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1.  Love makes smell blind: mating suppresses pheromone attraction in Drosophila females via Or65a olfactory neurons 
Scientific Reports  2014;4:7119.
In Drosophila, the male sex pheromone cis-vaccenyl acetate (cVA) elicits aggregation and courtship, through the odorant receptor Or67d. Long-lasting exposure to cVA suppresses male courtship, via a second channel, Or65a. In females, the role of Or65a has not been studied. We show that, shortly after mating, Drosophila females are no longer attracted to cVA and that activation of olfactory sensory neurons (OSNs) expressing Or65a generates this behavioral switch: when silencing Or65a, mated females remain responsive to cVA. Neurons expressing Or67d converge into the DA1 glomerulus in the antennal lobe, where they synapse onto projection neurons (PNs), that connect to higher neural circuits generating the attraction response to cVA. Functional imaging of these PNs shows that the DA1 glomerulus is inhibited by simultaneous activation of Or65a OSNs, which leads to a suppression of the attraction response to cVA. The behavioral role of postmating cVA exposure is substantiated by the observation that matings with starved males, which produce less cVA, do not alter the female response. Moreover, exposure to synthetic cVA abolishes attraction and decreases sexual receptivity in unmated females. Taken together, Or65a mediates an aversive effect of cVA and may accordingly regulate remating, through concurrent behavioral modulation in males and females.
doi:10.1038/srep07119
PMCID: PMC4236738  PMID: 25406576
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.  Modification of Male Courtship Motivation by Olfactory Habituation via the GABAA Receptor in Drosophila melanogaster 
PLoS ONE  2015;10(8):e0135186.
A male-specific component, 11-cis-vaccenyl acetate (cVA) works as an anti-aphrodisiac pheromone in Drosophila melanogaster. The presence of cVA on a male suppresses the courtship motivation of other males and contributes to suppression of male-male homosexual courtship, while the absence of cVA on a female stimulates the sexual motivation of nearby males and enhances the male-female interaction. However, little is known how a male distinguishes the presence or absence of cVA on a target fly from either self-produced cVA or secondhand cVA from other males in the vicinity. In this study, we demonstrate that male flies have keen sensitivity to cVA; therefore, the presence of another male in the area reduces courtship toward a female. This reduced level of sexual motivation, however, could be overcome by pretest odor exposure via olfactory habituation to cVA. Real-time imaging of cVA-responsive sensory neurons using the neural activity sensor revealed that prolonged exposure to cVA decreased the levels of cVA responses in the primary olfactory center. Pharmacological and genetic screening revealed that signal transduction via GABAA receptors contributed to this olfactory habituation. We also found that the habituation experience increased the copulation success of wild-type males in a group. In contrast, transgenic males, in which GABA input in a small subset of local neurons was blocked by RNAi, failed to acquire the sexual advantage conferred by habituation. Thus, we illustrate a novel phenomenon in which olfactory habituation positively affects sexual capability in a competitive environment.
doi:10.1371/journal.pone.0135186
PMCID: PMC4529276  PMID: 26252206
4.  Requirement for Drosophila SNMP1 for Rapid Activation and Termination of Pheromone-Induced Activity 
PLoS Genetics  2014;10(9):e1004600.
Pheromones are used for conspecific communication by many animals. In Drosophila, the volatile male-specific pheromone 11-cis vaccenyl acetate (cVA) supplies an important signal for gender recognition. Sensing of cVA by the olfactory system depends on multiple components, including an olfactory receptor (OR67d), the co-receptor ORCO, and an odorant binding protein (LUSH). In addition, a CD36 related protein, sensory neuron membrane protein 1 (SNMP1) is also involved in cVA detection. Loss of SNMP1 has been reported to eliminate cVA responsiveness, and to greatly increase spontaneous activity of OR67d-expressing olfactory receptor neurons (ORNs). Here, we found the snmp11 mutation did not abolish cVA responsiveness or cause high spontaneous activity. The cVA responses in snmp1 mutants displayed a delayed onset, and took longer to reach peak activity than wild-type. Most strikingly, loss of SNMP1 caused a dramatic delay in signal termination. The profound impairment in signal inactivation accounted for the previously reported “spontaneous activity,” which represented continuous activation following transient exposure to environmental cVA. We introduced the silk moth receptor (BmOR1) in OR67d ORNs of snmp11 flies and found that the ORNs showed slow activation and deactivation kinetics in response to the BmOR1 ligand (bombykol). We expressed the bombykol receptor complex in Xenopus oocytes in the presence or absence of the silk moth SNMP1 (BmSNMP) and found that addition of BmSNMP accelerated receptor activation and deactivation. Our results thus clarify SNMP1 as an important player required for the rapid kinetics of the pheromone response in insects.
