anesthesia Array apparatus was developed to expose multiple Drosophila melanogaster samples to VGAs, and behavioral assays were carried out to determine pharmacokinetic and pharmacodynamic properties of VGAs. The VGAs isoflurane and sevoflurane were studied in four wild type strains from the Drosophila Genetic Reference Panel, two commonly used laboratory strains (Canton S and w1118), and a mutant in Complex I of the mitochondrial electron transport chain (ND2360114). In all seven strains, isoflurane was more potent than sevoflurane, as predicted by their relative lipid solubilities, and emergence from isoflurane was slower than from sevoflurane, reproducing cardinal pharmacokinetic and pharmacodynamic properties in mammals. In addition, ND2360114 flies were more sensitive to both agents, as observed in worms, mice, and humans carrying Complex I mutations. Moreover, substantial variability was found among the fly strains both in absolute and in relative pharmacokinetic and pharmacodynamic profiles of isoflurane and sevoflurane. These data indicate that naturally occurring genetic variations measurably influence cardinal pharmacologic properties of VGAs and that flies can be used to identify relevant genetic variations (Olufs, 2018).
General anaesthesia (GA) is implicated as a cause of postoperative sleep disruption and fatigue with part of the disturbance being attributed to a shift of the circadian clock. In this study, Drosophila melanogaster was used as a model to determine how Isoflurane affects the circadian clock at the behavioural and molecular levels. The response of the clock was measured at both of these levels caused by different durations and different concentrations of Isoflurane at circadian time 4 (CT4). Once characterized, the duration and concentration constants (at 2% in air for 6 h) were held and the phase responses were calculated over the entire circadian cycle in both activity and period expression. Phase advances in behaviour were observed during the subjective day, whereas phase delays were associated with subjective night time GA interventions. The corresponding pattern of gene expression preceded the behavioural pattern by approximately four hours. The implications of this effect for clinical and research practice are discussed (Li, 2020).
Propofol is the most commonly used general anesthetic in humans. Understanding of its mechanism of action has focused on its capacity to potentiate inhibitory systems in the brain. However, it is unknown whether other neural mechanisms are involved in general anesthesia. This study demonstrates that the synaptic release machinery is also a target. Using single-particle tracking photoactivation localization microscopy, it was shown that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility. Removing the interaction with the t-SNARE partner SNAP-25 abolishes propofol-induced Syntaxin1A confinement, indicating that Syntaxin1A and SNAP-25 together form an emergent drug target. Impaired Syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated Syntaxin1A construct that interacts with SNAP-25. These results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional Syntaxin1A nanoclusters (Bademosi, 2018).
This study demonstrates that clinical concentrations of a commonly used GABA-acting general anesthetic, propofol, also restrict syntaxin1A mobility on the plasma membrane. The contrast seen with the effect of propofol analogs is particularly striking, with the non-anesthetic analogs significantly increasing syntaxin1A mobility instead. These results indicate that propofol acts like its non-anesthetic analogs when the interaction between syntaxin1A and SNAP-25 is lost, suggesting that propofol targets this interaction to immobilize syntaxin1A. It seems plausible that syntaxin1A confinement to nanoclusters could lead to impaired neurotransmission, which was also observed under propofol. However, more work is needed to establish causality here. How exactly propofol impairs syntaxin1A mobility remains unclear, although the requirement for SNAP-25 interaction suggests the nanoclusters are composed of syntaxin1A/SNAP-25 heterodimers that have been blocked from proceeding to a subsequent step in SNARE complex formation due to the presence of the general anesthetic. It is also unclear how a truncated syntaxin1A protein might preserve this process against the effects of propofol on syntaxin1A mobility and exocytosis. The finding that the truncated syntaxin1A molecule simultaneously interacts with both SNAP-25 and wild-type syntaxin1A suggests occupancy of a site that might otherwise be targeted by propofol. In this regard, future experiments with other truncation constructs employing propofol resistance as a readout will be helpful toward determining whether the effects on syntaxin1A mobility and exocytosis are indeed correlated (Bademosi, 2018).
In addition to identifying an alternative target process for this widely used sedative, the current findings may provide a more complete understanding of general anesthesia. Every neuron communicates with other neurons by way of syntaxin1A-mediated neurotransmission, which is highly conserved from worms to humans. Although these experiments were focused on the intravenous drugs propofol and etomidate, it will be interesting to see in future studies whether other general anesthetics have the same effect on syntaxin1A mobility. There is already considerable evidence that a broader range of general anesthetics affect synaptic release mechanisms, and a previous study using nuclear magnetic resonance found that clinical concentrations of these drugs interact with syntaxin1A and SNAP-25, but not VAMP2, which is consistent with the conclusion that propofol acts before completed SNARE formation. One hypothesis consistent with these findings would be that a general anesthetic target emerges only when syntaxin1A and SNAP-25 interact on the plasma membrane and that the association of propofol with this emergent target interferes with subsequent steps in SNARE formation. This would lead to a 'traffic jam' of syntaxin1A/SNAP-25 heterodimers (or another pre-SNARE moiety), which would manifest as syntaxin1A nanoclusters in this analysis. Another explanation for the decrease in syntaxin1A mobility could be that propofol promotes its recruitment into nonfunctional SNARE complexes that do not promote vesicle fusion. Whereas the data suggest interactions in the membrane, this need not be the only explanation for altered syntaxin1A mobility. An alternative possibility is that anesthetics might alter syntaxin1A mobility by more specifically interfering with other key protein interactions leading to SNARE formation, such as between syntaxin1A/SNAP-25 and Munc-13, which is a crucial mediator in forming the final tetrameric complex with VAMP2. Future experiments testing the effects of mutating candidate residues in the syntaxin1A SNARE motif should reveal the exact nature of this propofol-binding target, as has been revealed for other propofol targets, such as GABAA receptors (Bademosi, 2018).
Like sleep, general anesthesia resembles a reversible switch, and the search for mechanisms of anesthesia has justifiably focused on proteins that exert major effects on neuronal excitability, such as inhibitory GABAA receptors, which are indeed targets of many general anesthetics. However, the current results and the work of others show that clinically relevant concentrations of general anesthetics also compromise neurotransmitter release, and the current set of results with intravenous drugs suggests this may be consequence of effects on syntaxin1A mobility in the plasma membrane. However, general anesthetics do not abolish neurotransmission; they only decrease quantal content. So how could this be relevant to the behavioral endpoint that is general anesthesia? With most animal brains comprising anywhere between millions and trillions of synapses, it seems plausible that normal brain functions would be compromised if syntaxin1A mobility became globally restricted across a variety of synapses following exposure to general anesthetics. While a decrease in quantal content may not significantly impair some muscular (or spinal cord) functions, it is likely that a similar effect on central synapses would dramatically change temporal dynamics in the brain, leading to a loss of functional connectivity. In support of this view, recent electroencephalogram (EEG) and fMRI studies have shown that functional connectivity throughout the brain is significantly altered in patients undergoing general anesthesia. Thus, other manipulations that compromise presynaptic communication, including effects on presynaptic excitability , might fall into the same category of anesthetic mechanisms as the syntaxin1A-mediated effects described in this study, that may be considered a class of effects that is distinct from GABAergic sleep-related mechanisms. One possibility, which has been raised previously, is that GABAergic processes (e.g., sedation and loss of consciousness) are induced at lower drug doses (e.g., < 1 µM propofol), while the presynaptic processes discussed in this study are affected at the slightly higher concentrations required for surgery. It remains unknown however whether other general anesthetics target presynaptic mechanisms. A recent study using hippocampal cultures found that isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx rather than Ca2+-exocytosis coupling. In contrast, the current results suggest that propofoland etomidate-mediated presynaptic effects might be directly coupled to the exocytosis machinery. Whether this is a difference between intravenous and volatile anesthetics is unclear. Nevertheless, a set of distinct presynaptic mechanisms linked to exocytosis might explain why recovery from general anesthesia appears to involve a different process than anesthesia induction; re-establishing functional connectivity after neurotransmission has returned to normal levels across the brain would likely involve a different process than falling asleep or waking up. It will be interesting in future research to use transgenic syntaxin1A animals to link the local effects found at the presynapse with consequent changes in global readouts, such as whole-brain connectivity and coherence (Bademosi, 2018).
Syntaxin1A is a presynaptic molecule that plays a key role in vesicular neurotransmitter release. Mutations of syntaxin1A result in resistance to both volatile and intravenous anesthetics. Truncated syntaxin1A isoforms confer drug resistance in cell culture and nematode models of anesthesia Resistance to isoflurane anesthesia can be produced by transiently expressing truncated syntaxin1A proteins in adult Drosophila flies. Electrophysiologic and behavioral studies in Drosophila show that mutations in syntaxin1A facilitate recovery from isoflurane anesthesia. These observations suggest that presynaptic mechanisms, via syntaxin1A-mediated regulation of neurotransmitter release, are involved in general anesthesia maintenance and recovery Mutations in the presynaptic protein syntaxin1A modulate general anesthetic effects in vitro and in vivo. Coexpression of a truncated syntaxin1A protein confers resistance to volatile and intravenous anesthetics, suggesting a target mechanism distinct from postsynaptic inhibitory receptor processes. Hypothesizing that recovery from anesthesia may involve a presynaptic component, whether synatxin1A mutations facilitated recovery from isoflurane anesthesia in Drosophila melanogaster was tested. The same neomorphic syntaxin1A mutation that confers isoflurane resistance in cell culture and nematodes also produces isoflurane resistance in Drosophila. Resistance in Drosophila is, however, most evident at the level of recovery from anesthesia, suggesting that the syntaxin1A target affects anesthesia maintenance and recovery processes rather than induction. The absence of truncated syntaxin1A from the presynaptic complex suggests that the resistance-promoting effect of this molecule occurs before core complex formation (Troup, 2019).
There is growing evidence that general anesthetics target presynaptic mechanisms in addition to postsynaptic receptors. For example, clinical concentrations of both intravenous and volatile anesthetics have been found to impair neurotransmission. Syntaxin1A plays a key role in neurotransmission, presenting a crucial endpoint for synaptic vesicle release, without which neurotransmission could not occur. Mutations in this protein produce both hypersensitivity and resistance to volatile anesthetics in nematode worms and Drosophila flies, suggesting that the protein may be proximal to a presynaptic target for these drugs. Coexpression of a truncated syntaxin1A protein has been shown to produce resistance to volatile anesthetics in nematode worms and mammalian neurosecretory cells,mas well as resistance to the intravenous anesthetic propofol in mammalian cells. How exactly this neomorphic syntaxin1A protein protects synaptic release from the effect of general anesthetics remains unclear, although an interaction with other presynaptic-related proteins seems likely (Troup, 2019).
Most explanations of general anesthesia relate to postsynaptic targets. However, should general anesthesia comprise at least two distinct target domains, one postsynaptic and one presynaptic, this is likely to be reflected in the different kinetics of anesthesia induction and recovery. Many general anesthetics have rapid induction and slower recovery kinetics. This recovery inertia has been proposed as evidence that different processes might be involved during induction and recovery. One idea that has been proposed is that induction is rapid because it primarily reflects the sedative properties of these drugs, and the loss of consciousness associated with sleep is a rapid process. However, recovery time can vary significantly, and some patients report incomplete recovery for days or even months after the procedure. It has been have proposed that recovery inertia reflects in part presynaptic processes, in contrast to the rapid induction kinetics which are understood to reflect postsynaptic processes. Therefore, manipulations of presynaptic proteins that preserve neurotransmission under isoflurane anesthesia in vitro should reduce the recovery time required after the procedure, in vivo. This study tested this in an animal model, Drosophila melanogaster (Troup, 2019).
General anesthesia is fundamentally a behavioral endpoint, and understanding its mechanisms of action requires methods to probe behavioral responsiveness in behaving animals. Because the sedative component of general anesthesia most likely engages sleep-promoting pathways in the brain, it was decided to use Drosophila, which have sleep-promoting neurons that have been found to be involved in isoflurane anesthesia (Troup, 2013). Drosophila is an established model to study general anesthesia. Assays for probing sleep intensity or behavioral responsiveness are also well developed for Drosophila, providing an effective way of assessing the role of syntaxin1A in isoflurane induction and recovery. A tagged, truncated version of syntaxin1A was developed could be expressed in fly neurons, to determine how this affected isoflurane induction and recovery for behavioral endpoints as well as for neurotransmission. It was hypothesized that syntaxin1A effects on recovery from isoflurane anesthesia would be reflected across these different levels of investigation (Troup, 2019).
This study found that deletion mutations in syntaxin1A, when coexpressed alongside wild-type syntaxin1A in Drosophila melanogaster, significantly reduce the recovery time after isoflurane anesthesia. Whereas resistance to isoflurane was also evident during anesthesia induction, this effect was weaker compared with effects on recovery, in adult flies. This suggests that recovery from isoflurane anesthesia depends at least in part on syntaxin1A function. It was surprising how long adult wild-type flies required to regain normal levels of behavioral responsiveness after the procedure, compared with regaining locomotion; behavioral responsiveness still remained impaired even after two hours. In contrast, the syntaxin1A mutants could recover behavioral responsiveness after 10min. Delayed recovery of behavioral responsiveness in Drosophila may therefore be a promising model for studying cognitive impairments following general anesthesia in humans, which also often follow a longer time course than simply regaining consciousness. A full restoration of presynaptic functions across the brain is probably a complex problem in any animal, and the extremely conserved nature of synaptic release mechanisms suggests this might be a common mechanism (Troup, 2019).
syntaxin1A manipulations improved anesthesia recovery times across entirely different levels of analysis. Examination of effects on neurotransmission at the fly neuromuscular junction corroborated behavioral findings: recovery of quantal content occurred within 5min in the syntaxin1A mutant animals. Although it is unknown what neurotransmission recovery dynamics might be like in adult brain synapses, it seems likely that similar effects on recovery might be present because the same syntaxin1A protein is involved in the brain as at the larval neuromuscular junction, or in all animal synapses. Mutant Drosophila larvae also recovered faster than controls behaviorally (within 5 min), indicating an effect that transcends brains at different levels of complexity (the larval brain has an order of magnitude fewer neurons than the adult fly brain). It remains unknown, however, whether adult brain synapses are affected in the same way as motor synapses. Behavioral recovery dynamics in adult mutant animals remain more sluggish than recovery of quantal content at the larval neuromuscular junction. This suggests that brain synapses might recover function differently than motor nerve terminals in larvae (Troup, 2019).
One limitation of this study was the use of only female flies for behavioral experiments in adults. Sex-specific effects in Drosophila have been observed during recovery from anesthesia, although these effects generalize to cold-shock and oxygen deprivation anesthesia, which are likely unrelated to the presynaptic mechanisms described in this study. Given the lack of sexual dimorphism in synaptic proteins, it is unlikely that the phenotypes described in this study would be different using male flies, although this remains to be tested experimentally. In the larval studies both male and female animals were used. Despite any potential sexual differentiation in larval motor nerve terminals, significant effects were still found in the larval isoflurane experiments with expression of the truncated syntaxin1A protein (Troup, 2019).
How might a coexpressed syntaxin1A truncation construct be conferring a rapid recovery from isoflurane anesthesia? This same manipulation has now been shown to produce resistance to diverse general anesthetics across a variety of systems, in vitro and in vivo. Because syntaxin1A is a key player in SNARE-mediated exocytosis, it was therefore surprising to find that the HA-tagged truncated protein syx227 was not present in soluble nethylmaleimide sensitive factor attachment protein receptor complexes, at least in Drosophila. Because syx227 has been shown to interact with synaptosomal associated protein 25 (SNAP25) and wild-type syntaxin1A, this suggests an effect immediately before SNARE formation, meaning the truncated protein is probably ejected upon full soluble n-ethylmaleimide sensitive factor attachment protein receptor formation (when vesicle-associated membrane protein 2 links with syntaxin1A and SNAP25 to form a release ready tetrameric complex). This would imply that the protective effect of this protein is required before SNARE formation, and accordingly that the anesthetic effect on syntaxin1A function is also prior to soluble nethylmaleimide sensitive factor attachment protein receptor formation. This view is consistent with recent findings using super-resolution microscopy to track the mobility of single syntaxin1A molecules under propofol anesthesia (Troup, 2019).
It was found that clustering of syntaxin1A caused by propofol was dependent on an interaction with SNAP25, but not with vesicle-associated membrane protein 2, thereby suggesting a mechanism of action (for propofol) immediately before full SNARE formation. Work in other systems also suggests an interaction between volatile anesthetics and syntaxin1A/SNAP25 suggesting that this might indeed by a general anesthetic target. On the other hand, work in Caenorhabditis elegans, where the effect of the truncated syntaxin1A was discovered, points to unc-13 as a likely mediator of this resistance-promoting mechanism. unc-13 is understood to be associated with presynaptic active zones, where the SNAREs ultimately reside, so one interpretation of these diverse findings is that the drug-mediated clustering of syntaxin1A/SNAP25 occurs at these active zones, and that these pre-SNARE complexes are prevented from transforming into full SNAREs because unc-13 is less able to catalyze the next step. In this regard, it will be especially interesting to investigate what role unc-13 plays in this process; unc-13 has been shown to keep syntaxin1A in a closed conformation, until interaction with unc-13 opens syntaxin1A to promote complete full SNARE formation. One hypothesis consistent with this model is that general anesthetics promote a closed syntaxin1A conformation, by for example impairing the capacity of unc-13 to catalyze SNARE formation. One hypothesis for how syx227 affords resistance then is that the truncated (or deletion) proteins might promote open syntaxin1A moieties, and in this way remove an emergent target (the closed syntaxin1A-unc-18 complex). Interactions with vesicle-bound vesicle-associated membrane protein 2 would then lead to an energetically more favorable ternary complex, effectively ejecting syx227 upon SNARE formation. Future biochemical experiments should determine whether syx227 promotes an open syntaxin1A conformation, and to what level unc-13 and unc-18 are involved in this process (Troup, 2019).
One of the most striking observations in this this study is the prolonged duration of recovery from isoflurane anesthesia in wild-type flies, and how syntaxin1A mutations significantly reduce this recovery time. If syx227 is acting before SNARE formation, then how might this lead to faster recovery? One possibility following from the hypothesis proposed above is that syx227 provides more efficient access to already open pre-SNARE complexes that are ready to be incorporated into fully formed SNAREs. If general anesthetics produce a traffic jam of nonfunctional pre-SNARE nanoclusters, as suggested by single-molecule imaging experiments, then the time required for dissolving this proteinaceous traffic jam might take longer than clearance of the anesthetic drugs themselves. Consistent with this view, imaging work showed that expression of syx227 in mammalian cells prevented the syntaxin1A clustering effects of another general anesthetic, propofol. In contrast to these sluggish presynaptic recovery effects, the postsynaptic effects of general anesthetics such as isoflurane and propofol are most likely rapid, as they primarily linked to gamma-aminobutyric acid receptor function. General anesthesia induction is a rapid process, as this probably engages potent inhibitory systems in the brain that are designed to promote a rapid loss of consciousness. However, a rapid reversal of the effect on gamma-aminobutyric acid receptors after removal of these drugs might have little consequence on recovery from anesthesia until presynaptic processes across the brain have been fully restored. The data on syntaxin1A fly mutants exposed to isoflurane support this view of general anesthesia, with the largest effects seen for recovery rather than induction. However, the fact that these mutants are also resistant to isoflurane upon induction suggests that presynaptic effects might play a role during anesthesia induction as well. It will be interesting in future experiments to combine genetic manipulations that promote anesthetic resistance at both a pre and postsynaptic levels, in animals that have both target mechanisms (i.e., sleep/wake pathways and SNAREs). Such experiments will allow better dissecting of the relative contributions of either target process, and to determine whether some circuits or neurotransmitter systems are more affected by the presynaptic mechanisms this study has uncovered (Troup, 2019).
