The Interactive Fly

Zygotically transcribed genes

Courtship Learning and Behavior, Control of Ovulation and Post-mating Behavior

Courtship Learning and Behavior
  • Courtless, the Drosophila UBC7 Homolog, is involved in male courtship behavior and spermatogenesis
  • dissatisfaction encodes a Tailless-like nuclear receptor expressed in a subset of CNS neurons controlling Drosophila sexual behavior
  • The relative roles of vision and chemosensation in mate recognition of Drosophila
  • Opposing dopaminergic and GABAergic neurons control the duration and persistence of copulation in Drosophila
  • Novel role for ecdysone in Drosophila conditioned behavior: Linking GPCR-mediated non-canonical steroid action to cAMP signaling in the adult brain
  • Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila
  • The neural circuitry that functions as a switch for courtship versus aggression in Drosophila males
  • Effect of competitive cues on reproductive morphology and behavioral plasticity in male fruitflies
  • Cross-generational comparison of reproductive success in recently caught strains of Drosophila melanogaster
  • Multimodal chemosensory circuits controlling male courtship in Drosophila
  • Temporal mating isolation driven by genetic variation in period
  • Courtless, the Drosophila UBC7 Homolog, is involved in male courtship behavior and spermatogenesis
  • Cholinergic control of male reproductive characters in Drosophila
  • Increased dopamine level enhances male-male courtship in Drosophila
  • Active and passive sexual roles that arise in Drosophila male-male courtship are modulated by dopamine levels in PPL2ab neurons
  • Excitatory and inhibitory switches for courtship in the brain
  • Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila
  • Fruitless directs neural circuitry that governs Drosophila male courtship behavior
  • Drosophila Life Span and Physiology Are Modulated by Sexual Perception and Reward
  • Excitation and inhibition onto central courtship neurons biases mate choice
  • Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate
  • Modulation of Drosophila male behavioral choice; neuromodulator octopamine is necessary for males to coordinate sensory cue information presented by a second male and respond with the appropriate behavior
  • Gr33a modulates Drosophila male courtship preference
  • Obp56h modulates mating behavior in Drosophila melanogaster
  • Juvenile hormone is required in adult males for Drosophila courtship
  • Neuromodulation of courtship drive through tyramine-responsive neurons in the Drosophila brain
  • Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain
  • Specialized cells tag sexual and species identity in Drosophila melanogaster
  • Magnetoreception regulates male courtship activity in Drosophila
  • Odorant responses and courtship behaviors influenced by at4 neurons in Drosophila
  • Neural circuitry coordinating male copulation
  • Pleiotropic effects of loss of the Dα1 subunit in Drosophila melanogaster: Implications for insecticide resistance
  • Serotonergic neuronal death and concomitant serotonin deficiency curb copulation ability of Drosophila platonic mutants
  • Mate choice in fruit flies is rational and adaptive
  • Genomic responses to socio-sexual environment in male Drosophila melanogaster exposed to conspecific rivals
  • Quantitative analysis of visually induced courtship elements in Drosophila subobscura
  • Drosophila courtship conditioning as a measure of learning and memory
  • The physical environment mediates male harm and its effect on selection in females
  • Methyl-CpG binding domain proteins inhibit interspecies courtship and promote aggression in Drosophila
  • Tissue-specific insulin signaling mediates female sexual attractiveness
  • SIK3-HDAC4 signaling regulates Drosophila circadian male sex drive rhythm via modulating the DN1 clock neurons
  • Direct and trans-generational effects of male and female gut microbiota in Drosophila melanogaster

  • The role of cuticular pheromones in courtship conditioning of Drosophila males
  • Pickpocket 25, a sodium channel subunit required for activation of courtship behavior by chemosensory perception of female pheromones
  • Hormonal modulation of pheromone detection enhances male courtship success
  • Drosophila melanogaster females restore their attractiveness after mating by removing male anti-aphrodisiac pheromones
  • Dopamine neurons modulate pheromone responses in Drosophila courtship learning
  • An inhibitory sex pheromone tastes bitter for Drosophila males
  • Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster
  • Mutations at the Darkener of Apricot locus modulate pheromone production and sex behavior in Drosophila melanogaster
  • Genes involved in sex pheromone discrimination in Drosophila melanogaster and their background-dependent effect
  • Electrical synapses mediate synergism between pheromone and food odors in Drosophila melanogaster
  • A Drosophila female pheromone elicits species-specific long-range attraction via an olfactory channel with dual specificity for sex and food
  • Male mate choice via cuticular hydrocarbon pheromones drives reproductive isolation between Drosophila species

    Courtship Song
  • Motor control of Drosophila courtship song
  • Sensorimotor transformations underlying variability in song intensity during Drosophila courtship
  • Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality in Drosophila
  • Reported Drosophila courtship song rhythms are artifacts of data analysis
  • Experimental and statistical reevaluation provides no evidence for Drosophila courtship song rhythms
  • Failure to reproduce period-dependent song cycles in Drosophila is due to poor automated pulse-detection and low-intensity courtship
  • The sex-determination genes fruitless and doublesex specify a neural substrate required for courtship song
  • The role of courtship song in female mate choice in South American Cactophilic Drosophila

    Control of Ovulation, Female Sexual Behavior and Post-mating Behavior
  • Sensory neurons in the Drosophila genital tract regulate female reproductive behavior
  • Sex-peptide decreases female receptivity and stimulates egg production in the first mating of virgin females
  • Feeding regulates sex pheromone attraction and courtship in Drosophila females
  • Differential effects of male nutrient balance on pre- and post-copulatory traits, and consequences for female reproduction in Drosophila melanogaster
  • Male age affects female mate preference, quantity of accessory gland proteins, and sperm traits and female fitness in D. melanogaster
  • Abdominal-B neurons control Drosophila virgin female receptivity
  • Drosophila egg-laying site selection as a system to study simple decision-making processes
  • Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling
  • Mating-induced increase in germline stem cells via the neuroendocrine system in female Drosophila
  • Neural circuitry underlying Drosophila female postmating behavioral responses
  • Postmating circuitry modulates salt taste processing to increase reproductive output in Drosophila
  • Mechanosensitive neurons on the internal reproductive tract contribute to egg-laying-induced acetic acid attraction in Drosophila
  • Egg-laying demand induces aversion of UV light in Drosophila females
  • Fecal-derived phenol induces egg-laying aversion in Drosophila
  • Cancer brings forward oviposition in the fly Drosophila melanogaster
  • Dietary protein content alters both male and female contributions to Drosophila melanogaster female post-mating response traits
  • apterous brain neurons control receptivity to male courtship in Drosophila melanogaster females
  • Insulin signaling in the peripheral and central nervous system regulates female sexual receptivity during starvation in Drosophila

    Genes involved in courtship behavior

    The role of cuticular pheromones in courtship conditioning of Drosophila males

    Courtship conditioning is an associative learning paradigm in Drosophila melanogaster, wherein male courtship behavior is modified by experience with unreceptive, previously mated females. While the training experience with mated females involves multiple sensory and behavioral interactions, it is hypothesized that female cuticular hydrocarbons function as a specific chemosensory conditioned stimulus in this learning paradigm. The effects of training with mated females were determined in courtship tests with either wild-type virgin females as courtship targets, or with target flies of different genotypes that express distinct cuticular hydrocarbon (CH) profiles. Results of tests with female targets that lacked the normal CH profile, and with male targets that expressed typically female CH profiles, indicate that components of this CH profile are both necessary and sufficient cues to elicit the effects of conditioning. Results with additional targets indicate that the female-specific 7,11-dienes, which induce naive males to court, are not essential components of the conditioned stimulus. Rather, the learned response is significantly correlated with the levels of 9-pentacosene (9-P), a compound found in both males and females of many Drosophila strains and species. Adding 9-P to target flies showed that it stimulates courting males to attempt to copulate, and confirmed its role as a component of the conditioned stimulus by demonstrating dose-dependent increases in the expression of the learned response. Thus, 9-P can contribute significantly to the conditioned suppression of male courtship toward targets that express this pheromone (Siwicki, 2005).

    Based on the reasoning that trained males would exhibit a learned response only in courtship tests with targets that expressed a conditioned stimulus, flies with different combinations of putative female aphrodisiac cues were used as test targets, and assessed for their ability to elicit a learned response from Canton-S (CS) males. After 1 h of experience courting a previously mated CS female, the effects of this experience were measured in courtship assays with different test targets. For example, two variants on the CH profile were flies bearing pGAL4 insert in the desaturase 1 gene on Chromosome III (desat11573) and hs-tra virgin females, which are severely depleted of all CHs. Differences in the extent and duration of conditioned courtship suppression in tests with different targets were attributed to differences in the testing conditions, that is, in the combinations of aphrodisiac and anti-aphrodisiac cues expressed by the test targets (Siwicki, 2005).

    Target behavior was eliminated as a source of the observed differences in male courtship suppression because the targets were immobilized by decapitation. Nor did the presence of female abdominal anatomy correlate with a target's ability to elicit a learned response: Targets that were anatomically female and male elicited strong learned responses, while the target genotypes that elicited the weakest learned responses (desat11573 virgins and hs-tra virgins) were anatomically female. Therefore, the abdominal anatomy of a courtship target is not an essential element of the conditioned stimulus. This conclusion also is supported by the fact that visual stimuli are not required for courtship conditioning (Siwicki, 2005).

    The courtship activity [mean cortship index (CI)] of naive males was not well correlated with the hydrocarbon profiles of the different test targets, confirming the results of prior studies showing that female CHs are sufficient, but not necessary, to stimulate naive male courtship. In particular, naive males courted hydrocarbon-depleted hs-tra virgins and males depleted of unsaturated CHs as actively as they courted targets with typically female CH profiles. This is consistent with the view that naive males respond to a combination of aphrodisiac and anti-aphrodisiac pheromones on a decapitated target fly. In contrast, the conditioned suppression of courtship resulting from experience with mated females was closely correlated with the hydrocarbon profiles of the test targets. Results of testing with hydrocarbon-depleted female targets indicated that CHs are essential stimuli for the conditioned suppression of courtship after experience with mated females. Tests with feminized males (pGAL4/+; UAS-tra/+) that expressed typically female CH profiles revealed that this profile is sufficient both to stimulate naive male courtship and to elicit conditioned suppression of courtship in trained male subjects. Together, these results strongly support the hypothesis that components of the female CH profile function as the conditioned stimulus in courtship conditioning (Siwicki, 2005).

    This raises the question of whether specific compounds or the overall female profile are responsible for the learned response. The data indicate that a target's ability to elicit a learned response is most strongly correlated with the levels of a single monoene, 9-pentacosene (9-P). Specifically, Learning Indexes (LIs) based on tests either 5 min or 60 min after training were significantly correlated with target levels of a single unsaturated hydrocarbon, 9-pentacosene (9-P). Surprisingly, the LIs were not significantly correlated with levels of any principal compound previously known to possess aphrodisiac properties, that is, the 7,11-dienes or 7-P (Siwicki, 2005).

    Interpretation of this result requires consideration of previous evidence concerning the aphrodisiac properties of individual CHs. Two strategies have been used to assess the aphrodisiac and anti-aphrodisiac potencies of individual compounds in the cuticular profile. By applying synthetic compounds to CH-stripped dummy targets, previous studies have found 7,11-HD to be the most bioactive, stimulating aggressive male courtship with a threshold of ~100 ng/fly. This was confirmed by genetic manipulations to produce live targets with distinctive CH profiles; even low levels of 7,11-ND (10-15 ng) strongly potentiate the aphrodisiac effects of 7,11-HD. Both strategies produced evidence for the anti-aphrodisiac properties of 7-T and the aphrodisiac effects of 7-P. Prior evidence regarding the putative aphrodisiac properties of 9-P is limited to a positive correlation with the frequency of copulation attempts. When applied to hydrocarbon-stripped targets, 9-P failed to stimulate male courtship (Siwicki, 2005 and references therein).

    In the present study, one clear effect of adding exogenous, pure (Z)-9-pentacosene to courtship chambers was to increase the fraction of CS males that attempted to copulate with CS virgin females, and with CH-depleted virgins of the hs-tra and desat1 genotypes. This result strongly supports the hypothesis that the main aphrodisiac effect of 9-P is to increase the likelihood of copulation attempts. Interestingly, 9-P did not increase the mean CI in most of these test groups: Only naive males tested with desat1 females had a higher mean CI in the presence of 9-P. This selective effect on the mean CI of naive males with desat1 females, but not trained males, as well as the dose-dependent effects of 9-P on the Learning Index, clearly establish a role for 9-P as a component of the conditioned stimulus. With most targets, however, 9-P did not stimulate overall courtship activity; rather, it increased the probability for courting males to attempt copulations. Moreover, because this effect was elicited by the lower dose of 9-P for naive males, and only at the higher dose for trained males, it suggests that a specific effect of training with mated females is to reduce the responsiveness of trained males to the aphrodisiac effects of this pheromone (Siwicki, 2005).

    These results indicate a dual role for 9-P: (1) increasing the probability that courting males will attempt to copulate, and (2) contributing to the difference between naive and trained males in overall courtship. Yet, depending on the target genotype, different doses of 9-P induce different effects on the global amount of courtship (CI) and on the frequency of copulation attempts. For example, 9-P only increases the CI of naive males when present at 2 µg on desat1 females, but both 0.2-µg and 2-µg doses tended to increase the probability to attempt copulation when combined with any of the four targets. This difference can be explained because the duration of a copulation attempt is very brief compared to the wing vibration and licking behaviors that make up most of the CI measure. The selective effect of 9-P on CI with desat1 females suggests that its effect could be potentiated by other CHs that are present in these targets but absent in hs-tra females and immature males. At least two relatively abundant CHs fit this criterion: n-tricosane and 2-methyl-tetracosane. In contrast, it is likely that 9-P has minimal effects on CIs with CS females because all the CHs necessary to induce high levels of courtship are already present, and the exogenous 9-P does not increase the overall sex appeal of these targets. It was also with the three CH-depleted genotypes, but not with CS females, that dose-dependent effects of 9-P were found on LI5' (LIs for groups tested 5 min after training), a measure of the relative suppression of overall courtship in trained males, whereas 0.2 µg of 9-P was sufficient to elicit the maximum effect of training on the probability to attempt copulation. It is worth noting that the 0.2-µg dose of 9-P (applied to a filter paper under the target fly) is very similar to the biological dose found on CS females (50-70 ng). Thus, the results demonstrate that CS males can detect and respond to physiologically relevant levels of this pheromone (Siwicki, 2005).

    It is likely that 9-P is one component of a combination of CHs that function as the conditioned stimulus. This is supported by the fact that 9-P was not sufficient to elicit effects of training on courtship of CH-depleted hs-tra females. It is also supported by the modest, albeit significant, correlation between the LIs and 9-P levels in different test targets. When possible synergistic effects of other unsaturated CHs with 9-P were examined by running multiple regressions, there were no cases in which the levels of 9-P plus another CH predicted the LI-values significantly better than did 9-P alone. Thus, the identities of the putative interacting components of the conditioned stimulus remain to be determined (Siwicki, 2005).

    The surprising result of this study is that this particular compound emerged as a conditioned stimulus. The levels of 7,11-dienes in a courtship target were not correlated with the expression of the males' learned response, indicating that conditioning does not dramatically alter the potency of these highly abundant, female-specific pheromones. In contrast, 9-P is a relatively minor component of the Canton-S CH profile, and is expressed by both mature males and females (Siwicki, 2005).

    Having identified a role for 9-P as a chemosensory stimulus that is particularly susceptible to conditioning during courtship of mated females, the results may shed new light on the question of whether courtship conditioning ability is relevant to the behavior of males in the wild. While it is likely to be maladaptive for males to suppress their courtship toward conspecific virgin females, it has been proposed that courtship toward mated females might be decreased to a greater degree, thus improving male selectivity for virgins. Since 9-P is not very specific (it is found in cuticular extracts of both males and females of several Drosophila species), it is possible that experience-dependent modification of the responsiveness to this widely expressed hydrocarbon might provide a male with the ability to learn to discriminate appropriate from inappropriate courtship targets. For example, females of Drosophila affinis were reported to express much higher levels of 9-P than D. melanogaster females. These two species are sympatric in some areas of North America, and they have been observed courting in interspecific groups gathered on food sources. These observations suggest that the ability of CS males to learn to be less responsive to 9-P might allow them to learn to avoid courting D. affinis females. In an environment where misdirected courtship of heterospecific females is likely to occur, there would presumably be some advantage for males that could learn to become less sensitive to particular pheromones that are more abundant in heterospecific females than in con-specifics (Siwicki, 2005).

    D. melanogaster males also court immature males and females, which express distinctly different blends of CHs than mature flies. Experience courting immature males results in habituation to the aphrodisiac effects of specific 31C monoenes in the immature male CH blend. The CH profiles of immature males and females are similar to each other in that they are comprised mostly of longer 27C-37C compounds and contain only trace amounts of compounds smaller than 27C. It is difficult to predict how experience courting a mated female, and the corresponding decrease in aphrodisiac potency of 9-P, might affect a male's subsequent courtship of immature flies with little or no 9-P and an abundance of other CHs with unknown behavioral effects. This difficulty is compounded by the possibility that some longer-chain CHs of immature flies may be detected and processed through some of the same sensory pathways as mature CH pheromones, a possibility that is reinforced by recent evidence that many gustatory receptor neurons express combinations of receptors. At present, therefore, it is not possible to interpret the effects of mated-female training on male courtship of immature males and females in terms of specific modifications of responses to specific CHs (Siwicki, 2005).

    The present results suggest an additional level of complexity in the functional organization of the neural systems that control and modulate male courtship. Some CHs (7,11-dienes and 7-P) stimulate naive males to court actively; and others (7-T) inhibit naive courtship, suggesting that there are at least two distinct gustatory pathways by which contact pheromones influence courtship control centers. Males repeatedly sample all of these pheromones during training with mated females, yet the experience-dependent changes in subsequent courtship behavior are strongly correlated only with a target level of 9-P. This suggests that the sensorimotor circuits by which 7,11-dienes and 7-P stimulate courtship may be less modifiable than pathways activated by 9-P. It follows from this hypothesis that these pheromones may be detected and processed by distinct neural pathways, a prediction that can be tested by manipulating the genes and cells involved in pheromone detection (Siwicki, 2005).

    This study provides direct support for the hypothesis that the conditioned stimulus in courtship conditioning of D. melanogaster males is a chemical, rather than behavioral or anatomical, cue provided by the female. The results indicate that components of the female cuticular hydrocarbon profile function as the conditioned stimulus in courtship conditioning. In particular, the most relevant stimulus is 9-pentacosene, an unsaturated hydrocarbon found on the cuticle of both males and females. Together with previous evidence concerning the relative potencies of various cuticular substances at eliciting male courtship, the present results suggest that naive males respond to a combination of aphrodisiacs and anti-aphrodisiacs on a target fly, while conditioned males are less responsive than naives to the aphrodisiac effects of 9-P. Identifying the sensory pathways responsible for detection of this chemical conditioned stimulus will allow for a more definitive analysis of the neural mechanisms responsible for this form of associative learning (Siwicki, 2005).

    The relative roles of vision and chemosensation in mate recognition of Drosophila

    Animals rely on sensory cues to classify objects in their environment and respond appropriately. However, the spatial structure of those sensory cues can greatly impact when, where, and how they are perceived. This study examined the relative roles of visual and chemosensory cues in the mate recognition behavior of fruit flies (Drosophila melanogaster) by using a robotic fly dummy that was programmed to interact with individual males. By pairing male flies with dummies of various shapes, sizes, and speeds, or coated with different pheromones, it was determined that visual and chemical cues play specific roles at different points in the courtship sequence. Vision is essential for determining whether to approach a moving object and initiate courtship, and males were more likely to begin chasing objects with the same approximate dimensions as another fly. However, whereas males were less likely to begin chasing larger dummies, once started, they would continue chasing for a similar length of time regardless of the dummy's shape. The presence of female pheromones on the moving dummy did not affect the probability that males would initiate a chase, but it did influence how long they would continue chasing. Male pheromone both inhibits chase initiation and shortens chase duration. Collectively, these results suggest that male Drosophila use different sensory cues to progress through the courtship sequence: visual cues are dominant when deciding whether to approach an object whereas chemosensory cues determine how long the male pursues its target (Agrawal, 2014).

    Hormonal modulation of pheromone detection enhances male courtship success

    During the lifespans of most animals, reproductive maturity and mating activity are highly coordinated. In Drosophila melanogaster, for instance, male fertility increases with age, and older males are known to have a copulation advantage over young ones. The molecular and neural basis of this age-related disparity in mating behavior is unknown. This study shows that the Or47b odorant receptor is required for the copulation advantage of older males. Notably, the sensitivity of Or47b neurons to a stimulatory pheromone, palmitoleic acid, is low in young males but high in older ones, which accounts for older males' higher courtship intensity. Mechanistically, this age-related sensitization of Or47b neurons requires a reproductive hormone, juvenile hormone, as well as its binding protein Methoprene-tolerant in Or47b neurons. Together, this study identifies a direct neural substrate for juvenile hormone that permits coordination of courtship activity with reproductive maturity to maximize male reproductive fitness (LinCao, 2016).

    Drosophila melanogaster females restore their attractiveness after mating by removing male anti-aphrodisiac pheromones

    Males from many species ensure paternity by preventing their mates from copulating with other males. One mate-guarding strategy involves marking females with anti-aphrodisiac pheromones (AAPs), which reduces the females' attractiveness and dissuades other males from courting. Since females benefit from polyandry, sexual conflict theory predicts that females should develop mechanisms to counteract AAPs to achieve additional copulations, but no such mechanisms have been documented. This study shows that during copulation Drosophila melanogaster males transfer two AAPs: cis-Vaccenyl Acetate (cVA) to the females' reproductive tract, and 7-Tricosene (7-T) to the females' cuticle. A few hours after copulation, females actively eject cVA from their reproductive tract, which results in increased attractiveness and re-mating. Although 7-T remains on those females, it was shown that it is the combination of the two chemicals that reduces attractiveness. Thus, female AAP ejection provides the first example of a female mechanism that counter-acts chemical mate-guarding (Laturney, 2016).

    Opposing dopaminergic and GABAergic neurons control the duration and persistence of copulation in Drosophila

    Behavioral persistence is a major factor in determining when and under which circumstances animals will terminate their current activity and transition into more profitable, appropriate, or urgent behavior. This study shows that, for the first 5 min of copulation in Drosophila, stressful stimuli do not interrupt mating, whereas 10 min later, even minor perturbations are sufficient to terminate copulation. This decline in persistence occurs as the probability of successful mating increases and is promoted by approximately eight sexually dimorphic, GABAergic interneurons of the male abdominal ganglion. When these interneurons were silenced, persistence increased and males copulated far longer than required for successful mating. When these interneurons were stimulated, persistence decreased and copulations were shortened. In contrast, dopaminergic neurons of the ventral nerve cord promote copulation persistence and extend copulation duration. Thus, copulation duration in Drosophila is a product of gradually declining persistence controlled by opposing neuronal populations using conserved neurotransmission systems (Crickmore, 2013).

    Some behaviors are reflexive, occurring in specific circumstances regardless of internal states or competing external stimuli. In contrast, many behaviors are influenced by motivational processes, of which there are three main classes: selection, intensity, and persistence. Although the extent to which terms from human psychology such as 'motivation' and 'persistence' are appropriate for insects is unclear, this study shows that male Drosophila display flexible, persistence- like behavior in determining whether or not to terminate copulation given the conditions internal and external to the animal (Crickmore, 2013).

    The GABAergic and dopaminergic neurons that this study has identified work in opposition to set copulation duration by changing the behavioral state of the male fly over time. Neither population has any demonstrable control over the transfer of reproductive fluids. Previously reported populations of neurons in the male abdominal ganglion control copulation duration as well as reproductive fluid transfer, although Tayler (2012) experimentally dissociated the control of copulation duration and reproductive fluid transfer. The neurons studied in this study also differ from known populations of copulation neurons because their sexual dimorphism is instructed by dsx and not fru. Thus there are several functionally, anatomically, and molecularly distinct neuronal inputs into copulation duration in the fly (Crickmore, 2013).

    Some neurons that influence copulation duration supply information regarding relevant internal and external circumstances. For example, in addition to the in copulo flexibility documented in this study, mating time in Drosophila has been shown to increase 10%-20% if the male is housed in the presence of 'competitor' males, changes modestly within the first 3 days of male adulthood, is extended in period and timeless circadian rhythm mutants, and is subject to alteration by artificial selection. Thus copulation persistence may emerge from interactions between neuronal populations carrying information about internal states and life history, as well as about the progress of copulation. The current data suggest a role for the GABAergic interneurons as a central node in this circuitry that integrates information from these inputs to produce a cohesive behavioral state that changes as copulation progresses (Crickmore, 2013).

    The decline in copulation persistence proceeds as the probability that the mating has been successful increases. Under adverse conditions such as the heat or wind used in this study, persistence provides an important component of a cost-benefit analysis that determines whether or not to truncate the copulation. It is proposed that GABA release causes persistence to decline until it reaches a threshold at which other internal drives or opportunities are sufficient to reorient the male toward other behavioral goals. These GABAergic interneurons may operate over a relatively broad dynamic range, contributing both to copulation duration under calm conditions but also instructing premature copulation in the face of stressful external conditions (Crickmore, 2013).

    Although the decline in copulation persistence occurs on a timescale that ensures productive mating, it appears to be regulated by an interval timing mechanism independent of reproductive fluid transfer. This raises the possibility that a long-sought mechanism for neural interval timing may be found by further study of this simple behavior. The involvement of conserved neurotransmission systems increases confidence that copulation duration is a useful model for investigating the poorly understood phenomena of interval timing and persistence (Crickmore, 2013).

    Dopamine neurons modulate pheromone responses in Drosophila courtship learning

    Learning through trial-and-error interactions allows animals to adapt innate behavioural 'rules of thumb' to the local environment, improving their prospects for survival and reproduction. Naive Drosophila melanogaster males, for example, court both virgin and mated females, but learn through experience to selectively suppress futile courtship towards females that have already mated. This study shows that courtship learning reflects an enhanced response to the male pheromone cis-vaccenyl acetate (cVA), which is deposited on females during mating and thus distinguishes mated females from virgins. Dissociation experiments suggest a simple learning rule in which unsuccessful courtship enhances sensitivity to cVA. The learning experience can be mimicked by artificial activation of dopaminergic neurons, and this study identified a specific class of dopaminergic neuron that is critical for courtship learning. These neurons provide input to the mushroom body (MB) γ lobe, and the DopR1 dopamine receptor is required in MBγ neurons for both natural and artificial courtship learning. This work thus reveals critical behavioural, cellular and molecular components of the learning rule by which Drosophila adjusts its innate mating strategy according to experience (Keleman, 2012).

    Mature virgin Drosophila females are usually willing to mate, whereas those that have recently mated are generally recalcitrant to further mating attempts. A male thus increases his overall mating success if he concentrates his courtship efforts on virgins. Given geographic and seasonal fluctuations in the relative abundance of virgins and mated females, and the cues that distinguish them, the optimal courtship strategy is unlikely to be a species universal. A heuristic for approaching this optimum could, however, be universal, allowing evolution to select for genes that implement such a learning rule in the fly's brain (Keleman, 2012).

    A male's courtship behaviour can be quantified by a courtship index (CI), and his ability to discriminate virgins from mated females by a discrimination index (DI), the relative reduction in the mean CI in single-pair assays with mated versus virgin females: DI = [CIv-CIm]/CIv. In courtship assays, naive males courted mated females only marginally less vigorously than they courted virgins, whereas males that had experienced rejection from mated females were subsequently much less active when courting mated females than virgins. The relative difference between the mean CIs of experienced (CI+) and naive (CI) males gives rise to a learning index: LI = [CI-CI+<]/CI. For males trained with mated females, the LI was just 7.8% in tests with virgin females but 48.2% when tested with mated females. Similar results were obtained when decapitated virgins were used as trainers, suggesting that male behaviour is conditioned by the failure to mate, not by active rejection from the female (Keleman, 2012).

    To discriminate mated females from virgins, a male might detect either the subtle changes in female pheromones on mating or the telltale vestiges of male pheromones that linger on mated females. The male-specific pheromone cVA is transferred to the female cuticle on mating. It is not detectable on the cuticle of either males or virgin females. Naive Or67d mutant males, which are unable to detect cVA, courted virgin and mated females equally (DI = −0.4%) and did not benefit from training. In contrast, analogous mutations in either of two other candidate pheromone receptor genes, Or47b and Gr68a, did not impair discrimination or learning. cVA detection is therefore crucial for naive and experienced males to discriminate mated females from virgins (Keleman, 2012).

