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1.  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
2.  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
3.  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
4.  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
5.  Hierarchical chemosensory regulation of male-male social interactions in Drosophila 
Nature neuroscience  2011;14(6):757-762.
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.
doi:10.1038/nn.2800
PMCID: PMC3102769  PMID: 21516101
6.  Receptors and neurons for fly odors in Drosophila 
Current biology : CB  2007;17(7):606-612.
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.
doi:10.1016/j.cub.2007.02.043
PMCID: PMC1876700  PMID: 17363256
7.  Multimodal Sensory Integration of Courtship Stimulating Cues in Drosophila melanogaster 
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.
doi:10.1111/j.1749-6632.2009.04367.x
PMCID: PMC2795581  PMID: 19686165
Drosophila; olfaction; gustation; hearing; cis-vaccenyl acetate; pheromone
8.  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
9.  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
10.  A Female-Emitted Pheromone Component Is Associated with Reduced Male Courtship in the Parasitoid Wasp Spalangia endius 
PLoS ONE  2013;8(11):e82010.
During courtship interactions, the courted individual may not always be prepared to mate. For example, mating or courtship may be detrimental to its fitness and resistance is expected under these circumstances. As such, various resistance strategies have evolved, from physically fending off courting individuals to producing behavioural signals of unreceptivity. In the parasitoid wasp Spalangia endius, females rarely re-mate and mated females are avoided by males in favour of virgin females. Further, mated females appear to advertise their mating status by the release of a pheromone component (methyl 6-methylsalicylate), but direct evidence of the nature of this release is lacking. Here we used real-time chemical analysis to track the emission of the pheromone component during courtship interactions between virgin males and either virgin or mated females. We found that females actively release methyl 6-methylsalicylate when courted and that significantly greater concentrations are released by previously mated females. Further, high concentrations of this component are associated with both the prevention and termination of courtship.
doi:10.1371/journal.pone.0082010
PMCID: PMC3835669  PMID: 24278468
11.  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
12.  Substrate-Borne Vibratory Communication during Courtship in Drosophila melanogaster 
Current Biology  2012;22(22):2180-2185.
Summary
Courtship in Drosophila melanogaster has become an iconic example of an innate and interactive series of behaviors [1–11]. The female signals her acceptance of copulation by becoming immobile in response to a male's display of stereotyped actions. The male and female communicate via vision, air-borne sounds, and pheromones [1, 2], but what triggers the female's immobility is undetermined. Here, we describe an overlooked and important component of Drosophila courtship. Video recordings and laser vibrometry show that the male abdomen shakes (“quivers”), generating substrate-borne vibrations at about six pulses per second. We present evidence that the female becomes receptive and stops walking because she senses these vibrations, rather than as a response to air-borne songs produced by the male fluttering the wings [1, 2, 12]. We also present evidence that the neural circuits expressing the sex-determination genes fruitless and doublesex [8] drive quivering behavior. These abdominal quivers and associated vibrations, as well as their effect on female receptivity, are conserved in other Drosophila species. Substrate-borne vibrations are an ancient form of communication that is widespread in animals. Our findings in Drosophila open a door to study the neuromuscular circuitry responsible for these signals and the sensory systems needed for their reception.
Highlights
► Courting Drosophila males vibrate their abdomen generating substrate-borne signals ► Females respond to these signals by stopping movement, to allow copulation ► This substrate-borne mode of communication is conserved among Drosophila species ► fruitless and doublesex expressing neurons underlie the abdominal vibration behavior
doi:10.1016/j.cub.2012.09.042
PMCID: PMC3502867  PMID: 23103187
13.  Sequential Learning of Pheromonal Cues Modulates Memory Consolidation in Trainer-Specific Associative Courtship Conditioning 
Current biology : CB  2005;15(3):194-206.
Summary
Background
Associative memory formation requires that animals choose predictors for experiences they need to remember. When an artificial odor is paired with an aversive experience, that odor becomes the predictor. In more natural settings, however, animals can have multiple salient experiences that need to be remembered and prioritized. The mechanisms by which animals deal with multiple experiences are incompletely understood.