The physical basis of consciousness remains one of the most elusive concepts in current science. One influential conjecture is that consciousness is to do with some form of causality, measurable through information. The integrated information theory of consciousness (IIT) proposes that conscious experience, filled with rich and specific content, corresponds directly to a hierarchically organised, irreducible pattern of causal interactions; i.e. an integrated informational structure among elements of a system. This study tested this conjecture in a simple biological system (fruit flies), estimating the information structure of the system during wakefulness and general anesthesia. Consistent with this conjecture, it was found that integrated interactions among populations of neurons during wakefulness collapsed to isolated clusters of interactions during anesthesia. Classification analysis to quantify the accuracy of discrimination between wakeful and anesthetised states, and found that informational structures inferred conscious states with greater accuracy than a scalar summary of the structure, a measure which is generally championed as the main measure of IIT. In stark contrast to a view which assumes feedforward architecture for insect brains, especially fly visual systems, rich information structures were found, which cannot arise from purely feedforward systems, occurred across the fly brain. Further, these information structures collapsed uniformly across the brain during anesthesia. The results speak to the potential utility of the novel concept of an "informational structure" as a measure for level of consciousness, above and beyond simple scalar values (Leung, 2021).
Aggressive interactions are costly, such that individuals should display modified aggression in response to environmental stress. Many organisms experience frequent periods of food deprivation, which can influence an individual's capacity and motivation to engage in aggression. However, because food deprivation can simultaneously decrease an individual's resource-holding potential and increase its valuation of food resources, its net impact on aggression is unclear. This study tested the influence of increasingly prolonged periods of adult food deprivation on inter-male aggression in pairs of fruit flies, Drosophila melanogaster. Males displayed increased aggression following periods of food deprivation longer than a day. Increased aggression in food-deprived flies occurred despite their reduced mass. This result is probably explained by an increased attraction to food resources, as food deprivation increased male occupancy of central food patches, and food patch occupancy was positively associated with aggression. These findings demonstrate that aggressive strategies in male D. melanogaster are influenced by nutritional experience, highlighting the need to consider past nutritional stresses to understand variation in aggression (Edmunds, 2021).
The mitochondrial electron transport chain (mETC) contains molecular targets of volatile general anesthetics (VGAs), which places carriers of mutations at risk for anesthetic complications. The ND-2360114 and mt:ND2del1 lines of fruit flies (Drosophila melanogaster) that carry mutations in core subunits of Complex I of the mETC replicate numerous characteristics of Leigh syndrome (LS) caused by orthologous mutations in mammals and serve as models of LS. ND-2360114 flies are behaviorally hypersensitive to volatile anesthetic ethers and develop an age- and oxygen-dependent anesthetic-induced neurotoxicity (AiN) phenotype after exposure to isoflurane but not to the related anesthetic sevoflurane. The goal of this paper was to investigate whether the alkane volatile anesthetic halothane and other mutations in Complex I and in Complexes II-V of the mETC cause AiN. It was found that (1) ND-2360114 and mt:ND2del1 were susceptible to toxicity from halothane; (2) in wild-type flies, halothane was toxic under anoxic conditions; (3) alleles of accessory subunits of Complex I predisposed to AiN; and (iv) mutations in Complexes II-V did not result in an AiN phenotype. It is concluded that AiN is neither limited to ether anesthetics nor exclusive to mutations in core subunits of Complex I (Borchardt, 2023)
Sevoflurane is the primary inhaled anesthetic used in pediatric surgery. It has been the focus of research since animal models studies found that it was neurotoxic to the developing brain two decades ago. However, whether pediatric general anesthesia can lead to permanent cognitive deficits remained a subject of heated debate. Therefore, this study aims to determine the lifetime neurotoxicity of early long-time sevoflurane exposure using a short-life-cycle animal model, Drosophila melanogaster. To investigate this question, the lifetime changes of two-day-old flies' learning and memory abilities after anesthesia with 3 % sevoflurane for 6 h by the T-maze memory assay. Apoptosis, levels of ATP and ROS, and related genes were evaluated in the fly head. The results suggest that 6 h 3 % sevoflurane exposure at a young age can only induce transient neuroapoptosis and cognitive deficits around the first week after anesthesia. But this brain damage recedes with time and vanishes in late life. It was also found that the mRNA level of caspases and Bcl-2, ROS level, and ATP level increased during this temporary neuroapoptosis process. And mRNA levels of antioxidants, such as SOD2 and CAT, increased and decreased simultaneously with the rise and fall of the ROS level, indicating a possible contribution to the recovery from the sevoflurane impairment. In conclusion, these results suggest that one early prolonged sevoflurane-based general anesthesia can induce neuroapoptosis and learning and memory deficit transiently but not permanently in Drosophila (Liu, 2023).
General anesthetics cause a profound loss of behavioral responsiveness in all animals. In mammals, general anesthesia is induced in part by the potentiation of endogenous sleep-promoting circuits, although "deep" anesthesia is understood to be more similar to coma. Surgically relevant concentrations of anesthetics, such as isoflurane and propofol, have been shown to impair neural connectivity across the mammalian brain, which presents one explanation why animals become largely unresponsive when exposed to these drugs. It remains unclear whether general anesthetics affect brain dynamics similarly in all animal brains, or whether simpler animals, such as insects, even display levels of neural connectivity that could be disrupted by these drugs. This study used whole-brain calcium imaging in behaving female Drosophila flies to investigate whether isoflurane anesthesia induction activates sleep-promoting neurons, and then inquired how all other neurons across the fly brain behave under sustained anesthesia. It was possible to track the activity of hundreds of neurons simultaneously during waking and anesthetized states, for spontaneous conditions as well as in response to visual and mechanical stimuli. Whole-brain dynamics and connectivity were compared under isoflurane exposure to optogenetically induced sleep. Neurons in the Drosophila brain remain active during general anesthesia as well as induced sleep, although flies become behaviorally inert under both treatments. This study identified surprisingly dynamic neural correlation patterns in the waking fly brain, suggesting ensemble-like behavior. These become more fragmented and less diverse under anesthesia but remain wake-like during induced sleep (Troup, 2023).
Pathogens and parasites can manipulate their hosts to optimize their own fitness. For instance, bacterial pathogens have been shown to affect their host plants' volatile and non-volatile metabolites, which results in increased attraction of insect vectors to the plant, and, hence, to increased pathogen dispersal. Behavioral manipulation by parasites has also been shown for mice, snails and zebrafish as well as for insects. This study shows that infection by pathogenic bacteria alters the social communication system of Drosophila melanogaster. More specifically, infected flies and their frass emit dramatically increased amounts of fly odors, including the aggregation pheromones methyl laurate, methyl myristate, and methyl palmitate, attracting healthy flies, which in turn become infected and further enhance pathogen dispersal. Thus, olfactory cues for attraction and aggregation are vulnerable to pathogenic manipulation, and the alteration of social pheromones can be beneficial to the microbe while detrimental to the insect host. Behavioral manipulation of host by pathogens has been observed in vertebrates, invertebrates, and plants. This study shows that in Drosophila, infection with pathogenic bacteria leads to increased pheromone release, which attracts healthy flies. This process benefits the pathogen since it enhances bacterial dispersal, but is detrimental to the host (Keesey, 2017).
Many microbes induce striking behavioral changes in their animal hosts, but how they achieve this is poorly understood, especially at the molecular level. Mechanistic understanding has been largely constrained by the lack of an experimental system amenable to molecular manipulation. A strain of the behavior-manipulating fungal pathogen Entomophthora muscae infects wild Drosophila, and methods were established to infect D. melanogaster in the lab. Lab-infected flies manifest the moribund behaviors characteristic of E. muscae infection: hours before death, they climb upward, extend their proboscides, affixing in place, then raise their wings, clearing a path for infectious spores to launch from their abdomens. E. muscae was found to invade the nervous system, suggesting a direct means by which the fungus could induce behavioral changes. Given the vast molecular toolkit available for D. melanogaster, this new system will enable rapid progress in understanding how E. muscae manipulates host behavior (Elya, 2018).
Many species are able to share information about their environment by communicating through auditory, visual, and olfactory cues. In Drosophila melanogaster, exposure to parasitoid wasps leads to a decline in egg laying, and exposed females communicate this threat to naive flies, which also depress egg laying. This study found that species across the genus Drosophila respond to wasps by egg laying reduction, activate cleaved caspase in oocytes, and communicate the presence of wasps to naive individuals. Communication within a species and between closely related species is efficient, while more distantly related species exhibit partial communication. Remarkably, partial communication between some species is enhanced after a cohabitation period that requires exchange of visual and olfactory signals. This interspecies "dialect learning" requires neuronal cAMP signaling in the mushroom body, suggesting neuronal plasticity facilitates dialect learning and memory. These observations establish Drosophila as genetic models for interspecies social communication and evolution of dialects (Kacsoh, 2018).
The influence of oncogenic phenomena on the ecology and evolution of animal species is becoming an important research topic. Similar to host-pathogen interactions, cancer negatively affects host fitness, which should lead to the selection of host control mechanisms, including behavioral traits that best minimize the proliferation of malignant cells. Social behavior is suggested to influence tumor progression. While the ecological benefits of sociality in gregarious species are widely acknowledged, only limited data are available on the role of the social environment on cancer progression. This study exposed adult Drosophila, with colorectal-like tumors, to different social environments. Subtle variations in social structure have dramatic effects on the progression of tumor growth. Finally, it is revealed that flies can discriminate between individuals at different stages of tumor development and selectively choose their social environment accordingly. This study demonstrates the reciprocal links between cancer and social interactions and how sociality may impact health and fitness in animals and its potential implications for disease ecology (Dawson, 2018).
Isolation profoundly influences social behavior in all animals. Longer-term analysis of large groups of flies is hampered by the lack of effective and reliable tools. In this study a new imaging arena was built and the existing tracking algorithm was improved to reliably follow a large number of flies simultaneously. Next, based on the automatic classification of touch and graph-based social network analysis, an algorithm was designed to quantify changes in the social network in response to prior social isolation. It was observed that isolation significantly and swiftly enhanced individual and local social network parameters depicting near-neighbor relationships. The genome-wide molecular correlates of these behavioral changes were explored, and it was found that whereas behavior changed throughout the six days of isolation, gene expression alterations occurred largely on day one. These changes occurred mostly in metabolic genes, and the metabolic changes were varified by showing an increase of lipid content in isolated flies. In summary, this study describes a highly reliable tracking and analysis pipeline for large groups of flies that were use to unravel the behavioral, molecular and physiological impact of isolation on social network dynamics in Drosophila (Liu, 2018).
Cooperative behavior emerges in biological systems through coordinated actions among individuals. Although widely observed across animal species, the cellular and molecular mechanisms underlying the establishment and maintenance of cooperative behaviors remain largely unknown. To characterize the circuit mechanisms serving the needs of independent individuals and social groups, this study investigated cooperative digging behavior in Drosophila larvae. Although chemical and mechanical sensations are important for larval aggregation at specific sites, an individual larva's ability to participate in a cooperative burrowing cluster relies on direct visual input as well as visual and social experience during development. In addition, vision modulates cluster dynamics by promoting coordinated movements between pairs of larvae. To determine the specific pathways within the larval visual circuit underlying cooperative social clustering, larval photoreceptors (PRs) and the downstream local interneurons (lOLPs) were examined using anatomical and functional studies. The results indicate that rhodopsin-6-expressing-PRs (Rh6-PRs) and lOLPs are required for both cooperative clustering and movement detection. Remarkably, visual deprivation and social isolation strongly impact the structural and functional connectivity between Rh6-PRs and lOLPs, while at the same time having no effect on the adjacent rhodopsin-5-expressing PRs (Rh5-PRs). Together, these findings demonstrate that a specific larval visual pathway involved in social interactions undergoes experience-dependent modifications during development, suggesting that plasticity in sensory circuits could act as the cellular substrate for social learning, a possible mechanism allowing an animal to integrate into a malleable social environment and engage in complex social behaviors (Dombrovski, 2019).
Drosophila species communicate the threat of parasitoid wasps to naive individuals. Communication of the threat between closely related species is efficient, while more distantly related species exhibit a dampened, partial communication. Partial communication between D. melanogaster and D. ananassae about wasp presence is enhanced following a period of cohabitation, suggesting that species-specific natural variations in communication 'dialects' can be learned through socialization. This study identified six regions of the Drosophila brain essential for dialect training. Subgroups of neurons in these regions were identified, including motion detecting neurons in the optic lobe, layer 5 of the fan-shaped body, the D glomerulus in the antennal lobe, and the odorant receptor Or69a, where activation of each component is necessary for dialect learning. These results reveal functional neural circuits that underlie complex Drosophila social behaviors, and these circuits are required for integration several cue inputs involving multiple regions of the Drosophila brain (Kacsoh, 2019).
Many animals exhibit an astonishing ability to form groups of large numbers of individuals. The dynamic properties of such groups have been the subject of intensive investigation. The actual grouping processes and underlying neural mechanisms, however, remain elusive. This study established a social clustering paradigm in Drosophila to investigate the principles governing social group formation. Fruit flies spontaneously assembled into a stable cluster mimicking a distributed network. Social clustering was exhibited as a highly dynamic process including all individuals, which participated in stochastic pair-wise encounters mediated by appendage touches. Depriving sensory inputs resulted in abnormal encounter responses and a high failure rate of cluster formation. Furthermore, the social distance of the emergent network was regulated by ppk-specific neurons, which were activated by contact-dependent social grouping. Taken together, these findings revealed the development of an orderly social structure from initially unorganised individuals via collective actions (Jiang, 2020).
Theory predicts that social interactions can induce an alignment of behavioral asymmetries between individuals (i.e., population-level lateralization), but evidence for this effect is mixed. To understand how interaction with other individuals affects behavioral asymmetries, this study systematically manipulated the social environment of Drosophila melanogaster, testing individual flies and dyads (female-male, female-female and male-male pairs). In these social contexts individual and population asymmetries in individual behaviors (circling asymmetry, wing use) and dyadic behaviors (relative position and orientation between two flies) were measured in five different genotypes. It was reasoned that if coordination between individuals drives alignment of behavioral asymmetries, greater alignment at the population-level should be observed in social contexts compared to solitary individuals. It was observed that the presence of other individuals influenced the behavior and position of flies but had unexpected effects on individual and population asymmetries: individual-level asymmetries were strong and modulated by the social context but population-level asymmetries were mild or absent. Moreover, the strength of individual-level asymmetries differed between strains, but this was not the case for population-level asymmetries. These findings suggest that the degree of social interaction found in Drosophila is insufficient to drive population-level behavioral asymmetries (Versace, 2020).
Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. This study shows that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. The regulation of GABA availability by mitochondrial activity was identified as a biologically relevant mechanism, and its contribution to social behavior was identified. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. Aralar was identified as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions (Kanellopoulos, 2020).
Animals interact with each other in species-specific reproducible patterns. These patterns of organization are captured by social network analysis, and social interaction networks (SINs) have been described for a wide variety of species including fish, insects, birds, and mammals. The aim of this study is to understand the evolution of social organization in Drosophila. Using a comparative ecological, phylogenetic, and behavioral approach, the different properties of SINs formed by 20 drosophilids were compared. Whether drosophilid network structures arise from common ancestry, a response to the species' past climate, other social behaviors, or a combination of these factors was investigated. This study shows that differences in past climate predicted the species' current SIN properties. The drosophilid phylogeny offered no value to predicting species' differences in SINs through phylogenetic signal tests. This suggests that group-level social behaviors in drosophilid species are shaped by divergent climates. However, it was found that the social distance at which flies interact correlated with the drosophilid phylogeny, indicating that behavioral elements of SINs have remained largely unchanged in their evolutionary history. A significant correlation was found of leg length to social distance, outlining the interdependence of anatomy and complex social structures. Although SINs display a complex evolutionary relationship across drosophilids, this study suggests that the ecology, and not common ancestry, contributes to diversity in social structure in Drosophila (Jezovit, 2020).
Many organisms, when alone, behave differently from when they are among a crowd. Drosophila similarly display social behaviour and collective behaviour dynamics within groups not seen in individuals. In flies, these emergent behaviours may be in response to the global size of the group or local nearest-neighbour density. This study investigated i) which aspect of social life flies respond to: group size, density, or both and ii) whether behavioural changes within the group are dependent on olfactory support cells. Behavioural assays demonstrate that flies adjust their interactive behaviour to group size but otherwise compensate for density by achieving a standard rate of movement, suggesting that individuals are aware of the number of others within their group. Olfactory support cells are necessary for flies to behave normally in large groups. These findings shed insight into the subtle and complex life of Drosophila within a social setting (Rooke, 2020).
Living in a group creates a complex and dynamic environment in which behavior of individuals is influenced by and affects the behavior of others. Although social interaction and group living are fundamental adaptations exhibited by many organisms, little is known about how prior social experience, internal states, and group composition shape behavior in groups. This study presents an analytical framework for studying the interplay between social experience and group interaction in Drosophila melanogaster. The complexity of interactions in a group was simplified using a series of experiments in which the social experience and motivational states of individuals were controlled to compare behavioral patterns and social networks of groups under different conditions. Social enrichment promotes the formation of distinct group structure that is characterized by high network modularity, high inter-individual and inter-group variance, high inter-individual coordination, and stable social clusters. Using environmental and genetic manipulations, this study showed that visual cues and cVA-sensing neurons are necessary for the expression of social interaction and network structure in groups. Finally, the formation of group behavior and structure was exploited in heterogenous groups composed of flies with distinct internal states, and emergent structures were documented that are beyond the sum of the individuals that constitute it. These results demonstrate that fruit flies exhibit complex and dynamic social structures that are modulated by the experience and composition of different individuals within the group. This paves the path for using simple model organisms to dissect the neurobiology of behavior in complex social environments (Bentzur, 2020).
Social interactions are thought to be a critical driver in the evolution of cognitive ability. Cooperative interactions, such as pair bonding, rather than competitive interactions have been largely implicated in the evolution of increased cognition. This is despite competition traditionally being a very strong driver of trait evolution. Males of many species track changes in their social environment and alter their reproductive strategies in response to anticipated levels of competition. This study predicts this to be cognitively challenging. Using a Drosophila melanogaster model, it was possible to distinguish between the effects of a competitive environment versus generic social contact by exposing flies to same-sex same-species competition versus different species partners, shown to present non-competitive contacts. Males increase olfactory learning/memory and visual memory after exposure to conspecific males only, a pattern echoed by increased expression of synaptic genes and an increased need for sleep. For females, largely not affected by mating competition, the opposite pattern was seen. The results indicate that specific social contacts dependent on sex, not simply generic social stimulation, may be an important evolutionary driver for cognitive ability in fruit flies (Rouse, 2020).
