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1.  Drosophila Pheromone-Sensing Neurons Expressing the ppk25 Ion Channel Subunit Stimulate Male Courtship and Female Receptivity 
PLoS Genetics  2014;10(3):e1004238.
As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25 is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating courtship and mating.
Author Summary
Drosophila mating behaviors serve as an attractive model to understand how external sensory cues are detected and used to generate appropriate behavioral responses. Pheromones present on the cuticle of Drosophila have important roles in stimulating male courtship toward females and inhibiting male courtship directed at other males. Recently, stimulatory pheromones emitted by females and inhibitory pheromones emitted by males have been shown to stimulate distinct subsets of gustatory neurons on the legs. We have previously shown that a DEG/ENaC ion channel subunit, ppk25, is involved in male courtship toward females but not in inhibition of male-male courtship. Here we show that ppk25 is specifically expressed and functions in a subset of gustatory neurons that mediate physiological and behavioral responses to female-specific stimulatory pheromones. Furthermore, ppk25 is also required for the function of those neurons to activate male courtship in response to other pheromones that are not female-specific. In addition to their roles in males, we find that ppk25, and the related DEG/ENaC subunits ppk23 and ppk29, also stimulate female mating behavior. In conclusion, these results show that, in both sexes, ppk25 functions in a group of neurons with a specialized role in stimulating mating behaviors.
doi:10.1371/journal.pgen.1004238
PMCID: PMC3967927  PMID: 24675786
2.  A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in Drosophila 
BMC Biology  2012;10:56.
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.
doi:10.1186/1741-7007-10-56
PMCID: PMC3414785  PMID: 22715942
carboxylesterase; esterase 6; olfaction; pheromone; signal termination
3.  Pheromonal and Behavioral Cues Trigger Male-to-Female Aggression in Drosophila 
PLoS Biology  2010;8(11):e1000541.
By genetically manipulating both pheromonal profiles and behavioral patterns, we find that Drosophila males showed a complete reversal in their patterns of aggression towards other males and females
Appropriate displays of aggression rely on the ability to recognize potential competitors. As in most species, Drosophila males fight with other males and do not attack females. In insects, sex recognition is strongly dependent on chemosensory communication, mediated by cuticular hydrocarbons acting as pheromones. While the roles of chemical and other sensory cues in stimulating male to female courtship have been well characterized in Drosophila, the signals that elicit aggression remain unclear. Here we show that when female pheromones or behavior are masculinized, males recognize females as competitors and switch from courtship to aggression. To masculinize female pheromones, a transgene carrying dsRNA for the sex determination factor transformer (traIR) was targeted to the pheromone producing cells, the oenocytes. Shortly after copulation males attacked these females, indicating that pheromonal cues can override other sensory cues. Surprisingly, masculinization of female behavior by targeting traIR to the nervous system in an otherwise normal female also was sufficient to trigger male aggression. Simultaneous masculinization of both pheromones and behavior induced a complete switch in the normal male response to a female. Control males now fought rather than copulated with these females. In a reciprocal experiment, feminization of the oenocytes and nervous system in males by expression of transformer (traF) elicited high levels of courtship and little or no aggression from control males. Finally, when confronted with flies devoid of pheromones, control males attacked male but not female opponents, suggesting that aggression is not a default behavior in the absence of pheromonal cues. Thus, our results show that masculinization of either pheromones or behavior in females is sufficient to trigger male-to-female aggression. Moreover, by manipulating both the pheromonal profile and the fighting patterns displayed by the opponent, male behavioral responses towards males and females can be completely reversed. Therefore, both pheromonal and behavioral cues are used by Drosophila males in recognizing a conspecific as a competitor.
