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Most living organisms use pheromones for inter-individual communication. In Drosophila melanogaster flies, several pheromones perceived either by contact/at a short distance (cuticular hydrocarbons, CHs), or at a longer distance (cis-vaccenyl acetate, cVA), affect courtship and mating behaviours. However, it has not previously been possible to precisely identify all potential pheromonal compounds and simultaneously monitor their variation on a time scale. To overcome this limitation, we combined Solid Phase Micro-Extraction with gas-chromatography coupled with mass-spectrometry. This allowed us (i) to identify 59 cuticular compounds, including 17 new CHs; (ii) to precisely quantify the amount of each compound that could be detected by another fly, and (iii) to measure the variation of these substances as a function of aging and mating. Sex-specific variation appeared with age, while mating affected cuticular compounds in both sexes with three possible patterns: variation was (i) reciprocal in the two sexes, suggesting a passive mechanical transfer during mating, (ii) parallel in both sexes, such as for cVA which strikingly appeared during mating, or (iii) unilateral, presumably as a result of sexual interaction. We provide a complete reassessment of all Drosophila CHs and suggest that the chemical conversation between male and female flies is far more complex than is generally accepted. We conclude that focusing on individual compounds will not provide a satisfactory understanding of the evolution and function of chemical communication in Drosophila.

Summary

Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. Here we show that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the male’s strategy to suppress reproduction by competing males.

Pheromonal communication is crucial with regard to mate choice in many animals including insects. Drosophila melanogaster flies produce a pheromonal bouquet with many cuticular hydrocarbons some of which diverge between the sexes and differently affect male courtship behavior. Cuticular pheromones have a relatively high weight and are thought to be — mostly but not only — detected by gustatory contact. However, the response of the peripheral and central gustatory systems to these substances remains poorly explored. We measured the effect induced by pheromonal cuticular mixtures on (i) the electrophysiological response of peripheral gustatory receptor neurons, (ii) the calcium variation in brain centers receiving these gustatory inputs and (iii) the behavioral reaction induced in control males and in mutant desat1 males, which show abnormal pheromone production and perception. While male and female pheromones induced inhibitory-like effects on taste receptor neurons, the contact of male pheromones on male fore-tarsi elicits a long-lasting response of higher intensity in the dedicated gustatory brain center. We found that the behavior of control males was more strongly inhibited by male pheromones than by female pheromones, but this difference disappeared in anosmic males. Mutant desat1 males showed an increased sensitivity of their peripheral gustatory neurons to contact pheromones and a behavioral incapacity to discriminate sex pheromones. Together our data indicate that cuticular hydrocarbons induce long-lasting inhibitory effects on the relevant taste pathway which may interact with the olfactory pathway to modulate pheromonal perception.

Pheromones regulate male social behaviors in Drosophila, but the identities and behavioral role(s) of these chemosensory signals, and how they interact, are incompletely understood. Here we show that (Z)-7-tricosene (7-T), a male-enriched cuticular hydrocarbon (CH) previously shown to inhibit male-male courtship, is also essential for normal levels of aggression. The opposite influences of 7-T on aggression and courtship are independent, but both require the gustatory receptor Gr32a. Surprisingly, sensitivity to 7-T is required for the aggression-promoting effect of 11-cis-vaccenyl acetate (cVA), an olfactory pheromone, but 7-T sensitivity is independent of cVA. 7-T and cVA therefore regulate aggression in a hierarchical manner. Furthermore, the increased courtship caused by depletion of male CHs is suppressed by a mutation in the olfactory receptor Or47b. Thus, male social behaviors are controlled by gustatory pheromones that promote and suppress aggression and courtship, respectively, and whose influences are dominant to olfactory pheromones that enhance these behaviors.

Summary

Background

In many insect species, cuticular hydrocarbons serve as pheromones that can mediate complex social behaviors. In Drosophila melanogaster, several hydrocarbons including the male sex pheromone 11-cis-vaccenyl acetate (cVA) and female-specific 7,11-dienes influence courtship behavior and can function as cues for short-term memory associated with the mating experience. Behavioral and physiological studies suggest that other unidentified chemical communication cues are likely to exist. To more fully characterize the hydrocarbon profile of the D. melanogaster cuticle, we applied direct ultraviolet laser desorption/ionization orthogonal time-of-flight mass spectrometry (UV-LDI-o-TOF MS) and analyzed the surface of intact fruit flies at a spatial resolution of approximately 200 μm.

