Pheromone Perception Research Articles

Morphological characterization of antennal sensilla of Earias vittella (Fabricius) (Lepidoptera: Nolidae)

The shoot and fruit borer, Earias vittella Fab. (Lepidoptera: Nolidae) is an important and most devastating insect pest on okra and cotton. The pest mainly responsible for causing significant direct damage to tender shoots and fruits of okra, flowers and green bolls of cotton causing net yield loss in both crops. Many non-chemical control strategies have been developed under the insect pest management program, A complete knowledge on the antennal morphology of E. vitella is essential for future electrophysiological and behavioural studies. In the present study, the antennal morphology and types of sensilla on the antennae of both sexes of E. vitella were examined using light and scanning electron microscopy. Nine distinct types of sensilla were identified on the antennae of both sexes: sensilla trichodea, sensilla basiconica, sensilla coeloconica, sensilla auricillica (multiporous), sensilla chaetica, uniporous peg sensilla (uniporous), sensilla styloconica, sensilla squamiformia and bohm bristles (aporous). Among all sensilla, the most widespread are multiporous sensilla trichodea with 42.90 ± 1.77/flagellomere in male and 37.38 ± 1.38/flagellomere in female. Sensilla basiconica were the second most common sensillum type in male antennae with 15.67 ± 1.92/flagellomere. Other multiporous sensilla such as auricillica (11.90 ± 0.99) and coeloconica (4.57 ± 0.25) were significantly more abundant in female than in male antennae. Results of the study provide morphological evidence that E. vitella antennae possess microscopic cuticular structures that can play a role in perception of pheromones, plant odours and other chemical stimulants. This will open up opportunities to assess the possibility of using pheromones and plant-derived compounds for the monitoring or management of E. vittella moths in the agricultural landscapes.

Pheromonal Regulation of the Reproductive Division of Labor in Social Insects.

The reproductive altruism in social insects is an evolutionary enigma that has been puzzling scientists starting from Darwin. Unraveling how reproductive skew emerges and maintains is crucial to understand the reproductive altruism involved in the consequent division of labor. The regulation of adult worker reproduction involves conspecific inhibitory signals, which are thought to be chemical signals by numerous studies. Despite the primary identification of few chemical ligands, the action modes of primer pheromones that regulate reproduction and their molecular causes and effects remain challenging. Here, these questions were elucidated by comprehensively reviewing recent advances. The coordination with other modalities of queen pheromones (QPs) and its context-dependent manner to suppress worker reproduction were discussed under the vast variation and plasticity of reproduction during colony development and across taxa. In addition to the effect of QPs, special attention was paid to recent studies revealing the regulatory effect of brood pheromones. Considering the correlation between pheromone and hormone, this study focused on the production and perception of pheromones under the endocrine control and highlighted the pivotal roles of nutrition-related pathways. The novel chemicals and gene pathways discovered by recent works provide new insights into the understanding of social regulation of reproductive division of labor in insects.

Functional characteristic analysis of three odorant-binding proteins from the sweet potato weevil (Cylas formicarius) in the perception of sex pheromones and host plant volatiles.

The sweet potato weevil, Cylas formicarius, is the most serious pest of sweet potato worldwide. The molecular mechanism of sex pheromone recognition in C. formicarius has not been reported. Odorant-binding proteins (OBPs) play a critical role in selectively binding and transporting pheromones or other odors to the surface of olfactory receptor neurons through the aqueous sensillar lymph, therefore the function of sweet potato OBPs is worth studying. Herein, the CforOBP1-3 genes encoding three classical OBPs were cloned in C. formicarius by reverse transcription-polymerase chain reaction. Phylogenetic analysis showed that CforOBP1-3 were homologous genes, but the relationship between CforOBP2 and CforOBP3 was closest among the three genes. In addition, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays demonstrated that the expression of CforOBP1 was higher in the antennae and legs of female and male insects, while CforOBP2 and CforOBP3 were mainly expressed in the antennae of male insects. The fluorescent competitive binding assay results indicated that CforOBP1-3 had strong binding affinities to sex pheromones and other tested ligands. Finally, the mRNA expression of CforOBP1-3 was successfully inhibited by RNA interference, and in vivo behavioral experiments showed that CforOBP1-3-deficient C. formicarius was partly anosmic and lost some of its ability to locate sex pheromones and host plant volatiles. These results suggested that CforOBP1 was shown to be involved in the process of weevils feeding and finding sweet potato, and CforOBP2-3 were mainly involved in the mating behavior of adult male weevils.

