The Interactive Fly

Zygotically transcribed genes

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

Courtship Learning, Memory and Behavior
  • The relative roles of vision and chemosensation in mate recognition of Drosophila
  • Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila
  • Imaging brain activity during complex social behaviors in Drosophila with Flyception2
  • 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
  • Cholinergic control of male reproductive characters in Drosophila
  • Dopamine and serotonin are both required for mate-copying in Drosophila melanogaster
  • Drosophila Life Span and Physiology Are Modulated by Sexual Perception and Reward
  • Motivation, perception, and chance converge to make a binary decision
  • Neural circuit mechanisms of sexual receptivity in Drosophila females
  • Cell assembly analysis of neural circuits for innate behavior in Drosophila melanogaster using an immediate early gene stripe/egr-1
  • 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
  • Juvenile hormone is required in adult males for Drosophila courtship
  • Asexuality in Drosophila juvenile males is organizational and independent of juvenile hormone
  • Magnetoreception regulates male courtship activity in Drosophila
  • Pleiotropic effects of loss of the Dα1 subunit in Drosophila melanogaster: Implications for insecticide resistance
  • Mate choice in fruit flies is rational and adaptive
  • Genomic responses to socio-sexual environment in male Drosophila melanogaster exposed to conspecific rivals
  • GABA-mediated inhibition in visual feedback neurons fine-tunes Drosophila male courtship
  • Quantitative analysis of visually induced courtship elements in Drosophila subobscura
  • The physical environment mediates male harm and its effect on selection in females
  • Social experience and pheromone receptor activity reprogram gene expression in sensory neurons
  • Tissue-specific insulin signaling mediates female sexual attractiveness
  • Direct and trans-generational effects of male and female gut microbiota in Drosophila melanogaster
  • Mating system manipulation and the evolution of sex-biased gene expression in Drosophila
  • Flexible memory controls sperm competition responses in male Drosophila melanogaster
  • Antennae sense heat stress to inhibit mating and promote escaping in Drosophila females
  • The behavior of adult Drosophila in the wild
  • Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate
  • Drosophila courtship conditioning as a measure of learning and memory
  • Persistent activity in a recurrent circuit underlies courtship memory in Drosophila
  • Threshold-based ordering of sequential actions during Drosophila courtship
  • Social effects on fruit fly courtship song
  • Courtship behavior induced by appetitive olfactory memory
  • Generating parallel representations of position and identity in the olfactory system
  • Unsupervised identification of the internal states that shape natural behavior
  • Drosophila sexual attractiveness in older males is mediated by their microbiota
  • Fitness consequences of redundant cues of competition in male Drosophila melanogaster
  • Mate-finding dispersal reduces local mate limitation and sex bias in dispersal
  • Light dependent courtship behavior in Drosophila simulans and D. melanogaster
  • Female mating experience and genetic background independently influence male mating success in fruit flies
  • Regulation of Drosophila Long-Term Courtship Memory by Ecdysis Triggering Hormone
  • Hsp70 affects memory formation and behaviorally relevant gene expression in Drosophila melanogaster
  • A sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior
  • No water, no mating: Connecting dots from behaviour to pathways
  • Sexual arousal gates visual processing during Drosophila courtship
  • Sperm depletion in relation to developmental nutrition and genotype in Drosophila melanogaster
  • The nuclear receptor Hr46/Hr3 is required in the blood brain barrier of mature males for courtship
  • Divergence and correlated evolution of male wing spot and courtship display between Drosophila nepalensis and D. trilutea
  • Dorsal-lateral clock neurons modulate consolidation and maintenance of long-term memory in Drosophila
  • Behavioral signatures of structured feature detection during courtship in Drosophila
  • Nutrients and pheromones promote insulin release to inhibit courtship drive
  • Genes Responsible for H(2)S Production and Metabolism Are Involved in Learning and Memory in Drosophila melanogaster
  • Experimental evolution under varying sex ratio and nutrient availability modulates male mating success in Drosophila melanogaster
  • Plastic responses of males and females interact to determine mating behavior
  • Evidence for stronger sexual selection in males than females using an adapted method of Bateman's classic study of Drosophila melanogaster
  • Cryptic male mate choice for high-quality females reduces male postcopulatory success in future matings
  • Social group composition modulates the role of last male sperm precedence in post-copulatory sexual selection
  • Divergence of a speciation trait through artificial selection: Insights into constraints, by-product effects and sexual isolation
  • Integrating lipid metabolism, pheromone production and perception by Fruitless and Hepatocyte nuclear factor 4
  • The Effect of Chromosomes on Courtship Behavior in Sibling Species of the Drosophila virilis Group
  • Courtship suppression in Drosophila melanogaster: The role of mating failure
  • Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster
  • LIM-kinase 1 effects on memory abilities and male courtship song in Drosophila depend on the neuronal type

    Genes and courtship 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
  • Novel role for ecdysone in Drosophila conditioned behavior: Linking GPCR-mediated non-canonical steroid action to cAMP signaling in the adult brain
  • Temporal mating isolation driven by genetic variation in period
  • Gr33a modulates Drosophila male courtship preference
  • Obp56h modulates mating behavior in Drosophila melanogaster
  • Methyl-CpG binding domain proteins inhibit interspecies courtship and promote aggression in Drosophila
  • Contact chemoreceptors mediate male-male repulsion and male-female attraction during Drosophila courtship
  • Myc suppresses male-male courtship in Drosophila
  • Pleiotropic effects of loss of the Dα1 subunit in Drosophila melanogaster: Implications for insecticide resistance
  • The role of miRNAs in Drosophila melanogaster male courtship behavior
  • The yellow gene influences Drosophila male mating success through sex comb melanization
  • Amplification of Drosophila olfactory responses by a DEG/ENaC Channel promotes male courtship behavior
  • Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons
  • The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila
  • Mating experience modifies locomotor performance and promotes episodic motor activity in Drosophila melanogaster
  • Does sexual experience affect the strength of male mate choice for high-quality females in Drosophila melanogaster?
  • Drosophila oocyte proteome composition covaries with female mating status
  • Condition-dependent interaction between mating success and competitive fertilization success in Drosophila melanogaster
  • Sight of parasitoid wasps accelerates sexual behavior and upregulates a micropeptide gene in Drosophila
  • Biochemical evidence accumulates across neurons to drive a network-level eruption
  • CaMKII measures the passage of time to coordinate behavior and motivational state
  • Drosophila females receive male substrate-borne signals through specific leg neurons during courtship
  • Orcokinin neuropeptides regulate reproduction in the fruit fly, Drosophila melanogaster
  • Drosophila Lysophospholipase Gene swiss cheese Is Required for Survival and Reproduction
  • Regulation of Drosophila courtship behavior by the Tlx/tailless-like nuclear receptor, dissatisfaction
  • Pre-copulatory reproductive behaviours are preserved in Drosophila melanogaster infected with bacteria
  • Mutations in trpgamma, the homologue of TRPC6 autism candidate gene, causes autism-like behavioral deficits in Drosophila
  • Silencing Doublesex expression triggers three-level pheromonal feminization in Nasonia vitripennis males
  • Functional Dissection of Protein Kinases in Sexual Development and Female Receptivity of Drosophila
  • Effects of mitochondrial haplotype on pre-copulatory mating success in male fruit flies (Drosophila melanogaster)
  • The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship

    Neural circuits and courtship behavior
  • The neural circuitry that functions as a switch for courtship versus aggression in Drosophila males
  • Multimodal chemosensory circuits controlling 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
  • Excitation and inhibition onto central courtship neurons biases mate choice
  • 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
  • Odorant responses and courtship behaviors influenced by at4 neurons in Drosophila
  • SIK3-HDAC4 signaling regulates Drosophila circadian male sex drive rhythm via modulating the DN1 clock neurons
  • The PKA-C3 catalytic subunit is required in two pairs of interneurons for successful mating of Drosophila
  • Visual projection neurons mediating directed courtship in Drosophila
  • Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila
  • Activity-dependent visualization and control of neural circuits for courtship behavior in the fly Drosophila melanogaster
  • The effects of target contrast on Drosophila courtship
  • Neurons that function within an integrator to promote a persistent behavioral state in Drosophila
  • Drosulfakinin signaling in fruitless circuitry antagonizes P1 neurons to regulate sexual arousal in Drosophila
  • Drosulfakinin signaling modulates female sexual receptivity in Drosophila
  • A feedforward circuit regulates action selection of pre-mating courtship behavior in female Drosophila
  • A neural circuit encoding mating states tunes defensive behavior in Drosophila
  • Circuit and Behavioral Mechanisms of Sexual Rejection by Drosophila Females
  • Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons
  • Hormonal control of motivational circuitry orchestrates the transition to sexuality in Drosophila
  • Sexually dimorphic peripheral sensory neurons regulate copulation duration and persistence in male Drosophila
  • Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster
  • Increased dopamine level enhances male-male courtship in Drosophila
  • Dopaminergic neurons mediate male Drosophila courtship motivational state

  • 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
  • High fat diet alters Drosophila melanogaster sexual behavior and traits: decreased attractiveness and changes in pheromone profiles
  • The role of cuticular hydrocarbons in mate recognition in Drosophila suzukii
  • Evolution of a central neural circuit underlies Drosophila mate preferences
  • Pre-imaginal conditioning alters adult sex pheromone response in Drosophila
  • Olfactory landmark-based communication in interacting Drosophila
  • A novel sex difference in Drosophila contact chemosensory neurons unveiled using single cell labeling
  • Social context enhances hormonal modulation of pheromone detection in Drosophila
  • The genetics of male pheromone preference difference between Drosophila melanogaster and Drosophila simulans>
  • Mate discrimination among subspecies through a conserved olfactory pathway
  • Hormonal modulation of pheromone detection enhances male courtship success
  • Mating increases Drosophila melanogaster females' choosiness by reducing olfactory sensitivity to a male pheromone
  • Large-scale characterization of sex pheromone communication systems in Drosophila
  • The female sex pheromone (Z)-4-undecenal mediates flight attraction and courtship in Drosophila melanogaster
  • "Scent of a fruit fly": Cuticular chemoprofiles after mating in differently fed Drosophila melanogaster (Diptera: Drosophilidae) strains
  • A pleiotropic chemoreceptor facilitates the production and perception of mating pheromones
  • Male size does not affect the strength of male mate choice for high-quality females in Drosophila melanogaster

    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
  • Female Drosophila melanogaster respond to song-amplitude modulations
  • Light is required for proper female mate choice between winged and wingless males in Drosophila
  • Discovery of a new song mode in Drosophila reveals hidden structure in the sensory and neural drivers of behavior
  • Auditory sensitivity, spatial dynamics, and amplitude of courtship song in Drosophila melanogaster
  • The genetics of mating song evolution underlying speciation: Linking quantitative variation to candidate genes for behavioral isolation
  • A single male auditory response test to quantify auditory behavioral responses in Drosophila melanogaster
  • Calmodulin-binding transcription factor shapes the male courtship song in Drosophila
  • Shared song detector neurons in Drosophila male and female brains drive sex-specific behaviors
  • Evolution of sexual size dimorphism in the wing musculature of Drosophila
  • Behavioral Evolution of Drosophila: Unraveling the Circuit Basis
  • Neural network organization for courtship-song feature detection in Drosophila
  • Drosophila females have an acoustic preference for symmetric males

  • Female copulation song is modulated by seminal fluid
  • Serotonergic neuronal death and concomitant serotonin deficiency curb copulation ability of Drosophila platonic mutants
  • Opposing dopaminergic and GABAergic neurons control the duration and persistence of copulation in Drosophila
  • Neural circuitry coordinating male copulation
  • A neural circuit encoding the experience of copulation in female Drosophila
  • Characterization of the sexually dimorphic fruitless neurons that regulate copulation duration
  • The desaturase1 gene affects reproduction before, during and after copulation in Drosophila melanogaster
  • Ovipositor extrusion promotes the transition from courtship to copulation and signals female acceptance in Drosophila melanogaster

    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
  • A double-negative gene regulatory circuit underlies the virgin behavioral state
  • 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
  • The effect of mating and the male Sex peptide on group behaviour of post-mated female Drosophila melanogaster
  • Sex peptide regulates female receptivity through serotoninergic neurons in Drosophila
  • Sexual conflict drives male manipulation of female postmating responses in Drosophila melanogaster
  • 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
  • A male's seminal fluid increases later competitors' productivity
  • Divergence in transcriptional and regulatory responses to mating in male and female fruitflies
  • The Drosophila post-mating response: Gene expression and behavioral changes reveal perdurance and variation in cross-tissue interactions
  • Structural variation in Drosophila melanogaster spermathecal ducts and its association with sperm competition dynamics
  • Temporal and sequential order of nonoverlapping gene networks unraveled in mated female Drosophila
  • Memory of social experience affects female fecundity via perception of fly deposits

    Ageing and Sexual Traits
  • Ageing desexualizes the Drosophila brain transcriptome

    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).

    Neurons that function within an integrator to promote a persistent behavioral state in Drosophila

    Innate behaviors involve both reflexive motor programs and enduring internal states, but how these responses are coordinated by the brain is not clear. In Drosophila, male-specific P1 interneurons promote courtship song, as well as a persistent internal state that prolongs courtship and enhances aggressiveness. However, P1 neurons themselves are not persistently active. This study identified pCd neurons as persistently active, indirect P1 targets that are required for P1-evoked persistent courtship and aggression. Acute activation of pCd neurons alone is inefficacious but enhances and prolongs courtship or aggression promoted by female cues. Brief female exposure induces a persistent increase in male aggressiveness, an effect abrogated by interruption of pCd activity. pCd activity is not sufficient but necessary for persistent physiological activity, implying an essential role in a persistence network. Thus, P1 neurons coordinate both command-like control of courtship song and a persistent internal state of social arousal mediated by pCd neurons (Jung, 2019).

    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).

    Characterization of the sexually dimorphic fruitless neurons that regulate copulation duration

    Male courtship in Drosophila melanogaster is a sexually dimorphic innate behavior that is hardwired in the nervous system. Understanding the neural mechanism of courtship behavior requires the anatomical and functional characterization of all the neurons involved. Courtship involves a series of distinctive behavioral patterns, culminating in the final copulation step, where sperms from the male are transferred to the female. The duration of this process is tightly controlled by multiple genes. The fruitless (fru) gene is one of the factors that regulate the duration of copulation. Using several intersectional genetic combinations to restrict the labeling of GAL4 lines, this study found that a subset of a serotonergic cluster of fru neurons co-express the dopamine-synthesizing enzyme, tyrosine hydroxylase, and provide behavioral and immunological evidence that these neurons are involved in the regulation of copulation duration (Jois, 2018).

    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).

    A male's seminal fluid increases later competitors' productivity

    Polyandrous females allow for sexual selection to persist after mating. In the event that females successfully mate with more than one male, sperm competition can occur. Seminal fluid proteins can indirectly affect a male's success in sperm competition through reducing the remating behaviour of females, and can directly influence sperm competition through directly displacing competitor sperm or inducing females to eject it. These direct effects on competitor sperm are thought to contribute to the 'second male advantage', whereby the second male to mate sires the majority of offspring. This study shows an additional mechanism where seminal proteins already present within a mated female appear to enhance offspring production of later competitor males, and contribute to second male advantage. Counter to expectation, increased offspring production was not due to a priming effect of greater early female productivity, nor was it through a general and consistent increase in offspring production. Instead, enhanced productivity was solely through lengthening the time that offspring are sired by the second male, indicating that seminal proteins from the first male to mate may enhance second male advantage through a presumably unintended protective effect on subsequent competitor sperm (Nguyen, 2018).

    The Drosophila post-mating response: Gene expression and behavioral changes reveal perdurance and variation in cross-tissue interactions

    Examining cross-tissue interactions is important for understanding physiology and homeostasis. In animals, the female gonad produces signaling molecules that act distally. This study examined gene expression in Drosophila melanogaster female head tissues in 1) virgins without a germline compared to virgins with a germline, 2) post-mated females with and without a germline compared to virgins, and 3) post-mated females mated to males with and without a germline compared to virgins. In virgins, the absence of a female germline results in expression changes in genes with known roles in nutrient homeostasis. At one- and three-days post-mating, genes that change expression are enriched with those that function in metabolic pathways, in all conditions. Female post-mating impacts were systematically examined on sleep, food preference and re-mating, in the strains and time points used for gene expression analyses and compare to published studies. Post-mating, gene expression changes vary by strain, prompting an examination of variation in female re-mating. A genome-wide association study was performed that identified several DNA polymorphisms, including four in/near Wnt signaling pathway genes. Together, these data reveal how gene expression and behavior in females are influenced by cross-tissue interactions, by examining the impact of mating, fertility, and genotype (Newell, 2020).

    Structural variation in Drosophila melanogaster spermathecal ducts and its association with sperm competition dynamics

    The ability of female insects to retain and use sperm for days, months, or even years after mating requires specialized storage organs in the reproductive tract. In most orders, these organs include a pair of sclerotized capsules known as spermathecae. This study reports that some Drosophila melanogaster females exhibit previously uncharacterized structures within the distal portion of the muscular duct that links a spermatheca to the uterus.These 'spermathecal duct presences' (SDPs) may form in either or both ducts and can extend from the duct into the sperm-storing capsule itself. It was further found that the incidence of SDPs varies significantly between genotypes, but does not change significantly with the age or mating status of females, the latter indicating that SDPs are not composed of or stimulated by sperm or male seminal proteins. SDPs affect neither the number of first male sperm held in a spermatheca nor the number of offspring produced after a single mating. However, evidence was found that SDPs are associated with a lack of second male sperm in the spermathecae after females remate. This raises the possibility that SDPs provide a mechanism for variation in sperm competition outcome among females (Hopkins, 2020).

    Temporal and sequential order of nonoverlapping gene networks unraveled in mated female Drosophila

    This study reanalyzed available datasets of gene expression changes in female Drosophila head induced by mating. Mated females present metabolic phenotypic changes and display behavioral characteristics that are not observed in virgin females, such as repulsion to male sexual aggressiveness, fidelity to food spots selected for oviposition, and restriction to the colonization of new niches. This study characterized gene networks that play a role in female brain plasticity after mating using AMINE, a novel algorithm to find dysregulated modules of interacting genes. The uncovered networks of altered genes revealed a strong specificity for each successive period of life span after mating in the female head, with little conservation between them. This finding highlights a temporal order of recruitment of waves of interconnected genes which are apparently transiently modified: the first wave disappears before the emergence of the second wave in a reversible manner and ends with few consolidated gene expression changes at day 20. This analysis might document an extended field of a programmatic control of female phenotypic traits by male seminal fluid (Pasquier, 2022).

    Memory of social experience affects female fecundity via perception of fly deposits

    Animals can exhibit remarkable reproductive plasticity in response  to their social surroundings, with profound fitness consequences. The presence of same-sex conspecifics can signal current or future expected competition for resources or mates. Plastic responses to elevated sexual competition caused by exposure to same-sex individuals have been well-studied in males. However, much less is known about such plastic responses in females, whether this represents sexual or resource competition, or if it leads to changes in investment in mating behaviour and/or reproduction. Drosophila melanogaster to measure the impact of experimentally varying female exposure to other females prior to mating on fecundity before and after mating. Then physical and genetic methods were deployed to manipulate the perception of different social cues and sensory pathways and reveal the potential mechanisms involved. The results showed that females maintained in social isolation prior to mating were significantly more likely to retain unfertilised eggs before mating, but to show the opposite and lay significantly more fertilised eggs in the 24h after mating. More than 48h of exposure to other females was necessary for this social memory response to be expressed. Neither olfactory nor visual cues were involved in mediating fecundity plasticity-instead, the relevant cues were perceived through direct contact with the non-egg deposits left behind by other females. The results demonstrate that females show reproductive plasticity in response to their social surroundings and can carry this memory of their social experience forward through mating. Comparisons of these results with previous work show that the nature of female plastic reproductive responses and the cues they use differ markedly from those of males. The results emphasise the deep divergence in how each sex realises its reproductive success (Fowler, 2022).

    Ageing desexualizes the Drosophila brain transcriptome

    General evolutionary theory predicts that individuals in low condition should invest less in sexual traits compared to individuals in high condition. Whether this positive association between condition and investment also holds between young (high condition) and senesced (low condition) individuals is however less clear, since elevated investment into reproduction may be beneficial when individuals approach the end of their life. To address how investment into sexual traits changes with age, genes were studied with sex-biased expression in the brain, the tissue from which sexual behaviours are directed. Across two distinct populations of Drosophila melanogaster, it was found that old brains display fewer sex-biased genes, and that expression of both male-biased and female-biased genes converges towards a sexually intermediate phenotype owing to changes in both sexes with age. It was further found that sex-biased genes in general show heightened age-dependent expression in comparison to unbiased genes and that age-related changes in the sexual brain transcriptome are commonly larger in males than females.The results hence show that ageing causes a desexualization of the fruit fly brain transcriptome and that this change mirrors the general prediction that low condition individuals should invest less in sexual phenotypes (Malacrino, 2022).

    Divergence in transcriptional and regulatory responses to mating in male and female fruitflies

    Mating induces extensive physiological, biochemical and behavioural changes in female animals of many taxa. In contrast, the overall phenotypic and transcriptomic consequences of mating for males, hence how they might differ from those of females, are poorly described. Post mating responses in each sex are rapidly initiated, predicting the existence of regulatory mechanisms in addition to transcriptional responses involving de novo gene expression. That post mating responses appear different for each sex also predicts that the genome-wide signatures of mating should show evidence of sex-specific specialisation. In this study, we used high resolution RNA sequencing to provide the first direct comparisons of the transcriptomic responses of male and female Drosophila to mating, and the first comparison of mating-responsive miRNAs in both sexes in any species. As predicted, the results revealed the existence of sex- and body part-specific mRNA and miRNA expression profiles. More genes were differentially expressed in the female head-thorax than the abdomen following mating, whereas the opposite was true in males. Indeed, the transcriptional profile of male head-thorax tissue was largely unaffected by mating, and no differentially expressed genes were detected at the most stringent significance threshold. A subset of ribosomal genes in females were differentially expressed in both body parts, but in opposite directions, consistent with the existence of body part-specific resource allocation switching. Novel, mating-responsive miRNAs in each sex were also identified, and a miRNA-mRNA interactions analysis revealed putative targets among mating-responsive genes. This study has shown th structure of genome-wide responses by each sex to mating is strongly divergent, and provide new insights into how shared genomes can achieve characteristic distinctiveness (Fowler, 2019).

    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).

    Imaging brain activity during complex social behaviors in Drosophila with Flyception2

    Optical in vivo recordings from freely walking Drosophila are currently possible only for limited behaviors. This study expanded the range of accessible behaviors with a retroreflective marker-based tracking and ratiometric brain imaging system, permitting brain activity imaging even in copulating male flies. P1 neurons, active during courtship, are inactive during copulation, whereas GABAergic mAL neurons remain active during copulation, suggesting a countervailing role of mAL in opposing P1 activity during mating (Grover, 2020).

    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).

    The effect of mating and the male Sex peptide on group behaviour of post-mated female Drosophila melanogaster

    Sleep is a highly conserved state in animals, but its regulation and physiological function is poorly understood. Drosophila melanogaster is an excellent model for studying sleep regulation and has been used to investigate how sex and social interactions can influence wake-sleep profiles. Previous work has shown that copulation has a profound effect on day time activity and quiescence (siesta sleep) of individual post-mated females. The effect of mating and the transfer of the 36 amino acid sex peptide in the seminal fluid was studied on the behavior of mated female Drosophila populations, where there will be on-going social interactions. The locomotor activity and sleep patterns of virgin and post-mated female D. melanogaster from three laboratory strains (Oregon-R, Canton-S and Dahomey) were recorded in social groups of 20 individuals in a 12-12 h light-dark cycle. Virgin female populations from all three fly strains displayed consolidated periods of low activity in between two sharp peaks of activity, corresponding to lights-on and lights-off. Similar light-correlated peaks were recorded for the mated female populations, however, the low afternoon activity and siesta seen in virgin populations was abolished after mating in all three strains. In contrast, night activity appeared unaffected. This post-mating effect was sustained for several days and was dependent on the male SP acting as a pheromone. Evidence from mixed populations of virgin and mated females suggests that the siesta of non-mated females is not easily disturbed by the presence of highly active post-mated females (Isaac, 2019).

    Sex peptide regulates female receptivity through serotoninergic neurons in Drosophila

    The courtship ritual is a dynamic interplay between males and females. Courtship successfully leading to copulation is determined by the intention of both parties which is conveyed by complex action sequences. In Drosophila, the neural mechanisms controlling the female's willingness to mate, or sexual receptivity, have only recently become the focus of investigations. This study reports that pre-mating sexual receptivity in females requires activity within a subset of serotonergic projection neurons (SPNs), which positively regulate courtship success. Of interest, a male-derived sex peptide, SP, which was transferred to females during copulation acted to inhibit the activity of SPN and suppressed receptivity. Downstream of 5-HT, subsets of 5-HT7 receptor neurons played critical roles in SP-induced suppression of sexual receptivity. Together, this study reveals a complex serotonin signaling system in the central brain of Drosophila which manages the female's desire to mate (Yang, 2023).

    Sexual conflict drives male manipulation of female postmating responses in Drosophila melanogaster

    In many animals, females respond to mating with changes in physiology and behavior that are triggered by molecules transferred by males during mating. In Drosophila melanogaster, proteins in the seminal fluid are responsible for important female postmating responses, including temporal changes in egg production, elevated feeding rates and activity levels, reduced sexual receptivity, and activation of the immune system. It is unclear to what extent these changes are mutually beneficial to females and males or instead represent male manipulation. This study used an experimental evolution approach in which females are randomly paired with a single male each generation, eliminating any opportunity for competition for mates or mate choice and thereby aligning the evolutionary interests of the sexes. After >150 generations of evolution, males from monogamous populations elicited a weaker postmating stimulation of egg production and activity than males from control populations that evolved with a polygamous mating system. Males from monogamous populations did not differ from males from polygamous populations in their ability to induce refractoriness to remating in females, but they were inferior to polygamous males in sperm competition. Mating-responsive genes in both the female abdomen and head showed a dampened response to mating with males from monogamous populations. Males from monogamous populations also exhibited lower expression of genes encoding seminal fluid proteins, which mediate the female response to mating. Together, these results demonstrate that the female postmating response, and the male molecules involved in eliciting this response, are shaped by ongoing sexual conflict (Hollis, 2019).

    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).

    A double-negative gene regulatory circuit underlies the virgin behavioral state

    Virgin females of many species conduct distinctive behaviors, compared with post-mated and/or pregnant individuals. In Drosophila, this post-mating switch is initiated by seminal factors, implying that the default female state is virgin. However, it was recently shown that loss of miR-iab-4/miR-iab-8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgins to execute mated behaviors. This study used genomic analysis of mir-iab-4/8 deletion and hth-microRNA (miRNA) binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. Dsx and Hth proteins are highly complementary in CNS, and Dsx is downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose sensitive to developmental dsx function. Strikingly, depletion of Dsx from very restricted abdominal neurons (SAG-1 cells) abrogates female virgin conducts, in favor of mated behaviors. Thus, a double-negative regulatory pathway in the VNC (miR-iab-4/8 -| Hth -| Dsx) specifies the virgin behavioral state (Garaulet, 2021).

    Females of diverse invertebrate and vertebrate species coordinate multiple behavioral programs with their reproductive state. Mature female virgins are receptive to male courtship and copulation, but following mating and/or pregnancy, they decrease sexual activity and modulate behaviors to generate and foster their children. Behavioral remodeling associated with the female reproductive state includes increased aggression and nest building in avians and mammals and decreased male acceptance, increased egg-laying, and appetitive/metabolic changes in insects. The genetic and neurological control of this process has been intensively studied in fruit flies, where sexual activity induces the post-mating switch, a host of behavioral changes collectively known as post-mating responses (PMRs) (Garaulet, 2021).

    In Drosophila, as in other species, 'virgin' is typically considered the default behavioral state, because factors that induce PMRs are transferred in seminal fluids during copulation. Among these, Sex Peptide (SP) is necessary and sufficient to drive most female post-mated behaviors. SP signals via uterine SP sensory neurons (SPSNs). Some SPSN+ neurons contact abdominal interneurons in the ventral nerve cord (VNC) that express myoinhibitory peptide, which input into a restricted population of ascending neurons (SP abdominal ganglion [SAG] neurons) that project to the posterior brain, including pC1 neurons. This outlines an ascending flow of information for how a seminal fluid peptide can alter female brain activity. The brain integrates this with auditory and visual cues to coordinate diverse behaviors mediated by distinct lineages of descending neurons and VNC populations that modulate specific behaviors according to internal state and external stimuli (Garaulet, 2021).

    Recently, it was found that post-transcriptional suppression of the homeobox gene homothorax (hth) within the VNC is critical to implement the virgin behavioral state. Of note, deletion of the Bithorax Complex (BX-C) locus mir-iab-4/8, point mutations of their binding sites in hth, or deletion of the hth neural-specific 3' UTR extension bearing many of these microRNA (miRNA) sites all cause mutant female virgins to perform mated behaviors. Thus, the failure to integrate two post-transcriptional regulatory inputs at a single target gene prevents females from appropriately integrating their sexual internal state with external behaviors (Garaulet, 2021).

    Recognition of the transcription factor Hth as a target of regulatory circuits for virgin behavior implies that downstream loci may serve as a functional output for this process. This study used molecular genetic profiling to identify a critical requirement for Doublesex (Dsx) to implement the female virgin behavioral state. Dsx has been well studied with respect to differentiation of sexually dimorphic traits, but its roles in post-mitotic neurons are little known. This study found that expression of Dsx in the VNC mediates virgin behavior, and that modulation of Dsx in only a few abdominal VNC neurons is sufficient to convert the suite of female virgin behaviors into mated conducts (Garaulet, 2021).

    Recent work established how miRNA mediated suppression of the transcription factor Hth to safeguard the virgin female behavioral state. Using engineered alleles and spatio-temporal hth manipulations, this study demonstrated a developmental requirement for post-transcriptional regulation of Hth within the abdominal ganglion of the CNS for female behavior. However, Hth was not required in otherwise wild-type VT-switch neurons for execution of virgin behaviors, implying that expression of Hth in the abdominal VNC must normally be prevented. This involves integration of two mechanisms: a high density of BX-C miRNA binding sites (miR-iab-4/8) within the hth-hΔ3' UTR, as well as a neural-specific 3' UTR elongation, which unveils many of these sites only on neural hth isoforms (Garaulet, 2021).

    This study has extended this regulatory axis by showing that loss of BX-C miRNAs, acting through derepressed Hth, leads to downregulation of the Dsx in the abdominal VNC. Dsx is well-known as a master sex determination transcription factor, and it shows localized expression in specific CNS domains. However, although the activity of Dsx-expressing neurons per se has been implicated in the switch in females, the functions of Dsx in post-mitotic neurons are less well defined. This work reveals that Dsx itself is a central component in specifying virgin behavior, because its restricted suppression in as few as four (SAG-1+) neurons is sufficient to induce post-mated behaviors. It remains to be better defined how SAG-1 neurons are affected by depletion of Dsx. No overt differentiation defects were observed, but an effect of masculinization cannot be ruled out. Otherwise, the recent work suggests an activity defect in a general population of switch neurons in the miRNA mutant, but more direct analysis of dsx-depleted SAG-1 neurons awaits (Garaulet, 2021).

    Altogether, in contrast with highly branched regulatory networks that are bioinformatically inferred to lie downstream of individual miRNAs, this study revealed a linear, double-negative regulatory cascade comprising miRNAs and two transcription factors (see SAG-1 neurons specifically require Dsx for a suite of female virgin behaviors). These findings provide impetus to assess possible direct regulation of Dsx by Hth, as well as to elucidate Dsx targets that are relevant to female behavioral control. Overall, this study expands a genetic hierarchy that is essential for females to couple the virgin internal state with appropriate behaviors (Garaulet, 2021).

    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).

    Dopamine and serotonin are both required for mate-copying in Drosophila melanogaster

    Mate-copying is a form of social learning in which the mate-choice decision of an individual (often a female) is influenced by the mate-choice of conspecifics. Drosophila melanogaster females are known to perform such social learning, and in particular, to mate-copy after a single observation of one conspecific female mating with a male of one phenotype, while the other male phenotype is rejected. This study shows that this form of social learning is dependent on serotonin and dopamine. Using a pharmacological approach, dopamine or serotonin synthesis in adult virgin females with 3-iodotyrosine (3-IY) and DL-para-chlorophenylalanine (PCPA), respectively, and then their mate-copying performance was tested. While control females without drug treatment copied the choice of the demonstrator, drug-treated females with reduced dopamine or serotonin chose randomly. To ensure the specificity of the drugs, the direct precursors of the neurotransmitters, either the dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) or the serotonin precursor 5-L-hydroxytryptophan (5-HTP) were given together with the drug, (respectively 3-IY and PCPA) resulting in a full rescue of the mate-copying defects. This indicates that dopamine and serotonin are both required for mate-copying. These results give a first insight into the mechanistic pathway underlying this form of social learning in D. melanogaster (Monier, 2018).

    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).

    Motivation, perception, and chance converge to make a binary decision

    This study reveals a central role for chance neuronal events in the decision of a male fly to court, which can be modeled as a coin flip with odds set by motivational state. The decision is prompted by a tap of a female with the male's pheromone-receptor-containing foreleg. Each tap evokes competing excitation and inhibition onto P1 courtship command neurons. A motivating dopamine signal desensitizes P1 to the inhibition, increasing the fraction of taps that successfully initiate courtship. Once courtship has begun, the same dopamine tone potentiates recurrent excitation of P1, maintaining the courtship of highly motivated males for minutes and buffering against termination. Receptor diversity within P1 creates separate channels for tuning the propensities to initiate and sustain courtship toward appropriate targets. These findings establish a powerful invertebrate system for cue-triggered binary decisions and demonstrate that noise can be exploited by motivational systems to make behaviors scalable and flexible (Zhang, 2018).

    Neural circuit mechanisms of sexual receptivity in Drosophila females

    Choosing a mate is one of the most consequential decisions a female will make during her lifetime. A female fly signals her willingness to mate by opening her vaginal plates, allowing a courting male to copulate. Vaginal plate opening (VPO) occurs in response to the male courtship song and is dependent on the mating status of the female. How these exteroceptive (song) and interoceptive (mating status) inputs are integrated to regulate VPO remains unknown. This study characterize the neural circuitry that implements mating decisions in the brain of female Drosophila melanogaster. VPO is controlled by a pair of female-specific descending neurons (vpoDNs). The vpoDNs receive excitatory input from auditory neurons (vpoENs), which are tuned to specific features of the D. melanogaster song, and from pC1 neurons, which encode the mating status of the female. The song responses of vpoDNs, but not vpoENs, are attenuated upon mating, accounting for the reduced receptivity of mated females. This modulation is mediated by pC1 neurons. The vpoDNs thus directly integrate the external and internal signals that control the mating decisions of Drosophila females (Wang, 2020).

    Drosophila males woo potential mates by vibrating their wings to produce a species-specific courtship song. The male song induces deflections of the female aristae, thereby activating auditory sensory neurons that project to the central brain. Several types of song-responsive neurons have been identified in the female brain, but it is unknown whether and how these neurons regulate sexual receptivity. How a female responds to the song of a male is highly dependent on whether or not she has previously mated. Once mated, females store sperm for days to weeks, and during this time are reluctant to mate again. A male seminal fluid peptide (sex peptide) binds to sperm and signals the presence of sperm in the female reproductive tract through an ascending pathway from the sex peptide sensory neurons (SPSNs) in the uterus via the sex peptide abdominal ganglion (SAG) neurons in the ventral nerve cord to the pC1 neurons in the brain. Sex peptide attenuates neuronal activity in the SPSN, SAG and pC1 neurons, thereby reducing sexual receptivity after mating. This study sought to investigate how these distinct external and internal signals are integrated in the female brain to control VPO, the motor output that signals the willingness of the female to mate (Wang, 2020).

    Female receptivity is impaired by blocking the activity of the approximately 2,000 neurons that express either of the two sex-determination genes, fruitless (fru) or doublesex (dsx). This class of neurons includes the fru+ dsx+ SPSNs, the dsx+ SAGs and the dsx+ pC1 cells. To search for other fru+ or dsx+ neurons that contribute to female receptivity, a collection of 234 sparse driver lines specific for various fru+ or dsx+ cell types was screened. These driver lines were used to genetically silence each of these cell types, and virgin females were assayed for their frequency of copulation within 10 min of being individually paired with naive wild-type males. Of the seven lines with the strongest reduction in receptivity, two labelled the SPSNs, one labelled the SAGs and one labelled the pC1 cells. The other three lines targeted a pair of female-specific descending neurons, which were named vpoDNs. These neurons are dsx+ , fru- and cholinergic. Their dendrites arborize primarily in the lateral protocerebrum and their axons project to multiple regions of the ventral nerve cord, including the abdominal ganglion (Wang, 2020).

    Acute optogenetic silencing or genetic ablation of the vpoDNs rendered virgin females unreceptive, markedly reducing the frequency of VPO but not the intensity of male courtship. Conversely, photoactivation of vpoDNs reliably triggered VPO in isolated virgin females. no peripheral expression was driven by the vpoDN lines and by severing the abdominal nerve, confirmed that the VPO response is indeed due to activation of central neurons (Wang, 2020).

    Mated females are less receptive than virgins, which was found to correlate with a lack of VPO. To assess whether the failure to perform VPO accounts for the low receptivity of mated females, the vpoDNs were activated in mated females as they were being courted by wild-type males. Whereas control females never copulated during a 1-h assay, approximately 30%-50% of the vpoDN-activated females did remate. A similar remating frequency was observed upon vpoDN activation in mated females paired with wingless males, which court but cannot sing. Thus, direct activation of vpoDNs bypasses the need for both the internal state (virginity) and the external cue (song) that normally combine to elicit VPO (Wang, 2020).

