InteractiveFly: GeneBrief

Or65a: Biological Overview | References

Gene name - Odorant receptor 65a

Synonyms -

Cytological map position-65A14-65A14

Function - receptor

Keywords - odorant receptor, courtship behavior, aversive response, cis-vaccenyl acetate

Symbol - Or65a

FlyBase ID: FBgn0041625

Genetic map position - 3L: 6,313,711..6,315,198 [+]

Classification - G-protein coupled receptor

Cellular location - surface transmembrane

NCBI link: EntrezGene
Or65a orthologs: Biolitmine

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. This study shows 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 (Ejima, 2007).

In Drosophila, unsuccessful courtship decreases subsequent courtship. When the initial courtship object (trainer) is a virgin female, suppression has been shown to be the result of formation of an associative memory linking the failure to copulate with volatile stimulatory courtship cues specific to the age of the female trainer. Exposure to a mated female, on the other hand, results in a suppression of courtship toward all types of females and is believed to require an aversive pheromone. The cuticular hydrocarbon profiles of mature and immature females differ significantly, but these types of females also differ behaviorally. Mature virgins are receptive to courtship, while immature virgins and mated females show characteristic rejection behaviors. Immature females kick, fend, and run away, while mated females extrude their ovipositors. To determine whether female behavior or appearance had any role in the development of age-specific or general courtship suppression, males were trained and tested with decapitated females in dim red light. Memory index was expressed as a ratio of the courtship index (CI) during the 10 min test period to the mean CI of a sham-trained males tested with the same type of female. The use of a ratio allows direct comparison of the strength of memory between conditions, with a value of CItest/mCIsham = 1 indicating no memory (Ejima, 2007).

Consistent with results with mobile trainer females, decapitated virgins provoke an age-specific suppression, while decapitated mated female trainers cause general suppression of courtship. These data indicate that the specificity of learning with different trainer types does not stem from behavioral differences in the trainer female's response to courtship or from visual cues specific to the trainer type. Generalization of learning with a mated female trainer is therefore the result of chemosensory cues. In all subsequent experiments, decapitated trainers and testers were used, except where noted (Ejima, 2007).

In the previous experiment, males were placed in the same chamber as the trainer female and therefore could obtain both olfactory and gustatory information about that female. To investigate the nature of the generalization cue, attempts were made to reconstitute generalized learning with virgin trainers and mated female extracts. Placing a filter containing a hexane extract of mated female in the chamber with either a mature or immature trainer female caused a generalization of learning, as demonstrated by the ability of mature trainers to generate memory against immature testers and vice versa. To determine whether the active component of the mated female bouquet was volatile, a two-compartment courtship chamber was used, and a pheromone source (fly corpse or filter with extract) was placed across a mesh from the side of the chamber containing the male and the trainer female. For both mature and immature trainers, the presence of volatile compounds from either a mated female or a male was sufficient to cause generalization of courtship suppression, although the effects of these pheromones appeared more potent with mature trainers. In the absence of a courtship object, the presence of a filter with extract or a corpse did not generate suppression of courtship toward tester females (Ejima, 2007).

Next the identity of the generalization cue was addressed. The mated female and mature virgin trainers that were used were of the same age (4-5 days old) and might be expected to have similar cuticular hydrocarbon profiles, so any compound that differed between these two classes of females might have a role in generalization. Hexane washes of 4- to 5-day-old virgins and 4- to 5-day-old mated females that had been mated 24 hr before extraction were compared by using gas chromatography-flame ionization detection and mass spectrometry. Qualitatively, the two types of females appear identical with the exception of one peak, cis-vaccenyl acetate (cVA), which is undetectable in virgins but present at significant levels in mated females. cVA is a major component of mature male cuticular hydrocarbon and is not synthesized by females. Its presence in both males and mated females makes it a good candidate for being the generalization cue for courtship learning (Ejima, 2007).