Author Summary
As in other species, the fruit fly Drosophila melanogaster uses chemical signals in the form of pheromones to recognize the species and sex of another individual. Males typically fight with other males and do not attack females. While the roles of pheromonal and other sensory cues in stimulating courtship towards females have been extensively studied, the signals that elicit aggression towards other males remain unclear. In this work, we use genetic tools to show that masculinization of female pheromones is sufficient to trigger aggression from wild type males towards females. Surprisingly, males also attacked females that displayed male patterns of aggression, even if they show normal female pheromonal profiles, indicating that pheromones are not the only cues important for identifying another animal as an opponent. By simultaneously manipulating pheromones and behavioral patterns of opponents, we can completely switch the behavioral response of males towards females and males. These results demonstrate that not only pheromonal but also behavioral cues can serve as triggers of aggression, underlining the importance of behavioral feedback in the manifestation of social behaviors.
doi:10.1371/journal.pbio.1000541
PMCID: PMC2990703  PMID: 21124886
4.  Volatile Drosophila Cuticular Pheromones Are Affected by Social but Not Sexual Experience 
PLoS ONE  2012;7(7):e40396.
Recognition of conspecifics and mates is based on a variety of sensory cues that are specific to the species, sex and social status of each individual. The courtship and mating activity of Drosophila melanogaster flies is thought to depend on the olfactory perception of a male-specific volatile pheromone, cis-vaccenyl acetate (cVA), and the gustatory perception of cuticular hydrocarbons (CHs), some of which are sexually dimorphic. Using two complementary sampling methods (headspace Solid Phase Micro-Extraction [SPME] and solvent extraction) coupled with GC-MS analysis, we measured the dispersion of pheromonal CHs in the air and on the substrate around the fly. We also followed the variations in CHs that were induced by social and sexual interactions. We found that all CHs present on the fly body were deposited as a thin layer on the substrate, whereas only a few of these molecules were also detected in the air. Moreover, social experience during early adult development and in mature flies strongly affected male volatile CHs but not cVA, whereas sexual interaction only had a moderate influence on dispersed CHs. Our study suggests that, in addition to their role as contact cues, CHs can influence fly behavior at a distance and that volatile, deposited and body pheromonal CHs participate in a three-step recognition of the chemical identity and social status of insects.
doi:10.1371/journal.pone.0040396
PMCID: PMC3394786  PMID: 22808151
5.  Activation of the T1 Neuronal Circuit is Necessary and Sufficient to Induce Sexually Dimorphic Mating Behavior 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.
doi:10.1523/JNEUROSCI.4819-09.2010
PMCID: PMC3426441  PMID: 20164344
lush; Or67d; cVA; courtship; pheromone; olfaction
6.  Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate 
Current biology : CB  2007;17(7):599-605.
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.
doi:10.1016/j.cub.2007.01.053
PMCID: PMC1913718  PMID: 17363250
Learning and memory; olfaction; Drosophila; pheromones; cis-vaccenyl acetate
7.  A new male sex-pheromone and novel cuticular cues for chemical communication in Drosophila 
Current biology : CB  2009;19(15):1245-1254.
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.
doi:10.1016/j.cub.2009.06.037
PMCID: PMC2726907  PMID: 19615904
8.  Peripheral, Central and Behavioral Responses to the Cuticular Pheromone Bouquet in Drosophila melanogaster Males 
PLoS ONE  2011;6(5):e19770.
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.
doi:10.1371/journal.pone.0019770
PMCID: PMC3098836  PMID: 21625481
9.  miR-124 controls male reproductive success in Drosophila 
eLife  2013;2:e00640.
Many aspects of social behavior are controlled by sex-specific pheromones. Gender-appropriate production of the sexually dimorphic transcription factors doublesex and fruitless controls sexual differentiation and sexual behavior. miR-124 mutant males exhibited increased male–male courtship and reduced reproductive success with females. Females showed a strong preference for wild-type males over miR-124 mutant males when given a choice of mates. These effects were traced to aberrant pheromone production. We identified the sex-specific splicing factor transformer as a functionally significant target of miR-124 in this context, suggesting a role for miR-124 in the control of male sexual differentiation and behavior, by limiting inappropriate expression of the female form of transformer. miR-124 is required to ensure fidelity of gender-appropriate pheromone production in males. Use of a microRNA provides a secondary means of controlling the cascade of sex-specific splicing events that controls sexual differentiation in Drosophila.
