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1.  Activation of the T1 Neuronal Circuit is Necessary and Sufficient to Induce Sexually Dimorphic Mating Behavior in Drosophila 
The molecular and cellular events mediating complex behaviors in animals are largely unknown. Elucidating the circuits underlying behaviors in simple model systems may shed light on how these circuits function. In Drosophila, courtship behavior provides a tractable model for studying the underlying basis of innate behavior. The male-specific pheromone 11-cis-vaccenyl acetate (cVA) modulates courtship behavior and is detected by T1 neurons, located on the antenna of male and female flies. The T1 neurons express the odorant receptor Or67d, and are exquisitely tuned to cVA pheromone. However, cVA-induced changes in mating behavior have also been reported upon manipulation of olfactory neurons expressing odorant receptor Or65a. These findings raise the issue of whether multiple olfactory-driven circuits underlie cVA-induced behavioral responses, and what role these circuits play in behavior. Here, we engineered flies in which the Or67d circuit is specifically activated in the absence of cVA in order to determine the role of this circuit in behavior. We created transgenic flies that express a dominant-active, pheromone-independent variant of the extracellular pheromone receptor, LUSH. We found that, similar to the behaviors elicited by cVA, engineered male flies have dramatically reduced courtship, while engineered females showed enhanced courtship. Furthermore, cVA exposure did not enhance the dominant LUSH-triggered effects on behavior in the engineered flies. Finally, we show the effects of both cVA and dominant LUSH on courtship are reversed by genetically removing Or67d. These findings demonstrate that the T1/Or67d circuit is necessary and sufficient to mediate sexually dimorphic courtship behaviors.
doi:10.1523/JNEUROSCI.4819-09.2010
PMCID: PMC3426441  PMID: 20164344
lush; Or67d; cVA; courtship; pheromone; olfaction
2.  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
3.  Receptors and neurons for fly odors in Drosophila 
Current biology : CB  2007;17(7):606-612.
Summary
Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila [1]. We systematically examine one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, the trichoid sensilla, by electrophysiological analysis. We find that none respond strongly to food odors, but all respond to fly odors. Two subtypes of trichoid sensilla contain ORNs that respond to cis-vaccenyl acetate (cVA), an anti-aphrodisiac pheromone present in males and transferred to females during mating [2–4]. All trichoid sensilla yield responses to a male extract; a subset yield responses to a virgin female extract as well. Thus males can be distinguished from virgin females by the activity they elicit among the trichoid ORN population. We then systematically test all members of the Odor receptor (Or) gene family [5–7] that are expressed in trichoid sensilla [8], using an in vivo expression system [9]. Four receptors respond to fly odors in this system: two respond to extracts of both males and virgin females, and two respond to cVA. We propose a model for how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic.
doi:10.1016/j.cub.2007.02.043
PMCID: PMC1876700  PMID: 17363256
4.  Degradation of Pheromone and Plant Volatile Components by a Same Odorant-Degrading Enzyme in the Cotton Leafworm, Spodoptera littoralis 
PLoS ONE  2011;6(12):e29147.
Background
Odorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant.
Methodology
Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis, a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants.
Conclusion
SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.
doi:10.1371/journal.pone.0029147
PMCID: PMC3246455  PMID: 22216190
5.  Characterization of an Antennal Carboxylesterase from the Pest Moth Spodoptera littoralis Degrading a Host Plant Odorant 
PLoS ONE  2010;5(11):e15026.
Background
Carboxyl/cholinesterases (CCEs) are highly diversified in insects. These enzymes have a broad range of proposed functions, in neuro/developmental processes, dietary detoxification, insecticide resistance or hormone/pheromone degradation. As few functional data are available on purified or recombinant CCEs, the physiological role of most of these enzymes is unknown. Concerning their role in olfaction, only two CCEs able to metabolize sex pheromones have been functionally characterized in insects. These enzymes are only expressed in the male antennae, and secreted into the lumen of the pheromone-sensitive sensilla. CCEs able to hydrolyze other odorants than sex pheromones, such as plant volatiles, have not been identified.