    The salient feature of training might be the presence of cVA on the mated female, the lack of courtship success, or an association formed between the two. A dissociation experiment was designed to distinguish between these possibilities. Female post-mating behaviour, including courtship rejection, is triggered by sex peptide (SP), a male seminal fluid peptide transferred to the female during mating. Virgin females in which SP is transgenically expressed in the nervous system reject courting males (pseudomated females), whereas females that have mated with SP-null mutant males are still receptive (pseudovirgins). As expected, cVA was detected on the cuticle of both mated females and pseudovirgins, but not on virgins or pseudomated females. Thus, with pseudomated and pseudovirgin females the presence of cVA and sexual receptivity are fully dissociated (Keleman, 2012).

    Pseudomated females were just as effective as genuinely mated females when used as trainers, whereas pseudovirgin females were not. In contrast, pseudovirgin but not pseudomated females were as effective as mated females when used as testers. Indeed, robust courtship learning was observed when males were trained with pseudomated females and tested with pseudovirgins, but not vice versa. It is therefore concluded that the salient feature of training is simply the lack of courtship success, not its association with cVA, and that training alters the male’s response to cVA or some other vestige of previous contact with another male (Keleman, 2012).

    To test whether training does indeed alter sensitivity to cVA, varying doses of cVA were applied to pseudomated females and presented as testers to naive and experienced males. As expected, high doses of cVA inhibited courtship by both naive and experienced males. However, males trained with either mated or pseudomated females were inhibited by much lower doses of cVA than naive males were. Courtship training did not enhance sensitivity to an unrelated aversive odorant (Keleman, 2012).

    Dopamine is thought to provide a learning signal in a variety of different models and species, including aversive olfactory learning and conditioned suppression of male–male courtship in Drosophila. If dopamine also encodes an instructive signal during courtship learning, then artificial stimulation of dopaminergic neurons might mimic training with a mated female. To test this, the warmth-activated TrpA1 channel was expressed in most dopaminergic neurons, and attempts were made to'train' naive isolated males by warming them briefly to 30°C. When subsequently returned to 25°C and tested with mated females, the courtship activity of these males was indeed markedly reduced in comparison with that of control males. This suppression was specific for courtship towards mated but not virgin females, was dependent on a functional Or67d receptor, and was correlated with an increased sensitivity to cVA. In these respects, activation of dopaminergic neurons thus mimics a specific courtship learning signal rather than a non-specific punishment signal that might be expected to suppress courtship more generally. Experiments in which various subsets of dopaminergic neurons further suggest that the neurons involved in courtship learning are distinct from those previously implicated in various forms of aversive olfactory learning were selectively activated (Keleman, 2012).

    Many aspects of male courtship behaviour have been linked to the set of neurons that express the fruitless (fru) gene . Among these are the Or67d olfactory neurons (OSNs) and MBγ neurons, both of which function in courtship learning. It was speculated that the dopaminergic neurons involved in courtship learning might also be fru+. To test this hypothesis synaptic transmission of fru+ dopaminergic neurons was acutely blocked by using shits, which inhibits synaptic vesicle recycling at 30°C but not at 22°C. Such males showed significantly impaired learning when trained at 30°C and tested at 22°C, but not vice versa. These data thus establish a requirement for dopaminergic neurons in memory formation, not recall, and further indicate that the relevant cells are fru+ (Keleman, 2012).

    Previous studies identified two distinct classes of fru+ dopaminergic neurons: aSP4 and aSP13. To test whether aSP4 and/or aSP13 neurons contribute to courtship learning, synaptic transmission was chronically inhibited in these neurons with tetanus toxin light chain (TNT), using drivers selective for either aSP4 or aSP13. With each of five independent aSP13 drivers, learning was reduced by about 50% compared with control males that carried an inactive version of the TNT transgene in the same genetic background. A similar learning deficit was observed in positive controls in which TNT was targeted to both aSP13 and aSP4, to Or67d+ OSNs, or to MBγ neurons. In contrast, courtship learning was unimpaired in assays using either of two driver lines expressed in aSP4 but not aSP13. It is concluded that synaptic transmission of aSP13 neurons is crucial for courtship learning (Keleman, 2012).

    The presynaptic termini of aSP13 neurons are located at the tip of the MB γ lobe, indicating that they might convey a dopamine learning signal to MBγ neurons. If so, then a dopamine receptor should be required specifically in MBγ neurons for courtship learning. DopR1 and DopR2 receptors were considered as candidates, and homologous recombination was used to generate analogous loss-of-function alleles for each gene (DopR1attP and DopR2attP, respectively). Both mutants are viable and fertile and homozygous naive males court at normal levels. However, courtship learning was significantly impaired in DopR1attP but not DopR2attP mutants, as was 'fictive learning' induced by thermogenetic activation of dopaminergic neurons. Nevertheless, learning was not completely eliminated in these DopR1 mutants, indicating that other dopamine receptors might also contribute. To confirm that the learning deficit in the DopR1attP mutant was indeed due to loss of DopR1 function, the deleted genomic region was reintegrated by site-specific transgenesis. Males homozygous for this repaired DopR1 allele, DopR1Res, performed just as well as wild-type males in courtship learning assays (Keleman, 2012).

    Finally, RNA-mediated interference (RNAi) knockdown and rescue experiments were performed to test whether DopR1 function is indeed required in MBγ neurons. Expression of a DopR1 RNAi transgene selectively in MBγ neurons significantly reduced DopR1 expression levels in the γ lobe and impaired courtship learning. Conversely, the learning disability of DopR1attP mutants was fully alleviated by expressing a DopR1 transgene specifically in MBγ neurons. It is therefore postulate that DopR1 acts in MBγ neurons to transduce a dopamine learning signal provided by aSP13 neurons (Keleman, 2012).

    To maximize his reproductive success, a Drosophila male should be highly attuned to those cues that discriminate receptive from unreceptive females. A male that is too selective may miss mating opportunities; a male that is too promiscuous may waste resources on futile courtship. The optimal tuning is likely to vary from place to place and from time to time, depending for example on local and seasonal fluctuations in the abundance and quality of mating partners and the pheromone signals that they provide. This study defines a simple heuristic that could allow the male to learn an effective courtship strategy in his local environment: be promiscuous at first, but become more selective if a mating attempt fails. Furthermore, this study has identified key elements that implement this learning rule in the fly's brain. It is proposed that, when a mating attempt fails, aSP13 dopaminergic neurons convey a learning signal to MBγ neurons through the DopR1 receptor, and that this induces lasting changes in the internal processing of the cVA signal that discriminates mated females from virgins. Further studies of this genetically defined and tractable circuit should provide a detailed understanding of how a relatively simple learning circuit, embedded within decision-making centres of the brain, endows plasticity on an innate behaviour (Keleman, 2012).

    Novel role for ecdysone in Drosophila conditioned behavior: Linking GPCR-mediated non-canonical steroid action to cAMP signaling in the adult brain

    DopEcR, a G-protein coupled receptor for ecdysteroids, is involved in activity- and experience-dependent plasticity of the adult central nervous system. Remarkably, a courtship memory defect in rutabaga (Ca2+/calmodulin-responsive adenylate cyclase) mutants is rescued by DopEcR overexpression or acute 20E feeding, whereas a memory defect in dunce (cAMP-specific phosphodiestrase) mutants is counteracted when a loss-of-function DopEcR mutation is introduced. A memory defect caused by suppressing dopamine synthesis is also restored through enhanced DopEcR-mediated ecdysone signaling, and rescue and phenocopy experiments revealed that the mushroom body (MB) - a brain region central to learning and memory in Drosophila - is critical for the DopEcR-dependent processing of courtship memory. Consistent with this finding, acute 20E feeding induced a rapid, DopEcR-dependent increase in cAMP levels in the MB. The multidisciplinary approach demonstrates that DopEcR mediates the non-canonical actions of 20E and rapidly modulates adult conditioned behavior through cAMP signaling, which is universally important for neural plasticity. This study provides novel insights into non-genomic actions of steroids, and opens a new avenue for genetic investigation into an underappreciated mechanism critical to behavioral control by steroids (Ishimoto, 2013).

    Steroid hormones are essential modulators of a broad range of biological processes in a diversity of organisms across phyla. In the adult nervous system, the functions of steroids such as estrogens and glucocorticoids are of particular interest because they have significant effects on the resilience and adaptability of the brain, playing essential roles in endocrine regulation of behavior. Reflecting their importance in neural functions, steroid hormones are implicated in the etiology and pathophysiology of various neurological and psychiatric disorders, and are thus often targeted in therapies. The biological actions of steroids are mediated mainly by nuclear hormone receptors - a unique class of transcription factors that activate or repress target genes in a steroid-dependent manner. Substantial evidence suggests, however, that steroid hormones can also exert biological effects quickly and independently of transcriptional regulation, by modulating intracellular signaling pathways. Such 'non-genomic' effects might be induced by direct allosteric regulation of ion channels, including receptors for GABA and NMDA. Alternatively, in certain contexts, non-genomic steroid signaling could be mediated by classical nuclear hormone receptors acting as effector molecules in the cytosol (Ishimoto, 2013).

    G-protein coupled receptors (GPCRs) that directly interact with steroids have the potential to play an important role in non-genomic steroid signaling. So far, however, only few GPCRs have been identified as bona fide steroid receptors in vertebrates. The G-protein coupled estrogen receptor 1 (GPER, formally known as GPR30) is the best studied GPCR that is responsive to steroids. Pharmacological and gene knockout approaches suggest that this protein has widespread roles in the reproductive, nervous, endocrine, immune and cardiovascular systems (Prossnitz, 2011). Although other G-protein coupled receptors were predicted to be responsive to steroids (e.g., the Gq-coupled membrane estrogen receptor and estrogen receptor-X), their molecular identity is not known (Qiu, 2006; ToranAllerand, 2002). Overall, the physiological roles of the GPCR-mediated actions of steroids and the underlying molecular mechanisms remain poorly understood, and sometimes controversial, in spite of their importance. In particular, it is unknown how this non-canonical steroid mechanism influences neural functions and complex behaviors (Ishimoto, 2013).

    Drosophila genetics has been extensively used to study the roles and mechanisms of action of steroid hormones in vivo. The major steroid hormone in Drosophila is the molting hormone 20-hydroxy-ecdysone (20E), which orchestrates a wide array of developmental events, including embryogenesis, larval molting and metamorphosis. Recent studies revealed that 20E also plays important roles in adult flies, regulating: the innate immune response, stress resistance, longevity, the formation of long-term courtship memory and the active/resting state. In general, the functions of 20E during development and adulthood are thought to be executed by ecdysone receptors (EcRs), members of the evolutionarily conserved nuclear hormone receptor family (Ishimoto, 2013).

    In addition to canonical ecdysone signaling via EcRs, Srivastava (2005) identified a novel GPCR called DopEcR, and showed that it propagates non-genomic ecdysone signaling in vitro. DopEcR shares a high level of amino-acid sequence similarity with vertebrate β-adrenergic receptors. In situ hybridization and microarray data revealed that DopEcR transcripts are preferentially expressed in the nervous system. In heterologous cell culture systems, DopEcR is localized to the plasma membrane and responds to dopamine as well as ecdysteroids (ecdysone and 20E), modulating multiple, intracellular signaling cascades (Srivastava, 2005). Furthermore, Inagaki (2012) recently detected DopEcR expression in the sugar-sensing gustatory neurons of adult flies, and showed that DopEcR-mediated dopaminergic signaling enhances the proboscis extension reflex during starvation. Nonetheless, little is known about whether DopEcR functions as a steroid receptor in vivo, and about how it drives responses in the central nervous system (CNS) to modulate complex behaviors. This study reports that DopEcR mediates non-genomic ecdysone signaling in the adult brain, and that it is critical for memory processing. It was also shown that, during memory processing, DopEcR transmits information via novel steroid signals that interact with the cAMP pathway, a signaling cascade that is universally important for neuronal and behavioral plasticity. This genetic study thus uncovers underappreciated GPCR-mediated functions and mechanisms of action that employ non-canonical steroid signaling to regulate the adult nervous system and, thereby, behavior (Ishimoto, 2013).

    This study used genetic, pharmacological, and behavioral approaches in Drosophila to demonstrate that the steroid hormone 20E rapidly regulates behavioral plasticity via a non-genomic mechanism that is mediated by the GPCR-family protein DopEcR. This non-canonical steroid signaling pathway was found to have strong functional interactions with the classical 'memory genes' rut and dnc, which encode the central components of the cAMP pathway. The identification of 20E as an important modulator of cAMP signaling in the adult Drosophila brain reveals an unprecedented opportunity - that of taking advantage of fly genetics to dissect the molecular and cellular mechanisms responsible for the non-genomic steroid signaling that underlies neuronal and behavioral plasticity (Ishimoto, 2013).

    Electrophysiological analyses revealed that the adult giant-fiber (GF) pathway of DopEcR mutant flies is more resistant to habituation than that of control flies. Direct excitation of GF or its downstream elements would lead to a short-latency response of the dorsal longitudinal flight muscle (DLM), which could follow high-frequency stimuli up to several hundred Hz. In contrast, the afferent input to the GF leads to a long-latency response that is labile and fails to follow repetitive stimulation well below 100 Hz and displays habituation even at 2-5 Hz. Although there is the possibility that DopEcR-positive thoracic neurons may modulate thoracic motor outputs and contribute to certain parameters of the habituation process not characterized in this study, the more effective modulation would occur in the more labile element afferent to the GF circuit rather than the robust GF-PSI-DLMn downstream pathway (PSI referring to peripherally synapsing interneuron), which is responsible for the reliability of the escape reflex. Thus, the mutant phenotype in habituation indicates that DopEcR positively controls activity-dependent suppression of neuronal circuits afferent to the GF neurons in the brain (Ishimoto, 2013).

    Moreover, the finding that DopEcR and rut mutants have a similar GF habituation phenotype raises the possibility that DopEcR positively regulates cAMP levels in the relevant neurons following repetitive brain stimulation. Besides GF habituation, Drosophila displays olfactory habituation, which is mediated by the neural circuit in the antennal lobe. Interestingly, Das (2011) found that olfactory habituation is induced by enhancement of inhibitory GABAergic transmission, and that rut function is required for this neuronal modulation. Similar modulation of GABAergic transmission may also be responsible for habituation of the GF pathway. It will be interesting to examine whether and how DopEcR contributes to the regulation of rut and enhanced GABAergic transmission in GF habituation (Ishimoto, 2013).

    Several studies already suggested that 20E has rapid, EcR-independent effects in Drosophila and other invertebrate species. For example, 20E was shown to reduce the amplitude of excitatory junction potentials at the dissected Drosophila larval neuromuscular junction (NMJ), and to do so within minutes of direct application (Ruffner, 1999). Whereas treatment with 20E did not change the size and shape of the synaptic currents generated by spontaneous release, it led to a reduction in the number of synaptic vesicles released by the motor nerve terminals following electrical stimulation. A similar effect of 20E was observed in crayfish, and it was suggested that the suppression of synaptic transmission by 20E may account for the quiescent behavior of molting insects and crustaceans. These observations suggested that 20E suppresses synaptic efficacy under certain conditions by modulating presynaptic physiology through a non-genomic mechanism. It is possible that such actions of 20E are mediated by DopEcR. To detail the mechanisms underlying DopEcR-dependent neural plasticity, it will be worthwhile to determine if and how DopEcR contributes to 20E-induced, rapid synaptic suppression at the physiologically accessible larval NMJ, and to determine the extent to which non-genomic mechanisms of steroid actions are shared between the larval NMJ and the adult brain (Ishimoto, 2013).

    One surprising finding made in this study is that ecdysone signaling can modify the phenotypes associated with mutations in the classic 'memory genes', namely rut and dnc, through the actions of DopEcR. rut and dnc encode central components of the cAMP pathway, which is required for memory processing in vertebrates as well as invertebrates. The demonstration that genetically and/or pharmacologically enhancing DopEcR-mediated ecdysone signaling restores the courtship memory phenotype of loss-of-function rut mutants suggests that 20E-mediated DopEcR activation triggers an outcome similar to rut activation, i.e., increased cAMP levels. This assumption is supported by the finding that loss-of-function dnc mutants restore courtship memory when DopEcR activity is suppressed. A similar restoration of the dnc memory phenotype also occurs in a dnc and rut double mutant, again supporting the idea that DopEcR positively regulates cAMP production (Ishimoto, 2013).

    The results of rescue and phenocopy experiments indicate that the MB is critical for the DopEcR-dependent processing of courtship memory. Although the endogenous expression pattern of DopEcR is not known, DopEcR is thus likely to modulate cAMP levels in the MB in response to 20E during courtship conditioning. A new Gal4 line has been generated in which a portion of the first coding exon of DopEcR is replaced with a DNA element that contains the Gal4 cDNA whose translation initiation codon is positioned exactly at the DopEcR translation start site. When this line was used to drive UAS-GFP, the reporter gene was widely expressed in the adult brain with prominent signals in the MB. This preliminary result strongly indicates the endogenous expression of DopEcR in the MB. It has also been directly shown that cAMP levels in the MB increase rapidly in flies fed 20E, and that this increase does not occur when DopEcR expression is down-regulated specifically in the MB. Taken together, these findings suggest that DopEcR expressed in the MB responds to 20E and acts upstream of cAMP signaling in a cell-autonomous manner (Ishimoto, 2013).

    Surprisingly, enhancement of DopEcR-mediated ecdysone signaling restored courtship memory in flies harboring a strong hypomorphic allele of rut (rut1084). A similar result was obtained even in mutants harboring a presumptive rut null allele rut1. These results suggest that, upon stimulation by 20E, DopEcR may be able to signal via another adenylyl cyclase that can compensate for the lack of Rut. This interesting possibility requires further investigation (Ishimoto, 2013).

    This study has focused on the roles and mechanisms of action of DopEcR-mediated, non-genomic ecdysone signaling. Since it has been found that 20E levels rise in the head during courtship conditioning (Ishimoto, 2009), the data presented in this study suggest that DopEcR is activated by 20E during conditioning, triggers a rise in cAMP levels and induces physiological changes that subsequently suppress courtship behavior. This interpretation assumes that 20E directly activates DopEcR to increase cAMP levels. Previous cell-culture studies suggested that DopEcR also responds to dopamine to modulate intracellular signaling (Srivastava, 2005). Furthermore, Inagaki (2012) has demonstrated that flies respond to starvation by sensitizing gustatory receptor neurons to sugar via dopamine/DopEcR signaling. It is therefore necessary to consider whether dopamine is directly involved in the processing of courtship memory through DopEcR. There is a possibility that 20E initially stimulates the production and/or release of dopamine, and that it in turn activates DopEcR and elevates cAMP levels to induce courtship memory. This possibility is thought unlikely because even when courtship memory is disrupted by pharmacological suppression of dopamine synthesis, 20E feeding can compensate for decreased dopamine and allow restoration of memory. Although dopamine plays a significant role in courtship memory, the results suggest that DopEcR does not act as the major dopamine receptor in this particular learning paradigm. The possibility is thus favored that dopamine contributes to courtship memory in parallel with, or upstream of, DopEcR-mediated ecdysone signaling. Consistent with this view, Keleman (2012) reported that the formation of courtship memory depends on the MB γ neurons, which express DopR1 dopamine receptors, receiving dopaminergic inputs. Notably, the current results indicate that the processing of courtship memory requires DopEcR expression in the αβ, but not γ, neurons of the MB, which makes it unlikely that DopEcR is directly influenced by the dopaminergic neurons innervating γ neurons (Ishimoto, 2013).

    Ecdysone signaling through nuclear EcRs is necessary for forming long-term courtship memory that lasts at least 5 days, but appears not to have a significant effect on short-term courtship memory (Ishimoto, 2009). In contrast, we found that DopEcR-mediated ecdysone signaling is critical for habituation and 30-minute courtship memory. These findings suggest that DopEcR and EcRs control distinct physiological responses to courtship conditioning, and that the former regulates short-term memory, while the latter regulates long-term memory. Although non-genomic actions of steroid hormones have been implicated in vertebrate learning and memory, such actions have been attributed mainly to the classical nuclear hormone receptors that function outside of the nucleus and exert roles distinct from those of steroid-activated transcription factors. Although recent evidence has shown that membrane-bound receptors independent of the classical estrogen receptors are involved in estradiol-induced consolidation of hippocampal memory, the molecular identities of these proteins have not been established. The current findings in this study provide a novel framework for dissecting GPCR-mediated steroid signaling at the molecular and cellular levels. Furthermore, future analysis of the functional interplay between genomic and non-genomic steroid signaling pathways is expected to reveal novel mechanisms through which steroid hormones regulate plasticity of the nervous system and other biological phenomena (Ishimoto, 2013).

    apterous brain neurons control receptivity to male courtship in Drosophila melanogaster females

    Courtship behaviours allow animals to interact and display their qualities before committing to reproduction. In fly courtship, the female decides whether or not to mate and is thought to display receptivity by slowing down to accept the male. Very little is known on the neuronal brain circuitry controlling female receptivity. This study used genetic manipulation and behavioural studies to identify a novel set of neurons in the brain that controls sexual receptivity in the female without triggering the postmating response. These neurons, defined by the expression of the transcription factor Apterous, affect the modulation of female walking speed during courtship. Interestingly, it was found that the Apterous neurons required for female receptivity are neither Doublesex nor Fruitless positive suggesting that Apterous neurons are not specified by the sex-determination cascade. Overall, these findings identify a neuronal substrate underlying female response to courtship and highlight the central role of walking speed in the receptivity behaviour (Aranha, 2017).

    Reproductive behaviours are essential for the survival and fitness of the species. In Drosophila melanogaster, as in many other species, the decision of whether or not to mate is made by the female. However, understanding about the behaviour displayed by the virgin female and the neuronal circuits underlying it is still poor. This study set out to identify neurons involved in the response of virgin females to courting males. Female virgins with compromised activity in apterous neurons in the brain display a substantial reduction in copulation. What is specifically changed in the premating behaviour of apterous-silenced females? apterous-silenced females do not slow down during courtship even though they do recognize they are in the presence of a courting male because they extrude the ovipositor. Ovipositor extrusion is a display that occurs exclusively in the context of courtship. A caveat of this work is the large number of neurons that are affected by this manipulation. Is the low copulation rate a result of an issue created by silencing large numbers of neurons? The capacity to recognize the male partner does not seem to be affected and the phenotypes that were observed are revealed only in the context of courtship, which strongly suggests that parts of the natural receptivity circuit were specifically affected (Aranha, 2017).

    How can changes in activity of apterous neurons affect female velocity during courtship? The best understood cue from the male that shapes female velocity is the song. It is unlikely though that apterous-silenced females have impaired hearing. apGAL4 labels very faintly the region that is innervated by first and second order auditory neurons, AMMC. Third order auditory neurons innervate the ventral lateral protocerebrum (VLP). The VLP neurons (and a few other neurons) that express apterous were silenced, and no receptivity phenotype was observed. Moreover, flies with impaired hearing do not copulate within the time of the analysis. Most likely apterous neurons are involved in integrating sensory cues provided by the male that would lead to a receptive state of the female and over time result in a decrease of female velocity. (Aranha, 2017).

    This study also uncovered a role for apterous neurons in controlling egg laying, another critical aspect of reproductive behaviour. Females that have mated and then have their apterous neurons in the brain inhibited lay very few eggs, unlike control females. Classic gynandromorph studies pointed to an egg laying suppression focus in the brain, but this study seems to have identified a focus that promotes egg laying when active. A recent study implicates the doublesex-positive and female-specific PMN2 descending neurons in oviposition behaviou. apterous neurons are neither doublesex positive nor descending, so it is reasonable to assume that this study identified a novel set of neurons (Aranha, 2017).

    In conclusion, these findings contribute to understanding female receptivity and highlight the central role of female speed modulation during courtship. It will be interesting to elucidate in the future the role of the different apterous clusters and reveal how they interact with the sexual specification circuits (Aranha, 2017).

    Insulin signaling in the peripheral and central nervous system regulates female sexual receptivity during starvation in Drosophila

    Many animals adjust their reproductive behavior according to nutritional state and food availability. Drosophila females for instance decrease their sexual receptivity following starvation. Insulin signaling, which regulates many aspects of insect physiology and behavior, also affects reproduction in females. This study shows that insulin signaling is involved in the starvation-induced reduction in female receptivity. More specifically, females mutant for the insulin-like peptide (dilp5) were less affected by starvation compared to the other dilp mutants and wild-type flies. Knocking-down the insulin receptor, either in all fruitless-positive neurons or a subset of these neurons dedicated to the perception of a male aphrodisiac pheromone, decreased the effect of starvation on female receptivity. Disrupting insulin signaling in some parts of the brain, including the mushroom bodies even abolished the effect of starvation. In addition, Fruitless-positive neurons in the dorso-lateral protocerebrum and in the mushroom bodies co-expressing the insulin receptor were identified. Together, these results suggest that the interaction of insulin peptides determines the tuning of female sexual behavior, either by acting on pheromone perception or directly in the central nervous system (Lebreton, 2017a).

    Drosophila females need nutrients to produce eggs and a nutrient rich substrate to lay their eggs. When food is scarce it would therefore be beneficial for flies to decrease their sexual behavior and to focus on food searching instead. On the other hand, female flies can store sperm and use it several days later when conditions are suitable. It could therefore be optimal for females to remain receptive for short periods of food deprivation. Several insulin peptides produced in specific spatiotemporal patterns acting through one single receptor enables a fine-scale regulation of behaviors in response to changes in physiology. The expression of the different dilps is differentially affected by food quality or food deprivation. For instance, both starvation and dietary restriction reduce the expression of dilp5 but increase the expression of dilp6, while the expression of dilp2 is not affected by either condition. The results suggest that DILP5 might be involved in the decrease of receptivity during non-feeding stages. Indeed, dilp5 mutant females were less affected by starvation than other dilp mutants. The effect of the lack of DILP5 was no longer observed in the simultaneous absence of DILP2 and DILP3. Although, background mutation effects cannot be completely ruled out, this suggests that DILP5 might interact with other DILPs to finely tune female sexual receptivity (Lebreton, 2017a).

    Insulin is known to act on the olfactory system to modulate odor sensitivity after feeding. Moreover, normal InR expression in Or67d-expressing (Fruitless-positive) OSNs is necessary for fed females to be attracted to a blend of food odors and cVA, a pheromone promoting sexual receptivity. The results suggest that insulin signaling in Fruitless-positive neurons, and more specifically in Or67d OSNs may decrease sexual receptivity during starvation (Lebreton, 2017a).

    Fruitless-positive cells other than pheromone-sensing neurons can also be involved. Different Fruitless-positive cells in the protocerebrum were found that strongly express InR. First of all, a large number of Kenyon cells in the calyx of the mushroom bodies express both Fruitless and the insulin receptor. Additionally, one pair of neurons was found with somata located in the anterior dorso-lateral protocerebrum. It was not possible to trace any processes from these somata, and thus it is not known what neuropils they innervate. However, the fact that InR immunostaining was observed in Fruitless neurons, most of which were Kenyon cells, corroborate the behavioral results. Indeed, the sexual receptivity of females in which insulin signaling was knocked down in the mushroom bodies was not affected by starvation. Interestingly, the mushroom bodies are not required for virgin females to be receptive, suggesting that these structures may regulate the activity of neuronal networks inducing sexual receptivity. However, this result must be take with caution, given the fact that the Gal4 line that were used to target the mushroom bodies also drive expression to some extent in other brain tissues. Further experiments will be necessary to confirm that the mushroom bodies are indeed responsible for this effect (Lebreton, 2017a).

    Insulin signaling not only modulates neuronal activity in adults but also shapes neuronal networks during development. The effects observed in this study may therefore be the consequence of a developmental defect of specific neuronal circuitry rather than a direct effect of insulin on these neurons during starvation. However, Fruitless-positive neurons being required for females to be receptive, fed females would be expected to be unreceptive if the disruption of insulin signaling had altered the connectivity of these neurons during development, which was not the case. This suggests that insulin acts on these neurons during adult stage to modulate sexual receptivity. This is different for the mushroom bodies, which are not necessary for females to be receptive. Knocking down InR specifically during development or specifically in adults will be necessary to disentangle these two possible modes of action of insulin (Lebreton, 2017a).