DOI: http://dx.doi.org/10.7554/eLife.00640.001
eLife digest
Like many animals, the fruit fly Drosophila uses pheromones to influence sexual behaviour, with males and females producing different versions of these chemicals. One of the pheromones produced by male flies, for example, is a chemical called 11-cis-vaccenyl-acetate (cVA), which is an aphrodisiac for female flies and an anti-aphrodisiac for males.
The production of the correct pheromones in each sex is genetically controlled using a process called splicing that allows a single gene to be expressed as two or more different proteins. A variety of proteins called splicing factors ensures that splicing results in the production of the correct pheromones for each sex. Sometimes, however, the process by which sex genes are expressed as proteins can be ‘leaky’, which results in the wrong proteins being produced for one or both sexes.
Small RNA molecules called microRNAs act in some genetic pathways to limit the leaky expression of genes, and a microRNA called miR-124 carries out this function in the developing brain Drosophila. Now, Weng et al. show that miR-124 also helps to regulate sex-specific splicing and thereby to control pheromone production and sexual behaviour.
Mutant male flies lacking miR-124 were less successful than wild-type males at mating with female flies, and were almost always rejected if a female fly was given a choice between a mutant male and a wild-type male. Moreover, both wild-type and mutant male flies were more likely to initiate courtship behaviour towards another male if it lacked miR-124 than if it did not.
The mutant male flies produced less cVA than wild-type males, but more of other pheromones called pentacosenes, which is consistent with the observed behaviour because cVA attracts females and repels males, whereas pentacosenes act as aphrodisiacs for male flies in large amounts. Weng et al. showed that these changes in the production of pheromones were caused by an increased expression of the female version of a splicing factor called transformer in the mutant males, but further work is needed to understand this process in detail.
DOI: http://dx.doi.org/10.7554/eLife.00640.002
doi:10.7554/eLife.00640
PMCID: PMC3679528  PMID: 23795292
microRNA; pheromome; behaviour; genetics; selection; evolution; D. melanogaster
10.  Identification of an aggression-promoting pheromone and its receptor neurons in Drosophila 
Nature  2009;463(7278):227-231.
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.
doi:10.1038/nature08678
PMCID: PMC2999963  PMID: 19966787
11.  Octopamine Neuromodulation Regulates Gr32a-Linked Aggression and Courtship Pathways in Drosophila Males 
PLoS Genetics  2014;10(5):e1004356.
Chemosensory pheromonal information regulates aggression and reproduction in many species, but how pheromonal signals are transduced to reliably produce behavior is not well understood. Here we demonstrate that the pheromonal signals detected by Gr32a-expressing chemosensory neurons to enhance male aggression are filtered through octopamine (OA, invertebrate equivalent of norepinephrine) neurons. Using behavioral assays, we find males lacking both octopamine and Gr32a gustatory receptors exhibit parallel delays in the onset of aggression and reductions in aggression. Physiological and anatomical experiments identify Gr32a to octopamine neuron synaptic and functional connections in the suboesophageal ganglion. Refining the Gr32a-expressing population indicates that mouth Gr32a neurons promote male aggression and form synaptic contacts with OA neurons. By restricting the monoamine neuron target population, we show that three previously identified OA-FruM neurons involved in behavioral choice are among the Gr32a-OA connections. Our findings demonstrate that octopaminergic neuromodulatory neurons function as early as a second-order step in this chemosensory-driven male social behavior pathway.
Author Summary
To mate or fight? When meeting other members of their species, male fruit flies must determine whether a second fly is male or female and proceed with the appropriate behavioral patterns. The taste receptor, Gr32a, has been reported to respond to chemical messages (pheromones) that are important for gender recognition, as eliminating Gr32a function impairs both male courtship and aggressive behavior. Here we demonstrate that different subsets of Gr32a-expressing neuron populations mediate these mutually exclusive behaviors and the male Gr32a-mediated behavioral response is amplified through neurons that contain the neuromodulator octopamine (OA, an invertebrate equivalent of norepinephrine). Gr32a-expressing neurons connect functionally and synaptically with distinct OA neurons indicating these amine neurons may function as early as a second-order step in a chemosensory-driven circuit. Our results contribute to understanding how an organism selects an appropriate behavioral response upon receiving external sensory signals.