Author Summary
Pheromones are chemicals produced and released by animals for social communication with other members of their species. For example, male fruit flies produce a volatile pheromone that is sensed by both males and females, and which functions in gender recognition. This volatile male pheromone, called 11-cis vaccenyl acetate, is detected by olfactory neurons housed in hair-like appendages on the insect antenna. To effectively sense the pheromone, especially during navigation, the olfactory neurons must respond rapidly, and then quickly inactivate after the stimulation ceases. We found that a CD36-related protein referred to as sensory neuron membrane protein 1 (SNMP1) was required by olfactory neurons for the rapid on and off responses to 11-cis vaccenyl acetate. Loss of SNMP1 reduced the initial sensitivity to the pheromone, and then caused a strikingly slower termination of the response after removal of the pheromone. Our findings demonstrate that SNMP1 is a critical player that allows olfactory neurons to achieve sensitive and rapid on and off responses to a pheromone that is critical for social interactions in insects.
doi:10.1371/journal.pgen.1004600
PMCID: PMC4177743  PMID: 25255106
5.  Feeding regulates sex pheromone attraction and courtship in Drosophila females 
Scientific Reports  2015;5:13132.
In Drosophila melanogaster, gender-specific behavioural responses to the male-produced sex pheromone cis-vaccenyl acetate (cVA) rely on sexually dimorphic, third-order neural circuits. We show that nutritional state in female flies modulates cVA perception in first-order olfactory neurons. Starvation increases, and feeding reduces attraction to food odour, in both sexes. Adding cVA to food odour, however, maintains attraction in fed females, while it has no effect in males. Upregulation of sensitivity and behavioural responsiveness to cVA in fed females is paralleled by a strong increase in receptivity to male courtship. Functional imaging of the antennal lobe (AL), the olfactory centre in the insect brain, shows that olfactory input to DA1 and VM2 glomeruli is also modulated by starvation. Knocking down insulin receptors in neurons converging onto the DA1 glomerulus suggests that insulin-signalling partly controls pheromone perception in the AL, and adjusts cVA attraction according to nutritional state and sexual receptivity in Drosophila females.
doi:10.1038/srep13132
PMCID: PMC4530334  PMID: 26255707
6.  Activation of Latent Courtship Circuitry in the Brain of Drosophila Females Induces Male-like Behaviors 
Current Biology  2016;26(18):2508-2515.
Summary
Courtship in Drosophila melanogaster offers a powerful experimental paradigm for the study of innate sexually dimorphic behaviors [1, 2]. Fruit fly males exhibit an elaborate courtship display toward a potential mate [1, 2]. Females never actively court males, but their response to the male’s display determines whether mating will actually occur. Sex-specific behaviors are hardwired into the nervous system via the actions of the sex determination genes doublesex (dsx) and fruitless (fru) [1]. Activation of male-specific dsx/fru+ P1 neurons in the brain initiates the male’s courtship display [3, 4], suggesting that neurons unique to males trigger this sex-specific behavior. In females, dsx+ neurons play a pivotal role in sexual receptivity and post-mating behaviors [1, 2, 5, 6, 7, 8, 9]. Yet it is still unclear how dsx+ neurons and dimorphisms in these circuits give rise to the different behaviors displayed by males and females. Here, we manipulated the function of dsx+ neurons in the female brain to investigate higher-order neurons that drive female behaviors. Surprisingly, we found that activation of female dsx+ neurons in the brain induces females to behave like males by promoting male-typical courtship behaviors. Activated females display courtship toward conspecific males or females, as well other Drosophila species. We uncovered specific dsx+ neurons critical for driving male courtship and identified pheromones that trigger such behaviors in activated females. While male courtship behavior was thought to arise from male-specific central neurons, our study shows that the female brain is equipped with latent courtship circuitry capable of inducing this male-specific behavioral program.