    In contrast with Fruitless neurons and the mushroom bodies, no effect was observed of the corpora allata in the insulin-dependent control of sexual receptivity, whereas these structures have been linked to the development of receptivity in virgin females. This result should however be taken with caution, considering the behavioral variability displayed by the different transgenic lines, which would have prevented observing of subtle changes. Nonetheless, the results suggest that the structures that generate behaviors (such as the corpora allata) and those modulating these behaviors (for example the mushroom bodies) can be different and the underlying mechanisms uncoupled (Lebreton, 2017a).

    Taken together, Drosophila flies adjust their sexual behavior to match their nutritional state. Together with other hormonal pathways, insulin regulates some aspects of sexual activity, both after food intake and after a period of starvation. The results suggest that specific insulin peptides regulate female receptivity, possibly by acting on pheromone perception at the periphery or directly in the central nervous system. Indeed, the mushroom bodies probably play a major role in the insulin-dependent effect of starvation on female sexual receptivity. The next step will be to untangle the specific neuronal circuitry involved (Lebreton, 2017a).

    Motor control of Drosophila courtship song

    Many animals utilize acoustic signals-or songs-to attract mates. During courtship, Drosophila melanogaster males vibrate a wing to produce trains of pulses and extended tone, called pulse and sine song, respectively. Courtship songs in the genus Drosophila are exceedingly diverse, and different song features appear to have evolved independently of each other. How the nervous system allows such diversity to evolve is not understood. This study identified a wing muscle in D. melanogaster (hg1) that is uniquely male-enlarged. The hg1 motoneuron and the sexually dimorphic development of the hg1 muscle, whose dimorphic character is regulated by doublesex but not male specific fruitless, are required specifically for the sine component of the male song. In contrast, the motoneuron innervating a sexually monomorphic wing muscle, ps1, is required specifically for a feature of pulse song. Thus, individual wing motor pathways can control separate aspects of courtship song and may provide a 'modular' anatomical substrate for the evolution of diverse songs (Shirangi, 2013).

    This study has shown that the hg1 wing muscle and its sexually dimorphic development are required for the sine component of courtship song, whereas the ps1 wing muscle is required for a specific aspect of pulse song, but not sine song. The sexual size dimorphism in hg1 is analogous to the sexual differences in the size and physiology of the laryngeal muscles of singing Xenopus laevis frogs. Contraction of hg1 pulls the posterior notal wing process in an anterioventral direction, but how this event relates to the wing motions underlying sine song is not clear. The observation that feminizing hg1 reduces the amplitude of sine song suggests that hg1 may provide power to the wing strokes that generate sine song. Although hg1 is essential for sine song, it obviously does not work alone and the performance of this song component involves the synergistic actions of other wing muscles. Given the role of ps1 in linking the pleural and sternal apophyses, these results further suggest that thoracic rigidity regulates pulse carrier frequency. The findings echo a burgeoning idea that complex behaviors are composed of 'modules' that allow discrete aspects of a behavior to evolve independently of others. The results demonstrate that pulse and sine song are produced in part by separate sets of wing muscles, suggesting that the wing periphery is to a certain extent modular. By 'modular' it is meant that discrete features of the behavior can be functionally mapped to morphologically discrete subunits in the motor periphery. Given that the wing periphery consists of a relatively small number of muscles, the modularity observed may be due to the biomechanical constraints intrinsic to the wing musculoskeletal system. Species of the genus Drosophila display extensive diversity in courtship song, and different song features appear to evolve independently of each other. The apparent specialization of wing motor pathways for different aspects of song may provide a modular anatomical template for the evolution of different components of courtship song (Shirangi, 2013).

    Sensorimotor transformations underlying variability in song intensity during Drosophila courtship

    Diverse animal species, from insects to humans, utilize acoustic signals for communication. Studies of the neural basis for song or speech production have focused almost exclusively on the generation of spectral and temporal patterns, but animals can also adjust acoustic signal intensity when communicating. For example, humans naturally regulate the loudness of speech in accord with a visual estimate of receiver distance. The underlying mechanisms for this ability remain uncharacterized in any system. This study shows that Drosophila males modulate courtship song amplitude with female distance. The study investigates each stage of the sensorimotor transformation underlying this behavior, from the detection of particular visual stimulus features and the timescales of sensory processing to the modulation of neural and muscle activity that generates song. The results demonstrate an unanticipated level of control in insect acoustic communication and uncover novel computations and mechanisms underlying the regulation of acoustic signal intensity (Coen, 2016).

    Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality in Drosophila

    The indirect flight muscles (IFMs) of Drosophila and other insects with asynchronous flight muscles are characterized by a crystalline myofilament lattice structure. The high-order lattice regularity is considered an adaptation for enhanced power output, but supporting evidence for this claim is lacking. This study shows that IFMs from transgenic flies expressing flightin with a deletion of its poorly conserved N-terminal domain (flnΔN62) have reduced inter-thick filament spacing and a less regular lattice. This resulted in a decrease in flight ability by 33% and in skinned fibre oscillatory power output by 57%, but had no effect on wingbeat frequency or frequency of maximum power output, suggesting that the underlying actomyosin kinetics is not affected and that the flight impairment arises from deficits in force transmission. Moreover, flnΔN62 males were shown to produced an abnormal courtship song characterized by a higher sine song frequency and a pulse song with longer pulses and longer inter-pulse intervals (IPIs), the latter implicated in male reproductive success. When presented with a choice, wild-type females chose control males over mutant males in 92% of the competition events. These results demonstrate that flightin N-terminal domain is required for optimal myofilament lattice regularity and IFM activity, enabling powered flight and courtship song production. As the courtship song is subject to female choice, it is proposed that the low amino acid sequence conservation of the N-terminal domain reflects its role in fine-tuning species-specific courtship songs (Chakravorty, 2017).

    Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila

    Intraspecific male-male aggression, which is important for sexual selection, is regulated by environment, experience and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, a behavioral assay was established in Drosophila melanogaster, and the relationship between sexual experience and aggression was investigated. In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel Ppk29 and was mediated by male-specific GABAergic neurons acting on the GABAA receptor RDL in target cells. Silencing or activating this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. It is proposed that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression (Yuan, 2013).

    Aggression is a complex behavior that is regulated by various internal and external stimuli. To date, however, studies have remained largely focused on the sensory pathways involved in regulating baseline aggression, with less examination of the central components of the underlying neural pathway. Moreover, the close relationship between sex and aggression has been a fascinating topic in both biology and literature, but their intertwined nature and the underlying neurobiological basis have remained elusive. Using a behavioral genetics approach, this study identified a previously unknown neural pathway that underlies the modulation of sex-related male-male aggression in Drosophila by prior contacts with females (Yuan, 2013).

    These results suggest that prior female encounter through direct physical contacts activates the pheromone-sensing ppk29 neurons, resulting in inhibition of the central aggression circuit via GABAergic mechanisms involving the RDL GABAA receptor, thereby suppressing the behavioral output for male-male aggression. The three levels of the neural pathway involved in this experience-dependent behavior modification all exhibited sexual dimorphism, consistent with the notion that morphological differences in male and female brains correlate with their distinct behavioral needs. It was possible to modify the aggressive behavior output by manipulating the circuit at each of these three steps, which possibly represented the sequence of the information relay involved in the native behaviors, the sensory input, the information processing and the execution of the behavior. However, it is recognized that the circuit components elucidated by these experiments are clearly only parts of the machinery responsible for aggression modulation. In addition, by identifying RDL as a molecular target for aggression regulation, this study provides an entry point for characterizing the missing link of aggression studies, namely the central neurons that respond to experience-dependent modulation and mediate the execution of aggressive behaviors. Thus, this work provides new insights regarding the intricate interactions between sexual experience and aggression and delineates the underlying mechanisms to inform potential means to suppress excessive aggression (Yuan, 2013).

    The female contact-dependent suppression of male aggression may also be viewed as a form of learning-induced plasticity. The learning procedure in this case requires extended physical interactions between the male and the female (over 10 h), which could consist of repeated sessions of male courtship attempts and female rejection. As no obvious defects were observed in aggression suppression in genetic mutants with deficits in courtship conditioning, such as homer, eag, Shaker and orb2 mutants, this experience-induced suppression of aggression is likely different from conventional courtship conditioning. Another interesting feature of this suppression is that it is long term, yet reversible, lasting up to 2 d after the female encounter. However, it also differs from well-studied long-term memory formation, as no defect was observed in amn mutants, which is required for long-term memory formation. The results implicate the fru+ d5-HT1B+ and GABA+ cluster of neurons in the central brain as the regulators of this suppression, but it remains to be determined whether these neurons are involved in the initiation, acquisition, execution or consolidation phase(s) of this behavior, what takes form as the underlying 'memory trace' and whether plasticity is manifested at the level of the number of neurons activated, neurite arborization, neuronal activity or some other aspect of neuronal signaling (Yuan, 2013).

    Notwithstanding the emergence of Drosophila as a successful genetic model for aggression studies and the extensive characterization of its stereotypical motor display of aggression, the strong influence of genetic background over baseline aggression and locomotor activity often complicates Drosophila aggression studies. The current assay avoids such difficulties by consistently eliciting aggression in naive male flies in the presence of females and by inducing a strong suppression of aggression in males with prior female encounter. The small variations among different genetic backgrounds in the behavioral assay make it possible to identify critical cellular and molecular components involved in the regulation of aggression by experience (Yuan, 2013).

    One purpose of studying aggression regulation in animal models is to eventually understand the basis of human violence and establish venues to reduce or prevent it. Psychophysiological studies suggest that the failure to maintain an appropriate level of aggression in humans is associated with impaired executive cognitive processes or emotion registration. As an innate behavior built largely on predetermined neural pathways, aggression in Drosophila males can be modulated by prior exposure to females through GABAergic inhibition. This study raises the possibility that an ancient and basic machinery of the central neuronal circuitry, GABAergic inhibition, could be part of a conserved mechanism to modulate the level of aggression in males and ensure proper balance between reproductive competition and individual survival (Yuan, 2013).

    Active and passive sexual roles that arise in Drosophila male-male courtship are modulated by dopamine levels in PPL2ab neurons

    The neurology of male sexuality has been poorly studied owing to difficulties in studying brain circuitry in humans. Dopamine (DA) is essential for both physiological and behavioural responses, including the regulation of sexuality. Previous studies have revealed that alterations in DA synthesis in dopaminergic neurons can induce male-male courtship behaviour, while increasing DA levels in the protocerebral posteriolateral dopaminergic cluster neuron 2ab (PPL2ab) may enhance the intensity of male courtship sustainment in Drosophila. This study reports that changes in the ability of the PPL2ab in the central nervous system (CNS) to produce DA strongly impact male-male courtship in D. melanogaster. Intriguingly, the DA-synthesizing abilities of these neurons appear to affect both the courting activities displayed by male flies and the sex appeal of male flies for other male flies. Moreover, the observed male-male courtship is triggered primarily by target motion, yet chemical cues can replace visual input under dark conditions. This is interesting evidence that courtship responses in male individuals are controlled by PPL2ab neurons in the CNS. This study provides insight for subsequent studies focusing on sexual circuit modulation by PPL2ab neurons (Chen, 2017).

    The neural circuitry that functions as a switch for courtship versus aggression in Drosophila males

    Courtship and aggression are induced in a mutually exclusive manner in male Drosophila melanogaster, which quickly chooses one of these behavioral repertoires to run depending on whether the encountered conspecific is a female or male, yet the neural mechanism underlying this decision making remains obscure. By targeted excitation and synaptic blockage in a subset of brain neurons, this study demonstrates that the fruitless (fru)-negative subfraction (approximately 20 cells) of a doublesex-positive neural cluster, pC1, acts as the aggression-triggering center whereas the fru-positive subfraction (approximately 20 cells) of pC1 acts as the courtship-triggering center, and that the mutually exclusive activation of these two centers is attained by a double-layered inhibitory switch composed of two fru single-positive clusters, LC1 and mAL. This is the first report to unravel the cellular identity of the neural switch that governs the alternative activation of aggression and courtship in the animal kingdom (Koganezawa, 2016).

    Effect of competitive cues on reproductive morphology and behavioral plasticity in male fruitflies

    Phenotypic plasticity will be favored whenever there are significant fitness benefits of responding to environmental variation. The extent and nature of the plasticity that evolves depends on the rate of environmental fluctuations and the capacity to track and respond to that variability. Reproductive environments represent one arena in which changes can be rapid. The finding that males of many species show morphological, physiological, and behavioral plasticity in response to premating and postmating reproductive competition (RC) suggests that plasticity is broadly beneficial. The developmental environment is expected to accurately predict the average population level of RC but to be a relatively poor indicator of immediate RC at any particular mating. Therefore, this study predicts that manipulation of average RC during development should cause a response in plasticity "set" during development (e.g., size of adult reproductive structures), but not in flexible plasticity determined by the immediate adult environment (e.g., behavioral plasticity in mating duration). This prediction was tested in Drosophila melanogaster males by manipulating 2 independent cues of average RC during development: 1) larval density and 2) the presence or absence of adult males within larval culture vials. Consistent with the prediction, both manipulations result in the development of males with significantly larger adult accessory glands (although testis size decreases when males are added to culture vials). There is no effect on adult plasticity (mating duration, extended mating in response to rivals). The results suggest that males have evolved independent responses to long- and short-term variation in RC (Bretman, 2016).

    Cross-generational comparison of reproductive success in recently caught strains of Drosophila melanogaster

    Males and females often have opposing strategies for increasing reproductive fitness. Males that out-compete others higher lifetime reproductive success. Females that mate with a high quality male receive either direct benefits through productivity or acquisition of additional resources. These components may be in conflict: factors that increase offspring fitness may decrease a female's productivity, and alleles that are beneficial in one sex may be detrimental in the opposite sex. This study used a multigenerational study with recently caught strains of Drosophila melanogaster to examine the relationship between parental, male offspring, and female offspring fitness when fitness is measured in a basal non-competitive environment. Synergy was found between parental and offspring lifetime reproductive success, indicating a lack of parent-offspring conflict, and a synergy was found between son and daughter reproductive success, indicating a lack of intersexual conflict. Interestingly, inbreeding significantly reduced the lifetime reproductive success of daughters, but did not have a significant effect on short-term productivity measures of daughters, sons or parents. It is concluded that in wild-caught flies, there appears to be no parent-offspring conflict or intersexual conflict for loci influencing offspring production in a non-competitive environment (Nguyen, 2017).

    Abdominal-B neurons control Drosophila virgin female receptivity

    Female sexual receptivity offers an excellent model for complex behavioral decisions. The female must parse her own reproductive state, the external environment, and male sensory cues to decide whether to copulate. In the fly Drosophila melanogaster, virgin female receptivity has received relatively little attention, and its neural circuitry and individual behavioral components remain unmapped. Using a genome-wide neuronal RNAi screen, this study identified a subpopulation of neurons responsible for pausing, a novel behavioral aspect of virgin female receptivity characterized in this study. Abdominal-B (Abd-B), a homeobox transcription factor, was shown to be required in developing neurons for high levels of virgin female receptivity. Silencing adult Abd-B neurons significantly decreased receptivity. Two components of receptivity were characterized that are elicited in sexually mature females by male courtship: pausing and vaginal plate opening. Silencing Abd-B neurons decreased pausing but did not affect vaginal plate opening, demonstrating that these two components of female sexual behavior are functionally separable. Synthetic activation of Abd-B neurons increased pausing, but male courtship song alone was not sufficient to elicit this behavior. These results provide an entry point to the neural circuit controlling virgin female receptivity. The female integrates multiple sensory cues from the male to execute discrete motor programs prior to copulation. Abd-B neurons control pausing, a key aspect of female sexual receptivity, in response to male courtship (Bussell, 2014).

    Female receptivity is a complex behavior comprising multiple motor programs and requiring the integration of sensory cues across several modalities. Nevertheless, Drosophila mating behavior is innate, and receptivity is likely controlled by hardwired neural circuits. This study identified seven candidate genetic markers of receptivity neurons by using a neuronal RNAi screen. The data suggest a central role for one of these, the transcription factor Abd-B, in forming a neural circuit that functions in receptivity (Bussell, 2014).

    This study has refined the behavioral components of receptivity beyond mere copulation acceptance. Vaginal plate opening occurs throughout courtship and depends on sexual maturity. The historically noted slowing down of receptive females is attributed to punctuated bouts of pausing during courtship rather than decreased walking speed. Pausing behavior is specific to female receptivity: it is decreased in both unreceptive females and in mature virgin females not being actively courted by a male. The increased level of pausing associated with receptivity requires the integration of multiple sensory inputs, including song, from a courting male. Abd-BLDN neuronal activity is both necessary for this pausing response and sufficient to induce it, thus establishing direct function of these neurons within the receptivity circuit (Bussell, 2014).

    How do Abd-B neurons control pausing? The Abd-BLDN neurons important for receptivity are not themselves motor neurons, and females with silenced Abd-BLDN neurons are not generally deficient in movement or posture. This suggests that Abd-BLDN neurons play a role downstream of the sensation of individual male courtship sensory inputs but upstream of motor output. The abdominal ganglion is emerging as a potential locus coordinating female-specific behavior, and Abd-BLDN neurons there are well-positioned to interact with other neurons involved in female behavior, including the postmating response. These neurons could therefore potentially function to integrate male courtship cues and internal inputs and promote pausing (Bussell, 2014).

    Silencing Abd-BLDN neurons affects pausing, but not vaginal plate opening, which demonstrates that it is possible to uncouple these two aspects of receptivity. However, activation of Abd-BLDN neurons affects both pausing and the movement of the vaginal plates. It is therefore possible that Abd-BLDN neurons, or subsets within them, function in both of these aspects of receptivity. There are likely to be additional circuit components involved in plate opening that might be able to act redundantly in the absence of Abd-BLDN neurons, and the involvement of additional neurons in the control of the vaginal plates is consistent with the fact that Abd-BLDN activation does not induce periodic vaginal plate opening but rather locks the plates in the open position. How the receptivity circuitry coordinates vaginal plate opening with pausing and male copulation attempts remains unknown. Abd-BLDN neurons provide an important entry point to dissect the two female motor programs. It was observed that vaginal plate opening occurs both while the female is moving and while she is stationary. Female movement has been shown to provide feedback to the male during courtship, and it could be that pausing provides an important connection between the sexes within the context of the courtship duet (Bussell, 2014).

    Abd-B is required in neurons during development for females to become highly receptive to male courtship. How does the Abd-B protein affect the receptivity circuitry? Abd-BLDN > Abd-B RNAi experiments show that Abd-BLDN- Gal4 labels the neurons in which Abd-B functions during development to affect receptivity. However, it is possible that these developmental Abd-BLDN neurons are not identical to the adult Abd-BLDN neurons that function in receptivity. In developing neuroblasts, Abd-B can have different, even opposing, functions, promoting cell death or promoting a particular cell fate or repressing it, depending on neuroblast identity and context. In Abd-BLDN > Abd-B RNAi experiments, no obvious changes were observed in either the number or projections of Abd-BLDN neurons in the adult, but this does not exclude the possibility of subtle anatomical changes or changes in cell identity. Finally, it is noted that modularity in the control of complex innate behavior has been found across a variety of species and systems. From flies to mice, both aggression and mating are controlled by eliciting different modules in a sexually dimorphic way. Thus, female fly receptivity fits into a larger pattern of sex-specific control of innate behavioral components (Bussell, 2014).

    Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila

    Males of most species are more aggressive than females, but the neural mechanisms underlying this dimorphism are not clear. This study identified a neuron and a gene that control the higher level of aggression characteristic of Drosophila melanogaster males. Males, but not females, contain a small cluster of FruM+ neurons that express the neuropeptide tachykinin (Tk). Activation and silencing of these neurons increased and decreased, respectively, intermale aggression without affecting male-female courtship behavior. Mutations in both Tk and a candidate receptor, Takr86C, suppressed the effect of neuronal activation, whereas overexpression of Tk potentiated it. Tk neuron activation overcame reduced aggressiveness caused by eliminating a variety of sensory or contextual cues, suggesting that it promotes aggressive arousal or motivation. Tachykinin/Substance P has been implicated in aggression in mammals, including humans. Thus, the higher aggressiveness of Drosophila males reflects the sexually dimorphic expression of a neuropeptide that controls agonistic behaviors across phylogeny (Asahina, 2014).

    Aggression is an innate, species-typical social behavior that is widespread in animal phylogeny. Expression of agonistic behavior is commonly observed between conspecific males in conflict over access to reproductively active females, food, territory, or other resources. In many animal species, aggression is often quantitatively higher in males than in females. In humans, violent aggression constitutes a major public health problem and its incidence is overwhelmingly higher among males than females. In addition, the behavioral expression of aggression is often qualitatively different between males and females, and may differ in the contexts in which it is exhibited (Asahina, 2014).

    Despite recent progress, the neurobiological mechanisms underlying the evolutionarily conserved sexual dimorphism in aggressiveness remain poorly understood. Pheromones are known to play an important role in intermale aggression. However, in cases where the relevant receptors are known, dimorphic expression of these molecules does not appear to explain sex differences in aggressiveness. Studies in numerous vertebrate species have identified sexual dimorphisms in the size of brain nuclei or their constituent neuronal subpopulations that are controlled by gonadal steroid hormones in a manner that parallels the influence of these hormones on aggressive behavior. Recent studies have shown that genetic ablation of hypothalamic neurons expressing the progesterone receptor decreases both aggression and mounting in males, and mating behavior in females (Yang, 2013). These neurons display sexual dimorphisms in their projections, but whether this dimorphism is causally responsible for sex differences in levels of aggressiveness is not yet clear. As in other species, Drosophila males flies are more aggressive than females and also exhibit qualitative differences in agonistic behavior. These sex differences in aggression are known to be under the control of fruitless (fru), a master regulator of sexual differentiation of the brain. Although some efforts have been made to identify circuits through which fru exerts its influence on aggressive behavior, FruM+ neurons that are necessary, sufficient, and specific for male-type aggression have not yet been identified (Asahina, 2014).

    This study has identified a small group of sexually dimorphic, FruM+ neurons that promote aggressiveness in Drosophila males but have no influence on male-female courtship behavior. These neurons enhance aggression, at least in part, through the release of a neuropeptide, Drosophila tachykinin (DTK). Tachykinin/Substance P has been implicated in certain forms of aggression in several mammalian species. Thus, the higher level of aggression that is characteristic of Drosophila males is promoted by sexually dimorphic neurons, which express a neuropeptide that regulates agonistic behavior across phylogeny (Asahina, 2014).

    This study has identified a sexually dimorphic neuron and a gene that play a critical and specific role in the expression of intermale aggression in Drosophila. The gene encodes a neuropeptide homologous to mammalian Substance P, and its release from the identified neurons is important for aggression. Substance P has been implicated in aggression in several mammalian systems. Together, the data suggest that the higher level of aggressiveness in Drosophila males may be controlled by the expression in sexually dimorphic neurons of a neuropeptide that regulates forms of agonistic behavior across phylogeny (Asahina, 2014).

    Previous studies have investigated the role of FruM+ neurons in aggression versus courtship. Selective masculinization of certain groups of neurons in females masculinized courtship behavior, but not aggression, suggesting that distinct subsets of FruM neurons may control these behaviors; however, a selective masculinization of aggression, but not courtship, was not observed. Feminization of most or all octopaminergic (OA) or cholinergic neurons, via expression of UAS-Tra, altered the balance between male-male courtship and aggression, or enhanced aggression, respectively. Feminization of a small subset of OA neurons increased male-male courtship, but not aggression. Specific OA and dopaminergic neurons that influence aggression have been identified, but these neurons are not sexually dimorphic. The present results identify sexually dimorphic Tk-GAL4FruM neurons that are necessary, sufficient, and specific for the quantitatively higher level of aggressiveness that is characteristic of Drosophila males. The neurons responsible for the qualitative sex-specific differences in the behavioral expression of aggression remain to be identified (Asahina, 2014).

    Studies in mice have localized aggression-promoting neurons to the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl). Genetic ablation of anatomically dimorphic neurons within VMHvl that express the progesterone receptor (PR) was shown to partially reduce aggressive behavior. However, this effect of this ablation was not specific to aggression, since male mating behavior and female mating behavior were attenuated as well. In contrast, the Tk-GAL4FruM neurons identified in this study control aggression, but not mating behavior. Unlike PR+ neurons, moreover, these cells are not detectable in females (Asahina, 2014).

    The fact that the Tk-GAL4FruM neurons were not observed in females suggests that either the developmental generation of these neurons and/or their expression of the neuropeptide is male specific. Whatever the case, the absence of these neural elements from the female brain is likely to contribute to their lower level of aggressive behavior. The data suggest that sex-typical features of some innate behaviors in Drosophila may be achieved, at least in part, by the sexually dimorphic expression in specific neurons of neuropeptides that coordinate males-pecific behavioral subprograms. Dimorphic populations of FruM-expressing neurons also regulate sexually dimorphic behaviors through the release of classical fast neurotransmitters that act on sexually dimorphic chemical synapses (Asahina, 2014).

    Several lines of evidence presented in this study argue that Tk-GAL4FruM neurons influence aggressive arousal or motivation, rather than simply acting as 'command neurons' for aggressive actions. First, activation of these neurons did not trigger a single aggressive action, as would be expected for a command neuron, but rather increased the frequency of multiple agonistic behaviors, including wing-threat, lunging, and tussling. Second, thermogenetic activation of these neurons supervened the requirement for several aggression-permissive conditions and cues, some of which (such as male-specific pheromones) could be construed as 'releasing signals'. The activation of Tk-GAL41 neurons was even able to promote lunging toward a moving dummy fly (albeit in a minority of trials). To the extent that increased arousal decreases the requirement for specific releasing signals to evoke innate behaviors, activation of Tk-GAL4FruM neurons may generate an arousal-like state that is specific for aggression. Alternatively, Tk-GAL4FruM neurons may enhance behavioral sensitivity to multiple releasing signals that characterize an attackable object, either at the level of parallel sensory processing pathways or at a locus downstream of the integration of these multisensory cues, analogous to the neuropeptide regulation of feeding behavior in C. elegans (Asahina, 2014).

    Several lines of evidence presented in this study suggest that the release of DTK peptides indeed contributes to the aggression-promoting function of Tk-GAL4FruM neurons. Nevertheless, the release of a classical neurotransmitter, probably acetylcholine, likely contributes to the behavioral influence of Tk-GAL4FruM neurons as well. Furthermore, while the data implicate Takr86C as a receptor for Tk in the control of aggression, they do not exclude a role for Takr99D. Among three species of vertebrate Tachykinin neuropeptides, Substance P has been implicated, directly or indirectly, in various forms of aggression, including defensive rage and predatory attack in cats, and intermale aggression in rats. Although not all functions of Substance P are necessarily conserved (such as nociception in mammals and olfactory modulation in the fly, these data suggest that this neuropeptide is broadly involved in the control of agonistic behavior in both vertebrates and invertebrates. They therefore add to the growing list of neuropeptide systems that show a remarkable evolutionary conservation of functions in the regulation of innate 'survival behaviors' such as feeding and mating. Biogenic amines also control aggression across phylogeny. However, in the case of serotonin, the directionality of its influence is opposite in flies and humans (Asahina, 2014).

    The findings of this study indicate that studies of agonistic behavior in Drosophila can identify aggression-regulating genes with direct relevance to vertebrates. Interestingly, in humans, the concentration of Substance P-like immunoreactivity in cerebrospinal fluid has been positively correlated with aggressive tendencies in patients with personality disorders. Substance P antagonists have been tested in humans as anxiolytic and antidepressant agents, although they failed to show efficacy . The present findings, taken together with mammalian animal studies, suggest that it may be worthwhile to investigate the potential of these antagonists for reducing violent aggression in humans (Asahina, 2014).

    Drosophila Life Span and Physiology Are Modulated by Sexual Perception and Reward

    Sensory perception modulates aging and physiology across taxa. This study found that perception of female sexual pheromones through a specific gustatory receptor expressed in a subset of foreleg neurons in male fruit flies rapidly and reversibly decreases fat stores, reduces resistance to starvation, and limits life span together with neurons that express the reward-mediating neuropeptide F. High-throughput RNA-seq experiments revealed a set of molecular processes that were impacted by the activity of the longevity circuit, thereby identifying new candidate cell non-autonomous aging mechanisms. Mating reversed the effects of pheromone perception, suggesting a model where life span is modulated through integration of sensory and reward circuits and where healthy aging may be compromised when the expectations defined by sensory perception are discordant with ensuing experience (Gendron, 2013).