Quantitation of mature virgin and mated female hydrocarbon levels shows a significant difference in cVA levels. There is also a small, but statistically insignificant, increase in 7-tricosene (7-C23:1). 7-tricosene is a major component of the male cuticular hydrocarbon and is believed to have inhibitory effects on male-male courtship. Transfer of 7-tricosene to females has been shown to occur via cuticular contact during copulation, but it is largely gone by 8 hr after mating. Consistent with this, larger amounts of 7-tricosene are seen on virgins that have been courted, but not copulated, when they are extracted immediately after the courtship. Mature virgins and mated females that have been aged 24 hr after copulation have lower and statistically indistinguishable levels of 7-tricosene. The loss over time (presumably through passive transfer and grooming) of 7-tricosene and the nonvolatile nature of this compound make it an unlikely candidate for the generalizing cue. It is also significant to note that no decreases in mated females are seen of hydrocarbons such as 7,11-heptacosadiene (7,11-nC27:2), 7,11-nonacosadiene (7,11-nC29:2), and 9-pentacosene (9-C25:1) that are believed to be stimulatory pheromones for courtship conditioning. Thus, the only consistent mated female-specific difference in hydrocarbon content that was found was in cVA (Ejima, 2007).

How does cVA, a male lipid, become part of the mated female pheromonal profile? Like 7-tricosene, cVA could be transferred directly by contact during courtship and/or copulation. Alternatively, the presence of cVA in the male ejaculatory bulb suggests that it can be transferred with sperm during copulation. To determine the major mode of cVA transmission, cVA levels were measured on virgin females, virgin females that were courted in a small chamber and extracted immediately, and females that were extracted 24 hr after complete copulation or disrupted copulation. Only females that copulated long enough to receive ejaculate have significant levels of cVA. Females that did not copulate and were merely courted by the male had virtually no cVA, even though they had significant amounts of passively acquired 7-tricosene. This suggests that transfer of cVA occurs via ejaculate and that mated females store cVA (Ejima, 2007).

These data support a role for cVA as a generalizing cue, but the presence of other volatile compounds in mated female and male extracts might still be required. To test the sufficiency of cVA, varying amounts of purified cVA were applied to filters across the mesh in a two compartment courtship chamber, males were trained with either mature or immature virgins, and then tested with a virgin of the other age. In both cases, cVA was sufficient to generalize memory. With mature virgin testers, 0.2 ng of cVA was enough to generalize memory. The average amount of cVA present on a mated female 24 hr after mating is 9.3 ± 3.5 ng, so the effects of synthetic cVA are occurring in the biologically relevant dose range. In contrast to results with mature virgins, pairing cVA with immature trainers is less effective. Only large amounts of cVA (200 μg) produce generalized learning. cVA alone (with no trainer female) is ineffective, as is cis-vaccenol (cVOH), a putative metabolite of cVA with either trainer type (Ejima, 2007).

The circuitry underlying generalization is of great interest for understanding this behavior. As a first step, attempts were made to identify the olfactory receptor neurons that carry the aversive cVA signal. cVA has been shown to be sensed by a subset of trichoid sensilla in the Drosophila antenna, which includes the T1 type sensillum that expresses Or67d. By using the 'empty neuron' preparation, which allows the decoding of odor specificity for Drosophila olfactory receptors (ORs), it was found that there is an additional cVA-responsive receptor, Or65a, and that Or65a and Or67d differ in their response to cVOH, with Or67d responding strongly and Or65a not responding. Or65a is one of the several ORs expressed in neurons of the T3 sensilla (Ejima, 2007).

With this information, the role was investigated of the olfactory receptor neurons that express these two receptors in sensing the aversiveness of cVA. Initial courtship levels provide a simple assay for this property of cVA. Naive males show lower levels of courtship toward mated females than toward virgins of the same age. This effect can be reproduced by addition of a cVA-laced filter across the mesh in the two-compartment courtship chamber with a mature virgin courtship object in the upper chamber with the male. Expression of tetanus toxin (TNT), which blocks synaptic release, under control of Or65a-GAL4, but not Or67d-GAL4, abolished the ability of cVA to inhibit initial courtship. Males heterozygous for Or65a-GAL4 or the UAS-TNT transgene showed cVA-dependent courtship suppression as did males expressing inactive toxin (TNT-VA) under control of Or65a-GAL4. These results indicated that ORNs expressing Or65a-GAL4, but not Or67d-GAL4, are required for sensing cVA as an aversive cue (Ejima, 2007).