Methodology
In Spodoptera littoralis, a major crop pest, a diversity of antennal CCEs has been previously identified. We have employed here a combination of molecular biology, biochemistry and electrophysiology approaches to functionally characterize an intracellular CCE, SlCXE10, whose predominant expression in the olfactory sensilla suggested a role in olfaction. A recombinant protein was produced using the baculovirus system and we tested its catabolic properties towards a plant volatile and the sex pheromone components.
Conclusion
We showed that SlCXE10 could efficiently hydrolyze a green leaf volatile and to a lesser extent the sex pheromone components. The transcript level in male antennae was also strongly induced by exposure to this plant odorant. In antennae, SlCXE10 expression was associated with sensilla responding to the sex pheromones and to plant odours. These results suggest that a CCE-based intracellular metabolism of odorants could occur in insect antennae, in addition to the extracellular metabolism occurring within the sensillar lumen. This is the first functional characterization of an Odorant-Degrading Enzyme active towards a host plant volatile.
doi:10.1371/journal.pone.0015026
PMCID: PMC2993938  PMID: 21124773
6.  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
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.  Annotation and expression of carboxylesterases in the silkworm, Bombyx mori 
BMC Genomics  2009;10:553.
Background
Carboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. It plays important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. Previous studies mainly used Dipteran Drosophila and mosquitoes as model organisms to investigate the roles of the insect COEs in insecticide resistance. However, genome-wide characterization of COEs in phytophagous insects and comparative analysis remain to be performed.
Results
Based on the newly assembled genome sequence, 76 putative COEs were identified in Bombyx mori. Relative to other Dipteran and Hymenopteran insects, alpha-esterases were significantly expanded in the silkworm. Genomics analysis suggested that BmCOEs showed chromosome preferable distribution and 55% of which were tandem arranged. Sixty-one BmCOEs were transcribed based on cDNA/ESTs and microarray data. Generally, most of the COEs showed tissue specific expressions and expression level between male and female did not display obvious differences. Three main patterns could be classified, i.e. midgut-, head and integument-, and silk gland-specific expressions. Midgut is the first barrier of xenobiotics peroral toxicity, in which COEs may be involved in eliminating secondary metabolites of mulberry leaves and contaminants of insecticides in diet. For head and integument-class, most of the members were homologous to odorant-degrading enzyme (ODE) and antennal esterase. RT-PCR verified that the ODE-like esterases were also highly expressed in larvae antenna and maxilla, and thus they may play important roles in degradation of plant volatiles or other xenobiotics.
Conclusion
B. mori has the largest number of insect COE genes characterized to date. Comparative genomic analysis suggested that the gene expansion mainly occurred in silkworm alpha-esterases. Expression evidence indicated that the expanded genes were specifically expressed in midgut, integument and head, implying that these genes may have important roles in detoxifying secondary metabolites of mulberry leaves, contaminants in diet, and odorants. Our results provide some new insights into functions and evolutionary characteristics of COEs in phytophagous insects.
doi:10.1186/1471-2164-10-553
PMCID: PMC2784812  PMID: 19930670
9.  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
10.  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
11.  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
12.  Moth Sex Pheromone Receptors and Deceitful Parapheromones 
PLoS ONE  2012;7(7):e41653.