Results
Here we show that Drosophila males can be trained to discriminate between different types of female pheromones; they suppress courtship specifically to the type of female that was associated with unsuccessful courtship. Such “trainer-specific” learning is mediated by hydrocarbon olfactory cues and modifies the male’s processing of those cues. Animals that are unable to use olfactory cues can still learn by using other sensory modalities, but memory in this case is not specific to the trainer female’s maturation state. Concurrent and serial presentation of different pheromones demonstrates that the ability to consolidate memory of pheromonal cues can be modified by the temporal order in which they appear.
Conclusion
Suppression of memory by new learning demonstrates that the dynamics of memory consolidation are subject to plasticity in Drosophila. This type of metaplasticity is essential for navigation of experience-rich natural environments.
doi:10.1016/j.cub.2005.01.035
PMCID: PMC2805828  PMID: 15694302
14.  Sexual conflict and anti-aphrodisiac titre in a polyandrous butterfly: male ejaculate tailoring and absence of female control. 
Males of the green-veined butterfly Pieris napi synthesize and transfer the volatile methyl salicylate (MeS) to females at mating, a substance that is emitted by non-virgin females when courted by males, curtailing courtship and decreasing the likelihood of female re-mating. The volatile is released when females display the 'mate-refusal' posture with spread wings and elevated abdomen, when courted by conspecific males. Here, we assess how the amount of MeS released by courted females changes over time since mating, and whether it is influenced by the frequency with which females display the mate-refusal posture. We also assess whether males tailor the anti-aphrodisiac content of ejaculates with respect to the expected degree of sperm competition, by comparing how males allocate MeS proportionately to first and second ejaculates in relation to ejaculate mass. The results show that females housed for 5 days in individual cages where they were able to fly and oviposit normally, released similar amounts of MeS. However, females housed together for the same period of time, causing them to frequently display the mate-refusal posture, released significantly lower levels of MeS than the individually housed females. This indicates that female display of the mate-refusal posture depletes their anti-aphrodisiac stores, and suggests that females are unable to voluntarily control their release of the anti-aphrodisiac. A comparison of relative proportion of MeS transferred by males in their first and second ejaculates showed that proportionately more MeS was allocated to the first ejaculate, in accordance with the idea that these are tailored to delay female re-mating.
doi:10.1098/rspb.2003.2671
PMCID: PMC1691801  PMID: 15315890
15.  Quantitative analysis of pheromone-binding protein specificity 
Insect molecular biology  2012;22(1):31-40.
Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed.
doi:10.1111/j.1365-2583.2012.01167.x
PMCID: PMC3552018  PMID: 23121132
olfaction; 11-cis vaccenyl acetate; LUSH; pheromone-binding proteins; cyclodextrin; tryptophan fluorescence
16.  The effects of mating status and time since mating on female sex pheromone levels in the rice leaf bug, Trigonotylus caelestialium 
Die Naturwissenschaften  2014;101:153-156.
Although mating status affects future mating opportunities, the biochemical changes that occur in response to mating are not well understood. This study investigated the effects of mating status on the quantities of sex pheromone components found in whole-body extracts and volatile emissions of females of the rice leaf bug, Trigonotylus caelestialium. When sampled at one of four time points within a 4-day postmating period, females that had copulated with a male had greater whole-body quantities of sex pheromone components than those of virgin females sampled at the same times. The quantities of sex pheromone components emitted by virgin females over a 24-h period were initially high but then steadily decreased, whereas 24-h emissions were persistently low among mated females when measured at three time points within the 4 days after mating. As a result, soon after mating, the mated females emitted less sex pheromones than virgin females, but there were no significant differences between mated and virgin females at the end of the experiment. Thus, postmating reduction in the rate of emission of sex pheromones could explain previously observed changes in female attractiveness to male T. caelestialium.