Identification of olfactory genes of a forensically important blow fly, Aldrichina grahami (Diptera: Calliphoridae).

BackgroundThe time-length between the first colonization of necrophagous insect on the corpse and the beginning of investigation represents the most important forensic concept of minimum post-mortem inference (PMImin). Before colonization, the time spent by an insect to detect and locate a corpse could significantly influence the PMImin estimation. The olfactory system plays an important role in insect food foraging behavior. Proteins like odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs) and sensory neuron membrane proteins (SNMPs) represent the most important parts of this system. Exploration of the above genes and their necrophagous products should facilitate not only the understanding of their roles in forging but also their influence on the period before PMImin. Transcriptome sequencing has been wildly utilized to reveal the expression of particular genes under different temporal and spatial condition in a high throughput way. In this study, transcriptomic study was implemented on antennae of adult Aldrichina grahami (Aldrich) (Diptera: Calliphoridae), a necrophagous insect with forensic significance, to reveal the composition and expression feature of OBPs, CSPs, ORs, IRs and SNMPs genes at transcriptome level.MethodAntennae transcriptome sequencing of A. grahami was performed using next-generation deep sequencing on the platform of BGISEQ-500. The raw data were deposited into NCBI (PRJNA513084). All the transcripts were functionally annotated using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Differentially expressed genes (DEGs) were analyzed between female and male antennae. The transcripts of OBPs, CSPs, ORs, IRs and SNMPs were identified based on sequence feature. Phylogenetic development of olfactory genes of A. grahami with other species was analyzed using MEGA 5.0. RT-qPCR was utilized to verify gene expression generated from the transcriptome sequencing.ResultsIn total, 14,193 genes were annotated in the antennae transcriptome based on the GO and the KEGG databases. We found that 740 DEGs were differently expressed between female and male antennae. Among those, 195 transcripts were annotated as candidate olfactory genes then checked by sequence feature. Of these, 27 OBPs, one CSPs, 49 ORs, six IRs and two SNMPs were finally identified in antennae of A. grahami. Phylogenetic development suggested that some olfactory genes may play a role in food forging, perception of pheromone and decomposing odors.ConclusionOverall, our results suggest the existence of gender and spatial expression differences in olfactory genes from antennae of A. grahami. Such differences are likely to greatly influence insect behavior around a corpse. In addition, candidate olfactory genes with predicted function provide valuable information for further studies of the molecular mechanisms of olfactory detection of forensically important fly species and thus deepen our understanding of the period before PMImin.

Chromatin-based reprogramming of a courtship regulator by concurrent pheromone perception and hormone signaling.

To increase fitness, animals use both internal and external states to coordinate reproductive behaviors. The molecular mechanisms underlying this coordination remain unknown. Here, we focused on pheromone-sensing Drosophila Or47b neurons, which exhibit age- and social experience-dependent increase in pheromone responses and courtship advantage in males. FruitlessM (FruM), a master regulator of male courtship behaviors, drives the effects of social experience and age on Or47b neuron responses and function. We show that simultaneous exposure to social experience and age-specific juvenile hormone (JH) induces chromatin-based reprogramming of fruM expression in Or47b neurons. Group housing and JH signaling increase fruM expression in Or47b neurons and active chromatin marks at fruM promoter. Conversely, social isolation or loss of JH signaling decreases fruM expression and increases repressive marks around fruM promoter. Our results suggest that fruM promoter integrates coincident hormone and pheromone signals driving chromatin-based changes in expression and ultimately neuronal and behavioral plasticity.