Highlights
•Activation of brain dsx-pC1 neurons promote male-like courtship in females•Activated females court conspecific males and females and other Drosophila species•Methyl pheromones trigger male courtship behaviors in activated females•The female brain is equipped with latent circuitry underlying male-like behavior
Rezával et al. found that activation of specific neurons in the brain induces female fruit flies to display male-like courtship behaviors and identified pheromones that induce such behaviors. Thus, this study shows that the female fly brain is equipped with latent courtship circuitry that is capable of inducing a male-specific behavioral program.
doi:10.1016/j.cub.2016.07.021
PMCID: PMC5049544  PMID: 27568592
7.  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
8.  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
9.  Odorant Responses and Courtship Behaviors Influenced by at4 Neurons in Drosophila 
PLoS ONE  2016;11(9):e0162761.
In insects, pheromones function as triggers to elicit complex behavior programs, such as courtship and mating behavior. In most species, the neurons tuned to pheromones are localized in a specific subset of olfactory sensilla located on the antenna called trichoid sensilla. In Drosophila there are two classes of trichoid sensilla, at1 sensilla that contain the dendrites of a single neuron that is specifically tuned to the male-specific pheromone 11-cis vaccenyl acetate (cVA), and at4 sensilla that contain three neurons with relatively poorly defined chemical specificity and function. Using a combination of odorant receptor mutant analysis, single sensillum electrophysiology and optogenetics, we have examined the chemical tuning and behavioral consequences of the three at4 olfactory neuron classes. Our results indicate that one class, Or65abc neurons, are unresponsive to cVA pheromone, and function to inhibit courtship behaviors in response to an unknown ligand, while the other two neuron classes, Or88a and Or47b neurons, are sensitive to a diverse array of fly and non-fly odors, and activation of these neurons has little direct impact on courtship behaviors.
doi:10.1371/journal.pone.0162761
PMCID: PMC5019410  PMID: 27617442
10.  Activation of Pheromone-Sensitive Neurons Is Mediated by Conformational Activation of Pheromone-Binding Protein 
Cell  2008;133(7):1255-1265.
Summary
Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. Here, we show that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-biing protein, LUSH. We show that LUSH undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in LUSH that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, LUSHD118A, produces a dominant-active LUSH protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of LUSHD118A reveals that it closely resembles cVA-bound LUSH. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants.
doi:10.1016/j.cell.2008.04.046
PMCID: PMC4397981  PMID: 18585358
11.  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
12.  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
13.  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
14.  Central neural circuitry mediating courtship song perception in male Drosophila 
eLife  null;4:e08477.
Animals use acoustic signals across a variety of social behaviors, particularly courtship. In Drosophila, song is detected by antennal mechanosensory neurons and further processed by second-order aPN1/aLN(al) neurons. However, little is known about the central pathways mediating courtship hearing. In this study, we identified a male-specific pathway for courtship hearing via third-order ventrolateral protocerebrum Projection Neuron 1 (vPN1) neurons and fourth-order pC1 neurons. Genetic inactivation of vPN1 or pC1 disrupts song-induced male-chaining behavior. Calcium imaging reveals that vPN1 responds preferentially to pulse song with long inter-pulse intervals (IPIs), while pC1 responses to pulse song closely match the behavioral chaining responses at different IPIs. Moreover, genetic activation of either vPN1 or pC1 induced courtship chaining, mimicking the behavioral response to song. These results outline the aPN1-vPN1-pC1 pathway as a labeled line for the processing and transformation of courtship song in males.