doi:10.1371/journal.pgen.1004356
PMCID: PMC4031044  PMID: 24852170
12.  The smell of love in Drosophila 
Odors are key sensory signals for social communication and food search in animals including insects. Drosophila melanogaster, is a powerful neurogenetic model commonly used to reveal molecular and cellular mechanisms involved in odorant detection. Males use olfaction together with other sensory modalities to find their mates. Here, we review known olfactory signals, their related olfactory receptors, and the corresponding neuronal architecture impacting courtship. OR67d receptor detects 11-cis-Vaccenyl Acetate (cVA), a male specific pheromone transferred to the female during copulation. Transferred cVA is able to reduce female attractiveness for other males after mating, and is also suspected to decrease male-male courtship. cVA can also serve as an aggregation signal, maybe through another OR. OR47b was shown to be activated by fly odors, and to enhance courtship depending on taste pheromones. IR84a detects phenylacetic acid (PAA) and phenylacetaldehyde (PA). These two odors are not pheromones produced by flies, but are present in various fly food sources. PAA enhances male courtship, acting as a food aphrodisiac. Drosophila males have thus developed complementary olfactory strategies to help them to select their mates.
doi:10.3389/fphys.2013.00072
PMCID: PMC3617446  PMID: 23576993
courtship; Drosophila; olfaction; receptor; nervous system
13.  Courtship Initiation Is Stimulated by Acoustic Signals in Drosophila melanogaster 
PLoS ONE  2008;3(9):e3246.
Finding a mating partner is a critical task for many organisms. It is in the interest of males to employ multiple sensory modalities to search for females. In Drosophila melanogaster, vision is thought to be the most important courtship stimulating cue at long distance, while chemosensory cues are used at relatively short distance. In this report, we show that when visual cues are not available, sounds produced by the female allow the male to detect her presence in a large arena. When the target female was artificially immobilized, the male spent a prolonged time searching before starting courtship. This delay in courtship initiation was completely rescued by playing either white noise or recorded fly movement sounds to the male, indicating that the acoustic and/or seismic stimulus produced by movement stimulates courtship initiation, most likely by increasing the general arousal state of the male. Mutant males expressing tetanus toxin (TNT) under the control of Gr68a-GAL4 had a defect in finding active females and a delay in courtship initiation in a large arena, but not in a small arena. Gr68a-GAL4 was found to be expressed pleiotropically not only in putative gustatory pheromone receptor neurons but also in mechanosensory neurons, suggesting that Gr68a-positive mechanosensory neurons, not gustatory neurons, provide motion detection necessary for courtship initiation. TNT/Gr68a males were capable of discriminating the copulation status and age of target females in courtship conditioning, indicating that female discrimination and formation of olfactory courtship memory are independent of the Gr68a-expressing neurons that subserve gustation and mechanosensation. This study suggests for the first time that mechanical signals generated by a female fly have a prominent effect on males' courtship in the dark and leads the way to studying how multimodal sensory information and arousal are integrated in behavioral decision making.
doi:10.1371/journal.pone.0003246
PMCID: PMC2531232  PMID: 18802468
14.  A DROSOPHILA DEG/ENaC SUBUNIT FUNCTIONS SPECIFICALLY IN GUSTATORY NEURONS REQUIRED FOR MALE COURTSHIP BEHAVIOR 
The Journal of Neuroscience  2012;32(13):4665-4674.
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.
doi:10.1523/JNEUROSCI.6178-11.2012
PMCID: PMC3324785  PMID: 22457513
15.  An Inhibitory Sex Pheromone Tastes Bitter for Drosophila Males 
PLoS ONE  2007;2(8):e661.
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.
doi:10.1371/journal.pone.0000661
PMCID: PMC1937024  PMID: 17710124
16.  A Drosophila gustatory receptor essential for aversive taste and inhibiting male-to-male courtship 
Current biology : CB  2009;19(19):1623-1627.