    It was observed that wing extension is the most frequent male action just before female VPO, and that both VPO and copulation rates are reduced if males are muted by removing their wings or females deafened by removing their aristae. These results suggested that the vpoDNs might be activated by male song. Indeed, in two-photon calcium-imaging experiments, a robust increase of calcium levels was detected in the neurites of vpoDNs in virgin females upon playback of male courtship song, but not in response to white noise. The response to courtship song was lost when the aristae were immobilized to deafen the female. Song responses have also been reported for the pMN2 neurons, which are morphologically similar to vpoDNs and also dsx+ , although their reported functions differ (Wang, 2020).

    The vpoDN dendrites lie mostly in the superior lateral protocerebrum, with no obvious arborizations within the antennal mechanosensory centre (AMMC), the primary auditory neuropil, or in the wedge region, a secondary auditory neuropil known to include song-responsive neurons. Therefore this study sought to trace potential pathways from these auditory centres to the vpoDNs within the electron microscopy volume of a full adult female brain18 (FAFB). A single vpoDN was identified in each hemisphere and the vpoDN was extensively traced in the right hemisphere as well as its presynaptic partners, identifying a total of 45 neurons with at least 10 synapses impinging onto vpoDN (Wang, 2020).

    None of the vpoDN input neurons innervate the AMMC, but at least two cell types have extensive arborizations within the wedge. Multiple split-GAL4 driver lines specific for these two cell types were obtained. Fluorescence in situ hybridization predicted, and whole-cell recording confirmed, that one of these cell types is excitatory and the other is inhibitory. Accordingly, these two cell types were named the vpoENs and vpoINs, respectively. Within FAFB there are two vpoEN cells and 14 vpoIN cells in each hemisphere (Wang, 2020).

    Optogenetic silencing and activation experiments were performed to examine the roles of vpoENs and vpoINs in VPO and receptivity. Acute inhibition of the vpoENs significantly reduced the frequency of copulation and VPO when virgin females were paired with males. Conversely, strong optogenetic activation of vpoENs elicited VPO in isolated females, mimicking activation of vpoDNs. In virgin females paired with males, activating vpoINs suppressed mating and VPO, whereas silencing vpoINs had no effect. Thus, vpoENs and vpoINs promote and suppress, respectively, both VPO and receptivity (Wang, 2020).

    Using two-photon calcium imaging, it was found that both vpoENs and vpoINs, as with vpoDNs, responded to playback of male courtship songs. The courtship song varies considerably between different Drosophila species and is the primary cue the female uses for species recognition. To test whether the vpoDNs, vpoENs and vpoINs are specifically tuned to the D. melanogaster courtship song, natural courtship songs were presented from seven other Drosophila species, selecting two representative audio clips from each species. The vpoDNs showed little or no response to any of these songs, the vpoENs responded to one or two clips from five species, and the vpoINs responded to all but one clip from one species (Wang, 2020).

    The Drosophila song comprises two main components: brief trains of high-amplitude pulses (pulse song) and continuous low-amplitude oscillations (sine song). The pulse song is the primary basis for species recognition, and in D. melanogaster consists of a series of pulses with an inter-pulse interval (IPI) of approximately 35 ms and a carrier frequency of 200-400 Hz. This study generated synthetic D. melanogaster pulse songs in which systematically varied the IPI from 10 ms to 300 ms and the carrier frequency from 100 Hz to 800 Hz. Both vpoDNs and vpoENs responded robustly only to pulse songs with an IPI near 35 ms, and preferred lower carrier frequencies. Neither vpoDNs nor vpoENs responded to white noise or synthetic sine song, even if its amplitude was increased to match that of the pulse song. The vpoINs were much more broadly tuned, responding to pulse songs across a wide range of IPIs and with higher carrier frequencies. They also responded weakly to both sine song and white noise (Wang, 2020).

    Artificial pulse songs were generated for each of the other species, again systematically altering the IPI from 10 ms to 300 ms. Notably, the vpoDNs responded to the pulse songs of five other species once their IPI was shifted to match that of the D. melanogaster song. Together, these data establish that the vpoDNs are finely tuned to the D. melanogaster pulse song, owing to their selectivity for an IPI of about 35 ms. This narrow tuning may arise through a combination of strong excitation from highly selective vpoENs and weak inhibition from broadly responsive vpoINs (Wang, 2020).

    Having determined how auditory input controls VPO and sexual receptivity, how this response is modulated by the mating status of the female was examined. VPO may be attenuated after mating either because the vpoENs and vpoDNs are less potent at eliciting VPO, or because they are less excited by song. In optogenetic activation and calcium-imaging experiments, it was found that vpoDNs are equally potent in mated and virgin females, whereas both the basal calcium levels and the response to courtship song were lower in mated females than in virgins. By contrast, the vpoENs were significantly less potent at eliciting VPO in mated than in virgin females. Although basal fluorescence of vpoENs was slightly higher in mated females than in virgins, their song responses were indistinguishable. Calcium levels were imaged in vpoINs; the basal fluorescence and song responses of these cells were similar in mated and virgin females. Thus, these data show that vpoENs have a similar response to song in mated females as they do in virgins, but they are less able to excite vpoDNs in mated females (Wang, 2020).

    The cell type with the most synaptic inputs to the vpoDNs was the pC1 cells. Photoactivation of pC1 cells elicited a strong depolarization and action potentials in vpoDNs. The pC1 cells receive input from the SPSN-SAG pathway, which is silenced upon mating. The reduced excitability of vpoDNs after mating may therefore be explained at least in part by the lower activity of pC1 cells, one of their major excitatory inputs. In support of this hypothesis, it was found that acutely silencing either SAG or pC1 neurons reduced the frequency of VPO in virgin females to that of mated females. Conversely, photoactivation of pC1 cells in mated females restored both VPO and sexual receptivity in response to courtship by intact but not wingless males. Moreover, transient (5-s) photoactivation of the pC1 neurons in mated females increased the sensitivity of vpoDNs to courtship song, demonstrating that pC1 cells control vpoDN excitability. This effect persisted for up to 25 s after photoactivation of pC1 cells (Wang, 2020).

    It is concluded that the decision of the female fly to mate or not to mate is largely determined by how the vpoDNs integrate signals from two direct synaptic inputs: the vpoENs, which are selectively tuned to the conspecific male courtship song, and the pC1 cells, which encode the mating status of the female. When the male sings, female vpoENs are activated; whether or not this leads to vpoDN activation and hence VPO depends on the level of pC1 activity, which is higher in virgins than in mated females. The neural computation that underlies this state-dependent sensorimotor transformation remains to be determined; this will require methods for simultaneously manipulating and recording from all three cell types. One possibility is that the pC1 inputs gate the vpoEN inputs in a nonlinear fashion. No obvious spatial segregation of vpoEN and pC1 synapses onto the vpoDN dendrites were noted, as might be expected if these inputs are indeed processed hierarchically. Alternatively, vpoDNs might simply use a sum-to-threshold mechanism, in which the combined input from vpoENs and pC1s must exceed a certain level to elicit action potentials in vpoDNs. In this scenario, the lower pC1 activity after mating would necessitate a stronger vpoEN input to activate the vpoDNs. This model may account for the observation that wild-caught females are often multiply mated, consistent with the prediction from evolutionary theory that a mated female would increase her reproductive fitness by remating when she is courted by a male of higher quality than her first partner (Wang, 2020).

    The many other, as yet uncharacterized, inputs to pC1, vpoEN and vpoDN cells may convey additional signals that modulate female receptivity. For example, pC1 cells are reported to respond to a male pheromone, which may serve to enhance the receptivity of both virgin and mated females. The persistent enhancement of vpoDN song responses upon transient activation of pC1 cells resembles the persistent state of courtship arousal induced in males by transient activation of the male pC1 counterparts. The female pC1 cells may therefore encode both mating status and, as with their male counterparts, a lasting state of mating arousal induced by sensory cues from potential mates. The ensuing interaction between the two sexes involves a coordinated sequence of signals and responses, as exemplified by the male singing to elicit female VPO. In both sexes, these sensorimotor transformations may not be directly mediated by pC1 cells, as commonly thought, but rather modulated by the arousal states they encode. The neural architecture that is reported in this study for the control of Drosophila female sexual receptivity may thus also serve as a paradigm for understanding male sexual behaviour, and perhaps more generally for the state-dependent signal processing that underlies behavioural decisions across a range of species (Wang, 2020).

    Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons

    Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both 'classical' and 'modulatory' signals that could produce diverse and/or complementary effects in associated circuits. This study establishes that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identifed the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. These findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network (Sherer, 2020).

    Addressing the functional complexities of 'one neuron, multiple transmitters' is critical to understanding how neuron communication, circuit computation, and behavior can be regulated by a single neuron. Over many decades, significant progress has been made elucidating the functional properties of neurons co-expressing neuropeptides and small molecule neurotransmitters, where the neuropeptide acts as a co-transmitter and modulates the action of the neurotransmitter. Only recently have studies begun to examine the functional significance of co-transmission by a fast-acting neurotransmitter and a slow-acting monoamine (Sherer, 2020).

    This study has demonstrated that OA neurons express dVGLUT and has utilized a new genetic tool to remove dVGLUT in OA-glutamate neurons. Quantifying changes in the complex social behaviors of aggression and courtship revealed that dVGLUT in brain OGNs is required to promote aggressive behavior and a specific behavioral pattern, the lunge. In contrast, males deficient for dVGLUT function do not exhibit an increase in inter-male courtship. These results establish a previously undetermined role for dVGLUT in OA neurons located in the adult brain and reveal glutamate uncouples aggression from inter-male courtship. It has been suggested that classical neurotransmitters and monoamines present in the same neuron modulate each other's packaging into synaptic vesicles or after release via autoreceptors. For example, a reduction of dVGLUT in DA-glutamate neurons resulted in decreased AMPH-stimulated hyperlocomotion in Drosophila and mice suggesting a key function of dVGLUT is the mediation of vesicular DA content. In this study, the independent behavioral changes suggests enhancing the packaging of OA into vesicles is not the sole function of dVGLUT co-expression and suggests differences in signaling by OA from OGNs on courtship-related circuitry (Sherer, 2020).

    Co-transmission can generate distinct circuit-level effects via multiple mechanisms. One mechanism includes spatial segregation; the release of two neurotransmitters or a neurotransmitter and monoamine from a single neuron occurring at different axon terminals or presynaptic zones. Recent studies examining this possible mechanism have described (1) the release of GLU and DA from different synaptic vesicles in midbrain dopamine neurons and (2) the presence of VMAT and VGLUT microdomains in a subset of rodent mesoaccumbens DA neurons. This study expressed a new conditionally expressed epitope-tagged version of VMAT in OGNs and visualized endogenous dVGLUT via antibody labeling. Within OGNs, the colocalization of VMAT and dVGLUT puncta was not complete suggesting the observed behavioral phenotype differences may be due to spatial differences in OA signaling (Sherer, 2020).

    A second mechanism by which co-transmission may generate unique functional properties relies on activating distinct postsynaptic receptors. In Drosophila, recent work has identified a small population of male-specific neurons that express the alpha-like adrenergic receptor, OAMB, as aggression-promoting circuit-level neuronal targets of OA modulation independent of any effect on arousal and separately knockdown of the Rdl GABAa receptor in a specific doublesex+ population stimulated male aggression (Watanabe, 2017). Future experiments identifying downstream targets that express both glutamate and octopamine receptors would be informative, as well as using additional split-Gal4 lines to determine if segregation of transporters is a hallmark of the majority of OGNs. Finally, a third possible mechanism is Glu may be co-released from OGNs and act on autoreceptors to regulate presynaptic OA release (Sherer, 2020).

    Deciphering the signaling complexity that allows neural networks to integrate external stimuli with internal states to generate context-appropriate social behavior is a challenging endeavor. Neuromodulators including monoamines are released to signal changes in an animal's environment and positively or negatively reinforce network output. In invertebrates, a role for OA in responding to external chemosensory cues as well as promoting aggression has been well-established. In terms of identifying specific aggression circuit-components that utilize OA, previous results determined OA neurons directly receive male-specific pheromone information and the aSP2 neurons serve as a hub through which OA can bias output from a multi-functional social behavior network towards aggression. The ability of OA to bias behavioral decisions based on positive and negative reinforcement was also recently described for food odors. In vertebrates, it has been proposed that DA-GLU cotransmission in the NAc medial shell might facilitate behavioral switching. The finding that the majority of OA neurons are glutamatergic, suggests that the complex social behavior of aggression may rely on small subsets of neurons that both signal the rapid temporal coding of critical external stimuli as well as the frequency coding of such stimuli resulting in the enhancement of this behavioral network. One implication of the finding regarding the separable OA-dependent inhibition of inter-male courtship is the possibility of identifying specific synapses or axon terminals that when activated gate two different behavioral outcomes. A second implication is that aggressive behavior in other systems may be modified by targeting GLU function in monoamine neurons (Sherer, 2020).

    Finally, monoamine-expressing neurons play key roles in human behavior including aggression and illnesses that have an aggressive component such as depression, addiction, anxiety, and Alzheimer's. While progress is being made in addressing the functional complexities of dual transmission, the possible pathological implications of glutamate co-release by monoamine neurons remains virtually unknown. Analyzing the synaptic vesicle and release properties of monoamine-glutamate neurons could offer new possibilities for therapeutic interventions aimed at controlling out-of-context aggression (Sherer, 2020).

    Cell assembly analysis of neural circuits for innate behavior in Drosophila melanogaster using an immediate early gene stripe/egr-1

    Innate behavior, such as courtship behavior, is controlled by a genetically defined set of neurons. To date, it remains challenging to visualize and artificially control the neural population that is active during innate behavior in a whole-brain scale. Immediate early genes (IEGs), whose expression is induced by neural activity, can serve as powerful tools to map neural activity in the animal brain. IEGs were screened for in the vinegar fly Drosophila melanogaster and stripe/egr-1 was identified as a potent neural activity marker. Focusing on male courtship as a model of innate behavior, it was demonstrated that stripe-GAL4-mediated reporter expression can label fruitless (fru)-expressing neurons involved in courtship in an activity (experience)-dependent manner. Optogenetic reactivation of the labeled neurons elicited sexual behavior in males, whereas silencing of the labeled neurons suppressed courtship and copulation. Further, by combining stripe-GAL4-mediated reporter expression and detection of endogenous Stripe expression, methods were establised that can label neurons activated under different contexts in separate time windows in the same animal. The cell assembly analysis of fru neural population in males revealed that distinct groups of neurons are activated during interactions with a female or another male. These methods will contribute to building a deeper understanding of neural circuit mechanisms underlying innate insect behavior (Takayanagi-Kiya, 2023).

    Hormonal control of motivational circuitry orchestrates the transition to sexuality in Drosophila

    Newborns and hatchlings can perform incredibly sophisticated behaviors, but many animals abstain from sexual activity at the beginning of life. Hormonal changes have long been known to drive both physical and behavioral changes during adolescence, leading to the largely untested assumption that sexuality emerges from organizational changes to neuronal circuitry. This study shows that the transition to sexuality in male Drosophila is controlled by hormonal changes, but this regulation is functional rather than structural. In very young males, a broadly acting hormone directly inhibits the activity of three courtship-motivating circuit elements, ensuring the complete suppression of sexual motivation and behavior. Blocking or overriding these inhibitory mechanisms evokes immediate and robust sexual behavior from very young and otherwise asexual males. Similarities to mammalian adolescence suggest a general principle in which hormonal changes gate the transition to sexuality not by constructing new circuitry but by permitting activity in otherwise latent motivational circuit elements (Zhang, 2021).

    The identification and evaluation of a potential mating partner require multisensory integration, but, in the experimental paradigm used in this study, the decision to court is ultimately triggered by a male tapping a female with his pheromone receptor-bearing leg. The tap delivers parallel excitatory and inhibitory inputs to the male's P1 courtship command neurons, which initiate and maintain courtship when sufficiently stimulated. The sensitivity of P1 neurons to the inhibitory input from a tap is decreased by a local dopamine signal, which, in mature males, is tuned to reflect recent mating history. If a male has not mated for several days, the dopamine tone is high, increasing the probability that a tap will lead to courtship. Once courtship has commenced, the same dopamine signal maintains courtship bouts for the tens of seconds to several minutes required before mating starts. This motivating dopaminergic activity is maintained for days in the absence of females, is decremented by each mating, and slowly recovers over 3 to 4 days (Zhang, 2021).

    Matings promote satiety by activating a set of copulation reporting neurons (CRNs). These neurons, as a population, project dendrites to the external genitalia and send axons to the brain, where they reduce the activity of fruitless-positive neuropeptide F (NPF) producing neurons. This decrease is relayed through the NPF receptor to the motivation-promoting dopamine neurons, reducing their activity. After a few matings, substantial satiety is induced, and reproductive motivation remains low for several days. Mating drive has an intrinsic tendency to recover because of recurrent excitation: NPF neurons excite and are excited by Doublesex-positive pCd neurons, forming a loop that holds and gradually accumulates activity during periods of abstinence. Loop activity is prevented from immediately rebounding by the activity-dependent transcription factor CREB2, which, during the period of high motivation that precedes mating, transcribes inhibitory genes (e.g., potassium channels) that will sustain the decremented activity state, forcing recovery to proceed on a biochemical, rather than electrical, time scale (Zhang, 2021).

    This circuit architecture suggests several hypotheses for the prevention of courtship in newly eclosed males. For example, a juvenile male might not recognize or tap females; the CRNs may be constantly active, inducing overwhelming satiety; or, as in the organizational hypothesis, some or all of the courtship circuitry may not be fully developed. This study presents evidence refuting all these hypotheses. Instead, this study found that a spike in juvenile hormone levels at eclosion directly and selectively imposes long-lasting activity suppression on all known motivation-promoting circuit elements: both populations of loop neurons and the downstream dopaminergic neurons. Overriding any of these repressive mechanisms evokes robust courtship from extremely young males, providing clear evidence that much of the reproductive circuitry is developed and functional but lays dormant in juveniles. The multitiered suppression of motivational circuitry appears to be the key difference between the complete inactivation of mating drive in early life and the fluctuations experienced in adulthood (Zhang, 2021).

    The story of Eden indicates that the diverse behavioral changes coinciding with reproductive maturity had been fascinating and frustrating humans thousands of years before they were known to involve the brain. The hormonal signaling pathways that trigger these changes in mammals are now understood, but little is known about their mechanistic impact on behavioral and motivational circuitry. Teleological explanations for delays in the onset of sexual behavior likely vary across species, but the pervasiveness of this phenomenon suggests the possibility of a core mechanistic conservation that transcends idiosyncrasies of duration, purpose, and even the molecular nature of the hormonal trigger. The system described in this paper for analyzing the transition to sexuality allows rapid insight, with principles that suggest molecular and circuit hypotheses in other animals and other late-emerging brain functions (Zhang. 2021).

    One obvious but likely superficial difference in the control of behavioral maturation between Drosophila and mammals is the sign of the regulation by circulating hormones. Although some gonadal hormones can suppress mating drive, in juvenile male rodents, experimental elevation of testosterone causes precocious mating behavior, whereas this study found that juvenile hormone suppresses mating behavior. This discordance is reconciled early in signal transduction, as loss of the Met receptor for juvenile hormone decreases sexual behavior, similar to the effects of androgen (testosterone) receptor antagonists in humans. The receptors for testosterone, estrogen, and progesterone are all transcription factors, as are the Met and Gce receptors for juvenile hormone. Unlike ablating the source of juvenile hormone or overriding its action at the level of neuronal activity or CREB activation, removing the suppressive receptor, Gce, does not cause substantial juvenile courtship. This points to the likely existence of yet another juvenile hormone receptor, the identification of which will provide a deeper understanding of the suppressive mechanisms used to completely, selectively, and transiently inactivate this fundamental drive (Zhang. 2021).

    In mature animals, the recurrent loop that drives the male to court also primes itself for satiety through activity-induced, CREB-mediated production of suppressive TASK7-containing potassium channel complexes. This motivation-suppressing module is also used in juveniles, with hormonal signaling causing CREB2 activation. Although additional suppressive effectors have not yet been identified in juveniles, evidence is seen of fast-acting suppression by juvenile hormone and direct suppression of the dopaminergic neurons, neither of which can be accounted for by CREB activity in the pCd/NPF loop. Parallel fast and slow responses have also been found in the response to mammalian gonadal hormones. Multiple repressive mechanisms are likely necessary to completely inactivate mating drive, since individually removing or overriding individual suppressive effectors allows juvenile courtship. This is again similar to findings in mammals, where administering testosterone to either the preoptic area of the hypothalamus or to the medial amygdala suffices to restore mating behaviors in castrated males (Zhang. 2021).

    A second clear difference between Drosophila and vertebrate juvenile stages is the time scale. The fly system requires suppression of sexual behavior for days, not months or years [even 150 years as suggested in Greenland sharks]. However, several nonneuronal structures are still maturing in juvenile flies, such as the abdominal musculature, the cuticle, and the ejaculatory bulb, indicating that the neuronal circuitry that will eventually animate these features may require structural development as well. Although the kind of detailed anatomical analysis required to argue for or against fine-scale structural rearrangements, courtship circuitry appears grossly normal in juveniles, and the stimulation of several circuit elements (P1, dopaminergic, NPF, and pCd) can rapidly drive coherent courtship. While it is not yet known whether the courtship performed by juvenile males is identical to that of mature males (e.g., in song production),the current findings serve as strong evidence against a strict developmental ontogeny for the posteclosion emergence of reproductive motivation and behavior (Zhang. 2021).

    A recent report found structural changes in a key hypothalamic population during estrus and showed that these neurons could not drive sexual behaviors when activated in ovariectomized mice. It is noted that it was not possible to restore sexual behavior in male flies if subpopulations of individually necessary dopaminergic neurons are stimulated, a consideration that leaves open the possibility that the motivational and behavioral circuitry may be at least partially functional, but suppressed, in nonestrus female mice. The most parsimonious explanation may be that both structural and activational changes take place between asexual and sexual periods of life. Increased connectivity in motivational circuitry could be a result, rather than a cause, of increased activity, as has been reported in a growing number of systems. No reports were found of neuronal stimulation eliciting juvenile sexual behavior in the mammalian literature, but stimulation of hypothalamic neurons can produce coherent and robust parenting behaviors from otherwise nonparental mice, demonstrating latent but functional circuitry for these late-emerging behaviors (Zhang. 2021).

    Recently, new functions for pCd and NPF neurons have been reported in male-specific behaviors. In the Anderson laboratory, pCd neurons were found to sustain sexual and aggressive behaviors after P1 activation. This sustained excitation is remarkably similar to the role of pCd in the accrual and maintenance of sexual motivation in mature male flies. Since P1 activity is constantly required to sustain courtship, it is suggested that subsets of the eight pCd neurons may be specialized for maintaining recurrent activity over minutes (together with P1 neurons in sustaining courtship and aggression) and over days (together with NPF neurons in sustaining reproductive motivation). In work from the Montell laboratory, male-specific NPF neurons were reported to decrease sexual motivation generally and to prevent male-male courtship in particular. These conclusions appear to be in direct contrast with the courtship-promoting roles for these same NPF neurons described in earlier work. The results obtained across these studies are derived from a variety of assays and genetic manipulations, complicating direct comparisons, although they clearly point to multiple roles for NPF in courtship target selection and motivation. While several experiments have argued for various forms of hypersexuality resulting from decreased NPF signaling, the lone NPF neuronal stimulation experiment to yield a reduction in courtship behavior assessed courtship toward decapitated females and the effect was modest. The robust effects seen in this study make the authors confident that increasing the output of NPF neurons promotes a net increase in sexual motivation in otherwise asexual juveniles and in satiated males recovering from ad libitum access to virgin females (Zhang. 2021).

    These results provide an alternative explanation for what has been the strongest evidence in favor of the organizational hypothesis for the maturation of reproductive behaviors in mammals: the delay between hormonal changes and the behaviors they induce. In the fly, the long-lasting, CREB-imposed effects of an earlier suppressive hormonal state must decay away to allow loop activity to ramp up and promote sexual behavior. Enduring and distributed suppressive effects may explain why the awakening of sexuality triggered by hormonal changes is gradual and halting in both flies and mammals (Zhang. 2021).

    Sexually dimorphic peripheral sensory neurons regulate copulation duration and persistence in male Drosophila

    Peripheral sensory neurons are the gateway to the environment across species. In Drosophila, olfactory and gustatory senses are required to initiate courtship, as well as for the escalation of courtship patterns that lead to copulation. To be successful, copulation must last long enough to ensure the transfer of sperm and seminal fluid that ultimately leads to fertilization. The peripheral sensory information required to regulate copulation duration is unclear. This study employed genetic manipulations that allow driving gene expression in the male genitalia as a tool to uncover the role of these genitalia specific neurons in copulation. The fly genitalia contain sex-specific bristle hairs innervated by mechanosensory neurons. To date, the role of the sensory information collected by these peripheral neurons in male copulatory behavior is unknown. These MSNs are cholinergic and co-express both fru and dsx. The sensory information received by the peripheral sensory neurons from the front legs (GRNs) and mechanosensory neurons (MSNs) at the male genitalia contribute to the regulation of copulation duration. Moreover, the results show that their function is required for copulation persistence, which ensures copulation is undisrupted in the presence of environmental stress before sperm transfer is complete (Jois, 2022).

    Dopaminergic neurons mediate male Drosophila courtship motivational state

    Motivational states are important determinants of behavior. In Drosophila melanogaster, courtship behavior is robust and crucial for species continuation. However, the motivation of courtship behavior remains unexplored. This study first found the phenomenon that courtship behavior is modulated by motivational state. A male fly courts another male fly when it first courts a decapitated female fly, however, male-male courtship behavior rarely occurs under normal conditions. Therefore, in this phenomenon, the male fly's courtship motivational state is induced by its exposure to female flies. Blocking dopaminergic neurons synaptic transmission by expressing Tetanus toxin light chain (TNTe) decreases motivational state induced male-male courtship behavior without affecting male-female courtship behavior. Vision cues are another key component in sexually driven Drosophila male-male courtship behavior. This study has identified a base theory that the inner motivational state could eventually decide Drosophila behavior (Wang, 2022).

    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).

    Asexuality in Drosophila juvenile males is organizational and independent of juvenile hormone

    Sexuality is generally prevented in newborns and arises with organizational rewiring of neural circuitry and optimization of fitness for reproduction competition. Recent studies reported that sex circuitry in Drosophila melanogaster is developed in juvenile males but functionally inhibited by juvenile hormone (JH). This study found that the fly sex circuitry, mainly expressing the male-specific fruitless (fruM) and/or doublesex (dsx), is organizationally undeveloped and functionally inoperative in juvenile males. Artificially activating all fruM neurons induces substantial courtship in solitary adult males but not in juvenile males. Synaptic transmissions between major courtship regulators and all dsx neurons are strong in adult males but either weak or undetectable in juvenile males. It was further found that JH does not inhibit male courtship in juvenile males but instead promotes courtship robustness in adult males. These results indicate that the transition to sexuality from juvenile to adult flies requires organizational rewiring of neural circuitry (Ji, 2023).

    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).

    Female Drosophila melanogaster respond to song-amplitude modulations

    Males in numerous animal species use mating songs to attract females and intimidate competitors. This study demonstrates that modulations in song amplitude are behaviourally relevant in the fruit fly Drosophila. D. melanogaster females prefer amplitude modulations typical of melanogaster song over other modulations, which suggests that amplitude modulations are processed auditorily by D. melanogaster. This work demonstrates that receivers can decode messages in amplitude modulations, complementing the recent finding that male flies actively control song amplitude. To describe amplitude modulations, the concept of song amplitude structure (SAS) is proposed and similarities and differences to amplitude modulation with distance (AMD) are discussed (Bruggemeier, 2018).

    Light is required for proper female mate choice between winged and wingless males in Drosophila

    In many animal species, females choose potential mating partners according to their own preferences. Thus, female preference-based mate choice affects intraspecific mating success and prevents interspecific mating. To clarify the neuronal basis of female mate choice, it is essential to identify the important relevant sensory cues. In the fruitfly Drosophila melanogaster, the courtship song of males promotes female sexual receptivity. When wild-type virgin females can freely choose one of two types of courting males (winged or wingless males), they prefer to mate with winged males. This study reports a crucial sensory cue relevant to this female mate choice. In a female choice test, female receptivity toward winged and wingless males was markedly reduced when females had auditory impairments, although females with visual or olfactory impairments showed normal receptivity similar to wild-type females. However, females with visual impairments did not show clear mate preference toward winged males. Thus, these findings suggest that females utilize visual cues in mate choice between winged and wingless males in Drosophila (Watanabe, 2018).

    Discovery of a new song mode in Drosophila reveals hidden structure in the sensory and neural drivers of behavior

    Deciphering how brains generate behavior depends critically on an accurate description of behavior. If distinct behaviors are lumped together, separate modes of brain activity can be wrongly attributed to the same behavior. Alternatively, if a single behavior is split into two, the same neural activity can appear to produce different behaviors. This study addresses this issue in the context of acoustic communication in Drosophila. During courtship, males vibrate their wings to generate time-varying songs, and females evaluate songs to inform mating decisions. For 50 years, Drosophila melanogaster song was thought to consist of only two modes, sine and pulse, but using unsupervised classification methods on large datasets of song recordings, this study now establishes the existence of at least three song modes: two distinct pulse types, along with a single sine mode. This seemingly subtle distinction affects the interpretation of the mechanisms underlying song production and perception. Specifically, this study showed that visual feedback influences the probability of producing each song mode and that male song mode choice affects female responses and contributes to modulating his song amplitude with distance. At the neural level, it was demonstrated how the activity of four separate neuron types within the fly's song pathway differentially affects the probability of producing each song mode. These results highlight the importance of carefully segmenting behavior to map the underlying sensory, neural, and genetic mechanisms (Clemens, 2018).

    Auditory sensitivity, spatial dynamics, and amplitude of courtship song in Drosophila melanogaster

    Acoustic communication is an important component of courtship in Drosophila melanogaster. It takes the form of courtship song produced by males through the unilateral extension and vibration of a wing. Following the paradigm of sender-receiver matching, song content is assumed to match tuning in the auditory system, however, D. melanogaster audition is nonlinear and tuning dependent upon signal amplitude. At low stimulus amplitudes or in the absence of sound the antenna is tuned into song frequency, but as amplitude increases the antenna's resonance is shifted up by hundreds of Hertz. Accurate measurements of song amplitude have been elusive because of the strong dependency of amplitude upon the spatial geometry between sender and receiver. In this study, D. melanogaster auditory directional sensitivity and the geometric position between the courting flies are quantified. It is shown that singing occurs primarily from positions resulting in direct stimulation of the female antenna. Using this information, it is established that the majority of song is louder than theoretically predicted and at these sound levels the female antenna should not amplify or be tuned into song. The study implies that Drosophila hearing, and, in particular, its active mechanisms, could function in a broader context than previously surmised (Morley, 2018).

    The genetics of mating song evolution underlying speciation: Linking quantitative variation to candidate genes for behavioral isolation

    Differences in mating behaviors evolve early during speciation, eventually contributing to reproductive barriers between species. Knowledge of the genetic and genomic bases of these behaviors is therefore integral to a causal understanding of speciation. Acoustic behaviors are often part of the mating ritual in animal species. The temporal rhythms of mating songs are notably species-specific in many vertebrates and arthropods and often underlie assortative mating. Despite discoveries of mutations that disrupt the temporal rhythm of these songs, little is known about genes affecting naturally occurring variation in the temporal pattern of singing behavior. In the rapidly speciating Hawaiian cricket genus Laupala, the striking species variation in song rhythms constitutes a behavioral barrier to reproduction between species. This study mapped the largest-effect locus underlying interspecific variation in song rhythm between two Laupala species to a narrow genomic region, wherein no known candidate genes were found affecting song temporal rhythm in Drosophila. Whole genome sequencing, gene prediction and functional annotation of this region reveal an exciting and promising candidate gene, the putative cyclic nucleotide-gated ion channel-like gene, for natural variation in mating behavior. Identification and molecular characterization of the candidate gene reveals a non-synonymous mutation in a conserved binding domain, suggesting the hypothesis that ion channels are important targets of selection of rhythmic signaling during establishment of behavioral isolation and rapid speciation (Xu, 2019).

    A single male auditory response test to quantify auditory behavioral responses in Drosophila melanogaster

    During courtship, males of the fruit fly Drosophila melanogaster and related species produce a courtship song comprised of sine and pulse songs by vibrating their wings. The pulse song increases female receptivity and male courtship activity, indicating that it functions as a sexual signal. One song parameter, interpulse interval (IPI), varies among closely related species. In D. melanogaster, a song with a conspecific IPI induces a stronger behavioral response than heterospecific songs, indicating the ability of the flies to discriminate conspecific IPI. To quantify the individual ability to discriminate a conspecific song, this study systematically analyzed the auditory response of single male flies to sound with various parameters. By quantifying the locomotor activity of single D. melanogaster males during sound exposure, increased locomotor activity was detected in response to pulse songs, but not to white noise or pure tone. The conspecific song evoked stronger response than the heterospecific songs, and ablation of their antennal receivers severely suppressed the locomotor increase. A pulse song with a small IPI variation evoked a continuous response, while the response to songs with highly variable IPIs tends to be rapidly decayed. This provides the first evidence that fruit flies discriminate IPI variations, which possibly inform the age and social contexts of the singer. Sister species, D. sechellia, exhibited a locomotor response to pulse song, while D. simulans exhibited no behavioral response. This suggests that auditory and other stimuli that elicit this behavioral response are diversified among Drosophila species (Ishikawa, 2019).

    Calmodulin-binding transcription factor shapes the male courtship song in Drosophila

    Males of the Drosophila melanogaster mutant croaker (cro) generate a polycyclic pulse song dissimilar to the monocyclic songs typical of wild-type males during courtship. However, cro has not been molecularly mapped to any gene in the genome. This study demonstrates that cro is a mutation in the gene encoding the Calmodulin-binding transcription factor (Camta) by genetic complementation tests with chromosomal deficiencies, molecular cloning of genomic fragments that flank the cro-mutagenic P-insertion, and phenotypic rescue of the cro mutant phenotype by Camta+-encoding cDNA as well as a BAC clone containing the gene for Camta. It was shown that knockdown of the Camta-encoding gene phenocopies cro mutant songs when targeted to a subset of fruitless-positive neurons that include the mcALa and AL1 clusters in the brain. cro-GAL4 and an anti-Camta antibody labeled a large number of brain neurons including mcALa. It is concluded that the Camta-encoding gene represents the cro locus, which has been implicated in a species-specific difference in courtship songs between D. sechellia and simulans (Sato, 2019).

    Shared song detector neurons in Drosophila male and female brains drive sex-specific behaviors

    Males and females often produce distinct responses to the same sensory stimuli. How such differences arise-at the level of sensory processing or in the circuits that generate behavior-remains largely unresolved across sensory modalities. This issue was addressed in the acoustic communication system of Drosophila. During courtship, males generate time-varying songs, and each sex responds with specific behaviors. Male and female behavioral tuning was characterized for all aspects of song, and feature tuning was shown to be similar between sexes, suggesting sex-shared song detectors drive divergent behaviors. Higher-order neurons in the Drosophila brain, called pC2, were identified that are tuned for multiple temporal aspects of one mode of the male's song and drive sex-specific behaviors. Neurons were also uncovered that are specifically tuned to an acoustic communication signal and reside at the sensory-motor interface, flexibly linking auditory perception with sex-specific behavioral responses (Deutsch, 2019).

    Evolution of sexual size dimorphism in the wing musculature of Drosophila

    Male courtship songs in Drosophila are exceedingly diverse across species. While much of this variation is understood to have evolved from changes in the central nervous system, evolutionary transitions in the wing muscles that control the song may have also contributed to song diversity. Focusing on a group of four wing muscles that are known to influence courtship song in Drosophila melanogaster, this study investigated the evolutionary history of wing muscle anatomy of males and females from 19 Drosophila species. Three of the wing muscles have evolved sexual dimorphisms in size multiple independent times, whereas one has remained monomorphic in the phylogeny. These data suggest that evolutionary changes in wing muscle anatomy may have contributed to species variation in sexually dimorphic wing-based behaviors, such as courtship song. Moreover, wing muscles appear to differ in their propensity to evolve size dimorphisms, which may reflect variation in the functional constraints acting upon different wing muscles (Tracy, 2020).

    Behavioral Evolution of Drosophila: Unraveling the Circuit Basis

    Behavior is a readout of neural function. Therefore, any difference in behavior among different species is, in theory, an outcome of interspecies diversification in the structure and/or function of the nervous system. However, the neural diversity underlying the species-specificity in behavioral traits and its genetic basis have been poorly understood. This article discusses potential neural substrates for species differences in the courtship pulse song frequency and mating partner choice in the Drosophila melanogaster subgroup. Possible neurogenetic mechanisms are discussed whereby a novel behavioral repertoire emerges based on the study of nuptial gift transfer, a trait unique to D. subobscura in the genus Drosophila. The conserved central circuit composed primarily of fruitless-expressing neurons (the fru-circuit) serves for the execution of courtship behavior, whereas the sensory pathways impinging onto the fru-circuit or the motor pathways downstream of the fru-circuit are susceptible to changes associated with behavioral species differences (Sato, 2020).

    Neural network organization for courtship-song feature detection in Drosophila

    Animals communicate using sounds in a wide range of contexts, and auditory systems must encode behaviorally relevant acoustic features to drive appropriate reactions. How feature detection emerges along auditory pathways has been difficult to solve due to challenges in mapping the underlying circuits and characterizing responses to behaviorally relevant features. This paper describes auditory activity in the Drosophila melanogaster brain and investigated feature selectivity for the two main modes of fly courtship song, sinusoids and pulse trains. Twenty-four new cell types of the intermediate layers of the auditory pathway were identified, and using a new connectomic resource, FlyWire, all synaptic connections between these cell types, in addition to connections to known early and higher-order auditory neurons were mapped-this represents the first circuit-level map of the auditory pathway. The sign (excitatory or inhibitory) was determined of most synapses in this auditory connectome. Auditory neurons were found to display a continuum of preferences for courtship song modes and that neurons with different song-mode preferences and response timescales are highly interconnected in a network that lacks hierarchical structure. Nonetheless, this study found that the response properties of individual cell types within the connectome are predictable from their inputs. This study thus provides new insights into the organization of auditory coding within the Drosophila brain (Baker, 2022).