DOI: http://dx.doi.org/10.7554/eLife.08477.001
eLife digest
The seemingly simple fruit fly engages in an intricate courtship ritual before it mates. Male flies use their wings to ‘sing’ a complex song that makes females more willing to mate. The song also encourages nearby males to start courting, and these males may then intervene to compete for the female. Each species of fruit fly has its own song, and it is important for both males and females to detect the right song.
The sounds of the courtship song are detected by vibration-sensitive neurons on the flies' antennae. These neurons send signals to the fly's brain. But little is known about how this information is then processed, or how information about the song can be integrated with other courtship cues.
Zhou et al. have now identified a pathway of neurons in male flies that is responsible for hearing the courtship song. This pathway stretches from the antennae to neurons deep within the brain. These neural pathways are different in males and females, suggesting that the two sexes use different circuits of neurons for hearing courtship songs. Zhou et al. then used genetic techniques to show that males need every neuron in this pathway to hear courtship songs.
Further experiments revealed that stimulating the ‘deep layer’ neurons caused male flies to respond as if they are hearing the courtship song. These neurons are likely to integrate the song with information from other senses and may encode a general signal for arousal.
These findings now pave the way to deepen our understanding of how information from different senses—for example, courtship songs, visual cues, and pheromones—can be integrated to drive specific behaviors. The next challenge is to explore how species-specific songs are detected and recognized, a goal that has yet to be achieved in any species.
DOI: http://dx.doi.org/10.7554/eLife.08477.002
doi:10.7554/eLife.08477
PMCID: PMC4575990  PMID: 26390382
courtship; courtship song; auditory sensation; D. melanogaster
15.  Pleiotropic actions of the male pheromone cis-vaccenyl acetate in Drosophila melanogaster 
The male-specific lipid, cis-vaccenyl acetate (cVA) has multiple functions in intra-species communication in Drosophila melanogaster. The presence of cVA in a male suppresses courtship motivation of other males and averts male–male courtship. Meanwhile, aggression behaviors between males are promoted by a high amount of cVA caused by increased densities of male flies. cVA also works as a modifier of courtship memory, which is suppressed courtship motivation driven by previous unsuccessful courtship experience. Conversely, cVA in the courting male stimulates female reproductive motivation and increases the probability of copulation success. It also works as an aggregation pheromone, attracting both males and females at the gathering spot. Thus, cVA is a unique example of a single molecule leading to different behaviors in response to the social context. However, despite recent advances in understanding the molecular and neural machinery for cVA sensing, it is still largely unknown how cVA triggers a specific behavior as the situation demands. In this review article, I discuss two potential machineries that might determine cVA actions for behavior selection at the sensory level.
doi:10.1007/s00359-015-1020-9
PMCID: PMC4552767  PMID: 26105507
cVA; Pheromone; Courtship; Aggression; Copulation; Aggregation; Concentration; Odor context
16.  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
17.  Loss of Drosophila pheromone reverses its role in sexual communication in Drosophila suzukii 
The Drosophila pheromone cis-11-octadecenyl acetate (cVA) is used as pheromone throughout the melanogaster group and fulfils a primary role in sexual and social behaviours. Here, we found that Drosophila suzukii, an invasive pest that oviposits in undamaged ripe fruit, does not produce cVA. In fact, its production site, the ejaculatory bulb, is atrophied. Despite loss of cVA production, its receptor, Or67d, and cognate sensillum, T1, which are essential in cVA-mediated behaviours, were fully functional. However, T1 expression was dramatically reduced in D. suzukii, and the corresponding antennal lobe glomerulus, DA1, minute. Behavioural responses to cVA depend on the input balance of Or67d neurons (driving cVA-mediated behaviours) and Or65a neurons (inhibiting cVA-mediated behaviours). Accordingly, the shifted input balance in D. suzukii has reversed cVA's role in sexual behaviour: perfuming D. suzukii males with Drosophila melanogaster equivalents of cVA strongly reduced mating rates. cVA has thus evolved from a generic sex pheromone to a heterospecific signal that disrupts mating in D. suzukii, a saltational shift, mediated through offsetting the input balance that is highly conserved in congeneric species. This study underlines that dramatic changes in a species' sensory preference can result from rather ‘simple’ numerical shifts in underlying neural circuits.
doi:10.1098/rspb.2014.3018
PMCID: PMC4375874  PMID: 25716789
Drosophila suzukii; pheromone; evolution; ejaculatory bulb; fruitless; sensory drive
18.  The neuropeptide tachykinin is essential for pheromone detection in a gustatory neural circuit 
eLife  null;4:e06914.