Results

We report the chemical and spatial characterization of 28 species of cuticular hydrocarbons, including a new major class of oxygen-containing compounds. Using UV-LDI MS, pheromones previously shown to be expressed exclusively by one sex, e.g. cVA, 7,11-heptacosadiene, and 7,11-nonacosadiene, appear to be found on both male and female flies. In males, cVA co-localizes at the tip of the ejaculatory bulb with a second acetylated hydrocarbon named CH503. We describe the chemical structure of CH503 as 3-O-acetyl-1,3-dihydroxy-octacosa-11,19-diene and show one behavioral role for this compound as a long-lived inhibitor of male courtship. Like cVA, CH503 is transferred from males to females during mating. Unlike cVA, CH503 remains on the surface of females for at least 10 days.

Conclusions

Oxygenated hydrocarbons comprise one major previously undescribed class of compounds on the Drosophila cuticular surface. In addition to cVA, a newly-discovered long chain acetate, CH503, serves as a mediator of courtship-related chemical communication.

Aggression is regulated by pheromones in many animal species1,2,3. However in no system have aggression pheromones, their cognate receptors and corresponding sensory neurons been identified. Here we show that 11-cis-vaccenyl acetate (cVA), a male-specific volatile pheromone, robustly promotes male-male aggression in the vinegar fly Drosophila melanogaster. The aggression-promoting effect of synthetic cVA requires olfactory sensory neurons (OSNs) expressing the receptor Or67d4,5,6, as well as the receptor itself. Activation of Or67d-expressing OSNs, either by genetic manipulation of their excitability or by exposure to male pheromones in the absence of other classes of OSNs, is sufficient to promote aggression. High densities of male flies can promote aggression through release of volatile cVA. In turn, cVA-promoted aggression can promote male fly dispersal from a food resource, in a manner dependent upon Or67d-expressing OSNs. These data suggest that cVA may mediate negative feedback control of male population density, through its effect on aggression. Identification of a pheromone-OSN pair controlling aggression in a genetic organism opens the way to unraveling the neurobiology of this evolutionarily conserved behavior.

Sexual behavior requires animals to distinguish between the sexes and to respond appropriately to each of them. In Drosophila melanogaster, as in many insects, cuticular hydrocarbons are thought to be involved in sex recognition and in mating behavior, but there is no direct neuronal evidence of their pheromonal effect. Using behavioral and electrophysiological measures of responses to natural and synthetic compounds, we show that Z-7-tricosene, a Drosophila male cuticular hydrocarbon, acts as a sex pheromone and inhibits male-male courtship. These data provide the first direct demonstration that an insect cuticular hydrocarbon is detected as a sex pheromone. Intriguingly, we show that a particular type of gustatory neurons of the labial palps respond both to Z-7-tricosene and to bitter stimuli. Cross-adaptation between Z-7-tricosene and bitter stimuli further indicates that these two very different substances are processed by the same neural pathways. Furthermore, the two substances induced similar behavioral responses both in courtship and feeding tests. We conclude that the inhibitory pheromone tastes bitter to the fly.

Rapid evolution of gene expression patterns responsible for pheromone production in 24 species of Drosophila was mapped to simple mutations within the regulatory domain of the desatF gene.

A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We show that across the genus Drosophila, the expression of this enzyme is correlated with long-chain diene production and has undergone an extraordinary number of evolutionary transitions, including six independent gene inactivations, three losses of expression without gene loss, and two transitions in sex-specificity. Furthermore, we show that evolutionary transitions from monomorphism to dimorphism (and its reversion) in desatF expression involved the gain (and the inactivation) of a binding-site for the sex-determination transcription factor, DOUBLESEX. In addition, we documented a surprising example of the gain of particular cis-regulatory motifs of the desatF locus via a set of small deletions. Together, our results suggest that frequent changes in the expression of pheromone-producing enzymes underlie evolutionary transitions in chemical communication, and reflect changing regimes of sexual selection, which may have contributed to speciation among Drosophila.