SUMMARY
Contact chemosensation is required for several behaviors that promote the survival of fruit flies and other insects. These include evasive behaviors such as the suppression of feeding on dangerous repellent compounds, referred to as antifeedants. Contact chemosensation also contributes to the suppression of male-male courtship. However, the identities of the gustatory receptors (GRs) required for the responses to non-volatile avoidance chemicals are largely unknown. Exceptions include GR66a and GR93a, which are required specifically to prevent ingestion of caffeine [1, 2], and GR32a, which is necessary for inhibiting male-male courtship [3]. However, GR32a is dispensable for normal taste. Thus, distinct GRs may function in the sensing of avoidance pheromones and in detecting different subsets of antifeedants. Here, we describe the requirements for GR33a, which is expressed widely in gustatory receptor neurons (GRNs) that respond to aversive chemicals. Gr33a mutant flies were impaired in the avoidance responses to all non-volatile repellents tested, ranging from quinine to denatorium, lobeline and caffeine. Gr33a mutant males also displayed increased male-to-male courtship implying that it functioned in the detection of a repulsive male pheromone. In contrast to the broadly required olfactory receptor (OR) OR83b, which is essential for trafficking of other ORs [4], GR66a and GR93a were localized normally in Gr33a mutant GRNs. Thus, rather than regulating trafficking of GRs, GR33a may function as co-receptor required for sensing all non-volatile repulsive chemicals, including tastants and pheromones.
doi:10.1016/j.cub.2009.07.061
PMCID: PMC2762023  PMID: 19765987
17.  Feminization of pheromone-sensing neurons affects mating decisions in Drosophila males 
Biology Open  2014;3(2):152-160.
Summary
The response of individual animals to mating signals depends on the sexual identity of the individual and the genetics of the mating targets, which represent the mating social context (social environment). However, how social signals are sensed and integrated during mating decisions remains a mystery. One of the models for understanding mating behaviors in molecular and cellular terms is the male courtship ritual in the fruit fly (Drosophila melanogaster). We have recently shown that a subset of gustatory receptor neurons (GRNs) that are enriched in the male appendages and express the ion channel ppk23 play a major role in the initiation and maintenance of male courtship via the perception of cuticular contact pheromones, and are likely to represent the main chemosensory pathway that influences mating decisions by males. Here we show that genetic feminization of ppk23-expressing GRNs in male flies resulted in a significant increase in male–male sexual attraction without an apparent impact on sexual attraction to females. Furthermore, we show that this increase in male–male sexual attraction is sensory specific, which can be modulated by variable social contexts. Finally, we show that feminization of ppk23-expressing sensory neurons lead to major transcriptional shifts, which may explain the altered interpretation of the social environment by feminized males. Together, these data indicate that the sexual cellular identity of pheromone sensing GRNs plays a major role in how individual flies interpret their social environment in the context of mating decisions.
doi:10.1242/bio.20147369
PMCID: PMC3925318  PMID: 24463366
Fruit fly; Courtship; ppk23; Poxn; transformer; DEG/ENaC
18.  Drosophila Cuticular Hydrocarbons Revisited: Mating Status Alters Cuticular Profiles 
PLoS ONE  2010;5(3):e9607.
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.
doi:10.1371/journal.pone.0009607
PMCID: PMC2834761  PMID: 20231905
19.  Rapid Evolution of Sex Pheromone-Producing Enzyme Expression in Drosophila 
PLoS Biology  2009;7(8):e1000168.
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.