Gustatory pheromones play an essential role in shaping the behavior of many organisms. However, little is known about the processing of taste pheromones in higher order brain centers. Here, we describe a male-specific gustatory circuit in Drosophila that underlies the detection of the anti-aphrodisiac pheromone (3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol (CH503). Using behavioral analysis, genetic manipulation, and live calcium imaging, we show that Gr68a-expressing neurons on the forelegs of male flies exhibit a sexually dimorphic physiological response to the pheromone and relay information to the central brain via peptidergic neurons. The release of tachykinin from 8 to 10 cells within the subesophageal zone is required for the pheromone-triggered courtship suppression. Taken together, this work describes a neuropeptide-modulated central brain circuit that underlies the programmed behavioral response to a gustatory sex pheromone. These results will allow further examination of the molecular basis by which innate behaviors are modulated by gustatory cues and physiological state.
DOI: http://dx.doi.org/10.7554/eLife.06914.001
eLife digest
In many species of animals, the male decides to pursue a potential female mate based on how she smells and tastes. Powerful chemical signals known as pheromones control this decision. When a male fruit fly mates with a female fruit fly, he often leaves behind an anti-aphrodisiac pheromone that, when males taste it, deters them from mating with the female. Until recently, however, little was known about how the brain processes information from such taste pheromones.
Now, Shankar et al. have investigated this problem in a series of experiments with normal and genetically modified flies. In the first experiment normal male flies were exposed to the chemical on its own, to the chemical on a sample of female skin, and to the chemical on actual female flies. The male flies did not respond to the pheromone on its own, but they did respond to it in the other two scenarios.
Next, Shankar et al. used genetic techniques to eliminate individual neurons in the male flies and then observed how the loss of specific neurons influenced the response of the fly to the pheromone. These experiments showed that male flies have a special group of sensory neurons in their legs that detect the chemical and then send an electrical signal to the brain. Shankar et al. then went on to identify a brain circuit consisting of 8–10 neurons that responds to this signal and to show that the release of a neurochemical called Tachykinin is essential in communicating the signal.
In a final set of experiments, Shankar et al. introduced two sensors—one in the sensory neurons in the legs, the other in the 8–10 neurons in the brain—that light up when the neurons in that region are close enough to each other to form connections. The results suggest that the sensory neurons in the legs form connections with the 8–10 neurons in the brain.
A challenge for the future is to understand how the nervous system combines different social cues and information about the physiological state of the animal, and how this influences the decision to mate.
DOI: http://dx.doi.org/10.7554/eLife.06914.002
doi:10.7554/eLife.06914
PMCID: PMC4491540  PMID: 26083710
CH503; courtship; behavior; anti-aphrodisiac; NPF; calcium imaging; D. melanogaster
19.  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
20.  Excitation and inhibition onto central courtship neurons biases Drosophila mate choice 
eLife  null;4:e11188.
The ability to distinguish males from females is essential for productive mate selection and species propagation. Recent studies in Drosophila have identified different classes of contact chemosensory neurons that detect female or male pheromones and influence courtship decisions. Here, we examine central neural pathways in the male brain that process female and male pheromones using anatomical, calcium imaging, optogenetic, and behavioral studies. We find that sensory neurons that detect female pheromones, but not male pheromones, activate a novel class of neurons in the ventral nerve cord to cause activation of P1 neurons, male-specific command neurons that trigger courtship. In addition, sensory neurons that detect male pheromones, as well as those that detect female pheromones, activate central mAL neurons to inhibit P1. These studies demonstrate that the balance of excitatory and inhibitory drives onto central courtship-promoting neurons controls mating decisions.