Author Summary

Mate selection is a complex process involving communication between potential partners on many levels, such as visual, aural, and olfactory cues. Many animals use chemical signals in the form of pheromones to communicate and correctly recognize individuals of the appropriate species and sex during reproduction. Evolutionary changes in the production of these chemicals have been suggested to contribute to speciation. Yet, the molecular mechanisms governing these transitions have seldom been addressed. Here, we show that expression of the gene desatF, which encodes an enzyme involved in the production of the Drosophila pheromones known as dienes, is highly variable and rapidly evolving across Drosophila species. Changes in desatF gene expression correlate with changes in sex- and species-specific production of dienes. Further, these changes in diene production can be explained by simple modifications in the regulatory regions of the desatF gene, providing a molecular level understanding of the evolution of pheromone production in Drosophila.

The molecular and cellular events mediating complex behaviors in animals are largely unknown. Elucidating the circuits underlying behaviors in simple model systems may shed light on how these circuits function. In Drosophila, courtship behavior provides a tractable model for studying the underlying basis of innate behavior. The male-specific pheromone 11-cis-vaccenyl acetate (cVA) modulates courtship behavior and is detected by T1 neurons, located on the antenna of male and female flies. The T1 neurons express the odorant receptor Or67d, and are exquisitely tuned to cVA pheromone. However, cVA-induced changes in mating behavior have also been reported upon manipulation of olfactory neurons expressing odorant receptor Or65a. These findings raise the issue of whether multiple olfactory-driven circuits underlie cVA-induced behavioral responses, and what role these circuits play in behavior. Here, we engineered flies in which the Or67d circuit is specifically activated in the absence of cVA in order to determine the role of this circuit in behavior. We created transgenic flies that express a dominant-active, pheromone-independent variant of the extracellular pheromone receptor, LUSH. We found that, similar to the behaviors elicited by cVA, engineered male flies have dramatically reduced courtship, while engineered females showed enhanced courtship. Furthermore, cVA exposure did not enhance the dominant LUSH-triggered effects on behavior in the engineered flies. Finally, we show the effects of both cVA and dominant LUSH on courtship are reversed by genetically removing Or67d. These findings demonstrate that the T1/Or67d circuit is necessary and sufficient to mediate sexually dimorphic courtship behaviors.

We have established a new simple behavioral paradigm in Drosophila melanogaster to determine how genes and the environment influence the behavior of flies within a social group. Specifically, we measure social space as the distance between two flies. The majority of Canton-s flies, regardless of their gender, are within two-body lengths from each other. Their social experience affects this behavior, with social isolation reducing and mating enhancing social space respectively, in both males and females. Unlike several other social behaviors in the fly, including the formation of social groups themselves (a well described behavior - Bartelt et al., 1985, De Gee et al., 2008, Lof et al., 2008, Lof et al., 2009), social space does not require the perception of the previously identified aggregation pheromone cis-Vaccenyl Acetate (Bartelt et al., 1985). Conversely, performance of the assay in darkness or mutations in the eye pigmentation gene white increased social space. Our results establish a new assay for the genetic dissection of a fundamental mode of social interaction.

Sex pheromones are chemical signals frequently required for mate choice, but their reciprocal role on mate preference has rarely been shown in both sexes. In Drosophila melanogaster flies, the predominant cuticular hydrocarbons (CHs) are sexually dimorphic: only females produce 7,11-dienes, whereas 7-tricosene (7-T) is the principal male CH. Males generally prefer females with 7,11-dienes, but the role of 7-T on female behaviour remains unclear. With perfumed males, control females mated faster and more often with males carrying increased levels of 7-T showing that this CH acts as a chemical stimulant for D. melanogaster females. Control females—but not antenna-less females—could detect small variation of 7-T. Finally, our finding that desat1 mutant female showed altered response towards 7-T provides an additional role for this gene which affects the production and the perception of pheromones involved in mate choice, in both sexes.

Detection of specific female pheromones stimulates courtship behavior in Drosophila melanogaster males, but the chemosensory molecules, cells and mechanisms involved remain poorly understood. Here we show that ppk25, a DEG/ENaC ion channel subunit required for normal male response to females, is expressed at highest levels in a single sexually dimorphic gustatory neuron of most taste hairs on legs and wings, but not in neurons that detect courtship-inhibiting pheromones or food. Synaptic inactivation of ppk25-expressing neurons, or knockdown of ppk25 expression in all gustatory neurons significantly impairs male response to females, whereas gustatory expression of ppk25 rescues the courtship behavior of ppk25 mutant males. Remarkably, the only other detectable albeit significantly weaker expression of ppk25 occurs in olfactory neurons implicated in modulation of courtship behavior. However, expression of ppk25 in olfactory neurons is not required for male courtship under our experimental conditions. These data show that ppk25 functions specifically in peripheral taste neurons involved in activation of courtship behavior, an unexpected function for this type of channel. Furthermore, our work identifies a small subset of gustatory neurons with an essential role in activation of male courtship behavior, most likely in response to female pheromones.