A plant volatile alters the perception of sex pheromone blend ratios in a moth.

Mate finding in most moths is based on male perception of a female-emitted pheromone whose species specificity resides in component chemistry and proportions. Components are individually detected by specialized olfactory receptor neurons (ORNs) projecting into the macroglomerular complex (MGC) of the male brain. We asked how robust ratio recognition is when challenged by a plant volatile background. To test this, we investigated the perception of the pheromone blend in Agrotis ipsilon, a moth species whose females produce a blend of Z7-dodecenyl acetate (Z7-12:Ac), Z9-tetradecenyl acetate (Z9-14:Ac), and Z11-hexadecenyl acetate in a 4:1:4 ratio optimally attractive for males. First, we recorded the responses of specialist ORNs for Z7 and Z9 and showed that heptanal, a flower volatile, activated Z7 but not Z9 neurons. Then, we recorded intracellularly the responses of MGC neurons to various ratios and showed that heptanal altered ratio responses of pheromone-sensitive neurons. Finally, we analyzed the behavior of males in a wind tunnel and showed that their innate preference for the 4:1:4 blend was shifted in the presence of heptanal. Pheromone ratio recognition may thus be altered by background odorants. Therefore, the olfactory environment might be a selective force for the evolution of pheromone communication systems.

Evolution of sex pheromone receptors in Dendrolimus punctatus Walker (lepidoptera: Lasiocampidae) is divergent from other moth species

Dendrolimus punctatus Walker (Lepidoptera: Lasiocampidae) is a pine caterpillar moth distributed in most areas of southern China and is an economically important pest of pine, due to its defoliation activity. Understanding fundamental sex pheromone perception mechanisms in D. punctatus may provide effective and sustainable options for novel control strategies. However, the identification and function of pheromone receptors, key genes that receipt the pheromone of this pest, are both unclear now. Previous researches suggested several candidate pheromone receptors whose expression levels were male antennae bias in D. punctatus. In this study, we cloned six candidate pheromone receptors (DpunOR 20/45/46/51/54/58) and Orco from D. punctatus. Phylogenetic tree analysis showed that lepidopteran PRs tend to be conserved and clustered together; however, D. punctatus candidate PRs were located in a distinct clade. Motif analysis of PRs showed clear sequences differences between Dendrolimus spp. and other tested moth species. To illustrate the ligand response properties of the candidate PRs of D. punctatus, each of the six genes was expressed with an Orco gene in Xenopus oocytes and using two-electrode voltage-clamp recordings. Finally, we successfully identified two sex pheromone receptors (PR45 and PR46). Our study, which identified a novel lineage of PRs tuned to Type I pheromones in Lepidoptera, provides evidence for the new evolution origin of sex pheromone communication in moths, and lays a foundation for the development of novel control strategies of D. punctatus.

The fruitless gene affects female receptivity and species isolation.

Female mate rejection acts as a major selective force within species, and can serve as a reproductive barrier between species. In spite of its critical role in fitness and reproduction, surprisingly little is known about the genetic or neural basis of variation in female mate choice. Here, we identify fruitless as a gene affecting female receptivity within Drosophila melanogaster, as well as female Drosophila simulans rejection of male D. melanogaster. Of the multiple transcripts this gene produces, by far the most widely studied is the sex-specifically spliced transcript involved in the sex determination pathway. However, we find that female rejection behaviour is affected by a non-sex-specifically spliced fruitless transcript. This is the first implication of fruitless in female behaviour, and the first behavioural role identified for a fruitless non-sex-specifically spliced transcript. We found that this locus does not influence preferences via a single sensory modality, examining courtship song, antennal pheromone perception, or perception of substrate vibrations, and we conclude that fruitless influences mate choice via the integration of multiple signals or through another sensory modality.