doi:10.1371/journal.pbio.1000168
PMCID: PMC2711336  PMID: 19652700
20.  Two Drosophila DEG/ENaC channel subunits have distinct functions in gustatory neurons that activate male courtship 
Trimeric sodium channels of the DEG/ENaC family have important roles in neurons, but the specific functions of different subunits present in heteromeric channels are poorly understood. We previously reported that the Drosophila DEG/ENaC subunit Ppk25 is essential in a small subset of gustatory neurons for activation of male courtship behavior, likely through detection of female pheromones. Here we show that, like mutations in ppk25, mutations in another Drosophila DEG/ENaC subunit, nope, specifically impair male courtship of females. nope regulatory sequences drive reporter gene expression in gustatory neurons of the labellum, wings and legs, including all gustatory neurons in which ppk25 function is required for male courtship of females. In addition, gustatory-specific knockdown of nope impairs male courtship. Further, the impaired courtship response of nope mutant males to females is rescued by targeted expression of nope in the subset of gustatory neurons in which ppk25 functions. However, nope and ppk25 have non-redundant functions, as targeted expression of ppk25 does not compensate for the lack of nope and vice versa. Moreover, Nope and Ppk25 form specific complexes when co-expressed in cultured cells. Together, these data indicate that the Nope and Ppk25 subunits have specific, non-redundant functions in a subset of gustatory neurons required for activation of male courtship in response to females, and suggest the hypothesis that Nope and Ppk25 function as subunits of a heteromeric DEG/ENaC channel required for gustatory detection of female pheromones.
doi:10.1523/JNEUROSCI.1376-12.2012
PMCID: PMC3460529  PMID: 22915128
21.  Caste-Specific Expression Patterns of Immune Response and Chemosensory Related Genes in the Leaf-Cutting Ant, Atta vollenweideri 
PLoS ONE  2013;8(11):e81518.
Leaf-cutting ants are evolutionary derived social insects with elaborated division of labor and tremendous colony sizes with millions of workers. Their social organization is mainly based on olfactory communication using different pheromones and is promoted by a pronounced size-polymorphism of workers that perform different tasks within the colony. The size polymorphism and associated behaviors are correlated to distinct antennal lobe (AL) phenotypes. Two worker phenotypes differ in number of olfactory glomeruli in the AL and the presence or absence of an extremely large glomerulus (macroglomerulus), involved in trail-pheromone reception. The males' AL contains three macroglomeruli which are presumably involved in detection of sex-pheromone components. We investigated the antennal transcriptome data of all major castes (males, queens and workers) and two worker subcastes (large and tiny workers). In order to identify putative odorant receptor genes involved in pheromone detection, we identified differentially expressed odorant receptor genes (OR-genes) using custom microarrays. In total, we found 185 OR-gene fragments that are clearly related to ORs and we identified orthologs for 70 OR-genes. Among them one OR-gene differs in relative expression between the two worker subcastes by a factor of >3 and thus is a very promising candidate gene for the trail-pheromone receptor. Using the relative expression of OR-genes in males versus queens, we identified 2 candidates for sex-pheromone receptor genes in males. In addition, we identified genes from all other chemosensory related gene families (13 chemosensory protein genes, 8 odorant binding protein genes, 2 sensory-neuron membrane protein genes, 7 ionotropic receptor genes, 2 gustatory receptor genes), and we found ant-specific expansions in the chemosensory protein gene family. In addition, a large number of genes involved in immune defense exhibited differential expression across the three different castes, and some genes even between the two worker subcastes.
doi:10.1371/journal.pone.0081518
PMCID: PMC3829964  PMID: 24260580
22.  Contact Chemoreceptors Mediate Male-Male Repulsion and Male-Female Attraction during Drosophila Courtship 
Cell  2012;149(5):1140-1151.
Summary
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. Here, we identify two ion channel genes belonging to the degenerin/epithelial sodium channel 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.
doi:10.1016/j.cell.2012.03.045
PMCID: PMC3365544  PMID: 22632976
23.  Contribution of oenocytes and pheromones to courtship behaviour in Drosophila 
BMC Biochemistry  2009;10:21.
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.
doi:10.1186/1471-2091-10-21
PMCID: PMC2734525  PMID: 19671131
24.  ppk23-Dependent Chemosensory Functions Contribute to Courtship Behavior in Drosophila melanogaster 
PLoS Genetics  2012;8(3):e1002587.