DOI: http://dx.doi.org/10.7554/eLife.11188.001
eLife digest
Courtship displays are seen throughout the animal kingdom. For example, male birds-of-paradise are perhaps best known for the elaborate dances they use to attract a mate. Male fruit flies, belonging to the species Drosophila melanogaster, also perform courtship toward female flies. However, male flies do not court other males. Previous studies have shown that sex-specific chemical signals (or pheromones) are important cues that males use to direct courtship towards females. Researchers have previously identified two sets of sensory neurons that detect pheromones: one set detects female pheromones and promotes courtship, while the other detects male pheromones and inhibits courtship. However it was unclear how these sensory neurons controlled courtship behavior.
Now, Kallman et al. have studied the circuits of neurons in the fruit fly that promote or inhibit courtship when a fly detects a pheromone. The experiments identified several pathways of neurons in the brain of male Drosophila that respond to female and male pheromones. These pathways send signals that either excite or inhibit a central target, called P1 neurons. Female pheromones activated a pathway that activates the P1 neurons, whereas male pheromones activate another pathway that inhibits the P1 neurons. Kallman et al. suggest that the balance of these excitatory and inhibitory signals controls a fly’s decision to court.
Following on from this work one of the next challenges will be to identify the neural circuits that act downstream of the P1 neurons to control courtship. Future studies could also explore how P1 neurons integrate signals from different senses.
DOI: http://dx.doi.org/10.7554/eLife.11188.002
doi:10.7554/eLife.11188
PMCID: PMC4695383  PMID: 26568316
pheromones; neural circuits; sensory processing; mating behavior; D. melanogaster
21.  Chromatin Modulatory Proteins and Olfactory Receptor Signaling in the Refinement and Maintenance of Fruitless Expression in Olfactory Receptor Neurons 
PLoS Biology  2016;14(4):e1002443.
During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to generate defined motor outputs, or behavior. This integration requires a developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown. Here, we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs) involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless (fru). The first mode involves a developmental program that coordinately restricts to appropriate ORNs the expression of fru and two olfactory receptors (Or47b and Ir84a) involved in sex-specific behaviors. This regulation requires the chromatin modulatory protein Alhambra (Alh). The second mode relies on the signaling from the olfactory receptors through CamK and histone acetyl transferase p300/CBP to maintain ORN-specific fru expression. Our results highlight two feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs. Such a dual mechanism of fru regulation in ORNs might be a trait of neurons driving plastic aspects of sex-specific behaviors.
The fruitless (fru) gene regulates sexual behavior in flies, but what regulates fru? This study shows that the chromatin modulator Alhambra restricts fru expression to specific neurons in the developing Drosophila olfactory system. In adults, olfactory receptor signaling and the histone acetyltransferase p300 are required to maintain fru expression.
Author Summary
How do individual neurons know what type of a circuit they must integrate into? To correctly assemble neural circuits during development, the identities of neurons must be coordinated with the identities of the circuits into which they will be integrated. How is this process regulated? We have used the olfactory circuits that regulate sex-specific behaviors in Drosophila to answer this question. In Drosophila, the fruitless (fru) gene, which encodes a transcription factor, acts as a molecular marker that labels circuits regulating sex-specific behaviors. Fru is both a necessary and sufficient regulator of sex-specific behaviors like courtship and aggression and is expressed in only about 2,000 interconnected neurons in the fly nervous system. Even though fru expression and function is so critical to sex-specific behavior, the mechanisms regulating its expression in each of the neurons within the circuit are not known. Here, we revealed two different modes of transcriptional regulation of fru during the development of the olfactory receptor neurons that are involved in sex-specific behaviors. In the first mode, the putative chromatin modulator Alhambra (Alh) coregulates both olfactory receptors and fru expression in two olfactory receptor neuron classes during development. In adult flies, the second mode maintains fru expression through the activity of olfactory receptors, calcium signaling, and chromatin modulatory proteins in these olfactory receptor neurons. These two genetic programs separate the developmentally hardwired and the olfactory receptor activity-mediated regulation of fru expression in olfactory circuits, and might represent the molecular mechanisms that mediate the innate and adaptable aspects of odor-guided social behaviors.
doi:10.1371/journal.pbio.1002443
PMCID: PMC4836687  PMID: 27093619
22.  Cellular and Behavioral Functions of fruitless Isoforms in Drosophila Courtship 
Current Biology  2014;24(3):242-251.