    Sensory perception can modulate aging and physiology in multiple species. In Drosophila, exposure to food-based odorants partially reverses the anti-aging effect of dietary restriction, whereas broad reduction in olfactory function promotes longevity and alters fat metabolism. Even the well-known relation between body temperature and life span may have a sensory component (Gendron, 2013).

    To identify sensory cues and neuronal circuitry that underlie the effects of sensory perception on aging, this study focused on the perception of potential mates. Social interactions are prevalent throughout nature, and the influence of social context on health and longevity is well-known in several species, including humans. Such influences include behavioral interactions with mates and broader physiological 'costs of reproduction,' which often form the basis for evolutionary models of aging (Gendron, 2013).

    In Drosophila, the presence of potential mates is perceived largely through non-volatile cuticular hydrocarbons, which are produced by cells called oenocytes and are secreted to the cuticular surface where they function as pheromones. To test whether differential pheromone exposure influenced life span or physiology, 'experimental' flies of the same genotype were housed with 'donor' animals of the same sex that either expressed normal pheromone profiles or were genetically engineered to express pheromone profiles characteristic of the opposite sex. Donor males with feminized pheromone profiles were generated by targeting expression of the sex determination gene, tra, to the oenocytes (via OK72-GAL4 or Prom-E800-Gal4), whereas masculinization of female flies was accomplished by expressing tra-RNAi in a similar way. This design allowed manipulation of the experimental animals' perceived sexual environment without introducing complications associated with mating itself (Gendron, 2013).

    In Drosophila, sensory manipulations can affect life span, fat storage (as determined by baseline measures of triacylglyceride-TAG), and certain aspects of stress resistance. This study has found that flies exposed to pheromones of the opposite sex showed differences in these phenotypes. Experimental male flies exposed to male donor pheromone had higher amounts of TAG, were substantially more resistant to starvation, and exhibited a significantly longer life span than genetically identical male siblings exposed to female donor pheromone. Females exhibited similar phenotypes in response to male donor pheromone, but the magnitude of the effects was smaller. Subsequent experiments were therefore focused on males (Gendron, 2013).

    The characteristics of pheromone exposure were indicative of a mechanism involving sensory perception. Effects were similar in several genetic backgrounds, including a strain recently collected in the wild, and were largely unaffected by cohort composition. Pheromone-induced phenotypes were detected after as little as two days exposure to donor animals, persisted with longer manipulations, and were progressively reversed when female donor pheromone was removed. Pheromone effects appeared not to be mediated by aberrant or aggressive interactions with donor flies because no significant differences were observed in such behaviors and because continuous, vigorous agitation of the vials throughout the exposure period, which effectively disrupted observed behaviors, had no effect on the impact of donor pheromone. Furthermore, exposure of experimental males to the purified female pheromone 7-11-heptacosadiene (7-11 HD) produced physiological changes in the absence of donor animals (Gendron, 2013).

    To explore the sensory modality through which donor pheromone exerts its effects, this study tested whether a broadly-expressed olfactory co-receptor, Or83b, whose loss of function renders flies largely unable to smell, was required for pheromone effects. Or83b mutant flies exhibited similar changes in starvation resistance in response to donor pheromone as did control animals, indicating that olfaction was not required. To test whether taste perception was involved, flies were tested that were mutant for the gene Pox neuro (Poxn), a null mutation that putatively transforms all chemosensory neurons into mechanosensory neurons. Drosophila taste neurons are present in the mouthparts and distributed on different body parts including the wings, legs, and genitals, which allow sensation by contact. When the Poxn null mutation is coupled with a partially rescuing transgene, Poxn ΔM22-B5-ΔXB, flies are generally healthy, but gustatory perception is eliminated in the labelum, the legs, and the wing margins. Poxn ΔM22-B5XB flies showed no pheromone-induced changes in starvation resistance, TAG amounts, or life span. However, Poxn mutant flies that carried a transgene that restores taste function to the legs and wing margins (but not labelum; PoxnΔM22-B5-Full1 responses were similar to those of control flies. Thus, the effects of pheromone exposure appear to be mediated by taste perception through gustatory neurons outside of the mouthparts (Gendron, 2013).

    To identify specific gustatory receptors and neurons that might mediate the pheromone effects, candidate pheromone receptors were tested. Of the mutants that were examined, only flies that carried a loss of function mutation in the gene pickpocket 23 (ppk23) were resistant to the effects of pheromone exposure. Further analysis verified that ppk23 was required for the effects of pheromone exposure on starvation resistance, TAG amounts, and life span. Silencing ppk23-expressing neurons only during exposure to donor males by expressing a temperature-sensitive dominant negative allele of the dynamin gene shibire (via ppk23-GAL4; UAS-shits) also eliminated the differential response to pheromones. In male Drosophila, the transcription factor fruitless (fru) is expressed with ppk23 in pheromone-sensing neurons located in the animals' forelegs, and silencing fru-expressing neurons during exposure (via fru-GAL4;UAS-shits) abrogated pheromone effects. Consistent with a requirement for these neurons, it was found that surgical amputation of the forelegs, but not injury alone, was sufficient to reproducibly eliminate the effects of pheromone exposure. Moreover, acute, targeted activation of ppk23-expressing neurons using a temperature-sensitive TRPA1 channel (ppk23-GAL4;UAS-TRPA1) was sufficient to mimic the effects of female pheromone without exposure. Together, these data indicate that pheromone-sensing neurons in the foreleg of the male fly that express the gustatory receptor, ppk23, and the transcription factor, fruitless, influence stress resistance, physiology, and life span in response to perception of female pheromones (Gendron, 2013).

    To examine brain circuits that may function in transducing pheromone perception, UAS-shits was selectively expressed to block synaptic transmission in various neuro-anatomical regions with the goal of disrupting the physiological effects of donor pheromone exposure. The effects were abrogated when UAS-shits was driven in neurons characterized by expression of neuropeptide F (NPF, as represented by npf-GAL4). Further analysis verified that pheromone-induced changes in starvation resistance and TAG abundance were lost following silencing of npf-expressing neurons. Consistent with a possible role in transducing pheromone information, npf expression was significantly increased by 30% in experimental males after exposure to feminized donor males, and activation of npf-expressing neurons was sufficient to decrease life span in the absence of pheromone exposure (Gendron, 2013).

    NPF may function as a mediator of sexual reward in Drosophila, and its mammalian counterpart, neuropeptide Y (NPY), has been associated with sexual motivation and psychological reward. Tests were performed to see whether the effects of pheromone perception might be rescued by allowing males to successfully mate with females. Neither a small number of conjugal visits with virgin females nor housing with wild-type females in a 1:1 ratio was sufficient to ameliorate the effects of pheromone exposure. In this context, decreased longevity may be a consequence of pheromone perception and not of mating itself. Male Drosophila are willing and able to copulate up to five times in rapid succession before requiring a refractory period. It was found that supplementing donor cohorts with an excess of mating females (in a 5:1 ratio) was sufficient to significantly reduce the effects on mortality and TAG caused by female donor pheromone early in life. The benefits of mating on age-specific mortality decreased with age, suggesting that aging may reduce mating efficiency or may diminish effective mating reward (Gendron, 2013).

    To identify how sexual perception and reward may alter physiological responses in peripheral tissues, changes in gene expression were examined using whole-genome RNA-seq technology. 195 genes were found with significantly different expression (using an experiment-wise error rate of 0.05) in control male flies that were exposed to feminized or control donor males for 48 hours. Nearly all (188/195 = 96%) of the changes appeared to be due to pheromone perception because they were not observed in identical experiments using ppk23 mutant flies. Males exposed to female pheromones decreased transcription of genes encoding odorant-binding proteins and increased transcription of several genes with lipase activity. A significant enrichment was observed in secreted molecules, which includes genes encoding proteins mediating immune- and stress-responses. Many of these genes and pathways were highlighted in a recent meta-analysis of gene expression changes in response to stress and aging (Gendron, 2013).

    Activities of insulin and target of rapamycin (TOR) signaling, which modulate aging across taxa, increase sexual attractiveness in flies. The current demonstration that perception of sexual characteristics is sufficient to modulate life span and physiology suggests aging pathways in one individual may modulate health and life span in another. These types of indirect genetic effects have the potential to be influential agents of natural selection, suggesting that expectation/reward imbalance may have broad effects on health and physiology in humans and may present a potent evolutionary force in nature (Gendron, 2013).

    Excitation and inhibition onto central courtship neurons biases mate choice

    The ability to distinguish males from females is essential for productive mate selection and species propagation. Recent studies in Drosophila have identified different classes of contact chemosensory neurons that detect female or male pheromones and influence courtship decisions. This study examined central neural pathways in the male brain that process female and male pheromones using anatomical, calcium imaging, optogenetic, and behavioral studies. Sensory neurons were found that detect female pheromones, but not male pheromones, activate a novel class of neurons in the ventral nerve cord to cause activation of P1 neurons, male-specific command neurons that trigger courtship. In addition, sensory neurons that detect male pheromones, as well as those that detect female pheromones, activate central mAL neurons to inhibit P1. These studies demonstrate that the balance of excitatory and inhibitory drives onto central courtship-promoting neurons controls mating decisions (Kallman, 2015).

    Reported Drosophila courtship song rhythms are artifacts of data analysis

    In a series of landmark papers, Kyriacou, Hall, and colleagues reported that the average inter-pulse interval of Drosophila melanogaster male courtship song varies rhythmically (KH cycles), that the period gene controls this rhythm, and that evolution of the period gene determines species differences in the rhythm's frequency. Several groups failed to recover KH cycles, but this may have resulted from differences in recording chamber size. Using recording chambers of the same dimensions as used by Kyriacou and Hall, this study found no compelling evidence for KH cycles at any frequency. By replicating the data analysis procedures employed by Kyriacou and Hall, this study found that two factors (data binned into 10-second intervals and short recordings) imposed non-significant periodicity in the frequency range reported for KH cycles. Randomized data showed similar patterns. All of the results related to KH cycles are likely to be artifacts of binning data from short songs. Reported genotypic differences in KH cycles cannot be explained by this artifact and may have resulted from the use of small sample sizes and/or from the exclusion of samples that did not exhibit song rhythms (Stern, 2014).

    Experimental and statistical reevaluation provides no evidence for Drosophila courtship song rhythms

    From 1980 to 1992, a series of influential papers reported on the discovery, genetics, and evolution of a periodic cycling of the interval between Drosophila male courtship song pulses. The molecular mechanisms underlying this periodicity were never described. To reinitiate investigation of this phenomenon, automated segmentation of songs has been performed, but that study failed to detect the proposed rhythm. Various studies have reported that the previous study failed to detect song rhythms because (i) the flies did not sing enough and (ii) the segmenter did not identify many of the song pulses. Another study manually annotated a subset of the previous recordings and reported that two strains displayed rhythms with genotype-specific periodicity, in agreement with the original reports. Attempts to replicate this finding have failed and show that the manually annotated data, the original automatically segmented data, and a new dataset provide no evidence for either the existence of song rhythms or song periodicity differences between genotypes. Furthermore, the methods and analysis were reexamined and it was found that the automated segmentation method was not biased to prevent detection of putative song periodicity. It is concluded that there is no evidence for the existence of Drosophila courtship song rhythms (Stern, 2017).

    Failure to reproduce period-dependent song cycles in Drosophila is due to poor automated pulse-detection and low-intensity courtship

    Stern, 2014 (BMC Biol 12:38) has criticized a body of work from several groups that have independently studied the so-called "Kyriacou and Hall" courtship song rhythms of male Drosophila melanogaster, claiming that these ultradian approximately 60-s cycles in the interpulse interval (IPI) are statistical artifacts that are not modulated by mutations at the period (per) locus. This study has scrutinized Stern's raw data and observed that his automated song pulse-detection method identifies only approximately 50% of the IPIs found by manual (visual and acoustic) monitoring. This critical error is further compounded by Stern's use of recordings with very little song, the large majority of which do not meet the minimal song intensity criteria which Kyriacou and Hall used in their studies. Consequently most of Stern's recordings only contribute noise to the analyses. Of the data presented by Stern, only perL and a small fraction of wild-type males sing vigorously, so this study limited reanalyses to these genotypes. Stern's raw song recordings were manually reexamined, and IPI rhythms were analyzed using several independent time-series analyses. It was observed that perL songs show significantly longer song periods than wild-type songs, with values for both genotypes close to those found in previous studies. These per-dependent differences disappear when the song data are randomized. It is conclude that Stern's negative findings are artifacts of his inadequate pulse-detection methodology coupled to his use of low-intensity courtship song records (Kyriacou, 2017).

    Multimodal chemosensory circuits controlling male courtship in Drosophila
    Throughout the animal kingdom, internal states generate long-lasting and self-perpetuating chains of behavior. In Drosophila, males instinctively pursue females with a lengthy and elaborate courtship ritual triggered by activation of sexually dimorphic P1 interneurons. P1 interneurons, located in the dorsal posterior brain near the mushroom body, is composed of 20 interneurons, each of which has a primary transversal neurite with extensive ramifications in the bilateral protocerebrum. P1 is fated to die in females through the action of a feminizing protein, DsxF. A masculinizing protein Fru is required in the male brain for correct positioning of the terminals of P1 neurites. Gustatory pheromones are thought to activate P1 neurons but the circuit mechanisms that dictate their sensory responses to gate entry into courtship remain unknown. This study used circuit mapping and in vivo functional imaging techniques to trace gustatory and olfactory pheromone circuits to their point of convergence onto P1 neurons and reveals how their combined input underlies selective tuning to appropriate sexual partners. Inhibition, even in response to courtship-promoting pheromones, was identified as a key circuit element that tunes and tempers P1 neuron activity. The results suggest a circuit mechanism in which balanced excitation and inhibition underlie discrimination of prospective mates and stringently regulate the transition to courtship in Drosophila (Clowney, 2015).

    A Wolbachia-sensitive communication between male and female pupae controls gamete compatibility in Drosophila

    Gamete compatibility is fundamental to sexual reproduction. Wolbachia are maternally inherited endosymbiotic bacteria that manipulate gamete compatibility in many arthropod species. In Drosophila, the fertilization of uninfected eggs by sperm from Wolbachia-infected males often results in early developmental arrest. This gamete incompatibility is called cytoplasmic incompatibility (CI). CI is highest in young males, suggesting that Wolbachia affect sperm properties during male development. This study shows that Wolbachia modulate testis development. Unexpectedly, this effect was associated with Wolbachia infection in females, not males. This raised the possibility that females influenced testis development by communicating with males prior to adulthood. Using a combinatorial rearing protocol, evidence is provided for such a female-to-male communication during metamorphosis. This communication involves the perception of female pheromones by male olfactory receptors. This communication determines the compatibility range of sperm. Wolbachia interfere with this female-to-male communication through changes in female pheromone production. Strikingly, restoring this communication partially suppressed CI in Wolbachia-infected males. A reciprocal male-to-female communication at metamorphosis was identified that restricts the compatibility range of female gametes. Wolbachia also perturb this communication by feminizing male pheromone production. Thus, Wolbachia broaden the compatibility range of eggs, promoting thereby the reproductive success of Wolbachia-infected females. It is concluded that pheromone communication between pupae regulates gamete compatibility and is sensitive to Wolbachia in Drosophila (Pontier, 2015).

    This study explores how Wolbachia manipulate gamete compatibility during development in Drosophila. Two key conclusions can be drawn from this work. First, gamete compatibility is regulated through a novel pheromone-mediated communication occurring between males and females during metamorphosis. Second, Wolbachia manipulate gamete compatibility by perturbing pheromone production in males and females, thereby preventing proper between-sex communication (Pontier, 2015).

    Pheromone perception modulates social behavior and reproduction across taxa. Sexual pheromones—the pheromones that are produced by an individual to attract or influence individuals from the opposite sex-elicit innate rituals, decisions, and learning processes that determine mate choice and copulation in sexually mature adults. These pheromones are usually considered to mediate between-sex communication upon adult emergence. Thus, the observations that gamete compatibility depends on a communication between male and female pupae constitute the first indication that a between-sex communication occurs prior to adulthood. Importantly, this communication appears to provoke an adaptive process, which controls the properties of gametes, akin to a sexual imprinting phenomenon. These results also raise the possibility that some of the sexual properties of young adults that are usually considered as innate might in fact be acquired (Pontier, 2015).

    Further work will be required to fully understand the mechanisms underlying the plasticity of the sexual development of both females and males. The results indicate that in the case of the males, the plasticity observed at the testis level is induced by female pheromones and depends on male olfactory receptors expressed in neurons, presumably in antennae. Future studies are needed to link pheromone reception at the level of olfactory neurons to testis development (in males) and molecular control of gamete compatibility (in both sexes) (Pontier, 2015).

    The identification of a between-sex communication during metamorphosis in laboratory conditions also questions the relevance of this communication in wild populations. The observation that in nature, larvae tend to aggregate in close vicinity to each other around pupariation in several Drosophila species suggests that communication between males and females might also occur during development in wild populations. Given, however, that rearing laboratory conditions tend to select for the animals that are less sensitive to overcrowding and to high concentrations of pheromones in their environment, it is speculation that this communication will importantly influence the biology and the evolution of wild Drosophila populations (Pontier, 2015).

    In the cases in this study, it was observed that male-to-female and female-to-male communications had very distinct biological outcomes. Indeed, female pheromones appeared beneficial to male reproduction while the presence of males limited the compatibility range of eggs and thereby the fertility of females. This situation whereby a phenomenon benefits the reproductive potential of males is reminiscent of situations of sexual conflict. This also suggests that communication between pupae could significantly influence the gene flow of a given population. This also implies that any perturbations in the ability of males to communicate with females during their respective development, as this study observed, for example, upon Wolbachia infection, should directly affect this gene flow and hence influence the evolutionary trajectory of populations. Overall, these results illustrate how the influence of sexual pheromones extends far beyond the regulation of pre-mating and courting events, starting with the proper development and production of compatible gametes of the future mating partners (Pontier, 2015).

    The key finding that Wolbachia can regulate communication between male and female pupae also has important consequences to understanding of how these bacteria manipulate reproduction in Drosophila. In the modification-rescue model for CI, the ability of Wolbachia-infected males to successfully mate with Wolbachia-infected females while being incapable to mate with uninfected females is explained according to a two-component system: (1) some 'modifications' induced by the bacteria in sperm and (2) some 'rescuing modifications' induced by the bacteria within eggs. The results obtained in D. melanogaster and D. simulans suggest a significantly different model whereby Wolbachia manipulate gamete compatibility by perturbing the pheromone production of both females and males. Thus, Wolbachia infection prevents females from inducing sperm 'modifications' in males and limits the capacity of males to restrict the egg compatibility range of females. Since pheromone production varies with metabolic state, the model could account for the high sensitivity of Wolbachia-induced CI to environmental factors, including nutrition and overcrowding (Pontier, 2015).

    This study experimentally manipulated pheromone signaling only during metamorphosis. This is because the combinatorial rearing protocol started with LL3. Whether such male-female communication also takes place at larval stages remains to be tested. If so, this might explain why separating D. simulans wRi-infected males from their female siblings at late larval stages, i.e., after the onset of male-female communication, had only a significant but limited impact on the level of CI (Pontier, 2015).

    Finally, the data support the idea that Wolbachia can feminize the pheromone production of male pupae. While Wolbachia can fully or partially feminize several arthropod species, no such case was reported in Drosophila. Nevertheless, it is noted that Wolbachia was reported to suppress the defective female fertility associated with mutations in the sex determination gene Sex lethal (Pontier, 2015).

    In conclusion, this study provides the first indication that gamete compatibility is regulated in Drosophila through a Wolbachia-sensitive communication between male and female pupae. These findings led to the proposal of a novel model for the regulation of Wolbachia-induced CI. They also have important practical consequences. Indeed, several strategies currently developed to control pest insect species rely on the costly production and release of massive amount of sterile adult males that were grown separated from their female siblings. These males often exhibit delayed sexual maturity and poor mating performances compared to wild-type males. The results strongly suggest that providing female pheromones during the development of these males might enhance their sexual maturation and their reproductive potential and should thereby improve the efficiency of pest control strategies (Pontier, 2015).

    Central neural circuitry mediating courtship song perception in male Drosophila

    Animals use acoustic signals across a variety of social behaviors, particularly courtship. In Drosophila, song is detected by antennal mechanosensory neurons and further processed by second-order aPN1/aLN(al) neurons. However, little is known about the central pathways mediating courtship hearing. This study identified a male-specific pathway for courtship hearing via third-order ventrolateral protocerebrum Projection Neuron 1 (vPN1) neurons and fourth-order pC1 neurons. Genetic inactivation of vPN1 or pC1 disrupts song-induced male-chaining behavior. Calcium imaging reveals that vPN1 responds preferentially to pulse song with long inter-pulse intervals (IPIs), while pC1 responses to pulse song closely match the behavioral chaining responses at different IPIs. Moreover, genetic activation of either vPN1 or pC1 induced courtship chaining, mimicking the behavioral response to song. These results outline the aPN1-vPN1-pC1 pathway as a labeled line for the processing and transformation of courtship song in males (Zhou, 2015).

    Courtship behavior of Drosophila males provides a fundamental model for understanding how species-specific courtship signals may be processed and integrated to drive stereotyped motor outputs. Using anatomical, behavioral, and physiological approaches, this study outlines a male-specific pathway for courtship hearing, which processes and transforms song stimuli to activate central fruM+ or dsx+ neurons that support multimodal integration and drive courtship behavior (Zhou, 2015).

    fru and dsx are two key transcription factors with restricted expression patterns that specify the potential for sexual behaviors in Drosophila. fruM expression in primary auditory, tactile, gustatory, and visual neurons as well as the central brain may suggest that there are multiple fruM-labeled pathways conveying and integrating diverse sensory signals related to courtship, and an appealing hypothesis is that fruM labels interconnected neurons in a circuit that is dedicated to courtship. For example, the male-specific pheromone cVA is processed by a four-neuron pathway extending from sensory neurons through to the ventral nerve cord. This circuit appears to function as an olfactory labeled line, in that neurons in this circuit are functionally connected and selectively responsive to cVA (Zhou, 2015).

    This study has focused on elucidating the auditory pathway underlying courtship song perception. The aPN1-vPN1-pC1 pathway is a labeled line for courtship hearing, by fulfilling four criteria: (1) these neurons are functionally connected; (2) these neurons respond preferentially to courtship song; (3) these neurons are necessary for the behavioral response to courtship song in male flies, and (4) activation of this labeled line provides a fictive stimulus, observable by the chaining response elicited upon CsChrimson activation. Strikingly, this labeled line appears to be specified by the expression of fruM or dsx (Zhou, 2015).

    The auditory labeled line for courtship hearing begins with fruM-expressing JONs and second-order auditory neurons aPN1/aLN(al) in the antennal mechanosensory and motor center (AMMC). Silencing either fruM JONs or fruM aPN1 neurons reduced male song-induced responses. In addition, this study has also demonstrated the connectivity, response patterns, necessity, and sufficiency of fruM vPN1 and pC1 neurons in this pathway, thus, delineating a labeled line of fruM neurons leading directly from sensory neuron to multimodal integration (Zhou, 2015).

    The inclusion of vPN1 in this pathway is supported by three lines of evidence. First, vPN1 projections extensively overlap with projections extended by aPN1 neurons within WED, and functional connectivity was observed between aPN1 and vPN1. Second, silencing vPN1 reduced pulse song-induced chaining in male flies, while optogenetic activation of vPN1 neurons mimicked a song signal to induce male chaining. Third, GCaMP recordings reveal that vPN1 responds strongly to both pulse song and sine song. It is therefore concluded that fruM+ vPN1 neurons are the third-order neurons mediating courtship hearing (Zhou, 2015).

    vPN1 may provide its output via innervation of the lateral protocerebrum (LPC), a region receiving multimodal input that is likely to be a site for multi-sensory integration. This area is heavily innervated by dsx+ pC1 neurons, which include most of the male-specific fruM+ P1 neuron. While the broader pC1 population is important for both male courtship and female receptivity, the P1 neurons play a critical role in the initiation of male courtship and respond to both male and female pheromones. These neurons appear to be the downstream targets of vPN1, based on three lines of evidence. First, the arborizations of pC1 neurons match very closely with the projection of vPN1 neurons in the LPC, and optogenetic activation of vPN1 generates robust activity in pC1. Second, pC1 neurons show calcium responses to pulse song stimuli, with IPI tuning that matches that of the behavioral response. Third, silencing pC1 neurons in male flies almost completely abolishes song-induced chaining, while activation induces robust chaining in the absence of song. It is therefore concluded that vPN1 may carry song stimuli to activate pC1, where these stimuli are integrated with other sensory modalities such as pheromonal olfactory and gustatory cues to modulate the courtship level in males (Zhou, 2015).

    Taken together, the neural circuit identified in this study suggests that song information flows via a labeled line of fruM neurons from the antenna to AMMC, to WED, and then to LPC, providing a functional explanation of how pulse song induces male courtship behavior (Zhou, 2015).

    IPI is a key parameter of courtship song that exhibits great variation across Drosophila species. D. melanogaster not only produces song with a specific IPI, but also behaviorally recognizes song with that conspecific IPI in both males and females. This study also shows that song-induced male-chaining behavior is most responsive to a 35-ms IPI, although longer IPIs (35–65 ms) are still able to induce robust chaining behavior (Zhou, 2015).

    While Drosophila has behavioral preferences toward the conspecific IPI, it has not been clear how IPIs are represented in the nervous system or how the fly discriminates specific IPIs. The results suggest there is a significant change in pulse song representation across the ascending aPN1-vPN1-pC1 pathway. For aPN1, the GCaMP ΔF/F responses in female flies reflect an integration of pulse rate at IPIs longer than 25 ms. In contrast, vPN1 responses observed here are low-passed and preferentially tuned to longer IPIs. Interestingly, the vPN1 response saturates above ∼35-ms IPI, consistent with the saturating response observed when comparing dendritic and axonal GCaMP signals in aPN1. Notably, however, neither the aPN1 nor vPN1 response corresponds well with the behavioral sensitivity to IPI observed in male or female flies (Zhou, 2015).

    In contrast, the IPI sensitivity of pC1 reflects a band-pass response to IPI that closely matches the behavioral sensitivity of the chaining response. Indeed, the correlation between pC1 response and chaining behavior is significantly higher than the correlation observed for vPN1. Thus, while the mechanistic details remain unclear, the IPI sensitivity appropriate for species-appropriate responses is likely to be generated through a multi-stage transformation of song stimuli (Zhou, 2015).

    Sexual dimorphism at multiple levels in the Drosophila brain may give rise to sex-specific differences in sensory processing and multimodal integration. The central integrators of courtship-related sensory cues in male and female flies, the pC1 neurons, are themselves sexually dimorphic in both cell number and morphology. pC1 neurons arborize within the triangular lateral junction of the LPC in both sexes, where integration of multiple sensory modalities may occur, but they also show male-specific innervation of the LPC arch and male-specific contralateral projections (Zhou, 2015).

    For courtship hearing, pC1 neurons are stimulated by pulse song in both sexes, but are also stimulated by sine song in females. This result is consistent with the behavioral observation that both males and females are responsive to pulse song, while females are also responsive to sine song. However, the pC1 auditory response cannot be easily explained by the dimorphism of vPN1, which responds to pulse song in males but is absent in females. Moreover, the absence of vPN1 in females begs the question of how pC1 receives song information in females. One explanation comes from the observation that vPN1 is a subset of the fru+ aSP-k clone. aSP-k shows arborization in VLP and the LPC ring in both male and females, as well as male-specific innervation of the LPC arch that corresponds with vPN1 morphology. These neurons, including non-fru+ neurons in the same lineage, may compose a parallel pathway for female hearing (Zhou, 2015).

    More generally, this study observed a gradient of sexual dimorphism across the ascending pathway for both olfaction and audition. In both cases, only limited sexual dimorphism was noticed in second-order neurons (DA1 and aPN1, respectively), but dramatic changes in third-order neurons (aSP-f/aSP-g and vPN1) and integrative neurons (pC1), which show significant dimorphisms in cell number and morphology. This gradient may reflect a general rule for the flexible assembly of sexually dimorphic circuits on an evolutionary timescale (Zhou, 2015).