The insect's olfactory system is so selective that male moths, for example, can discriminate female-produced sex pheromones from compounds with minimal structural modifications. Yet, there is an exception for this “lock-and-key” tight selectivity. Formate analogs can be used as replacement for less chemically stable, long-chain aldehyde pheromones, because male moths respond physiologically and behaviorally to these parapheromones. However, it remained hitherto unknown how formate analogs interact with aldehyde-sensitive odorant receptors (ORs). Neuronal responses to semiochemicals were investigated with single sensillum recordings. Odorant receptors (ORs) were cloned using degenerate primers, and tested with the Xenopus oocyte expression system. Quality, relative quantity, and purity of samples were evaluated by gas chromatography and gas chromatography-mass spectrometry. We identified olfactory receptor neurons (ORNs) housed in trichoid sensilla on the antennae of male navel orangeworm that responded equally to the main constituent of the sex pheromone, (11Z,13Z)-hexadecadienal (Z11Z13-16Ald), and its formate analog, (9Z,11Z)-tetradecen-1-yl formate (Z9Z11-14OFor). We cloned an odorant receptor co-receptor (Orco) and aldehyde-sensitive ORs from the navel orangeworm, one of which (AtraOR1) was expressed specifically in male antennae. AtraOR1•AtraOrco-expressing oocytes responded mainly to Z11Z13-16Ald, with moderate sensitivity to another component of the sex pheromone, (11Z,13Z)-hexadecadien-1-ol. Surprisingly, this receptor was more sensitive to the related formate than to the natural sex pheromone. A pheromone receptor from Heliothis virescens, HR13 ( = HvirOR13) showed a similar profile, with stronger responses elicited by a formate analog than to the natural sex pheromone, (11Z)-hexadecenal thus suggesting this might be a common feature of moth pheromone receptors.
doi:10.1371/journal.pone.0041653
PMCID: PMC3401280  PMID: 22911835
13.  Identification of Odor-Processing Genes in the Emerald Ash Borer, Agrilus planipennis 
PLoS ONE  2013;8(2):e56555.
Background
Insects rely on olfaction to locate food, mates, and suitable oviposition sites for successful completion of their life cycle. Agrilus planipennis Fairmaire (emerald ash borer) is a serious invasive insect pest that has killed tens of millions of North American ash (Fraxinus spp) trees and threatens the very existence of the genus Fraxinus. Adult A. planipennis are attracted to host volatiles and conspecifics; however, to date no molecular knowledge exists on olfaction in A. planipennis. Hence, we undertook an antennae-specific transcriptomic study to identify the repertoire of odor processing genes involved in A. planipennis olfaction.
Methodology and Principal Findings
We acquired 139,085 Roche/454 GS FLX transcriptomic reads that were assembled into 30,615 high quality expressed sequence tags (ESTs), including 3,249 isotigs and 27,366 non-isotigs (contigs and singletons). Intriguingly, the majority of the A. planipennis antennal transcripts (59.72%) did not show similarity with sequences deposited in the non-redundant database of GenBank, potentially representing novel genes. Functional annotation and KEGG analysis revealed pathways associated with signaling and detoxification. Several odor processing genes (9 odorant binding proteins, 2 odorant receptors, 1 sensory neuron membrane protein and 134 odorant/xenobiotic degradation enzymes, including cytochrome P450s, glutathione-S-transferases; esterases, etc.) putatively involved in olfaction processes were identified. Quantitative PCR of candidate genes in male and female A. planipennis in different developmental stages revealed developmental- and sex-biased expression patterns.
Conclusions and Significance
The antennal ESTs derived from A. planipennis constitute a rich molecular resource for the identification of genes potentially involved in the olfaction process of A. planipennis. These findings should help in understanding the processing of antennally-active compounds (e.g. 7-epi-sesquithujene) previously identified in this serious invasive pest.
doi:10.1371/journal.pone.0056555
PMCID: PMC3570424  PMID: 23424668
14.  A Presynaptic Gain Control Mechanism Fine-Tunes Olfactory Behavior 
Neuron  2008;59(2):311-321.