Summary
Background
Male-specific products of the fruitless (fru) gene control the development and function of neuronal circuits that underlie male-specific behaviors in Drosophila, including courtship. Alternative splicing generates at least three distinct Fru isoforms, each containing a different zinc-finger domain. Here, we examine the expression and function of each of these isoforms.
Results
We show that most fru+ cells express all three isoforms, yet each isoform has a distinct function in the elaboration of sexually dimorphic circuitry and behavior. The strongest impairment in courtship behavior is observed in fruC mutants, which fail to copulate, lack sine song, and do not generate courtship song in the absence of visual stimuli. Cellular dimorphisms in the fru circuit are dependent on FruC rather than other single Fru isoforms. Removal of FruC from the neuronal classes vAB3 or aSP4 leads to cell-autonomous feminization of arborizations and loss of courtship in the dark.
Conclusions
These data map specific aspects of courtship behavior to the level of single fru isoforms and fru+ cell types—an important step toward elucidating the chain of causality from gene to circuit to behavior.
Highlights
•fru A, B, and C isoforms have largely overlapping expression in the male fly CNS•All three fru isoforms contribute to male courtship, with fruC being the most critical•FruC specifies sexual dimorphisms in neuron number and arborizations•FruC is required in defined neuronal classes for male-specific anatomy and behavior
Drosophila express three male-specific isoforms (A, B, and C) of the putative transcription factor fruitless (fru) in an overlapping pattern. von Philipsborn et al. show that isoform-mutant flies exhibit defects in male courtship, copulation success, and song production. FruC specifies sexual dimorphisms in defined neuronal classes potentially crucial for pheromone processing.
doi:10.1016/j.cub.2013.12.015
PMCID: PMC3969150  PMID: 24440391
23.  Neural circuitry coordinating male copulation 
eLife  null;5:e20713.
Copulation is the goal of the courtship process, crucial to reproductive success and evolutionary fitness. Identifying the circuitry underlying copulation is a necessary step towards understanding universal principles of circuit operation, and how circuit elements are recruited into the production of ordered action sequences. Here, we identify key sex-specific neurons that mediate copulation in Drosophila, and define a sexually dimorphic motor circuit in the male abdominal ganglion that mediates the action sequence of initiating and terminating copulation. This sexually dimorphic circuit composed of three neuronal classes – motor neurons, interneurons and mechanosensory neurons – controls the mechanics of copulation. By correlating the connectivity, function and activity of these neurons we have determined the logic for how this circuitry is coordinated to generate this male-specific behavior, and sets the stage for a circuit-level dissection of active sensing and modulation of copulatory behavior.
DOI: http://dx.doi.org/10.7554/eLife.20713.001
eLife digest
Idioms and love songs often euphemistically refer to “the birds and the bees”. Yet for neurobiologists interested in uncovering basic facts about sex and reproduction, the fruit fly has proven much more informative.
Male fruit flies court females with a series of “hard-wired” or genetically programmed behaviors. One gene called doublesex generates differences in the anatomy and behavior of males and females in many animal species. In male fruit flies, the doublesex gene is active in roughly 650 neurons, with specific groups of cells controlling distinct steps of the courtship ritual. However, it was not understood how the different steps involved in copulation were coordinated to ensure a successful mating.
Pavlou et al. have now identified a circuit of doublesex-expressing neurons that controls copulation itself. The circuit, which is in the fruit fly’s equivalent of the spinal cord, is made up of three types of neurons: motor neurons, inhibitory interneurons and mechanosensory neurons. The motor neurons coordinate the joining of the male’s genitals with those of the female. The inhibitory interneurons promote the release of the male’s genitals by opposing the motor neurons, while the mechanosensory neurons possibly coordinate the activity of the other neurons to generate the correct sequence of events needed for copulation. Pavlou et al. also showed that the mechanism that controls how the male attaches to and detaches from the female is independent of ejaculation, indicating that the mechanics of copulation are separate from those of reproduction.