Functional Characterization of Sex Pheromone Receptors in the Fall Armyworm (Spodoptera frugiperda)

Pheromone receptors (PRs) found in the antennae of male moths play a vital role in the recognition of sex pheromones released by females. The fall armyworm (FAW), Spodoptera frugiperda, is a notorious invasive pest, but its PRs have not been reported. In this report, six candidate PRs (SfruOR6, 11, 13, 16, 56 and 62) suggested by phylogenetic analysis were cloned, and their tissue–sex expression profiles were determined by quantitative real-time PCR (qPCR). All six genes except for SfruOR6 were highly and specifically expressed in the antennae, with SfruOR6, 13 and 62 being male-specific, while the other three (SfruOR11, 16 and 56) were male biased, suggesting their roles in sex pheromone perception. A functional analysis by the Xenopus oocyte system further demonstrated that SfruOR13 was highly sensitive to the major sex pheromone component Z9-14:OAc and the pheromone analog Z9,E12-14:OAc, but less sensitive to the minor pheromone component Z9-12:OAc; SfruOR16 responded weakly to pheromone component Z9-14:OAc, but strongly to pheromone analog Z9-14:OH; the other four candidate PRs did not respond to any of the four pheromone components and four pheromone analogs. This study contributes to clarifying the pheromone perception in the FAW, and provides potential gene targets for developing OR-based pest control techniques.

Response of different population sizes to traps and effect of spinosad on the trap catch and progeny adult emergence in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Tribolium castaneum is a major pest in stored products throughout the world. Male T. castaneum adults produce aggregation pheromone 4,8-dimethyldecanal (4,8 DMD) which is synthetically developed and available for monitoring T. castaneum populations but low trapping by 4,8 DMD is reported. Effects of biorational insecticides on orienting to pheromone and progeny production is shown but such tests using spinosad has areas for further discovery. Therefore the objective of this research was to determine the effect of population size and spinosad exposure on the trap catch and progeny production in T. castaneum adults. In the first experiment, T. castaneum adults were released at different population sizes and the attraction to the pheromone traps was determined. The second experiment tested attraction of T. castaneum adults pre-exposed to spinosad to the pheromone traps. In the third experiment, progeny production of T. castaneum adults exposed to spinosad was assessed. The trapping of T. castaneum is increased at high population sizes and pre-exposure to spinosad while progeny was reduced on exposure to spinosad showing sex-based effects. This study concludes with management opportunities for different population levels of T. castaneum through effects of spinosad on pheromone perception and progeny production.

Modeling Communication Processes in the Human Connectome through Cooperative Learning

Communication processes within the human brain at different cognitive states are neither well understood nor completely characterized. We assess communication processes in the human connectome using ant colony-inspired cooperative learning algorithm, starting from a source with no a priori information about the network topology, and cooperatively searching for the target through a pheromone-inspired model. This framework relies on two parameters, namely pheromone perception and edge perception , to define the cognizance and subsequent behaviour of the ants on the network and, overall, the communication processes happening between source and target nodes. Simulations obtained through different configurations allow the identification of path-ensembles that are involved in the communication between node pairs. These path-ensembles may contain different number of paths depending on the perception parameters and the node pair. In order to assess the different communication regimes displayed on the simulations and their associations with functional connectivity, we introduce two network measurements, effective path-length and arrival rate. These communication features are tested as individual as well as combined descriptors of functional connectivity during different tasks. Finally, different communication regimes are found in different specialized functional networks. Overall, this framework may be used as a test-bed for different communication regimes on top of an underlaying topology.