Or65a has been reported to be expressed solely in ORNs that innervated the DL3 glomerulus of the antennal lobe with anti-GFP immunohistochemistry in animals expressing GFP under control of Or65a-GAL4 promoter fusions (Couto, 2005; Fishilevich; 2005). Or65a-GAL4, while it has strong expression in DL3, shows a somewhat broader pattern, with significant expression in VA1v, DC1, and DA4m. By using confocal microscopy to directly visualize GFP from a UAS-mCD8-GFP transgene in unfixed brains, Or65a-GAL4 line was compared to other published Or65a-GAL4 lines. The Or65a-GAL4 line used in this study was many times stronger than that published by Fishilevich (2005), which has predominant expression in DL3. GFP fluorescence in the Couto (2005) GAL4 line was barely detectable. To determine whether the weak, but more DL3-specific, Fishilevich driver would also block cVA effects, it was used to express active and inactive tetanus toxin. Consistent with the results with the Or65a-GAL4 used in this study, active tetanus toxin significantly abrogated the ability of cVA to suppress initial courtship, although the effect appeared weaker than with the line used in this study. Inactive tetanus toxin had no effect on cVA-mediated suppression. It is concluded that the aversive effects of cVA on initial courtship are most likely mediated by ORNs expressing Or65a (Ejima, 2007).

Since its identification as a male-specific lipid in the Drosophila ejaculatory bulb, there has been interest in cVA as a potential modifier of reproductive behavior. In this study, it has been shown pairing of cVA and a virgin female trainer is sufficient to reproduce the unique effects of exposure to a mated female: generalized suppression of male-female courtship. This study has identified neurons expressing Or65a-GAL4 as responsible for the aversive effects of cVA. These results provided the first molecular/genetic insights into the pheromones responsible for courtship learning (Ejima, 2007).

The results also provide some insight into the controversial nature of cVA's role in Drosophila behavior. The literature on cVA has posited roles for this lipid as both as an attractant and as an antiaphrodisiac, although this last function has been disputed. The social attractant role of cVA makes sense because it is deposited on eggs at feeding sites by females, and congregation at such sites is advantageous in terms of finding food and mates. The aversive role is equally plausible in light of cVA's transfer to females during mating, which would make it a marker of previous copulation. Understanding the molecular basis of cVA function and the circuitry subserving its behavioral effects will be necessary to completely unravel its multiple roles, but several important findings have emerged (Ejima, 2007).

First, there are multiple cVA receptors, and they appear to have different behavioral roles. Or67d, which is expressed in T1, singly innervated sensilla, has a role in sensing the attractive properties of cVA (Xu, 2005; Ha, 2006). This receptor is not required for the courtship inhibitory role of cVA; this function appears to be served by Or65a, which is expressed in one of the three neurons of the T3 trichoid sensilla. These data suggest that Or67d is an 'appetitive' cVA receptor while Or65a is an 'aversive' receptor. Segregating the hedonic effects of this lipid by activating two independent receptors is an interesting way of establishing, at an early step, independent behavioral circuits for attraction and repulsion. The lack of behavioral redundancy between Or65a and Or67d neurons is also interesting in light of findings with the nonpheromonal olfactory receptor Or43b, where elimination of the receptor does not change the behavioral response to its preferred odorant. The interpretation of this result was that other olfactory receptors that recognized the odorant, but projected to different antennal glomeruli, could signal the same behavioral response. These results suggest that for some pheromone odorants the antennal lobe circuitry they connect to is critical to the behavioral output they engender (Ejima, 2007 and references therein).

Second, responses to cVA appear to be context dependent. Having multiple sensory channels for cVA does not itself help the animal decide how to respond to this chemical; there must be some mechanism by which the environment or other cues can tell the animals which sensory channel is relevant for a particular situation. One way to achieve this would be to have the cVA channels be linked to other, situation-relevant, odor cues. In the case of both attraction and aversion, this appears to be the case. The first report of cVA as an attractant found that cVA was not attractive unless presented with food or food-associated odors. This studies assay set-up was designed to measure fast (in minutes) attractive responses in an open arena, as opposed to the long-term (days) maze/trap assays used by another, which did not uncover a role for food odor. The two paradigms may differ in sensitivity and relevance to particular behaviors, but the issue remains to be fully explored (Ejima, 2007 and references therein).