    Drosophila females have an acoustic preference for symmetric males

    Theoretically, symmetry in bilateral animals is subject to sexual selection, since it can serve as a proxy for genetic quality of competing mates during mate choice. This study reports female preference for symmetric males in Drosophila, using a mate-choice paradigm where males with environmentally or genetically induced wing asymmetry were competed. Analysis of courtship songs revealed that males with asymmetric wings produced songs with asymmetric features that served as acoustic cues, facilitating this female preference. Females experimentally evolved in the absence of mate choice lost this preference for symmetry, suggesting that it is maintained by sexual selection (Vijendravarma, 2022).

    Female copulation song is modulated by seminal fluid

    In most animal species, males and females communicate during sexual behavior to negotiate reproductive investments. Pre-copulatory courtship may settle if copulation takes place, but often information exchange and decision-making continue beyond that point. This study shows that female Drosophila sing by wing vibration in copula. This copulation song is distinct from male courtship song and requires neurons expressing the female sex determination factor DoublesexF. Copulation song depends on transfer of seminal fluid components of the male accessory gland. Hearing female copulation song increases the reproductive success of a male when he is challenged by competition, suggesting that auditory cues from the female modulate male ejaculate allocation. These findings reveal an unexpected fine-tuning of reproductive decisions during a multimodal copulatory dialog. The discovery of a female-specific acoustic behavior sheds new light on Drosophila mating, sexual dimorphisms of neuronal circuits and the impact of seminal fluid molecules on nervous system and behavior (Kerwin, 2020).

    This study reports a novel acoustic behavior during Drosophila reproduction, female specific copulation song. It occurs in D. melanogaster as well as in its sibling species D. simulans, D. mauritiana and D. sechellia. While the acoustic parameters of male courtship song display marked inter-species differences, female song structure in D. melanogaster, D. simulans and D. mauritiana species is very similar. This is in line with the proposed function of male song as a prezygotic isolating barrier. In contrast, such a function seems unlikely for female copulation song, which occurs after a mating partner has been chosen. The results revise the notion that only male Drosophila melanogaster sings. This study has identified neuronal components of a female song circuit with shared and distinct elements compared to its male counterpart. Silencing of a specific motor output neuronal class required for flight, dlm mns, abolishes female song completely, whereas it only decreases the amplitude of male song. Male song depends critically on dsx+ fru+ neurons, many of which are male specific or sexually dimorphic. For female song, dsx+ fru- neurons of the ventral nerve cord, but not dsx+ fru+ neurons necessary for song. Future work dissecting thoracic circuits will reveal how sex specific wing motor patterning is generated and how neuronal dimorphisms explain the different acoustic parameters of male and female song (Kerwin, 2020).

    The composition of male ejaculate, critically affects female singing behavior, whereas the quality of male pre-copulatory courtship (visual, olfactory and gustatory input) is likely to have little impact. Absence of sperm in the presence of seminal fluid increases singing. Males generally depleted of ejaculate or specifically lacking secondary cell products (SCPs) in their seminal fluid barely elicit any female song. More song in the absence of sperm could be due to a potential increase in seminal fluid or a greater accessibility of SCPs, some of which are normally bound to sperm. Alternatively, sperm might suppress female singing. Sperm is transferred in a discrete, ~1 min long bout around 7-8 min after start of copulation. In contrast, seminal fluid transfer starts immediately after the initiation of copulation and is thought to continue until disengagement. This transfer pattern might explain the higher probability of female song at the beginning and end of copulation (Kerwin, 2020).

    Since females mutant for two mechanosensory channels expressed in the female reproductive tract (Ppk and Piezo) still sing in copula, it is unlikely that transfer of ejaculate elicits female singing via mechanical stimulation. It is hypothesized that SCPs provide a chemical cue for the female, analogous to female pheromones triggering male courtship. It will be interesting to unravel if a single SCP or a more complex mixture is needed for female song initiation, by which receptor(s) and sensory neurons the transfer is detected and how the signal is relayed to motor patterning circuits (Kerwin, 2020).

    These experiments demonstrate that female copulation song influences female remating and reproductive success when females have the possibility to remate. How could hearing female song exert such an effect? Female remating probability is impacted by the composition of male ejaculate. The latter is not fixed, but can be modulated by the male to adjust the amount of seminal fluid and sperm transferred to the presence of rivals and female condition (so-called strategic ejaculate allocation). Since seminal fluid is depleted after several matings, strategic allocation has been predicted to be adaptive for males. Based on these previous findings, it is proposed that female copulation song directly affects male seminal fluid allocation and by this decreases female remating. Since an effect of song playback is seen, it is assumeed that males detect female song with their auditory system. However, this does not rule out that males can also detect female song by mechanosensation. Female wing vibrations during singing might also dissipate pheromones and thereby influence olfaction (Kerwin, 2020).

    It is further proposed that a feedback loop might coordinate female singing and fine-tune ejaculate transfer. At the beginning of copulation, male SCPs trigger female song, cueing the male that his partner is responsive to seminal fluid components. During the subsequent course of copulation, female song influences further ejaculate transfer. Here, the function of female song could be to entice allocation of costly components from the male. Alternatively, female song might help to proportion overall ejaculate composition to match individual physiological needs. Females might not be able to predict male ejaculate composition by assessing male pre-copulatory courtship. There is no evidence that females can prematurely terminate copulations. Copulation song might thus be a way for females to give feedback to and influence males with whom they have chosen to copulate, modulating allocation behavior to their benefit (Kerwin, 2020).

    This first investigation of female copulation song has not yet unraveled its evolutionary significance, and it is only possible to speculate about potential roles in sexual conflict and sexual selection. This study found evidence that female song can delay remating. So far, it is not known if this is the only or most important function of female singing or might be only a secondary effect of changed ejaculate composition. Delayed remating could be adaptive for females under certain conditions, when it leads to efficient use of the sperm from the first male before it is replaced by the ejaculate of a subsequent mate. This might be in the interest of females, since it allows for mixed paternity and genetic diversity of their offspring (Kerwin, 2020).

    In a working model, the SCPs stimulating female singing are not necessarily identical with the seminal fluid components that are differentially transferred. In the future, comprehensive screening of the numerous SCPs which are altered in expression levels in iab-6cocu mutant males, which have defective secondary cells, as well as analysis of seminal fluid composition in song playback vs. silence copulations by ELISA or quantitative proteomics are needed to test these hypotheses. Identifying the factors which are differentially transferred in response to female song is crucial for building hypotheses about the adaptive value of female song (Kerwin, 2020).

    In general, it can be in the interest of females to influence ejaculate allocation, receipt of seminal fluid components, and, ultimately, paternity of their offspring by active signaling. This study proposes copulation song as a new mechanism by which male reproductive competition, most likely via male allocation behavior, can be influenced by females (Kerwin, 2020).

    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).

    The behavior of adult Drosophila in the wild

    Little is known about how Drosophila adults behave in the wild, including mating, allocation of food and space, and escape from predators. This lack of information has negative implications for the ability to understand the capabilities of the nervous system to integrate sensory cues necessary for the adaptation of organisms in natural conditions. This study characterized a set of behavioral routines of D. melanogaster and D. simulans adults in three ecologically different orchards: grape, apple and prickly pear. How the flies identify conspecifics and aliens in the wild were also investigated to better understand relationships between group formation and adaptation of Drosophila to breeding sites. The locations were characterized by recording in each orchard humidity, temperature, illumination conditions, pH of fruits, the presence/absence of other Drosophila species and the predator ant Linepithema humile. The findings suggest that the home range of these species of Drosophila includes decaying fruits and, principally, a variety of microhabitats that surround the fruits. The ecological heterogeneity of the orchards and odors emitted by adult D. melanogaster and D. simulans influence perch preferences, cluster formation, court and mating, egg-laying site selection, and use of space. This is one of the first large examinations of the association between changing, complex environments and a set of adult behaviors of Drosophila. Therefore, these results have implications for understanding the genetic differentiation and evolution of populations of species in the genus Drosophila (Soto-Yeber, 2018).

    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).

    GABA-mediated inhibition in visual feedback neurons fine-tunes Drosophila male courtship

    Vision is critical for the regulation of mating behaviors in many species. This study discovered that the Drosophila ortholog of human GABA (A) -receptor-associated protein (GABARAP; Atg8a) is required to fine-tune male courtship by modulating the activity of visual feedback neurons, lamina tangential cells (Lat). GABARAP is a ubiquitin-like protein that regulates cell-surface levels of GABA (A) receptors. Knocking down GABARAP or GABA (A) receptors in Lat neurons or hyperactivating them induces male courtship toward other males. Inhibiting Lat neurons, on the other hand, delays copulation by impairing the ability of males to follow females. Remarkably, the human ortholog of Drosophila GABARAP restores function in Lat neurons. Using in vivo two-photon imaging and optogenetics, Lat neurons were shown to be functionally connected to neural circuits that mediate visually-guided courtship pursuits in males. This work reveals a novel physiological role for GABARAP in fine-tuning the activity of a visual circuit that tracks a mating partner during courtship (Mabuchi, 2023).

    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).

    Social experience and pheromone receptor activity reprogram gene expression in sensory neurons

    Social experience and pheromone signaling in olfactory neurons affect neuronal responses and male courtship behaviors in Drosophila. Previous work has shown that social experience and pheromone signaling modulate chromatin around behavioral switch gene fruitless, which encodes a transcription factor necessary and sufficient for male sexual behaviors. To identify the molecular mechanisms driving social experience-dependent changes in neuronal responses, RNA-seq was performed from antennal samples of mutants in pheromone receptors and fruitless, as well as grouped or isolated wild-type males. Genes affecting neuronal physiology and function, such as neurotransmitter receptors, ion channels, ion and membrane transporters, and odorant binding proteins are differentially regulated by social context and pheromone signaling. While this study found that loss of pheromone detection only has small effects on differential promoter and exon usage within fruitless gene, many of the differentially regulated genes have Fruitless binding sites or are bound by Fruitless in the nervous system. Recent studies showed that social experience and juvenile hormone signaling co-regulate fruitless chromatin to modify pheromone responses in olfactory neurons. Interestingly, genes involved in juvenile hormone metabolism are also misregulated in different social contexts and mutant backgrounds. These results suggest that modulation of neuronal activity and behaviors in response to social experience and pheromone signaling likely arise due to large-scale changes in transcriptional programs for neuronal function downstream of behavioral switch gene function (Deanhardt, 2023).

    Persistent activity in a recurrent circuit underlies courtship memory in Drosophila

    Recurrent connections are thought to be a common feature of the neural circuits that encode memories, but how memories are laid down in such circuits is not fully understood. This study presents evidence that courtship memory in Drosophila relies on the recurrent circuit between mushroom body γ (MBγ), M6 output, and aSP13 dopaminergic neurons. Persistent neuronal activity of aSP13 neurons was demonstrated; it transiently potentiates synaptic transmission from MBγ>M6 neurons. M6 neurons in turn provide input to aSP13 neurons, prolonging potentiation of MBγ>M6 synapses over time periods that match short-term memory. These data support a model in which persistent aSP13 activity within a recurrent circuit lays the foundation for a short-term memory (Zhao, 2018).

    As animals pursue their goals, their behavioral decisions are shaped by memories that encompass a wide range of time scales: from fleeting working memories relevant to the task at hand, to short-term and long-term memories of contingencies learned hours, days, or even years in the past. Working memory is thought to reflect persistent activity generated within neural networks, including recurrent circuits. In contrast, short-term memory (STM) and long-term memory (LTM) involves changes in synaptic efficacy due to functional and structural modification of synaptic connections. However, the neural circuit mechanisms involved in the formation, persistence and transitions between these distinct forms of memory are not fully known (Zhao, 2018).

    A robust form of memory in Drosophila is courtship memory, which can last from minutes to days, depending on the duration and intensity of training. Naïve Drosophila males eagerly court both virgin females, which are generally receptive, and mated females, which are not. However, upon rejection by mated females, they become subsequently less likely to court other mated females. This selective suppression of courtship towards mated females, called courtship conditioning, can be attributed to the enhanced sensitivity of experienced males to an inhibitory male pheromone deposited on the female during mating, cis-vaccenyl acetate (cVA) (Zhao, 2018).

    Olfactory memory in insects relies on the function of a central brain structure called the mushroom body (MB). The principal MB cells, the cholinergic Kenyon cells (KCs), receive input from sensory pathways in the dendritic calyx region and from dopaminergic neurons (DANs) in the axonal lobes of the MB. These MB lobes are compartmentalized, with each compartment innervated by specific classes of DANs and MB output neurons (MBONs). MBONs receive input from both KCs and DANs (Zhao, 2018).

    Previously work established that short-term courtship conditioning is mediated by the aSP13 class of DANs (also known as the PAM-γ5 neurons (Aso, 2014), which innervate the MBγ5 compartment. The activity of aSP13 neurons is essential for courtship conditioning in experienced males and sufficient to induce conditioning in naïve males (Keleman, 2012). This study demonstrates that courtship memory also requires the corresponding MBγ KCs and the MBγ5 MBONs, the glutamatergic M6 neurons (also known as MBON-γ5&β;'2a neurons). Furthermore, this study presents evidence that MBγ, M6, and aSP13 neurons form a recurrent circuit and that persistent activity of the aSP13 neurons mediates plasticity at the MBγ to M6 synapses that can last from minutes to hours. Consistent with this model, M6 activity is required not only for memory readout but also, like aSP13, for memory formation. These data support a model in which persistent aSP13 activity within the MBγ>M6>aSP13 recurrent circuit lays the foundation for short-term courtship memory (Zhao, 2018).

    This study has identified and characterized a tripartite MBγ>M6>aSP13 recurrent circuit that is essential for courtship memory in Drosophila. Behavioral and physiological data suggest the following model for the function of this feedback loop in short-term courtship memory. When a naïve male courts a mated female, the aSP13 and MBγ neurons may both be activated, perhaps in response to behavioral rejection and olfactory stimuli presented by the female, respectively. Dopamine released by aSP13 neurons potentiates transmission from MBγ to M6 neurons, which in turn provide a recurrent excitatory glutamatergic input back onto aSP13 neurons. Upon activation by M6, aSP13 activity persists for several minutes, providing a short time window during which continued MBγ activity can further drive M6 and aSP13. Thus sustained, aSP13 activity can lead to a longer-lasting accumulation of dopamine in the γ5 compartment, facilitating MBγ>M6 neurotransmission for up to 2-3 hr (Zhao, 2018).

    The timescales for these physiological processes in ex vivo brain preparations broadly match the dynamics of courtship training and short-term memory formation. In the standard training paradigm, the male typically courts the female over several minutes, during which he performs a series of courtship bouts, each lasting for several seconds. As a result, a behavioral memory forms that lasts for several hours (Keleman, 2012). Memory formation during training requires both M6 and aSP13, consistent with the notion that it reflects activation of the recurrent circuit. Memory readout requires M6 but not aSP13, and so evidently does not involve the recurrent circuit. It is infered that M6 suppresses courtship through other, aSP13-independent, pathways, and that its ability to do so is independent of experience. The consequence of training is to provide MBγ neurons with access to this M6-dependent courtship suppression pathway (Zhao, 2018).

    Two important open questions are, first, what mechanism underlies the persistent calcium response in aSP13, and second, how does potentiation of MBγ>M6 synapses result in enhanced sensitivity to cVA (cis-vaccenyl acetate, cVA, a major component of the male cuticular hydrocarbon profile). The persistent calcium response the hallmark of courtship memory (Keleman, 2012). The persistent response in aSP13 is evidently not an intrinsic property of aSP13, as it is not induced when aSP13 neurons themselves are activated. This observation would also likely exclude reciprocal excitation between aSP13 and other DANs. Persistent aSP13 activity is induced in response to transient M6 activation, and is not associated with any persistent activity of M6 neurons themselves. Thus, it is also unlikely to involve feedback from aSP13 and M6, although aSP13 >M6 synapses likely do exist. One possibility is that aSP13 persistence reflects unusually prolonged activation of the glutamatergic M6 >aSP13 synapses, or perhaps lies within interposed but still unidentified circuit elements (Zhao, 2018).

    Given that M6 neurons activate a courtship suppression pathway, the potentiation of MBγ>M6 neurotransmission may explain why MBγ activation suppresses courtship in trained but not naïve flies. But MBγ neurons likely do not specifically respond to cVA, so this change alone cannot account for the enhanced sensitivity of trained flies to cVA. A small and variable subset of MB γneurons do receive input from the olfactory pathway that processes cVA, but cVA is not required during training and it is difficult to envision any other mechanism by which aSP13-dependent plasticity could be specifically restricted to the cVA-responsive MBγ neurons. It is formally possible that, despite the broad potentiation of MBγ output synapses upon training, it is only the contribution of the cVA-responsive MBγ neurons that drives courtship suppression when the male subsequently encounters as mated female. Alternatively, it has been suggested that M6 neurons encode a generic aversive signal, and so specificity to cVA might instead arise in downstream circuits that selectively integrate M6 output with the innate cVA-processing pathway from the lateral horn. In this regard, it is interesting to note that other MBONs have been implicated in courtship learning (Montague, 2016) or general aversion, but M6 is the only MBON common to both (Zhao, 2018).

    Late activation of the same aSP13 neurons in the time window of 8-10 hr after training is both necessary and sufficient to consolidate STM to LTM (Krüttner, 2015). Thus, in the time window when STM would otherwise decay, reactivation of the same MBγ>M6>aSP13 recurrent circuit may instead consolidate it into LTM. The mechanism by which aSP13 neurons are reactivated is unknown, but is evidently dependent upon their activation within the MBγ>M6>aSP13 recurrent circuit during training. It will be interesting to find out how this late aSP13 reactivation mechanism might relate to the mechanism that underlies persistent aSP13 activity during training (Zhao, 2018).

    In summary, the data suggest that a brief persistent activity of aSP13 neurons represents a neural correlate of courtship working memory, while the prolonged potentiation of MBγ>M6 synapses corresponds to STM. It is proposed that persistent activity of the dopaminergic neurons in the MBγ>M6>aSP13 feedback loop lays the foundation for formation of short-term courtship memory in Drosophila, and that later reactivation of the same recurrent circuit consolidates STM into LTM. Thus, in contrast to the prevailing view of memory progression in the Drosophila MB that distinct memory phases are located in different compartments or lobes, the current data suggest that in the context of courtship conditioning, working memory, STM, and LTM all reside in the same γ5 compartment. These conclusions do not preclude however, the involvement of other MB neurons in courtship memory (Montague, 2016) as it is conceivable that modulation, potentially of the opposite sign, of the appetitive memory pathways could be critical for courtship learning. This study therefore envisions that distinct courtship memory types are not located in distinct circuits, but rather mediated by distinct processes within a common circuit. Encoding distinct memory phases within a common circuit may be an efficient mechanism for encoding memories for which the behavioral consequence is largely independent of timing and context (Zhao, 2018).

    Threshold-based ordering of sequential actions during Drosophila courtship

    Goal-directed animal behaviors are typically composed of sequences of motor actions whose order and timing are critical for a successful outcome. Although numerous theoretical models for sequential action generation have been proposed, few have been supported by the identification of control neurons sufficient to elicit a sequence. This study identified a pair of descending neurons that coordinate a stereotyped sequence of engagement actions during Drosophila melanogaster male courtship behavior. These actions are initiated sequentially but persist cumulatively, a feature not explained by existing models of sequential behaviors. Evidence was found consistent with a ramp-to-threshold mechanism, in which increasing neuronal activity elicits each action independently at successively higher activity thresholds (McKellar, 2019).

    The role of miRNAs in Drosophila melanogaster male courtship behavior

    Drosophila melanogaster courtship, although stereotypical, continually changes based on cues received from the courtship subject. Such adaptive responses are mediated via rapid and widespread transcriptomic reprogramming, a characteristic now widely attributed to microRNAs (miRNAs) along with other players. This study conducted a large-scale miRNA knockout screen to identify miRNAs that affect various parameters of male courtship behavior. Apart from identifying miRNAs that impact male-female courtship, it was observed that miR-957 mutants performed significantly increased male-male courtship and chaining behavior whereby groups of males court one another. The effect of miR-957 reduction in specific neuronal cell clusters was tested, identifying miR-957 activity in Doublesex (DSX)-expressing and mushroom body clusters as an important regulator of male-male courtship interactions. The behavior of miR-957 mutants was further characterized, and these males were found to court male subjects vigorously but do not elicit courtship. Moreover, they fail to lower courtship efforts towards females with higher levels of anti-aphrodisiac pheromones. At the level of individual pheromones, miR-957 males show a reduced inhibitory response to both 7-Tricosene (7-T) and cis-vaccenyl acetate (cVA), with the effect being more pronounced in case of 7-T. Overall these results indicate that a single miRNA can contribute to regulation of complex behaviors, including detection or processing of chemicals that control important survival strategies such as chemical mate-guarding and maintenance of sex and species-specific courtship barriers (Iftikhar, 2019).

    Social effects on fruit fly courtship song

    Courtship behavior in Drosophila has often been described as a classic innate behavioral repertoire. This study examined courtship song plasticity of two species in the Drosophila melanogaster subgroup. Sexual isolation between the species is influenced by two male song traits, the interpulse interval (IPI) and sinesong frequency (SSF). Neither of these showed plasticity when males had prior experience of con- and heterospecific social partners. However, males of both species produced longer bursts of song during courtship when they were exposed to social partners (either con- or heterospecific) than when they were reared in isolation. D. melanogaster carrying mutations affecting short- or medium-term memory showed a similar response to the social environment, not supporting a role for learning. These results demonstrate that the amount of song a male produces during courtship is plastic depending on the social environment, which might reflect the advantage of being able to respond to variation in intrasexual competition, but that song structure itself is relatively inflexible, perhaps due to strong selection against hybridization (Marie-Orleach, 2019).

    Courtship behavior induced by appetitive olfactory memory

    Reinforcement signals such as food reward and noxious punishment can change diverse behaviors. This holds true in fruit flies, Drosophila melanogaster, which can be conditioned by an odor and sugar reward or electric shock punishment. Despite a wide variety of behavior modulated by learning, conditioned responses have been traditionally measured by altered odor preference in a choice, and other memory-guided behaviors have been only scarcely investigated. This study analyzed detailed conditioned odor responses of flies after sugar associative learning by employing a video recording and semi-automated processing pipeline. Trajectory analyses revealed that multiple behavioral components were altered along with conditioned approach to the rewarded odor. Notably, it was found that lateral wing extension, a hallmark of courtship behavior of D. melanogaster, was robustly increased specifically in the presence of the rewarded odor. Strikingly, genetic disruption of the mushroom body output did not impair conditioned courtship increase, while markedly weakening conditioned odor approach. These results highlight the complexity of conditioned responses and their distinct regulatory mechanisms that may underlie coordinated yet complex memory-guided behaviors in flies (Onodera, 2019).

    Generating parallel representations of position and identity in the olfactory system

    In Drosophila, a dedicated olfactory channel senses a male pheromone, cis-vaccenyl acetate (cVA), promoting female courtship while repelling males. This study shows that separate cVA-processing streams extract qualitative and positional information. cVA sensory neurons respond to concentration differences in a 5-mm range around a male. Second-order projection neurons encode the angular position of a male by detecting inter-antennal differences in cVA concentration, which are amplified through contralateral inhibition. At the third circuit layer, 47 cell types were identified with diverse input-output connectivity. One population responds tonically to male flies, a second is tuned to olfactory looming, while a third integrates cVA and taste to coincidentally promote female mating. The separation of olfactory features resembles the mammalian what and where visual streams; together with multisensory integration, this enables behavioral responses appropriate to specific ethological contexts (Taisz, 2023).

    The yellow gene influences Drosophila male mating success through sex comb melanization

    Drosophila melanogaster males perform a series of courtship behaviors that, when successful, result in copulation with a female. For over a century, mutations in the yellow gene, named for its effects on pigmentation, have been known to reduce male mating success. Prior work has suggested that yellow influences mating behavior through effects on wing extension, song, and/or courtship vigor. This study ruled out these explanations, as well as effects on the nervous system more generally, and found instead that the effects of yellow on male mating success are mediated by its effects on pigmentation of male-specific leg structures called sex combs. Loss of yellow expression in these modified bristles reduces their melanization, which changes their structure and causes difficulty grasping females prior to copulation. These data illustrate why the mechanical properties of anatomy, not just neural circuitry, must be considered to fully understand the development and evolution of behavior (Massey, 2019).

    Unsupervised identification of the internal states that shape natural behavior

    Internal states shape stimulus responses and decision-making, but methods to identify them are lacking. To address this gap, an unsupervised method was developed to identify internal states from behavioral data, and it was applied to a dynamic social interaction. During courtship, Drosophila melanogaster males pattern their songs using feedback cues from their partner. The model uncovers three latent states underlying this behavior and is able to predict moment-to-moment variation in song-patterning decisions. These states correspond to different sensorimotor strategies, each of which is characterized by different mappings from feedback cues to song modes. A pair of neurons previously thought to be command neurons for song production are sufficient to drive switching between states. These results reveal how animals compose behavior from previously unidentified internal states, which is a necessary step for quantitative descriptions of animal behavior that link environmental cues, internal needs, neuronal activity and motor outputs (Calhoun, 2019).

    Drosophila sexual attractiveness in older males is mediated by their microbiota

    Age is well known to be a basis for female preference of males. However, the mechanisms underlying age-based choices are not well understood, with several competing theories and little consensus. The idea that the microbiota can affect host mate choice is gaining traction, and in this study it was examine whether the male microbiota influences female preference for older individuals in the fruit fly Drosophila pseudoobscura. An intact microbiota was found to be a key component of attractiveness in older males. However, no evidence was found that this decrease in older male attractiveness was simply due to impaired microbiota generally reducing male quality. Instead, it is suggested that the microbiota underlies an honest signal used by females to assess male age, and that impaired microbiota disrupt this signal. This suggests that age-based preferences may break down in environments where the microbiota is impaired, for example when individuals are exposed to naturally occurring antibiotics, extreme temperatures, or in animals reared in laboratories on antibiotic supplemented diet (Heys, 2020).

    Fitness consequences of redundant cues of competition in male Drosophila melanogaster

    Phenotypic plasticity can allow animals to adapt their behavior, such as their mating effort, to their social and sexual environment. However, this relies on the individual receiving accurate and reliable cues of the environmental conditions. This can be achieved via the receipt of multimodal cues, which may provide redundancy and robustness. Male Drosophila melanogaster detect presence of rivals via combinations of any two or more redundant cue components (sound, smell, and touch) and respond by extending their subsequent mating duration, which is associated with higher reproductive success. Although alternative combinations of cues of rival presence have previously been found to elicit equivalent increases in mating duration and offspring production, their redundancy in securing success under sperm competition has not previously been tested. This was explicitly tested by exposing male D. melanogaster to alternative combinations of rival cues, and examine reproductive success in both the presence and absence of sperm competition. The results supported previous findings of redundancy of cues in terms of behavioral responses. However, there was no evidence of reproductive benefits accrued by extending mating duration in response to rivals. The lack of identifiable fitness benefits of longer mating under these conditions, both in the presence and absence of sperm competition, contrasted with some previous results, but could be explained by (a) damage sustained from aggressive interactions with rivals leading to reduced ability to increase ejaculate investment, (b) presence of features of the social environment, such as male and female mating status, that obscured the fitness benefits of longer mating, and (c) decoupling of behavioral investment with fitness benefits (Dore, 2020).

    Mate-finding dispersal reduces local mate limitation and sex bias in dispersal

    Sex-biased dispersal (SBD) often skews the local sex ratio in a population. This can result in a shortage of mates for individuals of the less-dispersive sex. Such mate limitation can lead to Allee effects in populations that are small or undergoing range expansion, consequently affecting their survival, growth, stability and invasion speed. Theory predicts that mate shortage can lead to either an increase or a decrease in the dispersal of the less-dispersive sex. However, neither of these predictions have been empirically validated. To investigate how SBD-induced mate limitation affects dispersal of the less-dispersive sex, Drosophila melanogaster populations with varying dispersal propensities were used. To rule out any mate-independent density effects, the behavioral plasticity of dispersal was examined in presence of mates as well as same-sex individuals with differential dispersal capabilities. In the presence of high-dispersive mates, the dispersal of both male and female individuals was significantly increased. However, the magnitude of this increase was much larger in males than in females, indicating that the former show greater mate-finding dispersal. Moreover, the dispersal of either sex did not change when dispersing alongside high- or low-dispersive individuals of the same sex. This suggested that the observed plasticity in dispersal was indeed due to mate-finding dispersal, and not mate-independent density effects. Strong mate-finding dispersal can diminish the magnitude of sex bias in dispersal. This can modulate the evolutionary processes that shape range expansions and invasions, depending on the population size. In small populations, mate-finding dispersal can ameliorate Allee effects. However, in large populations, it can dilute the effects of spatial sorting (Mishra, 2020).

    Light dependent courtship behavior in Drosophila simulans and D. melanogaster

    Differences in courtship signals and perception are well-known among Drosophila species. One such described difference is the dependency on light, and thus presumably vision, for copulation success. Many studies have described a difference in light-dependent copulation success between D. melanogaster and D. simulans, identifying D. simulans as a light-dependent species, and D. melanogaster as a light-independent one. However, many of these studies use assays of varying design and few strains to represent the entire species. This study attempted to better characterize this purported difference using 11 strains of each species, paired by collection location, in behavioral assays conducted at two different exposure times. While there is a species-wide difference in magnitude of light-dependent copulation success, D. melanogaster copulation success is, on average, still impaired in the dark at both exposure times measured. Additionally, there is significant variation in strain-specific ability to copulate in the dark in both species across two different exposure times. This variation correlates strongly with longitude in D. melanogaster, but not in D. simulans. It is hypothesized that differences in species history and demography may explain behavioral variation. Finally, courtship assays were used to show that light-dependent copulation success in one D. simulans strain is driven in part by both males and females. Potential differences in courtship signals and/or signal importance between these species are discussed and potential for further comparative studies for functional characterization (Shahandeh, 2020).

    Female mating experience and genetic background independently influence male mating success in fruit flies

    When the reproductive interests of males and females conflict, males can evolve traits that are harmful to females, and females can coevolve traits to resist this harm. This study tested how the genetic background of a female and her previous mating experience interact to affect the mating success of focal males. In the experience phase, females from 28 distinct genetic backgrounds were placed individually either with a single male (low conflict) or with three males (high conflict) for 48 hr. In the subsequent test phase, the mating and post-mating fertilization success was measured of focal males paired individually with each female. focal males paired with females from the high-conflict treatment were less successful at mating, took longer to mate when they were successful, and had a lower proportion of paternity share. Furthermore, significant female genetic variation associated with male mating success was identified. These results indicate that female experience, along with intrinsic genetic factors, can independently influence different fitness components of her subsequent mates and has implications for understanding of plastic female mating strategies and the evolution of sexually antagonistic traits in males and females (Filice, 2020).

    Regulation of Drosophila Long-Term Courtship Memory by Ecdysis Triggering Hormone

    Endocrine state is an important determinant of learning and memory in animals. In Drosophila, rejection of male courtship overtures by mated females leads to an aversive response manifested as courtship memory. This study reports that ecdysis triggering hormone (ETH) is an obligatory enabler of long-term courtship memory (LTM). ETH deficiency suppresses LTM, whereas augmented ETH release reduces the minimum training period required for LTM induction. ETH receptor knockdown either in the mushroom body (MB) γ lobe or in octopaminergic dorsal-anterior-lateral (DAL) neurons impairs memory performance, indicating its direct action in these brain areas. Consistent with these findings, brain exposure to ETH mobilizes calcium in MB γ lobe neuropils and DAL neurons. ETH receptor (ETHR) knockdown in the corpus allatum (CA) to create juvenile hormone (JH) deficiency also suppresses LTM, as does knockdown of the JH receptor Met in the MB γ lobe, indicating a convergence of ETH and JH signaling in this region of the brain. These findings identify endocrine-enabled neural circuit components in the brain that are critical for persistent behavioral changes resulting from aversive social experience (Lee, 2021).

    Hsp70 affects memory formation and behaviorally relevant gene expression in Drosophila melanogaster

    Heat shock proteins, in particular Hsp70, play a central role in proteostasis in eukaryotic cells. Due to its chaperone properties, Hsp70 is involved in various processes after stress and under normal physiological conditions. In contrast to mammals and many Diptera species, inducible members of the Hsp70 family in Drosophila are constitutively synthesized at a low level and undergo dramatic induction after temperature elevation or other forms of stress. In the courtship suppression paradigm used in this study, Drosophila males that have been repeatedly rejected by mated females during courtship are less likely than naive males to court other females. Although numerous genes with known function were identified to play important roles in long-term memory, there is no direct evidence implicating Hsp70 in this process. To elucidate a possible role of Hsp70 in memory formation, D. melanogaster strains containing different hsp70 copy numbers were used, including strains carrying a deletion of all six hsp70 genes. This investigations exploring the memory of courtship rejection paradigm demonstrated that a low constitutive level of Hsp70 is apparently required for learning and the formation of short and long-term memories in males. The performed transcriptomic studies demonstrate that males with different hsp70 copy numbers differ significantly in the expression of a few definite groups of genes involved in mating, reproduction, and immunity in response to rejection. Specifically, thus analysis reveals several major pathways that depend on the presence of hsp70 in the genome and participate in memory formation and consolidation, including the cAMP signaling cascade (Zatsepina, 2021).

    Experimental evolution under varying sex ratio and nutrient availability modulates male mating success in Drosophila melanogaster

    Biased population sex ratios can alter optimal male mating strategies, and allocation to reproductive traits depends on nutrient availability. However, there is little information on how nutrition interacts with sex ratio to influence the evolution of pre-copulatory and post-copulatory traits separately. To address this omission, this study tested how male mating success and reproductive investment evolve under varying sex ratios and adult diet in Drosophila melanogaster, using experimental evolution. Sex ratio and nutrient availability were found to interact to determine male pre-copulatory performance. Males from female-biased populations were slow to mate when they evolved under protein restriction. By contrast, direct and non-interacting effects of sex ratio and nutrient availability on post-copulatory success were found. Males that evolved under protein restriction were relatively poor at suppressing female remating. Males that evolved under equal sex ratios fathered more offspring and were better at supressing female remating, relative to males from male-biased or female-biased populations. These results support the idea that sex ratios and nutrition interact to determine the evolution of pre-copulatory mating traits, but independently influence the evolution of post-copulatory traits (Sepil, 2022).

    Plastic responses of males and females interact to determine mating behavior

    Individuals can respond plastically to variation in their social environment. However, each sex may respond to different cues and contrasting aspects of competition. Theory suggests that the plastic phenotype expressed by one sex can influence evolutionary dynamics in the other, and that plasticity simultaneously expressed by both sexes can exert sex-specific effects on fitness. However, data are needed to test this theory base. This study examined whether the simultaneous expression of adaptive plasticity by both sexes of Drosophila melanogaster fruit flies in response to their respective social environments interacts to determine the value of key reproductive traits (mating latency, duration, and fecundity). To vary social environments, males were kept alone, or with same sex rivals, and females were kept alone, in same-sex, or mixed-sex groups. Matings were then conducted between individuals from all of these five social treatments in all combinations, and the resulting reproductive traits measured in both "choice" and "no-choice" assays. Mating latency was determined by an interaction between the plastic responses of both sexes to their social environments. Interestingly, the mating latency response occurred in opposing directions in the different assays. In females exposed to same-sex social treatments, mating latency was more rapid with rival treatment males in the choice assays, but slower with those same males in no-choice assays. In contrast, mating duration was determined purely by responses of males to their social environments, and fecundity purely by responses of females. Collectively, the results show that plastic responses represent an important and novel facet of sexual interactions (Fowler, 2022).

    Evidence for stronger sexual selection in males than females using an adapted method of Bateman's classic study of Drosophila melanogaster

    Bateman's principles, originally a test of Darwin's theoretical ideas, has since become fundamental to sexual selection theory and vital to contextualising the role of anisogamy in sex differences of precopulatory sexual selection. Despite this, Bateman's principles have received substantial criticism, and researchers have highlighted both statistical and methodological errors, suggesting that Bateman's original experiment contains too much sampling bias for there to be any evidence of sexual selection. This study uses Bateman's original method as a template, accounting for two fundamental flaws in his original experiments, (i) viability effects and (ii) a lack of mating behaviour observation. Experimental populations of Drosophila melanogaster consisted of wild-type focal individuals and non-focal individuals established by backcrossing the brown eye (bw  -) eye-colour marker - thereby avoiding viability effects. Mating assays included direct observation of mating behaviour and total number of offspring, to obtain measures of mating success, reproductive success, and standardised variance measures based on Bateman's principles. The results provide observational support for Bateman's principles, particularly that (i) males had significantly more variation in number of mates compared to females and (ii) males had significantly more individual variation in total number of offspring. Significantly steeper Bateman gradient was found for males compared to females, suggesting that sexual selection is operating more intensely in males. However, female remating was limited, providing the opportunity for future study to further explore female reproductive success in correlation with higher levels of remating (Davies, 2023).

    Cryptic male mate choice for high-quality females reduces male postcopulatory success in future matings

    Cryptic male mate choice occurs when males differentially allocate resources to females during or after copulation. When male resources are limited, males may benefit by strategically allocating more resources toward higher-quality females. In the fruit fly, Drosophila melanogaster, males mate for longer and may transfer more sperm and more seminal proteins when they mate with larger females compared to smaller females. It is unclear, however, whether this increased investment in large females has any impact on the males' later matings. D. melanogaster males were mated sequentially to females of large or small body size in all possible combinations to test whether cryptic male mate choice for large females is costly to the males' subsequent matings. Second matings were shorter for males compared to their first matings, but there were no differences in fecundity between females mated first or second by a male. Interestingly, male success at defensive sperm competition declined between his first and second matings only when his first mating had been with a large female. This suggests that the higher initial investment in large females reduced male postcopulatory success in their subsequent matings. Cryptic male mate choice may carry underappreciated costs to males that could limit their reproductive potential (Anastasio, 2023).