Early sensory processing can play a critical role in sensing environmental cues. We have investigated the physiological and behavioral function of gain control at the first synapse of olfactory processing in Drosophila. We report that olfactory receptor neurons (ORNs) express the GABAB receptor (GABABR) and its expression expands the dynamic range of ORN synaptic transmission that is preserved in projection neuron responses. Strikingly, we find that different ORN channels have unique baseline levels of GABABR expression. ORNs that sense the aversive odorant CO2 do not express GABABRs nor exhibit any presynaptic inhibition. In contrast, pheromone-sensing ORNs express a high level of GABABRs and exhibit strong presynaptic inhibition. Furthermore, a behavioral significance of presynaptic inhibition was revealed by a courtship behavior in which pheromone-dependent mate localization is impaired in flies that lack GABABRs in specific ORNs. Together, these findings indicate that different olfactory receptor channels may employ heterogeneous presynaptic gain control as a mechanism to allow an animal’s innate behavioral responses to match its ecological needs.
doi:10.1016/j.neuron.2008.07.003
PMCID: PMC2539065  PMID: 18667158
Drosophila; olfaction; GABAB; presynaptic inhibition; gain control; dynamic range; two-photon imaging
15.  Drosophila Pheromone-Sensing Neurons Expressing the ppk25 Ion Channel Subunit Stimulate Male Courtship and Female Receptivity 
PLoS Genetics  2014;10(3):e1004238.
As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25 is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating courtship and mating.
Author Summary
Drosophila mating behaviors serve as an attractive model to understand how external sensory cues are detected and used to generate appropriate behavioral responses. Pheromones present on the cuticle of Drosophila have important roles in stimulating male courtship toward females and inhibiting male courtship directed at other males. Recently, stimulatory pheromones emitted by females and inhibitory pheromones emitted by males have been shown to stimulate distinct subsets of gustatory neurons on the legs. We have previously shown that a DEG/ENaC ion channel subunit, ppk25, is involved in male courtship toward females but not in inhibition of male-male courtship. Here we show that ppk25 is specifically expressed and functions in a subset of gustatory neurons that mediate physiological and behavioral responses to female-specific stimulatory pheromones. Furthermore, ppk25 is also required for the function of those neurons to activate male courtship in response to other pheromones that are not female-specific. In addition to their roles in males, we find that ppk25, and the related DEG/ENaC subunits ppk23 and ppk29, also stimulate female mating behavior. In conclusion, these results show that, in both sexes, ppk25 functions in a group of neurons with a specialized role in stimulating mating behaviors.
doi:10.1371/journal.pgen.1004238
PMCID: PMC3967927  PMID: 24675786
16.  Intensity invariant dynamics and odor-specific latencies in olfactory receptor neuron response 
Odors elicit spatio-temporal patterns of activity in the brain. Spatial patterns arise from the specificity of the interaction between odorants and odorant receptors expressed in different olfactory receptor neurons (ORNs). But the origin of temporal patterns of activity and their role in odor coding remain unclear. We investigate how physiological aspects of ORN response and physical aspects of odor stimuli give rise to diverse responses in Drosophila ORNs. We show that odor stimuli have intrinsic dynamics that depend on odor type and strongly affect ORN response. Using linear-nonlinear modeling to remove the contribution of the stimulus dynamics from the ORN dynamics we study the physiological properties of the response to different odorants and concentrations. For several odorants and receptor types the ORN response dynamics normalized by the peak response are independent of stimulus intensity for a large portion of the neuron’s dynamic range. Adaptation to a background odor changes the gain and dynamic range of the response but does not affect normalized response dynamics. Stimulating ORNs with various odorants reveals significant odor-dependent delays in the ORN response functions. These differences however can be dominated by differences in stimulus dynamics. In one case the response of one ORN to two odorants is predicted solely from measurements of the odor signals. Within a large portion of their dynamic range ORNs can capture information about stimulus dynamics independently from intensity while introducing odor-dependent delays. How insects might use odor-specific stimulus dynamics and ORN dynamics in discrimination and navigation tasks remains an open question.