Context also appears to be important for the aversive effects of cVA. Synthetic cVA is a very effective, and completely sufficient, generalizing cue when applied in small doses to mature virgin trainers, but is not very effective, requiring 104 times more, when used with immature virgin trainers. The potency of mated female extracts with immature trainers is also less than with mature trainers, but the difference is not as exaggerated. This strongly suggests that some component of the mature female hydrocarbon profile that is not shared with immature virgins acts in concert with cVA to generalize learning. With the immature trainer, the mated female extract is supplying a low dose of cVA, but it also may supply a mature female compound that enhances the cVA effect. The identity of the compound(s) is unknown, but given that mature male extract is also able to allow generalization with immature trainers, it may be a hydrocarbon that is shared between mature males and females (Ejima, 2007).

The requirement for concurrent mature fly chemical signals for cVA to be an effective aversive cue and generalizer of learning is not unreasonable from an evolutionary point of view. Under normal circumstances, cVA is found only on males or mated females. The meaning of cVA in the presence of male hydrocarbons is clear: males should suppress courtship of other males because it is wasted reproductive energy. If a male in the wild sees cVA in the context of an immature female pheromone profile, however, it is likely that he has encountered a virgin at a feeding site where cVA-laced eggs have been deposited, and he should not suppress courtship (Ejima, 2007).

The underlying logic of suppressing courtship when presented with cVA in the context of a mature (and theoretically receptive) female is less obvious from a male's point of view. Copulation with a previously mated female is not ideal because she is already storing sperm from her previous mate, but there is still marginal gain; the second male's sperm can displace the first male's sperm. From the female's reproductive point of view, remating might also be advantageous because she will have more genetically diverse offspring, but it comes at a cost: it is correlated with reduced life span. The only player for whom remating does not have some advantage is the first male. It has been well documented that components of seminal fluid in Drosophila alter female behavior and reproduction to decrease remating. Transfer of cVA may be another facet of the successful male's strategy to decrease reproduction by competitor males. The effect of cVA on initial courtship decreases a second male's chances of success with that particular mated female, but the aftereffect, generalized suppression of his courtship drive, eliminates him as a competitor for other virgin females. The ability of cVA to engage the intrinsic plasticity machinery that allows animals to adapt to and learn from change to bring about a long-lasting change in another male's behavior could provide selective advantage to successfully copulating males (Ejima, 2007).

Receptors for mate recognition in Drosophila; Or65a and Or67d detect male-specific pheromones

Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila. The trichoid sensilla, one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, were systematically examined by electrophysiological analysis. 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 transferred from males to females during mating. 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. All members of the Odor receptor (Or) gene family that are expressed in trichoid sensilla were then systematically tested by using an in vivo expression system. Four receptors respond to fly odors in this system: Two respond to extracts of both males and virgin females, and two respond to cVA. A model is proposed describing how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic (van der Goes van Naters, 2007).

The responses of ORNs in trichoid sensilla of the antenna were measured by single-unit electrophysiology. All three trichoid-sensilla subtypes, T1, T2, and T3, which contain one, two, and three ORNs, respectively, were tested. These three subtypes occupy distinct but overlapping regions of the antennal surface and together comprise more than 20% of the sensilla in the antennae. Initially, 86 compounds were tested, most of which are found in fruits or are fermentation products. These compounds were tested on 60 trichoid sensilla, 30 from males and 30 from females. The compounds were tested in mixtures, and no mixture elicited a response greater than 20 impulses/s, which represents less than 10% of the maximal response of these ORNs. Some mixtures inhibited the spontaneous activity of T2 and T3 sensilla and produced decreases of 10-20 impulses/s in the action-potential rate. The three most inhibitory odors were subsequently determined to be 1-hexanol, hexyl acetate, and butyl acetate. The paucity of strong excitatory responses to food odors is consistent with the results of an earlier screen with a limited number of chemicals; in this earlier screen, no strong responses were found, although modest responses were elicited by trans-2-hexenal and cis-vaccenyl acetate (cVA) (van der Goes van Naters, 2007).