    Social group composition modulates the role of last male sperm precedence in post-copulatory sexual selection

    In many species, the order in which males mate with a female explains much of the variation in paternity arising from post-copulatory sexual selection. Research in Drosophila suggests that mating order may account for the majority of the variance in male reproductive success. However, the effects of mating order on paternity bias might not be static but could potentially vary with social or environmental factors. To test this idea, an existing dataset was used, collated from an experiment that was previously published (Morimoto et al., PLoS One, 11, 2016, e0154468), with the addition of unpublished data from the same experiment. These previous experiments manipulated larval density in Drosophila melanogaster which generated variation in male and female body size, assembled groups of individuals of different sizes, and measured the mating success and paternity share of focal males. The data presented in this study provides information on each focal male's mating order and the frequency in which focal males remated with same females ('repetitive matings'). This information was combined with previously reported focal male reproductive success to partition variance in paternity into male mating order and repetitive matings across groups that differed in the body size composition of males and females. It was found, as expected, that male mating order explained a considerable portion of the variance in male paternity. However, it was also found that the impact of male mating order on male paternity was influenced by the body size composition of groups. Specifically, males that tended to mate last had a greater paternity advantage, and displayed lower variance, in groups containing a heterogenous mixture male body sizes than in groups with a single male body size. Repetitive mating only had a minor contribution to the variance in male paternity share across all experiments. Overall, these findings contribute to the growing body of research showing that post-copulatory sexual selection is subject to socio-ecological influences (Morimoto, 2023).

    Divergence of a speciation trait through artificial selection: Insights into constraints, by-product effects and sexual isolation

    Speciation and sexual isolation often occur when divergent female mating preferences target male secondary sexual traits. Despite the importance of such male signals, little is known about their evolvability and genetic linkage to other traits during speciation. To answer these questions, divergent artificial selection was imposed for 10 non-overlapping generations on the Inter-Pulse-Interval (IPI) of male courtship songs; which has been previously shown to be a major species recognition trait for females in the Drosophila athabasca species complex. Focusing on one of the species, Drosophila mahican (previously known as EA race), IPI's were examined: (1) rate of divergence, (2) response to selection in different directions, (3) genetic architecture of divergence and (4) by-product effects on other traits that have diverged in the species complex. Rapid and consistent response was found for for higher IPI but less response to lower IPI; implying asymmetrical constraints. Genetic divergence in IPI differed from natural species in X versus autosome contribution and in dominance, suggesting that evolution may take different paths. Finally, selection on IPI did not alter other components of male songs, or other ecological traits, and did not cause divergence in female preferences, as evidenced by lack of sexual isolation. This suggests that divergence of male courtship song IPI is unconstrained by genetic linkage with other traits in this system. This lack of linkage between male signals and other traits implies that female preferences or ecological selection can co-opt and mould specific male signals for species recognition free of genetic constraints from other traits (Yukilevich, 2023).

    Integrating lipid metabolism, pheromone production and perception by Fruitless and Hepatocyte nuclear factor 4

    Sexual attraction and perception, governed by separate genetic circuits in different organs, are crucial for mating and reproductive success, yet the mechanisms of how these two aspects are integrated remain unclear. In Drosophila, the male-specific isoform of Fruitless (Fru), Fru (M), is known as a master neuro-regulator of innate courtship behavior to control perception of sex pheromones in sensory neurons. This study shows that the non-sex specific Fru isoform (Fru (COM)) is necessary for pheromone biosynthesis in hepatocyte-like oenocytes for sexual attraction. Loss of Fru (COM) in oenocytes resulted in adults with reduced levels of the cuticular hydrocarbons (CHCs), including sex pheromones; adults showed altered sexual attraction and reduced cuticular hydrophobicity. Hepatocyte nuclear factor 4 (Hnf4) was identified as a key target of Fru (COM) in directing fatty acid conversion to hydrocarbons in adult oenocytes. fru- and Hnf4 -depletion disrupts lipid homeostasis, resulting in a novel sex-dimorphic CHC profile, which differs from doublesex - and transformer -dependent sexual dimorphism of the CHC profile. Thus, Fru couples pheromone perception and production in separate organs for precise coordination of chemosensory communication that ensures efficient mating behavior (Sun, 2023).

    The Effect of Chromosomes on Courtship Behavior in Sibling Species of the Drosophila virilis Group

    courtship behavior is not a typical quantitative trait that can be easily measured or quantified in both females and males, Each courtship element involves the participation of both female and male partners, making the genetic analysis of this behavior complex. This study employed a modified design to analysis of courtship behavior by introducing what is referred to as 'reference partners' during the testing of hybrid individuals from F1, F2, and backcrosses. These reference partners represented one of the parental species. This approach allowed categorization of all possible test combinations into four groups based on the reference partner's sex (female or male) and their constant genotype towards one of the parental species (D. virilis or D. americana). The genotype of the second partner in the within-group test combinations varied from completely conspecific to completely heterospecific, based on the parental chromosomal sets. To assess the contribution of partner genotypes to the variability of courtship-element parameters, structural equation modeling (SEM) was employed. SEM enabled estimation of the regression of the proportion of chromosomes of a specific species type on the value of each courtship-element parameter in partners with varying genotypes across different test combinations. The aim of the current study was to analyze the involvement of sex chromosomes and autosomes in the formation of courtship structure in D. virilis and D. americana. The genetic analysis was complemented by video recording and formalization of courtship-ritual elements. D. virilis was found to be more sensitive to mate stimuli compared to D. americana. The majority of species-specific parameters, such as latency and duration of courtship elements (e.g., male and female song, following, licking, and circling), were shown to be influenced by the D. virilis genotype. However, not all of these parameters significantly impact copulation success, with the male song, licking, and following being the most significant. In females, the female song was found to have a significant relationship only with copulation duration. The influence of the female genotype on the species-specific parameters of courtship elements is primarily related to autosomes, while the male genotype is associated with the X chromosomes. The study suggests that sexual selection primarily occurs through acoustic and chemoreceptor channels (Belkina, 2023).

    Courtship suppression in Drosophila melanogaster: The role of mating failure

    Drosophila melanogaster is a popular model organism in the study of memory due to a wide arsenal of methods used to analyze neuronal activity. The most commonly used tests in research of behavioral plasticity are shock avoidance associated with chemosensory cues and courtship suppression after mating failure. Many authors emphasize the value of courtship suppression as a model of behavior most appropriate to natural conditions. However, researchers often investigate courtship suppression using immobilized and decapitated females as targets of courtship by males, which makes the data obtained from such flies less valuable. In this study, courtship suppression towards immature mobile non-receptive females was evaluated after training with mated or immature females combined with an aversive stimulus (quinine). The previously described mechanisms of courtship suppression, as a result of the association of the courtship object with the repellent, as well as due to increased sensitivity to the anti-aphrodisiac cVA after mating failure, were not confirmed when immature mobile females were used. The reasons for the discrepancies between the current results and literature data, define the conditions to be met in the courtship suppression test if the aim is to analyze the natural forms of behavioral plasticity, and present data on the test modifications to approximate conditions to natural ones (Goncharova, 2023).

    Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster

    Males have finite resources to spend on reproduction. Thus, males rely on a 'time investment strategy' to maximize their reproductive success. For example, male Drosophila melanogaster extends their mating duration when surrounded by conditions enriched with rivals. This study reports a different form of behavioral plasticity whereby male fruit flies exhibit a shortened duration of mating when they are sexually experienced; this plasticity is referred to as 'shorter-mating-duration (SMD)'. SMD is a plastic behavior and requires sexually dimorphic taste neurons. Several neurons were identified in the male foreleg and midleg that express specific sugar and pheromone receptors. Using a cost-benefit model and behavioral experiments, it was further shown that SMD behavior exhibits adaptive behavioral plasticity in male flies. Thus, this study delineates the molecular and cellular basis of the sensory inputs required for SMD; this represents a plastic interval timing behavior that could serve as a model system to study how multisensory inputs converge to modify interval timing behavior for improved adaptation (Lee, 2023).

    LIM-kinase 1 effects on memory abilities and male courtship song in Drosophila depend on the neuronal type

    The signal pathway of actin remodeling, including LIM-kinase 1 (LIMK1) and its substrate cofilin, regulates multiple processes in neurons of vertebrates and invertebrates. Drosophila melanogaster is widely used as a model object for studying mechanisms of memory formation, storage, retrieval and forgetting. Previously, active forgetting in Drosophila was investigated in the standard Pavlovian olfactory conditioning paradigm. The role of specific dopaminergic neurons (DAN) and components of the actin remodeling pathway in different forms of forgetting was shown. This research investigated the role of LIMK1 in Drosophila memory and forgetting in the conditioned courtship suppression paradigm (CCSP). In the Drosophila brain, LIMK1 and p-cofilin levels appeared to be low in specific neuropil structures, including the mushroom body (MB) lobes and the central complex. At the same time, LIMK1 was observed in cell bodies, such as DAN clusters regulating memory formation in CCSP. GAL4 x UAS binary system was applied to induce limk1 RNA interference in different types of neurons. The hybrid strain with limk1 interference in MB lobes and glia showed an increase in 3-h short-term memory (STM), without significant effects on long-term memory. limk1 interference in cholinergic neurons (CHN) impaired STM, while its interference in DAN and serotoninergic neurons (SRN) also dramatically impaired the flies' learning ability. By contrast, limk1 interference in fruitless neurons (FRN) resulted in increased 15-60 min STM, indicating a possible LIMK1 role in active forgetting. Males with limk1 interference in CHN and FRN also showed the opposite trends of courtship song parameters changes. Thus, LIMK1 effects on the Drosophila male memory and courtship song appeared to depend on the neuronal type or brain structure (Zhuravlev, 2023).

    A sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    No water, no mating: Connecting dots from behaviour to pathways

    Insects hold considerable ecological and agricultural importance making it vital to understand the factors impacting their reproductive output. Environmental stressors are examples of such factors which have a substantial and significant influence on insect reproductive fitness. Insects are also ectothermic and small in size which makes them even more susceptible to environmental stresses. The present study assesses the consequence of desiccation on the mating latency and copulations duration in tropical Drosophila melanogaster. Flies were tested for these reproductive behavioral parameters at varying body water levels and with whole metabolome analysis in order to gain a further understanding of the physiological response to desiccation. The results showed that the duration of desiccation is positively correlated with mating latency and mating failure, while having no influence on the copulation duration. The metabolomic analysis revealed three biological pathways highly affected by desiccation: starch and sucrose metabolism, galactose metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. These results are consistent with carbohydrate metabolism providing an energy source in desiccated flies and also suggests that the phenylalanine biosynthesis pathway plays a role in the reproductive fitness of the flies. Desiccation is a common issue with smaller insects, like Drosophila and other tropical insects, and these findings indicate that this lack of ambient water can immediately and drastically affect the insect reproductive behaviour, which becomes more crucial because of unpredictable and dynamic weather conditions (Arya, 2021).

    Sexual arousal gates visual processing during Drosophila courtship

    Long-lasting internal arousal states motivate and pattern ongoing behaviour, enabling the temporary emergence of innate behavioural programs that serve the needs of an animal, such as fighting, feeding, and mating. However, how internal states shape sensory processing or behaviour remains unclear. In Drosophila, male flies perform a lengthy and elaborate courtship ritual that is triggered by the activation of sexually dimorphic P1 neurons, during which they faithfully follow and sing to a female. By recording from males as they court a virtual 'female', this study gained insight into how the salience of visual cues is transformed by a male's internal arousal state to give rise to persistent courtship pursuit. The gain of LC10a visual projection neurons is selectively increased during courtship, enhancing their sensitivity to moving targets. A concise network model indicates that visual signalling through the LC10a circuit, once amplified by P1-mediated arousal, almost fully specifies a male's tracking of a female. Furthermore, P1 neuron activity correlates with ongoing fluctuations in the intensity of a male's pursuit to continuously tune the gain of the LC10a pathway. Together, these results reveal how a male's internal state can dynamically modulate the propagation of visual signals through a high-fidelity visuomotor circuit to guide his moment-to-moment performance of courtship (Sten, 2021).

    Sperm depletion in relation to developmental nutrition and genotype in Drosophila melanogaster

    Nutrient limitation during development can restrict the ability of adults to invest in costly fitness traits, and genotypes can vary in their sensitivity to developmental nutrition. However, little is known about how genotype and nutrition affect male ability to maintain ejaculate allocation and achieve fertilization across successive matings. Using 17 isogenic lines of Drosophila melanogaster, This study investigated how variation in developmental nutrition affects males' abilities to mate, transfer sperm, and sire offspring when presented with successive virgin females. This study found that, with each successive mating, males required longer to initiate copulation, transferred fewer sperm, and sired fewer offspring. Males reared on a low-nutrient diet transferred fewer sperm than those reared on nutritionally superior diets, but the rate at which males depleted their sperm, as well as their reproductive performance, was largely independent of diet. Genotype and the genotype x diet interaction explained little of the variation in these male reproductive traits. Results show that sperm depletion can occur rapidly and impose substantial fitness costs for D. melanogaster males across multiple genotypes and developmental environments (Macartney, 2021).

    The nuclear receptor Hr46/Hr3 is required in the blood brain barrier of mature males for courtship

    The blood brain barrier (BBB) forms a stringent barrier that protects the brain from components in the circulation that could interfere with neuronal function. At the same time, the BBB enables selective transport of critical nutrients and other chemicals to the brain. Beyond these functions, another recently recognized function is even less characterized, specifically the role of the BBB in modulating behavior by affecting neuronal function in a sex-dependent manner. Notably, signaling in the adult Drosophila BBB is required for normal male courtship behavior. Courtship regulation also relies on male-specific molecules in the BBB. Previous studies have demonstrated that adult feminization of these cells in males significantly lowered courtship. In this study microarray analysis was carried out of BBB cells isolated from males and females. Findings revealed that these cells contain male- and female-enriched transcripts, respectively. Among these transcripts, nuclear receptor Hr46/Hr3 was identified as a male-enriched BBB transcript. Hr46/Hr3 is best known for its essential roles in the ecdysone response during development and metamorphosis. This study demonstrated that Hr46/Hr3 is specifically required in the BBB cells for courtship behavior in mature males. The protein is localized in the nuclei of sub-perineurial glial cells (SPG), indicating that it might act as a transcriptional regulator. These data provide a catalogue of sexually dimorphic BBB transcripts and demonstrate a physiological adult role for the nuclear receptor Hr46/Hr3 in the regulation of male courtship, a novel function that is independent of its developmental role (Lama, 2022).

    Divergence and correlated evolution of male wing spot and courtship display between Drosophila nepalensis and D. trilutea

    Male-specific wing spots are usually associated with wing displays in the courtship behavior of Drosophila and may play important roles in sexual selection. Two closely related species, D. nepalensis and D. trilutea, differ in wing spots and scissoring behavior. This study compared male morphological characters, pigmentation intensity of male wing spots, wing-scissoring behavior, courtship songs and reproductive isolation between two species. F1 fertile females and sterile males result from the cross between females of D. nepalensis and males of D. trilutea. The pigmentation of wing spots is significantly weaker in D. trilutea than in D. nepalensis and the F1 hybrid. Males scissor both wings in front of the female during courtship, with a posture spreading wings more widely, and at a faster frequency in D. nepalensis than in D. trilutea and the F1s. Males of D. trilutea vibrate wings to produce two types (A and B) of pulse songs, whereas D. nepalensis and the F1s sing only type B songs. The incidence of wing vibration and scissoring during courtship suggests that wing vibration is essential but scissoring is a facultative courtship element for successful mating in both species. The association between the darker wing spots with more elaborate scissoring might be the consequence of correlated evolution of these traits in D. nepalensis, however D. trilutea retains wing scissoring during courtship despite having weaker pigmentation of wing spots. The genetic architecture of two traits differs in the F1s, consistent with maternal or sex-linked effects for spots but non-additive effects for scissoring (Mo, 2021).

    Dorsal-lateral clock neurons modulate consolidation and maintenance of long-term memory in Drosophila

    A newly formed memory is initially unstable. However, if it is consolidated into the brain, the consolidated memory is stored as stable long-term memory (LTM). Despite the recent progress, the molecular and cellular mechanisms of LTM have not yet been fully elucidated. The fruitfly Drosophila melanogaster, for which various genetic tools are available, has been used to clarify the molecular mechanisms of LTM. Using the Drosophila courtship-conditioning assay as a memory paradigm, it was previously identified that the circadian clock gene period (per) plays a vital role in consolidating LTM, suggesting that per-expressing clock neurons are critically involved in LTM. However, it is still incompletely understood which clock neurons are essential for LTM. This study shows that dorsal-lateral clock neurons (LNds) play a crucial role in LTM. Using an LNd-specific split-GAL4 line, it wa confirmed that disruption of synaptic transmission in LNds impaired LTM maintenance. On the other hand, induction of per RNAi or the dominant-negative transgene of Per in LNds impaired LTM consolidation. These results reveal that transmitter release and Per function in LNds are involved in courtship memory processing (Suzuki, 2022).

    Behavioral signatures of structured feature detection during courtship in Drosophila

    Many animals detect other individuals effortlessly. In Drosophila, previous studies have examined sensory processing during social interactions using simple blobs as visual stimulation; however, whether and how flies extract higher-order features from conspecifics to guide behavior remains elusive. Arguing that this should be reflected in sensorimotor relations, this study developed unbiased machine learning tools for natural behavior quantification and applied these tools, which may prove broadly useful, to study interacting pairs. By transforming motor patterns with female-centered reference frames, this study established circling, where heading and traveling directions intersect, as a unique pattern of social interaction during courtship. Circling was found to be highly visual, with males exhibiting view-tuned motor patterns. Interestingly, males select specific wing and leg actions based on the positions and motions of the females' heads and tails. Using system identification, visuomotor transformation functions were derived indicating history-dependent action selection, with distance predicting action initiation and angular position predicting wing choices and locomotion directions. Integration of vision with somatosensation further boosts these sensorimotor relations. Essentially comprised of orchestrated wing and leg maneuvers that are more variable in the light, circling induces mutually synchronized conspecific responses stronger than wing extension alone. Finally, this study found that actions depend on integrating spatiotemporally structured features with goals. Altogether, we identified a series of sensorimotor relations during circling, implying that during courtship, flies detect complex spatiotemporally structured features of conspecifics, laying the foundation for a mechanistic understanding of conspecific recognition in Drosophila (Ninf, 2022).

    Nutrients and pheromones promote insulin release to inhibit courtship drive

    Food and reproduction are the fundamental needs for all animals. However, the neural mechanisms that orchestrate nutrient intake and sexual behaviors are not well understood. This study found that sugar feeding immediately suppresses sexual drive of male Drosophila, a regulation mediated by insulin that acts on insulin receptors on the courtship-promoting P1 neurons. The same pathway was co-opted by anaphrodisiac pheromones to suppress sexual hyperactivity to suboptimal mates. Activated by repulsive pheromones, male-specific PPK23 neurons on the leg tarsus release crustacean cardioactive peptide (CCAP) that acts on CCAP receptor on the insulin-producing cells in the brain to trigger insulin release, which then inhibits P1 neurons. These results reveal how male flies avoid promiscuity by balancing the weight between aphrodisiac and anaphrodisiac inputs from multiple peripheral sensory pathways and nutritional states. Such a regulation enables male animals to make an appropriate mating decision under fluctuating feeding conditions (Zhang, 2022).

    This work identified a neuropeptidergic neural circuit underlying mating decision, and a direct link is revealed between the 'metabolic center' and the 'sex center' in the Drosophila brain. First, sugar feeding largely suppressed male sexual drive toward a virgin female, and this metabolic state-dependent neural control relied on ILP2 and ILP5. A suppression on P1 neurons was induced in vitro with the activation of IPCs, which was triggered by loading sugar. This conclusion was further validated by the fact that the decrease of P1 neuron activity was weaker when InR was knock down specifically on P1 neurons. It was also demonstrated that leg tarsal PPK23 M cells release CCAP once contacting aversive pheromone and this inhibitory signal activates IPCs in the brain via CCAP-R to release ILP2/5. IPC-derived ILP2/5 furthermore suppresses the activity of courtship-promoting P1 neurons to ultimately shut down male's sexual drive toward inappropriate mating targets. Together with inhibition on P1 by the tandem connections of PPK23 M cells and mAL neurons, male flies maintain high selectivity against inappropriate mates (Zhang, 2022).

    As the central hub in control of male courtship, P1 neurons in the brain integrate both the external and internal information. The former includes sensory inputs from a potential mating target. While visual and olfactory cues are reported to trigger a male's propensity to court, contact pheromones can be either attractive or repulsive and gate the perception for other sensory pathways. The internal state, on the other hand, represents the male's readiness to mate, e.g., his mating history and nutritional state. P1 neurons constantly monitor the internal state of a male to evaluate the readiness to court or mate. For example, dopaminergic neurons control the mating drive of a male, and neuropeptide Drosulfakinin (DSK) and NPF impose an inhibitory tone on P1 neurons by encoding either mating experiences or nutritional state. In addition, sleep regulates mating via the functional interaction between circadian neurons and P1. Insulin signaling reportedly plays a well-established role in homeostatic regulation. This study revealed an unprecedented role of insulin in modulating male sexual behavior. On the basis of the fact that insulin level changes under numerous physiological conditions, such as feeding, temperature change, and sleep, the current findings raised the question whether, under such conditions, insulin regulates sexual activity via InR on P1. The present data support the existence of feeding/mating interaction via the insulin signaling (Zhang, 2022).

    Further investigation is needed to reveal the biological significance of courtship inhibition immediately after a sugar meal. In the studies of human and mouse, sugar intake reduces the level of testosterone, an effect likely mediated with insulin. This adversity of sugar intake on libido may be important for the animals' fitness. There is an immediate boost of the blood sugar level after a sugar meal. Insulin is then secreted to help move the sugar from the blood into the cells. During this process, the sexual activity may be momentarily inhibited to foster sugar uptake. It still needs further investigation how decreased sexual behaviors contribute to energy storage. Another interesting question is whether other nutrition-related hormones such as Adipokinetic hormone (Akh) and Unpaired 2 (Upd2) also regulate sexual activity in flies. It is already reported that DSK and NPF are both required to tune a male's sexual drive. Recently, it was reported that protein intake caused postprandial sleepiness that may be critical for protein metabolism, suggesting that animals' nutritional homeostasis is critical in maintaining the balance of their feeding, reproduction, and other behaviors (Zhang, 2022).

    Another intriguing question that warrants further investigation is whether insulin released upon contacting aversive pheromones would cause certain metabolic consequences to the males. It was reported that exposing male flies to female pheromone but preventing them from mating reduced males' life span. As deterring courtship is relatively fast, it would be interesting to look at the long-term effects after a male is exposed to aversive pheromones (Zhang, 2022).

    Insulin was implicated to regulate female sexual receptivity. The female flies with mutations in their insulin-like protein genes exhibit a higher sexual activity, a similar defect as seen in male flies in current study. However, the neural circuit controlling mating in male and female shows profound sexual dimorphism. It thus raises an interesting question: How does insulin regulate the sexual drive in females? A virgin female's receptivity is controlled by double-sex-positive neurons in the brain. Among them, clusters of neurons of pCd and pC1 play a determinant role in female's sex behaviors. It is still an open question whether these neurons express InR and, if so, under what circumstance are they inhibited by insulin release. Insulin is essential for vitellogenesis in female flies, suggesting that insulin signaling may play differential roles at different reproductive stages (Zhang, 2022).

    Genes Responsible for H(2)S Production and Metabolism Are Involved in Learning and Memory in Drosophila melanogaster

    The gasotransmitter hydrogen sulfide (H(2)S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H(2)S production and metabolism. This study investigated the role of H(2)S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. The learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation-reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes (Zatsepina, 2022).

    Amplification of Drosophila olfactory responses by a DEG/ENaC Channel promotes male courtship behavior

    Insect olfactory receptors operate as ligand-gated ion channels that directly transduce odor stimuli into electrical signals. However, in the absence of any known intermediate transduction steps, it remains unclear whether and how these ionotropic inputs are amplified in olfactory receptor neurons (ORNs). This study finds that amplification occurs in the Drosophila courtship-promoting ORNs through Pickpocket (PPK25), a member of the degenerin/epithelial sodium channel family (DEG/ENaC). Pharmacological and genetic manipulations indicate that, in Or47b and Ir84a ORNs, PPK mediates Ca(2+)-dependent signal amplification via an intracellular calmodulin-binding motif. Additionally, hormonal signaling upregulates PPK expression to determine the degree of amplification, with striking effects on male courtship. Together, these findings advance understanding of sensory neurobiology by identifying an amplification mechanism compatible with ionotropic signaling. Moreover, this study offers new insights into DEG/ENaC activation by highlighting a novel means of regulation that is likely conserved across species (Ng, 2019).

    Vertebrates detect odorants with G-protein-coupled receptors (GPCRs), the activation of which triggers subsequent metabotropic signaling cascades in the olfactory receptor neurons (ORNs) to transduce chemical stimuli into electrical signals. These series of transduction events also provide opportunities to amplify input signals. In contrast, insect olfaction is initiated by ligand-gated receptor channels that lack canonical G protein interacting domains. Although various G proteins and effectors have been implicated in the function of insect ORNs, it remains an open question whether those molecules play a specific role in olfactory transduction or a regulatory role in neuronal development, maintenance, and neuromodulation. Given that ligand-gated receptor channels can directly convert sensory stimuli to neuronal depolarization, it is unclear whether and how ionotropic inputs can be amplified in the absence of any known intermediate transduction steps (Ng, 2019).

    In earlier work, it was found that the responses of Or47b ORNs, which detect aphrodisiac fly odors in D. melanogaster, increase with age in male flies (Lin, 2016), pointing to the possibility of signal amplification downstream of insect olfactory receptors. This age-dependent plasticity therefore presents an opportunity to investigate the mechanisms by which ionotropic sensory inputs can be amplified. Intriguingly, Or47b ORNs express a degenerin/epithelial sodium channel (DEG/ENaC) subunit named Pickpocket (PPK25). Within invertebrate genomes, DEG/ENaCs constitute one of the largest ion channel families. In mechanosensory and gustatory neurons, PPK subunits are involved in touch, proprioception, nociception, salt taste, water sensation, and recognition of contact pheromones. However, the functional role of PPK in olfaction remains unknown (Ng, 2019).

    If PPK amplifies olfactory signals, how then is its activity regulated? Can it function as a transduction channel activated by intracellular second messengers downstream of receptor activation? Members of the DEG/ENaC superfamily, including the mammalian nonvoltage-gated sodium channels (SCNNs) and acid-sensing sodium ion channels (ASICs), are known to open in response to mechanical stimuli, extracellular ligands, or are otherwise constitutively active. In cultured cell lines, various intracellular signaling mechanisms can influence DEG/ENaC currents by regulating channel transcription, endocytosis, degradation, or translocation. Post-translational modification is also known to modulate DEG/ENaC function; for example, constitutive channel activity can be regulated by CaMKII-mediated phosphorylation or by protease-mediated cleavage of the extracellular domain. However, the possibility of DEG/ENaC activation by direct interaction with an intracellular ligand has not been explored (Ng, 2019).

    This study shows that signal amplification can occur downstream of ligand-gated receptor channels. The age-dependent response plasticity of Or47b ORNs arises through PPK25-mediated amplification. Additionally, this mechanism is employed in another type of courtship-promoting ORN expressing the Ir84a receptor. Interestingly, the degree of amplification is determined by PPK expression levels, which are in turn upregulated by a reproductive hormone. Thus, a common hormone regulates these two parallel olfactory pathways to coordinate courtship behavior. Mechanistically, PPK operates as a transduction channel: its activation requires odor-induced Ca2+ influx and a calmodulin binding motif (CBM) in the N-terminal intracellular domain. This result therefore highlights a novel mechanism whereby DEG/ENaCs can be activated by second messengers, a critical feature common to all transduction channels. Moreover, similar intracellular CBMs are predicted in multiple DEG/ENaCs across animal species, suggesting an evolutionarily conserved regulatory mechanism for channels in this superfamily (Ng, 2019).

    This study has demonstrated that ionotropic sensory inputs can be amplified in select Drosophila ORNs whose receptors are ligand-gated cation channels. Pharmacological and genetic experiments reveal a simple and elegant mechanism for this amplification. Upon odor stimulation, receptor excitation allows for direct influx of Ca2+, which serves as a second messenger to activate a DEG/ENaC channel, PPK25, and thereby amplify ORN responses (Ng, 2019).

    Ionotropic signal amplification, as described, affords remarkable versatility in sensory signaling when compared against G-protein-mediated metabotropic mechanisms. In vertebrate olfaction, separate families of metabotropic receptors typically couple to different G proteins, each engaging a unique downstream signaling cascade. In contrast, activation of specific G proteins is not required for Ca2+-mediated amplification, making it compatible with a wide variety of ionotropic receptor channels, so long as these receptors are permeable to Ca2+. As evidenced in this study, PPK can function downstream of Or47b, Ir84a, and ChR2, despite their low sequence similarity and distinct topologies because these receptors can flux Ca2+ (Ng, 2019).

    These findings highlight striking differences and commonalities between insect and vertebrate olfactory transduction. This study observed surprising heterogeneity within insect olfactory transduction: PPK is neither expressed nor functional in another ORN type expressing Or22a, which belongs to the same receptor family as Or47b. It is unclear whether input signals in Or22a ORNs are amplified. If so, it is likely through a different mechanism. This finding indicates that insect ORNs expressing the same family of receptors do not necessarily employ the same mechanism for amplification, in contrast with vertebrate olfaction where receptors from the same family share a common signaling pathway. Despite this difference, the activation mechanism and function of PPK are remarkably similar to those of Anoctamin (ANO2), a key transduction channel in vertebrate ORNs. Specifically, both PPK and ANO are activated by calcium to amplify olfactory inputs. Although the sources of Ca2+may differ-ligand-gated ion channels in insects or cyclic nucleotide-gated cation channels in vertebrates-Ca2+-mediated amplification may represent a shared signaling motif between the two olfactory systems (Ng, 2019).

    Interestingly, the impact of signal amplification on spike output differs between insect and vertebrate ORNs. Consistent with its role as a transduction channel, Ano mutation markedly reduces odor-evoked currents. However, the spike output of Ano knockout ORNs is higher than that of wild-type neurons. Vertebrate ORN spike number peaks when the transduction current is largely carried by ANO at intermediate odor concentrations, suggesting a strong depolarization block whereby amplification provides negative feedback to clamp total spike output. In contrast, the local field potential and spike responses of insect ORNs both peak at saturating odor concentrations. As such, blocking PPK25-mediated amplification not only reduces LFP response but also total spike number. Therefore, signal amplification in insect ORNs may predominantly serve to modulate the gain of neuronal output (Ng, 2019).

    What is the functional significance of PPK in the Or47b and Ir84a ORNs? Notably, these are the only ORN types known to promote courtship in D. melanogaster males, whose fertility and courtship drive increase and peak at about days of age. Male mating drive is highly influenced by external olfactory cues, including the availability of mates and food signaled by Or47b and Ir84a ORNs, respectively. Remarkably, the responses of both ORN types exhibit age-dependent plasticity, which is coordinated by the same reproductive hormone-juvenile hormone-through upregulation of PPK expression in older males. The expression level of PPK in turn determines the ORN response magnitude, with striking impacts on courtship. Therefore, flexibility over this biologically salient behavior is afforded by the dynamic regulation of PPK expression, which heightens males' sensitivity to food and mate odors at their age of peak fertility. This upregulation of PPK provides a molecular mechanism for how sex-specific refinements of olfactory circuits are achieved via hormonal signaling (Ng, 2019).

    The critical role of the intracellular CBM in PPK function argues that Ca2+/CaM activates the channel by directly interacting with this motif. Such regulation contrasts sharply with previously reported mechanisms, in which Ca2+/CaM indirectly modulates ENaC activity through intermediate proteins. For example, in cultured Xenopus cells, Ca2+/CaM can inhibit ENaC currents by interacting with MARCKS (myristoylated alanine-rich C kinase substrate) to modulate channel open probability and also by activating CaMKII to regulate ENaC apical trafficking. Together, these findings highlight the complexity of interactions between Ca2+/CaM and neuronal DEG/ENaC (Ng, 2019).

    The results described in this study further advance understanding of DEG/ENaC activation. The gating mechanisms for this family of sodium channels are known to be highly diverse: some open in response to mechanical stimuli; others to extracellular ligands; and still others are constitutively active. In light of these findings, it is possible that other members of the DEG/ENaC superfamily may also be directly activated by intracellular second messengers, allowing them to function as transduction channels to amplify sensory inputs. In support of this notion, similar N-terminal intracellular CBMs were bioinformatically identified in multiple members of the DEG/ENaC superfamily across species-including worm, fruit fly, mosquito, mouse, and human-suggesting that those channels have the potential to function as Ca2+-activated transduction channels (Ng, 2019).

    Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons

    For successful mating, a male animal must execute effective courtship behaviors toward a receptive target sex, which is female. Whether the courtship execution capability and upregulation of courtship toward females are specified through separable sex-determining genetic pathways remains uncharacterized. This study found that one of the two Drosophila sex-determining genes, doublesex (dsx), specifies a male-specific neuronal component that serves as an execution mechanism for courtship behavior, whereas fruitless (fru) is required for enhancement of courtship behavior toward females. The dsx-dependent courtship execution mechanism includes a specific subclass within a neuronal cluster that co-express dsx and fru. This cluster contains at least another subclass that is specified cooperatively by both dsx and fru. Although these neuronal populations can also promote aggressive behavior toward male flies, this capacity requires fru-dependent mechanisms. These results uncover how sex-determining genes specify execution capability and female-specific enhancement of courtship behavior through separable yet cooperative neurogenetic mechanisms (Ishii, 2020).

    This study has uncovered distinct yet cooperative roles of dsx and fru on male-type social behaviors through a specific subset of P1/pC1 neurons. For courtship behaviors, this study found that NP2631 ∩ dsxFLP neurons are specified in a fru-independent manner, and in males, their capacity to generate courtship behaviors does not require fruM. However, activation of NP2631 ∩ dsxFLP neurons in fruF males failed to increase courtship selectively toward female targets. These results suggest that dsx plays a major role in establishing a neuronal circuit that enables the male flies to execute courtship behavior, whereas fru is critical for enhancing courtship behavior toward females, likely through proper recognition of target sex. The fact that the sex of the target flies influences the function of P1/pC1 subsets implies that information about target sex can modulate the neural circuit units downstream of these neurons, and encourages revision of the linear circuit model for sexually dimorphic social behaviors. In contrast, the complete specification and courtship-promoting functions of P1a neurons require both dsx and fru, revealing genetic and functional heterogeneity within P1/pC1 neurons. Lastly, NP2631 ∩ dsxFLP and P1a neurons require a fruM-dependent mechanism to promote male-type aggressive behavior. This suggests that neither of these neurons are part of the execution mechanism for male-type aggressive behavior, and that the genetic mechanisms specifying execution components for courtship and aggressive behaviors are different (Ishii, 2020).

    Electrical stimulation of various parts of the brain has been known to elicit complex behaviors, including social behaviors, for almost a century. Recent technological advances have allowed researchers to identify specific, genetically labeled populations of neurons that can induce mating and aggressive behaviors upon acute optogenetic stimulation in both mice and in flies, even toward suboptimal targets (such as inanimate objects). These findings seem consistent with the idea that neuronal activation can override most contexts and generate specific behaviors depending on the identity of stimulated cells. However, interactions with a target animal can transmit important information which a tester animal may use to choose appropriate behaviors. In fact, attacks triggered by optogenetic stimulation of ventrolateral hypothalamus (VMH) in male mice tend to last longer toward castrated males than toward female targets, and chemogenetic activation of progesterone receptor-expressing VMH neurons appears to induce more attacks toward male than toward female targets. While effects on target sex are not consistently documented, the current results and previous observations in mice show that the target sex has a significant impact on behavioral choice even for optogenetically induced social behaviors. These results suggest that sensory or behavioral feedback from target animals can impact the operation of what may appear to be an 'execution mechanism' for a given behavior (Ishii, 2020).

    Identification of neural sites where the information about the target sex is integrated with the activity of both NP2631 ∩ dsxFLP and P1a neurons will be an important step in understanding how such context cues modulate ongoing neural activity and, ultimately, behavioral outcome. While a 'command'-like center that irreversibly executes courtship or aggressive behaviors, like recently characterized egg-laying controlling neurons, may exist, it is also possible that information about target sex (and its behavioral response) can be injected at multiple levels of a neural circuit, thereby ensuring the target sex-specific execution of sexually dimorphic social behaviors. This is conceptually analogous to the neural control of fine motions, which can be constantly adjusted by sensory feedback and efference copies all the way down to the motoneuron level (Ishii, 2020).

    Recently, the importance of addressing sex as a biological variable has been widely recognized. In the context of social behaviors, this variable in the tester animals can be critical for uncovering the underlying neural mechanisms (Ishii, 2020).

    The functional segregation of dsx and fru that was observed in this study can be considered analogous to the organizational and activation functions of sex hormones in mammals. Differential exposure to gonadal steroid hormones, mostly through estrogen receptors, specifies neural circuits that are necessary for sex-specific reproductive behaviors, whereas hormonal surges in the adult stage (such as testosterone or progesterone) orchestrate activation of sex-specific behaviors. It is postulated that dsx has an organizational function for the courtship execution circuit, whereas fru is important for the appropriate activation of the circuit (Ishii, 2020).