Sustained changes in mood or action require persistent changes in neural activity, but it has been difficult to identify the neural circuit mechanisms that underlie persistent activity and contribute to long-lasting changes in behavior. This study shows that a subset of Doublesex+ pC1 neurons in the Drosophila female brain, called pC1d/e, can drive minutes-long changes in female behavior in the presence of males. Using automated reconstruction of a volume electron microscopic (EM) image of the female brain, all inputs and outputs to both pC1d and pC1e were mapped. This reveals strong recurrent connectivity between, in particular, pC1d/e neurons and a specific subset of Fruitless+ neurons called aIPg. This study additionally found that pC1d/e activation drives long-lasting persistent neural activity in brain areas and cells overlapping with the pC1d/e neural network, including both Doublesex+ and Fruitless+ neurons. This work thus links minutes-long persistent changes in behavior with persistent neural activity and recurrent circuit architecture in the female brain (Deutsch, 2020).
Sociality is among the most important motivators of human behaviour. However, the neural mechanisms determining levels of sociality are largely unknown, primarily due to a lack of suitable animal models. This study reports the presence of a surprising degree of general sociality in Drosophila. A newly-developed paradigm to study social approach behaviour in flies reveal that social cues perceive through both vision and olfaction converged in a central brain region, the γ lobe of the mushroom body, which exhibit activation in response to social experience. The activity of these γ neurons control the motivational drive for social interaction. At the molecular level, the serotonergic system is critical for social affinity. These results demonstrate that Drosophila are highly sociable, providing a suitable model system for elucidating the mechanisms underlying the motivation for sociality (Sun, 2020).
Social behaviors are mediated by the activity of highly complex neuronal networks, the function of which is shaped by their transcriptomic and proteomic content. Contemporary advances in neurogenetics, genomics, and tools for automated behavior analysis make it possible to functionally connect the transcriptome profile of candidate neurons to their role in regulating behavior. This study used Drosophila melanogaster to explore the molecular signature of neurons expressing receptor for neuropeptide F (NPF), the fly homolog of neuropeptide Y (NPY). By comparing the transcription profile of NPFR neurons to those of nine other populations of neurons, this study discovered that NPFR neurons exhibit a unique transcriptome, enriched with receptors for various neuropeptides and neuromodulators, as well as with genes known to regulate behavioral processes, such as learning and memory. By manipulating RNA editing and protein ubiquitination programs specifically in NPFR neurons, this study demonstrated that the proper expression of their unique transcriptome and proteome is required to suppress male courtship and certain features of social group interaction. The results highlight the importance of transcriptome and proteome diversity in the regulation of complex behaviors and pave the path for future dissection of the spatiotemporal regulation of genes within highly complex tissues, such as the brain (Ryvkin, 2021).
Drosophila melanogaster displays social behaviors including courtship, mating, aggression, and group foraging. Recent studies employed social network analyses (SNAs) to show that D. melanogaster strains differ in their group behavior, suggesting that genes influence social network phenotypes. Aside from genes associated with sensory function, few studies address the genetic underpinnings of these networks. The foraging gene (for) is a well-established example of a pleiotropic gene that regulates multiple behavioral phenotypes and their plasticity. In D. melanogaster, there are two naturally occurring alleles of for called rover and sitter that differ in their larval and adult food-search behavior as well as other behavioral phenotypes. It was hypothesized that for affects behavioral elements required to form social networks and the social networks themselves. These effects are evident when gene dosage was manipulated. Flies of the rover and sitter strains were found to exhibit differences in duration, frequency, and reciprocity of pairwise interactions, and they form social networks with differences in assortativity and global efficiency. Consistent with other adult phenotypes influenced by for, rover-sitter heterozygotes show intermediate patterns of dominance in many of these characteristics. Multiple generations of backcrossing a rover allele into a sitter strain showed that many but not all of these rover-sitter differences may be attributed to allelic variation at for. These findings reveal the significant role that for plays in affecting social network properties and their behavioral elements in Drosophila melanogaster (Alwash, 2021).
Lifespan is modulated at distinct levels by multiple factors, including genetic backgrounds, the environment, behavior traits, metabolic status, and more interestingly, sensory perceptions. However, the effects of social perception between individuals living in the same space remain less clear. This study used the Drosophila model to study the influences of social perception on the lifespan of aged fruit flies. The lifespan of aged Drosophila was found to be markedly prolonged after being co-housed with young adults of the same gender. Moreover, the changes of lifespan were affected by several experimental contexts: (1) the ratios of aged and young adults co-housed, (2) the chronological ages of two populations, and (3) the integrity of sensory modalities. Together, it is hypothesize the chemical/physical stimuli derived from the interacting young adults are capable of interfering with the physiology and behavior of aged flies, ultimately leading to the alteration of lifespan (Cho, 2021).
The composition of the microbiome (the assemblage of symbiotic microorganisms within a host) is determined by environmental factors and the host's immune, endocrine and neural systems. The social environment could alter host microbiomes extrinsically by affecting transmission between individuals. Alternatively, intrinsic effects arising from interactions between the microbiome and host physiology (the microbiota-gut-brain axis) could translate social stress into dysbiotic microbiomes, with consequences for host health. This study investigated how manipulating social environments during larval and adult life-stages altered the microbiome composition of Drosophila melanogaster fruit flies. Social contexts that particularly alter the development and lifespan of males were used, predicting that any intrinsic social effects on the microbiome would therefore be sex-specific. The presence of adult males during the larval stage significantly altered the microbiome of pupae of both sexes. In adults, same-sex grouping increased bacterial diversity in both sexes. Importantly, the microbiome community structure of males was more sensitive to social contact at older ages, an effect partially mitigated by housing focal males with young rather than coaged groups. Functional analyses suggest that these microbiome changes impact ageing and immune responses. This is consistent with the hypothesis that the substantial effects of the social environment on individual health are mediated through intrinsic effects on the microbiome, and provides a model for understanding the mechanistic basis of the microbiota-gut-brain axis (Leech, 2021).
Mixed-species groups describe active associations among individuals of 2
or more species at the same trophic level. Mixed-species groups are
important to key ecological and evolutionary processes such as
competition and predation, and research that ignores the presence of
other species risks ignoring a key aspect of the environment in which social behavior
is expressed and selected. Despite the defining emphasis of active
formation for mixed-species groups, surprisingly little is known about
the mechanisms by which mixed-species groups form. Furthermore, insects
have been almost completely ignored in the study of mixed-species
groups, despite their taxonomic importance and relative prominence in
the study of single-species groups. In this study group formation
processes were measured in Drosophila melanogaster and its sister
species, Drosophila simulans. Each species was studied alone, and
together, and one population of D. melanogaster was also studied both
alone and with another, phenotypically distinct D. melanogaster
population, in a nested-factorial design. This approach differs from
typical methods of studying mixed-species groups in that group formation
could be quantitatively compared between single-population,
mixed-population, and mixed-species treatments. Surprisingly, no
differences were found between treatments in the number, size, or
composition of groups that formed, suggesting that single- and
mixed-species groups form through similar mechanisms of active
attraction. However, it was found that mixed-species groups showed
elevated interspecies male-male interactions, relative to
interpopulation or intergenotype interactions in single-species groups.
These findings expand the conceptual and taxonomic study of
mixed-species groups while raising new questions about the mechanisms of
group formation broadly (Girardeau, 2021).
Prolonged periods of forced social isolation is detrimental to well-being, yet little is known about which genes
regulate susceptibility to its effects. In the fruit fly, Drosophila
melanogaster, social isolation induces stark changes in behavior
including increased aggression, locomotor activity, and resistance to
ethanol sedation. To identify genes regulating sensitivity to isolation,
A collection of sixteen hundred P-element insertion lines was screened
for mutants with abnormal levels of all three isolation-induced
behaviors. The screen identified three mutants whose affected genes are
likely central to regulating the effects of isolation in flies. One
mutant, sex pistol (sxp), became extremely aggressive and resistant to ethanol sedation when socially isolated. sxp also had a high level of male-male courtship. The mutation in sxp
reduced the expression of two minor isoforms of the actin regulator hts
(adducin), as well as mildly reducing expression of CalpA, a
calcium-dependent protease. As a consequence, sxp also had increased expression of the insulin-like peptide, dILP5. Analysis of the social behavior of sxp suggests that these minor hts isoforms function to limit isolation-induced aggression, while chronically high levels of dILP5 increase male-male courtship (Eddison, 2021).
Social isolation and loneliness have potent effects on public health. Research in social psychology suggests that compromised sleep quality is a key factor that links persistent loneliness to adverse health conditions. Although experimental manipulations have been widely applied to studying the control of sleep and wakefulness in animal models, how normal sleep is perturbed by social isolation is unknown. This study reports that chronic, but not acute, social isolation reduces sleep in Drosophila. Quantitative behavioural analysis and transcriptome profiling were used to differentiate between brain states associated with acute and chronic social isolation. Although the flies had uninterrupted access to food, chronic social isolation altered the expression of metabolic genes and induced a brain state that signals starvation. Chronically isolated animals exhibit sleep loss accompanied by overconsumption of food. This resonates with anecdotal findings of loneliness-associated hyperphagia in humans. Chronic social isolation reduces sleep and promotes feeding through neural activities in the peptidergic fan-shaped body columnar neurons of the fly. Artificial activation of these neurons causes misperception of acute social isolation as chronic social isolation and thereby results in sleep loss and increased feeding. These results present a mechanistic link between chronic social isolation, metabolism, and sleep, addressing a long-standing call for animal models focused on loneliness (Li, 2021).
Fruit flies are social animals, and exhibit dynamic social network structures and collective behaviours, that contribute to environmental sensing, foraging, feeding, fighting, mating, oviposition, circadian time setting and even the existence of 'culture'. These important aspects of social interactions imply that insects can provide suitable models for studying how the objective absence of social relationships is perceived and represented in the brain (Li, 2021).
Social experience affects sleep need in Drosophila. This study revisited this finding by exploring how social isolation affects sleep in flies that have prior social experience. Sleep behaviour was tested after maintaining 1, 2, 5, 25 or 100 male flies in a food vial for 7 days. Group-housed flies, regardless of group size (2, 5, 25 or 100), exhibited similar sleep profiles. By contrast, flies housed in isolation displayed a significant loss of sleep, mainly distributed during the daytime (Li, 2021).
The duration of social isolation was manipulated: flies were either isolated or housed in a group of 25 flies for 1, 3, 5, or 7 days, before sleep was measured in a Drosophila activity monitor (DAM). Sleep profiles, which display the proportion of time spent sleeping in consecutive 30-min segments over 24 h, showed that chronic social isolation (5 or 7 days) changed sleep architecture primarily during the daytime and especially during an interval of several hours following dawn (lights on). Although short durations of social isolation (1 or 3 days) did not induce sleep loss, chronic social isolation (5 or 7 days) significantly reduced daily total sleep, daytime sleep and sleep between Zeitgeber time (ZT) 0 and ZT4 (corresponding to the first 4 h after lights-on in a light-dark (LD) cycle) (Li, 2021).
To assess how social isolation alters daytime sleep, all daytime sleep bouts were pooled from all animals tested for a given condition and their distributions were plotted as cumulative relative fractions for bout lengths. Acute social isolation (1 day) produced sleep bout distributions that were indistinguishable from those of 1-day group-housed flies. Flies that were socially isolated for 3 days showed slightly different sleep bout distributions from their group-housed counterparts. However, there was no deficit in total daily sleep, daytime sleep or ZT0-4 sleep in these flies. Flies that were isolated for longer periods (5 or 7 days) had sleep distributions that were significantly different from those of their group-housed counterparts. Cumulative relative frequency curves of daytime sleep bouts from chronically isolated flies climbed faster than those of their group-housed counterparts as shorter sleep bouts accumulated (5 or 7 days). Over seven days of isolation, daily total sleep, daytime sleep and ZT0-4 sleep all decreased progressively (Li, 2021).
Age-matched flies were used to rule out the possibility that chronic social isolation induced sleep loss because the flies were older. Chronic social isolation (7 days) induced sleep loss consistently in various isogenic strains, in aged (4-week-old) wild-type flies, and in sleep inbred panel (SIP) strains with different baseline levels of sleep (Li, 2021).
RNA sequencing (RNA-seq) libraries were prepared for three conditions: socially enriched flies (group treated, Grp), chronically isolated flies (isolated for 7 days, Iso_7D) and acutely isolated flies (isolated for 1 day, Iso_1D). Raster plots demonstrate sleep bouts of individual animals during a 24-h period measured immediately after group enrichment or social isolation. Daytime sleep was reduced and much more fragmented in chronically isolated flies than in group-housed or acutely isolated flies. Fly heads were collected between ZT0.5 and ZT2, a window of time within ZT0-4 that immediately preceded significant loss of daytime sleep in chronically isolated flies. Using differential gene expression analyses, intersectional and clustering strategies, candidate genes were identified for the sleep loss induced by chronic social isolation. These 214 candidate genes showed differences in expression in chronically isolated flies compared with both acutely isolated and group-housed flies and underwent unidirectional changes during the process of chronic social isolation. Gene ontology enrichment analysis suggested that these 214 genes are enriched for biological pathways associated with oxidation-reduction processes, one-carbon metabolic processes and carbohydrate metabolic processes. The rest of the gene ontology of biological pathways showed a strong preference for metabolic functions, such as fatty acid, pyruvate, glucose and amino acid metabolic processes. Consistent with the sleep loss phenotype, sleep was also among the most overrepresented gene ontologies for biological pathways (Li, 2021).
Among the top 20 genes in this list, two genes stood out: Limostatin (Lst, CG8317), expression of which increased 1.67-fold after chronic isolation, and Drosulfakinin (Dsk), expression of which decreased 2.03-fold after chronic isolation. Limostatin is a decretin hormone that is induced by starvation and suppresses insulin release. Drosulfakinin, a satiety-inducing cholecystokinin-like peptide, is secreted when the animal is fed. As a signal of satiety, drosulfakinin is depleted under starvation conditions. A third gene, target of brain insulin (tobi), also showed significantly increased expression (1.76-fold) during chronic social isolation. tobi encodes an α-glucosidase that is regulated by Drosophila insulin and glucagon analogues. In addition, 7 of these top 20 genes and 32 of the total 214 candidate genes were previously identified as being regulated in Drosophila heads after 24 h of starvation. Thus, from a transcriptomic perspective, the brain of a fly maintained in chronic social isolation closely resembles the brain of a starving fly, despite continuous access to food. It was reasoned that such a 'starvation brain state' might broadly affect gene expression associated with metabolic processes. Massive changes in mitochondrial functions and oxidation-reduction processes could be direct consequences of starvation and/or elevated feeding (Li, 2021).
The activity recording capillary feeder (ARC) assay, a video recording capillary feeder (CAFE) assay that monitors sleep and feeding behaviours simultaneously and continuously in individual Drosophila, was used. ARC assays validated the isolation-induced sleep loss phenotype previously observed with DAM assays: daily total sleep, daytime sleep and ZT0-4 sleep were reduced significantly after 7 days of social isolation. In addition, nighttime sleep was also reduced, probably owing to higher sensitivity in detecting movements using the positional tracking method, or differences in chamber shape and food source between the ARC and DAM systems. As predicted from the gene expression profiling results, increased feeding was observed in socially isolated animals compared to their group-treated counterparts. Flies isolated for 7 days showed significant increases in total food consumption, daytime food consumption, nighttime food consumption and ZT0-4 food consumption in comparison to flies that were group-housed for 7 days. Thus, chronic social isolation induces a starvation state in Drosophila at the levels of both gene expression in the brain and behaviour (Li, 2021).
The altered feeding pattern produced by chronic social isolation is not merely a consequence of sleep loss, because several classic sleep mutants all exhibited normal feeding behaviour. In addition, acutely isolated flies did not show a strong increase in food consumption (Li, 2021).
The candidate gene Lst is normally induced by nutrient restriction in endocrine neurons in the corpora cardiaca. However, the RNA profiling experiment suggested that there could be a previously unknown brain source for LST production. A resource of high-resolution transcriptomes of 100 GAL4 driver lines suggested that cells labelled by the driver line NPF-GAL4 (NPF, neuropeptide F [the fly homologue of neuropeptide Y)] are likely to express LST. Using a monoclonal antibody against LST, co-localized LST immunoreactivity and NPF-GAL4-driven GFP signals were detected. Among six known neuronal clusters that express NPF, LST immunoreactivity appeared to be co-localized with NPF-GAL4-driven GFP signals at the dorsal stratum of the fan-shaped body (dorsal fan-shaped body, dFB) and in a cluster of small cell bodies in the dorsal brain. Neurons without known function that comprise this cluster of NPF cells were previously named P2. A recent study used a split-GAL4 driver, SS0020-split-GAL4 (abbreviated as P2-GAL4 below), to strongly label the majority of P2 neurons that showed positive immunoreactivity for LST and NPF22 (Li, 2021).
Notably, the projections of the P2 neurons overlapped with the axonal projections of the dFB neurons labelled by R23E10-GAL4, which suggests that P2 neurons might signal to sleep-promoting dFB neurons. At the cell body level, P2 neurons differ from the R23E10-GAL4 labelled cells. A MultiColor FlpOut (MCFO) approach was used to stochastically decorate individual neurons labelled by P2-GAL4, and it was found that they are fan-shaped body columnar neurons. The hemibrain connectome allowed determination that P2 neurons include, as a dominant constituent, the hDeltaK cell type—a columnar cell class, where each neuron has a stereotypical dendritic input in the ellipsoid body (EB) in addition to the FB innervation. hDeltaK cells exhibit extensive synaptic connections with a known subset of R23E10-GAL4-labelled sleep-promoting dFB neurons27 . On the basis of the above connectome data and existing evidence that NPF/NPY is involved in animal metabolism and stress responses, focus was placed on P2 neurons (Li, 2021).
To test whether P2 neurons contribute to sleep loss induced by chronic social isolation, these neurons were chronically silenced by expressing the inward-rectifying potassium channel Kir2.1 under the control of P2-GAL4. In flies carrying both P2-GAL4 and UAS-Kir2.1, chronic isolation no longer induced an altered sleep profile when compared to their group-housed counterparts. The cumulative relative frequency curve of daytime sleep bouts for socially isolated animals no longer climbed faster than that of group-reared animals. Raster plots of sleep bouts in individual flies showed little difference between chronically isolated and group-housed flies. No difference was found between isolated and group-housed animals for daily total sleep, daytime sleep, or ZT0-4 sleep. By contrast, in heterozygous parental control animals carrying either the P2-GAL4 or the UAS-Kir2.1 transgene, chronic social isolation robustly induced sleep loss. Temporally silencing P2 neurons using UAS-shibirets1 during group enrichment or social isolation did not block social isolation-induced sleep loss. Although isolated flies carrying both P2-GAL4 and UAS-Kir2.1 still showed some overconsumption of food, they no longer showed excessive food consumption for ZT0-4, and the total increase in daytime food consumption was much smaller than in parental controls (Li, 2021).