Electronic supplementary material
The online version of this article (doi:10.1007/s00114-013-1141-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00114-013-1141-3
PMCID: PMC3933747  PMID: 24419707
Sex pheromones; Postmating behavior; Sexual selection; Trigonotylus caelestialium
17.  Song Choice Is Modulated by Female Movement in Drosophila Males 
PLoS ONE  2012;7(9):e46025.
Mate selection is critical to ensuring the survival of a species. In the fruit fly, Drosophila melanogaster, genetic and anatomical studies have focused on mate recognition and courtship initiation for decades. This model system has proven to be highly amenable for the study of neural systems controlling the decision making process. However, much less is known about how courtship quality is regulated in a temporally dynamic manner in males and how a female assesses male performance as she makes her decision of whether to accept copulation. Here, we report that the courting male dynamically adjusts the relative proportions of the song components, pulse song or sine song, by assessing female locomotion. Male flies deficient for olfaction failed to perform the locomotion-dependent song modulation, indicating that olfactory cues provide essential information regarding proximity to the target female. Olfactory mutant males also showed lower copulation success when paired with wild-type females, suggesting that the male’s ability to temporally control song significantly affects female mating receptivity. These results depict the consecutive inter-sex behavioral decisions, in which a male smells the close proximity of a female as an indication of her increased receptivity and accordingly coordinates his song choice, which then enhances the probability of his successful copulation.
doi:10.1371/journal.pone.0046025
PMCID: PMC3458092  PMID: 23049926
18.  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
19.  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
20.  No evidence for learned mating discrimination in male Drosophila pseudoobscura 
Background
Since females often pay a higher cost for heterospecific matings, mate discrimination and species recognition are driven primarily by female choice. In contrast, frequent indiscriminate matings are hypothesized to maximize male fitness. However, recent studies show that previously indiscriminate males (e.g., Drosophila melanogaster and Poecilia reticulata) can learn to avoid heterospecific courtship. This ability of males to discriminate against heterospecific courtship may be advantageous in populations where two species co-occur if courtship or mating is costly.
Results
Here, we tested whether Drosophila pseudoobscura males learn to discriminate against heterospecific females after being exposed to and rejected by D. persimilis females. In most of our assays, we failed to observe differences in D. pseudoobscura courtship intensity of heterospecific females by males that had previously courted heterospecific females vs. males that had been maintained in isolation.
Conclusion
We conclude that learning to avoid heterospecific courtship may not be universal, even within the genus Drosophila, and may possibly be dependent on the natural history of the species.
doi:10.1186/1471-2148-6-54
PMCID: PMC1534054  PMID: 16824212
21.  The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females 
Genes, brain, and behavior  2009;8(5):546-557.
Transient receptor potential (TRP) channels play crucial roles in sensory perception. Expression of the Drosophila painless (pain) gene, a homolog of the mammalian TRPA1/ANKTM1 gene, in the peripheral nervous system is required for avoidance behavior of noxious heat or wasabi. Here we report a novel role of the Pain TRP channel expressed in the nervous system in the sexual receptivity in Drosophila virgin females. Compared with wild-type females, pain mutant females copulated with wild-type males significantly earlier. Wild-type males showed comparable courtship latency and courtship index toward wild-type and pain mutant females. Therefore, the early copulation observed in wild-type male and pain mutant female pairs is the result of enhanced sexual receptivity in pain mutant females. Involvement of pain in enhanced female sexual receptivity was confirmed by rescue experiments in which expression of a pain transgene in a pain mutant background restored the female sexual receptivity to the wild-type level. Targeted expression of pain RNAi in putative cholinergic or GABAergic neurons phenocopied the mutant phenotype of pain females. On the other hand, target expression of pain RNAi in dopaminergic neurons did not affect female sexual receptivity. In addition, conditional suppression of neurotransmission in putative GABAergic neurons resulted in a similar enhanced sexual receptivity. Our results suggest that Pain TRP channels expressed in cholinergic and/or GABAergic neurons are involved in female sexual receptivity.
doi:10.1111/j.1601-183X.2009.00503.x
PMCID: PMC2728068  PMID: 19531155
painless; TRP channel; mating; sexual receptivity; Drosophila; brain; GAL4/UAS; GAL80
22.  Sensory Integration Regulating Male Courtship Behavior in Drosophila 
PLoS ONE  2009;4(2):e4457.