    The results do not mean that fru is not necessary for the establishment of all neuronal components involved in courtship. Nonetheless, the result suggests that the wing extension execution circuit that connects NP2631 ∩ dsxFLP neurons and relevant motoneurons is specified even in the absence of fruM, which is consistent with previous observations that fruF males are capable of expressing at least a part of courtship behavior. While a specification role for dsx on P1/pC1 neurons has been previously reported, the current study showed for the first time the behavioral role of a specific P1/pC1 subset (NP2631 ∩ dsxFLP neurons) in fruF males. dsx is important for the specification of a few other behaviorally relevant sexual dimorphisms in the Drosophila nervous system. For instance, the sexually dimorphic axon development of leg gustatory receptor neurons, which includes aphrodisiac pheromone sensors, requires dsx function. The neural connectivity and function of TN1 neurons, which are pre-motor neurons important for the production of pulse song, are also specified by dsx. Several classes of abdominal ganglia neurons involved in male copulation also express dsx. Although relatively few in number, these examples display the importance of dsx in key neuronal populations for organizing circuit components that are essential for the execution of courtship behaviors. It is noteworthy that dsx is involved in sex-determination across a variety of animal phyla, whereas fru's role in sex-determination seems confined to insects. This suggests that dsx may be evolutionarily more ancient in the context of sex-determination than fru, which can account for its dominance over fru when specifying sexually dimorphic neurons that co-express dsx and fru (Ishii, 2020).

    The proposal that fruM may be important for enhancing courtship behavior specifically towards females is consistent with the fact that many characterized fru-expressing neurons are involved in processing sex- and species-specific sensory cues. Namely, P1a neurons, as well as more broadly defined P1/pC1 neurons accessed by different genetic reagents, are known to respond to sex-specific chemical cues, underscoring their critical role in sensory integration for courtship. fruM can play the 'activation' role for courtship by establishing sensory circuits that transmit sex-specific sensory information to P1/pC1 neurons, or by enabling P1/pC1 neurons to properly integrate and transform such neural inputs. Neuroanatomical defects of P1a neurons in fruF males could disrupt either process (Ishii, 2020).

    Gain control of sex-specific sensory cues can be one neuronal mechanism for the 'activation' function, but courtship behavior can be enhanced in other ways as well. For instance, behavioral persistence or context-dependent intensity adjustment can result in an increase of the overall courtship vigor. Recently, a new class of fru-expressing neurons downstream of P1a neurons has been found to mediate the persistence of courtship behavior triggered by P1a neuronal activation (Jung, 2020). Even if fruM is not absolutely necessary for the formation of the minimal wing extension execution circuit, it can have a significant impact on the generation of effective wing extension toward female target flies (Ishii, 2020).

    While it is concluded that the role of fru is not necessarily to specify the execution mechanism for courtship behavior, fruM females can still perform wing extensions or dsx- expressing neurons in females can elicit wing extensions, suggesting that the residual execution mechanism for at least a part of courtship behavior may be specified in a sex-invariant manner. The presence of a latent mating execution circuit in female brains is also suggested in mice. Because the courtship songs produced by females or fruM females are defective, male-type splicing of dsx nonetheless seems to be instrumental in organizing the proper execution mechanism for Drosophila courtship behavior (Ishii, 2020).

    In striking contrast to wing extensions, this study found that activation of neither NP2631 ∩ dsxFLP nor P1a neurons in fruF males induced lunges. This result points to the existence of a fruM-dependent execution mechanism for male-type aggressive behaviors, likely downstream of these neurons. In Wohl (2020), it was found that at least one group of fruM-dependent neurons can promote male-type aggressive behaviors independent of dsx. Therefore, a separation of the courtship execution mechanism and the aggression execution mechanism by two sex-determining genes is likely accomplished by a partial separation of underlying neural circuits. The aggression-promoting function of NP2631 ∩ dsxFLP and P1a neurons likely reflects their roles to coordinate aggression and courtship depending on internal and external conditions, instead of a simple decision switch that triggers fixed types of behavior (Ishii, 2020).

    Both dsx and fru encode transcription factors. The sexually dimorphic morphology and wiring specificity of many fru-expressing neurons are determined in a cell-autonomous manner, suggesting that dsx and fru define Drosophila sex at a cellular level through regulation of a specific set of target genes. Mammalian sex hormones ultimately exert their effects through nuclear steroid receptors, which serve as transcription factors. Thus, both in flies and in mammals, organismal sex can be regarded as a collective phenotype of genetic 'sexes' that can be reduced down to the cellular leve. To understand how sex at the neuronal level influences sexually dimorphic behaviors, cell-type specific manipulation of sex-determining genes is required. The current study focused on neural functions in a whole animal mutant, which prevents addressing the role of either dsx or fru specifically within NP2631 ∩ dsxFLP or P1a neurons. For example, it is ot known whether NP2631 ∩ dsxFLP neurons in fruF males failed to enhance courtship behavior toward female targets because of the absence of fruM within this population, or because of the lack of fruM in other neuronal populations, or both. In addition, the current approach does not address if it is the presence of dsxM or the absence of dsxF that is important for the specification of male-type NP2631 ∩ dsxFLP neurons or P1a neurons (Ishii, 2020).

    It is important to note that sex specification is a developmental process of transformation. Both at genetic and organismal levels, one sex is not a loss-of-function mutant of the other. Loss-of-function manipulations at the cellular level, by cell type-specific RNA interference or CRISPR interference -based approaches, may show that either dsx or fru is necessary for the proper development or function of the given neurons, but may be insufficient to illuminate the genetic origin of the sex-specific transformation at the cellular level. In addition, temporally and spatially precise manipulation of genes during development remains difficult. This can create a difficulty interpreting the effects of either knock-down or over-expression of sex determining genes, which are dynamically regulated from early developmental stages. Creation of neuronal mutant clones may circumvent this problem, but the tra mutation, which has been previously used to convert a 'neuronal sex', cannot dissociate the roles of dsx and fru (Ishii, 2020).

    Faced with these often overlooked limitations of cell-type specific gene manipulations, it would be informative to characterize what types of transformations are observed in mutants of sex-specific splicing at an organismal level, as in this and other studies. Although a constitutive mutants have above-mentioned limitations, they nonetheless establish fundamental functional differences among sex-determining genes, as well as benchmarks for the efficacy for cell-specific manipulations techniques. Although clearly out of the scope of the current study, electron microscopy-based connectome reconstructions of fruF male and fruM female brains could provide useful information for understanding the transformative nature of sex specification in the brain (Ishii, 2020).

    Lastly, the serendipitous finding that NP2631 ∩ dsxFLP and P1a neurons contain genetically and functionally distinct populations underscores the importance of characterizing neuronal cell types in greater detail. How to determine cell types remains a challenge in neuroscience, but genetic access to a finely defined population of neurons even within what is considered as a single class of neurons can be the key to understand how a neural circuit generates complex behaviors such as social behaviors (Ishii, 2020).

    In the posterior part of male brains, 'P1' neurons, as defined by fru-expressing cluster, and pC1 neurons, as defined by dsx-expressing cluster, extensively overlap. This raises a question about the distinction between 'P1' and 'pC1' neurons. Furthermore, recent single cell level analyses of the neurons that belong to the male 'P1' cluster or 'pC1' cluster revealed surprising neuroanatomical and functional diversity, raising a possibility that P1/pC1 neurons may be functionally heterogeneous as well (Ishii, 2020).

    Surprisingly, this study found that behaviorally relevant NP2631 ∩ dsxFLP and P1a neurons, as well as NP2631 ∩ fruFLP and P1a neurons, seldom overlap. Optogenetic stimulation of NP2631 ∩ dsxFLP and P1a neurons triggers social behaviors in temporally distinct manners. Moreover, fru has a different impact on the specification and function of these two neuron groups, suggesting that little overlap of NP2631 ∩ dsxFLP and P1a neurons does not necessarily reflect arbitrary labeling bias within a single homogeneous neuronal population by different genetic reagents. Instead, these observations support the idea that of P1/pC1 neurons consist of functionally diverse subtypes (Ishii, 2020).

    It is acknowledged that the genetic reagents used in this study are likely insufficient to resolve the possible heterogeneity within either NP2631 ∩ dsxFLP or P1a neurons. Differential expression patterns of FruM proteins within both clusters alone suggest that such heterogeneity almost certainly exists. Recent advances in whole-brain neural reconstruction using electron microscopy images will provide a foundation for precise characterization of Drosophila neurons, as has been recently used for the female-type 'pC1' cluster. A large number of 'split-GAL4' collections will allow universal access to the specific subpopulations. These types of tools will facilitate cross-study comparisons of neuroanatomical and behavioral data, and will serve as a catalyst to understand the logic of neural control of behavior in general. With the advance of single cell-level genetic and epigenetic profiling techniques, the importance of precisely characterizing the targeted neuronal types will only grow not only in Drosophila, but in every model organism. Reproducible access to each neuronal type can uncover functional units for a given behavior at even finer detail, which will be fundamental for deconstructing the dynamics of neural circuits that are responsible for generating social behaviors in a context-dependent manner. Such knowledge will be also critical for establishing theoretical models that account for brain operations and population-level dynamics of animals engaging in social interactions (Ishii, 2020).

    The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila

    Fruitless alternative pre-mRNA splicing (AS) isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, this study shows that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ~1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior (Wang, 2016).

    How gene regulation modulates neuronal activities leading to cognition and behavior is an important question in biology. Although many behavior-associated genes and neuronal cell types have been identified, a detailed understanding that links the molecular events of gene regulation to specific behaviors is still lacking. Alternative pre-mRNA splicing (AS) is a crucial gene regulatory mechanism that enables a single gene to generate functionally distinct messenger RNA transcripts (mRNAs) and protein products. The nervous system makes extensive use of AS to generate diverse and complex neural mRNA expression patterns that determine numerous neuronal cell types and functions. AS is regulated by the small nuclear ribonucleoprotein complexes (snRNPs) that compose the spliceosome for intron recognition and removal, as well as a large repertoire of non-snRNP RNA-binding proteins that affect decisions on splice site use. This dynamic and complex AS regulatory network modulates diverse neuronal functions, like synaptic transmission and signal processing, hence further impacting higher brain functions, such as cognition and behavioral control (Wang, 2016).

    The Drosophila KH-domain RNA binding splicing factor P-element somatic inhibitor (PSI) is best known for regulating tissue-specific AS of the Drosophila P-element transposon transcripts to restrict transposition activity to germ-line tissues. PSI directly interacts with the U1 snRNP through a 70-aa tandem direct repeat domain at the C terminus of the PSI protein (termed the 'AB' domain). Deletion of the AB domain in transgenic flies resulted in male sterility and male courtship defects. U1 snRNP, as an essential component of the spliceosome that binds to 5' splice sites (5'SS), defines exon-intron boundaries, and initiates spliceosome assembly for intron removal. U1 snRNP further affects AS decisions and suppresses pre-mRNA premature cleavage and polyadenylation through binding to pseudo-5'SS (5'SS-like motifs that are not used for splicing) that are abundantly distributed throughout the transcriptome. It remains a mystery how U1 snRNP differentiates the vast number of functional 5'SS and pseudo-5'SS in the transcriptome that leads to functionally distinct AS patterns. In the case of Drosophila P-element transposon AS regulation, the PSI-U1 snRNP interaction enables PSI to modulate the competitive binding of U1 snRNP between the accurate 5'SS in the third intron and an upstream pseudo-5'SS in the transposon pre-mRNA, and thus influence the final AS decision. It is possible that PSI may play a more general role in specifically localizing U1 snRNP to the transcriptome for AS regulation beyond the P-element transposon, and thus exert a more broad influence over fruit fly physiology (Wang, 2016).

    AS patterns are often controlled by the interaction of RNA binding proteins (RBPs) with nascent pre-mRNA transcripts. These RNA-protein interactions can determine where the spliceosomal U1 and U2 snRNPs bind to the transcriptome, and thus dictate AS decisions and constitute an important mechanism for gene regulation. RBPs, such as PSI or TIA-1, which directly interact with U1 snRNP, are good candidates for proteins controlling AS patterns in this manner, and changes in these RBP-snRNP associations can have profound phenotypic effects. For example, this study shows that a subtle mutation that abolishes the PSI-U1 snRNP interaction dramatically changed the AS patterns of hundreds neuronal pre-mRNAs and resulted in highly abnormal male courtship behaviors. Given the diverse number of cell types, gene-expression patterns, and the extensive AS that occurs in animal nervous systems, it is anticipated that AS regulation will play critical roles in both the normal physiological or disease states of neurons (Wang, 2016).

    The PSI-U1 snRNP interaction may further play crucial roles in other pre-mRNA processing pathways. For example, U1 snRNP was recently ascribed a new function in regulating global mRNA 3' end termination and suppression of premature pre-mRNA cleavage and polyadenylation near the 5' ends of transcripts in humans, mice, and Drosophila through selective binding to 5'SS-like motifs, a process called telescripting. It has remained a mystery how U1 snRNP discriminates the numerous potential 5'SS sites across the transcriptome. PSI may be one example of RBP regulators that alter the binding of U1 snRNP to pre-mRNA sites through direct protein-protein interactions, and thus changing pre-mRNA splicing, polyadenylation, or other pre-mRNA processing patterns (Wang, 2016).

    These findings further reveal that even broadly expressed RBPs, such as PSI, can affect gene regulation in restricted subsets of neurons in the Drosophila brain that modulate specific behaviors, such as courtship and mating. The work presented here also provides the first identification of the PSI protein as a transacting RNA splicing factor controlling male-specific fruitless splicing (Wang, 2016).

    Taken together, these results link the molecular interaction between PSI and U1 snRNP to specific phenotypic effects on Drosophila courtship behavior through the coordination of an AS program in the brain. These results provide important insights into the mechanisms controlling gene activity in the nervous system, leading to the precise control of complex animal behaviors (Wang, 2016).

    Mating experience modifies locomotor performance and promotes episodic motor activity in Drosophila melanogaster

    Sexual behavior is a routine among animal species. Sexual experience has several behavioral consequences in insects, but its physiological basis is less well-understood. The episodic motor activity with a periodicity around 19 s was unintentionally observed in the wildtype Canton-S flies and was greatly reduced in the white-eyed mutant w(1118) flies. Episodic motor activity co-exists with several consistent locomotor performances in Canton-S flies whereas reduced episodic motor activity is accompanied by neural or behavioral abnormalities in w(1118) flies. The improvements of both episodic motor activity and locomotor performance are co-inducible by a pulsed light illumination in w(1118). This study shows that mating experience of w(1118) males promoted fast and consistent locomotor activities and increased the power of episodic motor activities. Compared with virgin males, mated ones showed significant increases of boundary preference, travel distance over 60 s, and increased path increments per 0.2 s. In contrast, mated males of Canton-S showed decreased boundary preference, increased travel distance over 60 s, and increased path increments per 0.2 s. Additionally, mated males of w(1118) displayed increased power amplitude of periodic motor activities at 0.03-0.1 Hz. These data indicated that mating experience promoted fast and consistent locomotion and improved episodic motor activities in w(1118) male flies (Qiu, 2020).

    Does sexual experience affect the strength of male mate choice for high-quality females in Drosophila melanogaster?

    Although females are traditionally thought of as the choosy sex, there is increasing evidence in many species that males will preferentially court or mate with certain females over others when given a choice. In the fruit fly, Drosophila melanogaster, males discriminate between potential mating partners based on a number of female traits, including species, mating history, age, and condition. Interestingly, many of these male preferences are affected by the male's previous sexual experiences, such that males increase courtship toward types of females that they have previously mated with and decrease courtship toward types of females that have previously rejected them. D. melanogaster males also show courtship and mating preferences for larger females over smaller females, likely because larger females have higher fecundity. It is unknown, however, whether this preference shows behavioral plasticity based on the male's sexual history as is seen for other male preferences. This study manipulated the sexual experience of D. melanogaster males and tested whether this manipulation has any effect on the strength of male mate choice for large females. Sexually inexperienced males were found to have a robust courtship preference for large females that is unaffected by previous experience mating with, or being rejected by, females of differing sizes. Given that female body size is one of the most common targets of male mate choice across insect species, these experiments with D. melanogaster may provide insight into how these preferences develop and evolve (Sinclair, 2021).

    Drosophila oocyte proteome composition covaries with female mating status

    Oocyte composition can directly influence offspring fitness, particularly in oviparous species such as most insects, where it is the primary form of parental investment. Oocyte production is also energetically costly, dependent on female condition and responsive to external cues. This study investigated whether mating influences mature oocyte composition in Drosophila melanogaster using a quantitative proteomic approach. The analyses robustly identified 4,485 oocyte proteins and revealed that stage-14 oocytes from mated females differed significantly in protein composition relative to oocytes from unmated females. Proteins forming a highly interconnected network enriched for translational machinery and transmembrane proteins were increased in oocytes from mated females, including calcium binding and transport proteins. This mating-induced modulation of oocyte maturation was also significantly associated with proteome changes that are known to be triggered by egg activation. It is proposed that these compositional changes are likely to have fitness consequences and adaptive implications given the importance of oocyte protein composition, rather than active gene expression, to the maternal-to-zygotic transition and early embryogenesis (McDonough-Goldstein, 2021).

    Condition-dependent interaction between mating success and competitive fertilization success in Drosophila melanogaster

    In many species, larger (high-condition) males gain higher mating success through male-male competition and female choice, and female condition can affect the extent of both female mate choice and male investment in courtship or ejaculates. This study manipulated the larval diet of male and female Drosophila melanogaster to study how body size variation in both sexes biases competitive outcomes at different reproductive stages, from mating to paternity. Not difference was found in mate preference or mating latency between females of different conditions, nor any interaction between male and female conditions. However, large males were more successful in gaining matings, but only when in direct competition, whereas mating latencies were shorter for low-condition males in noncompetitive settings. Small males also transferred more sperm to nonvirgin females, displaced a larger proportion of resident sperm, and achieved higher paternity shares per mating than large males. In agreement with existing theory, it is suggested that small males might partially compensate for their low mating success by strategically investing in larger sperm numbers and potentially other, unmeasured ejaculate traits, when they do have a mating opportunity (De Nardo, 2021).

    Sight of parasitoid wasps accelerates sexual behavior and upregulates a micropeptide gene in Drosophila

    Parasitoid wasps inflict widespread death upon the insect world. Hundreds of thousands of parasitoid wasp species kill a vast range of insect species. Insects have evolved defensive responses to the threat of wasps, some cellular and some behavioral. This study found an unexpected response of adult Drosophila to the presence of certain parasitoid wasps: accelerated mating behavior. Flies exposed to certain wasp species begin mating more quickly. The effect is mediated via changes in the behavior of the female fly and depends on visual perception. The sight of wasps induces the dramatic upregulation in the fly nervous system of a gene that encodes a 41-amino acid micropeptide. Mutational analysis reveals that the gene is essential to the behavioral response of the fly. This work provides a foundation for further exploration of how the activation of visual circuits by the sight of a wasp alters both sexual behavior and gene expression (Ebrahim, 2021).

    CaMKII measures the passage of time to coordinate behavior and motivational state

    Electrical events in neurons occur on the order of milliseconds, but the brain can process and reproduce intervals millions of times longer. This study presents what is believed to be the first neuronal mechanism for timing intervals longer than a few seconds. The activation and gradual relaxation of calcium-independent CaMKII measure a 6-min time window to coordinate two male-specific events during Drosophila mating: sperm transfer and a simultaneous decrease in motivation. These functions were localized to four neurons whose electrical activity is necessary only to report the conclusion of the decline in CaMKII's activity, not for the measurement of the interval. The computation of elapsed time is therefore largely invisible to standard methods of monitoring neuronal activity. Its broad conservation, ubiquitous expression, and tunable duration of activity suggest that CaMKII may time a wide variety of behavioral and cognitive processes (Thornquist, 2020).

    Several lines of evidence argue that CaMKII is the timer itself rather than an effector of some other sustained signal: (1) the core timing mechanism does not require electrical activity; (2) selectively impairing calcium-dependent CaMKII activity (via the T287A mutation) shortens the timer, indicating that it relies on CaMKII autophosphorylation rather than sustained calcium; (3) elevating calcium-independent CaMKII activity (via the T287D mutation) prevents the timer from running down; (4) acute inhibition of CaMKII concludes the time interval; and (5) the dynamics of CaMKII in the Crz neurons match the timed interval. The gradual decline in CaMKII's sustained activity therefore appears to be the central timing mechanism in this system, but this work provides less specific information about the processes that initiate and report the conclusion of the timer or the factors that control the rate of CaMKII's decline over time (Thornquist, 2020).

    Because the Crz neurons do not extend projections outside of the abdominal ganglion, upstream neurons must exist that detect the onset of mating and relay this information to start the timer. Voltage changes in the Crz neurons are not required to start the timer, suggesting that it may be initiated by G-protein coupled receptor signaling and liberation of internal calcium stores, although other, non-calcium-mediated forms of CaMKII activation are also possible. Activated CaMKII then delays the output of the Crz neurons for a duration that depends on the ability of autophosphorylated subunits to sustain activity against inactivating factors (e.g., phosphatases). When CaMKII activity eventually returns to baseline, the Crz neurons use a voltage-dependent process to signal conclusion of the timer to downstream neurons. Inhibiting CaMKII outside of the mating context does not induce sperm transfer, one of several lines of evidence suggesting a constant excitatory force acting on the Crz neurons during mating. CaMKII activity opposes this excitation through a mechanism that remains obscure but that likely involves suppression of sustained calcium elevation. A sustained response may amplify the input signal over time, explaining the ~75 s of membrane voltage required for the Crz neurons to signal after the CaMKII timer has run down. In this model, a high and sustained level of electrical activity may be necessary to gate the release of the neurotransmitter(s) that trigger(s) sperm transfer and the motivational switch. Localization of the timer to four neurons, identification of the core timekeeping mechanism, the ability to infer the activity of the timer through a robust behavioral readout, and a system for automated scoring should allow rapid resolution of these molecular- and circuit-level details (Thornquist, 2020).

    Downstream of the Crz neurons, the sperm transfer signal is likely processed by serotonergic neurons that innervate the ejaculatory bulb, whereas the motivational output may adjust the activity of previously described dopaminergic and/or GABAergic neurons in the abdominal ganglion that regulate the changing motivational state during mating. The Crz, dopaminergic, and GABAergic neurons clearly affect motivation as opposed to motor function because experimentally altering their activity biases the tendency to terminate the mating in response to competing stimuli (as opposed to simply inducing termination) and suppressing their signaling does not obviously affect motor functions. It was initially surprising to find these neurons in a region of the nervous system most often called the ventral nerve cord (recently updated to the ventral nervous system [VNS]. Like the spinal cord, the VNS is situated in the thorax and contains motor neurons. But unlike the spinal cord, the VNS does not physically resemble a cord and is comparable in size to the central brain (if the optic lobes are not considered). The VNS is far more interconnected with the fly brain than its vertebrate counterparts (e.g., there are ~105 descending neurons in mice of ~108 brain neurons [0.1%], whereas there are ~103 descending neurons in flies of their ~105 brain neurons [1%]). Although there are many anatomical similarities between invertebrates and vertebrates, there are important differences; for example, insects enclose their entire bodies, not only their CNS, in a hard shell. This, together with their generally much smaller body size, may lead to different spatial constraints in the positioning of neurons with various functions in the CNS, including the location of a motivational control center in the abdominal ganglion of the VNS (Thornquist, 2020).

    How extensively is this type of fixed time measurement used in the nervous system? Perhaps more so than currently appreciated. For example, a 6-min timer nested within the 23-min mating duration was not anticipated when this study began. Unlike traditional sensory modalities, any neuron can, in principle, detect the passage of time directly and use that information to organize or synchronize circuit functions. The duration of CaMKII's sustained activity is differentially tuned in different contexts; sub-threshold activation experiments show that, even within the same neurons, CaMKII can produce a range of time intervals that reflect the level of input. Although not apparent in this system, a CaMKII-based timer could also be adjusted in real time by modulatory inputs that increase or decrease phosphorylation at T286/7. Since CaMKII activity could be readout at any or all points during its decay, the mechanism described here may be used beyond the measurement of fixed time intervals, generating a range of solutions for translating neuronal timescales into behavioral ones. Temporal intervals are implicit in many functions of the nervous system, especially those organizing behavior. There are hints that CaMKII may be involved in timing functions in other animals and behaviors; for example, in the precisely timed circalunar eclosion of midges and the intestine-controlled rhythmic defecation of C. elegans every 45 s (Thornquist, 2020).

    CaMKII is most often studied in the nervous system for its role in memory formation and storage, with a particular focus on the potentiation of synaptic transmission in the hippocampus, but CaMKII evolutionarily predates the ionotropic glutamate receptors that underlie this form of synaptic plasticity by hundreds of millions of years. This study shows that CaMKII delays the output of Crz neurons in a memory-independent interval timing system. Although the idea that biochemical reactions progress over reliable timescales of many seconds or minutes is certainly not new, theoretical work on interval timing has focused largely on electrical network mechanisms. Similarly, early hypotheses regarding circadian timing were developed around the production of oscillations via electrical and synaptic mechanisms before Drosophila genetics revealed its molecular nature. The current work points to timing mechanisms that may not even be detectable by standard electrical recording and calcium imaging techniques (Thornquist, 2020).

    Single-celled organisms use biochemical computations to respond to their environment in sophisticated ways; for example, the cyanobacterial circadian clock keeps time using autophosphorylation in a process strikingly similar to CaMKII. It is unlikely that neurons have abandoned these evolutionarily ancient strategies, given that they enable processing of much more information than immediate electrical input provides. Although, in principle, many biochemical processes might function to measure intervals of time, the long-recognized function of CaMKII as a molecular memory of earlier events makes it especially suitable for timekeeping. Its broad expression, strict evolutionary conservation, and tunable duration of its activity make CaMKII a candidate for timing functions and slowly evolving dynamics underlying a wide variety of behavioral states and other emergent neuronal network properties (Thornquist, 2020).

    Drosophila females receive male substrate-borne signals through specific leg neurons during courtship

    Substrate-borne vibratory signals are thought to be one of the most ancient and taxonomically widespread communication signals among animal species, including Drosophila flies. During courtship, the male Drosophila abdomen tremulates to generate vibrations in the courting substrate. These vibrations coincide with nearby females becoming immobile, a behavior that facilitates mounting and copulation. It was unknown how the Drosophila female detects these substrate-borne vibratory signals. This study confirmed that the immobility response of the female to the tremulations is not dependent on any air-borne cue. Substrate-borne communication is used by wild Drosophila and the vibrations propagate through those natural substrates (e.g., fruits) where flies feed and court. Transmission of the signals through a variety of substrates was examined, and how each of these substrates modifies the vibratory signal during propagation and affects the female response is described. Moreover, the main sensory structures and neurons that receive the vibrations were identified in the female legs; the mechanically gated ion channels Nanchung and Piezo (but not Trpγ) that mediate sensitivity to the vibrations. Together, these results show that Drosophila flies, like many other arthropods, use substrate-borne communication as a natural means of communication, strengthening the idea that this mode of signal transfer is heavily used and reliable in the wild. These findings also reveal the cellular and molecular mechanisms underlying the vibration-sensing modality necessary for this communication (McKelvey, 2021).

    Orcokinin neuropeptides regulate reproduction in the fruit fly, Drosophila melanogaster

    In animals, neuropeptidergic signaling is essential for the regulation of survival and reproduction. In insects, Orcokinins are poorly studied, despite their high level of conservation among different orders. In particular, there are currently no reports on the role of Orcokinins in the experimental insect model, the fruit fly, Drosophila melanogaster. The present work made use of the genetic tools available in this species to investigate the role of Orcokinins in the regulation of different innate behaviors including ecdysis, sleep, locomotor activity, oviposition, and courtship. RNAi-mediated knockdown of the orcokinin gene caused a disinhibition of male courtship behavior, including the occurrence of male to male courtship, which is rarely seen in wildtype flies. In addition, orcokinin gene silencing caused a reduction in egg production. Orcokinin is emerging as an important neuropeptide family in the regulation of the physiology of insects from different orders. In the case of the fruit fly, these results suggest an important role in reproductive success (Silva, 2021).

    Drosophila Lysophospholipase Gene swiss cheese Is Required for Survival and Reproduction

    Drosophila is widely used to analyse functions of different genes. The phosphatidylcholine lysophospholipase gene swiss cheese was initially shown to be important in the fruit fly nervous system. However, the role of this gene in non-nervous cell types has not been elucidated yet, and the evolutional explanation for the conservation of its function remains elusive. This study analyses the expression pattern and some aspects of the role of the swiss cheese gene in the fitness of Drosophila melanogaster. The spatiotemporal expression of swiss cheese throughout the fly development is described and the survival and productivity of swiss cheese mutants were analyzed. swiss cheese was found to be expressed in salivary glands, midgut, Malpighian tubes, adipocytes, and male reproductive system. Dysfunction of swiss cheese results in severe pupae and imago lethality and decline of fertility, which is impressive in males. The latter is accompanied with abnormalities of male locomotor activity and courtship behavior, accumulation of lipid droplets in testis cyst cells and decrease in spermatozoa motility. These results suggest that normal swiss cheese is important for Drosophila melanogaster fitness due to its necessity for both specimen survival and their reproductive success (Melentev, 2021).

    Regulation of Drosophila courtship behavior by the Tlx/tailless-like nuclear receptor, dissatisfaction

    Sexually dimorphic courtship behaviors in Drosophila melanogaster develop from the activity of the sexual differentiation genes, doublesex (dsx) and fruitless (fru), functioning with other regulatory factors that have received little attention. The dissatisfaction (dsf) gene encodes an orphan nuclear receptor homologous to vertebrate Tlx and Drosophila tailless that is critical for the development of several aspects of female- and male-specific sexual behaviors. This study reports the pattern of dsf expression in the central nervous system and shows that the activity of sexually dimorphic abdominal interneurons that co-express dsf and dsx is necessary and sufficient for vaginal plate opening in virgin females, ovipositor extrusion in mated females, and abdominal curling in males during courtship. dsf activity results in different neuroanatomical outcomes in females and males, promoting and suppressing, respectively, female development and function of these neurons depending upon the sexual state of dsx expression. It is positted that dsf and dsx interact to specify sex differences in the neural circuitry for dimorphic abdominal behaviors (Duckhorn, 2022).

    Pre-copulatory reproductive behaviours are preserved in Drosophila melanogaster infected with bacteria

    The activation of the immune system upon infection exerts a huge energetic demand on an individual, likely decreasing available resources for other vital processes, like reproduction. The factors that determine the trade-off between defensive and reproductive traits remain poorly understood. This study exploited the experimental tractability of the fruit fly Drosophila melanogaster to systematically assess the impact of immune system activation on pre-copulatory reproductive behavior. Contrary to expectations, it was found that male flies undergoing an immune activation continue to display high levels of courtship and mating success. Similarly, immune-challenged female flies remain highly sexually receptive. By combining behavioural paradigms, a diverse panel of pathogens and genetic strategies to induce the fly immune system, this study shows that pre-copulatory reproductive behaviours are preserved in infected flies, despite the significant metabolic cost of infection (Rose, 2022).

    Mutations in trpgamma, the homologue of TRPC6 autism candidate gene, causes autism-like behavioral deficits in Drosophila

    Autism Spectrum Disorder (ASD) is characterized by impaired social communication, restricted interests, and repetitive and stereotyped behaviors. The TRPC6 (transient receptor potential channel 6) represents an ASD candidate gene under an oligogenic/multifactorial model based on the initial description and cellular characterization of an individual with ASD bearing a de novo heterozygous mutation disrupting TRPC6, together with the enrichment of disruptive TRPC6 variants in ASD cases as compared to controls. This study performed a clinical re-evaluation of the initial non-verbal patient, and also present eight newly reported individuals ascertained for ASD and bearing predicted loss-of-function mutations in TRPC6. In order to understand the consequences of mutations in TRPC6 on nervous system function, the fruit fly, Drosophila melanogaster, was used to show that null mutations in transient receptor gamma (trpγ the fly gene most similar to TRPC6), cause a number of behavioral defects that mirror features seen in ASD patients, including deficits in social interactions (based on courtship behavior), impaired sleep homeostasis (without affecting the circadian control of sleep), hyperactivity in both young and old flies, and defects in learning and memory. Some defects, most notably in sleep, differed in severity between males and females and became normal with age. Interestingly, hyperforin, a TRPC6 agonist and the primary active component of the St. John's wort antidepressant, attenuated many of the deficits expressed by trpγ mutant flies. In summary, these results provide further evidence that the TRPC6 gene is a risk factor for ASD. In addition, they show that the behavioral defects caused by mutations in TRPC6 can be modeled in Drosophila, thereby establishing a paradigm to examine the impact of mutations in other candidate genes (Palacios-Munoz, 2022).

    Silencing Doublesex expression triggers three-level pheromonal feminization in Nasonia vitripennis males

    Doublesex (Dsx) has a conserved function in controlling sexual morphological differences in insects, but knowledge of its role in regulating sexual behaviour is primarily limited to Drosophila. This study shows with the parasitoid wasp Nasonia vitripennis that males whose Dsx gene had been silenced (NvDsx-i) underwent a three-level pheromonal feminization: (1) NvDsx-i males were no longer able to attract females from a distance, owing to drastically reduced titres of the long-range sex pheromone; (2) NvDsx-i males were courted by wild-type males as though they were females, which correlated with a lower abundance of alkenes in their cuticular hydrocarbon (CHC) profiles. Supplementation with realistic amounts of synthetic (Z)-9-hentriacontene (Z9C31), the most significantly reduced alkene in NvDsx-i males, to NvDsx-i males interrupted courtship by wild-type conspecific males. Supplementation of female CHC profiles with Z9C31 reduced courtship and mating attempts by wild-type males. These results prove that Z9C31 is crucial for sex discrimination in N. vitripennis; and (3) Nvdsx-i males were hampered in eliciting female receptivity and thus experienced severely reduced mating success, suggesting that they are unable to produce the to-date unidentified oral aphrodisiac pheromone reported in N. vitripennis males. It is concluded that Dsx is a multi-level key regulator of pheromone-mediated sexual communication in N. vitripennis (Wang, 2022).

    Functional Dissection of Protein Kinases in Sexual Development and Female Receptivity of Drosophila

    Protein phosphorylation is crucial for a variety of biological functions, but how it is involved in sexual development and behavior is rarely known. This study= performed a screen of RNA interference targeting 177 protein kinases in Drosophila and identified 13 kinases involved in sexual development in one or both sexes. It was further identified that PKA and CASK promote female sexual behavior while not affecting female differentiation. Knocking down PKA or CASK in about five pairs of pC1 neurons in the central brain affects the fine projection but not cell number of these pC1 neurons and reduces virgin female receptivity. PKA and CASK signaling is required acutely during adulthood to promote female sexual behavior. These results reveal candidate kinases required for sexual development and behaviors and provide insights into how kinases would regulate neuronal development and physiology to fine tune the robustness of sexual behaviors (Chen, 2022).

    Drosulfakinin signaling modulates female sexual receptivity in Drosophila

    Female sexual behavior as an innate behavior is of prominent biological importance for survival and reproduction. However, molecular and circuit mechanisms underlying female sexual behavior is not well understood. This study identified the Cholecystokinin-like peptide Drosulfakinin (DSK) to promote female sexual behavior in Drosophila. Loss of DSK function reduces female receptivity while overexpressing DSK enhances female receptivity. Two pairs of Dsk-expressing neurons in the central brain were found to promote female receptivity. DSK peptide acts through one of its receptors, CCKLR-17D3, to modulate female receptivity. Manipulation of CCKLR-17D3 and its expressing neurons alters female receptivity. It was further revealed that the two pairs of Dsk-expressing neurons receive input signal from pC1 neurons that integrate sex-related cues and mating status. These results demonstrate how a neuropeptide pathway interacts with a central neural node in the female sex circuitry to modulate sexual receptivity (Wang, 2022).

    This study systematically investigated DSK-mediated neuromodulation of female sexual receptivity. At the molecular level, it was revealed that DSK neuropeptide and its receptor CCKLR-17D3 are crucial for modulating female sexual receptivity. At the neuronal circuit level, it was identified that DSK neurons are the immediate downstream targets of sex-promoting pC1 neurons in controlling female sexual receptivity. Moreover, intersectional tools were employed to subdivide DSK neurons into medial DSK neurons (DSK-MP1) and lateral DSK neurons (DSK-MP3) and uncovered that DSK-MP1 neurons rather than DSK-MP3 neurons play essential roles in modulating female receptivity. Collectively, these findings illuminate a pC1-DSK-MP1-CCKLR-17D3 pathway that modulates female sexual behaviors in Drosophila (Wang, 2022).

    The female sexual behavior is a complex innate behavior. The decision for the female to accept a courting male or not depends on not only sensory stimulation but also internal states. If the female is willing to mate, she slows down, pauses, and opens her vaginal plates to accept a courting male, if not, she extrudes her ovipositor to deter a courting male or flies away. Tbe results of this study show that DSK signaling is crucial for virgin female receptivity but has no effect on ovipositor extrusion behavior. How exactly does the DSK signaling regulate virgin female receptivity is still not clear. One possibility is that DSK signaling regulates pausing behavior in response to male courtship, as DSK receptor CCKLR-17D3 expresses in the central complex that has been found to be crucial for locomotor behaviors. However, no change was observed in locomotor activity in DSK or CCKLR-17D3 mutant females. Note only locomotor behavior was studied in single females but not in females paired with courting males due to technical limit for analysis, and it is possible that the DSK signaling does not affect general locomotor behavior but regulates courtship-stimulated pausing behavior (Wang, 2022).