Using [Ca2+] imaging, it was found that the activity of P2 neurons was correlated with locomotor activity in both group-housed and isolated flies. One might expect that P2 neurons would be tonically more active in isolated flies than in group-housed flies, but this effect in baseline [Ca2+] levels could not be detected. Alternatively, it can be hypothesize that locomotion drives more P2 neuron total activity during 7 days of isolation than during 1 day of isolation (Li, 2021).
Therefore whether boosting activity in P2 neurons during acute social isolation (1 day) is sufficient to promote behavioural changes that resemble the effects of chronic social isolation (7 days) was measured. To activate P2 neurons, a Drosophila warmth-gated cation channel, UAS-dTRPA1, was expressed with P2-GAL4. The P2-GAL4-labelled neurons were activated by treating the flies at 28 °C during acute social isolation or group enrichment (1 day). Control experiments, using flies of the same genotype, were conducted by treating the flies at 22 °C during acute social isolation or group enrichment (1 day). Following these treatments, all flies were subsequently maintained at 22 °C for measurement of sleep or feeding behaviour. In animals carrying both P2-GAL4 and UAS-dTRPA1, there were significant interactions between temperature treatment and isolation status for total, daytime, and ZT0-4 sleep and food consumption: activation of P2 neurons during acute social isolation promoted significant sleep loss and excessive feeding, whereas activation of P2 neurons in group housing did not alter either sleep or feeding behaviour. In control experiments, no evidence was found for interactions between temperature treatment and isolation status in the heterozygous parental flies (Li, 2021).
Notably, P2 neurons are connected to the dFB neurons that are known to regulate sleep homeostasis and couple energy metabolism to sleep. Artificial activation of P2 neurons can produce a behavioural state that resembles the effects of chronic isolation after social isolation for a single day; however, activation of P2 neurons failed to produce these behaviours in the complete absence of social isolation. This indicates that both activity in P2 neurons and a status of being socially isolated are required to induce reduced sleep and increased feeding. Social isolation might be sensed by P2 neurons or elsewhere in the brain, but in either case appears to cause the activity of P2 neurons to be interpreted differently and thereby to generate novel behaviours. Downregulation of a secreted cytokine in a non-neural tissue, the fat body, suppresses sleep and promotes feeding in Drosophila. It would be interesting to determine whether these behavioural responses also depend on P2 neuronal activity (Li, 2021).
Modifications of feeding circuits appear to be crucial for the evolution of complex social behaviours. For example, in C. elegans, a single-residue difference in the neuropeptide Y receptors of naturally occurring strains determines whether the strains exhibit solitary or social feeding behaviour. As antibodies to neuropeptide F, the fly homologue of neuropeptide Y, label P2 neurons, future work may ascertain whether Drosophila's P2 neurons influence social patterns of feeding behaviour as well as mediating feeding and sleep responses to social isolation (Li, 2021).
In humans, social isolation promotes new emotional states that intensify with the passage of time.
Sleep loss in Drosophila is a faithful readout of the duration of social isolation, and this allowed identification of specific patterns of gene and behavioural states that emerge as social isolation becomes chronic. This unexpected association between social isolation, sleep and metabolism in an insect model is reminiscent of the connection observed by social psychologists between loneliness, sleep difficulties and hyperphagia. Such robust findings in Drosophila suggest that studies of animal models might identify conserved brain states, genes, and neural circuits that are associated with social isolation (Li, 2021).
How social interactions influence cognition is a fundamental question, yet rarely addressed at the neurobiological level. It is well established that the presence of conspecifics affects learning and memory performance, but the neural basis of this process has only recently begun to be investigated. In the fruit fly Drosophila melanogaster, the presence of other flies improves retrieval of a long-lasting olfactory memory. This study demonstrates that this is a composite memory composed of two distinct elements. One is an individual memory that depends on outputs from the α'β' Kenyon cells (KCs) of the mushroom bodies (MBs), the memory center in the insect brain. The other is a group memory requiring output from the αβ KCs, a distinct sub-part of the MBs. Social facilitation of memory increases with group size and is triggered by CO(2) released by group members. Among the different known neurons carrying CO(2) information in the brain, this study established that the bilateral ventral projection neuron (biVPN), which projects onto the MBs, is necessary for social facilitation. Moreover, it was demonstrated that CO(2)-evoked memory engages a serotoninergic pathway involving the dorsal-paired medial (DPM) neurons, revealing a new role for this pair of serotonergic neurons. Overall, this study identified both the sensorial cue and the neural circuit (biVPN>αβ>DPM>αβ) governing social facilitation of memory in flies. This study provides demonstration that being in a group recruits the expression of a cryptic memory and that variations in CO(2) concentration can affect cognitive processes in insects (Maria, 2021).
The ability of an individual to form distinct memories and refer to past experiences contributes to the survival of many species. Sensory stimuli from the environment are processed and integrated during memory formation and retrieval, sometimes impacting animal physiology over the very long term. In so-called social species, conspecifics are part of each individual's environment and constitute an important source of information that can lead to social learning. Although social learning has been widely examined in the literature, the influence of social context on memory retrieval has been poorly addressed, as most memory protocols are carried out on isolated individuals. This is not the case for the fruit fly Drosophila melanogaster, for which memory studies are generally carried out on groups and thus measure memory expression in a social context (Maria, 2021).
Despite a small brain of about 100,000 neurons, Drosophila can learn to associate and memorize different stimuli. A protocol leading to a measurable aversive olfactory memory is widely used in the literature. When exposed to one odor (conditioned stimulus plus; CS+) associated with electric shocks versus another odor (conditioned stimulus minus; CS-) without electric shock, flies learn the association between the CS+ odor and electrical shocks and form an aversive associative olfactory memory. Memory is then scored using a T maze offering a choice between two compartments enriched in the previously negatively reinforced CS+ odor versus the non-reinforced CS- odor. Memory is thus revealed by a selective avoidance of the CS+. After a single training protocol, this memory is short lasting. However, repeated training cycles generate a long-lasting memory that is measurable at least 24 h after training. Multiple training cycles without any resting period (i.e., massed training) form a consolidated memory that persists for at least 24 h and is independent of de novo protein synthesis. So far, this form of consolidated memory has been characterized as anesthesia-resistant memory (ARM) because it is resistant to a cold-shock anesthesia. Interestingly, memory after massed training is socially facilitated, as flies tested in groups perform better than individuals tested alone, which is not the case for short-lasting memory. After massed training, only flies that express ARM are influenced by the social context during memory retrieval, which implies that ARM formed after massed conditioning is required to reveal this socially facilitated memory (hereafter SFM). Another form of consolidated memory can be generated by multiple training cycles performed with a 15-min resting period between each cycle (i.e., spaced training), which leads to a robust memory dependent at least partly on de novo protein synthesis and defined as long-term memory (LTM). Recent work proposed that spaced training leads to a dual memory composed of a safety memory for the CS-, identified as the de novo protein synthesis LTM, and an aversive memory for the CS+, which displays similarities with ARM generated by a massed training.9 Unlike memory generated after massed conditioning, individual memory (i.e., memory performance of a fly tested alone) is much higher and not sensitive to the social context. The lack of influence of the social context after spaced training could be explained by the high individual memory, which would have reached a ceiling effect. Alternatively, the ARM generated by spaced conditioning might be different from that formed by massed training and not be subject to SFM or, although sharing similarities with ARM, the CS+ memory measured after spaced training might not be ARM as formally described in other studies. In any case, only memory formed after massed training is predisposed to SFM, for which memory performance increases in a social context. Although social facilitation of memory retrieval has been reported in humans, the increased memory performance of Drosophila tested in groups constitutes the first example of this phenomenon in invertebrates. Understanding the mechanisms underlying SFM could lead to insight into how social interactions influence cognition (Maria, 2021).
This study has shown that CO2 can act as a facilitating cue leading to an improvement in memory retrieval. Moreover, it was demonstrated that such improvement relies on the expression of ARM formed after a massed training, which is expressed distinctly from individual memory, and the neural network supporting the expression of this additional CO2-sensitive memory was identified. Memory retrieval within a group relies on the recruitment of a second neural network in addition to the one required when flies are tested alone. SFM is not a simple improvement of the expression of an individual memory but constitutes a memory expression in its own right. Therefore, the memory revealed in a social context is actually a composite memory consisting of two previously encoded memories, ASM and ARM, whose expression relies on distinct neural structures. Expression of these memories is indeed independent and additive given that the inhibition of one memory during the retrieval phase does not impair the expression of the other. Thus, this work has provided evidence that ASM is the memory expressed when flies are tested individually and is independent of CO2, whereas SFM has been characterized as the additional expression of ARM in a social context (Maria, 2021).
The predictability of an unconditioned stimulus (US) by an originally neutral stimulus becomes higher upon repetition of the stimulus pairing over extended periods. In Drosophila, two types of aversive long-lasting memories have been characterized. On the one hand, the composite memory described in the present study arises after massed training and is independent of protein synthesis. On the other hand, another form of consolidated memory occurs after spaced training and is dependent on de novo protein synthesis (LTM). Recently, this consolidated memory has been defined as the addition of LTM and ARM, an aversive memory independent of protein synthesis. ARM potentially generated by spaced training and the socially facilitated ARM generated by massed training would involve distinct molecular processes, as suggested by the distinct pathways recruited by spaced and massed trainings. Indeed, serotonin synthesis inhibitor para-chlorophenylalanine (pCPA) treatment, the Drk mutation, or the biVPN blockade (this study) impairs the memory formed after massed training but not the memory generated by spaced training. Like ARM measured after massed conditioning, the CS+ memory measured after spaced training is Radish dependent, which led to its characterization as ARM. However, the memory generated by spaced conditioning does not seem to share the other ARM characteristics detailed above and it should be considered that this CS+ memory would not be ARM in the classical sense, as supported by other studies. In any case, memory formed after spaced training is the most stable form of memory reported in Drosophila and can last up to 7 days post-training. It enables high individual retrieval performances but requires, at least in part, de novo protein synthesis (LTM) involving metabolically costly processes, which can occur at the expense of an animal's fitness under stressful conditions. Similar to aversive LTM formed after spaced training, long-lasting appetitive memory depends on de novo protein synthesis. Interestingly, neither aversive nor appetitive memory dependent on protein synthesis is socially facilitated. The SFM mechanism, purely independent of protein synthesis, would then allow flies to behave appropriately while reducing the costs of learning. Surprisingly, social context does not influence the formation of SFM but rather only its retrieval. This suggests that CO2 possibly released by flies during training does not foster individual learning. this would indicate that the training procedure used in this study generated sufficiently high levels of learning for the influence of the social context to become negligible. Because CO2 is not necessary for the retrieval of memory formed after aversive spaced training, it is concluded that CO2 does not play a general role as a memory enhancer. This aspect deserves further investigation (Maria, 2021).
Besides Drosophila, an influence of the social context on memory retrieval has been highlighted in humans, first addressed by Kenneth Spence in 1956 and summarized by the Drive theory. According to this theory, an individual's performance is potentiated by the presence of other individuals provided that the task performed has been correctly learned beforehand. Social facilitation of memory in Drosophila is consistent with this theory. Yet, because the studies in humans have focused only on short-term restitution, the influence of social context on long-lasting retrieval evinced in the current work remains to be addressed in other taxa, such as rodents or insects. Memory tests are typically conducted on individuals because the characterization of memory refers to an individual's acquisition, storage, and retrieval of information. Yet, in light of the current findings, it would be interesting to determine to what extent social context affects memory retrieval in other animal species (Maria, 2021).
This study showed that CO2 recruits additional circuits leading to the socially facilitated ARM expression. Flies emit and process more CO2 in a group, possibly integrating CO2 as a marker of stress. Therefore, CO2 can be conceived as a stress cue enhancing a fly's attention, changing its representation of the environment, and mediating the expression of an additive memory. Indeed, this study has provided evidence that exposure to CO2 alters the CS- response in DPM neurons, which could stimulate flies' awareness to the CS+ memory trace by inhibiting the responses to the irrelevant CS- stimulus. In vertebrates, moderate stress can promote aversive long-lasting memory. Although memory mechanisms described for vertebrates differ from those in the current model, the benefits of moderate stress on memory seem to be common across species (Maria, 2021).
So far, the role of CO2 in insect behavior has been mostly limited to naive avoidance and attraction. This study reveals an important role for CO2 as a facilitator of olfactory memory. In natural environments, CO2 is a ubiquitous cue, including within the nest of eusocial insects such as ants, termites, or bees8 that can be potentially significant and attractive. It is an attractive cue for insects at food sources and oviposition sites and also plays a key role in host detection for hematophagous insects such as tsetse flies or mosquitoes. Olfactory learning plays a significant role in host preference and disease transmission in blood-feeding insects. Thus, exploring the impact of CO2 on memory processes in these insects would be interesting to develop and improve control strategies to reduce the risk of disease transmission. These findings suggest that CO2 may have an unsuspected impact on the cognition of a broad spectrum of insect species (Maria, 2021).
Cooperative behavior often arises when a common exploitable resource is generated. Cooperation can provide equitable distribution and protection from raiding of a common resource such as processed food. Under crowded conditions in liquid food, Drosophila larvae adopt synchronized feeding behavior which provides a fitness benefit. A key for this synchronized feeding behavior is the visually guided alignment of a 1-2 s locomotion stride between adjacent larvae in a feeding cluster. The locomotion stride is thought to be set by embryonic incubation temperature. This raises a question as to whether sib larvae will only cluster efficiently if they hatch at the same temperature. To test this, larvae were first collected and incubated in outdoor conditions. Morning hatched lower temperature larvae move slower than their afternoon higher temperature sibs. Both temperature types synchronize but tend to exclude the other type of larvae from their clusters. In addition, fitness, as measured by adult wing size, is highest when larvae cluster with their own temperature type. Thus, the temperature at which an egg is laid sets a type of behavioral stamp or password which locks in membership for later cooperative feeding (Williamson, 2021).
The Drosophila model is used to investigate the effects of diet on physiology as well as the effects of genetic pathways, neural systems and environment on feeding behavior. Previous work showed that Blue 1 works well as a dye tracer to track consumption of agar-based media in Drosophila in a method called Consumption-Excretion (Con-Ex. This study describes Orange 4 as a novel dye for use in Con-Ex studies that expands the utility of this method. Con-Ex experiments using Orange 4 detect the predicted effects of starvation, mating status, strain, and sex on feeding behavior in flies. Orange 4 is consumed and excreted into vials linearly with time in Con-Ex experiments, the number of replicates required to detect differences between groups when using Orange 4 is comparable to that for Blue 1, and excretion of the dye reflects the volume of consumed dye. In food preference studies using Orange 4 and Blue 1 as a dye pair, flies decreased their intake of food laced with the aversive tastants caffeine and NaCl as determined using Con-Ex or a more recently described modification called EX-Q. These results indicate that Orange 4 is suitable for Con-Ex experiments, has comparable utility to Blue 1 in Con-Ex studies, and can be paired with Blue 1 to assess food preference via both Con-Ex and EX-Q (Shell, 2021).
Many behaviors exhibit ~24-h oscillations under control of an endogenous circadian timing system.Most circadian research in Drosophila has focused on the generation of locomotor activity rhythms, but a fundamental question is how the circadian clock orchestrates multiple distinct behavioral outputs. This study has investigated the cells and circuits mediating circadian control of feeding behavior. This study shows that the presence of feeding rhythms requires molecular clock function in the ventrolateral clock neurons of the central brain. This study further demonstrate that the speed of molecular clock oscillations in these neurons dictates the free-running period length of feeding rhythms. In contrast to the effects observed with central clock cell manipulations, This study shows that genetic abrogation of the molecular clock in the fat body, a peripheral metabolic tissue, is without effect on feeding behavior. Under these conditions, the period of feeding rhythms tracks with molecular oscillations in central brain clock cells, consistent with a primary role of the brain clock in dictating the timing of feeding behavior. Finally, despite a lack of effect of fat body selective manipulations, this study found that flies with simultaneous disruption of molecular clocks in multiple peripheral tissues (but with intact central clocks) exhibit decreased feeding rhythm strength and reduced overall food intake. This study concluded that both central and peripheral clocks contribute to the regulation of feeding rhythms, with a particularly dominant, pacemaker role for specific populations of central brain clock cells (Fulgham, 2021).
Animals, from insects to humans, exhibit obvious diurnal rhythmicity of feeding behavior. Serving as a genetic animal model, Drosophila has been reported to display feeding rhythms; however, related investigations are limited due to the lack of suitable and practical methods. This study presents a video recording-based analytical method, namely, Drosophila Feeding Rhythm Analysis Method (dFRAME). Using this newly developed computer program, FlyFeeding, the movement track of individual flies was extracted, and their food-approaching behavior was characterized. To distinguish feeding and no-feeding events, high-magnification video recording was used to optimize the method by setting cut-off thresholds to eliminate the interference of no-feeding events. Furthermore, it was verified that this method is applicable to both female and male flies and for all periods of the day. Using this method, long-term feeding status of wild-type and period mutant flies was analyzed. The results recaptured previously reported feeding rhythms and revealed detailed profiles of feeding patterns in these flies under either light/dark cycles or constant dark environments. Together, the dFRAME method enables a long-term, stable, reliable, and subtle analysis of feeding behavior in Drosophila. High-throughput studies in this powerful genetic animal model will gain great insights into the molecular and neural mechanisms of feeding rhythms (Niu, 2021).
Pavlovian conditioning is a broadly used learning paradigm where defined stimuli are associated to induce behavioral switching. To define a causal relationship between activity change in a single neuron and behavioral switching, this study took advantage of a 'command neuron' that connects cellular function to behavior. To examine the cellular and molecular basis of Pavlovian conditioning, previous work identified a pair of feeding command neurons termed 'feeding neurons' in the adult Drosophila brain using genetic screening and opto- and thermo-genetic techniques. The feeding neuron is activated by sweet signals like sucrose and induces the full complement of feeding behaviors, such as proboscis extension and food pumping. Ablation or inactivation of the pair of feeding neurons abolishes feeding behavior, suggesting that this single pair of neurons is indispensable for natural feeding behaviors. This study describes a novel conditioning protocol to associate a signal-mediating rod removal from legs (conditioned stimulus [CS]) to feeding behavior induced by sucrose stimulation (unconditioned stimulus [US]). Calcium imaging of the feeding neuron demonstrated it acquires responsiveness to CS during conditioning, with inactivation of the feeding neuron during conditioning suppressing plasticity. These results suggest conditioning alters signals flowing from the CS into the feeding circuit, with the feeding neuron functioning as a key integrative hub for Hebbian plasticity (Sakurai, 2021).