doi:10.1523/JNEUROSCI.0426-12.2013
PMCID: PMC3678969  PMID: 23575828
17.  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
18.  Expressed Sequence Tags from Cephalic Chemosensory Organs of the Northern Walnut Husk Fly, Rhagoletis suavis, Including a Putative Canonical Odorant Receptor 
Rhagoletis fruit flies are important both as major agricultural pests and as model organisms for the study of adaptation to new host plants and host race formation. Response to fruit odor plays a critical role in such adaptation. To better understand olfaction in Rhagoletis, an expressed sequence tag (EST) study was carried out on the antennae and maxillary palps of Rhagoletis suavis (Loew) (Diptera: Tephritidae), a common pest of walnuts in eastern United States. After cDNA cloning and sequencing, 544 ESTs were annotated. Of these, 66% had an open reading frame and could be matched to a previously sequenced gene. Based on BLAST sequence homology, 9% (49 of 544 sequences) were nuclear genes potentially involved in olfaction. The most significant finding is a putative odorant receptor (OR), RSOr1, that is homologous to Drosophila melanogaster Or49a and Or85f. This is the first tephritid OR discovered that might recognize a specific odorant. Other olfactory genes recovered included odorant binding proteins, chemosensory proteins, and putative odorant degrading enzymes.
doi:10.1673/031.010.5101
PMCID: PMC3014805  PMID: 20569128
host race; jugions nigra; olfaction; odorant receptor; Rhagoletis; Tephritidae; speciation
19.  Pheromonal and Behavioral Cues Trigger Male-to-Female Aggression in Drosophila 
PLoS Biology  2010;8(11):e1000541.
By genetically manipulating both pheromonal profiles and behavioral patterns, we find that Drosophila males showed a complete reversal in their patterns of aggression towards other males and females
Appropriate displays of aggression rely on the ability to recognize potential competitors. As in most species, Drosophila males fight with other males and do not attack females. In insects, sex recognition is strongly dependent on chemosensory communication, mediated by cuticular hydrocarbons acting as pheromones. While the roles of chemical and other sensory cues in stimulating male to female courtship have been well characterized in Drosophila, the signals that elicit aggression remain unclear. Here we show that when female pheromones or behavior are masculinized, males recognize females as competitors and switch from courtship to aggression. To masculinize female pheromones, a transgene carrying dsRNA for the sex determination factor transformer (traIR) was targeted to the pheromone producing cells, the oenocytes. Shortly after copulation males attacked these females, indicating that pheromonal cues can override other sensory cues. Surprisingly, masculinization of female behavior by targeting traIR to the nervous system in an otherwise normal female also was sufficient to trigger male aggression. Simultaneous masculinization of both pheromones and behavior induced a complete switch in the normal male response to a female. Control males now fought rather than copulated with these females. In a reciprocal experiment, feminization of the oenocytes and nervous system in males by expression of transformer (traF) elicited high levels of courtship and little or no aggression from control males. Finally, when confronted with flies devoid of pheromones, control males attacked male but not female opponents, suggesting that aggression is not a default behavior in the absence of pheromonal cues. Thus, our results show that masculinization of either pheromones or behavior in females is sufficient to trigger male-to-female aggression. Moreover, by manipulating both the pheromonal profile and the fighting patterns displayed by the opponent, male behavioral responses towards males and females can be completely reversed. Therefore, both pheromonal and behavioral cues are used by Drosophila males in recognizing a conspecific as a competitor.
Author Summary
As in other species, the fruit fly Drosophila melanogaster uses chemical signals in the form of pheromones to recognize the species and sex of another individual. Males typically fight with other males and do not attack females. While the roles of pheromonal and other sensory cues in stimulating courtship towards females have been extensively studied, the signals that elicit aggression towards other males remain unclear. In this work, we use genetic tools to show that masculinization of female pheromones is sufficient to trigger aggression from wild type males towards females. Surprisingly, males also attacked females that displayed male patterns of aggression, even if they show normal female pheromonal profiles, indicating that pheromones are not the only cues important for identifying another animal as an opponent. By simultaneously manipulating pheromones and behavioral patterns of opponents, we can completely switch the behavioral response of males towards females and males. These results demonstrate that not only pheromonal but also behavioral cues can serve as triggers of aggression, underlining the importance of behavioral feedback in the manifestation of social behaviors.
doi:10.1371/journal.pbio.1000541
PMCID: PMC2990703  PMID: 21124886
20.  An Expressed Sequence Tag collection from the male antennae of the Noctuid moth Spodoptera littoralis: a resource for olfactory and pheromone detection research 
BMC Genomics  2011;12:86.