Insects utilize diverse families of ion channels to respond to environmental cues and control mating, feeding, and the response to threats. Although degenerin/epithelial sodium channels (DEG/ENaC) represent one of the largest families of ion channels in Drosophila melanogaster, the physiological functions of these proteins are still poorly understood. We found that the DEG/ENaC channel ppk23 is expressed in a subpopulation of sexually dimorphic gustatory-like chemosensory bristles that are distinct from those expressing feeding-related gustatory receptors. Disrupting ppk23 or inhibiting activity of ppk23-expressing neurons did not alter gustatory responses. Instead, blocking ppk23-positive neurons or mutating the ppk23 gene delayed the initiation and reduced the intensity of male courtship. Furthermore, mutations in ppk23 altered the behavioral response of males to the female-specific aphrodisiac pheromone 7(Z), 11(Z)-Heptacosadiene. Together, these data indicate that ppk23 and the cells expressing it play an important role in the peripheral sensory system that determines sexual behavior in Drosophila.
Author Summary
Insects utilize diverse families of ion channels to respond to environmental cues and control mating, feeding, and the response to threats. Degenerin/epithelial sodium channels (DEG/ENaC) represent one of the largest families of ion channels in Drosophila melanogaster. However, the physiological functions of the majority of these proteins in Drosophila or any other animal are still unknown. We discovered that the DEG/ENaC channel ppk23 is expressed in a subpopulation of sexually dimorphic gustatory-like chemosensory neurons. We show that ppk23 and the neurons expressing it are important for normal male sexual behaviors, possibly via the detection of an aphrodisiac pheromone, but not for feeding decisions. Together, these data indicate that DEG/ENaC signaling plays an important role in the peripheral sensory system that determines sexual behavior in Drosophila.
doi:10.1371/journal.pgen.1002587
PMCID: PMC3305452  PMID: 22438833
25.  Plasticity of the Chemoreceptor Repertoire in Drosophila melanogaster 
PLoS Genetics  2009;5(10):e1000681.
For most organisms, chemosensation is critical for survival and is mediated by large families of chemoreceptor proteins, whose expression must be tuned appropriately to changes in the chemical environment. We asked whether expression of chemoreceptor genes that are clustered in the genome would be regulated independently; whether expression of certain chemoreceptor genes would be especially sensitive to environmental changes; whether groups of chemoreceptor genes undergo coordinated rexpression; and how plastic the expression of chemoreceptor genes is with regard to sex, development, reproductive state, and social context. To answer these questions we used Drosophila melanogaster, because its chemosensory systems are well characterized and both the genotype and environment can be controlled precisely. Using customized cDNA microarrays, we showed that chemoreceptor genes that are clustered in the genome undergo independent transcriptional regulation at different developmental stages and between sexes. Expression of distinct subgroups of chemoreceptor genes is sensitive to reproductive state and social interactions. Furthermore, exposure of flies only to odor of the opposite sex results in altered transcript abundance of chemoreceptor genes. These genes are distinct from those that show transcriptional plasticity when flies are allowed physical contact with same or opposite sex members. We analyzed covariance in transcript abundance of chemosensory genes across all environmental conditions and found that they segregated into 20 relatively small, biologically relevant modules of highly correlated transcripts. This finely pixilated modular organization of the chemosensory subgenome enables fine tuning of the expression of the chemoreceptor repertoire in response to ecologically relevant environmental and physiological conditions.
Author Summary
Rapid adaptation and phenotypic plasticity to the chemical environment are essential prerequisites for survival; and, consequently, large families of genes that mediate the recognition of olfactory and gustatory cues have evolved. We asked how flexible the expression of these genes is in the face of rapidly changing conditions encountered during an individual's lifetime. We used the fruit fly, Drosophila melanogaster, to address this question, since both the genetic composition and environmental rearing conditions can be controlled precisely in this experimentally amenable model organism. By measuring expression levels of all chemosensory genes simultaneously, we identified genes that show altered expression at different developmental stages, during aging, in males and females, following mating, and in different social conditions. We asked whether chemosensory genes are regulated independently or whether their regulation is structured. We found that chemosensory genes that are located in close proximity to one another on the chromosome are often regulated independently. However, statistical analysis showed that groups of chemosensory genes are coordinately expressed in response to a range of environmental conditions, revealing an underlying modular organization of the phenotypic plasticity of the chemosensory receptor repertoire.
doi:10.1371/journal.pgen.1000681
PMCID: PMC2750752  PMID: 19816562

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