This study demonstrate Pavlovian conditioning between tactile (CS) and gustatory (US) stimuli results in altered information processing by a pair of command neurons that control the Drosophila feeding circuit. This conditioning paradigm creates CS-induced excitement of the feeding neuron that commands feeding behavior in this animal, with the conditioned response requiring activity of the feeding neuron during pairing. Pioneering studies by Kandel and colleagues demonstrated the first synaptic and cellular mechanism underlying classical conditioning using the Aplysia gill withdrawal response. In Aplysia, the presynaptic terminal of a sensory neuron innervating the motor neuron was modulated by serotonin. Presynaptic modulation as a mechanism to generate Drosophila valence behaviors has been extensively studied, and recent progress indicates presynaptic terminals innervating mushroom body output neurons are modulated by dopaminergic neurons to establish Drosophila valence through appetitive and aversive olfactory association. Neither Aplysia plasticity nor Drosophila valence in these paradigms requires postsynaptic activity during learning. In contrast, Hebb proposed general principles to explain mechanisms for memory formation that better match results from commonly used mammalian experimental models, such as hippocampal long-term potentiation (LTP).Hebb postulated sequential firing of a presynaptic neuron and postsynaptic partner strengthens their connection. The requirement of feeding neuron activity for the conditioned response observed in this study fits well to a Hebbian mechanism if the underlying change is manifested in altered synaptic properties (although it cannot be excluded that inactivation of the feeding neuron and subsequent behavioral changes also alter neuromodulation, influencing memory formation). During association, CS-conveying neurons and the feeding neuron driven by sucrose stimulation would now fire together, resulting in strengthened connection between CS-conveying neurons and the feeding neuron according to a Hebbian mechanism. The response to US, however, did not change during conditioning, suggesting that connections between US-conveying neurons and the feeding neuron were not altered. Thus, one can hypothesize that the CS-feeding neuron circuit was newly established, whereas the pre-existing US-feeding neuron connection was not changed. These results suggest that Pavlovian conditioning is established through a change in information processing by the command neuron, which functions as the integrative hub of the feeding circuit (Sakurai, 2021).
This Pavlovian conditioning mechanism can also accommodate presynaptic modulation as demonstrated in Aplysia plasticity and Drosophila valence if reward signals are coupled to Hebbian plasticity through presynaptic neuromodulation. For Drosophila valance memory, reward signals consist of both sweet sensing and nutrition. Similar reward signals are likely to be relevant in vivo for Pavlovian conditioning, although the nutrition reward is eliminated in the current study due to removal of the esophagus from the preparation and application of a sucrose-wet paper strip only to the sensilla of the proboscis. Therefore, reward signals are likely constant between the groups that were tested, even for different US responses in the halorhodopsin experiments. Thus, differences in reward signal can be excluded from the altered conditioned responses observed between the groups. It is hypothesized that inactivation of the feeding neuron results in weaker memory due to postsynaptic activity in this neuron contributing to memory formation independent of changes in the reward signal. It is speculated that reward signals in the current model may also be mediated by dopamine, octopamine, or serotonin, similar to their role as reward signals in the mushroom bodies for Drosophila valence memory. In Aplysia, presynaptic adenylyl cyclase, which synthesizes cAMP, is believed to associate CS and US in this conditioning paradigm through US-driven serotonin modulation of the presynaptic terminal of the CS-conveying neuron. Adenylyl cyclase is encoded by rut, while dnc encodes a cAMP phosphodiesterase that degrades cAMP. As demonstrated in Aplysia and Drosophila, cAMP functions as a signal to modulate synaptic transmission. Given its role in LTP, cAMP is likely to play a critical role in Hebbian plasticity as well, consistent with the disruption of CS-US pairing in rut and dnc mutants. Considering the involvement of postsynaptic cells in Hebbian plasticity, retrograde signals from the postsynaptic cell can also be coupled to presynaptic cAMP signaling, as demonstrated previously at the Drosophila neuromuscular junction (Sakurai, 2021).
In the original experiment conducted by Pavlov, it is speculated there are groups of neurons that command feeding behavior in the dog. CS/US association may change responsiveness of a subset of those neurons that result in sound-induced saliva secretion, even in the absence of food signals. Electrophysiological studies have shown neural responses to CS are altered after Pavlovian conditioning in cat red nucleus and rabbit cerebellum, although how this kind of plastic change leads to alterations in command neuron function is unknown. Neurons with command function have been identified across many species. A command neuron is pivotally located within the sensorimotor watershed of a neuronal circuit and triggers a behavioral program after integrating numerous sensory inputs. Command neurons were first identified in crayfish through experiments where electrical stimulation of a certain neuron switched on or off behaviors, such as rhythmical movement of the swimmeret or escape responses. After identification of command neurons in invertebrate CNSs Mauthner cells were demonstrated to command escape behavior in fish. Recently, a group of neurons commanding feeding behavior have been identified in the mouse brain. Therefore, the scheme shown in Figure 4D may represent a common mechanism underlying Pavlovian conditioning across species, given the role of command neurons as an integrative hub within the sensorimotor watershed of neuronal circuits (Sakurai, 2021).
The feeding neuron in the Drosophila brain functions as a single command neuron pair that triggers the entire feeding program.3 This feature allowed reliable demonstration that CS-induced activation of the feeding neuron after conditioning was as robust as US-induced activation, suggesting the CS-induced activation of the feeding neuron can trigger the conditioned behavior. Thus, neurophysiological changes can be unambiguously correlated with behavioral change, making the causal relationship clear and allowing reliable manipulation. The current results are consistent with the assumption that both the CS signal and the US signal converge at a single identified neuron through a Hebbian mechanism. Taking advantage of the defined circuit with the feeding neuron at the center, it is not possible to define the cellular and molecular mechanisms for synaptic plasticity using this experimentally accessible neuron within the CNS. This approach, coupled with real-time live imaging, may allow tracking of changes in the structure or activity of identified synapses responsible for memory formation once CS-conveying neurons are defined in the experimental system. If so, it may be possible to directly observe pre- and/or postsynaptic changes mediating memory formation on the dendrite of the feeding neuron. Whether a new circuit is generated by strengthening a rudimentary pre-existing connection or a new connection forms de novo during associative conditioning will require future analysis. Molecular and cellular mechanisms underlying this plastic change can be investigated in detail as previously characterized at neuromuscular junctions. Taken together, the study of synaptic plasticity in the feeding neuron provides a model system to characterize basic principles of memory formation at the single-cell level (Sakurai, 2021).
Swallowing is an essential step of eating and drinking. However, how the quality of a food bolus is sensed by pharyngeal neurons is largely unknown. This study finds that mechanical receptors along the Drosophila pharynx are required for control of meal size, especially for food of high viscosity. The mechanical force exerted by the bolus passing across the pharynx is detected by neurons expressing the mechanotransduction channel NOMPC (no mechanoreceptor potential C) and is relayed, together with gustatory information, to IN1 neurons in the subesophageal zone (SEZ) of the brain. IN1 (ingestion neurons) neurons act directly upstream of a group of peptidergic neurons that encode satiety. Prolonged activation of IN1 neurons suppresses feeding. IN1 neurons receive inhibition from DSOG1 (descending subesophageal neurons) neurons, a group of GABAergic neurons that non-selectively suppress feeding. These results reveal the function of pharyngeal mechanoreceptors and their downstream neural circuits in the control of food ingestion (Yang, 2021).
Overconsumption is harmful for animals. Although the drive to ingest can be overwhelming for a hungry animal in the initial stage of a meal, inhibition becomes more dominant with the processes of food intake. This study found that food flowing across the pharynx accumulates the satiety state in the brain, demonstrating that multiple strategies are used by the nervous systems to avoid overeating. These pharyngeal sensory neurons are sensitive to sugar and mechanical force, serving as a flow meter that monitors food quality and amount so that the brain knows how much food is ingested even before the food reaches the intestine. This circuit may coordinate with other satiety signals, such as those conveyed by mechanical feedback from the intestine, to control feeding (Yang, 2021).
Gustatory and mechanosensory neurons are well separated on the fly labellum before their axons reach the SEZ, where they interact with each other to regulate the perception of food quality. In contrast, the sensory neurons in the pharynx seem to adapt a different coding mechanism. Some of the pharyngeal neurons are polymodal because they respond to chemical and mechanical stimuli, with PM neurons being an example. A 'generalist' versus 'specialist strategy has been found in other sensory organs too. Being able to evaluate multiple properties of a bolus in the pharynx allow the animals to effectively control the feeding amount. There are sensory neurons in the pharynx that may be tuned to gustatory or mechanosensory cues. For example, the R41E11-GAL4 and nompC-QF labeled approximately 10 pairs of neurons in LSOs along the pharynx, similar to the number observed for mechanosensory neurons. Most of those neurons are likely 'generalist' and are tuned to mechanical stimuli only. It would be valuable to determine the full repertoire of these sensory neurons to understand how the swallowing maneuver is initiated, sustained, and terminated (Yang, 2021).
It has been proposed that IN1 neurons may function as a key node of the feeding control circuits to govern rapid feeding decisions. Previous studies have revealed that IN1 neurons are directly downstream of pharyngeal GRNs and that activation of IN1 neurons to sugar stimulation is correlated with a fly's motivation to feed. Because activation of IN1 neurons triggers proboscis extension to food, they are likely upstream of the motor circuit that controls feeding. IN1 neurons thus appear to function as a hub that integrates sensory information to initiate food ingestion. This study found that IN1 neurons' activity is under control of the fly's feeding states. IN1 neurons are directly downstream of DSOG1 neurons that non-selectively suppress ingestion. In fed flies, DSOG1 neurons impart inhibition on IN1 neurons, resulting in a transient and moderate response to a sugar sip that triggers a robust and sustained calcium response in fasted flies (Yang, 2021).
It has been proposed that DSOG1 neurons impart constant inhibition on the neuronal circuits that initiate food ingestion. However, the upstream circuit of DSOG1 neurons has not been identified. A cohort of neuropeptide receptor genes has been screened, but none of them seemed to function on DSOG1 neurons in feeding control. This study found that interrupting signaling of the neuropeptide MIP phenocopied overfeeding in flies with silenced DSOG1 neurons. It is tantalizing to hypothesize that MIP neurons are upstream of the DSOG1 circuit, either directly or indirectly. Because the receptors of MIP have not yet been identified, further experiments are need to differentiate between the two possibilities (Yang, 2021).
Besides PM neurons, there are many NOMPC-expressing mechanosensory neurons along the fly pharynx. Because of the lack of specific driver lines and the technique to record a single neuron's activity during feeding, their roles in feeding regulation are interesting open questions and await in-depth investigation. Moreover, the receptors of MIP peptide have not been identified, especially the ones involved in feeding regulation, making it difficult to establish a connection between MIP neurons and DSOG1 neurons (Yang, 2021).
The mechanism through which the brain senses the metabolic state, enabling an animal to regulate food consumption, and discriminate between nutritional and non-nutritional foods is a fundamental question. Flies choose the sweeter non-nutritive sugar, L-glucose, over the nutritive D-glucose if they are not starved. However, under starvation conditions, they switch their preference to D-glucose, and this occurs independent of peripheral taste neurons. This study found that eliminating the TRPgamma channel impairs the ability of starved flies to choose D-glucose. This food selection depends on trpgamma expression in neurosecretory cells in the brain that express Diuretic hormone 44 (DH44). Loss of trpgamma increases feeding, alters the physiology of the crop, which is the fly stomach equivalent, and decreases intracellular sugars and glycogen levels. Moreover, survival of starved trpgamma flies is reduced. Expression of trpgamma in DH44 neurons reverses these deficits. These results highlight roles for TRPgamma in coordinating feeding with the metabolic state through expression in DH44 neuroendocrine cells (Dhakal, 2022).
Dietary restriction (DR) improves survival across a wide range of taxa yet remains poorly understood. The key unresolved question is whether this evolutionarily conserved response to temporary lack of food is adaptive. Recent work suggests that early-life DR reduces survival and reproduction when nutrients subsequently become plentiful, thereby challenging adaptive explanations. A new hypothesis maintains that increased survival under DR results from reduced costs of overfeeding. This study tested the adaptive value of DR response in an outbred population of Drosophila melanogaster fruit flies. DR females did not suffer from reduced survival upon subsequent re-feeding and had increased reproduction and mating success compared to their continuously fully fed (FF) counterparts. The increase in post-DR reproductive performance was of sufficient magnitude that females experiencing early-life DR had the same total fecundity as continuously FF individuals. These results suggest that the DR response is adaptive and increases fitness when temporary food shortages cease (Sultanova, 2021).
To study the behavior of Drosophila, it is often necessary to restrain and mount individual flies. This requires removal from food, additional handling, anesthesia, and physical restraint. A strong positive correlation was found between the length of time flies are mounted and their subsequent reflexive feeding response, where one hour of mounting is the approximate motivational equivalent to ten hours of fasting. In an attempt to explain this correlation, anesthesia side-effects, handling, additional fasting, and desiccation were ruled out. Respirometric and metabolic techniques coupled with behavioral video scoring were used to assess energy expenditure in mounted and free flies. A specific behavior was isolated capable of exerting large amounts of energy in mounted flies, and it was identified as an attempt to escape from restraint. A model is presented where physical restraint leads to elevated activity and subsequent faster nutrient storage depletion among mounted flies. This ultimately further accelerates starvation and thus increases reflexive feeding response. In addition, it was shown that the consequences of the physical restraint profoundly alter aerobic activity, energy depletion, taste, and feeding behavior, and suggest that careful consideration is given to the time-sensitive nature of these highly significant effects when conducting behavioral, physiological or imaging experiments that require immobilization (Gordon, 2021).
Cellular Insulin signaling (IS) shows a remarkable high molecular and functional conservation. Insulin-producing cells respond directly to nutritional cues in circulation and receive modulatory input from connected neuronal networks. Neuronal control integrates a wide range of variables including dietary change or environmental temperature. Although it is shown that neuronal input is sufficient to regulate Insulin-producing cells, the physiological relevance of this network remains elusive. In Drosophila melanogaster, Insulin-like peptide7-producing neurons are wired with Insulin-producing cells. The former cells regulate the latter to facilitate larval development at high temperatures, and to regulate systemic Insulin signaling in adults feeding on calorie-rich food lacking dietary yeast. These results demonstrate a role for neuronal innervation of Insulin-producing cells important for fruit flies to survive unfavorable environmental conditions (Prince, 2021).
This study has analyzed the role of dIlp7-producing neurons in different thermal treatments. D7Ns are active on yeast diets, but show no activity in animals kept on yeast-free corn food (CF). Activated D7Ns are required to respond to heat stress. In addition, dIlp7 produced by D7Ns regulates dIlp2/dIlp3-induced Insulin signaling (IS) on CF, and yeast products are able to supplement efficiently for the loss of this neuropeptide (Prince, 2021).
The generative cycle of Drosophila is divided into feeding and non-feeding stages. Due to the absence of food intake during embryonic and pupal development these stages highly rely on internal energy stores. In contrast, larvae and adults need to absorb food to survive and develop. The insulin signaling cascade is one metabolic circuit to regulate the absorption and internal turnover of macronutrients. In addition, the cascade is essential to provide thermal resistance for ectothermic insects. All feeding stages of Drosophila express four neuronal Insulin-like peptides, namely dIlp 2, 3, 5, and 7 . Larvae with functionally compromised Insulin-producing cells (IPCs) kept on yeast diets are heat sensitive, slow in development and small in size (Prince, 2021).
Dietary yeast increase intracellular Ca2+ levels of IPCs, elevate systemic IS and support survival at high temperatures. This study found that IPCs with high Ca2+ are not sufficient to rescue larval survival at high temperatures on yeast-free CF. Therefore, it was speculated that yeast products likely activate additional neurons involved in heat stress responses. It was shown that animals kept on yeast increase Ca2+ in D7Ns (Linneweber, 2014). D7Ns connect to IPCs and are able to stimulate the latter. This study shows that, on CF, D7Ns are low on Ca2+ with respect to yeast-fed animals and that induced Ca2+ levels in D7Ns improve larval heat resistance on CF. In addition, larvae with inactivated D7Ns kept on yeast show poor survival at high temperatures. Thus, D7Ns are one integral part of the heat response and it is speculated that these neurons directly communicate with IPCs. D7Ns secrete a multitude of neuropeptides including dIlp7. DIlp7 mutants kept on yeast food (YF) are slightly heat sensitive, and due to such relative high survival rates, is is deemed unlikely that dIlp7 is one main cue crucial to withstand thermal treatments (Prince, 2021).
D7Ns are inactive on CF and attempts were made to identify dIlp candidates responsible for IS on yeast-free diets. Interestingly, dIlp2, dIlp3, and dIlp7 were identified as essential for larval development. Moreover, genetic interactions revealed that δdIlp2,3 double mutants are unable to survive on CF. In stark contrast, δdilp2,7 and δdilp2-3,7 animals rescued the lethality shown by single mutants. These findings indicate a new metabolic link between dIlp7 and dIlp2 essential for larval development in yeast-free environments. However, wild larvae grow in microbe-rich environments, such as rotting fruits, and have likely access to dietary yeast. Adult flies sometimes feed on yeast-poor diets or avoid yeast in response to cold. Therefore, adults kept on CF were sampled. Adult δdilp7 flies show reduced IS levels and higher lethality rates with respect to genetic controls. Moreover, the combined absence of dIlp2 and dIlp7 pronounced the observed adult lethality on CF. Thus, larval and adult dIlp7 signaling is likely very different (Prince, 2021).
It was reported that dIlp7 is expressed in the subesophageal ganglion region of the brain and suggested that D7Ns regulate the feeding behavior (Cognigni, 2011). Therefore, reduced feeding of dIlp7 mutants could explain the lower IS levels on CF. This study has shown that, on CF, δdilp2, and δdilp7 mutants ingest food faster, have a longer retention time of the ingested material and are able to absorb macronutrients. Therefore, the idea that these flies are starving on CF is not favored. It is more likely that dIlp7 is required to stimulate IPCs to maintain basic dIlp levels in circulation. To test for this possibility, wthe predicted target receptor of dIlp7, the G-protein-coupled rector Lgr3 was knocked down. The loss of Lgr3 results in low IS levels on CF. In contrast, on YF, all tested genotypes show IS comparable to controls. Taken together, it is concluded that neuronal dIlp7/Lgr3 signaling controls IPCs in adults kept on yeast-free diets. As such dIlp7 secures a basic amount of systemic IS and therefore, likely contributes to thermal resistance of adult flies. However, required adult tracking on CF at low temperatures appeared impractical to confirm this idea (Prince, 2021).
Neuronal innervation of IPCs is established in many animals and modulates metabolic signals. The current findings indicate that food products can overwrite such neuronal stimulation. In Drosophila, a dual role for D7Ns was found: (1) these neurons facilitate the heat response of larvae feeding on yeast and (2) they form a metabolic circuit that enables adult flies to thrive on yeast-free diets if required. In mice and humans, pancreatic islets are directly innervated; however, the role of this neuronal stimulation in response to dietary cues is not well understood. This study has identified the importance of D7Ns and their product, dIlp7, in regulating IS in response to dietary quality. These findings provide new insights into the neuronal stimulation of IPCs within a given ecological context and provide a model to study neuronal innervation of insulin producing cells (Prince, 2021).
In flies, neuronal sensors detect prandial changes in circulating fructose levels and either sustain or terminate feeding, depending on internal state. This study describes a three-part neural circuit that imparts satiety-dependent modulation of fructose sensing. Dorsal fan-shaped body neurons display oscillatory calcium activity when hemolymph glucose is high, and these oscillations require glutamatergic input from SLP-AB or 'Janus' neurons projecting from the protocerebrum to the asymmetric body. Suppression of activity in this circuit, either by starvation or by genetic silencing, promotes specific drive for fructose ingestion. This is achieved through neuropeptidergic signaling by tachykinin, which is released from the fan-shaped body when glycemia is high. Tachykinin, in turn, signals to Gr43a-positive fructose sensors to modulate their response to fructose. Together, these results demonstrate how a three-layer neural circuit links the detection of two sugars to produce precise satiety-dependent control of feeding behavior (Musso, 2021).