Background
Nocturnal insects such as moths are ideal models to study the molecular bases of olfaction that they use, among examples, for the detection of mating partners and host plants. Knowing how an odour generates a neuronal signal in insect antennae is crucial for understanding the physiological bases of olfaction, and also could lead to the identification of original targets for the development of olfactory-based control strategies against herbivorous moth pests. Here, we describe an Expressed Sequence Tag (EST) project to characterize the antennal transcriptome of the noctuid pest model, Spodoptera littoralis, and to identify candidate genes involved in odour/pheromone detection.
Results
By targeting cDNAs from male antennae, we biased gene discovery towards genes potentially involved in male olfaction, including pheromone reception. A total of 20760 ESTs were obtained from a normalized library and were assembled in 9033 unigenes. 6530 were annotated based on BLAST analyses and gene prediction software identified 6738 ORFs. The unigenes were compared to the Bombyx mori proteome and to ESTs derived from Lepidoptera transcriptome projects. We identified a large number of candidate genes involved in odour and pheromone detection and turnover, including 31 candidate chemosensory receptor genes, but also genes potentially involved in olfactory modulation.
Conclusions
Our project has generated a large collection of antennal transcripts from a Lepidoptera. The normalization process, allowing enrichment in low abundant genes, proved to be particularly relevant to identify chemosensory receptors in a species for which no genomic data are available. Our results also suggest that olfactory modulation can take place at the level of the antennae itself. These EST resources will be invaluable for exploring the mechanisms of olfaction and pheromone detection in S. littoralis, and for ultimately identifying original targets to fight against moth herbivorous pests.
doi:10.1186/1471-2164-12-86
PMCID: PMC3045336  PMID: 21276261
21.  A Single Sex Pheromone Receptor Determines Chemical Response Specificity of Sexual Behavior in the Silkmoth Bombyx mori 
PLoS Genetics  2011;7(6):e1002115.
In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex pheromones. In most moth species, male moths rely heavily on species-specific sex pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible pheromone system, in which a single pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex pheromone receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex pheromone receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex pheromone receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex pheromone receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol receptor neurons alone is sufficient to trigger full sexual behavior. Thus, a single gene defines behavioral selectivity in sex pheromone communication in the silkmoth. Our findings show that a single molecular determinant can not only function as a modulator of behavior but also as an all-or-nothing initiator of a complex species-specific behavioral sequence.
Author Summary
Like many animal species, moths use chemical signals called sex pheromones to communicate with conspecific individuals of the opposite sex in the context of reproduction. Typically, male moths depend on sex pheromones emitted by conspecific females to identify and locate their mates. Therefore, the behavioral preference of male moths to conspecific pheromones is a critical factor for successful reproduction. Sex pheromone receptor proteins expressed in specialized antennal olfactory receptor neurons reportedly play a central role in sex pheromone discrimination. However, the causal relationship between sex pheromone receptor specificity and behavioral preference remains to be proven. We have addressed this question in a genetically tractable moth species, the silkmoth (Bombyx mori), because this species possesses the simplest possible pheromone system in which a single pheromone substance, bombykol, elicits full sexual behavior. Using transgenic silkmoths expressing a sex pheromone receptor from another moth species, we revealed that solely the chemical specificity of the odorant receptors in bombykol receptor neurons determines the behavioral preference in male silkmoths. Our results show that the initiation of a complex programmed sexual behavior can depend on the properties of a single pheromone receptor gene expressed in a population of olfactory receptor neurons.