The odor of live flies was tested. 50 flies were placed in a glass tube that was closed at both ends with a cotton mesh. Air was puffed through the tube toward the antenna of a fly mounted for electrophysiological recording. 75 individual trichoid sensilla, of all three subtypes, were tested for responses to the odors of both males and virgin females. Air passing over male flies elicited a strong response from ORNs in a large group of trichoid sensilla. These ORNs did not respond to the odor of virgin females. These sensilla correspond to the T1 subtype, each of which houses a single ORN. T1 sensilla are found on both male and female antennae; in both cases they respond to the odor of males but not of virgin females. The T2 and T3 sensilla did not produce responses to fly odors when they were tested in this paradigm (van der Goes van Naters, 2007).

These experiments showed that at least some trichoid sensilla respond to fly odors. However, whether other trichoid sensilla might show responses to fly odors was tested in a more sensitive assay. A new paradigm was developed. Because flies approach each other closely during courtship, it was reasoned that some pheromone-sensitive sensilla might be adapted for short-range information reception. Some of the chemical cues that influence courtship behavior in Drosophila are present in the cuticle, i.e., on the surface of the fly, and are long-chain unsaturated hydrocarbons of very limited volatility. Although some of these cues are believed to be detected via the taste system, it seemed possible that the olfactory system might also contribute to the reception of cuticular components at very close range during courtship (van der Goes van Naters, 2007).

Accordingly, rather than adding odor stimuli to an air stream directed at the fly from a distance, stimuli were presented by approaching the antenna with the tip of a glass capillary carrying the odor. This procedure was designed to simulate the proximity of two interacting flies. As an initial test of the feasibility of this paradigm, 500 pl of a solution of cVA was draw into the capillary. cVA has been shown to act as an anti-aphrodisiac pheromone in Drosophila; there is also evidence for its playing a role as an aggregation pheromone. As the capillary tip approached certain trichoid sensilla, the impulse rates of certain ORNs increased and reached a maximum of >200 impulses/s upon physical contact of the capillary tip with the sensillum shaft. Control stimuli prepared with the hexane solvent alone gave no response (van der Goes van Naters, 2007).

Having established a short-range delivery paradigm, the responses, initially to cVA, of trichoid sensilla were systematically examined across the entire antennal surface. Mature male flies contain approximately 1 μg of cVA, primarily in the ejaculatory bulb. A capillary tip was loaded with 5 ng of cVA (0.005 fly equivalent) and 189 trichoid sensilla were approached individually. Strong responses of >100 impulses/s in were observed 169 of the 189 sensilla. Previous reports had shown that the ORN in T1 sensilla responds to cVA, and this study confirmed this finding. Responses to 5 ng of cVA exceeded 200 impulses/s in T1 sensilla. Also in agreement with the previous reports, some sensilla immediately adjacent to the zone containing T1 did not respond to cVA. However, it was determined that, in addition to the T1 subtype, a large number of sensilla more distolateral on the antennal surface also contained ORNs that are sensitive to cVA in this paradigm. Neurons in the distolateral sensilla responded to the cVA stimulus with a rate increase of more than 100 impulses/s. Thus, there appear to be at least two populations of sensilla with ORNs that respond to this pheromone (van der Goes van Naters, 2007).

To expand the scope of this analysis from a single defined pheromone, cVA, to a broad representation of the cuticular pheromone profile, hexane extracts of males and virgin females were prepared. Approximately 500 pl of extract was drawn into the capillary tip; this amount is equal to 0.25% of the material extracted from a single fly (van der Goes van Naters, 2007).

When a male extract was used as the odor source, all 147 trichoid sensilla tested, from all regions of the antennal surface, yielded responses. Different ORNs began to respond to the approaching odor source at different distances. The T1 sensilla, which house a single ORN, appeared to be particularly sensitive; they showed responses greater than 20 impulses/s when the odor source came within a 1 cm radius. As the odor source became still closer, the impulse rates increased rapidly. ORNs in T2 and T3 sensilla appeared to be less sensitive and had impulse rates increasing only after the odor source approached a distance of 200 μm, as determined with an ocular micrometer. The responses were dose dependent; when the dose was increased from 0.25% fly equivalent to 5% fly equivalent, the response radius increased from 200 μm to 500 μm (van der Goes van Naters, 2007).