The courtship behavior of Drosophila melanogaster serves as an excellent model system to study how complex innate behaviors are controlled by the nervous system. To understand how the underlying neural network controls this behavior, it is not sufficient to unravel its architecture, but also crucial to decipher its logic. By systematic analysis of how variations in sensory inputs alter the courtship behavior of a naïve male in the single-choice courtship paradigm, we derive a model describing the logic of the network that integrates the various sensory stimuli and elicits this complex innate behavior. This approach and the model derived from it distinguish (i) between initiation and maintenance of courtship, (ii) between courtship in daylight and in the dark, where the male uses a scanning strategy to retrieve the decamping female, and (iii) between courtship towards receptive virgin females and mature males. The last distinction demonstrates that sexual orientation of the courting male, in the absence of discriminatory visual cues, depends on the integration of gustatory and behavioral feedback inputs, but not on olfactory signals from the courted animal. The model will complement studies on the connectivity and intrinsic properties of the neurons forming the circuitry that regulates male courtship behavior.
doi:10.1371/journal.pone.0004457
PMCID: PMC2636894  PMID: 19214231
23.  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
24.  Volatile pheromone signalling in Drosophila 
Physiological entomology  2012;37(1):10.1111/j.1365-3032.2011.00813.x.
Once captured by the antenna, 11-cis vaccenyl acetate (cVA) binds to an extracellular binding protein called LUSH that undergoes a conformational shift upon cVA binding. The stable LUSH–cVA complex is the activating ligand for pheromone receptors present on the dendrites of the aT1 neurones, comprising the only neurones that detect cVA pheromone. This mechanism explains the single molecule sensitivity of insect pheromone detection systems. The receptor that recognizes activated LUSH consists of a complex of several proteins, including Or67d, a member of the tuning odourant receptor family, Orco, a co-receptor ion channel, and SNMP, a CD36 homologue that may be an inhibitory subunit. In addition, genetic screens and reconstitution experiments reveal additional factors that are important for pheromone detection. Identification and functional dissection of these factors in Drosophila melanogaster Meigen should permit the identification of homologous factors in pathogenic insects and agricultural pests, which, in turn, may be viable candidates for novel classes of compounds to control populations of target insect species without impacting beneficial species.
doi:10.1111/j.1365-3032.2011.00813.x
PMCID: PMC3859522  PMID: 24347807
25.  Reduction of Dopamine Level Enhances the Attractiveness of Male Drosophila to Other Males 
PLoS ONE  2009;4(2):e4574.
Dopamine is an important neuromodulator in animals and its roles in mammalian sexual behavior are extensively studied. Drosophila as a useful model system is widely used in many fields of biological studies. It has been reported that dopamine reduction can affect female receptivity in Drosophila and leave male-female courtship behavior unaffected. Here, we used genetic and pharmacological approaches to decrease the dopamine level in dopaminergic cells in Drosophila, and investigated the consequence of this manipulation on male homosexual courtship behavior. We find that reduction of dopamine level can induce Drosophila male-male courtship behavior, and that this behavior is mainly due to the increased male attractiveness or decreased aversiveness towards other males, but not to their enhanced propensity to court other males. Chemical signal input probably plays a crucial role in the male-male courtship induced by the courtees with reduction of dopamine. Our finding provides insight into the relationship between the dopamine reduction and male-male courtship behavior, and hints dopamine level is important for controlling Drosophila courtship behavior.
doi:10.1371/journal.pone.0004574
PMCID: PMC2642723  PMID: 19238209

Results 1-25 (200983)