    These anatomical, behavioral, and physiological analyses have outlined the architecture of a system supporting species-specific courtship hearing, built upon genetically labeled lines expressing fruM or dsx within the fly. Although it is clear that courtship song representations are systematically transformed along the aPN1-vPN1-pC1 pathway, a circuit and synapse-level explanation for how this occurs, as well as an understanding of how pC1 activation gives rise to distinct and appropriate behavioral outputs in each sex, requires additional study (Zhou, 2015).

    Obp56h modulates mating behavior in Drosophila melanogaster

    Social interactions in insects are driven by conspecific chemical signals that are detected via olfactory and gustatory neurons. Odorant binding proteins (Obps) transport volatile odorants to chemosensory receptors, but their effects on behaviors remain poorly characterized. This study reports that RNAi knockdown of Obp56h gene expression in Drosophila melanogaster enhances mating behavior by reducing courtship latency. The change in mating behavior that results from inhibition of Obp56h expression is accompanied by significant alterations in cuticular hydrocarbon (CHC) composition, including reduction in 5-tricosene (5-T), an inhibitory sex pheromone produced by males that increases copulation latency during courtship. Whole genome RNA sequencing confirms that expression of Obp56h is virtually abolished in Drosophila heads. Inhibition of Obp56h expression also affects expression of other chemoreception genes, including upregulation of lush in both sexes and Obp83ef in females, and reduction in expression of Obp19b and Or19b in males. In addition, several genes associated with lipid metabolism, which underlies the production of cuticular hydrocarbons, show altered transcript abundances. These data show that modulation of mating behavior through reduction of Obp56h is accompanied by altered cuticular hydrocarbon profiles and implicate 5-T as a possible ligand for Obp56h (Shorter, 2016).

    Juvenile hormone is required in adult males for Drosophila courtship

    Juvenile Hormone (JH) has a prominent role in the regulation of insect development. Much less is known about its roles in adults, although functions in reproductive maturation have been described. In adult females, JH has been shown to regulate egg maturation and mating. To examine a role for JH in male reproductive behavior, this study generated males with reduced levels of Juvenile Hormone Acid O-Methyl Transferase (JHAMT) and tested them for courtship. JHAMT regulates the last step of JH biosynthesis in the Corpora Allata (CA), the organ of JH synthesis. Males with reduced levels of JHAMT show a reduction in courtship that can be rescued by application of Methoprene, a JH analog, shortly before performing the courtship assays. In agreement with this, reducing JHAMT conditionally in mature flies leads to courtship defects that are rescuable by Methoprene. The same result is also observed when the CA are conditionally ablated by the expression of a cellular toxin. These findings demonstrate that JH plays an important physiological role in the regulation of male mating behavior (Wijesekera, 2016).

    Neuromodulation of courtship drive through tyramine-responsive neurons in the Drosophila brain

    Neuromodulators influence the activities of collections of neurons and have profound impacts on animal behavior. Male courtship drive is complex and subject to neuromodulatory control. Using the fruit fly Drosophila melanogaster, this study identified neurons in the brain (inferior posterior slope; IPS) that impact courtship drive and are controlled by tyramine-a biogenic amine related to dopamine, whose roles in most animals are enigmatic. A tyramine-specific receptor, TyrR, which is expressed in IPS neurons, was knocked out. Loss of TyrR leads to a striking elevation in courtship activity between males. This effect occurrs only in the absence of females, as TyrRGal4 mutant males exhibit a wild-type preference for females. Artificial hyperactivation of IPS neurons causes a large increase in male-male courtship, whereas suppression of IPS activity decreases male-female courtship. The study concludes that TyrR is a receptor for tyramine, and suggests that it serves to curb high levels of courtship activity through functioning as an inhibitory neuromodulator (Huang, 2016).

    Neurotransmitters and neuromodulators that are derived from tyrosine are evolutionarily conserved, and are critical mediators of animal behavior. Dopamine and the related catecholamine norepinephrine are synthesized through a simple pathway that begins with conversion of tyrosine into dihydroxyphenylalanine (DOPA). Tyrosine is also a substrate for production of octopamine, which is structurally similar to norepinephrine. Octopamine is produced in both mammals and invertebrates, although its role as a neuromodulator and neurotransmitter is best characterized in insects, where it promotes an array of behaviors. These range from male aggression to learning and memory in flies, female post-mating behaviors, sleep, foraging, and others (Huang, 2016).

    The biosynthesis of octopamine is initiated by decarboxylation of tyrosine to produce tyramine, which is present at low levels in many mammalian tissues, including the brain. Due to its concentration in trace amounts, it has long been thought to serve primarily as a biosynthetic precursor of octopamine, and not as a neuroactive chemical in its own right. Nevertheless, the discovery of a specific family of G protein-coupled receptors (GPCRs), some members of which are activated primarily by tyramine, raises the possibility that tyramine may function independently as a neuromodulator. Indeed, the concentration of tyramine is altered in a variety of human neurological disorders, including schizophrenia, Parkinson's disease, attention deficit hyperactivity disorder, Tourette syndrome, and phenylketonuria. Nevertheless, the functions of tyramine are enigmatic, especially in mammals (Huang, 2016).

    The brains of the fruit fly harbor populations of neurons that produce tyramine, and not octopamine, arguing against a trivial role for tyramine exclusively as a metabolic intermediate. A few experiments in insects address this possibility. For example, a Drosophila mutation affecting a receptor for both octopamine and tyramine (Oct-TyrR) results in reduced odor avoidance (Kutsukake, 2000). However, it is unclear whether the phenotype reflects a role for octopamine or tyramine, because Oct-TyrR is activated by tyramine and octopamine with similar potency. Application of tyramine to Drosophila tissue, or injections of tyramine into the blowfly or moth, produces a variety of physiological responses. However, the tyramine might be metabolically converted to other biogenic amines that elicit function. At this time, there is no clear genetic evidence indicating a role for tyramine as an independent neuromodulator in Drosophila. Despite the presence of tyramine in the brains of animals that include mammals and insects, Caenorhabditis elegans is the only organism for which genetic evidence supports a role of tyramine as a neuromodulator (Huang, 2016).

    The Drosophila genome encodes multiple GPCRs that are activated by biogenic amines, one of which (TyrR) is activated specifically by tyramine, but not by the other biogenic amines tested, including octopamine, dopamine, serotonin, and histamine. This study has generated a null mutation in TyrR and found that the mutant males displayed a profound increase in male-male courtship but no change in gender preference. TyrR was expressed and functioned in a set of tyramine-responsive neurons in the Drosophila brain called the inferior posterior slope (IPS). Genetic hyperactivation of IPS neurons induced a significant elevation in male-male courtship, similar to the mutant males. Conversely, inactivation of these neurons decreased male-female courtship. It is concluded that basal IPS activity is required to permit sufficient levels of sexual drive for male-female courtship. In addition, through activation of TyrR, it is suggested that tyramine serves as an inhibitory neuromodulator to reduce sexual drive (Huang, 2016).

    Nearly all wild-type Drosophila males court and mate with females. However, among wild-type flies, the frequency of male-male courtship is low. Nevertheless, there are multiple mutations that increase male-male courtship. The changes in behavior are typically due to deficits in identifying males, such as occurs upon elimination of male pheromones or the corresponding receptors. This study found that TyrRGal4 mutant males exhibit a dramatic increase in male-male sexual activity. In contrast to previous mutations that increase male-male courtship, TyrRGal4 flies discriminate between male and females. When provided a choice between the two genders, the TyrRGal4 mutants select females at the same high proportion as wild-type males. These results suggest that the strong male-male courtship activity was not due to a deficit in sensing repulsive male pheromones. Furthermore, TyrRGal4 males also exhibited increased courtship toward young and aged females. These phenotypes were due to loss of TyrR and not potential effects of the mini-white transgene, because the TyrR phenotype with a wild-type TyrR transgene. Moreover, heterozygous control males harboring the mini-white gene (TyrR/+) display wild-type levels of courtship behavior. Furthermore, the increased male-male courtship was recapitulated by RNAi knockdown of TyrR. Consistent with the conclusion that tyramine modulates male courtship activity, Tdc2, but not Tβh, mutant males show elevated levels of male-male courtship (Huang, 2016).

    It is proposed that the TyrR-expressing neurons control overall male sexual drive. In support of this concept, suppressing the normal activity of TyrR-expressing neurons in wild-type males significantly reduced male-female courtship behavior. This manipulation slightly reduced male-male courtship behavior in wild-type. However, the effect was not statistically significant, because basal male-male courtship activity was very low. Nevertheless, silencing TyrR+ neurons in TyrRGal4 mutant males eliminated the high male-male courtship activity. Conversely, when the TyrR+ neurons were artificially activated in wild-type males, the animals displayed a strong elevation in male-male courtship behavior. Thus, the dramatic increase in male-male courtship reflected an increase in overall sexual activity, rather than an increase in same-sex preference. Based on GRASP studies, it is suggested that the TyrR+ neurons function through the FruM neural circuits. Thus, the normal low activity of TyrR-positive neurons is permissive for male-female courtship. Higher activity stimulates greater courtship drive such that the animals will also court males, but only if females are not present, because even at artificially elevated levels of activity the males still prefer females if both gender targets are available. This role for TyrR-expressing neurons differs from P1 neurons, which promote distinct behaviors, aggression and courtship, at low and high activity levels, respectively (Huang, 2016).

    The TyrRGal4 phenotype was due to a requirement for tyramine for controlling courtship behavior, because this study found that TyrR-expressing neurons were activated by tyramine, but not octopamine, consistent with in vitro data indicating that TyrR is activated specifically by tyramine (Cazzamali, 2005) Tyramine is most likely acting as a neuromodulator, rather than as a neurotransmitter, because TyrR is a GPCR rather than an ionotropic receptor. In further support of this model, no GRASP signals were detected using the Tdc2-LexA and TyrRGal4, suggesting that the tyramine-producing neurons are not in direct contact with the TyrR-expressing neurons. However, a caveat is that the Tdc2-LexA recapitulates only a subset of the Tdc2 neurons (Huang, 2016).

    A neuromodulator can either be excitatory or inhibitory, depending on the receptor that is activated. The data in this study suggest that as a consequence of activating TyrR, tyramine serves as an inhibitory rather than excitatory neuromodulator, which curbs sexual activity. In favor of this proposal are the genetic activation and inactivation experiments. Artificial stimulation of TyrR-expressing neurons increased male-male courtship, whereas inhibition of these neurons reduced male-female courtship (Huang, 2016).

    The model that TyrR is an inhibitory neuromodulator receptor in vivo is consistent with in vitro studies showing that tyramine reduces the amplitude of excitatory junction potential (EJP) in neuromuscular junctions (Ormerod, 2013; Nagaya, 2002). In vivo Ca2+ imaging results, as well as an in vitro analysis, indicate that TyrR is coupled to Gq, which typically leads to neuronal activation. However, loss of Gq/G11 signaling can increase neuronal activity as well. This could potentially occur through inhibiting glutamate release, gating of a Ca2+ -activated K+ channel, or promiscuous coupling of Gq/G11 -coupled receptors to Gi/o G proteins. In support of this latter possibility, two related Drosophila catecholamine receptors are coupled to both Gq and Gi proteins (Huang, 2016).

    This study found that TyrR activity was required in a small group of neurons (TyrRIPS) in the brain for controlling courtship drive. Tyramine-induced inhibition of TyrRIPS neurons was strictly dependent on TyrR, because the response was eliminated in TyrRGal4 mutant brains. Based on findings using the GRASP technique, it is proposed that TyrRIPS neurons may form synaptic connections with FruM neurons, which regulate courtship. It is proposed that courtship behavior is enhanced by release of acetylcholine from basal or highly activated TyrRIPS neurons. In support of this proposal, TyrRIPS cells expressed ChAT, and knockdown of ChAT in these cells reduced courtship behavior (Huang, 2016).

    In conclusion, the findings show that in Drosophila, tyramine is not simply a biosynthetic intermediate for octopamine. Rather, it has an important function in the neuromodulation of male courtship drive through its specific receptor, TyrR. However, it does not affect gender preference. Given the presence of tyramine as a trace monogenic amine in the mammalian brain, the question arises as to whether tyramine also functions in mammals as a neuromodulator of behavior (Huang, 2016).

    Specialized cells tag sexual and species identity in Drosophila melanogaster

    Social interactions depend on individuals recognizing each other, and in this context many organisms use chemical signals to indicate species and sex. Cuticular hydrocarbon signals are used by insects, including Drosophila, to distinguish conspecific individuals from others. These chemicals also contribute to intraspecific courtship and mating interactions. However, the possibility that sex and species identification are linked by common chemical signalling mechanisms has not been formally tested. This study provides direct evidence that a single compound is used to communicate female identity among flies, and to define a reproductive isolation barrier between Drosophila melanogaster and sibling species. A transgenic manipulation eliminated cuticular hydrocarbons by ablating the oenocytes (see Insect oenocytes: a model system for studying cell-fate specification by Hox genes), specialized cells required for the expression of these chemical signals. The resulting oenocyte-less (oe-) females elicited the normal repertoire of courtship behaviours from males, but were actually preferred over wild-type females by courting males. In addition, wild-type males attempted to copulate with oe- males. Thus, flies lacking hydrocarbons are a sexual hyperstimulus. Treatment of virgin females with the aversive male pheromone cis-vaccenyl acetate (cVA) significantly delayed mating of oe- females compared to wild-type females. This difference was eliminated when oe- females were treated with a blend of cVA and the female aphrodisiac (7Z,11Z)-heptacosadiene (7,11-HD), showing that female aphrodisiac compounds can attenuate the effects of male aversive pheromones. 7,11-HD also was shown to have a crucial role in heterospecific encounters. Specifically, the species barrier was lost because males of other Drosophila species courted oe- D. melanogaster females, and D. simulans males consistently mated with them. Treatment of oe(-) females with 7,11-HD restored the species barrier, showing that a single compound can confer species identity. These results identify a common mechanism for sexual and species recognition regulated by cuticular hydrocarbons (Billeter, 2009).

    D. melanogaster produces hydrocarbons of various chain lengths, including unbranched alkanes, methyl-branched alkanes, alkenes and derivatives thereof. The alkenes are expressed sex-specifically, and have been associated with both sex and species discrimination. Compared to females, males express high levels of the monoalkene (Z)-7-tricosene (7-T), which has been reported to increase females' receptivity to mating attempts. Moreover, 7-T is repulsive to other males and may prevent male-male interactions. In contrast, females produce sex-specific dienes such as (7Z,11Z)-heptacosadiene (7,11-HD) and (7Z,11Z)-nonacosadiene (7,11-ND), which act as aphrodisiac pheromones for D. melanogaster males. Hydrocarbons are strongly associated with sexual recognition, because wild-type males court males that have been genetically modified to express female hydrocarbons, indicating that the mutants are perceived as females hydrocarbons (Billeter, 2009).

    There are still large gaps in knowledge of the functions of individual hydrocarbons and the tissues where these compounds are synthesized. As in other insects, specialized cells called oenocytes, located on the inner surface of the abdominal cuticle, are thought to be the site of hydrocarbon biosynthesis in D. melanogaster. Consistent with this hypothesis, desaturase 1 (desat1), which encodes an enzyme involved in hydrocarbon synthesis, is expressed in Drosophila oenocytes (Marcillac, 2005). Previous studies have demonstrated that genetic feminization of these cells results in production of female hydrocarbons by male flies; however, these and other manipulations have been confounded by the concurrent feminization of cells in many other sexually dimorphic tissues, including the central nervous system. To test the hypothesis that these cells are required for production of chemical signals used in sexual and species recognition, the Gal4-UAS system was used to target transgene expression specifically to the adult oenocytes. An oenocyte Gal4 driver was generated, derived from the regulatory sequence of one of the desat1 promoters (Marcillac, 2005) that is expressed specifically in oenocytes of adult females. The driver is also expressed in the larval oenocytes and in the reproductive organs of adult males. This driver was used to ablate adult oenocytes by inducing expression of the pro-apoptotic gene head involution defective (hid). This approach initially caused lethality in larvae, probably due to the destruction of the larval oenocytes. To circumvent this problem blocked the driver's action was blocked during development using the Tubulin-Gal80ts transgene. Using this method, flies were generated without oenocytes (oe-). Analysis of whole-body hydrocarbon extracts confirmed that both oe- males and females were essentially devoid of these compounds, showing that the oenocytes are necessary for hydrocarbon display in D. melanogaster. The male pheromone cis-vaccenyl acetate (cVA) was unaffected in oe- males because this compound is synthesized in the ejaculatory bulb. The oe- transgenic strain therefore provided a 'blank slate' for evaluating the role of hydrocarbons in intra- and interspecific communication hydrocarbons (Billeter, 2009).

    Sexual behaviour of oe- flies was assayed to test hydrocarbon function during reproduction. Despite the association of hydrocarbon signals and Drosophila courtship, absence of these signals did not alter courtship behaviours per se. The oe- males displayed normal courtship behaviour towards wild-type females, but slightly less intense than control males. However, wild-type females were less receptive to oe- males than control males, with oe- males taking almost four times as long to achieve mating. Thus, hydrocarbons of males do not seem to affect their own courtship behaviour, but rather, influence the receptivity of females to their mating attempts. However, the influence of non-oenocyte cells within the male reproductive organs that may have been affected by the ablation cannot be excluded. Notably, oe- males elicited courtship and copulation attempts from both wild-type males and other oe- males, indicating that oe- males were perceived as females, even though all other male characteristics were present. The vigorous courtship of oe- males by each other resulted in unnatural behaviours such as engaging one another by rotating in a head-to-head orientation, and males attempting copulation with each other's heads. These behaviours were suppressed by treatment of oe- males with synthetic 7-T, confirming the function of 7-T in inhibiting male-male interactions hydrocarbons (Billeter, 2009).

    Wild-type males exhibited normal courtship behaviour towards oe- females, apparently undeterred by the lack of female. However, mating latency was significantly shorter, and when given a choice between an oe- and a control female, wild-type males preferred oe- females. Together, these data indicate that females lacking hydrocarbons are more attractive than those with a normal hydrocarbon profile. This suggests that female hydrocarbons normally act to slow down male mating attempts, facilitating assessment of a potential partner's species and fitness. Thus, any oe- fly, irrespective of its development as female or male, seems to sexually hyperstimulate males. It is hypothesized that hydrocarbons normally act to superimpose sexual identity on an otherwise attractive fly substrate hydrocarbons (Billeter, 2009).

    The results described above suggested that female attractiveness depends on a balance between attraction/stimulation and repulsion/deterrence. This was investigated by treating females with the aphrodisiac compound 7,11-HD, and with cVA, which males transfer to females via the ejaculate to deter further mating attempts by other males. Whereas cVA decreases the probability that females will re-mate, wild-caught females produce offspring from multiple sires, indicating that polyandry is common and that the effect of cVA is not absolute. O- flies were treated with doses of these compounds approximating wild-type levels. The mating latency of wild-type males with oe- females treated with 7,11-HD was not different from that with untreated oe- females, indicating that 7,11-HD alone does not affect attractiveness of oe- females. As expected, treating wild-type females with increasing doses of cVA delayed mating accordingly, and the effect was even more pronounced with oe- females treated with the same doses of cVA. This effect was not due to differences in the rates of release of cVA from the control and oe- flies, as shown by the profiles of cumulative loss of cVA over time for the two genotypes. Instead, the exaggerated effect of cVA on oe- females is consistent with the hypothesis that the aversive effects of this compound are normally moderated by the presence of other hydrocarbons. Indeed, when cVA and 7,11-HD were applied together, the mating latencies of oe- and wild-type females were indistinguishable. Apparently, 7,11-HD mitigated the deterrent effects of cVA. The data suggest that a male's perception of a female's availability is normally regulated by a mixture of attractive and aversive signals. From an evolutionary perspective, the combined effect of a female attractant with a male deterrent may illustrate an instance of post-copulatory sexual conflict in which the attractant solicits additional mates despite the first male's effort to render a female unattractive by marking her with cVA hydrocarbons (Billeter, 2009).

    In addition to mediating conspecific reproductive interactions, the hydrocarbons of female D. melanogaster have an important role in reproductive isolation between species. For example, within the nine species of the melanogaster subgroup, only D. melanogaster, D. sechellia and D. erecta produce female-specific dienes. Females in the rest of the subgroup express the same hydrocarbons as males. Males of species with non-sexually dimorphic hydrocarbons generally do not court females from dimorphic species, indicating that the dienes might act as reproductive isolation barriers between these species groups. Furthermore, males from dimorphic species do not vigorously court females from non-dimorphic species. In contrast, males of all species in the melanogaster subgroup have similar hydrocarbons, including abundant 7-T. Finally, D. melanogaster females lacking hydrocarbons are courted by at least two sibling species, D. simulans and D. mauritiana. The behaviour of males from other species in the melanogaster subgroup towards oe- females was tested, to assess the contribution of oenocytes and hydrocarbons to reproductive isolating mechanisms. D. simulans and D. yakuba were tested as test species because they represent species in which the females lack dienes. D. erecta was included because it differs from D. melanogaster in the pattern of dienes expressed hydrocarbons (Billeter, 2009).

    Males of all three species courted oe- D. melanogaster females, but exhibited limited or no courtship towards control D. melanogaster females. This indicates that oenocytes and their hydrocarbon products are major components of the reproductive isolation barrier, ensuring that courtship and mating attempts are only initiated between conspecifics. It has been proposed that 7,11-HD functions to create this barrier in D. melanogaster. To test this directly, oe- D. melanogaster females and wild-type females from the different species were treated with synthetic 7,11-HD. Treatment suppressed courtship by males of all three species, demonstrating that 7,11-HD alone is sufficient to create a species barrier. Interestingly, D. erecta males were blocked by 7,11-HD, despite the fact that hydrocarbons of D. erecta females include other dienes in common with those of D. melanogaster. Furthermore, D. melanogaster males actively courted D. erecta females, possibly because the diene 7,11-ND is also expressed by D. melanogaster females. D. simulans and D. yakuba females treated with 7,11-HD elicited strong courtship from D. melanogaster males. These results demonstrate the multifunctional role of 7,11-HD as an attractant and/or stimulant for some species and as a deterrent for others hydrocarbons (Billeter, 2009).

    Despite attempting copulation, D. erecta males never mated with oe- females, suggesting that signals other than hydrocarbons are required to induce receptivity in these females. However, within a 24-h period, nearly all oe- D. melanogaster females mated with D. simulans males, whereas no control D. melanogaster females mated with these males. Treatment of oe- females with 7,11-HD completely blocked interspecific mating with D. simulans males, even at a dose five times lower than the amount found in females of wild-type D. melanogaster strain. Similar treatment of D. simulans females with 7,11-HD only delayed mating by D. simulans males. It is hypothesized that 7-T counters the effect of 7,11-HD in D. simulans females. This is because 7-T functions as an aphrodisiac for D. simulans males and is expressed in higher quantities in D. simulans females than in D. melanogaster females. D. simulans males were assayed with oe- females treated with either 7-T alone, or in combination with 7,11-HD. Synthetic 7-T alone induced a slight decrease in mating latency, indicating that 7-T is an attractant for D. simulans males. However, the striking effect of 7-T was to reduce the effect of 7,11-HD in a dose-dependent manner. These data parallel the balancing effect of 7,11-HD on cVA for D. melanogaster males. Thus, this study has demonstrated that female hydrocarbons orchestrate mating both within and between the species. Whereas a single compound such as 7,11-HD may be enough to establish a species barrier, the effect of this compound is moderated by the relative quantity of other signals. Indeed, the effects of 7,11-HD are particularly noteworthy because it functions as an attractant in an intraspecific context, whereas in an interspecific context, it aids in species recognition, thereby placing social communication and speciation on the same continuum hydrocarbons (Billeter, 2009).

    The logic of pheromonal communication in Drosophila seems to be based on a foundation that imparts general attractiveness to a fly. In this study female oenocytes are the primary organ for communicating species and sex identity to males. Others have shown that males use species-specific acoustic tags within their love song for females during courtship. Thus, both acoustic and pheromonal tags establish a context for social interactions by regulating sex and species recognition. Given that individual flies regulate their own hydrocarbon display in accord with their social surroundings, it is plausible that these compounds also function in individual recognition hydrocarbons (Billeter, 2009).

    The role of courtship song in female mate choice in South American Cactophilic Drosophila

    Courtship songs have undergone a spectacular diversification in the Drosophila buzzatii cluster. Accordingly, it has been suggested that sexual selection has played a significant role in promoting rapid diversification, reproductive isolation and speciation. However, there is no direct evidence (i.e., song playback experiments with wingless males) supporting this tenet. Moreover, several studies have showed that the courtship song in the genus Drosophila is not always used in female mate choice decisions, nor plays the same role when it is taken into account. In this vein, this study used an approach that combines manipulative and playback experiments to explore the importance and the role of courtship song in female mate choice in four species of the D. buzzatii cluster and one species of the closely related D. martensis cluster for outgroup comparison. The importance of courtship song in sexual isolation in sympatry was also investigated between the only semi-cosmopolitan species, D. buzzatii, and the other species of the D. buzzatii cluster. This study revealed that the courtship song is used by females of the D. buzzatii cluster as a criterion for male acceptance or influences the speed with which males are chosen. In contrast, it was shown that this characteristic is not shared by D. venezolana, the representative species of the D. martensis cluster. It was also found that the studied species of the D. buzzatii cluster differ in the role that conspecific and heterospecific songs have in female mate choice and in sexual isolation. These findings support the hypothesis that divergence in female preferences for courtship songs has played a significant role in promoting rapid diversification and reproductive isolation in the D. buzzatii cluster. However, evidence from D. venezolana suggests that the use of the courtship song in female mate choice is not a conserved feature in the D. buzzatii complex (Iglesias, 2017).

    Magnetoreception regulates male courtship activity in Drosophila

    The possible neurological and biophysical effects of magnetic fields on animals is an area of active study. This study reports that courtship activity of male Drosophila increases in a magnetic field and that this effect is regulated by the blue light-dependent photoreceptor Cryptochrome (CRY). Naive male flies exhibited significantly increased courtship activities when they were exposed to a >/= 20-Gauss static magnetic field, compared with their behavior in the natural environment (0 Gauss). CRY-deficient flies, cryb and crym, did not show an increased courtship index in a magnetic field. RNAi-mediated knockdown of cry in cry-GAL4-positive neurons disrupted the increased male courtship activity in a magnetic field. Genetically expressing cry under the control of cry-GAL4 in the CRY-deficient flies restored the increase in male courtship index that occurred in a magnetic field. Interestingly, artificially activating cry-GAL4-expressing neurons, which include large ventral lateral neurons and small ventral lateral neurons, via expression of thermosensitive cation channel dTrpA1, also increased the male courtship index. This enhancement was abolished by the addition of the cry-GAL80 transgene. These results highlight the phenomenon of increased male courtship activity caused by a magnetic field through CRY-dependent magnetic sensation in CRY expression neurons in Drosophila (Wu, 2016).

    Odorant responses and courtship behaviors influenced by at4 neurons in Drosophila

    In insects, pheromones function as triggers to elicit complex behavior programs, such as courtship and mating behavior. In most species, the neurons tuned to pheromones are localized in a specific subset of olfactory sensilla located on the antenna called trichoid sensilla. In Drosophila there are two classes of trichoid sensilla, at1 sensilla that contain the dendrites of a single neuron that is specifically tuned to the male-specific pheromone 11-cis vaccenyl acetate (cVA), and at4 sensilla that contain three neurons with relatively poorly defined chemical specificity and function. Using a combination of odorant receptor mutant analysis, single sensillum electrophysiology and optogenetics, this study examined the chemical tuning and behavioral consequences of the three at4 olfactory neuron classes. The results indicate that one class, Or65abc neurons, are unresponsive to cVA pheromone, and function to inhibit courtship behaviors in response to an unknown ligand, while the other two neuron classes, Or88a and Or47b neurons, are sensitive to a diverse array of fly and non-fly odors, and activation of these neurons has little direct impact on courtship behaviors (Pitts, 2016).

    Neural circuitry coordinating male copulation

    Copulation is the goal of the courtship process, crucial to reproductive success and evolutionary fitness. Identifying the circuitry underlying copulation is a necessary step towards understanding universal principles of circuit operation, and how circuit elements are recruited into the production of ordered action sequences. This study identified key sex-specific neurons that mediate copulation in Drosophila, and define a sexually dimorphic motor circuit in the male abdominal ganglion that mediates the action sequence of initiating and terminating copulation. This sexually dimorphic circuit composed of three neuronal classes - motor neurons, interneurons and mechanosensory neurons - controls the mechanics of copulation. By correlating the connectivity, function and activity of these neurons, the logic was determined for how this circuitry is coordinated to generate this male-specific behavior, and sets the stage for a circuit-level dissection of active sensing and modulation of copulatory behavior (Pavlou, 2016).