Regulation of energy intake is a complex process involving food search, an animal's internal state, and the sensory qualities of food. In flies, fructose, either consumed directly or rapidly metabolized from precursors, promotes feeding through activation of a brain fructose sensor called Gr43a. This study describes how a neuronal network composed of neurons in the FB and asymmetric body contributes to energy homeostasis by detecting satiety-dependent changes in hemolymph glucose and modulating fructose drive (Musso, 2021).
The central complex, which is composed of the FB, the protocerebral bridge (PB), the ellipsoid body, and the noduli, is regarded as a center for sensorimotor integration that functions in goal-directed behavior. The FB is organized in nine horizontal layers and nine vertical columns. FB large field neurons of layers 1 to 3, and inputs to these layers from the PB, encode flight direction and general sensory orientation. FB layers 6 and 7 are well known to regulate sleep and arousal, locomotor control, courtship, visual memory, and decision-making related to taste. Layer 6 also plays a role in avoiding conditioned odors, while layers 1, 2, 4, and 5 respond to electric stimuli and are required for innate odor avoidance. However, the function of the most dorsal FB layers (8 and 9), mostly innervated local tangential neurons and AB-FBl8 (or vΔA_a), remained poorly understood. The results demonstrate a role for these layers in feeding regulation (Musso, 2021).
dFB oscillations were found to be require glutamatergic input from Janus neuron projections to the asymmetric body. Described for the first time in 2004, very little is known about AB function; 92.4% of flies display asymmetry in the AB, with the body present only in the right hemisphere, while 7.6% also have a body on the left side. It is noted that oscillations in the dFB display a tendency to be faster on the right side, with clearly asynchronous activity between the two sides that may reflect their asymmetric input from Janus neurons. The small proportion of flies displaying symmetry in the AB have defects in LTM, a process that is known to require energy. It is speculated that these symmetric flies may have a dysfunctional Janus neurons-to-dFB connection, resulting in impaired Tk release. This could affect LTM either directly or through changes in feeding. A role for TK in memory has been demonstrated in honeybees and mammals, and TkR86C appears to be expressed in serotonergic paired neurons known to interact with MB-MP1 neurons required for LTM formation. Tk also acts through TkR99D to modulate activity in neurons producing insulin-like peptides, which affect LTM formation (Musso, 2021).
Modulation of dFB oscillations by Janus neurons requires glutamatergic signaling through a group of glutamate receptors including KaiR1D, NmdaR1, NmdaR2, and GluClα, but not AMPA receptors. Both KaiR1D receptors, which are homomeric, and N-methyl-D-aspartate (NMDA) receptors, which are heteromeric complexes between subunits 1 and 2, pass Ca2+ current. NMDA receptors (NMDAR) are well known for their role in mediating synaptic plasticity and can also trigger oscillatory activity. NMDAR function as molecular coincidence detectors, requiring simultaneous ligand binding and membrane depolarization for activation. It is possible that dFB neuron oscillations are triggered by the coincident detection of glutamate from Janus neurons and glucose from the hemolymph; however, because the FB are receiving many inputs from other brain region, it is suspect that dFB oscillations require additional inputs as well. The chloride channel GluClα is also required for dFB oscillations. GluClα has been previously implicated in on/off responses of the visual system of flies and memory retention in honeybees, demonstrating a role in regulating cell excitability. Perhaps, GluClα functions in repolarization of the dFB neurons between calcium bursts. Further study will be required to fully understand how the suite of glutamate receptors function together to drive oscillations, along with the source of input to Janus neurons in the protocerebrum (Musso, 2021).
Because glucose is the primary circulating energy source, one might intuitively expect that enhancing feeding in response to postingestive glucose detection would be the most efficient means of optimizing energy uptake. However, using elevation of hemolymph glucose as a signal to continue feeding is problematic because glucose levels are tightly regulated and elevated glucose serves as a signal of satiety. On the other hand, internal fructose can vary widely in response to ingestion and can therefore be a more reliable indicator of recent sugar intake. Thus, the separation of glucose as a satiety indicator and fructose as marker of sugar consumption removes the potential ambiguity of each as a signal. Moreover, fructose typically coexists with other nutritive sugars in common food sources. Therefore, it may not be the case that flies specifically benefit from fructose intake but rather that fructose serves as an effective proxy for general carbohydrate ingestion. By using fructose and the narrowly tuned Gr43a fructose receptor to survey sugar consumption, flies can effectively benefit from both a fructose-mediated positive feedback loop and glucose-mediated negative feedback to co-operatively ensure appropriate energy intake (Musso, 2021).
The finding that dFB glucose sensing modulates fructose feeding via Gr43a brain neurons fits with the established model of Gr43a brain neurons as central fructose sensors. For this mechanism to effectively sustain feeding on a rich sugar source, ingested sugars must rapidly increase fructose signaling to Gr43a brain neurons, which then must acutely promote feeding. While the precise kinetics of internal fructose elevation after sugar consumption have not been quantified, fructose levels in the head rapidly increase 10-fold after fructose feeding and then return to baseline. The role of direct fructose sensing by Gr43a brain neurons is highlighted by the observation that Gr43a knockdown in those neurons results in markedly lower relative intake of fructose compared to glucose. Unexpectedly, knockdown of Gr64a, another sugar receptor expressed in the same neurons, produced the opposite effect. This could be because Gr64a contributes to modulation of Gr43a brain neurons by other sugar cues, and the absence of this activity makes Gr43a-mediated fructose responses more pronounced. Alternatively, Gr43a may be expressed more strongly after Gr64a knockdown, leading to an increased fructose response (Musso, 2021).
Little is known about the mechanisms downstream of Gr43a brain neurons that promote feeding. All Gr43a brain neurons express the peptide Crz, but knockdown of Crz expression produced no significant effect on fructose preference over glucose. This suggests an important functional role for another neurotransmitter, although it is also possible that the RNAi knockdown was not effective. Irrespective of mechanism, two experiments support the idea that activation of Gr43a neurons acutely enhances feeding. First, silencing of dFB neurons by genetic manipulation or prolonged starvation produces Gr43a-dependent fructose preference within the first 10 min of a flyPAD assay. Second, closed-loop optogenetic activation of Gr43a brain neurons was sufficient to produce a strong positive preference within 10 min in the STROBE (Musso, 2021).
The separable functions of glucose and fructose sensing in flies bear notable resemblance to the differential effects of these two sugars in the mammalian hypothalamus. In particular, AMPK expression in the arcuate nucleus of the hypothalamus is known to link energy levels to food drive. When glycemia is low, AMPK is activated and thereby promotes feeding through orexigenic AgRP/NPY neuron activity. Glucose administration suppresses activity in these peptidergic neurons, while fructose can have the opposite effect and promote further feeding. The first description of fly Gr43a neurons noted their orexinegenic activity and suggested a potential functional homology with the hypothalamus. In the present study, a multilayered neural system centered on a brain energy sensor (dFB), was uncovered whose activation by glucose leads to anorexigenic behavior through inhibition of the brain fructose sensor Gr43a. Thus, the results are consistent with at least partial functional homology between the mammalian hypothalamus and brain Gr43a neurons of the fly (Musso, 2021).
Taste cues regulate immediate feeding behavior, but their ability to modulate future behavior has been less well studied. Pairing one taste with another can modulate subsequent feeding responses through associative learning, but this requires simultaneous exposure to both stimuli. This study investigated whether exposure to one taste modulates future responses to other tastes even when they do not overlap in time. Using Drosophila, it was found that brief exposure to sugar enhanced future feeding responses, whereas bitter exposure suppressed them. This modulation relies on neural pathways distinct from those that acutely regulate feeding or mediate learning-dependent changes. Sensory neuron activity was required not only during initial taste exposure but also afterward, suggesting that ongoing sensory activity may maintain experience-dependent changes in downstream circuits. Thus, the brain stores a memory of each taste stimulus after it disappears, enabling animals to integrate information as they sequentially sample different taste cues that signal local food quality (Deere, 2022).
The feeding of pests is one of the important reasons for losses of agricultural crop yield. This study aimed to reveal how juvenile hormone participates in larval feeding regulation of the Asian corn borer Ostrinia furnacalis. Larvae of O. furnacalis exhibit a daily circadian rhythm on feeding, with a peak at ZT18 and a trough at ZT6 under both photoperiod (LD) and constant dark (DD) conditions, which may be eliminated by application of fenoxycarb, a juvenile hormone (JH) active analogue. JH negatively regulates larval feeding as a downstream factor of neuropeptide F (NPF), in which knocking down JH increases larval feeding amount along with body weight and length. The production of JH in the brain-corpora cardiaca-corpora allata (brain-CC-CA) is regulated by the brain NPF rather than gut NPF, which was demonstrated in Drosophila larvae through GAL4/UAS genetic analysis. In addition, feeding regulation of JH is closely related to energy homeostasis in the fat body by inhibiting energy storage and promoting degradation. The JH analogue fenoxycarb is an effective pesticide to O. furnacalis that controls feeding and metabolism. The brain NPF system regulates JH, with functions in food consumption, feeding rhythms, energy homeostasis and body size. This study provides an important basis for understanding the feeding mechanism and potential pest control (Yu, 2022).
Drosophila melanogaster, the fruit fly, is an excellent model organism for studying dopaminergic mechanisms and simple behaviors, but methods to measure dopamine during behavior are needed. This study developed fast-scan cyclic voltammetry (FSCV) to track in vivo dopamine during sugar feeding. First, acetylcholine stimulation was used to evaluate the feasibility of in vivo measurements in an awake fly. Next, sugar feeding was tested by placing sucrose solution near the fly proboscis. In the mushroom body medial tip, 1 pmol acetylcholine and sugar feeding released 0.49±0.04 μM and 0.31#177;0.06 μM dopamine, respectively but sugar-evoked release lasted longer than with acetylcholine. Administering the dopamine transporter inhibitor nisoxetine or D2 receptor antagonist flupentixol significantly increased sugar-evoked dopamine. This study develops FSCV to measure behaviorally evoked release in fly, enabling Drosophila studies of neurochemical control of reward, learning, and memory behaviors (Shin, 2022).
Foraging and feeding are indispensable for survival and their timing depends not only on the metabolic state of the animal but also on the availability of food resources in their environment. Since both these aspects are subject to change over time, these behaviors exhibit rhythmicity in occurrence. As the locomotor activity of an organism is related to its disposition to acquire food, and peak feeding in fruit flies has been shown to occur at a particular time of the day, it was asked if cyclic food availability can entrain their rhythmic activity. By subjecting flies to cyclic food availability, that is, feeding-starvation (FS) cycles, food cues were provided contrasting to the preferred activity times, and whether this imposed cycling in food availability could entrain the activity-rest rhythm was studied. Phase control, which is a property integral to entrainment, was not achieved despite increasing starvation duration of FS cycles (FS 12:12, FS 10:14, and FS 8:16). It was also found that flies subjected to T21 and T26 FS zeitgeber cycles were unable to match period of the activity rhythm to short or long T-cycles. Taken together, these results show that external food availability cycles do not entrain the activity-rest rhythm of fruit flies. However, it was found that starvation-induced hyperactivity causes masking which results in phase changes. In addition, T-cycle experiments resulted in minor period changes during FS treatment. These findings highlight that food cyclicity by itself may not be a potent zeitgeber but may act in unison with other abiotic factors like light and temperature to help flies time their activity appropriately (Singh, 2022).
To survive, animals maintain energy homeostasis by seeking out food. Compared to freely feeding animals, food-deprived animals may choose different strategies to balance both energy and nutrition demands, per the metabolic state of the animal. Serotonin mediates internal states, modifies existing neural circuits, and regulates animal feeding behavior, including in humans and fruit flies. However, an in-depth study on the neuromodulatory effects of serotonin on feeding microstructure has been held back for several technical reasons. Firstly, most feeding assays lack the precision of manipulating neuronal activity only when animals start feeding, which does not separate neuronal effects on feeding from foraging and locomotion. Secondly, despite the availability of optogenetic tools, feeding in adult fruit flies has primarily been studied using thermogenetic systems, which are confounded with heat. Thirdly, most feeding assays have used food intake as a measurement, which has a low temporal resolution to dissect feeding at the microstructure level. To circumvent these problems, OptoPAD assay, which provides the precision of optogenetics to control neural activity contingent on the ongoing feeding behavior, was utilized. Manipulating the serotonin circuit optogenetically affects multiple feeding parameters state-dependently. Food-deprived flies with optogenetically activated and suppressed serotonin systems feed with shorter and longer sip durations and longer and shorter inter-sip intervals, respectively. It was further shown that serotonin suppresses and enhances feeding via 5-HT1B and 5-HT7 receptors, respectively (Banu, 2022).
Active touch facilitates environments exploration by voluntary, self-generated movements. However, the neural mechanisms underlying sensorimotor control for active touch are poorly understood. During foraging and feeding, Drosophila gather information on the properties of food (texture, hardness, taste) by constant probing with their proboscis. This study identified a group of neurons (sd-L neurons) on the fly labellum< that are mechanosensitive to labellum displacement and synapse onto the sugar-sensing neurons via axo-axonal synapses to induce preference to harder food. These neurons also feed onto the motor circuits that control proboscis extension and labellum spreading to provide on-line sensory feedback critical for controlling the probing processes, thus facilitating ingestion of less liquified food. Intriguingly, this preference was eliminated in mated female flies, reflecting an elevated need for softer food. These results propose a sensorimotor circuit composed of mechanosensory, gustatory and motor neurons that enables the flies to select ripe yet not over-rotten food by active touch (Yu, 2023).
Consumption of food and water is tightly regulated by the nervous system to maintain internal nutrient homeostasis. Although generally considered independently, interactions between hunger and thirst drives are important to coordinate competing needs. In Drosophila, four neurons called the Interoceptive Subesophageal zone Neurons (ISNs) respond to intrinsic hunger and thirst signals to oppositely regulate sucrose and water ingestion. This study investigated the neural circuit downstream of the ISNs to examine how ingestion is regulated based on internal needs. Utilizing the recently available fly brain connectome, this study found that the ISNs synapse with a novel cell type Bilateral T-shaped neuron (BiT) that projects to neuroendocrine centers. In vivo neural manipulations revealed that BiT oppositely regulates sugar and water ingestion. Neuroendocrine cells downstream of ISNs include several peptide-releasing and peptide-sensing neurons, including insulin producing cells (IPC), crustacean cardioactive peptide (CCAP) neurons, and CCHamide-2 receptor isoform RA (CCHa2R-RA) neurons. These neurons contribute differentially to ingestion of sugar and water, with IPCs and CCAP neurons oppositely regulating sugar and water ingestion, and CCHa2R-RA neurons modulating only water ingestion. Thus, the decision to consume sugar or water occurs via regulation of a broad peptidergic network that integrates internal signals of nutritional state to generate nutrient-specific ingestion (Lez-Segarra, 2023).
Light exposure impacts several aspects of Drosophila development including the establishment of circadian rhythms, neuroendocrine regulation, life-history traits, etc. Introduction of artificial lights in the environment has caused almost all animals to develop ecological and physiological adaptations. White light which comprises different lights of differing wavelengths shortens the lifespan in fruit flies Drosophila melanogaster. The wavelength-specific effects of white light on Drosophila development remains poorly understood. This study shows that different wavelengths of white light differentially modulate Drosophila development in all its concomitant stages when maintained in a 12-h light: 12-h dark photoperiod. It was observed that exposure to different monochromatic lights significantly alters larval behaviours such as feeding rate and phototaxis that influence pre-adult development. Larvae grown under shorter wavelengths of light experienced an altered feedingrate. Similarly, larvae were found to avoid shorter wavelengths of light but were highly attracted to the longer wavelengths of light. Most of the developmental processes were greatly accelerated under the green light regime while in other light regimes, the effects were highly varied. Interestingly, pre-adult survivorship remained unaltered across all light regimes but light exposure was found to show its impact on sex determination. This study for the first time reveals how different wavelengths of white light modulate Drosophila development which in the future might help in developing non-invasive therapies and effective pest measures (Ramakrishnan, 2022).
Neprilysins are highly conserved ectoenzymes that hydrolyze and thus inactivate signaling peptides in the extracellular space. This study focused on Neprilysin 4 from Drosophila melanogaster and evaluate the existing knowledge on the physiological relevance of the peptidase. Particular attention is paid to the role of the neprilysin in regulating feeding behavior and the expression of insulin-like peptides in the central nervous system. In addition, this study assessed the function of the peptidase in controlling the activity of the sarcoplasmic and endoplasmic reticulum Ca(2+) ATPase in myocytes, as well as the underlying molecular mechanism in detail expression evolution structure and function (Buhr, 2023)
Appropriate nutritional intake is essential for organismal survival. In holometabolous insects such as Drosophila melanogaster, the quality and quantity of food ingested as larvae determines adult size and fecundity. This study has identified a subset of dopaminergic neurons (THD') that maintain the larval motivation to feed. Dopamine release from these neurons requires the ER Ca2+ sensor STIM. Larvae with loss of STIM stop feeding and growing, whereas expression of STIM in THD' neurons rescues feeding, growth and viability of STIM null mutants to a significant extent. Moreover STIM is essential for maintaining excitability and release of dopamine from THD' neurons. Optogenetic stimulation of THD' neurons activated neuropeptidergic cells, including median neuro secretory cells that secrete insulin-like peptides. Loss of STIM in THD' cells alters the developmental profile of specific insulin-like peptides including ilp3. Loss of ilp3 partially rescues STIM null mutants and inappropriate expression of ilp3 in larvae affects development and growth. In summary this study has identified a novel STIM-dependent function of dopamine neurons that modulates developmental changes in larval feeding behaviour and growth (Kasturacharya, 2023).
In Drosophila, as in other holometabolous insects, growth is restricted to the larval stages. In early stages of larval development cells exit mitotic quiescence and re-enter mitosis resulting in organismal growth. This change is accompanied by an increase in the feeding rate of the organism so as to provide sufficient nutrition for the accompanying growth in organismal size. In STIMKO larvae a loss of this ability to feed persistently was observed starting from early second instar larvae. The focus of this feeding deficit lies in a subset of central dopaminergic neurons that require STIM function to maintain excitability. Importantly, these dopaminergic neurons communicate with multiple neuropeptidergic cells in the brain to regulate appropriate changes in larval feeding behaviour. The identified dopaminergic cells also communicate with ilp producing neuropeptidergic cells, the MNSc, through which they appear to impact larval growth (Kasturacharya, 2023).
The THD' cells were identified as critical for larval feeding from their inability to function in the absence of the store-operated Ca2+ entry (SOCE) regulator STIM. Loss of excitability and the absence of dopamine release from THD' cells in STIMKO larvae suggests that voltage-dependent receptor activity is required to maintain growth in early 2nd instar larvae. Changes in expression of ion channels and presynaptic components have been observed earlier upon knockdown of STIM in Drosophila and mammalian neurons. Moreover, loss of STIM-dependent SOCE in Drosophila neurons effects their synaptic release properties. Partial rescue of viability in STIMKO organisms by over-expression of a bacterial sodium channel NaChBac and restoration of dopamine release upon rescue by STIM+ supports the idea that STIM-dependant SOCE maybe required for appropriate function and/or expression of ion channels and synaptic components in THD' neurons. Changes in ER-Ca2+ suggest that STIM is also required to maintain neuronal Ca2+ homeostasis (Kasturacharya, 2023).