Summary

Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila [1]. We systematically examine one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, the trichoid sensilla, by electrophysiological analysis. We find that none respond strongly to food odors, but all respond to fly odors. Two subtypes of trichoid sensilla contain ORNs that respond to cis-vaccenyl acetate (cVA), an anti-aphrodisiac pheromone present in males and transferred to females during mating [2–4]. All trichoid sensilla yield responses to a male extract; a subset yield responses to a virgin female extract as well. Thus males can be distinguished from virgin females by the activity they elicit among the trichoid ORN population. We then systematically test all members of the Odor receptor (Or) gene family [5–7] that are expressed in trichoid sensilla [8], using an in vivo expression system [9]. Four receptors respond to fly odors in this system: two respond to extracts of both males and virgin females, and two respond to cVA. We propose a model for how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic.

Summary

Insects such as the fruit fly, Drosophila melanogaster, rely on contact chemosensation to detect nutrient-rich foods, to avoid consuming toxic chemicals, and to select mates and hospitable zones to deposit eggs. Flies sense tastants and non-volatile pheromones through gustatory bristles and pegs distributed on multiple body parts including the proboscis, wing margins, legs and ovipositor. The sensilla house gustatory receptor neurons, which express members of the family of 68 gustatory receptors (GRs). In contrast to mammalian chemosensation or Drosophila olfaction, which are initiated by receptors composed of dimers of one or two receptor types, the functional Drosophila GRs may include three or more subunits. Several GRs appear to be expressed in multiple cell types that are not associated with contact chemosensation raising the possibility that these proteins may have roles that extend beyond the detection of tastants and pheromones.

In the fruitfly, Drosophila melanogaster, mate choice during courtship depends on detecting olfactory cues, sex pheromones, which are initially processed in the antennal lobe (AL), a primary olfactory centre of the brain. However, no sexual differences in the structure of the AL have been found in Drosophila. We compared the central brain anatomy of 37 species of Drosophilidae from the islands of the Hawaiian archipelago, uncovering an extreme sexual dimorphism within the AL in which two out of the 51 identifiable glomeruli were markedly enlarged in males. A phylogeny indicated that the sexual dimorphism of the homologous glomeruli arose 0.4-1.9 Myr ago independently in two species groups of Hawaiian endemic Drosophilidae. The corresponding glomeruli in D. melanogaster were also found to be sexually dimorphic. The formation of glomeruli of male size is prevented by the ectopic expression of female-type transformer (tra) cDNA in males, indicating that the glomerular sexual dimorphism is under the control of the sex-determination cascade of genes. It is suggested that a defined set of glomeruli in Drosophila can enlarge in response to sex-determination genetic signals, the mutations of which may result in species differences in sexual dimorphism of the brain.

Background

Insects respond to the spatial and temporal dynamics of a pheromone plume, which implies not only a strong response to 'odor on', but also to 'odor off'. This requires mechanisms geared toward a fast signal termination. Several mechanisms may contribute to signal termination, among which odorant-degrading enzymes. These enzymes putatively play a role in signal dynamics by a rapid inactivation of odorants in the vicinity of the sensory receptors, although direct in vivo experimental evidences are lacking. Here we verified the role of an extracellular carboxylesterase, esterase-6 (Est-6), in the sensory physiological and behavioral dynamics of Drosophila melanogaster response to its pheromone, cis-vaccenyl acetate (cVA). Est-6 was previously linked to post-mating effects in the reproductive system of females. As Est-6 is also known to hydrolyze cVA in vitro and is expressed in the main olfactory organ, the antenna, we tested here its role in olfaction as a putative odorant-degrading enzyme.

Results

We first confirm that Est-6 is highly expressed in olfactory sensilla, including cVA-sensitive sensilla, and we show that expression is likely associated with non-neuronal cells. Our electrophysiological approaches show that the dynamics of olfactory receptor neuron (ORN) responses is strongly influenced by Est-6, as in Est-6° null mutants (lacking the Est-6 gene) cVA-sensitive ORN showed increased firing rate and prolonged activity in response to cVA. Est-6° mutant males had a lower threshold of behavioral response to cVA, as revealed by the analysis of two cVA-induced behaviors. In particular, mutant males exhibited a strong decrease of male-male courtship, in association with a delay in courtship initiation.