    Mutations at the Darkener of Apricot locus modulate pheromone production and sex behavior in Drosophila melanogaster

    Mutations at the Darkener of Apricot (Doa) locus of Drosophila melanogaster alter sexual differentiation by disrupting sex-specific splicing of doublesex pre-mRNA, a key regulator of sex determination. This paper examined the effect of seven Doa alleles and several trans-heterozygous combinations on pheromones and courtship behavior . The cuticular hydrocarbon (CHC) profile was slightly masculinized in females, with an accumulation of shorter compounds (C23 and C25) and a reduction in longer compounds (C27 and C29). The profile was feminized in males. Female cuticular profiles showed fewer dienes and female pheromones in six alleles and in the trans-heterozygotes and showed more male pheromones (tricosene and pentacosene) in three alleles (DEM, E786 and HD) and in all trans-heterozygotes. Courtship was severely affected in Doa males; in particular, males made fewer copulation attempts and copulated less with both control and Doa females. These results suggest that Doa could modulate pheromone production and sex behavior by altering sexual differentiation in the cuticle and the nervous system (Fumey, 2017).

    Pleiotropic effects of loss of the Dα1 subunit in Drosophila melanogaster: Implications for insecticide resistance

    Nicotinic acetylcholine receptors (nAChRs) are a highly conserved gene family that form pentameric receptors involved in fast excitatory synaptic neurotransmission. The specific roles individual nAChR subunits perform in Drosophila melanogaster and other insects are relatively uncharacterized. Of the 10 D. melanogaster nAChR subunits, only three have described roles in behavioral pathways; Dα3 and Dα4 in sleep, and Dα7 in the escape response. Other subunits have been associated with resistance to several classes of insecticides. In particular, previous work has demonstrated that an allele of the Dα1 subunit is associated with resistance to neonicotinoid insecticides. This study used ends-out gene targeting to create a knockout of the Dα1 gene to facilitate phenotypic analysis in a controlled genetic background. This is the first report of a native function for any nAChR subunits known to be targeted by insecticides. Loss of Dα1 function was associated with changes in courtship, sleep, longevity, and insecticide resistance. While acetylcholine signaling had previously been linked with mating behavior and reproduction in D. melanogaster, no specific nAChR subunit had been directly implicated. The role of Dα1 in a number of behavioral phenotypes highlights the importance of understanding the biological roles of nAChRs and points to the fitness cost that may be associated with neonicotinoid resistance (Somers, 2017).

    Serotonergic neuronal death and concomitant serotonin deficiency curb copulation ability of Drosophila platonic mutants

    Drosophila platonic (plt) males court females, but fail to copulate. This study shows that plt is an allele of scribbler (sbb), a BMP signalling component. sbb knockdown in larvae leads to the loss of approximately eight serotonergic neurons, which express the sex-determinant protein Doublesex (Dsx). Genetic deprivation of serotonin (5-HT) from dsx-expressing neurons results in copulation defects. Thus, sbb+ is developmentally required for the survival of a specific subset of dsx-expressing neurons, which support the normal execution of copulation in adults by providing 5-HT. This study highlights the conserved involvement of serotonergic neurons in the control of copulatory mechanisms and the key role of BMP signalling in the formation of a sex-specific circuitry (Yilmazer, 2016).

    Mate choice in fruit flies is rational and adaptive

    According to rational choice theory, beneficial preferences should lead individuals to sort available options into linear, transitive hierarchies, although the extent to which non-human animals behave rationally is unclear. This study demonstrates that mate choice in the fruit fly Drosophila melanogaster results in the linear sorting of a set of diverse isogenic female lines, unambiguously demonstrating the hallmark of rational behaviour, transitivity. These rational choices are associated with direct benefits, enabling males to maximize offspring production. Furthermore, female behaviours and cues act redundantly in mate detection and assessment, as rational mate choice largely persists when visual or chemical sensory modalities are impaired, but not when both are impaired. Transitivity in mate choice demonstrates that the quality of potential mates varies significantly among genotypes, and that males and females behave in such a way as to facilitate adaptive mate choice (Arbuthnott, 2017).

    Genomic responses to socio-sexual environment in male Drosophila melanogaster exposed to conspecific rivals

    Socio-sexual environments have profound effects on fitness. Local sex ratios can alter the threat of sexual competition, to which males respond via plasticity in reproductive behaviours and ejaculate composition. In Drosophila melanogaster, males detect the presence of conspecific mating rivals prior to mating using multiple, redundant sensory cues. Males that respond to rivals gain significant fitness benefits by altering mating duration and ejaculate composition. This study investigated the underlying genome-wide changes involved. RNA-seq was used to analyse male transcriptomic responses 2, 26 and 50h after exposure to rivals, a time period that has previously been identified as encompassing the major facets of male responses to rivals. The results show a strong early activation of multiple sensory genes in the head-thorax (HT), prior to the expression of any phenotypic differences. This gene expression response is reduced by 26h, at the time of maximum phenotypic change, and shut off by 50h. In the abdomen (A) fewer genes change in expression and gene expression responses appear to increase over time. The results also suggest that different sets of functionally equivalent genes might be activated in different replicates. This could represent a mechanism by which robustness is conferred upon highly plastic traits. Overall, these data reveal that mRNA-seq can identify subtle genomic signatures characteristic of flexible behavioural phenotypes (Mohorianu, 2017).

    Quantitative analysis of visually induced courtship elements in Drosophila subobscura

    Fly Motion-detector with an Actuator-Coupled Stimulator (FlyMacs), in which the stimulation of a fly with a moving visual target and recording of induced behaviors are automated under computer control, was employed for the identification of motion features that trigger specific courtship elements in Drosophila subobscura. A female abdomen attached to the actuator, when moved in an appropriate pattern, evokes in the test male tapping-like foreleg motions, midleg swing and proboscis extension, which are considered to be elementary actions in male courtship behavior. Tapping is primarily induced when the target is moving, whereas midleg swing and proboscis extension are most frequently observed after the target stops moving. In contrast to midleg swing, which tends to occur immediately after target cessation (approximately 3000 ms), the incidence of proboscis extension gradually increases with time after target cessation, reaching a plateau at 3000 ms. The results suggest that tapping, midleg swing and proboscis extension are each induced by different movement features of the visual target. These findings do not support the view that a single key stimulus induces the entire courtship ritual. Rather, courtship behaviors in D. subobscura are correlated with movement and position of the target, which suggests that D. subobscura uses sensory information to pattern its courtship (Higuchi, 2017).

    Drosophila courtship conditioning as a measure of learning and memory

    Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male's copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to naive flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized in this study. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms (Koemans, 2017).

    The physical environment mediates male harm and its effect on selection in females

    Recent experiments indicate that male preferential harassment of high-quality females reduces the variance in female fitness, thereby weakening natural selection through females and hampering adaptation and purging. It is proposed that this phenomenon, which results from a combination of male choice and male-induced harm, should be mediated by the physical environment in which intersexual interactions occur. Using Drosophila melanogaster, this study examined intersexual interactions in small and simple (standard fly vials) versus slightly more realistic (small cages with spatial structure) environments. In these more realistic environments, sexual interactions are less frequent, are no longer biased towards high-quality females, and that overall male harm is reduced. Next, the selective advantage of high- over low-quality females was examined while manipulating the opportunity for male choice. Male choice weakens the viability advantage of high-quality females in the simple environment, consistent with previous work, but strengthens selection on females in the more realistic environment. Laboratory studies in simple environments have strongly shaped our understanding of sexual conflict but may provide biased insight. These results suggest that the physical environment plays a key role in the evolutionary consequences of sexual interactions and ultimately the alignment of natural and sexual selection (Yun, 2017).

    Methyl-CpG binding domain proteins inhibit interspecies courtship and promote aggression in Drosophila

    Reproductive isolation and speciation are driven by the convergence of environmental and genetic variation. The integration of these variation sources is thought to occur through epigenetic marks including DNA methylation. Proteins containing a methyl-CpG-binding domain (MBD) bind methylated DNA and interpret epigenetic marks, providing a dynamic yet evolutionarily adapted cellular output. This study reports the Drosophila MBD-containing proteins, dMBD-R2 and dMBD2/3, contribute to reproductive isolation and survival behavioral strategies. Drosophila melanogaster males with a reduction in dMBD-R2 specifically in octopamine (OA) neurons exhibit courtship toward divergent interspecies D. virilis and D. yakuba females and a decrease in conspecific mating success. Conspecific male-male courtship is increased between dMBD-R2-deficient males while aggression is reduced. These changes in adaptive behavior are separable as males with a hypermethylated OA neuronal genome exhibited a decrease in aggression without altering male-male courtship. These results suggest Drosophila MBD-containing proteins are required within the OA neural circuitry to inhibit interspecies and conspecific male-male courtship and indicate that the genetically hard-wired neural mechanisms enforcing behavioral reproductive isolation include the interpretation of the epigenome (Gupta, 2017).

    Tissue-specific insulin signaling mediates female sexual attractiveness

    Individuals choose their mates so as to maximize reproductive success, and one important component of this choice is assessment of traits reflecting mate quality. Previous work has shown that global manipulation of insulin signaling, a nutrient-sensing pathway governing investment in survival versus reproduction, affects female sexual attractiveness in Drosophila. This study demonstrates that these effects on attractiveness derive from insulin signaling in the fat body and ovarian follicle cells, whose signals are integrated by pheromone-producing cells called oenocytes. Functional ovaries were required for global insulin signaling effects on attractiveness, and manipulations of insulin signaling specifically in late follicle cells recapitulated effects of global manipulations. Interestingly, modulation of insulin signaling in the fat body produced opposite effects on attractiveness, suggesting a competitive relationship with the ovary. Furthermore, all investigated tissue-specific insulin signaling manipulations that changed attractiveness also changed fecundity in the corresponding direction, pointing to insulin pathway activity as a reliable link between fecundity and attractiveness cues. The cues themselves, cuticular hydrocarbons, responded distinctly to fat body and follicle cell manipulations, indicating independent readouts of the pathway activity from these two tissues. Thus, this study describes a system in which female attractiveness results from an apparent connection between attractiveness cues and an organismal state of high fecundity, both of which are created by lowered insulin signaling in the fat body and increased insulin signaling in late follicle cells (Fedina, 2017).

    SIK3-HDAC4 signaling regulates Drosophila circadian male sex drive rhythm via modulating the DN1 clock neurons

    The physiology and behavior of many organisms are subject to daily cycles. In Drosophila melanogaster the daily locomotion patterns of single flies are characterized by bursts of activity at dawn and dusk. Two distinct clusters of clock neurons-morning oscillators (M cells) and evening oscillators (E cells)-are largely responsible for these activity bursts. In contrast, male-female pairs of flies follow a distinct pattern, most notably characterized by an activity trough at dusk followed by a high level of male courtship during the night. This male sex drive rhythm (MSDR) is mediated by the M cells along with DN1 neurons, a cluster of clock neurons located in the dorsal posterior region of the brain. This study reports that males lacking Salt-inducible kinase 3 (SIK3) expression in M cells exhibit a short period of MSDR but a long period of single-fly locomotor rhythm (SLR). Moreover, lack of Sik3 in M cells decreases the amplitude of Period (Per) cycling in DN1 neurons, suggesting that SIK3 non-cell-autonomously regulates DN1 neurons' molecular clock. This study also shows that Sik3 reduction interferes with circadian nucleocytoplasmic shuttling of Histone deacetylase 4 (HDAC4), a SIK3 phosphorylation target, in clock neurons and that constitutive HDAC4 localization in the nucleus shortens the period of MSDR. Taking these findings together, it is concluded that SIK3-HDAC4 signaling in M cells regulates MSDR by regulating the molecular oscillation in DN1 neurons (Fujii, 2017).

    The physiology and behavior of most animals undergo daily oscillations, which are controlled by a small set of clock neurons in the brain. In mammals, a heterodimeric complex between CLOCK (CLK) and BMAL1 activates transcription of Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes, and their protein products in turn inhibit the activity of CLK/BMAL1. Likewise, Drosophila heterodimeric complexes between CLK and CYCLE (CYC) activate the genes period (per) and timeless (tim), and their respective protein products repress CLK/CYC. These conserved negative-feedback loops, which include several kinases, produce rhythmic transcription profiles in numerous genes (Fujii, 2017).

    The Drosophila brain contains ∼150 clock neurons which are divided into seven clusters based on their anatomical locations and functional characteristics: the small and large ventral lateral neurons (sLNvs and lLNvs), the dorsal lateral neurons (LNds), the lateral posterior neurons (LPNs), and three dorsal neuron clusters (DN1-3). Some of these clock neurons have distinct functions in circadian locomotor behavior. Specifically, four sLNvs, also referred to as 'morning' (M) cells, express the neuropeptide pigment-dispersing factor (PDF) and control the timing of morning locomotor activity during light:dark (LD) cycles; these neurons are also the key pacemaker neurons in constant darkness (DD). The fifth, PDF−, sLNv and the LNds, referred to as 'evening' (E) cells, are required for the generation of the evening activity peak in LD cycles. Communication between various groups of cells within this interconnected neural network enhances the synchrony of molecular oscillation in each neuron (Fujii, 2017).

    Ventral lateral neuron (LNv)-derived PDF plays a critical role in regulating the molecular clock. Specifically, PDF participates in synchronization of clock neurons by up-regulating cAMP, which activates PKA, which in turn regulates the stability of PER and TIM in PDF receptor (PDFR)-expressing target neurons. Thus, ion status and is regu neurosecretory signaling: In well-fed flies, SIK3 is thought to be indirectly activated by insulin-likeavioral rhythms even when flies are kept in DD (Fujii, 2017).

    Locomotor activity is the best-characterized circadian behavior in Drosophila, but numerous other behaviors, such as courtship and mating, sleep, and feeding, are under strong circadian influence. Previously work has shown that male-female pairs of flies exhibit activity patterns strikingly distinct from those of singly kept males or females (i.e., single-fly locomotor rhythm or SLR) or same-sex pairs of flies. The activity pattern of male-female pairs, which is referred to as 'male sex-drive rhythm' (MSDR), is characterized by a trough at subjective dusk, followed by a sharp increase in male-driven courtship activity (especially 'following' behavior) that peaks during the subjective night. A functional molecular clock in both Pdf+ LNvs and DN1 neurons is necessary and sufficient for proper MSDR. However, few other cellular and molecular components contributing to MSDR have been identified to date. Specifically, information is lacking about both the molecular and cellular identity of downstream effectors of the main clock components that are important for MSDR (Fujii, 2017).

    This study reports the identification of two downstream effectors of the molecular clock that play distinct roles in MSDR and SLR. Using an RNAi screen for kinases, this study shows that Salt-inducible kinase 3 (SIK3) is a critical component for circadian behavior. Sik3 knockdown in subsets of clock neurons (DN1 neurons or Pdf+ LNvs) causes a short period of MSDR, whereas the period length of SLR is slightly shortened with Sik3 knockdown in DN1 neurons and is slightly elongated with Sik3 knockdown in sLNvs. This study also found that transcriptional activity of Histone deacetylase 4 (HDAC4) is regulated by SIK3 in a circadian manner. Finally, Sik3 reduction in Pdf+ LNvs reduces the amplitude of PER oscillation in DN1 neurons and shortens the length of the MSDR period, suggesting that SIK3-HDAC4 signaling plays an important role in the determination of MSDR period by modulating the intercellular communication between clock neurons (Fujii, 2017).

    SIK-HDAC (class IIa) signaling is evolutionarily conserved from worm to mammals, operating in a number of tissues, including the nervous system, liver, and muscle. In mice, SIK1-HDAC signaling is important for muscle integrity by regulating the activity of the transcription factor MEF2 (Stewart, 2013). In the fly, SIK3-HDAC4 signaling was shown to control the expression of lipolytic and gluconeogenic genes in the fat body (Choi, 2015). Furthermore, both Drosophila HDAC4 and MEF2 have been implicated in circadian rhythm, as has the related HDAC5 gene in mice. This paper has established a critical role for SIK3 in two circadian behaviors, single-fly locomotor activity and male sex drive, respectively (Fujii, 2017).

    MSDR is mediated through the activity of Pdf+ LNvs and DN1 neurons (Fujii, 2010). This study specifically targeted SIK3 in either group of circadian neurons using RNAi. Strikingly, SIK3 knockdown in LNvs shortened the period length of MSDR but slightly yet reproducibly lengthened that of SLR. The loss of PER rhythm amplitude observed specifically in the DN1 neurons and its apparent phase advance on the second or third day of constant conditions would fit with these observations. The advance would be symptomatic of DN1 neurons free-running with a short period and thus presumably explains the short-period MSDR. The loss of amplitude could indicate that a small subset of DN1 neurons runs at a different pace, perhaps explaining the slightly long period of the SLR. Indeed, both SLR and MSDR depend on sLNvs driving DN1 neurons. The broader M peak might also be an early sign that DN1 neurons are not as coherent, even under LD, because the DN1 neurons function downstream of the sLNvs to control morning anticipatory activity. The same short-period DN1 neurons might drive the M peak and MSDR. Unfortunately, the amplitude of the M peak in DD was too low to be able to determine whether it free-runs with a short period (Fujii, 2017).

    Knockdown of SIK3 in DN1 neurons shortened the period length of MSDR that is well correlated with shortened PER oscillations in DN1 neurons, and these flies show subtle but significantly shortened period length in SLR. Together, these findings suggest that SIK3 is a key component in molecular oscillator coupling between sLNvs and DN1 neurons and that its role is especially important for maintaining an appropriate MSDR period length. However, the possibility cannot be excluded that SIK3 also influences the period of the circadian pacemaker in a neuron-specific manner (i.e., in the DN1 neurons), as was proposed for SGG and CKII. It was also demonstrated that HDAC4 cycles in a SIK3-dependent fashion between the cytoplasm and the nucleus in the M cells (Fujii, 2017).

    Because M-cell restricted overexpression of phosphorylation-defective, constitutively nuclear-located HDAC43A, but not wild-type HDAC4, mimics the phenotype of flies lacking SIK3 in these cells, it is suggested that HDAC4 is a critical component for the transduction of the circadian intercellular signal from M cells to DN1 neurons. However, the function of SIK3 in oscillator coupling is unlikely to be mediated by HDAC4 in DN1 neurons, because most of these neurons do not express HDAC4. Another potential SIK3 phosphorylation target such as CREB or CRTC, which are implicated in cAMP-mediated signaling and the circadian clock, may play a role in the regulation of oscillator coupling in DN1 neurons for MSDR (Fujii, 2017).

    SIK3-dependent circadian shuttling of HDAC4 in sLNvs implies that the activity of SIK3 is under circadian control. How is SIK3 activity regulated in sLNvs? In fat cells (and rat adipocytes) SIK3 activity is dependent on nutrition status and is regulated indirectly through neurosecretory signaling: In well-fed flies, SIK3 is thought to be indirectly activated by insulin-like peptides (ILPs), whereas in starved flies, it is inhibited by adipokinetic hormone (AKH). SIK3 activity itself is regulated via phosphorylation by AKT1 (activated by ILPs) and cAMP-dependent protein kinase A (PKA) (activated by AKH). These kinases target distinct but overlapping sets of serine and threonine residues, and thus it appears that SIK3 activity is dependent on the particular phosphorylation pattern at these sites. Intriguingly, it has been reported that PDF stabilizes PER by increasing cAMP levels and PKA activity in Pdfr+ clock neurons (including M cells) at dawn, a time when HDAC4 is activated and translocated into the nucleus. PDF thus could be an indirect circadian regulator of SIK3 activity via PKA. However, the reduction of PDF in M cells did not shorten the MSDR period length, suggesting that PDF signaling probably does not regulate SIK3. Moreover, RNAi-mediated knockdown of MEF2, which regulates SIK3-HDAC in the mouse, had no effect on MSDR. Future experiments will be needed to investigate how SIK3 activity is regulated and how HDAC4 controls intercellular communications between M cells and DN1 neurons (Fujii, 2017).

    How does the lack of SIK3 in M cells (i.e., sLNvs) alter the robustness of PER cycling in some (DN1 neurons) but not other (sLNvs and LNds) PDFR-expressing clock cells? One possibility might be the manner by which sLNvs communicate with other clock cells. Functional and anatomical studies including GFP Reconstitution Across Synaptic Partners strongly suggest that at least some DN1 neurons are direct downstream targets of sLNvs, and hence accurately timed communication between these neurons likely occurs through synapses, which is proposed to rely on SIK3 function in LNvs. In contrast, autocrine (sLNvs) and paracrine (LNds) communication likely occurs via untargeted release of PDF, a process that is suggested not to be dependent on SIK3. The projections of sLNvs to DN1 neurons, in addition to PDF-containing dense core vesicles, harbor small clear vesicles that house classical neurotransmitters, raising the possibility that communication between sLNvs and DN1 neurons pertinent to the robust amplitude of PER oscillation in DN1 neurons is mediated by an as yet unidentified HDAC4-dependent signal. Moreover, DN1 neurons are probably heterogeneous in function, and thus it is quite likely that only some of these cells respond to the sLNv-derived and SIK3-HDAC4-dependent signal, whereas another either overlapping or entirely distinct group of DN1 neurons is responsive to the sLNv-derived PDF. In this context, it is worth noting that sLNvs also express the small neuropeptide F (sNPF). Moreover, a discrete requirement for both PDF-mediated and classical neurotransmitter signaling has been proposed for distinct aspects of SLR, and glycine in sLNvs was recently proposed to coordinate locomotor behavior and appears either to accelerate or to slow down circadian oscillators in specific neuronal groups. Future studies will be necessary to identify the LNv-derived signal that maintains the appropriate amplitude and speed of the clock in DN1 neurons to coordinate MSDR and SLR (Fujii, 2017).

    It is surprising that the loss of SIK3 in sLNvs results in a long SLR and a short MSDR, whereas the loss of SIK3 in the DN1 neurons shortens both SLR and MSDR, because in either case it appears that the DN1 neurons are disconnected from the sLNvs. One explanation could be that SIK3 is differentially modulated in different subpopulations of DN1 neurons by the sLNv synchronizing cue, thus resulting in DN1 desynchronization in flies lacking SIK3 in the sLNvs. However, when SIK3 is missing in DN1 neurons, they all adopt a short period by default (Fujii, 2017).

    In summary, this work unexpectedly reveals the existence of a SIK3-HDAC4 regulatory pathway that allows the M cells -- the critical circadian pacemaker neurons of the fly brain -- to control specific circadian neurons and behaviors. This pathway could prove particularly important in explaining how circadian behaviors can be differentially modulated in response to environmental conditions or internal states. Indeed, the ability to tune and prioritize specific behaviors in a daily manner to minimize energy expenditure and to maximize fitness and reproductive output is critical for animals. Given the strong neural and molecular homologies between the circadian system of fruit flies and mammals, it will be particularly interesting to determine whether the SIK-HDAC pathway is also active in VIP (vasoactive intestinal polypeptide-expressing) neurons of the mammalian suprachiasmatic nucleus and, if so, whether it also controls specific circadian behaviors (Fujii, 2017).

    Genes involved in sex pheromone discrimination in Drosophila melanogaster and their background-dependent effect

    Mate choice is based on the comparison of the sensory quality of potential mating partners, and sex pheromones play an important role in this process. In Drosophila melanogaster, contact pheromones differ between male and female in their content and in their effects on male courtship, both inhibitory and stimulatory. To investigate the genetic basis of sex pheromone discrimination, this study experimentally selected males showing either a higher or lower ability to discriminate sex pheromones over 20 generations. This experimental selection was carried out in parallel on two different genetic backgrounds: wild-type and desat1 mutant, in which parental males showed high and low sex pheromone discrimination ability respectively. Male perception of male and female pheromones was separately affected during the process of selection. A comparison of transcriptomic activity between high and low discrimination lines revealed genes not only that varied according to the starting genetic background, but varied reciprocally. Mutants in two of these genes, Shaker and quick-to-court, were capable of producing similar effects on discrimination on their own, in some instances mimicking the selected lines, in others not. This suggests that discrimination of sex pheromones depends on genes whose activity is sensitive to genetic context and provides a rare, genetically defined example of the phenomenon known as 'allele flips,' in which interactions have reciprocal effects on different genetic backgrounds.

    Direct and trans-generational effects of male and female gut microbiota in Drosophila melanogaster

    There is increasing evidence of the far-reaching effects of gut bacteria on physiological and behavioural traits, yet the fitness-related consequences of changes in the gut bacteria composition of sexually interacting individuals remain unknown. To address this question, the gut microbiota of fruit flies, Drosophila melanogaster, were manipulated by monoinfecting flies with either Acetobacter pomorum (AP) or Lactobacillus plantarum (LP). Re-inoculated individuals were paired in all treatment combinations. LP-infected males had longer mating duration and induced higher short-term offspring production in females compared with AP-infected males. Furthermore, females of either re-inoculation state mated with AP-infected males were more likely to have zero offspring after mating, suggesting a negative effect of AP on male fertility. Finally, the effects of male and female gut bacteria interacted to modulate their daughters', but not sons' body mass, revealing a new trans-generational effect of parental gut microbiota. In conclusion, this study shows direct and trans-generational effects of the gut microbiota on mating and reproduction (Morimoto, 2017).

    Electrical synapses mediate synergism between pheromone and food odors in Drosophila melanogaster

    In Drosophila melanogaster, the sex pheromone produced by males, cis-vaccenyl acetate (cVA), evokes a stereotypic gender-specific behavior in both males and females. As Drosophila adults feed, mate, and oviposit on food, they perceive the pheromone as a blend against a background of food odors. Previous studies have reported that food odors enhance flies' behavioral response to cVA, specifically in virgin females. However, how and where the different olfactory inputs interact has so far remained unknown. This study has elucidated the neuronal mechanism underlying the response at an anatomical, functional, and behavioral level. The data show that in virgin females cVA and the complex food odor vinegar evoke a synergistic response in the cVA-responsive glomerulus DA1. This synergism, however, does not appear at the input level of the glomerulus, but is restricted to the projection neuron level only. Notably, it is abolished by a mutation in gap junctions in projection neurons and is found to be mediated by electrical synapses between excitatory local interneurons and projection neurons. As a behavioral consequence, this study demonstrated that virgin females in the presence of vinegar become receptive more rapidly to courting males, while male courtship is not affected. Altogether, these results suggest that lateral excitation via gap junctions modulates odor tuning in the antennal lobe and drives synergistic interactions between two ecologically relevant odors, representing food and sex (Das, 2017).

    A Drosophila female pheromone elicits species-specific long-range attraction via an olfactory channel with dual specificity for sex and food

    Mate finding and recognition in animals evolves during niche adaptation and involves social signals and habitat cues. Drosophila melanogaster and related species are known to be attracted to fermenting fruit for feeding and egg-laying, which poses the question of whether species-specific fly odours contribute to long-range premating communication. This study has discovered an olfactory channel in D. melanogaster with a dual affinity to sex and food odorants. Female flies release a pheromone, (Z)-4-undecenal (Z4-11Al), that elicits flight attraction in both sexes. Its biosynthetic precursor is the cuticular hydrocarbon (Z,Z)-7,11-heptacosadiene (7,11-HD), which is known to afford reproductive isolation between the sibling species D. melanogaster and D. simulans during courtship. Twin olfactory receptors, Or69aB and Or69aA, are tuned to Z4-11Al and food odorants, respectively. They are co-expressed in the same olfactory sensory neurons, and feed into a neural circuit mediating species-specific, long-range communication; however, the close relative D. simulans, which shares food resources with D. melanogaster, does not respond to Z4-11Al. The Or69aA and Or69aB isoforms have adopted dual olfactory traits. The underlying gene yields a collaboration between natural and sexual selection, which has the potential to drive speciation (Lebreton, 2017b).

    Male mate choice via cuticular hydrocarbon pheromones drives reproductive isolation between Drosophila species

    Mate discrimination is a key mechanism restricting gene flow between species. While studied extensively with respect to female mate choice, mechanisms of male mate choice between species are far less studied. Thus, there is little knowledge of the relative frequency, importance, or overall contribution of male mate discrimination to reproductive isolation. This study estimated the relative contributions of male and female choice to reproductive isolation between Drosophila simulans and D. sechellia, and showed that male mate discrimination accounts for the majority of the current isolation between these species. It was further demonstrate that males discriminate based on female cuticular hydrocarbon pheromones, and collect evidence supporting the hypothesis that male mate discrimination may alleviate the costs associated with heterospecific courtship and mating. These findings highlight the potentially significant contribution of male mate choice to the formation of reproductive isolating barriers, and thus the speciation process (Shahandeh, 2017).