While mechanisms that regulate developmental progression of Drosophila larvae have been extensively studied, neural control of essential changes in feeding behaviour that need to accompany each larval developmental stage have not been identified previously. Artificial manipulation of activity in the central dopaminergic neuron subset examined in this study (THD'), either by expression of an inward rectifying potassium channel (Kir2.1) or the bacterial sodium channel NaChBac, suggests an important role for THD' neurons during larval development. This idea is supported by the altered dynamics of muscarinic acetylcholine receptor (mAChR) stimulated Ca2+ release observed in THD' neurons between early, mid and late third instar larvae when larval feeding slows down and ultimately stops and re-iterates that signaling in and from these neurons drives larval feeding whereas lower carbachol-induced Ca2+ responses signal cessation of feeding. A weaker rescue of STIMKO larvae is also obtained from STIM+ expression in the THC' neuron subset. Taken together these observations suggest a neuromodulatory role for dopamine, where DA release from THD' neurons has a greater influence on feeding than the DA release from THC' neurons, possibly due to the DL1 and DL2 cluster (among THD' marked neurons) receiving more feeding and metabolic inputs. A role for cells other than THD', in maintaining kinetics of dopamine release required for feeding behaviour are also indicated because expression of STIM+ in THD' neurons did not revert kinetics of dopamine release to wild type levels. The prolonged dopamine release observed in wild-type THD' neurons may arise from synaptic/modulatory inputs to THD' neurons from other neurons that require STIM function (Kasturacharya, 2023).
Though the cells that provide cholinergic inputs to THD' cells have not been identified it is possible that such neurons sense the nutritional state. In this context, two pairs of cells in the THD' subset also motivate the search for food in hungry adult Drosophila. Starved flies with knock down of the mAChR on THD' neurons exhibit a decrease in food seeking behaviour. Cholinergic inputs to THD' neurons for sensing nutritional state/hunger may thus be preserved between larval and adult stages (Kasturacharya, 2023).
Interestingly, dopamine is also required for reward-based feeding, initiation, and reinforcement of feeding behaviour in adult mice. These findings parallel past studies where prenatal mice genetically deficient for dopamine (DA-/-), were unable to feed and died from starvation. Feeding could however be initiated upon either enforced supplementation or injection with L-DOPA allowing them to survive. More recent findings show that dopaminergic neurons in the ventral tegmental area (VTA), and not the substantia nigra, drive motivational behaviour and facilitate action initiation for feeding in adult mice (Kasturacharya, 2023).
Both activation and inhibition of specific classes of neuropeptidergic cells by optogenetic activation of THD' cells suggests a dual role for dopamine possibly due to the presence of different classes of DA receptors. The Drosophila genome encodes four DA receptors referred to as Dop1R1, Dop1R2, DD2R and a non-canonical DopEcR [66]. Dop1R1, Dop1R2 and DopEcR activate adenylate cyclase and stimulate cAMP signaling whereas DD2R is inhibitory. Cell specific differences among dopamine receptors have been observed in adults. Down regulation of Dop1R1 on AstA and NPF cells shifted preference towards sweet food whereas down regulation of DopEcR in DH44 cells shifted preference towards bitter food. In third instar larvae a dopaminergic-NPF circuit, arising from central dopaminergic DL2 neurons, two cells of which are marked by THD'GAL4, motivates feeding in presence of appetitive odours. The dopamine-neuropeptide axis identified in this study demonstrates a broader role for dopamine in regulating neuropeptide release and/or synthesis, in the context of larval feeding behaviour, perhaps similar to the mammalian circuit described above (Kasturacharya, 2023).
Of specific interest is the untimely upregulation of ilp3 transcripts in STIMKO larvae. Rescue of lethality in STIMKO larvae either by bringing back activity to THD' neurons or by reducing ilp3 levels suggests an interdependence of Dopamine-Insulin signaling that is likely conserved across organisms. Thr data suggest that ilp3 expression is suppressed during the feeding and growth stages of larvae, and once enough nutrition accumulates expression of ilp3 is up-regulated, concurrent with a reduction in carbachol-induced Ca2+ signals in THD' neurons, possibly followed by upregulation and release of ilp3. The idea of ilp3 as a metabolic signal whose expression is antagonistic to larval growth is supported by the observation that knock-down of ilp3 in the MNSc leads to larger pupae in wild type animals and larger larvae in STIMKO. This is the first report of ilp3 as a larval signal that is antagonistic to growth. Given that Drosophila encode a single Insulin receptor for ilp2, ilp3 and ilp5 the cellular mechanism of ilp3 action remains to be elucidated. Possibly, ilps with different affinity for the insulin receptor stimulate different cellular subsets and/or different intracellular signaling mechanisms, including ecdysone signaling that is essential for larval transition to pupae. Interestingly, in STIMKO larval brains there is a significant increase in expression of the Insulin Receptor. Further studies are needed to fully understand ilp3 function in larvae (Kasturacharya, 2023).
Expression of other neuropeptides did not show significant changes in STIMKO larval brains, suggesting that for neuropeptidergic cells in the LNC and SEZ, dopamine signals alter release properties rather than synthesis. However, it was not possible to to identify specific neuropeptides for cells in the LNC and the SEZ that responded upon activation of THD' (Kasturacharya, 2023).
The importance of dopamine for multiple aspects of feeding behaviour is well documented in juvenile and adult mice. Of interest are more recent findings linking dysregulation of dopamine-insulin signaling with the regulation of energy metabolism and the induction of binge eating. The identification of a simple neuronal circuit where dopamine-insulin signaling regulates feeding and growth could serve as a useful model for investigating new therapeutic strategies targeted towards the treatment of psychological disorders for obesity and metabolic syndrome (Kasturacharya, 2023).
Feeding behavior is essential for growth and survival of animals; however, relatively little is known about its intrinsic mechanisms. This study demonstrates that Gart is expressed in the glia, fat body, and gut and positively regulates feeding behavior via cooperation and coordination. Gart in the gut is crucial for maintaining endogenous feeding rhythms and food intake, while Gart in the glia and fat body regulates energy homeostasis between synthesis and metabolism. These roles of Gart further impact Drosophila lifespan. Importantly, Gart expression is directly regulated by the CLOCK/CYCLE heterodimer via canonical E-box, in which the CLOCKs (CLKs) in the glia, fat body, and gut positively regulate Gart of peripheral tissues, while the core CLK in brain negatively controls Gart of peripheral tissues. This study provides insight into the complex and subtle regulatory mechanisms of feeding and lifespan extension in animals (He, 2023).
Feeding is a necessary behavior for animals to grow and survive, with a characteristic of taking food regularly. The quality and quantity of feeding directly impact the normal growth and development of animals. Time-restricted feeding or fasting is beneficial for preventing obesity, alleviating inflammation, and attenuating cardiac diseases and even has antitumor effects. Metabolic syndrome has become a global health problem. Shift work and meal irregularity disrupt circadian rhythms, with an increased risk of developing metabolic syndrome. The maintenance of the daily feeding rhythm is very important in metabolic homeostasis.Irregular feeding perturbs circadian metabolic rhythms and results in adverse metabolic consequences and chronic diseases (He, 2023).
Most behaviors in animals are synchronized to a ~24 h (circadian) rhythm induced by circadian clocks in both the central nervous system and peripheral tissues. Circadian rhythmic behaviors, such as feeding and locomotion, are involved in complex connections and specific output pathways under the control of the circadian clock. Although the core clock feedback loop has been well established in recent decades, the crucial genes responsible for rhythmic feeding regulation as well as for the interrelation between the core clocks and feeding are still unclear (He, 2023).
To increase the understanding of how the circadian clock regulates feeding and metabolism, this study sought to identify the output genes in the circadian feeding and metabolism control network, in which the model animal Drosophila provides special advantages to explore the mechanistic underpinnings and the complex integration of these primitive responses. Previous studies confirmed that one of juvenile hormone receptors, methoprene tolerance (Met), is important for the control of neurite development and sleep behavior in Drosophila. Many genes related to metabolic regulation have attracted attention in the transcriptome data from Met27, a Met-deficient fly line, in which this study focused on the target genes regulated by CLOCK/CYCLE (CLK/CYC). As a basic Helix-Loop-Helix-Per-ARNT-Sim (bHLH-PAS) transcription factor with a canonical binding site “CACGTG," the CLK/CYC heterodimer is a crucial core oscillator that regulates circadian rhythms (He, 2023).
The Gart trifunctional enzyme, a homologous gene of adenosine-3 in mammals, is a trifunctional polypeptide with the activities of phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase (Tiong, 1990). Gart in astrocytes of vertebrates plays a role in the lipopolysaccharide-induced release of proinflammatory factors (Zhang, 2014), and Gart expressed in the liver and heart is required for de novo purine synthesis. However, there is no information yet for Gart's functions in feeding rhythm. In this study, Gart was identified as a candidate that was controlled by the core clock gene CLK/CYC heterodimer and was found to be related to feeding behavior in Drosophila. Thus this study focused Gart studies on the role of feeding rhythms and further regulatory mechanisms. This study provides a critical foundation for understanding the complex feeding mechanism. (He, 2023).
In animals, hundreds of genes exhibit daily oscillation under clock regulation, and some of them are involved in metabolic functions. This study identified a CLK/CYC-binding gene, Gart, which is involved in feeding rhythms and energy metabolism independent of locomotor rhythms. Previous research reported that blocking CLK in different tissues yields different phenotypes. This study found that MET, like CYC, can combine with CLK to regulate the transcription of Gart. Knocking down Gart in different tissues exhibits different phenotypes, and Gart in different tissues can rescue the phenotype caused by CLK deletion; thus, the phenomenon caused by CLK deletion is due to the change in Gart (He, 2023).
CLK regulates the feeding rhythms of Drosophila, and its loss can cause disorders of feeding rhythms and abnormal energy storage. Tim01, Cry01, and Per01 mutants have significantly lower levels of truactkglycerides (TAGs). The maintenance of energy homeostasis is achieved by a dynamic balance of energy intake (feeding), storage, and expenditure (metabolic rate), which are crucial factors for longevity and resistance to adverse environments in Drosophila. Additionally, studies have shown that mutations of Timeless and per shorten the adult lifespan of Drosophila. This study further reveals that peripheral CLKs control the oscillation of Gart among different peripheral tissues; however, core CLKs in the brain can negatively regulate Gart expression in peripheral tissues, indicating that a complex and refined network regulatory system exists between CLK and Gart in the brain and in different peripheral tissues to coordinate feeding behavior and energy homeostasis in Drosophila and that it further affects sensitivity to starvation and longevity. These novel findings enrich the network of regulatory mechanisms by the clocks-Gart pathway on feeding, energy homeostasis, and longevity (He, 2023).
Glial cells have vital functions in neuronal development, activity, plasticity, and recovery from injury. This study reveals that flies lacking Gart in glial cells display a significant decline in the viability of Drosophila under starvation, caused by a decrease in energy storage that puts flies under a state of energy deficit. This discovery extends the functions of glial cells in feeding, energy storage, and starvation resistance controlled by Gart (He, 2023).
The fat body is the primary energy tissue for the storage of fuel molecules, such as TAG and glycogen, which play an important role in the regulation of metabolic homeostasis and provide the most energy during starvation. Indeed, functional defects of the fat body increase starvation sensitivity in Drosophila. In this study, flies lacking Gart in the fat body led to decreased energy storage, which reduces the survival rate and the survival time under starvation conditions. However, flies lacking gut Gart still maintain normal energy storage, which is not sensitive to food shortage or starvation. In addition, this study found that although high temperature can stimulate the food intake of Drosophila, which is consistent with previous reports, it does not affect the feeding rhythm (He, 2023).
This study reveals that Gart in the glia and the fat body collectively regulate the homeostasis of energy intake, storage, and expenditure, thereby influencing the viability of flies under starvation stress. Although Gart in the gut strongly influences feeding behavior, it does not play similar functions as the glia and the fat body in adversity resistance. This occurs possibly because the gut has vital roles in digestion and absorption, while the fat body has crucial functions in energy metabolism. In addition, Gart in the glia and the fat body has biased roles in the synthesis of glycogen and TAG, despite having similar functions in energy storage. The biased role of the glia and the fat body may be coordinated to provide effective energy homeostasis. These findings provide new insight into how specific circadian coordination of various tissues modulates adversity resistance and aging (He, 2023).
Such robust findings in Drosophila suggest that a decrease in lifespan and an increase in sensitivity to starvation in Drosophila is a faithful readout of disordered feeding rhythms and abnormal metabolism. Gart effects on metabolism in both glia cells and the fat body indicate the intricacy of the circadian network and energy homeostasis. It is crucial for animals to have a well-organized network to coordinate and ensure that these various tissue regions are in a normal state (He, 2023).
This study has demonstrated that CLK regulates feeding, energy homeostasis, and longevity via Gart. Even though attempts were made to explore more comprehensively how Gart coordinates and regulates the physiological functions in different tissues of D. melanogaster, there are still some limitations. For instance, it is still unclear that how Gart achieves functional differentiation in different tissues, as well as whether Gart regulates lifespan through autophagy and/or bacterial content or not, which are two critical factors related to lifespan. These future studies are of great significance for understanding the relationship between feeding and longevity regulated by Gart (He, 2023).
This study describes a leaky integrate-and-fire computational model of the entire Drosophila brain, based on neural connectivity and neurotransmitter identity, to study circuit properties of feeding and grooming behaviors. Activation of sugar-sensing or water-sensing gustatory neurons in the computational model accurately predicts neurons that respond to tastes and are required for feeding initiation. Computational activation of neurons in the feeding region of the Drosophila brain predicts those that elicit motor neuron firing, a testable hypothesis that this study validated by optogenetic activation and behavioral studies. Moreover, computational activation of different classes of gustatory neurons makes accurate predictions of how multiple taste modalities interact, providing circuit-level insight into aversive and appetitive taste processing. This computational model predicts that the sugar and water pathways form a partially shared appetitive feeding initiation pathway, which calcium imaging and behavioral experiments confirmed. Additionally, this model was applied to mechanosensory circuits and found that computational activation of mechanosensory neurons predicts activation of a small set of neurons comprising the antennal grooming circuit that do not overlap with gustatory circuits, and accurately describes the circuit response upon activation of different mechanosensory subtypes. These results demonstrate that modeling brain circuits purely from connectivity and predicted neurotransmitter identity generates experimentally testable hypotheses and can accurately describe complete sensorimotor transformations (Shiu, 2023).
Animals form a behavioral decision by evaluating sensory evidence on the background of past experiences and the momentary motivational state. In insects, we understanding of how and at which stage of the recurrent sensory-motor pathway behavioral decisions are formed is still lacking. The mushroom body (MB), a central brain structure in insects and crustaceans, integrates sensory input of different modalities with the internal state, the behavioral state, and external sensory context through a large number of recurrent, mostly neuromodulatory inputs, implicating a functional role for MBs in state-dependent sensory-motor transformation. A number of classical conditioning studies in honeybees and fruit flies have provided accumulated evidence that at its output, the MB encodes the valence of a sensory stimulus with respect to its behavioral relevance. Recent work has extended this notion of valence encoding to the context of innate behaviors. This study co-analyzed a defined feeding behavior and simultaneous extracellular single-unit recordings from MB output neurons (MBONs) in the cockroach in response to timed sensory stimulation with odors. Clear neuronal responses occurred almost exclusively during behaviorally responded trials. Early MBON responses to the sensory stimulus preceded the feeding behavior and predicted its occurrence or non-occurrence from the single-trial population activity. These results therefore suggest that at its output, the MB does not merely encode sensory stimulus valence. It is hypothesized instead that the MB output represents an integrated signal of internal state, momentary environmental conditions, and experience-dependent memory to encode a behavioral decision (Arican, 2023)
Diet-induced obesity leads to dysfunctional feeding behavior. However, the precise molecular nodes underlying diet-induced feeding motivation dysregulation are poorly understood. Using a longitudinal high-sugar regime in Drosophila, this study sought to address how diet-induced changes in adipocyte lipid composition regulate feeding behavior. It was observed that subjecting adult Drosophila to a prolonged high-sugar diet degrades the hunger-driven feeding response. Lipidomics analysis reveals that longitudinal exposure to high-sugar diets significantly alters whole-body phospholipid profiles. By performing a systematic genetic screen for phospholipid enzymes in adult fly adipocytes, Phosphoethanolamine cytidylyltransferase (Pect) was identified as a critical regulator of hunger-driven feeding. Pect is a rate-limiting enzyme in the phosphatidylethanolamine (PE) biosynthesis pathway and the fly ortholog of human PCYT2. Disrupting Pect activity only in the Drosophila fat cells causes insulin resistance, dysregulated lipoprotein delivery to the brain, and a loss of hunger-driven feeding. Previously human studies have noted a correlation between PCYT2/Pect levels and clinical obesity. Now, these unbiased studies in Drosophila provide causative evidence for adipocyte Pect function in metabolic homeostasis. Altogether, this study has uncovered that PE phospholipid homeostasis regulates hunger response (Kelly, 2022).
Improper hunger-sensing underlies a multitude of eating disorders, including obesity. Yet, the cellular and molecular mechanisms governing the breakdown of the hunger-sensing system are poorly understood. In addition to lipid storage, adipocytes play a crucial endocrine role in maintaining energy homeostasis. Factors secreted by adipocytes impinge on several organs, including the brain, to regulate systemic metabolism and feeding behavior. Since lipids play a key role in signaling, adipocyte lipid composition is likely to regulate hunger perception and feeding behavior. Linking specific changes in adipocyte lipid composition to hunger perception and feeding behavior remains challenging (Kelly, 2022).
While the effects of neutral fat reserves such as triglycerides on feeding behavior have been extensively studied, less is known about the effects of phospholipids. Phospholipids comprise the lipid bilayer of the plasma membrane and anchor integral membrane proteins, including ion channels and receptors. They are essential components of cellular organelles, lipoproteins, and secretory vesicles. Changes to phospholipid composition can alter the permeability of cell membranes and disrupt intra- and intercellular signaling. Numerous clinical studies suggest an association between phospholipid composition and obesity. For example, insulin resistance, a hallmark of obesity-induced type 2 diabetes, is strongly associated with alterations in phospholipid composition. Additionally, key phospholipid biosynthesis enzymes are correlated with obesity in human genome-wide association studies. Despite these intriguing possibilities, a causative link between altered phospholipid composition and metabolic dysfunction is yet to be established. Furthermore, whether altered adipocyte phospholipid composition specifically leads to dysfunctional hunger-sensing is unknown (Kelly, 2022).