When an extract from virgin females was used as the stimulus, strong responses were observed in ORNs of all trichoid sensilla except T1. Thus, T1 sensilla appear to be tuned to male odor, whereas T2 and T3 sensilla yield strong responses to both males and virgin females. Sensitivity to male and virgin-female extracts was comparable in T2 and T3 sensilla. These in vivo recordings, taken together, demonstrate that trichoid sensilla respond to fly odors and that the odors of males and virgin females are registered differently across the ensemble of trichoid sensilla. A limitation of the analysis is that it is difficult to ascribe responses to individual ORNs within trichoid sensilla. With the exception of T1, trichoid sensilla contain multiple ORNs. In recordings, this is evident from summation and cancellation events between impulses in the traces. In most cases it was not possible to discriminate the activities of the individual ORNs because the action potentials, as recorded extracellularly, did not differ significantly in size or shape. Because of the inability to classify action potentials with confidence, it was not possible to determine whether there is a functional subdivision among the ORNs sharing a sensillum. To address this limitation, advantage was taken of another experimental system, the 'empty neuron' system, in an effort to analyze the responses of trichoid sensilla at a higher resolution (van der Goes van Naters, 2007).

Drosophila contains a family of 60 Or (Odor receptor) genes, and the following 12 family members have been reported to map to individual ORNs of trichoid sensilla: Or2a, Or19a, Or19b, Or23a, Or43a, Or47b, Or65a, Or65b, Or65c, Or67d, Or83c, and Or88a. Each of these 12 Or genes were expressed in the 'empty neuron' system, an in vivo expression system based on a mutant ORN, ab3A, that resides in a basiconic sensillum. The endogenous receptor genes of this ORN, Or22a and Or22b, are deleted, and the promoter of Or22a drives ectopic expression of another odor receptor in ab3A via the UAS-GAL4 system. The odor responses conferred upon ab3A by the ectopically expressed receptor are then measured by single-unit electrophysiology (van der Goes van Naters, 2007).

The 12 trichoid receptors were systematically tested in the empty-neuron system with a panel of fly-derived chemicals: hexane extracts of males and virgin females, material from the genital regions of flies (males, virgin females, and mated females), and cVA. The genital odors were obtained by drawing a glass capillary, with a tip pulled to a diameter of 3 μm, across the genital region of a fly such that material visibly coated the tip. Preliminary experiments showed that the responses could be quantified most reproducibly not during the approach of a stimulus to the antenna but after the capillary tip contacted the sensillum. Responses mediated by the trichoid receptors were were therefore quantified by determining impulse rates of the ORN after contact. The 12 receptors were expressed and tested in both male and female recipients with all six stimuli, and no differences between the responses of male and female flies were identified (van der Goes van Naters, 2007).

Of the 12 receptors, four mediated responses to fly odors in this system. All four, Or47b, Or65a, Or67d, and Or88a, responded to male extract, and their action-potential frequencies increased by 50-200 impulses/s. Two of these receptors, Or65a and Or67d, did not respond to extract from virgin females. The sex specificity of Or65a and Or67d is consistent with a role for these receptors in the detection of male-specific pheromones. The other two receptors, Or47b and Or88a, responded to extract from virgin females; these responses were comparable to those they gave to male extracts. It was noted that both Or47b and Or88a were previously tested in the empty-neuron system with a panel of 110 odors, most of which were present in fruits and were of widely varying chemical structures, and no excitatory responses were recorded. These results are consistent with the hypothesis that Or47b and Or88a detect a pheromone secreted by both males and females (van der Goes van Naters, 2007).

Male genital material elicited strong responses from Or65a, Or67d, and Or88a. Genital material from virgin females did not elicit a strong response from any of the 12 receptors. However, material from the genital region of females that were mated 1-4 hr previously produced responses from these three receptors, which, yielded firing rates comparable to those observed with male genital material. These results suggest that during copulation the male transfers compounds that activate these receptors (van der Goes van Naters, 2007).