    Differential effects of male nutrient balance on pre- and post-copulatory traits, and consequences for female reproduction in Drosophila melanogaster.

    Male fitness depends on the expression of costly traits involved in obtaining mates (pre-copulatory) and fertilization (post-copulatory). However, very little is known about the nutrient requirements for these traits and whether males compromise their diet to maximize one trait at the expense of another. This study used Nutritional Geometry to investigate macronutrient requirements for pre- and post-copulatory traits in Drosophila, when males were the first or second to mate with females. No significant effects of male diet on sperm competitiveness were found. However, although males self-regulate their macronutrient intake at a protein-to-carbohydrate ratio ("P:C ratio") of 1:1.5, this ratio does not coincide with their optima for several key reproductive traits: both the short-term (~24 hr) rate of offspring production after a female's first mating, as well as the total offspring number sired when males were second to mate are maximized at a P:C ratio of 1:9, whereas male attractiveness (latency to mate), are maximised at a P:C ratio of 1:1. These results suggest a compromised optimum diet, and no single diet that simultaneously maximizes all male reproductive traits. The protein intake of first males also negatively affected female offspring production following remating, suggesting a long-term intersexual effect of male nutrition (Morimoto, 2016). 

    Male age affects female mate preference, quantity of accessory gland proteins, and sperm traits and female fitness in D. melanogaster

    For species in which mating is resource-independent and offspring do not receive parental care, theoretical models of age-based female mate preference predict that females should prefer to mate with older males as they have demonstrated ability to survive. Thus, females should obtain a fitness benefit from mating with older males. However, male aging is often associated with reductions in quantity of sperm. The adaptive significance of age-based mate choice is therefore unclear. Various hypotheses have made conflicting predictions concerning this issue, because published studies have not investigated the effect of age on accessory gland proteins and sperm traits. D. melanogaster exhibits resource-independent mating, and offspring do not receive parental care, making this an appropriate model for studying age-based mate choice. The present study found that D. melanogaster females of all ages preferred to mate with the younger of two competing males. Young males performed significantly greater courtship attempts and females showed least rejection for the same than middle-aged and old males. Young males had small accessory glands that contained very few main cells that were larger than average. Nevertheless, compared with middle-aged or old males, the young males transferred greater quantities of accessory gland proteins and sperm to mated females. As a result, females that mated with young male produced more eggs and progeny than those that mated with older males. Furthermore, mating with young male reduced female's lifespan. These studies indicate that quantity of accessory gland proteins and sperm traits decreased with male age and females obtain direct fitness benefit from mating with preferred young males (Rezaei, 2016).

    Drosophila egg-laying site selection as a system to study simple decision-making processes

    The ability to select a better option from multiple acceptable ones is important for animals to optimize their resources. The mechanisms that underlie such decision-making processes are not well understood. This study found that selection of egg-laying site in Drosophila melanogaster is a suitable system to probe the neural circuit that governs simple decision-making processes. First, Drosophila females pursue active probing of the environment before depositing each egg, apparently to evaluate site quality for every egg. Second, Drosophila females can either accept or reject a sucrose-containing medium, depending on the context. Last, communication of the 'acceptability' of the sucrose-containing medium as an egg-laying option to the reproductive system depends on the function of a group of insulin-like peptide 7 (ILP7)-producing neurons. These findings suggest that selection of egg-laying site involves a simple decision-making process and provide an entry point toward a systematic dissection of this process (Yang, 2008).

    Decision-making, in one view, is the process by which animals deliberate whether to invest in one action or not by taking into account the values and costs associated with available options. Selection of egg-laying site by Drosophila provides a plausible system to investigate such decision-making process; egg production is costly, thus the ability to weigh egg-laying options might have been selected to ensure better survival of offspring in uncertain environment. Drosophila females are known to be selective towards egg-laying sites and will withhold eggs when there are no appropriate sites. It is less clear, however, whether Drosophila females value a given egg-laying site differently according to the availability of other laying options. This study examined the selection of egg-laying site of individual Drosophila females, in an attempt to find a genetically tractable system to study the molecular and cellular basis of simple decision-making processes (Yang, 2008).

    The first indications that egg-laying site selection may employ a simple decision-based process emerged from observations of females as they lay eggs. By reviewing hundreds of egg-laying events, it was found that immediately prior to each physical egg-expulsion, females stereotypically bend their abdomen downward until it is nearly perpendicular to the substrate surface to extrude and insert the ovipositor into the substrate, before initiating a series of back-and-forth movements to expel and insert a single egg into the substrate. This behavioral component always accompanies the physical deposition of an egg and typically lasts about 6 seconds on grape-agar. This behavioral component was called the 'ovipositor motor program' because it is reminiscent of the 'oviposition motor program' of grasshopper egg-laying. Two more behavioral components were found that regularly follow the ovipositor program: an animal always grooms its ovipositor with its hind legs for a few seconds and then stays immobile for a while as though it is resting. After the 'clean and rest program' and before the initiation of the next ovipositor program, the animals presumably have the opportunity to locate an appropriate site for the next egg. Indeed, despite being placed in a relatively attractive grape-agar environment, the animals in virtually all cases display a 'search-like' behavior by walking around and probing the substrates with their proboscis and ovipositor. Since the proboscis, legs, and ovipositor all contain sensory receptors, this 'search-like' program should aid the animals in identifying appropriate egg-laying sites. In 199 out of 200 cases observed (~20 egg-layings each by 10 animals), the 'search-like' behavior was detected preceding the ovipositor program, though its duration varied. Thus, Drosophila females deposit their eggs one at a time and nearly always follow the cycle of search, oviposit, and clean and rest, with the search-like program lasting for from a few seconds to several minutes while the other programs remain relatively constant in duration (Yang, 2008).

    What the females seek was examined by presenting them with different egg-laying options in the behavioral chamber. A sweet (sucrose-containing) and a bitter (lobeline-containing) soft agarose medium were placed in the chamber. These separated by a region of hard agarose that deters egg-laying and prevents simultaneous detection of the two soft media. Surprisingly, animals consistently laid more eggs on the lobeline side of the chamber. This bias against sucrose medium was not caused by intrinsic attraction to the lobeline medium for egg-laying, however, because animals tended to avoid lobeline when the other option was a plain medium, consistent with previous findings on quinine avoidance in egg-laying. Furthermore, females avoided laying eggs on the sucrose-containing medium in the chambers no matter whether the alternative was lobeline-containing, plain, or a substrate containing sodium chloride at the same concentration as the sucrose (Yang, 2008).

    To determine whether females actually encountered both options before laying an egg on the lobeline medium, individual animals were tracked for two hours, and it was found that for each egg deposited on the lobeline side, the animals paid an average of 1.5 prior visits to the sucrose option. Animals often probed the sucrose substrate actively but rarely activated the ovipositor program while they were still in contact with this medium; in contrast, activation of the ovipositor program was frequently seen when the animals were probing the lobeline medium. Thus, lack of significant egg-laying on the sucrose medium is not due to animals' not making regular contacts with this medium. General repulsion to the sucrose-containing medium in the chamber is not the cause either--animals were often seen actively feeding on the sucrose medium by extending onto it their proboscis. Moreover, the avoidance of egg-laying on the sucrose-medium (when it is paired with a plain one) is concentration dependent: it attenuates and can even turn into mild attraction as the concentration of sucrose decreases (Yang, 2008).

    The reliance on neuronal activities of the sweet taste receptor (Gr5a) neurons is evident in the experiments employing the GAL4-UAS method to express in these Gr5a-expressing neurons a hyperpolarizing Kir2.1 potassium channel. This manipulation significantly increased egg-laying on the sucrose-containing medium, suggesting that sucrose detection through the Gr5a neurons is important for the low egg-laying on this medium in the two-choice chamber (Yang, 2008).

    To determine if the desirability, or 'value', of the sucrose-containing medium for egg-laying might change according to context, egg-laying was examined in single-choice chambers that contain either only the sucrose-containing medium or the lobeline-containing medium. Interestingly, females lay comparable numbers of eggs in the two single-choice chambers; thus, in the absence of other options, females readily accept the sucrose-containing medium for egg-laying. Furthermore, the same sucrose-containing medium can even become a preferred option for egg-laying if it is paired with a more repulsive medium that contains twice the amount of sucrose (Yang, 2008).

    To explore further how context impacts the valuation of sucrose medium, the physical separation between the sucrose- and lobeline-containing media was varied in the two-choice chamber. It was found that when the distance between the two choices was increased to 3- or 10- fold, egg-laying on the sucrose medium progressively increased, and some of the individual animals tested actually laid more eggs on the sucrose-containing medium than on the lobeline one. This result suggests that the preference of sucrose medium over lobeline is not absolute and animals can weigh the 'desirability' of egg-laying sites versus the 'effort it takes to locate them' in selecting sites for egg-laying. Interestingly, this distance-dependent response to sucrose-containing medium was not altered by food deprivation prior to assays, suggesting that egg-laying and foraging are distinctive tasks with distinctive substrate preferences (Yang, 2008).

    Sequential placement of the same animals first in a two-choice chamber and then in a sucrose medium-only single choice chamber revealed speedy recovery of egg-laying interests on the sucrose-containing medium, suggesting that the substrate evaluation process is efficient and performed predominantly on an egg by egg (search by search) basis. However, compared to the number of eggs deposited in a lobeline medium-only chamber, there were fewer eggs in the sucrose medium-only chamber, while no difference was observed when naive flies were introduced to the single-choice chambers; it thus appears that prior experience may exert some influence in egg-laying decision. Taken together, these results suggest that Drosophila females possess some neural process that assigns 'acceptability' or 'value' to a given egg-laying substrate by taking readily into account the availability of other options. Such 'value' can then be used by the motor output systems to decode whether a particular option is appropriate to trigger the physical egg-laying action in the given context (Yang, 2008).

    To begin discerning the neural circuitry that underlies egg-laying site selection, attempts were made to identify specific neurons that regulate egg-laying rate as they might be engaged by the 'value system' to control egg-laying on a given substrate. Neurons that express an insulin-like neuropeptide, ILP7, are of interests since ILP7 shares some homology with Relaxin, an important reproductive hormone in mammals. Antibodies revealed that ILP7 is present in only very few cells in the larval and adult CNS with some cells sending projections to the sub-esophageal ganglion (SOG) and distinct positions in the ventral nerve cord, which are all sites where gustatory information relay might occur. Moreover, projection of ILP7 to the female internal reproductive tract can also be found, in support of the notion that ILP7 may be the Drosophila Relaxin. Interestingly, many of the ILP7 neurons are also positive for Fruitless expression in adult males. This result suggests a potential role of these neurons in male reproductive behavior since Fruitless has been shown to be a master regulator of male courtship behavior in Drosophila (Yang, 2008).

    An ILP7-GAL4 was created to specifically alter ILP7-neurons' function. Using the GAL4-UAS approach, it was found that females with hyperpolarized ILP7-neurons showed no discernable developmental defects but displayed virtually no ovipositor motor programs and are thus sterile. To distinguish between the possibility that ILP7-neurons are required merely for house-keeping processes to push the egg through the internal reproductive tract, which could account for the fully penetrant egg-jamming phenotype due to silencing ILP7-neurons, and the possibility that ILP7-neurons also participate in conveying the 'acceptability' of potential laying options to the reproductive system (e.g., the reproductive tract and the ovipositor motor program), the effect was examined of elevated ILP7 level--in ILP7-neurons or ubiquitously--on egg-laying choice. In both cases, the elevation of ILP7 level caused the animals to be more receptive to laying eggs on the sucrose medium in the 'regular' two-choice chambers. These results are consistent with the idea ILP7 might participate in the relay of the 'appropriateness' of a given option to the reproductive systems to execute egg-laying on that option (Yang, 2008).

    In summary, Drosophila females can accept or avoid a given sucrose-containing medium for egg-laying depending on context. Whereas such context-dependent avoidance of high sucrose medium shows little discernable advantage in the laboratory setting (embryo hatching rate on sucrose-containing and lobeline-containing media are comparable), it could have been selected for by virtue of predation avoidance and larval dietary balance (protein/carbohydrate ratio). The finding that Drosophila employs a simple decision-making process in selecting egg-laying site raises the possibility that fruit fly has the capacity to compare and assess available options by performing integrations and amplifications in its nervous system. Dopamine and octopamine are both candidates for mediating such amplification/integration processes: the former is important for decision-making in primates and flies and is used to signal the unconditional stimulus of 'punishment' during learning tasks in Drosophila; the latter is a reinforcing signal for appetitive conditioning in both Drosophila and honeybees and an important regulator of egg-laying in Drosophila. In addition, this work suggests that the ILP7-expressing neurons are important for proper execution of egg-laying decision, thus providing an additional anatomical and molecular entry point into dissecting the decision-making processes during egg-laying site selection (Yang, 2008).

    Neural circuitry underlying Drosophila female postmating behavioral responses

    After mating, Drosophila females undergo a remarkable phenotypic switch resulting in decreased sexual receptivity and increased egg laying. Transfer of male sex peptide (SP) during copulation mediates these postmating responses via sensory neurons that coexpress the sex-determination gene fruitless (fru) and the proprioceptive neuronal marker pickpocket (ppk) in the female reproductive system. Little is known about the neuronal pathways involved in relaying SP-sensory information to central circuits and how these inputs are processed to direct female-specific changes that occur in response to mating. This study demonstrates an essential role played by neurons expressing the sex-determination gene doublesex (dsx) in regulating the female postmating response. Shared circuitry was uncovered between dsx and a subset of the previously described SP-responsive fru+/ppk+-expressing neurons in the reproductive system. In addition, sexually dimorphic dsx circuitry was identified within the abdominal ganglion (Abg) that was critical for mediating postmating responses. Some of these dsx neurons target posterior regions of the brain while others project onto the uterus. It is proposed that dsx-specified circuitry is required to induce female postmating behavioral responses, from sensing SP to conveying this signal to higher-order circuits for processing and through to the generation of postmating behavioral and physiological outputs (Rezával, 2012).

    These results show that in the female, dsx neurons associated with the internal genitalia not only form a component part of the previously described fru+/ppk+ network, but in fact define a more minimal SP-responsive neural circuit capable of inducing postmating changes, such as reduced receptivity, increased levels of rejection, and egg deposition (Rezával, 2012).

    In addition to these 'classic' postmating behavioral responses, it was also noted that SP signaling to dsx neurons induces postmating changes in locomotor activity between unmated and mated females. Studies have shown that Drosophila males court immobilized females less than moving females; essentially, males react to changes in female locomotion, suggesting a causal link between female locomotion and increased courtship levels. It has been proposed that males are 'acoustically tuned' to signals generated by active females, stimulating increased courtship by changing the attention state of the male. Therefore, female mobility appears to contribute to her 'sex appeal' and decreased locomotion in mated females is likely to affect the male's willingness to copulate (Rezával, 2012).

    The female's nervous system must have the capacity to receive, and interpret, postcopulatory signals derived from the male seminal package to direct physiological and behavioral responses required for successful deposition of fertilized eggs. It was demonstrated that two dsx clusters, composed of three bilateral neurons of the uterus, comprise a more defined component of the SP-responsive sensory circuit. In addition, the majority of other dsx neurons originating on the internal genitalia were shown to coexpress ppk. As ppk neurons are mechanosensory, these may be acting as uterine stretch receptors, facilitating sperm and egg transport, fertilization, and oviposition. Silencing neural function of ppk neurons appears to inhibit egg deposition, presumably by impeding egg transport along the oviducts. Similarly, in dsxGal4 females expressing TNT no egg deposition is ever observed, with unfertilized eggs atrophying in the lateral oviducts. In contrast, when fru+ neurons are silenced, deposition of successfully fertilized eggs is still observed, suggesting that different subsets of the dsx+/fru+/ppk+ SP-responsive sensory circuit may direct distinct postmating behavioral responses. As SP has been detected in the hemolymph of mated females, it has been suggested that this peptide could pass from the reproductive tract into the hemolymph to reach CNS targets. The fact that neither receptivity nor oviposition was restored to control levels when ppk-Gal80 (or Cha-Gal80) was expressed in dsxGal4/UAS-mSP flies opens the possibility that SP expression might affect additional dsx neurons in the CNS (Rezával, 2012).

    Triggering of postmating responses via SP reception appears to occur via a small number of neurons expressing SPR on the female reproductive tract; however, SPR is also found on surface regions of the CNS as well as in endocrine glands and other reproductive tissues. Surprisingly, SPR may even be detected in the Drosophila male CNS, where no exposure to SP would be expected, and in insects that apparently lack SP-like. SPRs are therefore potentially responsive to other ligands, performing functions other than those associated with postmating responses in the diverse tissues in which SPRs are expressed (Rezával, 2012).

    Extensive coexpression was found of dsx-expressing cells and SPR in the epithelium of the lower oviduct and spermathecae in females. However, mSP expression (or SPR downregulation) specifically in spermathecal secretory cells (SSC) or oviduct epithelium cells had no effect on receptivity or egg laying. In agreement with rescue experiments using neuronal Gal80 drivers to intersect Gal4-responsive UAS expression in dsx cells, this suggests that these cells are neither neuronal nor directly involved in SP-mediated postmating behaviors. SPR staining in the CNS was more difficult to determine given the limitations of the antibody; while no colocalization in the brain was observed, apparent coexpression was observed between SPR and a small subset of ventral (Rezával, 2012).

    The results indicate that dsx-Abg neurons are required for the induction and regulation of specific components of the postmating response. It has been shown that inhibition of neurotransmission in apterous-expressing Abg neurons impairs SP-mediated postmating changes in receptivity and oviposition, emphasizing the importance of these neurons in the modulation of postmating responses (Rezával, 2012).

    The level of dsx neuronal expression within the Abg and their associated fascicles projecting to the brain, where they form extensive presynaptic arborizations within the SOG, coupled with the effects that impairment of function in these neurons has on postmating responses, speaks to the involvement of these neurons in relaying information from the reproductive tract to the brain. That dsx-Abg neurons also project, and form presynaptic arborizations on the uterus, and that the effects on postmating responses when their function is impaired again argue that these neurons play a direct role in mediating processes such as egg fertilization and oviposition. Interestingly, most dsx intersecting neurons are specific to females. Sex-specific behaviors can arise from either shared circuits between males and females that operate differently and/or sex-specific circuits that result from the presence/absence of unique circuit components in one sex versus the other. The results support the latter (Rezával, 2012).

    The VNC has been implicated in the modulation of postmating responses, with an identified focus specifically involved in ovulation and transfer of eggs into the uterus for fertilization. Octopaminergic modulatory neurons located at the distal tip of the VNC projecting to the reproductive tract are required for triggering ovulation, possibly by regulating muscle contractions in the ovaries and oviducts. Since earlier studies have shown that ablation of the pars intercerebralis revealed an additional focus for egg laying in the head, and the brain appears to be required for sexual behaviors, such that decapitated virgin females neither mate nor lay eggs, it seems likely that neurons in the Abg also require signals from the brain to regulate postmating responses such as egg transport, fertilization, and deposition (Rezával, 2012 and references therein).

    Higher-order circuits in the female brain must be capable of integrating sensory inputs from the olfactory, auditory, and reproductive systems to decide between the alternative actions of acceptance or rejection of the male. Early gynandromorph studies mapped a region of the dorsal brain that must be female for an animal to be receptive; it has been recently shown that the majority of dsx neuronal clusters are located in this region. While neurons coexpressing dsx and fru in male brains define a more restricted circuitry for determining male mating decisions, in females no overlap between dsx+ and fru+ neurons is observable in the brain. It is also important to note that the sex-specific Fru isoform is absent in females; thus any circuits that are actively specified in the female are likely to depend on the female isoform DsxF. Most dsx neurons in the brain are found in the lateral protocerebrum, a region where multiple sensory inputs are thought to be integrated and discrete motor actions selected and coordinated. Further high-resolution functional and connectivity mapping will help to define which neurons participate in specific pre- and postmating behaviors in the female, allowing circuit architecture to be integrated with underlying cellular and synaptic properties. Future experiments will define what activity patterns trigger these behaviors and what activity patterns correlate with these behaviors (Rezával, 2012).

    Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

    Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, the increase was studied in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg's release from the ovary and its passage into the oviduct. The seminal protein Ovulin (Accessory gland protein 26Aa) increases ovulation rates after mating. Tests were performed to see whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover a mating- dependent relaxation of oviduct musculature was identified, for which ovulin is a necessary and sufficient male contribution. It is further reported that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin's increasing of OA neuronal signaling. Finally, it was shown that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction (Rubinstein, 2013).

    Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating. Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology (Rubinstein, 2013).

    In Drosophila, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate. For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating, and the seminal protein Sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating. However, although a receptor for SP has been identified, along with elements of the neural circuit in which it is required, SP's mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses (Rubinstein, 2013)?

    This question was addressed by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics. In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine. Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates. Noradrenaline, the vertebrate structural and functional equivalent to OA important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders. This paper investigated the role of neurons that release OA and tyramine in ovulin's action. For simplicity, these neurons are referred as 'OA neurons' to reflect the well-established role of OA in ovulation behavior (Rubinstein, 2013).

    This study investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology. Ovulin was shown to increase ovulation and egg laying through OA neuronal signaling. Ovulin was found to relax oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects an ovulin-dependent increase was detected in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin's stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology (Rubinstein, 2013).

    To investigate whether ovulin acts through neurons that release OA, tests were performed to see whether ectopically increasing the activity of those neurons could rescue the ovulation defect that is observed when females are mated to males without ovulin. This phenotype was tested by examining the number of eggs in the lateral oviducts 3 h after the start of mating; at this early time, ovulation rate can still be measured without being confounded by the influence of increased postmating oogenesis. If ovulin acts through OA neuronal signaling, females with ectopically hyperactive OA neurons should not show an ovulin-dependent increase in ovulation behavior. If ovulin acts independent or downstream of OA neuronal signaling, females with hyperactive OA neurons should still require ovulin for their postmating increase in ovulation rate. The tdc2-GAL4 driver was used to express a dominant-negative ether-a-gogo potassium channel subunit (UAS-eagDN) in OA neurons (loss of function of eag is known to induce increased neuronal activity) (Rubinstein, 2013).

    As expected, control tdc2-GAL4 females had significantly more eggs in their lateral oviducts after mating with control males compared with mates of ovulin-deficient males; as expected, this increased ovulation also affected egg-laying rate. However, tdc2-GAL4/UAS-eagDN experimental females, which would have elevated activity of OA neurons, showed no difference in postmating ovulation rate regardless of whether or not they received ovulin from their mates. These results indicate that increasing OA neuronal activity in females compensates for a lack of ovulin received during mating. Thus, ovulin acts through OA neuronal signaling (Rubinstein, 2013).

    No difference was detected in ovulation rates between control females and females with elevated OA neuronal activity after either type of female had mated to control males. This suggests that the normal increase in a Drosophila female's ovulation rate after mating reflects OA neurons' maximal possible contribution to ovulation rate. In this scenario, ovulin's role would be to increase ovulation to OA neuron's maximal contribution. Although this study focused on the principal known ovulation regulator, OA, it is likely that other signaling factors could further increase ovulation and that variation among the females could also result in differences in timing for the initiation of ovulation; all of these factors may dynamically control ovulation rates after mating. Consistent with this idea, many females in these experiments did not show the maximum possible number of eggs in their lateral oviducts (Rubinstein, 2013).

    The tdc2-GAL4 driver was used to manipulate neurons that produce and release OA because tdc2-GAL4 expression strongly overlaps with immunoreactivity for OA and for TβH, the metabolic enzyme producing OA. These neurons also produce the OA metabolic precursor, tyramine, which can also affect behaviors. Although this study has shown that ovulin acts through OA neuronal signaling, the possibility cannot be excluded that ovulin might act through tyramine signaling in OA neurons. However, given the documented role of OA and the OA receptor OAMB in Drosophila ovulation, OA signaling is the most likely pathway for ovulin's action via OA neurons (Rubinstein, 2013).

    OA increases ovarian muscle contractions, while blocking contractions of oviduct musculature in Drosophila and other insects. This effect of OA is proposed to facilitate ovulation of an egg from the ovary into the relaxed oviduct. Because ovulin stimulates ovulation, it was of interest to determine whether ovulin contributes to the relaxation of the oviduct that occurs after mating. Ovulin could relax oviduct musculature either through decreasing rates or amplitudes of phasic muscle contraction twitches, or it could induce a more basally relaxed state by modulating muscle tonus. Because OA blocks contractions of the oviduct, it was reasoned that ovulin's action on the oviduct might be observable by examining muscle tonus (Rubinstein, 2013).

    Oviduct muscle tonus was assayed by measuring the average sarcomere length of oviduct myofibrils. To do this, females were used expressing Myosin heavy-chain fused to GFP. In these flies, GFP labels the A band of the sarcomere units. Thus, the distance between each GFP band represents the sarcomere length. Sarcomere lengths were measured in these females after mating to control or ovulin-deficient males and in virgin females. Although ovulin's most direct effect is to increase the release of an egg from the ovary to the lateral oviduct, it was not possible to obtain reliable measurements of sarcomere length in lateral oviducts, because sarcomere lengths varied along the length and circumference of the lateral oviduct (Rubinstein, 2013).

    Therefore, myofibril sarcomere lengths were measured at the adjacent upper (anterior) common oviduct, where it is possible to reproducibly measure from a standardized position in the reproductive tract across individuals (Rubinstein, 2013).

    A significant increase was observed in oviduct muscle sarcomere length between virgin females and control-mated females at 1.5 h after the start of mating, indicating that the oviduct relaxes after mating. Then, whether ovulin contributes to this relaxation was tested by measuring the sarcomere lengths of females mated to ovulin-null males. Sarcomere lengths of ovulin-null–mated females were significantly shorter than those of wild type-mated femalesa and were indistinguishable from those of virgin females. These data suggest that ovulin is necessary for the postmating relaxation of oviduct musculature (Rubinstein, 2013).

    Then whether ovulin is sufficient to increase oviduct sarcomere lengths in females, in the absence of anything else male-derived, was tested. Virgin females were generated that could ectopically express ovulin (which is normally a male-expressed gene; UAS-ovulin) under control of a heat shock promoter (hs-GAL4) and control females that could not (they only carried hs-GAL4). Both types of females were subjected to heat shock to induce ovulin expression in experimental females and then oviduct sarcomere lengths were measured. Females that ectopically expressed ovulin showed significantly increased sarcomere lengths compared with control females (Rubinstein, 2013).

    These results indicate that although male-derived ovulin is necessary for maximal ovulation and is sufficient to increase ovulation rates, ovulin is also a necessary and sufficient male contribution for relaxing the oviduct musculature. This suggests that ovulin's relaxation of oviduct musculature underlies its role in increasing ovulation rate (Rubinstein, 2013).

    Because OA is critical for proper ovulation and blocks oviduct muscle contraction and because ovulin works through OA neuronal signaling, the results above suggested that OA might also be required for postmating oviduct muscle relaxation. This hypothesis was tested by examining oviduct sarcomere lengths in tbhM18 mutant females, which are unable to produce OA and instead accumulate tyramine. In tbhM18 heterozygous control females that also carried the MHC-GFP construct, a mating-dependent increase in sarcomere length was detected, like that seen with wild-type females. However, in tbhM18 homozygous females, there was no detectable mating-dependent increase in sarcomere length. To determine whether tbhM18 homozygous females do not exhibit an increase in sarcomere length after mating because they lack OA, tbhM18 homozygous females were fed OA at 7.5 mg/mL and subsequently sarcomere lengths of virgin and mated females was measured. After tbhM18 homozygous females were fed OA, there was an increase in sarcomere length in females mated to wild-type males compared with virgin controls. Although other signaling molecules (i.e., tyramine and glutamate) likely also play a role in oviduct muscle contraction, this result indicates that OA plays a critical role in the postmating increase in oviduct sarcomere length (Rubinstein, 2013).

    To determine if oviduct sarcomere lengths depend on OA neuronal activity, thermally activated TrpA channels (UAS-TrpA) under control of tdc2-GAL4 were used to conditionally increase activity of OA neurons. At the nonactivating temperature of 22 oC, no difference in sarcomere lengths was seen between control virgin females that carried only the tdc2-GAL4 construct vs. experimental virgin females that carried tdc2-GAL4 and UAS-TrpA. However, at the TrpA-activating temperature of 32 oC, the experimental virgin females that carried both constructs had longer oviduct sarcomere lengths than control females. These data indicate that increasing the activity of OA neurons induces relaxation of oviduct muscle (Rubinstein, 2013).