Conclusions

Our study presents evidence that Est-6 plays a role in the physiological and behavioral dynamics of sex pheromone response in Drosophila males and supports a role of Est-6 as an odorant-degrading enzyme (ODE) in male antennae. Our results also expand the role of Est-6 in Drosophila biology, from reproduction to olfaction, and highlight the role of ODEs in insect olfaction.

The evolution of communication is a fundamental biological problem. The genetic control of the signal and its reception must be tightly coadapted, especially in inter-individual sexual communication. However, there is very little experimental evidence for tight genetic linkage connecting the emission of a signal and its reception. We found that a single genomic transposon inserted in the desat1 gene of Drosophila melanogaster simultaneously affected the emission and the perception of sex-specific signals. This mutation greatly decreased the production of unsaturated hydrocarbons on the cuticle of mature flies of both sexes. These substances represent the sex pheromones necessary for mate discrimination: control males could not discriminate the sex of mutant desat1 flies. Moreover, mutant males were unable to discriminate the sex pheromones of control flies. Expression of desat1 was found in the peripheral tissues that produce and detect sex pheromones. Excision of the transposon rescued both the production and discrimination phenotypes, but the two effects did not always coincide. This indicates that the emission and perception of pheromones are coded by different products of the same gene, reflecting the pleiotropic activity of desat1.

Background

Females in a wide range of taxa have been shown to base their choice of mates on pheromone signals. However, little research has focussed specifically on the form and intensity of selection that mate choice imposes on the pheromone signal. Using multivariate selection analysis, we characterise directly the form and intensity of sexual selection acting on cuticular hydrocarbons, chemical compounds widely used in the selection of mates in insects. Using the Australian field cricket Teleogryllus oceanicus as a model organism, we use three measures of male attractiveness to estimate fitness; mating success, the duration of courtship required to elicit copulation, and subsequent spermatophore attachment duration.

Results

We found that all three measures of male attractiveness generated sexual selection on male cuticular hydrocarbons, however there were differences in the form and intensity of selection among these three measures. Mating success was the only measure of attractiveness that imposed both univariate linear and quadratic selection on cuticular hydrocarbons. Although we found that all three attractiveness measures generated nonlinear selection, again only mating success was found to exert statistically significant stabilizing selection.

Conclusion

This study shows that sexual selection plays an important role in the evolution of male cuticular hydrocarbon signals.

Sexual selection has the potential to contribute to population divergence and speciation. Most studies of sexual selection in Drosophila have concentrated on a single signaling modality, usually either courtship song or cuticular hydrocarbons (CHCs), which can act as contact pheromones. We have examined the relationship between both signal types and reproductive success using F1–3 offspring of wild-collected flies, raised in the lab. We used two populations of the Holarctic species Drosophila montana that represent different phylogeographic clades that have been separate for ca. 0.5 million years (MY), and differ to some extent in both traits. Here, we characterize the nature and identify the targets of sexual selection on song, CHCs, and both traits combined within the populations. Three measures of courtship outcome were used as fitness proxies. They were the probability of mating, mating latency, and the production of rejection song by females, and showed patterns of association with different traits that included both linear and quadratic selection. Courtship song predicted courtship outcome better than CHCs and the signal modalities acted in an additive rather than synergistic manner. Selection was generally consistent in direction and strength between the two populations and favored males that sang more vigorously. Sexual selection differed in the extent, strength, and nature on some of the traits between populations. However, the differences in the directionality of selection detected were not a good predictor of population differences. In addition, a character previously shown to be important for species recognition, interpulse interval, was found to be under sexual selection. Our results highlight the complexity of understanding the relationship between within-population sexual selection and population differences. Sexual selection alone cannot predict differences between populations.

Mechanisms for identifying appropriate mating partners are required for any species to survive. In many types of animals, males employ multiple sensory modalities to initially search for females and to subsequently determine if they are fit and/or receptive. In this paper we will detail the multiple types of sensory information that are used to initiate and drive courtship in Drosophila melanogaster and discuss the importance of context in the interpretation of chemosensory cues. We find that food-related olfactory cues increase the salience of the aversive pheromone cis-vaccenyl acetate.