    The four pairs of DSK neurons are classified into two types (DSK-MP1 and DSK-MP3) based on the location of the cell bodies, and DSK-MP1 neurons extend descending fibers to ventral nerve cord. In this study, it was also found that activating DSK-MP1 neurons enhance female receptivity whereas inactivating DSK-MP1 neurons reduce female receptivity. Silencing adult Abd-B neurons and SAG neurons located in the abdominal ganglion inhibits female sexual receptivity. It has been found that Abd-B neurons control female pausing behavior, and it would be interesting to further investigate whether DSK-MP1 neurons relay information from Abd-B neurons to regulate pausing and receptivity in females. It is also noted that DSK-MP1 neurons extend projections to suboesophageal ganglion (SOG), and the SOG is the terminus of ascending projections from a subset of female reproductive tract sensory neurons labeled by pickpocket (ppk), fruitless (fru), and doublesex (dsx). It is also possible that DSK-MP1 neurons may integrate information directly from these sensory neurons to regulate female receptivity. Another study further classified the DSK-MP1 neurons into two types (MP1a and MP1b) based on the morphology of their neuritis. Future studies would further build genetic tools to uncover the function of each subset of DSK neurons in regulating distinct innate behaviors, such as male courtship, aggression, and female sexual behavior (Wang, 2022).

    Previous studies have revealed that pC1 neurons extend projections to lateral protocerebral complex (LPC) and this neural cluster responds to courtship song and cVA. Moreover, recent works have shown that DSK-MP1 neurons project to the region of LPC. This study used GRASP, trans-Tango, and patch-clamp techniques and revealed that DSK-MP1 neurons are direct downstream target of R71G01-GAL4 neurons that include pC1. EM reconstruction revealed that pC1 neurons have intense synaptic input on MP1b but not MP1a neurons, suggesting a crucial role of the single pair of MP1b neurons in female receptivity. Based on these findings, it is propose that: (1) pC1 neurons act as a central node for female sexual receptivity by integrating sex-related sensory cues (courtship song and cVA) and mating status; and (2) DSK-MP1 neurons may integrate internal states and pC1-encoded information to modulate female sexual behavior. Thus, it is of prime importance to further investigate how a neuropeptide pathway modulate a core neural node in the sex circuitry to fine-tune the female's willingness for sexual behavior in the future (Wang, 2022).

    Effects of mitochondrial haplotype on pre-copulatory mating success in male fruit flies (Drosophila melanogaster)

    While mitochondria have long been understood to be critical to cellular function, questions remain as to how genetic variation within mitochondria may underlie variation in general metrics of organismal function. To date, studies investigating links between mitochondrial genotype and phenotype have largely focused on differences in expression of genes and physiological and life-history traits across haplotypes. Mating display behaviours may also be sensitive to mitochondrial functionality and so may also be affected by sequence variation in mitochondrial DNA, with consequences for sexual selection and fitness. This study tested whether the pre-copulatory mating success of male fruit flies (Drosophila melanogaster) varies across six different mitochondrial haplotypes expressed alongside a common nuclear genetic background. A significant effect of mitochondrial haplotype was found on measuring of competitive mating success, driven largely by the relatively poor performance of males with one particular haplotype. This haplotype, termed 'Brownsville', has previously been shown to have complex and sex-specific effects, most notably including depressed fertility in males but not females. This study extends this disproportionate effect on male reproductive success to pre-copulatory aspects of reproduction. The results demonstrate that mutations in mitochondrial DNA can plausibly affect pre-copulatory mating success, with implications for future study into the subcellular underpinnings of such behaviours and the information they may communicate (Koch, 2022).

    The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship

    The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. This study shows that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. These findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior (Love, 2023).

    Biochemical evidence accumulates across neurons to drive a network-level eruption

    Neural network computations are usually assumed to emerge from patterns of fast electrical activity. Challenging this view, this study shows that a male fly's decision to persist in mating hinges on a biochemical computation that enables processing over minutes to hours. Each neuron in a recurrent network contains slightly different internal molecular estimates of mating progress. Protein kinase A (PKA) activity contrasts this internal measurement with input from the other neurons to represent accumulated evidence that the goal of the network has been achieved. When consensus is reached, PKA pushes the network toward a large-scale and synchronized burst of calcium influx that is called an eruption. Eruptions transform continuous deliberation within the network into an all-or-nothing output, after which the male will no longer sacrifice his life to continue mating. Here, biochemical activity, invisible to most large-scale recording techniques, is the key computational currency directing behavior and motivational state (Thornquist, 2021).

    The Corazonin-expressing (Crz) neurons of the male abdominal ganglion comprise an exceptionally tractable system for investigating neuronal networks and behavioral control. These four neurons drive two simultaneous and crucial events in the lives of the male and his mating partner: (1) the transfer of sperm from the male to the female and (2) a transition out of a period of insurmountably high motivation to continue mating. Both of these events occur 6 min after mating begins and are under the control of a molecular timer encoded by the slowly decaying autophosphorylation of the kinase CaMKII. This study shows that the Crz neurons use cyclic AMP (cAMP) signaling to average evidence about the passage of time across the network and generate an eruption that signals to downstream circuitry only when a consensus is reached. In addition to revealing this network phenomenon, these results explain the function of CaMKII in the only mechanism for neuronal interval timing yet to be described (Thornquist, 2021).

    Decisions and behavioral control are thought to arise from long-lasting composite dynamics of neuronal networks, such as the seconds-long ramping of firing rates observed in premotor centers. This study provides mechanisms for the accumulation and storage of network information over much longer timescales, as well as insight into a thresholding mechanism for reporting the outcome. Here, the information is a spatially distributed estimate of elapsed time that emerges from interwoven biochemical and electrical processes. Cell-intrinsic evidence is read out within each neuron from the immediate activity of CaMKII, and network-level information is received from the electrical activity of other Crz neurons. The common currency is cAMP signaling, which accumulates intracellularly at the level of cAMP itself, PKA activity, and/or the accumulation of phosphate groups on the targets of PKA. A stark thresholding operation transforms the graded and distributed representation of evidence into a binary decision variable: the presence or absence of a network-wide eruption (Thornquist, 2021).

    Eruption-like mechanisms seem well suited for diverse long-timescale computations. The linearity of evidence accumulation and dissipation allows undistorted evaluation, whether the system is near or far from its threshold. The magnitude of network activation distinguishes the information-gathering phase from the decisive output, allowing evidence to accumulate without triggering the downstream consequences. The accumulated signal persists even in the total absence of electrical input, enabling activity-silent memory that imparts the system with a history dependence that could not be discerned from purely electrophysiological measurements (Thornquist, 2021).

    Although there has been no previous description of anything closely resembling an eruption in the nervous system, islets of pancreatic β cells synchronize their activity to coordinate insulin release using a similar positive feedback loop between electrical activity and cAMP signaling. In the brain, much of the cortex has been hypothesized to operate at near-criticality, giving rise to brief but expansive neural avalanches. Early in cortical development, nascent neuronal networks exhibit spontaneous, network-wide synchronous activation driven by positive feedback that is sustained for tens of seconds. Neurons in the primate lateral intraparietal cortex show trial-averaged ramping responses during decision-making tasks, but closer inspection shows that individual neurons jump to high activity rates as evidence accumulates. On much longer timescales, neurons in the mammalian suprachiasmatic nucleus (SCN) undergo 10-fold changes in activity depending on the time of day. In the SCN, each neuron expresses a cell-intrinsic representation of the time of day (encoded by levels of circadian clock proteins), but, as in the Crz neurons, the reliability of behavior is a network output that is much greater than would be expected from its individual oscillators (Thornquist, 2021).

    This paper proposes to define an eruption as a thresholded jump in recurrent network activity, triggering downstream processes that had been blind to intranetwork computations. The eruption can be highly localized, as in the four-cell network examined in this study, and so it may have escaped detection by the population-level dimensionality reduction techniques used by most modern studies of neuronal decision making. Even in approaches that focus on individual cells, it is usually difficult or impossible to identify the network in which a given neuron functions. For example, the step changes in cortical spike rates observed during decision making in primates may indicate participation in an erupting network or reflect the enduring consequences of an upstream eruption (Thornquist, 2021).

    Whether in existing datasets or from new experimental designs, it is believed eruptions are worth searching for. They may help explain the transformation of continuous brain activity into our discretized actions and experience and would therefore change our thinking about emergent brain properties. This study found that profound behavioral and motivational changes emerge from the interplay of population dynamics with molecular processing in a remarkably small group of cells. It is therefore argued that presuming neurons to be simple processing elements underestimates their computational power. It is believed that the computational capacity of individual neurons stems as much from their rich intracellular signaling pathways as from their interconnectedness, especially for computations in the regime of cellular supremacy, where biomolecular computations are proposed to be more efficient than implementations in the classic von Neumann and Turing frameworks. Studying the molecular-electrical interface in relatively simple systems such as the Crz network will provide still more guiding principles for linking the operations of neurons to our thoughts, emotions, precepts, and actions (Thornquist, 2021).

    A major limitation of studying the Crz eruption has been an inability to monitor neuronal activity during mating. Nevertheless, the close fidelity between manipulations that alter the eruption in behaving animals and in ex vivo imaging preparation has allowed the authors to lay out the general structure of the Crz eruption. Still, many details remain unclear or are difficult to fully address with the data. Most prominent is the multifaceted role of calcium in this model. Calcium first activates the CaMKII timer, then drives the pre-eruption accumulation of cAMP, and finally a massive calcium influx essentially is the eruption. There are some clues as to how calcium could manage these diverse functions. Subcellular compartmentalization may explain some of this multifunctionality: the activation of CaMKII is voltage independent (Thornquist, 2020), whereas the eruption acts through VGCCs. Unlike the calcium-conducting ChR2-XXM, stimulation of the Crz neurons with the non-calcium conducting channel CsChrimson fails to activate CaMKII (Thornquist, 2020), further supporting the notion that VGCCs cannot activate CaMKII in this system. So, what does initiate the CaMKII timer? The most promising candidate may be the release of calcium from intracellular stores. The molecular screen presented here did not turn up hits in endoplasmic-reticular or mitochondrial calcium channels (or in the approximately two-thirds of the annotated GPCRs that were tested), but the screen will have missed manipulations that reduce CaMKII activation, as a premature eruption will not affect copulation duration. More directed explorations of the onset of the timer have so far failed to yield any clear leads, due either to limitations in tools or in the hypotheses (Thornquist, 2021).

    Like action potentials in individual neurons, the system described in this study resets after an eruption. This is not fully understand either, although it may involve re-activation of CaMKII: elevation of intracellular calcium following strong bPAC stimulation activates CaMKII, while inhibiting CaMKII results in a high frequency of successive eruptions. A possible mechanism for CaMKII's ability to both delay and reset the eruption is indicated by work in mammalian cardiomyocytes, where CaMKII phosphorylates and activates phosphodiesterase 4D (PDE4), which degrades cAMP. In the Crz neurons, RNAi knockdown of the PDE4 homolog dunce shortens the duration of the voltage requirement, activation of CaMKII dramatically reduces baseline cAMP levels, and a potential CaMKII phosphorylation site on PDE4 seems to be conserved on dunce. However, it would not be surprising if CaMKII acts at multiple levels to inhibit cAMP signaling (although without strongly affecting baseline membrane voltage (Thornquist, 2020; Thornquist, 2021).

    When CaMKII activity is low, cAMP activates PKA to drive the eruption, but what exactly does PKA do? In mammals, PKA is known to potentiate calcium influx through multiple calcium channels, suggesting a straightforward mechanism for driving the eruption that concludes the timer. However, other hits in the screen do not fit neatly into this pathway, suggesting further complexity and surprises upon deeper investigation (Thornquist, 2021).

    Finally, what is the signal that is released by an eruption and how does it affect downstream neurons? The output signal is most likely different from the (also unknown) signal(s) and receptor(s) used for intranetwork recurrent excitation, and could consist of one or more neuropeptides, since they often require sustained excitation for release. The screens so far have only identified Unc13, a component of vesicle release machinery for both classical neurotransmitters and neuropeptides. The immediate downstream targets of the Crz neurons are also unknown, but ultimately the eruption drives the transfer of sperm and seminal fluid through the activation of a set of serotonergic neurons and adjusts the properties of drive-integrating neurons that control the immediate responsiveness of the male to challenges that arise during mating (Thornquist, 2021).

    Drosulfakinin signaling in fruitless circuitry antagonizes P neurons to regulate sexual arousal in Drosophila

    Animals perform or terminate particular behaviors by integrating external cues and internal states through neural circuits. Identifying neural substrates and their molecular modulators promoting or inhibiting animal behaviors are key steps to understand how neural circuits control behaviors. This study identified the Cholecystokinin-like peptide Drosulfakinin (DSK) that functions at single-neuron resolution to suppress male sexual behavior in Drosophila. Dsk neurons physiologically interact with male-specific P neurons, part of a command center for male sexual behaviors, and function oppositely to regulate multiple arousal-related behaviors including sex, sleep and spontaneous walking. This study further found that the DSK- peptide functions through its receptor CCKLR-17D to suppress sexual behaviors in flies. Such a neuropeptide circuit largely overlaps with the fruitless-expressing neural circuit that governs most aspects of male sexual behaviors. Thus DSK/CCKLR signaling in the sex circuitry functions antagonistically with P neurons to balance arousal levels and modulate sexual behaviors (Wu, 2019).

    Male courtship in Drosophila melanogaster is one of the best-understood innate behaviors, and largely controlled by the fruitless (fru) gene and doublesex (dsx) gene, which encode sex-specific transcription factors (FRUM and DSXM in males and DSXF in females). FRUM is responsible for most aspects of male courtship, and DSXM is important for the experience-dependent acquisition of courtship in the absence of FRUM, and courtship intensity and sine song production in the presence of FRUM. FRUM is expressed in a dispersed subset of ca. 2000 neurons including sensory neurons, interneurons, and motor neurons that are potentially interconnected to form a sex circuitry controlling sexual behaviors. In contrast, DSXM is expressed in ca. 700 neurons in males, the majority of which also express FRUM, and are crucial for male courtship. Recently, substantial progress has been made into how external sensory cues are perceived and integrated by fruM and/or dsxM neurons to initiate male courtship, in particular, how a subset of male-specific fruM- and dsxM-expressing P1 neurons integrate olfactory and gustatory cues from female or male targets to initiate or terminate courtship. Such a neuronal pathway is also conserved in other Drosophila species (Wu, 2019).

    Behavioral decisions depend on both excitatory and inhibitory modulations. P1 neurons represent an excitatory center that integrates multiple (both excitatory and inhibitory) sensory cues and initiates courtship. However, whether there is an inhibitory counterpart that operates against P1 neurons to balance sexual activity is still unknown. Indeed, males do not absolutely court virgin females even if these females may provide the same visual, olfactory, and gustatory cues, depending on the male's internal states and past experiences. It has been previously shown that neuropeptide SIFamide acts on fruM-positive neurons and inhibits male-male but not male-female courtship, and SIFamide neurons also integrate multiple peptidergic neurons to orchestrate feeding behaviors, but whether SIFamide inhibits internal arousal states for sexual behaviors is not clear. Recently, it was found that sleep and sex circuitries interact mutually and demonstrate how DN1 neurons in the sleep circuitry and P1 neurons in the courtship circuitry function together to coordinate behavioral choices between sleep and sex. However, very little is known about the inhibitory pathway(s) that may represent internal arousal states and inhibit courtship toward females (Wu, 2019).

    This study sets out to identify courtship inhibitory neurons that express neuropeptides in Drosophila, as neuropeptides play key roles in adjusting animal behaviors based on environmental cues and internal needs. This study identifies that the neuropeptide Drosulfakinin (DSK), the fly ortholog of Cholecystokinin (CCK) in mammals, functions through its receptor CCKLR-17D3 in the fruM-expressing sex circuitry to inhibit male courtship toward females. It is further demonstrate dthat Dsk neurons and P1 neurons interact and oppositely regulate male sexual behaviors (Wu, 2019).

    The results identify, at single-neuron resolution, four pairs of fruM-expressing Dsk neurons (MP1 and MP3) that suppress male and female sexual behaviors. The suppression of male and female sexual behaviors depends on the secretion of the neuropeptide DSK-2, which then acts on one of its receptors CCKLR-17D3 that is expressed in many fruM neurons including P1 neurons and the mushroom bodies. Dsk neurons function antagonistically with courtship promoting P1 neurons to co-regulate male courtship, as well as sleep and spontaneous walking (Wu, 2019).

    Cholecystokinin (CCK) signaling appears well conserved over evolution and modulates multiple behaviors. In Drosophila, the CCK-like sulfakinin (DSK) is multifunctional and has been reported to be involved in regulating aspects of food ingestion and satiety, aggression, as well as escape-related locomotion and synaptic plasticity during neuromuscular junction development. In mammals, CCK generated from the intestine acts on its receptors in the nucleus of the solitary tract of the brain to transmit satiety signaling and thus inhibit feeding. Furthermore, CCK signaling in the nucleus accumbens modulates dopaminergic influences on male sexual behaviors in rats. CCK is also involved in nociception, learning and memory, aggression and depressive-like behaviors (Wu, 2019).

    Despite its significant and conserved roles in modulating multiple innate and learned behaviors, how CCK or DSK signaling responds to environment and/or internal changes and acts on specific neurons expressing its receptors to modulate multiple behaviors, is still rarely known. The finding that DSK/CCKLR signaling functions in the fruM- and/or dsx-expressing sex circuitry to inhibit male courtship is an effort to use Drosophila as a model to investigate how this conserved signaling modulates animal behaviors (Wu, 2019).

    The results uncovered a functional circuitry from many dsx neurons (including courtship-promoting P1 neurons) to four pairs of Dsk MP neurons via direct synaptic transmission, and these MP neurons then modulate CCKLR-17D3 neurons including many fruM and/or dsx neurons via secretion of DSK peptides. It is of particular interest to reveal how the four pairs of MP neurons integrate sensory information (in any), physiological states and past experiences in the future to better understand how this neuropeptide signaling modulate arousal states. It is still not known if these MP neurons receive sensory inputs, but since they receive inputs from many dsx neurons, including P1 neurons that integrate multiple chemosensory information, these sensory inputs will at least relay to Dsk MP neurons via P1. Whether there are other pathways from sensory inputs to MP neurons awaits further study. It is also noted that multiple physiological changes including feeding states, aging and sleep deprivation, as well as past housing conditions affect expression of DSK/CCKLR-17D3, but how they affect DSK signaling and behaviors is still unclear. This study showed that male-male group-housing increases DSK expression and thereby reduces male courtship at least under a restricted condition, and previous findings also revealed opposite effects of group-housing on the excitability of P1 neurons in males that have fruM function or lack fruM function. Such male-male housing experience may mildly reduce sexual arousal in a persistent manner, perhaps by increasing DSK expression, but how such housing condition affects physiological roles of Dsk MP neurons and P1 neurons awaits further functional imaging studies on a potential P1-Dsk-P1 functional loop (and a much complex dsx-Dsk-17D3 pathway) with sensory stimulation under different physiological states. In terms of the time scale that DSK functions to inhibit male courtship, the results indicate an immediate behavioral effect upon Dsk neuronal activation. It is also noted that activation of Dsk neurons inhibits male courtship and lasts for minutes, and previous findings also showed that activation of P1 neurons promoted wing extension and aggression and lasts for minutes. These persistent behavioral effects may represent persistent arousal states regulated by Dsk and P1 neurons in this study; however, how this persistency is generated both in the circuit level and behavioral level still needs further investigation (Wu, 2019).

    Dsk mutants do not have obvious courtship abnormality under the courtship assays. There are at least two possibilities: (1) DSK may function only in specific conditions (e.g., group-housing) that increase its expression to inhibit courtship; and (2) There are redundant inhibitory signals for courtship, such as another neuropeptide SIFamide that acts on fruM neurons, although they specifically inhibit male-male courtship. Further studies on how DSK/CCKLR signaling is activated under certain conditions, as well as how DSK, SIFamide and other inhibitory signals (if any) jointly modulate male courtship are needed to fully understand this. Nevertheless, that courtship inhibition by activation of Dsk neurons depends on DSK/CCKLR-17D3 signaling, and increasing such signaling through Cas9 activators in an otherwise wild-type male efficiently inhibited courtship, unambiguously reveal the role of DSK/CCKLR-17D3 signaling in suppressing sexual behaviors (Wu, 2019).

    As DSK signaling modulates multiple behaviors, one may argue that its role in male courtship is not specific, e.g., activation of Dsk neurons may drive a competing behavior that phenotypically shunting male courtship. Although such possibility cannot be excluded, a number of evidences are listed to support DSK's role with specificity in courtship inhibition: (1) DSK functions in four pairs of fruM-expressing neurons to inhibit courtship; (2) males with activated Dsk neurons rarely court virgin females, while they follow rotating visual objects normally; (3) Dsk neurons receive synaptic transmission from courtship promoting P1 neurons (and many other dsx-expressing neurons) in an experience-dependent manner; (4) Dsk and P1 neurons antagonistically modulate sexual behaviors and wakefulness; and (5) DSK receptor CCKLR-17D3 inhibits male courtship and expresses in many fruM and/or dsx neurons including P1 neurons. It is noted that CCKLR-17D3 is expressed broadly in the CNS including not only P1 neurons, but also mushroom bodies that regulate a range of behaviors including learning, locomotion and sleep. That DSK signaling is multifunctional is possibly due to broad expression of its receptors, and further studies on dissection of CCKLR function in subsets of neurons will help to understand how DSK/CCKLR signaling modulates multiple behaviors (Wu, 2019).

    The decision for male flies to court or not depends on not only environmental cues such as availability and suitability of potential mates (males, virgin females, or mated females), but also their internal states (e.g., thirsty or sleepy). It is proposed that there are at least four factors affecting such a decision: (1) external cues that inhibit courtship, referred to as Ex-In factor, such as the male-specific pheromone cVA4; (2) external cues that are excitatory for courtship, referred to as Ex-Ex factor, such as courtship song; (3) internal states that inhibit courtship, referred to as In-In factor; and (4) internal states that are excitatory for courtship, referred to as In-Ex factor. These factors dynamically change and jointly determine males' decision to court or not (Wu, 2019).

    Substantial progress has been made on how Ex-In and Ex-Ex factors jointly modulate the activity of male-specific P1 neurons, which is crucial for courtship initiation. In contrast, much less is understood on In-In and In-Ex factors. Recently, Zhang (2016; 2018) found that dopaminergic modulation of P1 neurons drives male courtship not only by desensitizing P1 to inhibition, but also by promoting recurrent P1 stimulation, thus may act as an In-Ex factor for male courtship. Note that all the three factors mentioned above converge on P1 neurons, making them a decision-making center for male courtship. The DSK/CCKLR signaling identified in this study is of particular interest, as it is likely to act as an In-In factor for male courtship, and above all, it does not simply act on P1 neurons like three other factors, but instead forms a potential functional loop with P1 neurons and antagonizes P1 function in modulating male courtship and wakefulness. That Dsk neurons receive synaptic transmission from P1 neurons and other dsx-expressing neurons in an experience-dependent manner further highlights a central role that the DSK/CCKLR signaling plays. These factors, excitatory vs. inhibitory, external vs. internal, jointly control appropriate performance of sexual behaviors, and further studies will reveal how P1 and other dsx-expressing neurons physiologically interact with Dsk neurons to balance behavioral output (Wu, 2019).

    A prominent feature of the neuronal control of male and female sexual behaviors in Drosophila is that, despite large similarity in sensory systems, central integrative neurons are sex-specific in the two sexes, with dsx-expressing pC1 neurons integrating olfactory and auditory cues and promoting receptivity to courting males, and fruM-expressing P1 neurons (largely overlapped with dsx-expressing pC1) integrating olfactory, gustatory, and auditory cues and promoting courtship to females. In contrast, the four pairs of Dsk-expressing MP neurons investigated in this study are sexually monomorphic and inhibit both male courtship and female receptivity. Thus DSK/CCKLR signaling may inhibit sexual behaviors in response to physiological changes that are common to both sexes, while sex-promoting central neurons integrating distinct sensory cues are sexually dimorphic. Interestingly, these Dsk neurons common to both sexes receive synaptic transmission from sexually dimorphic dsx neurons in both males and females, providing a simple solution to link sex-specific excitatory and sexually non-specific inhibitory control of sexual behaviors in males and females (Wu, 2019).

    A feedforward circuit regulates action selection of pre-mating courtship behavior in female Drosophila

    In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. This study shows that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset of neurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-d-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. This study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female (Ishimoto, 2020).

    Overall, the present findings provide evidence for a neural relation that regulates the action selection of pre-mating behaviors in female Drosophila. The PPM3, a subset of DA neurons, forms a circuit with the R neurons R2/R4m and R4d in the EB. These different types of R neurons require different types of DA receptors, Dop1R1/D2R and Dop1R2. The knockdown of each DA receptor type indicates that all of these receptors are required to activate the expressing neurons, although the D2R conventionally inhibits D2R-expressing neurons. R2/R4m and R4d are GABAergic and cholinergic, respectively. Synaptic GRASP analysis revealed a neuronal connection from R2/R4m to R4d. R4d inhibition via the GABAA receptor is required for the proper reduction of pre-mating rejection. In addition to DA regulation of R2/R4m, the potential retrograde regulation may facilitate the GABA transmission of R2/R4m, depending on the R4d activity, with the production of NO via NMDAR/NOS signaling. This potentiation-like regulation provides the activation order of each R neuron with flexibility for the neural circuit output, and therefore to the rejection response for controlling the pre-mating behavioral kinetics (Ishimoto, 2020).

    The pre-mating rejection should continue if the encounter does not match the female's criteria. Pheromones are important sexual cues provided by the male. Fruit flies produce cuticular hydrocarbons as pheromonal substances. cVA is a male-specific pheromonal cue that elicits female sexual arousal via the olfactory sensory system. This cVA signal activates pCd via a third-order olfactory interneuron, aSP-g. The SMP region contains aSP-g, pCd, and PPM3, although the direct connection between them has not been demonstrated. This aggregation of important components for female mating behavior suggests that pCd potentially integrates and verifies male information to execute the final decision for initiating the copulation. The female must resist the encounter until the evaluation process is complete-that is, the pre-mating rejection response controlled by the circuit found in the present study. These considerations lead to an assumption that the pCd and PPM3/R neuron circuits execute pre-mating computation in parallel. Many sexually dimorphic behaviors are reportedly mediated by sex-specific neural circuits (e.g., pCd, pC1). PPM3 and R neurons do not express fruitless or dsx, and no morphological sexual dimorphism has been detected. Thus, a non-sexually dimorphic circuit modulating sex-specific pre-mating behavior is proposed(Ishimoto, 2020).

    Genetic manipulations of R neurons altered the pre-mating kinetics in virgin female flies, leading to the question of whether the modulation of R neurons also affects post-mating behavior, which is induced by injection of the seminal fluid sex peptide from the male fly and sustained for several days. One of the remarkable characteristics of post-mating rejection is ovipositor extrusion controlled by a distinct class of neurons in post-mating females, but either suppression of R2/R4m neurons or activation of R4d neurons, both of which reduced receptivity, rarely induced ovipositor extrusion. Mated females with R4d suppression, which makes virgin females highly receptive, exhibited a large rejection response similar to that in mated control females, and no copulation was observed during the 1-h observation time. In addition to these observations, the virgin females with Rdl knockdown in R4d reduced their walking speed with the progression of the male courtship; however, the walking speed of the mated females increases with the progression of the male courtship. Although further studies are required to elucidate the whole picture of rejection control in females, neuronal regulation of the post-mating response is presumably parallel to the pre-mating rejection response modulated by R neurons in the EB (Ishimoto, 2020).

    This study found circuit functions that contain PPM3 DA subcluster neurons, and R neurons R2/R4m and R4d, were involved in the regulation of the kinetics of pre-mating behavior in virgin females. Pre-mating rejection is acutely elicited and then gradually decreased. A circuital feature of PPM3/R neurons may provide a theoretical action for controlling these pre-mating kinetics. PPM3 sends DA signals to both R2/R4m and R4d, and this recurrent circuit forms a feedforward motif with a repressor, the so-called incoherent type 1 feedforward loop (I1-FFL). I1-FFL is one of the most common network motifs implicated in gene and protein regulation networks, metabolic pathways, and neural networks from bacteria to humans. In the I1-FFL circuit, an activator X (e.g., PPM3) activates a target Z (e.g., R4d) and simultaneously activates another target Y (e.g., R2/R4m) that inhibits the target Z. Previous studies demonstrated that the I1-FFL accelerates the response of target Z, with a shorter response time. Moreover, upon input from X, Z activity increases and then, depending on the Y activity, it decreases toward basal levels. The I1-FFL circuit comprising PPM3, R2/R4m, and R4d would thus theoretically generate the acute activation of R4d, which promotes the pre-mating rejection behavior of females that would later be gradually attenuated by GABAergic signals from R2/R4m. This schema is consistent with the current findings, in which the circuit of PPM3/R neurons plays an important role in the action selection of pre-mating behavior (Ishimoto, 2020).

    Genetic analysis indicates that retrograde signals from R4d to R2/R4m, mediated by NO and NMDAR, would add some flexibility to the Y-to-Z regulation in the I1-FFL circuit, contributing to progressive attenuation of the rejection response in virgin females. Depolarization of R4d activates NMDAR by the coincident input of glutamate from R2/R4m. The retrograde signals induced by NMDAR/NOS/sGC probably facilitate the GABA transmission of R2/R4m, which progressively suppresses R4d activity via the GABAA receptors. Because NMDARs require simultaneous activation by glutamate and depolarization, the NMDAR pore opening may be insufficient until the activity of R4d neurons will reach a sufficient level. This may lead to keeping the GABAergic R2/R4m function at a lower level that is under a certain threshold required for GABA release, and it may induce a continuous pre-mating rejection response. Recently, new subdivision of R neurons (R5 and R6) were classified by clonal morphological analysis and cell lineage analysis. These new subclasses of R neurons may provide a novel function of the putative I1-FFL. To investigate this further, the physiological properties of each R neuron should be analyzed (Ishimoto, 2020).

    The distribution pattern of neurotransmitters in EB neurons has been documented. In the R2/R4m axonal ring structure, the glutamatergic population seems to be smaller than the GABAergic population, suggesting that the neuronal population in the R2/R4m is heterologous and contains few, if any, neurons that co-express both glutamate and GABA. In the vertebrate system, heterosynaptic regulation of GABAergic transmission is incorporated into inhibitory long-term potentiation. Inhibitory long-term potentiation of GABAergic neurons is induced at heterosynaptic sites containing glutamatergic and GABAergic neurons as presynaptic cells. In GABAergic inhibitory long-term potentiation, NO induced by glutamatergic activation of the NMDAR/NOS pathway retrogradely activates sGC, which augments cGMP levels to enhance GABA release. These intriguing analogies between vertebrate findings and the current results suggest that a similar molecular machinery potentiates GABAergic subsets of the R2/R4m neurons for attenuating the pre-mating rejection response, and hence for the action selection of pre-mating kinetics. Regarding the action selection for adaptive behaviors, others have proposed a correspondence of functions and neural architectures between vertebrate basal ganglia and the insect central complex, which contains EB neurons. In the basal ganglia, DA activates the neurons of the nucleus accumbens with modulation of glutamate and GABA release. The neurochemicals in the nucleus accumbens released by sexual interaction regulate female action selection for the affiliation of monogamous prairie voles (Microtus ochrogaster). The current genetic manipulations of particular DA, cholinergic, glutamatergic, and GABAergic systems in the central complex significantly affected the action selection for female fruit fly pre-mating behaviors, implying that the mechanisms are similar to those in the vertebrate system, which sheds light on the evolutionary parallels and diversities across the animal kingdom (Ishimoto, 2020).

    A neural circuit encoding mating states tunes defensive behavior in Drosophila

    Social context can dampen or amplify the perception of touch, and touch in turn conveys nuanced social information. However, the neural mechanism behind social regulation of mechanosensation is largely elusive. This study reports that fruit flies exhibit a strong defensive response to mechanical stimuli to their wings. In contrast, virgin female flies being courted by a male show a compromised defensive response to the stimuli, but following mating the response is enhanced. This state-dependent switch is mediated by a functional reconfiguration of a neural circuit labelled with the Tmc-L gene in the ventral nerve cord. The circuit receives excitatory inputs from peripheral mechanoreceptors and coordinates the defensive response. While male cues suppress it via a doublesex (dsx) neuronal pathway, mating sensitizes it by stimulating a group of uterine neurons and consequently activating a leucokinin-dependent pathway (see Lkr). Such a modulation is crucial for the balance between defense against body contacts and sexual receptivity (Liu, 2020).

    Touch can be pleasant or aversive. Animals' responses to external stimuli, including mechanical force, are tuned by internal states and social contexts. The perception of touch is affected with social context and in turn conveys nuanced social information. This versatility of mechanosensory behaviors is essential for animals to adapt to different environments. These modulations can occur at both the peripheral and central nervous systems. However, the neural mechanism behind social regulation of mechanosensation is largely elusive (Liu, 2020).

    In animals with intra-species interaction, response to body touch is actively regulated with social contexts. For example, activation of mechanosensory neurons on the fore-leg induced by tapping can trigger collective behavior in Drosophila. During courtship, a major form of social interaction between flies, the role of touch sensation is poorly understood. Unlike visual, auditory and chemical cues known to play important roles during sexual behaviors of flies, the tactile communication between a pair of flies during courtship is often overlooked. One reason is that body touch can occur to most parts of the body surface and the females' response is highly diverse, and unlike other sensory modes, there are multiple regions in the brain and the ventral nerve cord that receive mechanosensory inputs. Moreover, mechanical stimuli on the body surface are usually alert signals and flies tend to provoke escape or defensive response. This gives rise to a profound question: how do female flies adjust their responsive state to body touch according to the context of sexual activity? In female mice, different groups of neurons in the ventromedial hypothalamus seem to play different roles in regulating sexual receptivity and defensive behaviors, implicating that defensive response is affected with mating activities. However, the dissection of neural circuits that mediate the interplay between mating states and sensory inputs has not been achieved (Liu, 2020).

    In recent years, progress has been made to uncover the genetic and neuronal basis of sexual behaviors, although the studies on males far outnumber those on females. Female flies have well-established sexually dimorphic behaviors that are under rigorous control. For instance, the doublesex (dsx) circuits in the brain are activated with male pheromone and courtship song to slow down the female's locomotion. Meanwhile, an increase of receptivity causes a reduced level of kicking and fencing against male's tap or lick. In contrast, mating can trigger a switch of both behaviors and internal states in female, including reduced receptivity and changes in diet preference. They also become more aggressive when competing for food resources. Whether mated female flies become more responsive to or alert against body contact stimuli compared to pre-mated females is unknown (Liu, 2020).

    To answer these questions, this study combined genetic and behavioral approaches to investigate the versatility of female flies' defensive response to body contacts. A somatosensory center in the ventral nerve cord was identified that transmits somatosensory input to motor actions. The functional plasticity of this neural structure is responsible for the mating-induced switch in defensive response. This study reveals the neural basis for the sexual activity inducing modulation of touch sensitivity (Liu, 2020).

    Female flies show a profound behavioral switch after mating. For example, they become hypersensitive to many sensory stimuli after mating. This paper showns that the behavioral responses to tactile stimuli are also exaggerated. The sensitivity to a mechanical touch on the body in female flies was tuned by different mating states. Upon touch on the wing margins, the mechanical information was relayed to Central Tmc-L neurons (CTNs) to trigger the defensive response. The activity of CTNs was inhibited by a dsx neural circuit when male courtship cues were present; After copulation, a group of Gr32a neurons on the uterus were activated, culminating in the enhancement of defensive response, which presumably resulted from an elevated responsiveness of CTNs by the action of LK. The switch between pre-mating and post-mating states in the defensive response is fast and reversible and of decisive importance for female receptivity (Liu, 2020).

    Previous studies demonstrate that the post-mating response (PMR) is mainly mediated by male ejaculate. Suppression of re-mating, as a major PMR effect, emerges in two phases: the fast phase commences immediately after mating, whereas the slow phase takes hours after mating to appear and can last for as long as two weeks. A recent study identified a sensory (likely mechanosensory) pathway that directly encodes mating experience to suppress re-mating immediately after mating. This study has unraveled an additional pathway that originates from the uterus, yet is independent of SP signaling, to suppress female receptivity to mate, in which the uterine neurons (UNs) was involved in enhancing the defensive response and consequently reducing receptivity after mating. Gr32a is known to form, together with other Gr molecules, a gustatory receptor complex responsive to a wide array of bitter compounds including some male cuticular hydrocarbons that inhibits male courtship to conspecific males or females from other species. While it's highly plausible that UNs function as an internal sensor to detect male ejaculate, another intriguing possibility is that, during copulation, a male fly transmits to his mate certain cuticular hydrocarbons, which activate Gr32a in UNs, leading to a post-mating change in the defensive response in the recipient female (Liu, 2020).

    Why do females need this alternative pathway from the uterus to the CTNs to regulate their PMR? Some potential functions of this pathway are envisioned: 1) As sensory neurons directly innervating the uterus, the UNs are well positioned to sense the ingredients in the seminal fluid. Besides SP, the seminal fluid contains a cocktail of proteins and other molecules, many of which take actions on unidentified sites. It is tantalizing to hypothesize that Gr32a and UNs are involved in the detection of some of the molecules. 2) UNs may act as a compliment of the LASN pathway to mediate the short-term post-mating response (PMR). This notion is supported by the fact that females with their LASN silenced still had substantial experience index around 4 h after mating and ablating the Crz neurons can't block the transfer of all the components of seminal fluid. 3) As a downstream of UNs, ABLK neurons were found to regulate water homeostasis and food intake, implicating that the activation of UNs may be linked with changes of other physiological states that can last longer that hours. Although in this study focus was placed on the defensive behavior, it is conceivable that UNs' activation may impact other post-mating behaviors such as feeding, egg-laying or aggression (Liu, 2020).