doi:10.1371/journal.pgen.1002115
PMCID: PMC3128102  PMID: 21738481
22.  Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata 
The Mediterranean fruit fly (or medfly), Ceratitis capitata (Wiedemann; Diptera: Tephritidae), is a serious pest of agriculture worldwide, displaying a very wide larval host range with more than 250 different species of fruit and vegetables. Olfaction plays a key role in the invasive potential of this species. Unfortunately, the pheromone communication system of the medfly is complex and still not well established. In this study, we report the isolation of chemicals emitted by sexually mature individuals during the “calling” period and the electrophysiological responses that these compounds elicit on the antennae of male and female flies. Fifteen compounds with electrophysiological activity were isolated and identified in male emissions by gas chromatography coupled to electroantennography (GC–EAG). Within the group of 15 identified compounds, 11 elicited a response in antennae of both sexes, whilst 4 elicited a response only in female antennae. The binding affinity of these compounds, plus 4 additional compounds known to be behaviourally active from other studies, was measured using C. capitata OBP, CcapOBP83a-2. This OBP has a high homology to Drosophila melanogaster OBPs OS-E and OS-F, which are associated with trichoid sensilla and co-expressed with the well-studied Drosophila pheromone binding protein LUSH. The results provide evidence of involvement of CcapOBP83a-2 in the medfly's odorant perception and its wider specificity for (E,E)-α-farnesene, one of the five major compounds in medfly male pheromone emission. This represents the first step in the clarification of the C. capitata and pheromone reception pathway, and a starting point for further studies aimed towards the creation of new powerful attractants or repellents applicable in the actual control strategies.
Graphical abstract
Highlights
•The pheromone blend produced by medfly males during the calling period is investigated using air entrainment and GC–EAG.•Out of 15 chemicals, 11 elicited antennal responses in both sexes and 4 elicited the response only in female antennae.•The first medfly odorant binding protein CcapOBP83a-2 has been expressed and purified (87).•CcapOBP83a-2 showed binding affinity for (E,E)-α-farnesene, one of the components of medfly pheromone.•The ability of CcapOBP83a-2 to bind other electrophysiologically active compounds shows its broad binding specificity.
doi:10.1016/j.ibmb.2014.02.005
PMCID: PMC4003389  PMID: 24607850
Medfly; Ceratitis capitata; Olfaction; Odorant binding protein; Pheromone binding protein; Pheromone; Binding studies; Protein expression; Electroantennography; GC–EAG; Fluorescence displacement; OBP, Odorant binding protein; PBP, Pheromone binding protein; CSP, Chemosensory protein; OR, Odorant receptor; EAG, Electroantennography; GC–EAG, Gas chromatography coupled to electroantennography; SIT, Sterile Insect Technique
23.  Olfactory Processing and Behavior Downstream from Highly Selective Receptor Neurons 
Nature neuroscience  2007;10(5):623-630.
In either the vertebrate nose or the insect antenna, most olfactory receptor neurons (ORNs) respond to multiple odors. However, some ORNs respond to just a single odor, or at most to a few highly related odors. It has been hypothesized that narrowly-tuned ORNs project to narrowly-tuned neurons in the brain, and that these dedicated circuits mediate innate behavioral responses to a particular ligand. Here we have investigated neural activity and behavior downstream from two narrowly-tuned ORN types in Drosophila. We found that genetically ablating either of these ORN types impairs innate behavioral attraction to their cognate ligand. Neurons in the antennal lobe postsynaptic to one of these ORN types are, like their presynaptic ORNs, narrowly tuned to a pheromone. However, neurons postsynaptic to the second ORN type are broadly tuned. These results demonstrate that some narrowly-tuned ORNs project to dedicated central circuits, ensuring a tight connection between stimulus and behavior, whereas others project to central neurons which participate in the ensemble representations of many odors.
doi:10.1038/nn1881
PMCID: PMC2838507  PMID: 17417635
24.  Functional Roles for Synaptic-Depression within a Model of the Fly Antennal Lobe 
PLoS Computational Biology  2012;8(8):e1002622.