One compound that the male transfers to the female during copulation is cVA. The sensitivity of Or67d to cVA is consistent with previous observations; expression studies have shown that Or67d is expressed in T1 sensilla, which are sensitive to cVA, and ectopic expression of Or67d in other trichoid sensilla conferred sensitivity to cVA. However, the results indicate that there are multiple receptors for cVA. Both Or67d and Or65a responded most strongly to cVA among a panel of six related compounds. The two receptors differed in their specificities, however; Or67d gave a relatively stronger response than did Or65a to cis-vaccenyl alcohol, for example. It is noted that the detection of a second cVA receptor, which has not been reported previously, may reflect the sensitivity of the short-range delivery paradigm that was designed (van der Goes van Naters, 2007).

The response specificity of Or67d, as measured in the empty-neuron system, is nearly identical to that of the ORN in the T1 sensillum. However, it is noted that the magnitude of the response to cVA in the expression system is approximately half that in T1. Dose-response curves show that the response threshold is also lower in the native T1 sensillum; it appears as though the T1 neuron can detect a dose of approximately 10−4 ng, whereas the expressed Or67d receptor may require a dose of approximately 10−2 ng for detection. Slower rise and decay rates were also found, along with higher levels of spontaneous firing in the expression system. These results suggest that the expression system may lack a component that is present in the endogenous context; for example, the odorant-binding protein Lush was found to be required for normal response to cVA in T1 sensilla (van der Goes van Naters, 2007).

Whereas Or67d mediates responses to cVA in T1 sensilla, Or65a is expressed in the ORNs of trichoid sensilla that are more distolateral on the antenna and that also respond to cVA. It is noted that the Or65a gene is in close proximity to Or65b and Or65c and that the three genes are coexpressed in a single ORN. Although neither Or65b nor Or65c mediated responses to any of the fly odors tested in the empty-neuron system, the possibility was considered that they might contribute to the response of the ORN if they were coexpressed with Or65a, perhaps via heterodimer formation. Accordingly, all pairwise combinations of the three receptor genes were co-expressed. It was found that coexpression of Or65b or Or65c with Or65a did not increase the response mediated by Or65a to any stimulus or change the level of spontaneous activity. Coexpression of Or65b and Or65c yielded little, if any, response to any stimulus (van der Goes van Naters, 2007).

Finally, it is noted with interest that although Or88a conferred responses to male genital material, it did not mediate responses to cVA, suggesting that it detects an additional pheromone that is also transferred from males to females upon mating (van der Goes van Naters, 2007).

This study has identified four receptors that mediate responses to fly odors. Or47b and Or88a mediate responses to the odors of both males and virgin females. Or65a and Or67d mediate responses to cVA, a male-specific lipid that is present in male genital material, is presumably extracted in hexane extracts, and is transferred to females upon mating. Or88a also responds to a compound in male genitalia, but this compound is distinct from cVA (van der Goes van Naters, 2007).

The responses of these receptors suggest a working model of the olfactory basis of mate recognition by males. In this model, neural activity mediated by Or47b and Or88a reports the proximity of a fly, either male or female. This olfactory recognition may contribute to the recognition mediated by other sensory modalities; recognition of conspecifics is a prerequisite to successful courtship. The activity of Or65a, Or67d, or both would indicate that the partner is a male or a recently mated female; thus, when the antenna of a male is in close proximity to another fly, the activation of Or65a and/or Or67d would report that the other fly is unsuitable as a mate. The lack of a signal from these receptors would permit continued courtship activity by the male (van der Goes van Naters, 2007).

A well-documented phenomenon can be interpreted in terms of this model. Mature males not only court virgin females but also vigorously court newly eclosed males. Young males, like virgin females, lack cVA and would not be expected to activate Or65a and Or67d, allowing courtship to proceed (van der Goes van Naters, 2007).

Why would Or65a and Or67d not be activated in the antenna of a male by material in its own genital region? Perhaps very little of the internal genital material is released to the air unless the region is manipulated by a capillary tip or washed in hexane, and perhaps what little is released under natural conditions can normally be detected only at very close range; if cVA were released in large amounts and inhibited mating over a long range, then mating might be inhibited at sites where flies congregate and often mate, such as rich food sources. It is also possible that the fly adapts to the ambient level of cVA, produced by its own genital region, and is sensitive to increases above that level (van der Goes van Naters, 2007).