    A cohort of neuropeptides mediate the PMR. Leucokinin was initially found critical for body water balance as it is a neurohormone to increase Malpighian tubule fluid secretion and hindgut motility. It also plays a role in the regulation of meal size. This study may provide a link between the water/nutrition balance system and the mechanism for female receptivity via a common signaling channel mediated by LK and LKR. Although there are only a few of clusters of LK neurons in the fly, LKR is expressed broadly in the fly central nervous system and other tissues. Notably, among the 20 LK neurons in the VNC, at least 4-6 abdominal LK (ABLK) neurons also express DH44, a neuropeptide with established roles in female reproductive behaviors. It is an open question whether there is any functional heterogeneity among the ABLK neurons. Intriguingly, the LK receptor LKR is homologous to vertebrate tachykinin receptors. In rodents, tachykinins and all neurokinin receptors are present in the uterus and their abundance is regulated during pregnancy. It thus seems plausible that the LK pathway plays an evolutionarily conserved role in the reproductive behaviors of female animals (Liu, 2020).

    The fly VNC has attracted less attention as a site for neural integration for controlling behavior, despite that its critical role in generating motor output is well-documented. This study unequivocally demonstrated that the mating state-dependent, and LK-mediated changes in defensive response rely on neural plasticity occurring exclusively within the VNC. The CTNs neural circuit characterized in this study provides an entry point to delineate the neural mechanism whereby ongoing behavior is fine-tuned at every moment of actions by sensory-guided neural plasticity. Interestingly, the sheet-holder shaped arborization of CTNs in the accessary mesothoracic ganglion is very similar to the structure in the same VNC region reported to be the neural substrate that balances locomotion and feeding (Mann, 2013). In that study, the arborizations were visualized with E564-Gal4, which labels a group of brain-descending neurons involved in gustatory information processing. These descending neurons were also reported to be downstream of sensory afferents from multiple appendages. An attractive scenario is that the sheet-holder shaped arborization serves as the integrative module that coordinates feeding, locomotion and defensive response in a sexual activity-dependent manner with and/or without the brain involvement. Further study is needed to elucidate whether CTNs and E564 neurons connect to each other, and whether the regulatory dsx and LK inputs impinge on this specific neural structure (Liu, 2020).

    The neural circuits in the fly brain encoding mating rejection are poorly understood. The copulation rate or latency are commonly measured as a readout of receptivity. However, in view of the rich repertoire of the motor programs for rejection that includes kicking, fencing, wing flicking and ovipositor extrusion, it is conceivable that a multilayered neural network distributing the brain and VNC operates to organize rejection behaviors. One of the key neural elements controlling receptivity is the Dsx-positive pC1 cluster in the female brain, the male counterpart of which includes the P1 cluster, the neural center that makes the decision to court. Although this study has established a synaptic connection between a class of dsx neurons and CTNs, it remains to be examined whether pC1 has any role in controlling CTNs and if it does, which descending neurons deliver the pC1 output to CTNs (Liu, 2020).

    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).

    Mating system manipulation and the evolution of sex-biased gene expression in Drosophila

    Sex differences in dioecious animals are pervasive and result from gene expression differences. Elevated sexual selection has been predicted to increase the number and expression of male-biased genes, and experimentally imposing monogamy on Drosophila melanogaster has led to a relative feminisation of the transcriptome. This hypothesis was further tested by subjecting another polyandrous species, D. pseudoobscura, to 150 generations of experimental monogamy or elevated polyandry. Sex-biased genes were found to change in expression but, contrary to predictions, there is usually masculinisation of the transcriptome under monogamy, although this depends on tissue and sex. We also gene expression changes were identified and described following courtship experience. Courtship often influences gene expression, including patterns in sex-biased gene expression. These results confirm that mating system manipulation disproportionately influences sex-biased gene expression but show that the direction of change is dynamic and unpredictable (Veltsos, 2017).

    Contact chemoreceptors mediate male-male repulsion and male-female attraction during Drosophila courtship

    The elaborate courtship ritual of Drosophila males is dictated by neural circuitry established by the transcription factor Fruitless and triggered by sex-specific sensory cues. Deciphering the role of different stimuli in driving courtship behavior has been limited by the inability to selectively target appropriate sensory classes. This study identified two ion channel genes belonging to the degenerin/epithelial sodium channel/pickpocket (ppk) family, ppk23 and ppk29, which are expressed in Fruitless-positive neurons on the legs and are essential for courtship. Gene loss-of-function, cell-inactivation, and cell-activation experiments demonstrate that these genes and neurons are necessary and sufficient to inhibit courtship toward males and promote courtship toward females. Moreover, these cells respond to cuticular hydrocarbons, with different cells selectively responding to male or female pheromones. These studies identify a large population of pheromone-sensing neurons and demonstrate the essential role of contact chemosensation in the early courtship steps of mate selection and courtship initiation (Thistle, 2012).

    Myc suppresses male-male courtship in Drosophila

    Despite strong natural selection on species, same-sex sexual attraction is widespread across animals, yet the underlying mechanisms remain elusive. This study reports that the proto-oncogene Myc is required in dopaminergic neurons to inhibit Drosophila male-male courtship. Loss of Myc, either by mutation or neuro-specific knockdown, induced males' courtship propensity toward other males. Genetic screen identified DOPA decarboxylase (Ddc) as a downstream target of Myc. While loss of Ddc abrogated Myc depletion-induced male-male courtship, Ddc overexpression sufficed to trigger such behavior. Furthermore, Myc-depleted males exhibited elevated dopamine level in a Ddc-dependent manner, and their male-male courtship was blocked by depleting the dopamine receptor DopR1. Moreover, Myc directly inhibits Ddc transcription by binding to a target site in the Ddc promoter, and deletion of this site by genome editing was sufficient to trigger male-male courtship. Finally, drug-mediated Myc depletion in adult neurons by GeneSwitch technique sufficed to elicit male-male courtship. Thus, this study uncovered a novel function of Myc in preventing Drosophila male-male courtship, and supports the crucial roles of genetic factors in inter-male sexual behavior (Pan, 2022).

    The PKA-C3 catalytic subunit is required in two pairs of interneurons for successful mating of Drosophila

    Protein kinase A (PKA) has been shown to play a role in a plethora of cellular processes ranging from development to memory formation. Its activity is mediated by the catalytic subunits whereby many species express several paralogs. Drosophila encodes three catalytic subunits (PKA-C1-3) and whereas PKA-C1 has been well studied, the functions of the other two subunits were unknown. PKA-C3 is the orthologue of mammalian PRKX/Pkare and they are structurally more closely related to each other than to other catalytic subunits within their species. PRKX is expressed in the nervous system in mice but its function is also unknown. This study now shows that the loss of PKA-C3 in Drosophila causes copulation defects, though the flies are active and show no defects in other courtship behaviours. This phenotype is specifically due to the loss of PKA-C3 because PKA-C1 cannot replace PKA-C3. PKA-C3 is expressed in two pairs of interneurons that send projections to the ventro-lateral protocerebrum and the mushroom bodies and that synapse onto motor neurons in the ventral nerve cord. Rescue experiments show that expression of PKA-C3 in these interneurons is sufficient for copulation, suggesting a role in relaying information from the sensory system to motor neurons to initiate copulation (Cassar, 2018).

    Visual projection neurons mediating directed courtship in Drosophila

    Many animals rely on vision to detect, locate, and track moving objects. In Drosophila courtship, males primarily use visual cues to orient toward and follow females and to select the ipsilateral wing for courtship song. This study shows that the LC10 visual projection neurons of the Lobula Column convey essential visual information during courtship. Males with LC10 neurons silenced are unable to orient toward or maintain proximity to the female and do not predominantly use the ipsilateral wing when singing. LC10 neurons preferentially respond to small moving objects using an antagonistic motion-based center-surround mechanism. Unilateral activation of LC10 neurons recapitulates the orienting and ipsilateral wing extension normally elicited by females, and the potency with which LC10 induces wing extension is enhanced in a state of courtship arousal controlled by male-specific P1 neurons. These data suggest that LC10 is a major pathway relaying visual input to the courtship circuits in the male brain (Ribeiro, 2018).

    Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila

    Animals consolidate some, but not all, learning experiences into long-term memory. Across the animal kingdom, sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage. However, the underlying mechanisms of sleep dependent memory consolidation are poorly understood. This study shows that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition. Specific fan-shaped body neurons were identified that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation. Thus, this study provide a direct link between sleep, neuronal reactivation of dopaminergic neurons, and memory consolidation (Dag, 2019).

    The activity of DAN-aSP13s, which is essential for courtship memory acquisition, is also necessary during a discrete post-learning time window for LTM consolidation. Because neuronal reactivation occurs during sleep in rodents, it was hypothesized that post-learning activation of DAN-aSP13s involves a sleep-dependent mechanism. Using behavioral analysis and neuronal activity monitoring and perturbation approaches, thus study shows that DAN-aSP13s display an increased activity in freely behaving animals during sleep after a prolonged learning experience. This sleep is necessary for LTM consolidation, and it can be mediated by a specific class of sleep promoting neurons in the ventral layer of the fan shaped body (vFB). These vFB neurons consolidate courtship LTM in a discrete time window and provide an excitatory input to DAN-aSP13s. Thus, these data provide a causal link between sleep promoting neurons in the vFB, post-learning activation of dopaminergic neurons, and LTM consolidation (Dag, 2019).

    Based on these data, the following model is proposed for sleep-dependent consolidation of courtship LTM in Drosophila (see Post-learning activation of DAN-aSP13 neurons mediates LTM consolidation). During a prolonged learning experience, γKCs and DAN-aSP13s are repeatedly activated by olfactory and behavioral cues presented by an unreceptive female, respectively. Whereas prolonged wakefulness leads to an increase in homeostatic sleep drive in ellipsoid body neurons that in turn is conveyed to dFB, it is hypothesized that an extended learning experience generates a learning-dependent sleep drive that is transmitted to vFB. In turn, the vFB neurons enhance sleep after learning and provide an excitatory input back on DAN-aSP13s. It is thought that one potential site of a learning-dependent sleep drive are the MB neurons since they have been implicated in both memory formation and sleep regulation. Dopamine released upon DAN-aSP13 reactivation stimulates molecular processes in γKCs that are different from those engaged during STM acquisition and involve protein synthesis that is essential for LTM formation and persistence (Dag, 2019).

    Dopaminergic pathways are thought to convey information about whether an experience is rewarding or punishing and thus, worth remembering. Post-learning neuronal activity of the dopaminergic hippocampal inputs from the Ventral Tagmental Area (VTA) has been implicated in the consolidation of fear memory in rodents. Interestingly, this activity is critical during a discrete time window after learning. Post-learning activity of the VTA dopamine neurons has been also implicated in the reactivation during sleep of the hippocampal cells involved earlier in encoding of the spatial experience. This study shows that post-learning activation of DAN-aSP13s mediates the consolidation of courtship LTM in Drosophila. It is proposed that reactivation during sleep of the dopamine neurons that were previously active during memory acquisition ensures that spurious experiences are not admitted into LTM storage and thus only experiences that are either sufficiently salient or persistent become long-lasting memories. Specifically, reactivation of DAN-aSP13s during post-learning sleep enhances reactivation of the γKCs and cognate MBON-M6, which together with DAN-aSP13s form a recurrent circuit necessary for courtship memory acquisition. This study considers two hypotheses to account for this selectivity. During sleep, vFB neurons might selectively reactivate only the relevant DANs, or alternatively, they might activate all DANs but only the relevant subset is able to consolidate LTM. The selective-reactivation model would require some marker to distinguish which DANs were activated, whereas the selective-consolidation model would require a marker in the synapses of the γKCs that were earlier active during memory acquisition, for example translational regulator Orb2, which regulates translation upon neuronal activity during LTM consolidation (Dag, 2019).

    Activity of dopaminergic neurons regulates sleep-wake states in animals, including flies. Artificial activation of DAN-aSP13s has been shown to increase wakefulness. In contrast, the data presented in this study imply that activation of vFB neurons, although they activate DAN-aSP13s, do not promote wakefulness. These results suggest that activation of DAN-aSP13s by vFB neurons during sleep is qualitatively different from a direct optogenetic or thermogenetic activation used in previous studies. One potential explanation is that post-learning sleep involves activation of the vFB circuit, which provides both excitatory stimulus to DAN-aSP13s and inhibitory input to motor neurons. Another possibility is that post-learning sleep activates a subset od DAN-aSP13s that do not affect wakefulness (Dag, 2019).

    Thia study has identified a class of sleep-promoting neurons in the ventral layer of FB that are distinct from the well-studied sleep-promoting neurons in the dorsal layer of FB, which regulate sleep homeostasis. Given that vFB neurons enhance sleep and activate DAN-aSP13s for LTM consolidation, whereas dFB neurons are neither necessary nor sufficient for LTM consolidation, it is hypothesized that dFB and vFB neurons promote distinct components of sleep that have different functions. Homeostatic sleep is thought to facilitate memory encoding by downscaling synaptic weights and clearing metabolites from the brain accumulated during wakefulness. In contrast, the function of learning-dependent sleep might be to facilitate memory consolidation by strengthening synaptic connections that were engaged earlier during memory acquisition. Thus, the co-operation of homeostatic- and experience-dependent sleep would facilitate optimal conditions for learning new information and, if appropriate, incorporating it into long-term storage (Dag, 2019).

    Recent studies have implied that sleep in flies, as in humans and rodents, exhibits sleep stages characterized by distinct electrophysiological signatures. Interestingly, the signature of sleep that is induced by activation of the dFB neurons seems to have a simpler oscillatory pattern, recorded by local field potentials, than sleep that is induced by activation of the FB neurons comprising both dFB and vFB neurons. Thus, these data support the hypothesis that dFB and vFB neurons promote sleep with different properties and likely different functions (Dag, 2019).

    It is thought that sleep evolved in animals that are capable of complex learning which requires selective attention. Courtship learning is a multisensory form of learning that requires selective attention of a male to associate multiple learning cues presented by the mated female with the outcome of his own behavior. Accordingly, studies in bees have shown that sleep affects a complex form of learning such as spatial memory but has no role in the simple learning paradigm of proboscis extension. Hence, it would be interesting to investigate whether post-learning sleep is involved in the consolidation of other types of memory in Drosophila, such as the well-studied Pavlovian olfactory associative learning whereby animals associate an individual learning cue with a behavioral contingency (Dag, 2019).

    This work has established a functional link between a novel class of sleep-promoting neurons in the FB, post-learning reactivation of dopaminergic neurons and consolidation of courtship LTM. Moreover, the data suggest that sleep promoting vFB neurons mediate a learning-dependent regulation of sleep that is distinct from the homeostatic control which is facilitated by dFB neurons. Thus, this study uncovered a causal link between sleep-mediated neuronal reactivation and LTM consolidation in Drosophila. In addition, courtship LTM was establish in Drosophila as a tractable model to investigate the mechanisms that link learning-dependent sleep, neuronal reactivation and LTM consolidation (Dag, 2019).

    Activity-dependent visualization and control of neural circuits for courtship behavior in the fly Drosophila melanogaster

    Males of Drosophila melanogaster exhibit stereotypic courtship behavior through which they assess potential mates by processing multimodal sensory information. Although previous studies revealed important neural circuits involved in this process, the full picture of circuits that participate in male courtship remains elusive. This study established a genetic tool to visualize or optogenetically reactivate neural circuits activated upon specific behavior, exploiting promoter activity of a neural activity-induced gene Hr38. With this approach, neural circuits activated in the male brain and the ventral nerve cord were visualized when a male interacted with a female. The labeling of neural circuits was additively dependent on inputs from antennae and foreleg tarsi. In addition, neural circuits that express the sex-determining gene fruitless or doublesex were extensively labeled by interaction with a female. Furthermore, optogenetic reactivation of the labeled neural circuits induced courtship posture. With this mapping system, it was found that a fruitless-positive neural cluster aSP2 was labeled when a male interacted with a female, in addition to previously characterized neurons. Silencing of neurons including aSP2 led to frequent interruption of courtship and significant reduction of mating success rate without affecting latency to start courtship, suggesting that these neurons are required for courtship persistency important for successful copulation. Overall, these results demonstrate that activity-dependent labeling can be used as a powerful tool not only in vertebrates, but also in invertebrates, to identify neural circuits regulating innate behavior (Takayanagi-Kiya, 2019).

    A neural circuit encoding the experience of copulation in female Drosophila

    Female behavior changes profoundly after mating. In Drosophila, the mechanisms underlying the long-term changes led by seminal products have been extensively studied. However, the effect of the sensory component of copulation on the female's internal state and behavior remains elusive. This question was pursued by dissociating the effect of coital sensory inputs from those of male ejaculate. The sensory inputs of copulation were shown to cause a reduction of post-coital receptivity in females, referred to as the "copulation effect." Three layers of a neural circuit underlying this phenomenon were identified. Abdominal neurons expressing the mechanosensory channel Piezo convey the signal of copulation to female-specific ascending neurons, LSANs, in the ventral nerve cord. LSANs relay this information to neurons expressing myoinhibitory peptides in the brain. This study hereby provides a neural mechanism by which the experience of copulation facilitates females encoding their mating status, thus adjusting behavior to optimize reproduction (Shao, 2019).

    The effects of target contrast on Drosophila courtship

    Many animals use visual cues like object shape, color, and motion to detect and pursue conspecific mates. Contrast is another possibly informative visual cue, but has not been studied in great detail. This study presents male Drosophila melanogaster with small, fly-sized, moving objects painted either black, white, or grey to test if they use contrast cues to identify mates. Males were found to frequently chase grey objects and rarely chased white or black objects. Although males started chasing black objects as often as grey objects, the resulting chases were much shorter. To test whether the attraction to grey objects was mediated via contrast, black and grey behavioral chambers were fabricated. However, wildtype males almost never chased any objects in these darkly colored chambers. To circumvent this limitation, baseline levels of chasing were increased by thermogenetically activating P1 neurons to promote courtship. Males with thermogenetically activated P1 neurons maintained a similar preference for grey objects despite elevated levels of courtship behavior. When placed in a black chamber, males with activated P1 neurons switched their preference and chased black objects more than grey objects. Whether males use contrast cues to orient to particular parts of the female's body during courtship was also tested. When presented with moving objects painted two colors, males positioned themselves next to the grey half regardless of whether the other half was painted black or white. These results suggest that males can use contrast to recognize potential mates and to position themselves during courtship (Agrawa, 2019).

    Flexible memory controls sperm competition responses in male Drosophila melanogaster

    Males of many species use social cues to predict sperm competition (SC) and tailor their reproductive strategies, such as ejaculate or behavioural investment, accordingly. While these plastic strategies are widespread, the underlying mechanisms remain largely unknown. Plastic behaviour requires individuals to learn and memorize cues associated with environmental change before using this experience to modify behaviour. Drosophila melanogaster respond to an increase in SC threat by extending mating duration after exposure to a rival male. This behaviour shows lag times between environmental change and behavioural response suggestive of acquisition and loss of memory. Considering olfaction is important for a male's ability to assess the SC environment, it is hypothesized that an olfactory learning and memory pathway may play a key role in controlling this plastic behaviour. The role were assessed of genes and brain structures known to be involved in learning and memory. SC responses depend on anaesthesia-sensitive memory, specifically the genes rut and amn. The gamma lobes of the mushroom bodies are integral to the control of plastic mating behaviour. These results reveal the genetic and neural properties required for reacting to changes in the SC environment (Rouse, 2081).

    Antennae sense heat stress to inhibit mating and promote escaping in Drosophila females

    Environmental stress is a major factor that affects courtship behavior and evolutionary fitness. Although mature virgin females of Drosophila melanogaster usually accept a courting male to mate, they may not mate under stressful conditions. Above the temperature optimal for mating (20-25 degrees C), copulation success of D. melanogaster declines with increasing temperature although vigorous courtship attempts were observed by males, and no copulation takes place at temperatures over 36 degrees C. Attempts were made to identify the sensory pathway for detecting heat threat that drives a female to escape rather than to engage in mating that detects hot temperature and suppresses courtship behavior. The artificial activation of warmth-sensitive neurons ('hot cells') in the antennal arista of females completely abrogates female copulation success even at permissive temperatures below 32 degrees C. Moreover, mutational loss of the GR28b.d thermoreceptor protein caused females to copulate even at 36 degrees C. These results indicate that antennal hot cells provide the input channel for detecting the high ambient temperature in the control of virgin female mating under stressful conditions (Miwa, 2018).

    Somers, J., Luong, H. N., Mitchell, J., Batterham, P. and Perry, T. (2017). Pleiotropic effects of loss of the Dα1 subunit in Drosophila melanogaster: Implications for insecticide resistance. Genetics 205(1): 263-271. PubMed ID: 28049707

    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).

    Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor subfamily of ligand-gated ion channels (LGICs) that mediate the transduction of a chemical signal into an electrical signal. Activation in response to acetylcholine (ACh), the major excitatory neurotransmitter of the Drosophila melanogaster central nervous system, is on a micro- to submicrosecond timescale. nAChRs are expressed in a wide range of tissues, including but not limited to the nervous system. When expressed presynaptically, nAChRs can enhance neurotransmitter release, while postsynaptic expression mediates excitation. Like all LGICs, nAChRs are pentameric and can consist of several different subunits forming multiple receptor subtypes that vary in both their sensitivity to particular ligands and their permeability to particular cations. Individual subunits consist of a pore-forming transmembrane domain coupled to a large extracellular N-terminal domain, which forms the endogenous ligand-binding site with adjacent subunits. Upon ACh binding, a conformational change occurs opening the channel pore to permit the flow of cations (Somers, 2017).

    D. melanogaster has 10 nAChR subunits; however, only three of these have been associated with behavioral phenotypes, with specific roles described for Dα7 in the escape response and for both Dα3 and Dα4 in sleep behavior. Mutations in three D. melanogaster nAChR subunits confer resistance to two important classes of insecticides; Dα1 and Dβ2 to neonicotinoids, and Dα6 to spinosyns. Modification of an insecticide target protein can decrease the efficacy of an insecticide through altered affinity or pharmacological response. Modifications of this nature can provide a significant fitness boost to individuals in a population during insecticide treatment periods and can rapidly increase in frequency leading to control failures. However, allele frequencies will also be shaped by fitness costs if resistant mutations negatively impact reproductive output, either by reducing viability or the capacity to mate. This could be particularly true for alleles that directly modify the neonicotinoid-binding site as neonicotinoids occupy the same binding site as ACh. Loss-of-function mutants for the Dα1, Dα6, and Dβ2 genes are insecticide-resistant and viable. The viability phenotype suggests a level of functional redundancy among nAChR subunits. Questions remain as to whether individual subunits have other, nonredundant functions in controlling behavior or if the subtle pharmacological and physiological differences from compensating subunits manifests in altered behaviors. Impacts on mating behavior are of particular interest with respect to potential fitness costs (Somers, 2017).

    While invertebrate nAChR pharmacology and biochemistry with respect to insecticide binding has been examined in detail, little research has been devoted to the endogenous functions of these receptors. D. melanogaster is commonly used as a model insect system to study many facets of biology, including insecticide resistance. A large number of well-defined behavioral paradigms exist to investigate the roles of genes in traits, including sleep and cognition through to mating and auditory faculties (Somers, 2017).

    This study reports the creation of a Dα1 knockout mutant and a rescue system through transgene expression. This provided a consistent genetic background to analyze several behavioral paradigms. These reagents were used to identify roles for the D. melanogaster Dα1 subunit in mating, locomotion, and sleep, demonstrating the diverse pleiotropic influences that nAChRs can have on insect behavior. This research has relevance to the consideration of fitness costs that might be associated with resistance conferring mutations in Dα1 orthologs in pest insects, and the behavioral impact that exposure to neonicotinoids may have in beneficial species such as the western honey bee, Apis mellifera (Somers, 2017).

    To generate a Dα1 null mutant, a modified ends-out targeting scheme was used. A precise 57-kb deletion of the Dα1 genomic region was created, then validated by Southern blot and sequencing. This mutant is referred to as Dα1KO. Given that previously created Dα1 alleles are resistant to neonicotinoids (Perry, 2008), Dα1KO was screened on two different neonicotinoids (imidacloprid and nitenpyram) and was found to be highly resistant to both. Calculated LC50 values and resistance ratios were consistent with those measured for Dα1 mutants studied previously (Perry, 2008). Dα1 and orthologs in pest species are well-established targets for several different neonicotinoid insecticides. Heterologous expression and affinity chromatography studies have also implicated Dα1 in directly binding imidacloprid at a site that overlaps that normally occupied by the ligand, ACh. Therefore, these resistance data matched the prediction that a mutant with a genomic deletion of Dα1 would be resistant to neonicotinoid insecticides (Somers, 2017).

    The GAL4-UAS system was employed to create a phenotypic rescue in which a Dα1 cDNA clone was expressed in the Dα1KO background. Expression of the Dα1 clone using the pan-neuronal elav::GAL4 driver rescued sensitivity to both imidacloprid and nitenpyram. The reversion of the resistance phenotype indicates that the subunit expressed from the Dα1 transgene is assembling into functional nAChRs that bind these insecticides (Somers, 2017).

    The loss of Dα1 results in significant levels of resistance to neonicotinoids. However, the level of resistance is not of the same magnitude observed in the Dα6 knockout mutant, which is over 1000-fold resistant to spinosad. The Dα1KO mutant is still susceptible when exposed to a high enough dose, which may be due to expression of other neonicotinoid-sensitive nAChR subtypes. Mutations in the orthologs of Dα3 and Dβ1 have been identified in neonicotinoid-resistant strains of Nilaparvata lugens and Myzus persicae, respectively. While only one imidacloprid-binding site has been reported in adult D. melanogaster and other Dipteran and Lepidopteran species, multiple binding sites have been reported in several Hemipteran species. Unlike Hemipterans, Dipterans and Lepidopterans are holometabolous insects that undergo complete metamorphosis from larva to adult. It is possible that as yet undescribed imidacloprid-sensitive nAChR subtypes are expressed in the larval life stages. Another possibility is that a novel subtype is formed as a consequence of the loss of the Dα1 subunit that alters the mutant's sensitivity to neonicotinoids (Somers, 2017).

    RNAi knockdown of the Dα1 subunit resulted in defects in courtship and copulation behavior. Previous microarray studies, confirmed by RT-PCR, highlighted expression of Dα1 in both neuronal and reproductive tissues. Taken together, these lines of evidence suggested the potential for Dα1 to function in mating behavior (Somers, 2017).

    Mating behavior can be influenced by genetic background, for example expression of w is important for visual cues and misexpression of this gene can trigger male-male courtship. As the Dα1KO line was generated in the w1118 background, both the mutant and the control line have functionally null copies of w. Therefore, the w+ X chromosome from another isogenic line, RAL059, was used to replace the w1118 X chromosome present in both the Dα1KO mutant line and the w1118 background line. These lines will be referred to as the mutant and wild-type lines respectively (Somers, 2017).

    Courtship behavior was measured in terms of the latency of courtship initiation by males after female introduction into the mating chamber. Flies that failed to initiate courtship within the allowed 10-min period were given a maximum value. Wild-type males initiated courtship in every trial, regardless of the genotype of the female partner. Mutant males only initiated courtship 65% of the time with wild-type females and 79% of the time with mutant females. Mutant males also took significantly longer to initiate courtship than wild-type males when paired with either wild-type or mutant females (Somers, 2017).

    Copulation latency was also measured for the same flies. Mutant males rarely copulated within the 10-min period, only 15% of trials when paired with a wild-type female and only 3% when paired with a mutant female. Wild-type males were more successful, initiating copulation in 90% of the trials when paired with a wild-type female and in 56% of the trials when paired with a mutant female. No significant difference was observed in copulation latency of mutant males when paired with either wild-type or mutant females. In contrast, wild-type males did initiate copulation significantly faster with wild-type females compared to mutant females. This suggests that, unlike the latency of courtship initiation that is primarily influenced by the genotype of the male, both sexes contribute to the latency of copulation initiation (Somers, 2017).

    The rescue system was again employed to see if expression of a Dα1 transgene could rescue the courtship and copulation phenotypes observed for the Dα1KO mutant. Appropriate driver-only and UAS-only control flies were used for comparison to rescue flies. No discernible differences in courtship initiation were observed when wild-type males were crossed to control or rescue female flies. However, when crossed to wild-type female flies, male rescue flies were more successful in initiating courtship than male control flies. Male rescue flies also showed a significant decrease in courtship initiation when crossed to female wild-type compared to male control flies. Significant rescue of copulation initiation was also observed in both female and male rescue flies. Rescue flies were both more successful and faster at initiating copulation than the appropriate controls (Somers, 2017).

    It is clear from the data that Dα1 plays a role in Drosophila mating behavior; however, the underlying mechanism remains unknown. Defects in ACh signaling have previously been associated with abnormal mating behavior. Analysis of mosaic mutants, defective for cholinergic signaling, identified a neuropile in the mushroom body calyx critical for normal male courtship behavior. Male-specific, cholinergic neurons have also been identified in the abdominal ganglion, the disruption of which significantly decreased male fertility, potentially due to their innervation of the male reproductive system. The disruption of these neurons has been hypothesized to result in the uncoordinated or altered release of sperm, seminal fluid, and accessory proteins. The study, performed by Acebes (2004) used the presynaptic choline acetyltransferase marker to identify these neurons; however, the receptors receiving this signal were not identified. The data from mating experiments and expression analysis suggests that Dα1 may be one of the specific nAChR subunits expressed in this pathway. Another possibility to consider is a higher processing role for Dα1 in mating behavior. The phenotypes observed in the Dα1KO mutant are consistent with a defect in one or more sensory modalities. While there are specialized receptors responsible for detecting sensory stimuli, cholinergic signaling has been identified in connecting sensory circuitry to processing centers in the brain. Dα1 expression has previously been observed in the mushroom body calyx and lateral protocerebrum, which includes the lateral horn. Recently, the role of the lateral horn in locusts has been proposed to serve as a site for multimodal sensory integration. This may suggest that Dα1 plays a role in integrating multimodal courtship circuitry to higher sensory processing centers. Challenging Dα1KO mutants with sensory-specific behavioral paradigms and neuron-specific rescue could test this hypothesis (Somers, 2017).

    The possibility was explored that impaired locomotion may be contributing to the mating phenotype observed in the Dα1KO mutant. Over a 24-hr period, the Dα1KO mutant exhibited hyperactivity; however, it moved at a slower average speed. When average speed was binned in 3-hr intervals, the difference was significant during the middle two 3-hr bins of the day and the last three 3-hr bins of the night. Most importantly, there was no significant difference in average speed during the first 3-hr bin of the day, when courtship assays were performed, indicating that general locomotion deficits did not impact the measurement of mating behavior (Somers, 2017).

    The Dα1KO mutant also has an unusual pattern of sleep. Although there were no significant differences observed in total amount of sleep, mutant flies slept significantly less during the night, experiencing less sleep episodes of significantly shorter duration than wild-type controls. The total time of day sleep experienced by the mutant was significantly longer with a higher number of sleep episodes; however, there was no observable difference in episode length. Expression of the Dα1 transgene in the mutant background increased amount of sleep, both during the day and night. This increase was a result of longer sleep episodes, which may explain why both mutant and rescue flies both show less night sleep episodes. Rescue flies have less night sleep episodes due to the length of these episodes, whereas mutant flies may have trouble with sleep initiation and maintenance. The exceptional increase in episode length observed in rescue flies is likely due to nonnative expression of the transgene; however, it is clear Dα1 influences sleep (Somers, 2017).

    While activation of all cholinergic neurons inhibits sleep in flies, different neuronal groups can be wake-promoting or sleep-promoting. Individual nAChR subunits also appear to have different roles in sleep regulation. From this study, Dα1 seems to have net sleep-promoting effects, especially with sleep maintenance. The increase in daytime sleep observed in mutant flies may be a compensation mechanism to cope with the reduced amount of nighttime sleep. Further experiments are needed to determine whether the Dα1KO mutant has intact sleep homeostatic regulation, and whether loss of sleep affects sleep quality. Sleep deprivation has been linked to various detrimental effects, namely reduced life span and learning deficits (Somers, 2017).

    Longevity of Dα1KO mutants was measured, revealing a much shorter life span in the mutant compared to the wild-type. While longevity is not a direct measure of fitness, the mutants' reduced life span highlights the importance of the Dα1 subunit in D. melanogaster physiology. It is not clear if this effect is due to a single physiological role of Dα1, such as the subunits involvement in sleep, or cumulative effects of several impaired physiological roles that impact the mutant's longevity. In either case, it suggests that a resistance allele in the Dα1 subunit is likely to impact the fitness levels of the insect. It also supports the notion that sublethal exposures to insecticides that target receptor subunits orthologous to Dα1 encountered by beneficial insects, such as honey bees, are likely to affect their behavior in ways that may also impact their fitness (Somers, 2017).

    The Dα1KO mutant generated in this study provides a useful tool to study the role of Dα1 in insecticide resistance but, more significantly, to explore the endogenous roles of this gene (Somers, 2017).

    Based on prior evidence, it was expected that the Dα1KO mutant would be resistant to neonicotinoid insecticides (Perry, 2008). However, the data presented in this study allow a fresh evaluation of the value of Dα1 and orthologs in other species as insecticide targets. The ability of the Dα1 knockout flies to survive at all presents a potential issue for resistance evolution to compounds that target this receptor. Given that a wide range of mutations would lead to a total loss-of-function phenotype, such mutations will arise frequently, conferring insecticide resistance. The data presented in this study indicate that, looking beyond viability, there is a significant fitness cost associated with the total loss of Dα1 function, most obvious in terms of severe mating behavior defects, but possibly contributed to by the sleep and longevity phenotypes. Under optimal environmental conditions in the laboratory, large phenotypic differences between Dα1KO and control flies were observed. It is possible that the fitness cost associated with a null allele of this gene would be even greater under less ideal conditions experienced by natural populations. Therefore, while a nonsense mutation resulting in a resistance allele may be viable, it would likely be associated with fitness costs that would prevent it from persisting in the field. In contrast, mutations that may be null alleles have been found in Dα6 orthologs in spinosad-resistant insects in a number of species. Therefore, it is possible that the spectrum of mutations in Dα1 orthologs that can increase in frequency to confer insecticide resistance in a pest species are constrained by fitness costs (Somers, 2017).

    The potential impact of neonicotinoid insecticides on the behavior of beneficial insects, such as A. mellifera, has been intensively researched. Eleven nAChR subunits have been identified in A. mellifera, including a 1:1 ortholog of Dα1. While it cannot be assumed that the functional roles of the honey bee ortholog are identical to those of Dα1, it is likely to influence a range of behaviors that may be perturbed upon exposure to sufficient concentrations of neonicotinoid insecticides (Somers, 2017).

    The genetic resources that can be developed to study gene function in D. melanogaster, such as those described here, are extremely powerful. Research on nonmodel insects, both beneficial and pest species, is more challenging. Some functional analysis of Dα6 orthologs from pest species has been possible following the appropriate expression of these genes in a D. melanogaster Dα6 null mutant. A similar approach may be useful in the functional characterization of the orthologs of Dα1 (Somers, 2017).

    Insecticides can be powerful probes in neuroscience. To exert toxic effects, these chemicals bind to receptors that have crucial roles in neurotransmission. Research using pyrethroids and cyclodiene insecticides has been productive in elucidating the function of sodium channels and ligand-gated chloride channels, respectively. Similarly, the use of insecticides that target nAChRs has stimulated research on their function. The data demonstrates that such research will make a vital contribution in providing a detailed knowledge of the role neurotransmission in a wide range of behaviors. The variety of behavioral traits impacted by Dα1 loss-of-function indicates a high level of involvement of Dα1 in several behavioral neural circuits, which will require further investigation. Further analysis of Dα1 and the remaining members of the nAChR gene family with a range of paradigms is likely to reveal function in a wide range of insect behaviors (Somers, 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).

    High fat diet alters Drosophila melanogaster sexual behavior and traits: decreased attractiveness and changes in pheromone profiles

    Sexual traits convey information about individual quality to potential mates. Environmental and genetic factors affect sexual trait expression and perception via effects on animal condition and health. High fat diet (HFD) is one environmental factor that adversely affects Drosophila melanogaster health, and its effects on animal health are mediated through conserved metabolic signaling pathways. HFD decreases female attractiveness, resulting in reduced male mating behaviors toward HFD females. HFD also affects the ability of males to judge mate attractiveness and likely alters fly condition and sexual traits to impact mating behavior. This study shows that HFD affects both visual (body size) and non-visual (pheromone profiles) sexual traits, which likely contribute to decreased fly attractiveness. It was also demonstrated that adult-specific HFD effects on male mate preference can be rescued by changing metabolic signaling. These results demonstrate that HFD alters Drosophila sexual cues to reflect concurrent effects on condition and that less severe behavioral defects can be reversed by genetic manipulations that rescue fly health. This work expands on current knowledge of the role that metabolic signaling pathways play in linking animal health, sexual traits, and mating behavior, and provides a robust assay in a genetically tractable system to continue examining these processes (Schultzhaus, 2018).

    Similar to effects in other animals, overconsumption of lipids has severe detrimental effects on the health of D. melanogaster, and this decrease in fly health is correlated with alterations in mating behavior. In females, developmental HFD leads to decreased activity levels, body size, and fecundity, all of which reflect the worsened condition of the fly and ultimately lead to decreased female attractiveness. Therefore, some female sexual trait(s) evaluated by males during courtship must also be altered by HFD. Male flies are more resilient to the effects of HFD, as developmental HFD does not decrease male body size, activity levels, or attractiveness, but exposure to developmental HFD does affect the male's ability to discriminate between poor and good condition mates. This study has expanded understanding of how HFD affects D. melanogaster mating behavior by identifying dietary conditions that decrease male and female attractiveness by interrogating which sexual traits have been modified by HFD, and whether changes in mating behavior and attractiveness are mediated by conserved metabolic signaling pathways (Schultzhaus, 2018).