Several experiments indicate that there exists substantial synaptic-depression at the synapses between olfactory receptor neurons (ORNs) and neurons within the drosophila antenna lobe (AL). This synaptic-depression may be partly caused by vesicle-depletion, and partly caused by presynaptic-inhibition due to the activity of inhibitory local neurons within the AL. While it has been proposed that this synaptic-depression contributes to the nonlinear relationship between ORN and projection neuron (PN) firing-rates, the precise functional role of synaptic-depression at the ORN synapses is not yet fully understood. In this paper we propose two hypotheses linking the information-coding properties of the fly AL with the network mechanisms responsible for ORNAL synaptic-depression. Our first hypothesis is related to variance coding of ORN firing-rate information — once stimulation to the ORNs is sufficiently high to saturate glomerular responses, further stimulation of the ORNs increases the regularity of PN spiking activity while maintaining PN firing-rates. The second hypothesis proposes a tradeoff between spike-time reliability and coding-capacity governed by the relative contribution of vesicle-depletion and presynaptic-inhibition to ORNAL synaptic-depression. Synaptic-depression caused primarily by vesicle-depletion will give rise to a very reliable system, whereas an equivalent amount of synaptic-depression caused primarily by presynaptic-inhibition will give rise to a less reliable system that is more sensitive to small shifts in odor stimulation. These two hypotheses are substantiated by several small analyzable toy models of the fly AL, as well as a more physiologically realistic large-scale computational model of the fly AL involving glomerular channels.
Author Summary
Understanding the intricacies of sensory processing is a major scientific challenge. In this paper we examine the early stages of the olfactory system of the fruit-fly. Many experiments have revealed a great deal regarding the architecture of this system, including the types of neurons within it, as well as the connections those neurons make amongst one another. In this paper we examine the potential dynamics produced by this neuronal network. Specifically, we construct a computational model of this early olfactory system and study the effects of synaptic-depression within this system. We find that the dynamics and coding properties of this system depend strongly on the strength, and sources of, synaptic-depression. This work has ramifications for understanding the coding properties of other insect olfactory systems, and perhaps even other sensory modalities in other animals.
doi:10.1371/journal.pcbi.1002622
PMCID: PMC3426607  PMID: 22927802
25.  Plant odorants interfere with detection of sex pheromone signals by male Heliothis virescens 
In many insects, mate finding relies on female-released sex pheromones, which have to be deciphered by the male olfactory system within an odorous background of plant volatiles present in the environment of a calling female. With respect to pheromone-mediated mate localization, plant odorants may be neutral, favorable, or disturbing. Here we examined the impact of plant odorants on detection and coding of the major sex pheromone component, (Z)-11-hexadecenal (Z11-16:Ald) in the noctuid moth Heliothis virescens. By in vivo imaging the activity in the male antennal lobe (AL), we monitored the interference at the level of olfactory sensory neurons (OSN) to illuminate mixture interactions. The results show that stimulating the male antenna with Z11-16:Ald and distinct plant-related odorants simultaneously suppressed pheromone-evoked activity in the region of the macroglomerular complex (MGC), where Z11-16:Ald-specific OSNs terminate. Based on our previous findings that antennal detection of Z11-16:Ald involves an interplay of the pheromone binding protein (PBP) HvirPBP2 and the pheromone receptor (PR) HR13, we asked if the plant odorants may interfere with any of the elements involved in pheromone detection. Using a competitive fluorescence binding assay, we found that the plant odorants neither bind to HvirPBP2 nor affect the binding of Z11-16:Ald to the protein. However, imaging experiments analyzing a cell line that expressed the receptor HR13 revealed that plant odorants significantly inhibited the Z11-16:Ald-evoked calcium responses. Together the results indicate that plant odorants can interfere with the signaling process of the major sex pheromone component at the receptor level. Consequently, it can be assumed that plant odorants in the environment may reduce the firing activity of pheromone-specific OSNs in H. virescens and thus affect mate localization.
doi:10.3389/fncel.2012.00042
PMCID: PMC3465774  PMID: 23060749
pheromone detection; antennal lobe; pheromone receptor; pheromone binding protein; olfaction

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