    These results suggested that ovulin increases OA neuronal activity to increase ovulation rates and that such increased activity induces a relaxation of oviduct muscle. To test this directly, the UAS-eagDN transgene was used to determine whether ovulin affects oviduct sarcomere length through OA neuronal signaling. The same experimental design measured oviduct sarcomere lengths instead of egg numbers. A significant difference was observed between tdc2- GAL4/+ control females when they were mated to males with (+) or without (-) ovulin. There was no significant difference in oviduct sarcomere length between experimental tdc2-GAL4/UAS-eagDN females that did or did not receive ovulin from their mates. This indicates that ectopically increasing OA neuronal activity can compensate for a lack of ovulin. Taken together, these data indicate that ovulin increases oviduct sarcomere length through OA neuronal signaling and support the model that ovulin works through OA neurons to maximize ovulation rates (Rubinstein, 2013).

    To confirm whether increased oviduct sarcomere lengths confer an increased ability to accommodate an egg ovulated from the ovary, the circumference of oviduct musculature was measured through 3D reconstruction of the datasets used to determine sarcomere lengths, using a subset of control females (tbhM18 heterozygotes). Sarcomere length and musculature circumference showed a significant, positive, and linear relationship, suggesting that increased sarcomere lengths also indicate an increase in oviduct circumference and therefore an increased ability to accommodate an egg. Further, mating resulted in a 19.4% increase in sarcomere length (comparing virgins and females mated to control male), which corresponds to a 42.6% increase of oviduct cross-sectional area, reflecting a sizable increase in the oviduct's activity to accommodate an egg after mating (Rubinstein, 2013).

    By 6h after mating, increased synaptic development is detected for axons with type II boutons at the female reproductive tract. Type II synaptic boutons characterize OA neuronal release sites, and an increased number of axonal boutons, anatomical swellings indicative of neurotransmitter release sites, has been reported in these synapses by 6 h postmating. An increase in synaptic bouton number is often attributed to increased activity at that synapse, a form of synaptic plasticity whereby a heavily used synapse grows to meet signaling demands. Because the results suggest that ovulin increases OA neuronal signaling and because type II synaptic boutons characterize OA neuronal release sites, tests were performed to see whether ovulin could increase the synaptic development of OA neuronal– reproductive tract neuromuscular junctions (NMJs) after mating. This was assessed by labeling OA neurons with GFP and counting synaptic boutons at OA neuronal termini at the common oviduct after the start of mating (Rubinstein, 2013).

    Because it can take ∼2 h for changes in bouton number to become visible after neuronal stimulation, even though ovulin's effects on ovulation behavior are detectable by 1.5 h after the start of mating, initially 6 h postmating was chosen to examine ovulin effects on bouton numbers. At this time point, a significant increase was detected in the number of GFP-labeled boutons between unmated females and females mated to control males, suggesting that the type II boutons are in fact from OA neurons. To determine whether ovulin affected the bouton number, the number of OA neuron boutons in females mated to control vs. ovulin-null males, was tested at 6 h after the start of mating. It was observed that females that had mated to ovulin-null males had significantly fewer GFP-labeled boutons than females mated to control males. Indeed, the numbers of GFP-labeled boutons in females that had mated to ovulin-null males were not significantly different from virgin females. These results support the conclusion that ovulin acts to up-regulate OA neuronal signaling (Rubinstein, 2013).

    It was then asked whether the increase in the number of boutons might be evident at 1.5 h after the start of mating, when ovulin's function is detectable. It was reasoned that if ovulin induces the increase in synaptic boutons indirectly, i.e., by increasing activity of OA neurons, this process would involve biochemical transduction of increased activity; contributions from retrograde signaling; signal transduction of those signals; and finally mobilization of scaffolding, cell adhesion, and vesicle release machinery. Therefore, fully developed boutons would likely not be evident at 1.5 h after the start of mating, before the ∼2 h time window previously reported for the visible appearance of type II boutons. However, if ovulin directly affects cell-signaling or protein-mobilization pathways within OA neurons to induce an increase in bouton numbers, the process referred to above would not be required, and the ovulin-dependent increase in bouton counts might be visible at this early time point. No differences were detected in the number of GFP-labeled boutons between unmated females and females mated to control males at 1.5 h after the start of mating. Thus, neither mating nor any seminal protein (including ovulin) causes a detectable increase in bouton counts this early after mating. Because there is an ovulin-dependent increase in bouton number but it is only evident at a later time point, the increase in OA bouton number likely represents synaptic plasticity as an indirect response to ovulin-induced increase in OA neuronal activity, rather than a causative effect of ovulin directly on OA synaptic boutons. The data cannot distinguish whether the increased bouton number is the consequence of a direct effect of ovulin on OA neurons that leads to the ovulation increase or is caused by the passage of additional eggs through the oviducts (Rubinstein, 2013).

    Future studies to measure sarcomere length in eggless females will resolve this question, and additional further measures, such as measurement of bouton numbers in tdc-GAL4/UAS-eagDN females, may also help to define the precise mechanism by which ovulin increases the number of type II boutons after mating (Rubinstein, 2013).

    This study has reported that the Drosophila seminal protein, ovulin, acts through an endogenous signaling pathway in the female to affect reproductive physiology. Further, the relaxation of oviduct musculature by ovulin and its downstream regulator, OA neurons, provides a potential mechanism to explain how ovulin increases a female's ovulation rate after mating. The data also indicate that ovulin, likely indirectly, underlies the subsequent increase in the number of OA synaptic sites at the reproductive tract after mating. This provides another indication of ovulin's interaction with the OA neuronal signaling system. Moreover, the data suggest that the increased type II bouton counts reported after mating reflect, specifically, OA neurons and that the changes in mating-dependent synaptic regulation that had been measured by bouton counts and GFP-labeled vesicle contents can be caused by the action of specific seminal proteins such as ovulin. Further, these findings demonstrate a link between a seminal protein's postmating effect and a mechanism of action through known female regulators of reproductive physiology and behavior. It will be useful for future studies to determine whether ovulin acts centrally in the nervous system, on presynaptic terminals of the reproductive tract to increase OA synaptic output, or on nonneuronal tissue in the reproductive tract which signals back to the nervous system (Rubinstein, 2013).

    Although OA's regulation of ovulation and egg-laying behavior is conserved in many arthropods, the ovulin gene is evolving rapidly under strong positive selection. An evolutionarily labile regulator sitting atop an evolutionarily conserved regulatory 'core' (in this case, the OA signaling pathway) allows selection to act on innovations enhancing the core's output without disrupting the core itself; a similar idea has been suggested for sex determination mechanisms in Diptera. Applying this logic to the model of ovulin and OA neuronal signaling, the conserved control by OA of ovulation could ensure that this essential process occurs unfettered, whereas frequent changes in ovulin sequence could allow for small, yet selectively advantageous, increases in ovulation rate (Rubinstein, 2013).

    Because ovulation rates increase partially (but not to the full extent) after matings in which females do not receive ovulin, other signaling factors apart from ovulin-dependent OA neuronal signaling must also contribute to the postmating increase in ovulation. For example, in Drosophila and other insects, other signaling factors (e.g., females' glutamate, proctolin, and ILP7) are known to be important in ovulation and egg laying. However, two post-mating phenotypes described in this study, oviduct muscle tonus and synaptic plasticity, can be largely attributed to ovulin and its interaction with OA signaling. These results imply that other mating-related stimuli that increase ovulation might work through independent mechanisms, perhaps distinct from OA neuronal signaling (Rubinstein, 2013).

    Observing effects of ovulin on synaptic strength at 6 h postmating was intriguing because by this time, ovulin is nearly undetectable in mated females and because ovulin has been reported to affect egg laying only within the first 24 h after mating. It is possible that the increased synaptic strength seen at 6 h is simply a visible, but indirect, consequence of ovulin's earlier effects and/or that the synaptic growth/plasticity that ovulin induced is somehow transient. Alternatively, ovulin might be able to affect egg production even after it has disappeared by having induced a longer-lasting developmental effect at the oviduct NMJ. Such consequences of ovulin's earlier actions may not be detectable after the first postmating day if they are masked by later, larger effects from other sources. For example, ovulin's primary action has been proposed to be to stimulate release of the mature oocytes that had built up in females before mating; in such a model, once all those accumulated eggs have been released, effects of ovulin may not be apparent in the context of Sex Peptide's large stimulation of egg production rate (Rubinstein, 2013).

    This study found that a male seminal protein, ovulin, controls reproductive behavior in female Drosophila through neurons that release a canonical neuromodulator, OA. Understanding the female modulators of reproductive physiology thus provides a useful framework for addressing how male seminal proteins influence females. These findings provide support for the idea that seminal proteins modulate conserved endogenous physiological mechanisms and circuits (Rubinstein, 2013).

    Mating-induced increase in germline stem cells via the neuroendocrine system in female Drosophila

    Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. This study reports that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. It was shown that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. This study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating (Ameku, 2016).

    Mechanosensitive neurons on the internal reproductive tract contribute to egg-laying-induced acetic acid attraction in Drosophila

    Selecting a suitable site to deposit their eggs is an important reproductive need of Drosophila females. Although their choosiness toward egg-laying sites is well documented, the specific neural mechanism that activates females' search for attractive egg-laying sites is not known. This study shows that distention and contraction of females' internal reproductive tract triggered by egg delivery through the tract plays a critical role in activating such search. Females start to exhibit acetic acid (AA) attraction prior to depositing each egg but no attraction when they are not laying eggs. Artificially distending the reproductive tract triggers AA attraction in non-egg-laying females, whereas silencing the mechanosensitive neurons this study identified that can sense the contractile status of the tract eliminates such attraction. This work uncovers the circuit basis of an important reproductive need of Drosophila females and provides a simple model for dissecting the neural mechanism that underlies a reproductive need-induced behavioral modification (Gou, 2014 PubMed).

    Egg-laying demand induces aversion of UV light in Drosophila females

    Drosophila melanogaster females are highly selective about the chemosensory quality of their egg-laying sites, an important trait that promotes the survival and fitness of their offspring. How egg-laying females respond to UV light is not known, however. UV is a well-documented phototactic cue for adult Drosophila, but it is an aversive cue for larvae. This study shows that female flies exhibit UV aversion in response to their egg-laying demand. First, females exhibit egg-laying aversion of UV: they prefer to lay eggs on dark sites when choosing between UV-illuminated and dark sites. Second, they also exhibit movement aversion of UV: positional tracking of single females suggests that egg-laying demand increases their tendency to turn away from UV. Genetic manipulations of the retina suggest that egg-laying and movement aversion of UV are both mediated by the inner (R7) and not the outer (R1-R6) photoreceptors. Finally, the Dm8 amacrine neurons, a synaptic target of R7 photoreceptors and a mediator of UV spectral preference, were shown to be dispensable for egg-laying aversion but essential for movement aversion of UV. This study suggests that egg-laying demand can temporarily convert UV into an aversive cue for female Drosophila and that R7 photoreceptors recruit different downstream targets to control different egg-laying-induced behavioral modifications (Zhu, 2014).

    Fecal-derived phenol induces egg-laying aversion in Drosophila

    Feces is an abundant, rich source of energy, utilized by a myriad of organisms, not least by members of the order Diptera, i.e., flies. How Drosophila melanogaster reacts to fecal matter remains unclear. This study examined oviposition behavior toward a range of fecal samples from mammals native to the putative Southeast African homeland of the fly. D. melanogaster displays a strong oviposition aversion toward feces from carnivorous mammals but indifference or even attraction toward herbivore dung. A set of four predictor volatiles were identified that can be used to differentiate fecal from non-fecal matter, as well as separate carnivore from herbivore feces. Of these volatiles, phenol-indicative of carnivore feces-confers egg-laying aversion and is detected by a single class of sensory neurons expressing Or46a. The Or46a-expressing neurons are necessary and sufficient for oviposition site aversion. It was further demonstrated that carnivore feces-unlike herbivore dung-contain a high rate of pathogenic bacteria taxa. These harmful bacteria produce phenol from L-tyrosine, an amino acid specifically enriched in high protein diets, such as consumed by carnivores. Finally, it was demonstrated that carnivore feces, as well as phenol, is also avoided by a ball-rolling species of dung beetle, suggesting that phenol is a widespread avoidance signal because of its association with pathogenic bacteria (Mansourian, 2016).

    Cancer brings forward oviposition in the fly Drosophila melanogaster

    Hosts often accelerate their reproductive effort in response to a parasitic infection, especially when their chances of future reproduction decrease with time from the onset of the infection. Because malignancies usually reduce survival, and hence potentially the fitness, it is expected that hosts with early cancer could have evolved to adjust their life-history traits to maximize their immediate reproductive effort. Despite the potential importance of these plastic responses, little attention has been devoted to explore how cancers influence animal reproduction. This study used an experimental setup, a colony of genetically modified Drosophila melanogaster which develop colorectal cancer in the anterior gut, to show the role of cancer in altering life-history traits. Specifically, it was tested whether females adapt their reproductive strategy in response to harboring cancer. It was found that flies with cancer reach the peak period of oviposition significantly earlier (i.e., 2 days) than healthy ones, while no difference in the length and extent of the fecundity peak was observed between the two groups of flies. Such compensatory responses to overcome the fitness-limiting effect of cancer could explain the persistence of inherited cancer-causing mutant alleles in the wild (Arnal, 2017).

    Dietary protein content alters both male and female contributions to Drosophila melanogaster female post-mating response traits

    Males transfer sperm, proteins and other molecules to females during mating. In Drosophila melanogaster, these molecules contribute to the induction of egg maturation, ovulation, oviposition, sperm storage and changes in female receptivity. This suite of physiological and behavioral changes is referred to as the female post-mating response (PMR). Protein is a necessary macronutrient for both male and female reproduction, but imbalances in protein content can decrease reproductive potential. Dietary protein affects the production of proteins in the male ejaculate that are important for induction of the PMR, and female fecundity increases with dietary protein while lifetime mating rate decreases. The effects of dietary protein levels on other aspects of the female PMR and on male ability to induce the PMR are unknown. To investigate how protein content affects PMR, we raised flies on diets containing low, moderate or high levels of protein and mated females and males from each diet in a combinatorial manner. The mating duration for each pair, an indication of male reproductive investment, was measured, and then two aspects of the female PMR were evaluated, fecundity and female remating latency. Mating duration was negatively correlated with male dietary protein, and females that mated with high protein males laid fewer eggs. Female diet had no effect on mating duration, but females fed diets with higher protein content laid more eggs and remated sooner. Therefore, dietary protein levels can affect postcopulatory processes important for reproductive output in a sexually dimorphic manner (Schultzhaus, 2017).

    apterous brain neurons control receptivity to male courtship in Drosophila melanogaster females

    Courtship behaviours allow animals to interact and display their qualities before committing to reproduction. In fly courtship, the female decides whether or not to mate and is thought to display receptivity by slowing down to accept the male. Very little is known on the neuronal brain circuitry controlling female receptivity. This study used genetic manipulation and behavioural studies to identify a novel set of neurons in the brain that controls sexual receptivity in the female without triggering the postmating response. These neurons, defined by the expression of the transcription factor Apterous, affect the modulation of female walking speed during courtship. Interestingly, it was found that the Apterous neurons required for female receptivity are neither Doublesex nor Fruitless positive suggesting that Apterous neurons are not specified by the sex-determination cascade. Overall, these findings identify a neuronal substrate underlying female response to courtship and highlight the central role of walking speed in the receptivity behaviour (Aranha, 2017).

    Reproductive behaviours are essential for the survival and fitness of the species. In Drosophila melanogaster, as in many other species, the decision of whether or not to mate is made by the female. However, understanding about the behaviour displayed by the virgin female and the neuronal circuits underlying it is still poor. This study set out to identify neurons involved in the response of virgin females to courting males. Female virgins with compromised activity in apterous neurons in the brain display a substantial reduction in copulation. What is specifically changed in the premating behaviour of apterous-silenced females? apterous-silenced females do not slow down during courtship even though they do recognize they are in the presence of a courting male because they extrude the ovipositor. Ovipositor extrusion is a display that occurs exclusively in the context of courtship. A caveat of this work is the large number of neurons that are affected by this manipulation. Is the low copulation rate a result of an issue created by silencing large numbers of neurons? The capacity to recognize the male partner does not seem to be affected and the phenotypes that were observed are revealed only in the context of courtship, which strongly suggests that parts of the natural receptivity circuit were specifically affected (Aranha, 2017).

    How can changes in activity of apterous neurons affect female velocity during courtship? The best understood cue from the male that shapes female velocity is the song. It is unlikely though that apterous-silenced females have impaired hearing. apGAL4 labels very faintly the region that is innervated by first and second order auditory neurons, AMMC. Third order auditory neurons innervate the ventral lateral protocerebrum (VLP). The VLP neurons (and a few other neurons) that express apterous were silenced, and no receptivity phenotype was observed. Moreover, flies with impaired hearing do not copulate within the time of the analysis. Most likely apterous neurons are involved in integrating sensory cues provided by the male that would lead to a receptive state of the female and over time result in a decrease of female velocity. (Aranha, 2017).

    This study also uncovered a role for apterous neurons in controlling egg laying, another critical aspect of reproductive behaviour. Females that have mated and then have their apterous neurons in the brain inhibited lay very few eggs, unlike control females. Classic gynandromorph studies pointed to an egg laying suppression focus in the brain, but this study seems to have identified a focus that promotes egg laying when active. A recent study implicates the doublesex-positive and female-specific PMN2 descending neurons in oviposition behaviou. apterous neurons are neither doublesex positive nor descending, so it is reasonable to assume that this study identified a novel set of neurons (Aranha, 2017).

    In conclusion, these findings contribute to understanding female receptivity and highlight the central role of female speed modulation during courtship. It will be interesting to elucidate in the future the role of the different apterous clusters and reveal how they interact with the sexual specification circuits (Aranha, 2017).

    Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain

    The morphology and physiology of neurons are directed by developmental decisions made within their lines of descent from single stem cells. Distinct stem cells may produce neurons having shared properties that define their cell class, such as the type of secreted neurotransmitter. This study developed the transgenic cell class-lineage intersection (CLIn) system to assign cells of a particular class to specific lineages within the Drosophila brain. CLIn also enables birth-order analysis and genetic manipulation of particular cell classes arising from particular lineages. The power of CLIn was demonstrated in the context of the eight central brain type II lineages, which produce highly diverse progeny through intermediate neural progenitors. 18 dopaminergic neurons from three distinct clusters were mapped to six type II lineages that show lineage-characteristic neurite trajectories. In addition, morphologically distinct dopaminergic neurons are produced within a given lineage, and they arise in an invariant sequence. Type II lineages that produce doublesex- and fruitless-expressing neurons were identified, and whether female-specific apoptosis in these lineages accounts for the lower number of these neurons in the female brain was examined. Blocking apoptosis in these lineages results in more cells in both sexes with males still carrying more cells than females. This argues that sex-specific stem cell fate together with differential progeny apoptosis contribute to the final sexual dimorphism (Ren, 2016).

    The relationship between neuron classes and lineages is complex in the Drosophila brain, where analogous neurons of a given class may arise from distinct lineages and a single lineage can yield multiple neuron classes. Therefore, a method was developed that would enable mapping and and analysis of neuron classes with respect to lineage identity using intersectional transgenic strategies. Specifically, the neuron class of interest expresses the GAL4 transcriptional activator from a class-specific transgene, while the lineage(s) of interest expresses the KD recombinase from a lineage-specific transgene. The KD recombinase activity triggers production of another recombinase, Cre, under the control of the deadpan (dpn) promoter, which is active in all NBs. Cre recombinase activity then triggers the simultaneous production of the LexA::p65 transcriptional activator and loss of the GAL4 inhibitor, GAL80, in all subsequently born progeny within the lineage(s). The LexA::p65 activates reporter-A expression within lineages of interest via lexAop. Because all other neurons outside lineage(s) of interest express GAL80, GAL4 is only active in neurons of the LexA::p65-expressing lineage(s) and thus can positively mark these neurons by activating expression of a reporter-B under UAS control. One can therefore subdivide any complex set of neurons that express a class-specific GAL4 transgene based on their developmental lineage(s). Consequently, CLIn enables the unambiguous determination of the lineage origins of particular neuron classes, which is essential for understanding the development and organization of the Drosophila brain (Ren, 2016).

    The CLIn system unambiguously establishes the correspondence between cell classes and their lineage origins and enables the subdivision of a given neuronal class among certain NB lineages. It also allows interrogation of serially derived neuronal diversity. One can therefore map individual neurons of a given class with respect to their lineage and temporal origins in an effort to unravel the intricate neuron class-lineage relationships in the brain (Ren, 2016).

    Revealing diverse cell classes of a lineage, by carefully choosing different GAL4 drivers that each distinguish a particular cell class, will allow better characterization of progeny heterogeneity within a lineage. It is therefore possible to explore how cellular diversity is generated during development. For example, it will be interesting to determine whether a specific cell class develops from one fixed temporal window. Moreover, comparing the cell-class diversity of different lineages will provide insight into the developmental heterogeneity of stem cells (Ren, 2016).

    Conversely, for cell classes that originate from multiple lineages, CLIn analysis reveals the distribution of a cell class among different lineages. Vertebrate studies found that neurons of the same lineage origin, compared to neurons of the same class but different lineage origins, are more likely structurally connected via gap junction and have similar network functions. In Drosophila, lineage has been shown to be a developmental and a functional unit. Thus, assigning a cell class to different lineages may reveal the particular function of a neuronal subset within a cell class (Ren, 2016).

    Moreover, the CLIn system permits incorporations of additional effectors driven by the GAL4-UAS system or the LexA system to manipulate cell class or lineage, respectively. The toolkit of effectors for different purposes is growing rapidly over recent years. Multiple reporter constructs are available to label specific sub-domains of the cell (dendrite, axon, or synapse). Effectors that affect cell viability could eliminate or immortalize specific neurons or glia. Effectors that alter membrane activity can be used to modulate neural activity. In addition, CLIn enables distinguishing gene’s functions in whole lineage including stem and progenitor cells versus only in a subset of lineage progeny by independent gene manipulations via lexAop versus UAS systems (Ren, 2016).

    However, the CLIn system requires further improvement to reach its full potential. In particular, the drivers for targeting various NB subsets remain to be fully characterized. Moreover, their targeting efficiency and specificity could vary individually. Engineering drivers based on genes known to be expressed in defined subsets of embryonic NBs may provide an initial complete set of more reliable NB drivers. An additional challenge for the study of type II lineages is how to selectively target INP sublineages. Via the current dpn enhancer, the frequencies of INP1 sublineages are very low compared with that of NB lineages (Ren, 2016).

    Type II NBs yield supernumerary neurons plus glia, which are expected to be highly diverse in cell classes. CLIn unambiguously assigned various neuronal classes to common type II lineages. In this study, the majority of progeny remained negative for the drivers employed. Revealing the full spectrum of neuronal heterogeneity within type II lineages requires characterization of additional cell-class drivers (Ren, 2016).

    Diverse cell classes could arise from a single INP. Single-cell lineage analysis has shown that one INP can produce multiple morphological classes of neurons most likely pertaining to different functional classes. Temporal mapping by CLIn revealed the birth of both TH-GAL4 and dsxGAL4 neurons in early windows of larval type II lineages. This lends further support to the production of diverse neuronal classes by common INPs. Examining INP clones labeled by CLIn did validate that the first larval-born INP of the DM6 lineage makes one fruGAL4 neuron in addition to two TH-GAL4 neurons (Ren, 2016).

    Per the limited cell-lineage analysis along the NB axis of type II lineages, sibling INPs produce morphologically similar series of neurons that differ in subsets of terminal neurite elaborations. These phenomena indicate expansion of related neurons across sibling INP sublineages. Assuming production of about 50 sibling INPs and in the absence of apoptosis, one would expect composition of 50 cell units for each neuronal class made by one type II NB. Notably, rescuing apoptotic dsx- or fru-expressing neurons throughout lineage development did restore complements of 50 or so cells in several, but not all, type II lineages. However, most type II lineages yield very few, if any, TH-GAL4 neurons. For instance, the DL1 lineage produces two TH-GAL4 neurons that innervate the upper FB layers. Temporal mapping of the DL1 lineage reveals the existence of multiple (n > 3) morphologically distinct INP clones that contain neurons projecting to the FB upper layers, similar to the DL1 TH-GAL4 neurons. Thus, morphologically similar neurons may belong to different functional classes, highlighting the challenges in sorting out neuronal classes and their interrelationships in the brain (Ren, 2016).

    Pioneering studies in C. elegans showed that the acquisition of neurotransmitter identity could be achieved through distinct mechanisms. A shared regulatory signature consisting of three terminal-selector types of transcription factors regulates the terminal identity of all dopaminergic neurons. By contrast, different combinations of terminal selectors act in distinct subsets of glutamatergic neurons to initiate and maintain their glutamatergic identity. In the present study, it was observed that six type II lineages produce 18 dopaminergic neurons but all during early larval neurogenesis. The derivation of TH neurons from multiple neuronal lineages at similar temporal windows argues for their specification by combinations of different lineage-identity genes with common temporal factors (Ren, 2016).

    Previous analysis of fruGAL4 neurons has uncovered 62 discrete MARCM clones in the fly central brain that might arise from an equal number of lineages. Ten clones show dimorphic cell numbers, and 22 clones exhibit dimorphic trajectories. Contrasting the stochastic clonal labeling of only fruGAL4 neurons, CLIn allows determination among a collection of lineages of whether a given lineage yields any fruGAL4 neurons. Based on the additional lineage information, two clones (pIP-j and pIP-h) were attributed as being partial clones of another two full-sized clones (pIP-g and pMP-f). Moreover, a more focused approach reveals sexual dimorphism of fru-expressing neurons in all type II NB lineages (Ren, 2016).

    The presence of many more dsx- or fru-expressing neurons in male than female brains is proposed to result from selective loss of specific neurons in females through apoptosis. However, blocking apoptosis increased dsx- or fru-expressing neurons in both male and female lineages. This is consistent with a previous report showing that sex-independent apoptosis occur widely in type II lineages. Although the number of apoptosis-blocked female neurons was similar, if not identical, to that of the control male neurons, blocking apoptosis unexpectedly increased the number of male dsx- or fru-expressing neurons such that there were more neurons in the apoptosis-blocked male than female lineages. This unmasks the original potential of the male and female NBs to produce different numbers of dsx- or fru-expressing neurons in type II lineages (Ren, 2016).

    Distinct fates have been reported for male and female NBs in the abdominal ganglion of Drosophila CNS. In this study, the male isoform of Dsx, DsxM, promotes additional NB divisions in males relative to females. Similarly, it has been reported that DsxM specifies additional cell divisions in the male, relative to female, central brain NBs that give rise to the pC1 and pC2 clusters. The proliferation of Drosophila intestinal stem cells is also determined by their sexual identity, although this is controlled by genes other than dsx and fru. Consistent with the notion that male and female NBs may possess distinct fates, this study found that male type II lineages contain more neurons committed to express dsx or fru, which possibly results from the greater number of NB divisions in males, as shown in the previous study. Elucidating the underlying molecular mechanisms of sex-specific neuron numbers in the central brain will require additional studies of the sex-dependent production and specification of different dsx- or fru-expressing neurons in the apoptosis-blocked type II NB lineages (Ren, 2016).

    Lineage mapping based on morphology provides limited information about neuronal classes. Given the intricate relationship between neuronal classes and cell lineages, CLIn is needed to resolve the detail even in fly brains where invariant neuronal lineages exist. This is critical for fully understanding how cell lineages guide the formation of variant neural circuits with distinct combinations of neuronal classes and types (Ren, 2016).

    In mammalian brains, extensive neuronal migration obscures the roles of cell lineages in the global organization of neural networks. However, clonally related neurons preferentially make local connections. Moreover, ample evidence exists for the heterogeneity of mammalian neural stem cells and the control of neuronal identity by spatiotemporal patterning of neural progenitors. Untangling of a further sophisticated brain and its development may absolutely require examination of cell lineages and neuronal classes at the same time. Systems like CLIn with its emphasis on the relationship between cell class and lineage potentially aid greatly in the study of mammalian brain development, anatomy, and function (Ren, 2016).


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    list of genes

    Zygotically transcribed genes

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