    The major sexual traits that signal female attractiveness are body size, behavioral responses to male courtship, and pheromone profiles. Drosophila males prefer larger females, and female acceptance or rejection behaviors also provide feedback to courting males, leading them to increase or decrease their courtship efforts. It was previously demonstrated that HFD decreased female activity levels prior to mating, which may be an indication of increased receptivity. However, it is possible that less mobile females are judged by males as being less attractive. Pheromones are another important sexual cue that convey information about species, sex, age, and mating status. In particular, the long-chain hydrocarbon dienes 7,11-heptacosadiene and 7,11-nonacosadiene, characteristic of female cuticles, function as aphrodisiacs for males. Dietary sugar and protein levels are known to alter D. melanogaster pheromone profiles, so it is likely that dietary lipids also affect pheromone production, especially since pheromone biosynthesis partially relies upon precursors from diet-derived fatty acids. Therefore, there are a number of sexual traits that could be modified by HFD to decrease female attractiveness, and it is hypothesized that pheromone changes are important contributors (Schultzhaus, 2018).

    To demonstrate that HFD alters female pheromone profiles in a way that decreases female attractiveness, males were examined as they courted HFD females without the confounding effects of diet on female body size and behavior. Control males discriminated against HFD females in both light and dark conditions by delaying the start of courtship, indicating that nonvisual sexual cues are important for male judgment of female quality. Although expected changes were observed in courtship latency in the light and the dark and in courtship index in the light, where control males decreased performed less courtship towards HFD females, control male courtship index towards HFD females was not altered in the dark. In fact, courtship indices for all pairings in the dark with intact females were drastically reduced. During video analysis, it was clear that males could not accurately track females in the dark, resulting in sporadic courtship bursts as flies came in contact in the courtship chambers. The general difficulty in finding mates in the dark may explain why there was no decrease in control male courtship towards intact 3% HFD females (Schultzhaus, 2018).

    The light or dark condition assays were repeated with decapitated females, which will not respond to courtship advances, allowing for the removal of female behavioral feedback from male mate assessment. Additionally, decapitated females are less mobile, which should allow males to find females more easily in dark conditions. This study found that control males did not modify their behavior significantly towards decapitated HFD females in the light. In light conditions, males can visually examine the decapitated females, so that although they stand upright, the females may have appeared injured or the males may have noted their inactivity, leading to increased courtship latency and decreased courtship index towards all types of decapitated females compared to intact females. Such judgments cannot be made in dark conditions, where it was observed that control males increased courtship latency and decreased courtship index toward HFD females. These experiments demonstrate that control males discriminated against HFD females without visual assessment of body size or behavioral feedback from the female, indicating that changes in pheromonal profiles likely contribute to male discrimination against 3% HFD females (Schultzhaus, 2018).

    Developmental exposure to environmental factors can have dramatic, long-lasting effects on fly health. Larval crowding or malnutrition can cause numerous alterations in fly traits, including body size. To fully remove the potential confounding effect of HFD on fly body size, flies were raised on a similar control diet during development and only the adults were exposed to increasing amounts of fat. All levels of HFD caused changes in female activity levels, but the behavioral defects seen in the developmental treatment were only recapitulated with the 15% adult-only diet treatment. At this level, HFD females mated faster, indicating that they are less choosy or more receptive to mating. Control males took longer to begin courting HFD females, indicating that the 15% HFD females are less attractive due to changes in a trait other than body size (Schultzhaus, 2018).

    Interestingly, control males did not decrease their overall courtship effort towards the adult-only HFD females as they had towards the developmental HFD females. It is therefore possible that common changes in pheromones caused by the developmental and adult-only diets are important for initial male assessment of the female and influence his decision to begin courtship behavior, i.e., to enter an arousal state. Once in this state, visual assessment of smaller developmental HFD females may have led males to decrease courtship, which would not happen with the adult diet females where body size is unaltered. Yet, males decreased courtship toward decapitated developmental HFD females in the dark when visual assessment was not possible (Schultzhaus, 2018).

    Both the developmental and adult-only HFD caused similar decreases in the levels of cuticular monoenes and dienes (primarily 7,11-heptacosadiene) of females. Additionally, the overall amount of CHCs produced by adult-only HFD treated females, but not developmental HFD females, decreased. These differences may be due to larval adaptation to HFD, leading to changes in adult metabolism and responses to HFD during adulthood, as has been observed in D. melanogaster in response to other types of larval dietary stress. The loss of the aphrodisiac 7,11-heptacosadiene in females under the 3% developmental and 15% adult-only HFD regimes is consistent with a loss of attractiveness. However, while 7,11-heptacosadiene levels also decreased in the 3% adult-only HFD females, a corresponding change in female attractiveness was not observed, suggesting that although a change in pheromone levels may contribute to diet-induced alterations in attractiveness, a combination of sensory features may be used by males in the decision to court. Future experiments using CHC extracts from 15% HFD females to 'perfume' oenocyte-less flies may provide an avenue for direct tests of whether diet-induced changes in CHCs underlie the loss of female attractiveness (Schultzhaus, 2018).

    Neural circuits necessary for D. melanogaster mating behavior are established during development. For example, expression of ecdysone receptor (EcR) in fruitless (fru) expressing neurons is necessary for standard male courtship behavior. Reduction of EcR expression in fru neurons during adulthood has little effect on male courtship behavior, but disruption during development increased male courtship performance. However, reduced EcR signaling during adulthood in has been shown to increase male-male courtship. Previous work has shown that males exposed to HFD during development did not find HFD females unattractive, as control males had, and did not appear to distinguish between control and HFD females to any degree. HFD therefore appears to alter male physiological functions that are important for mate discrimination, resulting in less choosy males. Whether exposure to HFD during development is necessary for this alteration to occur, or whether the change could happen after neural circuits underlying courtship behavior are established is unknown. This question was addressed by raising flies on a common, control diet and then exposing the flies to increasing amounts of HFD in adulthood. Courtship latency of 15% HFD males towards 15% HFD and control females did not differ, even though control males found 15% HFD females unattractive. This result suggests that HFD does not necessarily affect neural development to alter male discrimination ability, but that HFD may affect neural function post-development instead. This possibility could be evaluated by examining the activity of pheromone-responsive neurons in HFD males compared to control males. In control males, these neurons would be expected to have lower activity when stimulated by unattractive pheromone profiles from HFD females, whereas in HFD males similar activity would be expected in response to both control and HFD profiles (Schultzhaus, 2018).

    Males appear to be more resistant to dietary lipid effects than females, as only changes in male mate perception occurred at lower fat doses, while a multitude of behavioral defects were seen in females treated with the same dose. Sex-specific physiology and responses to environmental factors are well documented in D. melanogaster. For example, females live and withstand starvation longer, and exercise only benefits male flies. While the 3% developmental and 15% adult-only HFD treatments decreased female attractiveness, these same treatments had no effect on male attractiveness. Male attractiveness was affected only when males were fed 30% adult-only diet. Both competitive and non-competitive assays revealed that 30% males were less attractive to control females. However, no differences in pheromone levels were identified in HFD males, and while song analyses did not identify a significant effect of male HFD on courtship song performance, there was a trend toward HFD males performing less pulse song than control diet males. Therefore, it is possible that a song that is slightly poorer in quality due to diet has some influence on how the female perceives the male (Schultzhaus, 2018).

    Understanding of how HFD affects D. melanogaster mating behavior centers on the idea that HFD alters fly physiology and health, which affects sexual traits, leading to changes in mating behavior. This argument would be strengthened if blocking the HFD effects on fly health rescues the effects of HFD on mating behavior and attractiveness. Upon exposure to HFD in wild-type flies, insulin and TOR signaling initially increase. Genetic manipulations that either block this initial induction of the insulin/TOR pathway or cause overexpression of a fat lipase, Brummer (Bmm), rescue health defects caused by HFD. Insulin signaling results in the repression of foxo expression and de-represses TOR signaling by deactivating TOR inhibitors. Overexpression of FOXO and a dominant-negative version of TOR (TORDN) therefore serve to decrease the downstream components of insulin signaling. Increased Bmm expression leads to increased breakdown of stored lipids, negating the effects caused by lipid accumulation. Genetic manipulations of conserved metabolic signaling pathways (insulin, TOR, and Brummer) have been shown to rescue certain HFD physiological defects, such as heart dysfunction, lipid accumulation, and insulin resistance, and female pheromone profiles and attractiveness are also regulated by insulin and TOR signaling. This study performed the same genetic manipulations on both developmental and adult-only HFD treatments, using the same Gal4 driver, to determine whether HFD effects on behavior and attractiveness could be rescued. No developmental HFD treatment defects were rescued, which may not be surprising as only adult-specific defects were examined and rescued in a previous study. Yet when evaluating adult-specific dietary effects on behavior, this study found that while female attractiveness was not rescued by any genetic manipulation, FOXO and Brummer overexpression rescued male discrimination ability. TOR-DN expression did not alter male phenotypes, possibly because TOR signaling induces a separate genetic cascade than FOXO signaling that may not be involved in male mate discrimination. These results provide evidence that repression of insulin signaling effects by overexpression of FOXO, which is inhibited by insulin signaling, and blocking fat accumulation by overexpression of Brummer, a fat lipase, can rescue male health sufficiently to rescue male-specific HFD behavioral defects but not female-specific effects. However, these results do not eliminate the possibility that conserved metabolic signaling pathways are involved in mediating the female response to HFD. HFD affects females more strongly than males, and the female-specific effects may be too severe to rescue with these genetic manipulations. Even though certain physiological defects caused by HFD can be rescued, female activity levels were not, and whether these manipulations rescue other defects, such as mortality, has not yet been examined. It is possible that the driver used in these studies (armadillo-Gal4) does not provide high enough expression to rescue drastic physiological defects caused by HFD that lead to the observed alterations in females (Schultzhaus, 2018).

    In conclusion, this study shows that HFD affects D. melanogaster behavioral interactions through alteration of both visual and non-visual sexual traits, and that females are more susceptible to defects caused by HFD. The less severe effects of HFD on males can be rescued by genetic manipulation of conserved metabolic signaling pathways. Further characterization of HFD impacts on D. melanogaster behavior could advance the understanding of how genetic and environmental factors interact to affect animal health and sexual selection, drawing together two often disparate fields of research to provide insights into the fluidity of sexual selection (Schultzhaus, 2018).

    The role of cuticular hydrocarbons in mate recognition in Drosophila suzukii

    Cuticular hydrocarbons (CHCs) play a central role in the chemical communication of many insects. In Drosophila suzukii, an economically important pest insect, very little is known about chemical communication and the possible role of CHCs. This study identified 60 CHCs of Drosophila suzukii and studied their changes in function of age (maturation), sex and interactions with the opposite sex. Age (maturation) was identified is the key factor driving changes in the CHC profiles. The effect on courtship behaviour and mating of six CHCs, five of which were positively associated with maturation and one negatively. The results of these experiments demonstrate that four of the major CHC peaks with a chain length of 23 carbons, namely 9-tricosene (9-C23:1), 7-tricosene (7-C23:1), 5-tricosene (5-C23:1) and tricosane (n-C23), negatively regulated courtship and mating, even though all these compounds were characteristic for sexually mature flies. The study then goes on to show that this effect on courtship and mating is likely due to the disruption of the natural ratios in which these hydrocarbons occur in Drosophila suzukii. Overall, these results provide key insights into the cuticular hydrocarbon signals that play a role in D. suzukii mate recognition (Snellings, 2018).

    Evolution of a central neural circuit underlies Drosophila mate preferences

    Courtship rituals serve to reinforce reproductive barriers between closely related species. Drosophila melanogaster and Drosophila simulans exhibit reproductive isolation, owing in part to the fact that D. melanogaster females produce 7,11-heptacosadiene, a pheromone that promotes courtship in D. melanogaster males but suppresses courtship in D. simulans males. This study compared pheromone-processing pathways in D. melanogaster and D. simulans males to define how these sister species endow 7,11-heptacosadiene with the opposite behavioural valence to underlie species discrimination. Males of both species were shown to detect 7,11-heptacosadiene using homologous peripheral sensory neurons, but this signal is differentially propagated to P1 neurons, which control courtship behaviour. A change in the balance of excitation and inhibition onto courtship-promoting neurons transforms an excitatory pheromonal cue in D. melanogaster into an inhibitory cue in D. simulans. These results reveal how species-specific pheromone responses can emerge from conservation of peripheral detection mechanisms and diversification of central circuitry, and demonstrate how flexible nodes in neural circuits can contribute to behavioural evolution (Seeholzer, 2018).

    The sensory periphery has been proposed to be the most evolutionarily labile element of the nervous system, as changes in the expression or tuning of sensory receptors can allow for the emergence of species-specific behaviours without necessitating potentially more complex developmental rewiring of central pathways in the brain. By contrast, the current results suggest that species-specific behavioural responses to 7,11-HD arise through functional alterations in how pheromone signals are propagated through a structurally conserved central circuit that consists of parallel excitatory and feedforward inhibitory branches. By reweighting the balance of excitatory vAB3 and inhibitory mAL signalling to P1 neurons, 7,11-HD is transformed from an excitatory signal that promotes courtship in D. melanogaster males into an inhibitory signal that suppresses courtship in D. simulans males. Although the analysis highlights the importance of this pheromone pathway in shaping species-specific mate preferences, the possibility cannot be excluded that additional inputs to P1 neurons or other targets of mAL and vAB3 neurons also contribute to divergent courtship decisions. Nevertheless, the data suggest that the branched architecture of pheromone-processing pathways serves as a substrate for the evolution of mate preferences, pointing to the potential existence of favourable sites within neural circuits to instantiate adaptive behavioural changes, analogous to how specific nodes within developmental regulatory networks contribute to morphological diversity (Seeholzer, 2018).

    The conserved tuning of Ppk23+ neurons suggests that D. simulans males dedicate this sensory pathway to the detection of a female pheromones, rather than sensing the chemical cues carried by conspecific females. Consistent with this idea, although P1 neurons are sufficient to elicit courtship in D. simulans males, they are not excited by the taste of a D. simulans female. Given that D. simulans cuticular pheromones are sexually monomorphic and offer ambiguous signals for mate recognition, males probably rely on additional sensory inputs for their arousal. Indeed, the fervent courtship exhibited by D. simulans ppk23 mutants towards females of different species demonstrates that D. simulans males can be aroused in the absence of any species-specific excitatory cue. Dienes, such as 7,11-HD, actively suppress this arousal, presumably through recruitment of strong mAL-mediated inhibition via Ppk23+ pathways. Together, these observations reinforce the notion that pheromone communication in Drosophila serves to focus a male's desire, such that flies lacking cuticular pheromones can be inherently attractive and appropriate mate choices are honed by specific inhibitory chemical cues (Seeholzer, 2018).

    Peripheral adaptations are likely to play an important role in the evolution of novel chemical sensitivities. In this case, however, preserving the sensory periphery while varying central circuits provides a mechanism to alter the behavioural valence of a single chemical cue. As D. melanogaster and D. simulans diverged, their reproductive isolation was probably strengthened by the ability of both species to detect the same pheromone but assign it a different meaning through these central circuit modifications. Notably, in D. melanogaster, P1 neuron excitability is regulated by the social history of a male. This suggests that both experience-dependent and evolutionary adaptations may act on the same neural substrate to modify sensory integration and mate choices, similar to how phenotypic plasticity may facilitate morphological evolution35. Therefore, functional reweighting of sensory inputs at flexible nodes in the nervous system, shaped by evolutionary selection or individual experience, may allow for alternative behavioural responses to the same sensory signal (Seeholzer, 2018).

    Pre-imaginal conditioning alters adult sex pheromone response in Drosophila

    Pheromones are chemical signals that induce innate responses in individuals of the same species that may vary with physiological and developmental state. In Drosophila melanogaster, the most intensively studied pheromone is 11-cis-vaccenyl acetate (cVA), which is synthezised in the male ejaculatory bulb and is transferred to the female during copulation. Among other effects, cVA inhibits male courtship of mated females. This study found that male courtship inhibition depends on the amount of cVA and this effect is reduced in male flies derived from eggs covered with low to zero levels of cVA. This effect is not observed if the eggs are washed, or if the eggs are laid several days after copulation. This suggests that courtship suppression involves a form of pre-imaginal conditioning, which is shown to occur during the early larval stage. The conditioning effect could not be rescued by synthetic cVA, indicating that it largely depends on conditioning by cVA and other maternally-transmitted factor(s). These experiments suggest that one of the primary behavioral effects of cVA is more plastic and less stereotypical than had hitherto been realised (Everaerts, 2018).

    Olfactory landmark-based communication in interacting Drosophila

    To communicate with conspecifics, animals deploy various strategies to release pheromones, chemical signals modulating social and sexual behaviors. Importantly, a single pheromone induces different behaviors depending on the context and exposure dynamics. Therefore, to comprehend the ethological role of pheromones, it is essential to characterize how neurons in the recipients respond to temporally and spatially fluctuating chemical signals emitted by donors during natural interactions. In Drosophila melanogaster, the male pheromone 11-cis-vaccenyl acetate (cVA) activates specific olfactory receptor neurons (ORNs) to regulate diverse social and sexual behaviors in recipients. Physicochemical analyses have identified this chemical on an animal's body and in its local environment. However, because these methods are imprecise in capturing spatiotemporal dynamics, it is poorly understood how individual pheromone cues are released, detected, and interpreted by recipients. This study developed a system based on bioluminescence to monitor neural activity in freely interacting Drosophila, and investigated the active detection and perception of the naturally emitted cVA. Unexpectedly, neurons specifically tuned to cVA did not exhibit significant activity during physical interactions between males, and instead responded strongly to olfactory landmarks deposited by males. These landmarks mediated attraction through Or67d receptors and allured both sexes to the marked region. Importantly, the landmarks remained attractive even when a pair of flies was engaged in courtship behavior. In contrast, female deposits did not affect the exploration pattern of either sex. Thus, Drosophila use pheromone marking to remotely signal their sexual identity and to enhance social interactions (Mercier, 2018).

    A novel sex difference in Drosophila contact chemosensory neurons unveiled using single cell labeling

    Among the sensory modalities involved in controlling mating behavior in Drosophila melanogaster, contact sex pheromones play a primary role. The key receptor neurons for contact sex pheromones are located on the forelegs, which are activated in males upon touching the female abdomen during tapping events in courtship actions. A fruitless (fru)-positive (fru [+]) male-pheromone sensing cell (M-cell) and a fru [+] female-pheromone sensing cell (F-cell) are paired in a sensory bristle on the legs, and some fru [+] chemoreceptor axons project across the midline in the thoracic neuromere in males but not in females. However, the receptor cells that form sexually dimorphic axon terminals in the thoracic ganglia remain unknown. By generating labeled single-cell clones, this study shows that only a specific subset of fru [+] chemosensory neurons have axons that cross the midline in males. It was further demonstrated that there exist two male-specific bristles, each harboring two chemosensory neurons; neither of which exhibits midline crossing, a masculine characteristic. This study reveals hitherto unrecognized sex differences in chemosensory neurons, imposing a reinvestigation of the pheromone input pathways that impinge on the central courtship circuit (Kimura, 2018).

    The desaturase1 gene affects reproduction before, during and after copulation in Drosophila melanogaster

    Desaturase1 (desat1) is one of the few genes known to be involved in the two complementary aspects of sensory communication - signal emission and signal reception - in Drosophila melanogaster. In particular, desat1 is necessary for the biosynthesis of major cuticular pheromones in both males and females. It is also involved in the male ability to discriminate sex pheromones. Each of these two sensory communication aspects depends on distinct desat1 putative regulatory regions. This study used (i) mutant alleles resulting from the insertion/excision of a transposable genomic element inserted in a desat1 regulatory region, and (ii) transgenics made with desat1 regulatory regions used to target desat1 RNAi. These genetic variants were used to study several reproduction-related phenotypes. In particular, this study investigated the fecundity of various mutant and transgenic desat1 females with regard to the developmental fate of their progeny. The mating performance in pairs of flies was compared with altered desat1 expression in various desat1-expressing tissues together with their inability to disengage at the end of copulation. Moreover, the developmental origin of altered sex pheromone discrimination was investigated in male flies. Attempts were made to map some of the tissues involved in these reproduction-related phenotypes. Given that desat1 is expressed in many brain neurons and in non-neuronal tissues required for varied aspects of reproduction, the data suggest that the regulation of this gene has evolved to allow the optimal reproduction and a successful adaptation to varied environments in this cosmopolitan species (Nojima, 2019).

    Ovipositor extrusion promotes the transition from courtship to copulation and signals female acceptance in Drosophila melanogaster

    Communication between male and female fruit flies during courtship is essential for successful mating, but, as with many other species, it is the female who decides whether to mate. This study shows a novel role for ovipositor extrusion in promoting male copulation attempts in virgin and mated females and signaling acceptance in virgins. It is first shown that ovipositor extrusion is only displayed by sexually mature females, exclusively during courtship and in response to the male song. A pair of descending neurons was identified that controls ovipositor extrusion in mated females. Genetic silencing of the descending neurons shows that ovipositor extrusion stimulates the male to attempt copulation. A detailed behavioral analysis revealed that during courtship, the male repeatedly licks the female genitalia, independently of ovipositor extrusion, and that licking an extruded ovipositor prompts a copulation attempt. However, if the ovipositor is not subsequently retracted, copulation is prevented, as it happens with mated females. This study reveals a dual function of the ovipositor: while its extrusion is necessary for initiating copulation by the male, its retraction signals female acceptance. This study thus uncovers the significance of the communication between male and female that initiates the transition from courtship to copulation (Mezzera, 2020).

    Courtship behaviors allow animals to interact and assess their qualities before they commit to reproduction. In fruit flies, as with many animal species, the male decides whether to court, and the female decides whether or not to mate with the courting male. Although this decision is crucial for reproduction, little is known on how the female communicates that she is receptive to copulation. Understanding the communication between males and females leading to copulation will shed light on this decision process. During courtship, the male performs a series of behaviors directed at the female consisting of tapping, following, vibrating a wing to generate the courtship song, quivering the abdomen, and licking. At some point, the male attempts copulation. How the transition from courtship to copulation attempt is reached has not been explored. During the male display, the female exhibits behaviors that can be interpreted as mild rejections, possibly to increase sampling time. These behaviors are walking away or jumping, kicking, fending, wing fluttering, and curling. In some Drosophila species, female behavioral displays associated with copulation have been identified. Such examples are observed in species in which, unlike melanogaster, the male courts in front of the female. Such signals include spreading the wings outward and upward or opening the labellum and 'kissing' the male's extended labellum. In Drosophila melanogaster, a few behaviors, such as partial ovipositor extrusion, abdominal preening, and droplet emission, have been associated with receptivity. In addition, a progressive reduction in walking speed and increase in pausing have been observed before mating. However, a conspicuous female acceptance signal has not been identified yet. The best candidate is ovipositor extrusion, since it is reported to occur before copulation. However, ovipositor extrusion is also thought to block copulation and repel the male. During ovipositor extrusion, the female pushes her vaginal plates posteriorly, which results in their spread and the formation of a temporary tube-like structure protruding from the tip of the abdomen. It is thought that the extruded member is the ovipositor. Flies extrude the ovipositor to different extents. These are referred as partial ovipositor extrusion (also called vaginal plate spreading) and full ovipositor extrusion, even though there is a continuum between the two as opposed to a sharp distinction. It is likely that the different extents of extrusion underlie the conflicting reports that ovipositor extrusion (1) predicts copulation, (2) blocks copulation, and (3) repels the male. Without the ability to manipulate the magnitude and dynamics of ovipositor extrusion, it has been difficult to establish its role in a conclusive manner (Mezzera, 2020).

    Recently, a collection of GAL4 driver lines that provide genetic access to subsets of descending neurons, which have their cell bodies in the brain and project to the ventral nerve cord (VNC, analog of the vertebrate spinal cord), has opened the possibility to address this issue. In this collection, a pair of descending neurons was identified that command full ovipositor extrusion, allowing manipulation of their activity and thus control the female's behavior. This study dissected the male-female interaction by manipulating the female's behavior. Ovipositor extrusion is shown to entice the male to attempt copulation, independently of whether it is a full or a partial extrusion. The crucial difference between ovipositor extrusion of virgin and mated females is that virgin females retract the ovipositor upon a copulation attempt, thus allowing copulation, whereas mated females do not, thus blocking copulation. Moreover, by removing female hearing or the male's ability to sing, ovipositor extrusion is displayed in response to song perception. On the male side, the male repeatedly probes the female genitalia with the proboscis (licking) independently of ovipositor extrusion display. It was then shown that it is the coincidence of ovipositor extrusion and licking that stimulates copulation attempt. Together, these findings identify a sequence of steps in the male-female communication during courtship, where courtship song leads to ovipositor extrusion and licking the extruded ovipositor prompts copulation attempt, which may, if the female is receptive, lead to copulation (Mezzera, 2020).

    These results shed light on the transition from courtship to copulation, which represents a dramatic behavioral switch from the appetitive to the consummatory stage of sexual behavior with consequences in the reproductive success of the animals (Mezzera, 2020).

    This work has shown that in fruit flies, male licking and female ovipositor extrusion are involved in this transition. An important feature of this interaction is that the female mating status determines whether this transition is complete. It was observed that virgin females retract the ovipositor upon a male's attempt, thus allowing copulation, whereas mated females do not, thus blocking copulation (Mezzera, 2020).

    Virgin and mated females use a variation of the same behavior to stimulate and prevent copulation, respectively. This variation requires different descending neurons probably acting on common circuits. This could be a versatile and economic strategy to mediate opposite responses when the same individual uses one or the other variation depending on the circumstances (Mezzera, 2020).

    Why would a mated female signal the male to attempt copulation while blocking intromission? In circumstances that were not addressed in this study, mated females re-mate. For a few hours after mating, and given the appropriate context, mated females will eject the sperm and re-mate in an attempt to increase fecundity and offspring genetic diversity. In this case, prompting the male to attempt copulation makes sense, as it could lead to copulation. An additional role for full ovipositor extrusion in mated females, which was not explored in this study, may be in announcing the female's current pheromonal composition. Ovipositor extrusion would be an efficient way of exposing the anti-aphrodisiac pheromones 3-O-acetyl-1,3-dihydroxyoctacosa-11,19-diene present in the tip of the ovipositor and cis-vaccenyl acetate, mostly in the reproductive system, which, together with 7-tricosene present in the cuticle, indicate to an approaching male that the female has mated, and, depending on the combination and intensity of the chemical cues, the male may or may not initiate courtship (Mezzera, 2020).

    This work has identified a pair of descending neurons that control full ovipositor extrusion. Full ovipositor extrusion can be induced in the virgin female by DNp13 activation, but activity in these neurons is not necessary for ovipositor extrusion in virgins. Although full and partial ovipositor extrusions do not have a sharp distinction, ovipositor extrusion is commanded by different neurons and controlled differently in virgin and mated flies. It remains to be elucidated which are the descending neurons controlling virgin ovipositor extrusion and how they interact with DNp13 to control similar behavior in females in different mating states (Mezzera, 2020).

    This work shows that, during the interaction between the sexes, the female responds to the male courtship song with ovipositor extrusion. However, it is apparent in the videos that song does not always lead to ovipositor extrusion. This response pattern suggests that ovipositor extrusion is not a reflexive reaction to courtship song, but rather arises from a temporal or multimodal integration. Further experiments are required to elucidate the nature of this association. How does the male verify that the song was heard? The results indicate that the male is sampling the female genitalia with the proboscis throughout courtship. Presumably, licking is intended to probe the chemical landscape of the female genitalia, which is likely to change when the ovipositor is extruded; in this way, the male could sense when the female is responding to the song. This study shows that licking of the ovipositor elicits a copulation attempt. In line with early suggestions that compounds are released during ovipositor extrusion to stimulate the male, it is speculated that a chemical compound is presented with the ovipositor by the female and sensed by taste neurons on the male proboscis. A gustatory signal, yet unidentified and common to virgin and mated females, would stimulate the male to attempt copulation. Having established that licking an extruded ovipositor is the starting point for the male to attempt copulation allows the same starting point to be used to study how the transition from courtship to copulation is processed in the male brain (Mezzera, 2020).

    In conclusion, this work highlights how both sexes contribute to continuous communication during courtship that culminates in copulation attempt gated by the female ovipositor extrusion. Moreover, findings open new avenues of study of the neuronal regulation of behaviors that lead to the transition from courtship to copulation and how this transition regulates neuronal activity (Mezzera, 2020).

    Social context enhances hormonal modulation of pheromone detection in Drosophila

    Critical to evolutionary fitness, animals regulate social behaviors by integrating signals from both their external environments and internal states. This study found that population density modulates the courtship behavior of male Drosophila melanogaster in an age-dependent manner. In a competitive mating assay, males reared in a social environment have a marked advantage in courting females when pitted against males reared in isolation. Group housing promotes courtship in mature (7-day) but not immature (2-day) males; this behavioral plasticity requires the Or47b pheromone receptor. Using single-sensillum recordings, this study found that group housing increases the response of Or47b olfactory receptor neurons (ORNs) only in mature males. The effect of group housing on olfactory response and behavior can be mimicked by chronically exposing single-housed males to an Or47b ligand. At the molecular level, group housing elevates Ca(2+) levels in Or47b ORNs, likely leading to CaMKI-mediated activation of the histone-acetyl transferase CBP. This signaling event in turn enhances the efficacy of juvenile hormone, an age-related regulator of reproductive maturation in flies. Furthermore, the male-specific Fruitless isoform (Fru(M)) is required for the sensory plasticity, suggesting that Fru(M) functions as a downstream genomic coincidence detector in Or47b ORNs-integrating reproductive maturity, signaled by juvenile hormone, and population density, signaled by CBP. In all, this study has identify a neural substrate and activity-dependent mechanism by which social context can directly influence pheromone sensitivity, thereby modulating social behavior according to animals' life-history stage (Sethi, 2019).

    The genetics of male pheromone preference difference between Drosophila melanogaster and Drosophila simulans

    Species of flies in the genus Drosophila differ dramatically in their preferences for mates, but little is known about the genetic or neurological underpinnings of this evolution. Recent advances have been made to understanding of one case: pheromone preference evolution between the species D. melanogaster and D. simulans. Males of both species are very sensitive to the pheromone 7,11-HD that is present only on the cuticle of female D. melanogaster. In one species this cue activates courtship, and in the other it represses it. This change in valence was recently shown to result from the modification of central processing neurons, rather than changes in peripherally expressed receptors, but nothing is known about the genetic changes that are responsible. This study shows that a 1.35 Mb locus on the X chromosome has a major effect on male 7,11-HD preference. Unfortunately, when this locus is divided, the effect is largely lost. Instead this study attempted to filter the 159 genes within this region using newfound understanding of the neuronal underpinnings of this phenotype to identify and test candidate genes. The results of these tests are presented, and the difficulty of identifying the genetic architecture of behavioral traits and the potential of connecting these genetic changes to the neuronal modifications that elicit different behaviors is discussed (Shahandeh, 2019).

    Mate discrimination among subspecies through a conserved olfactory pathway

    Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, this study identified two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. These results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner (Khallaf, 2020).

    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 (Lin, 2016).

    Mating increases Drosophila melanogaster females' choosiness by reducing olfactory sensitivity to a male pheromone

    Females that are highly selective when choosing a mate run the risk of remaining unmated or delaying commencing reproduction. Therefore, low female choosiness would be beneficial when males are rare but it would be maladaptive if males become more frequent. How can females resolve this issue? Polyandry would allow mating-status-dependent choosiness, with virgin females selecting their first mate with little selectivity and becoming choosier thereafter. This plasticity in choosiness would ensure timely acquisition of sperm and enable females to increase offspring quality during later mating. This study shows that Drosophila melanogaster females display such mating-status-dependent choosiness by becoming more selective once mated and identifies the underlying neurohormonal mechanism. Mating releases juvenile hormone, which desensitizes Or47b olfactory neurons to a pheromone produced by males, resulting in increased preference for pheromone-rich males. Besides providing a mechanism to a long-standing evolutionary prediction, these data suggest that intersexual selection in D. melanogaster, and possibly in all polyandrous, sperm-storing species, is mainly the domain of mated females since virgin females are less selective. Juvenile hormone influences behaviour by changing cue responsiveness across insects; the neurohormonal modulation of olfactory neurons uncovered in D. melanogaster provides an explicit mechanism for how this hormone modulates behavioural plasticity (Kohlmeier, 2021).

    Large-scale characterization of sex pheromone communication systems in Drosophila

    Insects use sex pheromones as a reproductive isolating mechanism to attract conspecifics and repel heterospecifics. Despite the profound knowledge of sex pheromones, little is known about the coevolutionary mechanisms and constraints on their production and detection. Using whole-genome sequences to infer the kinship among 99 drosophilids, this study investigated how phylogenetic and chemical traits have interacted at a wide evolutionary timescale. Through a series of chemical syntheses and electrophysiological recordings, this study identified 52 sex-specific compounds, many of which are detected via olfaction. Behavioral analyses reveal that many of the 43 male-specific compounds are transferred to the female during copulation and mediate female receptivity and/or male courtship inhibition. Measurement of phylogenetic signals demonstrates that sex pheromones and their cognate olfactory channels evolve rapidly and independently over evolutionary time to guarantee efficient intra- and inter-specific communication systems. These results show how sexual isolation barriers between species can be reinforced by species-specific olfactory signals (Khallaf, 2021).

    The female sex pheromone (Z)-4-undecenal mediates flight attraction and courtship in Drosophila melanogaster

    Specific mate communication and recognition underlies reproduction and hence speciation. This study provides new insights in Drosophila melanogaster premating olfactory communication. Mate communication evolves during adaptation to ecological niches and makes use of social signals and habitat cues. Female-produced, species-specific volatile pheromone (Z)-4-undecenal (Z4-11Al) and male pheromone (Z)-11-octadecenyl acetate (cVA) interact with food odour in a sex-specific manner. Furthermore, Z4-11Al, which mediates upwind flight attraction in both sexes, also elicits courtship in experienced males. Two isoforms of the olfactory receptor Or69a are co-expressed in the same olfactory sensory neurons. Z4-11Al is perceived via Or69aB, while the food odorant (R)-linalool is a main ligand for the other variant, Or69aA. However, only Z4-11Al mediates courtship in experienced males, not (R)-linalool. Behavioural discrimination is reflected by calcium imaging of the antennal lobe, showing distinct glomerular activation patterns by these two compounds. Male sex pheromone cVA is known to affect male and female courtship at close range, but does not elicit upwind flight attraction as a single compound, in contrast to Z4-11Al. A blend of the food odour vinegar and cVA attracted females, while a blend of vinegar and female pheromone Z4-11Al attracted males, instead. Sex-specific upwind flight attraction to blends of food volatiles and male and female pheromone, respectively, adds a new element to Drosophila olfactory premating communication and is an unambiguous paradigm for identifying the behaviourally active components, towards a more complete concept of food-pheromone odour objects (Borrero-Echeverry, 2022).

    "Scent of a fruit fly": Cuticular chemoprofiles after mating in differently fed Drosophila melanogaster (Diptera: Drosophilidae) strains

    In the world of complex smells in natural environment, feeding and mating represent two important olfactory-guided behaviors in Drosophila melanogaster (Diptera: Drosophilidae). Diet affects the chemoprofile composition of the individuals, which, indirectly, may significantly affect their mating success. In this study, chemoprofiles of recently mated flies belonging to four D. melanogaster strains, which were fed for many generations on different substrates (standard cornmeal-S strain; banana-B strain; carrot-C strain; tomato-T strain) were identified and quantified. In total, 67 chemical compounds were identified: 48 compounds were extracted from males maintained on banana and carrot, and 47 compounds from males maintained on cornmeal and tomato substrates, while total of 60 compounds were identified in females from all strains. The strains and the sexes significantly differed in qualitative nature of their chemoprofiles after mating. Significant differences in the relative amount of three major male pheromones (cis-vaccenyl acetate-cVA, (Z)-7-pentacosene, and (Z)-7-tricosene) and in female pheromone (Z,Z)-7,11-nonacosadiene among strains were also recorded. Furthermore, multivariate analysis of variance (MANOVA) pointed to significant differences between virgin and mated individuals of all strains and within both sexes. Differences in some of the well known sex pheromones were also identified when comparing their relative amount before and after mating. The presence of typical male pheromones in females, and vice versa may indicate their bidirectional transfer during copulation. These results confirm significant effect of mating status on cuticular hydrocarbon (CHC) phenotypes in differently fed D. melanogaster flies (Pavkovic-Lucic, 2020).

    A pleiotropic chemoreceptor facilitates the production and perception of mating pheromones

    Optimal mating decisions depend on the robust coupling of signal production and perception because independent changes in either could carry a fitness cost. However, since the perception and production of mating signals are often mediated by different tissues and cell types, the mechanisms that drive and maintain their coupling remain unknown for most animal species.This study shows that in Drosophila, behavioral responses to, and the production of, a putative inhibitory mating pheromone are co-regulated by Gr8a, a member of the Gustatory receptor gene family. Specifically, through behavioral and pheromonal data, this study found that Gr8a independently regulates the behavioral responses of males and females to a putative inhibitory pheromone, as well as its production in the fat body and oenocytes of males. Overall, these findings provide a relatively simple molecular explanation for how pleiotropic receptors maintain robust mating signaling systems at the population and species levels (Vernier, 2023).

    Male size does not affect the strength of male mate choice for high-quality females in Drosophila melanogaster

    Theory predicts that the strength of male mate choice should vary depending on male quality when higher-quality males receive greater fitness benefits from being choosy. This pattern extends to differences in male body size, with larger males often having stronger pre- and post-copulatory preferences than smaller males. This study sought to determine whether large males and small males differ in the strength (or direction) of their preference for large, high-fecundity females using the fruit fly, Drosophila melanogaster. Male courtship preferences and mating duration were measured to show that male body size had no impact on the strength of male mate choice; all males, regardless of their size, had equally strong preferences for large females. To understand the selective pressures shaping male mate choice in males of different sizes, the fitness benefits associated with preferring large females for both large and small males were also measured. Male body size did not affect the benefits that males received: large and small males were equally successful at mating with large females, received the same direct fitness benefits from mating with large females, and showed similar competitive fertilization success with large females. These findings provide insight into why the strength of male mate choice was not affected by male body size in this system. This study highlights the importance of evaluating the benefits and costs of male mate choice across multiple males to predict when differences in male mate choice should occur (Lev, 2023).

    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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