A future challenge will be to understand how command centres in the brain combine these signals with sensory feedback to enable males to execute and modify their copulation-related behaviors. Identifying neural circuits that drive behaviors in fruit flies provide insights into the universal principles by which a nervous system can coordinate complex motor behaviors such as walking and flying.
DOI: http://dx.doi.org/10.7554/eLife.20713.002
doi:10.7554/eLife.20713
PMCID: PMC5114013  PMID: 27855059
sexual behavior; sexual-dimorphism; copulation; doublesex; D. melanogaster
24.  Multimodal chemosensory circuits controlling male courtship in Drosophila 
Neuron  2015;87(5):1036-1049.
Summary
Throughout the animal kingdom, internal states generate long-lasting and self-perpetuating chains of behavior. In Drosophila, males instinctively pursue females with a lengthy and elaborate courtship ritual triggered by activation of sexually dimorphic P1 interneurons. Gustatory pheromones are thought to activate P1 neurons but the circuit mechanisms that dictate their sensory responses to gate entry into courtship remain unknown. Here, we use circuit mapping and in vivo functional imaging techniques to trace gustatory and olfactory pheromone circuits to their point of convergence onto P1 neurons and reveal how their combined input underlies selective tuning to appropriate sexual partners. We identify inhibition, even in response to courtship-promoting pheromones, as a key circuit element that tunes and tempers P1 neuron activity. Our results suggest a circuit mechanism in which balanced excitation and inhibition underlie discrimination of prospective mates and stringently regulate the transition to courtship in Drosophila.
doi:10.1016/j.neuron.2015.07.025
PMCID: PMC4560615  PMID: 26279475
25.  The KRÜPPEL-Like Transcription Factor DATILÓGRAFO Is Required in Specific Cholinergic Neurons for Sexual Receptivity in Drosophila Females 
PLoS Biology  2014;12(10):e1001964.
Female decision-making in Drosophila flies requires the expression of a transcription factor in a small number of cholinergic neurons in discrete brain regions.
Courtship is a widespread behavior in which one gender conveys to the other a series of cues about their species identity, gender, and suitability as mates. In many species, females decode these male displays and either accept or reject them. Despite the fact that courtship has been investigated for a long time, the genes and circuits that allow females to generate these mutually exclusive responses remain largely unknown. Here, we provide evidence that the Krüppel-like transcription factor datilógrafo (dati) is required for proper locomotion and courtship acceptance in adult Drosophila females. dati mutant females are completely unable to decode male courtship and almost invariably reject males. Molecular analyses reveal that dati is broadly expressed in the brain and its specific removal in excitatory cholinergic neurons recapitulates the female courtship behavioral phenotype but not the locomotor deficits, indicating that these are two separable functions. Clonal analyses in female brains identified three discrete foci where dati is required to generate acceptance. These include neurons around the antennal lobe, the lateral horn, and the posterior superior lateral protocerebrum. Together, these results show that dati is required to organize and maintain a relatively simple excitatory circuit in the brain that allows females to either accept or reject courting males.
Author Summary
Males of the fruit fly Drosophila melanogaster generate a series of courtship displays that convey visual, auditory, and olfactory information that females must decode in order to accept or reject mating. Despite the central role of female decision in sexual selection, relatively little is known about how genes and neural circuits generate this behavior. Here we show that the transcription factor datilografo (dati) is required to organize and maintain the neural circuitry required for acceptance in the central brain. Strikingly, dati is required in an excitatory circuit involving few neurons that express acetylcholine as their neurotransmitter and are located in the olfactory lobe, the first entry point for odor processing in the brain. In addition, dati is required in two other brain centers: a region where olfaction and presumably other senses are integrated and a novel region. Together these results show that a complex behavior can be generated by very few excitatory neurons, suggesting that the sharp cutoffs between acceptance and rejection may involve different thresholds of stimulation as postulated decades ago.
doi:10.1371/journal.pbio.1001964
PMCID: PMC4188565  PMID: 25291190

Results 1-25 (953428)