Phosphatidylethanolamine (PE) is the second most abundant phospholipid and is essential in membrane fission/fusion events. PE is synthesized through two main pathways in the endoplasmic reticulum (ER) and the mitochondria. Phosphatidylethanolamine cytidylyltransferase (Pcyt/Pect) is the rate-limiting enzyme of the ER-mediated PE biosynthesis pathway (Dobrosotskaya, 2002). Global dysregulation in Pcyt/Pect activity has been shown to cause metabolic dysfunction in animal models and humans. For example, Pyct/Pect deficiency in mice causes a reduction in PE levels, leading to obesity and insulin resistance). Similarly, human studies have found that obese individuals with insulin resistance have decreased Pcyt/Pect expression levels. Chronic exposure to a high-fat diet causes upregulation of Pcyt/Pect, associated with increased weight gain and insulin resistance. These findings suggest that disruptions in Pcyt/Pect activity, and consequently PE homeostasis, are a common underlying feature of obesity and metabolic disorders. What remains largely unknown is whether Pcyt/Pect activity in the adipose tissue directly regulates insulin sensitivity and feeding behavior (Kelly, 2022).
Like humans, chronic overconsumption of a high-sugar diet (HSD) results in insulin resistance, diet-induced obesity (DIO), and metabolic imbalance in flies. There is deep evolutionary conservation of feeding neural circuits regulating feeding behavior between flies and mammals, and multiple studies on feeding behavior in Drosophila have identified key neurons and receptors involved. Furthermore, like humans, Drosophila display altered feeding behavior in response to highly palatable foods. Additionally, given flies' short lifespan, feeding behavior in response to an obesogenic diet can be monitored throughout the adult fly's lifespan, providing temporal resolution of behavioral changes under DIO. Thus, using a chronic HSD feeding regime in adult flies allows for discovering specific mechanisms relevant to human biology (Kelly, 2022).
This study assesses the effects of chronic HSD consumption on flies' hunger-driven feeding (HDF) behavior across a 28-day time window. It is noted that while HSD-fed flies maintain their ability to mobilize fat stores on starvation, they lose their HDF response after 2 weeks of HSD treatment, suggesting an uncoupling of nutrient sensing and feeding behavior. This study revealed that changes in phospholipid concentrations in HSD-fed flies occur during HDF loss. It was further shown that genetic disruption of the key PE biosynthesis enzyme Pect in the fat body, the fly's adipose tissue, results in the loss of HDF even under normal food (NF) conditions. Significantly, Pect overexpression in the fat body is sufficient to protect flies from HSD-induced loss in HDF. These data suggest that adipocyte PE-phospholipid homeostasis is critical to maintaining insulin sensitivity and regulating hunger response (Kelly, 2022).
Several studies have shown a link between chronic sugar consumption and altered hunger perception. Although the neuronal circuits governing hunger and HDF behavior have been well studied, less is known about the impact of adipose tissue dysfunction on feeding behavior. Using a Drosophila DIO model, this study showed that phospholipids, specifically PE, play a crucial role in maintaining HDF behavior (Kelly, 2022).
The Drosophila model organism is a relevant model for human DIO and insulin resistance. Previous studies have performed measurements on taste preference, feeding behavior/intake, survival, etc., using an HSD-induced obesity model, and have found much in common with their mammalian counterparts. However, the longest measurement of adult feeding behavior has been capped at 7 days. A recent study by analyzed the fly lipidome on 3-week and 5-week HSD in a tissue-specific manner and identified changes in neutral fat stores in the cardiac tissue (Kelly, 2022).
This study defined that a 14-day exposure of adult Drosophila to an HSD regime disrupts hunger response. On evaluating HSD regime-induced lipid composition changes at this critical 14-day point, a critical requirement was uncovered for adipocyte PE homeostasis and a fat-specific role for the PE enzyme Pect in controlling HDF. Pect function in the adult fly adipocytes is critical for appropriate fat-to-brain lipoprotein delivery and the maintenance of systemic insulin sensitivity. In sum, this study identified that adipocyte-specific loss of Pect phenocopies the metabolic dysfunctions observed in a chronic HSD regime in adult flies. Therefore, it is proposed that PE homeostasis, specifically Pect activity in fat tissue, regulates HDF response (Kelly, 2022).
Changes in feeding behavior in both vertebrates and invertebrates occur via communication between peripheral organs responsible for digestion/energy storage and the brain. This communication is facilitated by factors that provide information on nutritional state. One example of such a factor is leptin, released from the adipose tissue and acts on neuronal circuits in the brain to promote satiety. While leptin has long been studied as a satiety hormone, recent work in mice and flies suggests that a key function of leptin and its fly homolog upd2 regulates starvation response. Indeed, previous work has shown that exposing flies to HSD alters synaptic contacts between Leptin/Upd2 sensing neurons and Insulin neurons. However, it resets within 5 days, suggesting that yet-to-be-defined mechanisms maintain homeostasis on surplus HSDs beyond 5 days (Kelly, 2022).
Feeding behavior was analyzed over time to delineate how HSD alters the starvation response.under normal diet conditions flies display a clear response to starvation in the form of elevated feeding that is termed 'hunger-driven feeding (HDF),' which was independent of age. In contrast, chronic exposure to HSD led to a progressive loss of HDF that began on day 14. It could be argued that loss of HDF is simply due to an elevation of TAG storage in HSD-fed flies, thus losing the need to feed on starvation. However, several pieces of evidence support the idea that HSD affects feeding behavior independently of nutrient sensing. Under the current experimental conditions, this study found basal feeding to be statistically similar between NF-fed and HSD-fed conditions at all timepoints with the exception of day 10. Note that it has been reported that on a 20% sucrose liquid diet for 7 days elevated food interactions. However, those studies are not comparable with the current study due to the large differences in experimental protocol. The previous study evaluated taste preference changes and feeding interactions on 5–30% sucrose liquid diet in 24-hr window over a period of 7 days. This study assessed food interaction in a 3-hr window, after providing a complex lab standard diet, to monitor HDF. Future studies would be needed to assess the effect of 14-day HSD on taste perception using the experimental design in this study. The HDF response of HSD-fed flies is significantly lower than that of NF-fed flies, but they sense energy deficit and mobilize fat stores accordingly. Hence, HSD-fed flies can calibrate their HDF to compensate only for the amount of fat lost in starvation. Nonetheless, this capacity of flies to couple energy sensing and feeding motivation is lost beyond day 14, as evidenced by the loss of HDF and continuous TAG breakdown. Strikingly, subjecting 14-day HSD-fed flies to prolonged starvation (up to 32 hr) was insufficient to induce increased HDF. While there was an uptick in feeding behavior at 20 hr of starvation, this hunger response was not sustained at 24 and 32 hr, even though flies continued to mobilize TAG reserves at 24 and 32 hr. Thus, prolonged exposure to HSD leads to uncoupling nutrient sensing and feeding behavior (Kelly, 2022).
Notably, fly and mammalian DIO models have striking differences and similarities. Mice show linear weight gain on obesogenic diets, but flies' rigid exoskeleton limits their capacity to store TAG beyond a certain point. However, similar to mammals, prolonged exposure to HSD, strongly associated with phospholipid dysregulation, leads to reduced insulin sensitivity. This study shows that the levels of Dilp5, the fly's insulin ortholog, are reduced in the IPCs of HSD-fed flies. However, no decrease in Dilp5 or Dilp2 mRNA levels was observed; this is suggestive of increased insulin secretion on HSD, similar to previously reported. Consistent with the idea that 14-day HSD triggers insulin resistance, elevated FOXO nuclear localization was observed in the fat bodies of the HSD-fed flies, despite a likely increase in Dilp5 secretion on HSD. Again, these findings align with mammalian studies showing that dysregulated FOXO signaling is implicated in insulin resistance, type 2 diabetes, and obesity (Kelly, 2022).
Changes in the lipidome are strongly correlated with insulin resistance and obesity . However, less is known about how the lipidome affects feeding behavior. To this end, the lipid profiles of NF and HSD-fed flies were examined over time. As expected, exposure to HSD increased the overall content of neutral lipids compared to the NF flies, with TAGs and DAGs increasing the most, which is consistent with other DIO models. Surprisingly, it was noted that 14 days of HSD treatment caused a decrease in FFAs and a rise in TAGs and DAGs. It is speculated that this reduction in FFA may be due to their involvement in TAG biogenesis. It was of interest to see whether the decrease in FFA correlated to a particular lipid species as PE and PC are made from DAGs with specific fatty acid chains. However, further analysis of FFAs at the species level did not reveal any distinct patterns. Most FFA chains decreased in HSD, including 12.0, 16.0, 16.1, 18.0, 18.1, and 18.2. This data was more suggestive of a global decrease in FFA, likely converted to TAG and DAG rather than depleting a specific fatty acid chain (Kelly, 2022).
On day 14 of HSD treatment, when HDF response begins to degrade, PE and PC levels rise dramatically, whereas LPE significantly decreases. Interestingly, similar patterns of phospholipid changes have been associated with diabetes, obesity, and insulin resistance in clinical studie, yet no causative relationship has been established. Intriguingly, this study found that PC balance appears dispensable for maintaining HDF-response. But both the mitochondrial and cytosolic PE pathways seem critical for HDF response. Multiple pathways synthesize PE. Studies have shown that in addition to the mitochondrial PISD and cytosolic CDP-ethanolamine Kennedy pathway, PE can be synthesized from LPE. This pathway is named the exogenous lysolipid metabolism (ELM) pathway. ELM can substitute for the loss of the PISD pathway in yeast and requires the activity of the enzyme lyso-PE acyltransferase (LPEAT) that converts LPE to PE. In this study, it is noted PE levels were upregulated on HSD while LPE levels were downregulated (Kelly, 2022).
In contrast, fat-specific Pect-KD caused PE levels to trend downward, whereas LPE was upregulated. Though the level changes for PE and LPE are contrasting between 14-day HSD lipidome and Pect-KD, under both states, there is an imbalance of phospholipids classes PE and LPE. Hence, it is propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to HDF and insulin sensitivity (Kelly, 2022).
The role of the minor phospholipid class LPE remains obscure. This study observes that the LPE imbalance occurs during prolonged HSD exposure and when fat body Pect activity is disrupted. This suggests that LPE balance likely plays a role in insulin sensitivity and the regulation of feeding behavior. It is anticipated that this observation will stimulate interest in studying this poorly understood minor phospholipid class. In future work, it would be interesting to test how the genetic interactions between the enzyme that converts LPE to PE, called LPEAT, and Pect manifest in HDF. Specifically, it will be interesting to ask whether reducing or increasing LPEAT will restore PE-LPE balance to improve the HDF response in HSD-fed flies and Pect-KD. Future studies should explore how LPE-PE balance can be manipulated to affect feeding behaviors (Kelly, 2022).
In addition to changes in phospholipid classes, this study found that HSD caused an increase in the concentration of PE and PC species with double bonds. Double bonds create kinks in the lipid bilayer, leading to increased lipid membrane fluidity, impacting vesicle budding, endocytosis, and molecular transport. Hence, a possible mechanism by which HSD induces changes to signaling by altering the membrane biophysical properties, such as by increased fluidity; this would impact various cellular processes, including synaptic firing and inter-organization vesicle transport. Consistent with this idea, a significant reduction was observed in the trafficking of ApoII-positive lipophorin particles from adipose tissue to the brain. Targeted experiments are required to understand how lipid membrane fluidity alters hunger response fully (Kelly, 2022).
To explore the idea that fat–brain communication may be perturbed under HSD and Pect knockdown, a fat-specific signal known to travel to the brain was examined. ApoLpp chaperones PE-rich vehicles called lipophorins traffic lipids from fat to all peripheral tissues, including the brain. ApoII, the Apolpp fragment harboring the lipid-binding domain, has been shown to regulate systemic insulin signaling by acting on a subset of neurons in the brain. This study found that both HSD treatment and Pect knockdown reduced ApoII levels in the brain. Given that ApoII acts as a ligand for lipophorin receptors in the brain, ApoII may be a direct regulator of feeding. Alternatively, it could ferry signaling molecules and PE/PC lipids. In the future, it would be important to explore whether lipoprotein trafficking from fat-to-brain directly impacts the hunger response (Kelly, 2022).
This study has uncovered a role for the phospholipid enzyme Pect as an important component in maintaining HDF. Future work should explore the precise mechanism of how Pect and the associated disruption in phospholipid homeostasis can impact adipose tissue signaling. In sum, this study lays the groundwork for further investigation into Pyct2/Pect as a potential therapeutic target for obesity and its associated comorbidities (Kelly, 2022).
A 24-h rhythm of feeding behavior, or synchronized feeding/fasting episodes during the day, is crucial for survival. Internal clocks and light input regulate rhythmic behaviors, but how they generate feeding rhythms is not fully understood. This study aimed to dissect the molecular pathways that generate daily feeding patterns. By measuring the semidiurnal amount of food ingested by single flies,it was demonstrated that the generation of feeding rhythms under light:dark conditions requires quasimodo (qsm) but not molecular clocks. Under constant darkness, rhythmic feeding patterns consist of two components: CLOCK (CLK) in digestive/metabolic tissues generating feeding/fasting episodes, and the molecular clock in neurons synchronizing them to subjective daytime. Although CLK is a part of the molecular clock, the generation of feeding/fasting episodes by CLK in metabolic tissues was independent of molecular clock machinery. These results revealed novel functions of qsm and CLK in feeding rhythms in Drosophila (Maruko, 2023).
Many organisms, including insects, fish, birds, rodents, and primates, show particular feeding patterns during the day. This study investigated the roles of molecular clock genes in food intake in Drosophila. In LD, light stimuli were sufficient to induce feeding via the QSM-mediated pathway, while feeding rhythms in DD can be dissected into two components: generation of feeding/fasting episodes by Clk/cyc in metabolic tissues and their synchronization by molecular clocks in neurons. These results suggest novel roles of qsm and clock molecules regulating feeding behavior (Maruko, 2023).
There has been a significant advance in understanding the circuits and neurotransmitters that mediate central clocks in the brain that control feeding behavior. As for the peripheral clocks, their functions in feeding behavior seem more diverse than those in the central clocks. In addition to contributing to the regulation of 24-h feeding rhythms, it was also reported that peripheral clocks negatively regulate food intake amount and feeding rhythm strength. However, it was elucidated whether the increase in food intake amount is caused by the disruption of the molecular clock machinery or other functions of CLK/CYC. The results suggest that fat body CLK regulates food intake amount independent of molecular clocks. Since flies with Clk/cyc disruption with various genotypes consistently show a reduction in fluctuation in food intake compared to their control (i.e., ClkJrk, cycout, cyc02, to>dnClk, cyc02, to > cyc), it is less likely that fluctuation in food intake is due to differences in the body size or other background effects. The suppression of CLK function in the fat body reduced fluctuations of feeding amount in per01 background, indicating that CLK/CYC in the metabolic tissues contributes to rhythmic feeding behavior in addition to its function as components of the molecular clock. It explains the different effects of clock genes on patterns of feeding rhythms: per- and tim-null mutants showed a shift in the peak of the feeding timing, and Clk- and cyc-null mutants ingest food constantly without peak of the feeding timing (Maruko, 2023).
The results suggest that the effect of CLK/CYC in the metabolic tissues to reduce feeding rhythm strength is likely to be mediated by suppression of feeding during the fasting period. Several factors that act on suppression of food intake in peripheral tissues have been reported, and expression of some of these genes, such as allatostatin A and its receptor, are regulated by peripheral CLK/CYC. In addition, rhythmic expression of the allatostatin A receptor-2 gene is affected by the disruption of the fat body clock. CLK is also associated with cAMP-responsive element binding protein (CREB), which is involved in the energy homeostasis of insects and mammals. Blocking CREB activity in the fat body increases food intake in flies, and Nejire, a homolog of CREB-binding protein (CBP)/p300, has been reported as a regulator of CLK/CYC-dependent transcription. Further studies on these pathways may reveal a novel signaling axis that constitute the feeding/fasting cycle (Maruko, 2023).
This study found the novel role of qsm in feeding behavior in LD. It has been reported that cry-null mutants do not display the morning peak in LD. It was also observed that cry-null mutants do not display the early morning peak in LD, while these flies still showed the synchronized feeding/fasting episodes in LD. In addition, the feeding/fasting episodes in DD were observed without the morning peak. Thus, the morning peak is not associated with the daytime feeding pattern that was the focus of this study. The results revealed that qsm is indispensable in the daytime feeding pattern. qsm encodes a ZP (Zona Pellucida) domain and constitutes part of CRY-independent light input to the circadian clock. QSM is expressed in many cells in the immediate proximity of clock neurons, and it is not clear in which cells QSM influences the feeding pattern for now. Further study to understand where and how the QSM regulates feeding rhythms in LD would help understand light-induced regulation of feeding behavior (Maruko, 2023).
In summary, these results revealed novel pathways that regulate the formation of feeding rhythms in Drosophila. Feeding/fasting rhythms coordinate metabolism and affect aging and life span. Further studies of these axes may contribute to human health (Maruko, 2023).
This study has successfully dissected the molecular pathways that regulate feeding patterns. However, these analyses are limited to several key components, and their upstream and downstream molecules remain to be elucidated. It was found that qsm regulates feeding rhythms under light:dark conditions, while its downstream signaling is unknown. This study also found that, under constant darkness conditions, CLK/CYC in digestive/metabolic tissues generates feeding/fasting episodes, and the molecular clock in neurons synchronizes them. However, molecular mechanisms that link those two components remain to be elucidated. Further studies are needed to understand the entire picture of the molecular pathways that generate feeding patterns (Maruko, 2023).
Sleep and feeding patterns lack a clear daily rhythm during early life. As diurnal animals mature, feeding is consolidated to the day and sleep to the night. Circadian sleep patterns begin with formation of a circuit connecting the central clock to arousal output neurons; emergence of circadian sleep also enables long-term memory (LTM). However, the cues that trigger the development of this clock-arousal circuit are unknown. This study identify a role for nutritional status in driving sleep-wake rhythm development in Drosophila larvae. In the 2(nd) instar (L2) period, sleep and feeding are spread across the day; these behaviors become organized into daily patterns by L3. Forcing mature (L3) animals to adopt immature (L2) feeding strategies disrupts sleep-wake rhythms and the ability to exhibit LTM. In addition, the development of the clock (DN1a)-arousal (Dh44) circuit itself is influenced by the larval nutritional environment. Finally, it was demonstrated that larval arousal Dh44 neurons act through glucose metabolic genes to drive onset of daily sleep-wake rhythms. Together, these data suggest that changes to energetic demands in developing organisms triggers the formation of sleep-circadian circuits and behaviors (Poe, 2023).
Although painful stimuli elicit defensive responses including escape behavior for survival, starved animals often prioritize feeding over escape even in a noxious environment. This behavioral priority is typically mediated by suppression of noxious inputs through descending control in the brain, yet underlying molecular and cellular mechanisms are incompletely understood. This study identified a cluster of GABAergic neurons in Drosophila larval brain, designated as SEZ-localized Descending GABAergic neurons (SDGs), that project descending axons onto the axon terminals of the peripheral nociceptive neurons and prevent presynaptic activity through GABA(B) receptors. Remarkably, glucose feeding to starved larvae causes sustained activation of SDGs through glucose-sensing neurons and subsequent insulin signaling in SDGs, which attenuates nociception and thereby suppresses escape behavior in response to multiple noxious stimuli. These findings illustrate a neural mechanism by which sugar sensing neurons in the brain engages descending GABAergic neurons in nociceptive gating to achieve hierarchical interaction between feeding and escape behavior (Nakamizo-Dojo, 2023).
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).
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).