Why are there two cVA receptors, expressed in two distinct ORNs, in different subtypes of trichoid sensilla? There is evidence that cVA serves two functions as a pheromone in Drosophila. (1) cVA has been shown to act as an anti-aphrodisiac, detering males from courting with a recently mated female. (2) cVA is deposited by females during egg laying, and there is evidence that it enhances the attractiveness of the oviposition substrate to other flies. Perhaps Or65a and Or67d activate two distinct behavioral circuits and thereby separately mediate two functions of cVA in conjunction with other cues (van der Goes van Naters, 2007).

Interestingly, no receptor for female-specific odors was identifed, although there is evidence that 7,11-heptacosadiene and 7,11-nonacosadiene, two female-specific hydrocarbons, act as aphrodisiacs. It is possible that some of the trichoid receptors respond to these compounds, which were not tested individually, or other female-specific compounds but do not function efficiently in the expression system. It is also possible that these compounds are detected by gustatory receptors, perhaps members of the Gr family. One class of gustatory neuron, which expresses Gr68a, has been shown to be required for normal courtship. Finally, the possibility is noted that some of the receptors that did not respond to the tested stimuli might detect pheromones of other Drosophila species (van der Goes van Naters, 2007).

It is striking that no differences were observed between males' and females' antennal responses to any of the fly odors tested. This similarity is in stark contrast to the extreme sexual dimorphism in antennal responses to pheromones in moths, such as Bombyx mori and Manduca sexta. The similarity in Drosophila peripheral olfactory responses suggests that in the fly, differences in male and female behavioral responses may be determined by differences in reception of other classes of sensory input, such as taste information, or by differences in the transmission or processing of olfactory information. It is possible that cVA, for example, is sensed through the same peripheral mechanisms in males and females but that only in males is the primary representation transformed in a way that accords it a negative valence (van der Goes van Naters, 2007).

In summary, a systematic analysis was carried out of the trichoid sensilla, one of the three major types of sensilla on the Drosophila antenna. These sensilla appear to be specialized for sensing fly odors, as opposed to food odors. The differential activity of ORNs in trichoid sensilla provides an olfactory basis for a male's ability to discriminate suitable from unsuitable mating partners. The molecular basis of these responses was further explored and four odor receptors were identified that mediate responses to fly odors. A model is proposed in which olfactory information flows through these receptors according to a simple logic. Although the full repertoire of pheromones and receptors has yet to be characterized, it is possible that the model may be richly elaborated without undergoing an alteration in its fundamental logic (van der Goes van Naters, 2007).

Love makes smell blind: mating suppresses pheromone attraction in Drosophila females via Or65a olfactory neurons

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. This study shows 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 (Lebreton, 2014 PubMed).


Search PubMed for articles about Drosophila Or65a

Couto, A., Alenius, M. and Dickson, B. J. (2005). Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr. Biol. 15: 1535-1547. PubMed ID: 16139208

Ejima, A., et al. (2007). Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate. Curr. Biol. 17(7): 599-605. PubMed ID: 17363250

Fishilevich, E. and Vosshall, L. B. (2005). Genetic and functional subdivision of the Drosophila antennal lobe. Curr. Biol. 15: 1548-1553. PubMed ID: 16139209

Ha, T. S. and Smith, D. P. (2006). A pheromone receptor mediates 11-cis-vaccenyl acetate-induced responses in Drosophila. J. Neurosci. 26(34): 8727-33. PubMed ID: 16928861

Lebreton, S., Grabe, V., Omondi, A. B., Ignell, R., Becher, P. G., Hansson, B. S., Sachse, S. and Witzgall, P. (2014). Love makes smell blind: mating suppresses pheromone attraction in Drosophila females via Or65a olfactory neurons. Sci Rep 4: 7119. PubMed ID: 25406576

van der Goes van Naters, W. and Carlson, J. R. (2007). Receptors and neurons for fly odors in Drosophila. Curr. Biol. 17(7): 606-12. PubMed ID: 17363256

Xu, P., Atkinson, R., Jones, D. N. and Smith, D. P. (2005). Drosophila OBP Lush is required for activity of pheromone-sensitive neurons. Neuron 45: 193-200. PubMed ID: 15664171

Biological Overview

date revised: 30 December 2014

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