Background

In Drosophila, cuticular sex pheromones are long-chain unsaturated hydrocarbons synthesized from fatty acid precursors in epidermal cells called oenocytes. The species D. melanogaster shows sex pheromone dimorphism, with high levels of monoenes in males, and of dienes in females. Some biosynthesis enzymes are expressed both in fat body and oenocytes, rendering it difficult to estimate the exact role of oenocytes and of the transport of fatty acids from fat body to oenocytes in pheromone elaboration. To address this question, we RNAi silenced two main genes of the biosynthesis pathway, desat1 and desatF, in the oenocytes of D. melanogaster, without modifying their fat body expression.

Results

Inactivation of desat1 in oenocytes resulted in a 96% and 78% decrease in unsaturated hydrocarbons in males and females, respectively. Female pheromones (dienes) showed a decrease of 90%. Inactivation of desatF, which is female-specific and responsible for diene formation, resulted in a dramatic loss of pheromones (-98%) paralleled with a two-fold increase in monoenes. Courtship parameters (especially courtship latency) from wild-type males were more affected by desat1 knocked-down females (courtship latency increased by four fold) than by desatF knocked-down ones (+65% of courtship latency).

The number of transcripts in oenocytes was estimated at 0.32 and 0.49 attomole/μg for desat1 in males and females, respectively, about half of the total transcripts in a fly. There were only 0.06 attomole/μg desatF transcripts in females, all located in the oenocytes.

Conclusion

Knock-down results for desat1 suggest that there must be very little transport of unsaturated precursors from fat body to the oenocytes, so pheromone synthesis occurs almost entirely through the action of biosynthesis enzymes within the oenocytes. Courtship experiments allow us to discuss the behavioral role of diene pheromones, which, under special conditions, could be replaced by monoenes in D. melanogaster. A possible explanation is given of how pheromones could have evolved in species such as D. simulans, which only synthesize monoenes.

Drosophila Cuticular Hydrocarbons (CH) influence courtship behaviour, mating, aggregation, oviposition, and resistance to desiccation. We measured levels of 24 different CH compounds of individual male D. melanogaster hourly under a variety of environmental (LD/DD) conditions. Using a model-based analysis of CH variation, we developed an improved normalization method for CH data, and show that CH compounds have reproducible cyclic within-day temporal patterns of expression which differ between LD and DD conditions. Multivariate clustering of expression patterns identified 5 clusters of co-expressed compounds with common chemical characteristics. Turnover rate estimates suggest CH production may be a significant metabolic cost. Male cuticular hydrocarbon expression is a dynamic trait influenced by light and time of day; since abundant hydrocarbons affect male sexual behavior, males may present different pheromonal profiles at different times and under different conditions.

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, we 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.

SUMMARY

Detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly non-volatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones [1–6]. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor [7–9], the functions of GRs involved in feeding are unknown. Here, we show that the Gr64 genes encode receptors for numerous sugars. We generated a fly strain that contained a deletion for all six Gr64 genes (ΔGr64) and showed that these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar [8, 9]. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, we present evidence that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for co-expression of multiple sugar receptors in the same taste neurons.

Most animals rely on olfaction to find sexual partners, food or a habitat. The olfactory system faces the challenge of extracting meaningful information from a noisy odorous environment. In most moth species, males respond to sex pheromone emitted by females in an environment with abundant plant volatiles. Plant odours could either facilitate the localization of females (females calling on host plants), mask the female pheromone or they could be neutral without any effect on the pheromone. Here we studied how mixtures of a behaviourally-attractive floral odour, heptanal, and the sex pheromone are encoded at different levels of the olfactory pathway in males of the noctuid moth Agrotis ipsilon. In addition, we asked how interactions between the two odorants change as a function of the males' mating status. We investigated mixture detection in both the pheromone-specific and in the general odorant pathway. We used a) recordings from individual sensilla to study responses of olfactory receptor neurons, b) in vivo calcium imaging with a bath-applied dye to characterize the global input response in the primary olfactory centre, the antennal lobe and c) intracellular recordings of antennal lobe output neurons, projection neurons, in virgin and newly-mated males. Our results show that heptanal reduces pheromone sensitivity at the peripheral and central olfactory level independently of the mating status. Contrarily, heptanal-responding olfactory receptor neurons are not influenced by pheromone in a mixture, although some post-mating modulation occurs at the input of the sexually isomorphic ordinary glomeruli, where general odours are processed within the antennal lobe. The results are discussed in the context of mate localization.