Functional characterization of one sex pheromone receptor (AlucOR4) in Apolygus lucorum (Meyer-Dür)

Traps baited with female-produced sex pheromones have been very effective in the monitoring and management of mirid bugs in numerous field trials. However, none of the target odorant receptors for sex pheromone components in Apolygus lucorum have been identified. Here, we identified one candidate sex pheromone receptor, AlucOR4, from A. lucorum. Quantitative real-time PCR (qPCR) analysis revealed that AlucOR4 was antennae-enriched and male-biased in adult A. lucorum. Xenopus oocyte expression system assays demonstrated that AlucOR4/AlucOrco was sensitive to two major sex pheromone constituents and exhibited high sensitivity to (E)-2-hexenyl butyrate (E2HB) and lower sensitivity to hexyl butyrate (HB). The expression level of target mRNA was significantly reduced (>80%) in dsAlucOR4-injected bugs after five days. The electroantennogram (EAG) responses of male antennae to E2HB and HB were also reduced significantly (~40%). Our findings suggest that AlucOR4 is essential to sex pheromone perception in A. lucorum.

CRISPR/Cas9 mediated gene knockout reveals a more important role of PBP1 than PBP2 in the perception of female sex pheromone components in Spodoptera litura

Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SlitPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SlitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SlitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SlitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.

Candidate chemosensory genes identified from the greater wax moth, Galleria mellonella, through a transcriptomic analysis

The greater wax moth, Galleria mellonella Linnaeus (Lepidoptera: Galleriinae), is a ubiquitous pest of the honeybee, and poses a serious threat to the global honeybee industry. G. mellonella pheromone system is unusual compared to other lepidopterans and provides a unique olfactory model for pheromone perception. To better understand the olfactory mechanisms in G. mellonella, we conducted a transcriptomic analysis on the antennae of both male and female adults of G. mellonella using high-throughput sequencing and annotated gene families potentially involved in chemoreception. We annotated 46 unigenes coding for odorant receptors, 25 for ionotropic receptors, two for sensory neuron membrane proteins, 22 for odorant binding proteins and 20 for chemosensory proteins. Expressed primarily in antennae were all the 46 odorant receptor unigenes, nine of the 14 ionotropic receptor unigenes, and two of the 22 unigenes coding for odorant binding proteins, suggesting their putative roles in olfaction. The expression of some of the identified unigenes were sex-specific, suggesting that they may have important functions in the reproductive behavior of the insect. Identification of the candidate unigenes and initial analyses on their expression profiles should facilitate functional studies in the future on chemoreception mechanisms in this species and related lepidopteran moths.

Chemosensory sensilla of the Drosophila wing express a candidate ionotropic pheromone receptor

The Drosophila wing was proposed to be a taste organ more than 35 years ago, but there has been remarkably little study of its role in chemoreception. We carry out a differential RNA-seq analysis of a row of sensilla on the anterior wing margin and find expression of many genes associated with pheromone and chemical perception. To ask whether these sensilla might receive pheromonal input, we devised a dye-transfer paradigm and found that large, hydrophobic molecules comparable to pheromones can be transferred from one fly to the wing margin of another. One gene, Ionotropic receptor (IR)52a, is coexpressed in neurons of these sensilla with fruitless, a marker of sexual circuitry; IR52a is also expressed in legs. Mutation of IR52a and optogenetic silencing of IR52a+ neurons decrease levels of male sexual behavior. Optogenetic activation of IR52a+ neurons induces males to show courtship toward other males and, remarkably, toward females of another species. Surprisingly, IR52a is also required in females for normal sexual behavior. Optogenetic activation of IR52a+ neurons in mated females induces copulation, which normally occurs at very low levels. Unlike other chemoreceptors that act in males to inhibit male–male interactions and promote male–female interactions, IR52a acts in both males and females, and can promote male–male as well as male–female interactions. Moreover, IR52a+ neurons can override the circuitry that normally suppresses sexual behavior toward unproductive targets. Circuit mapping and Ca2+ imaging using the trans-Tango system reveals second-order projections of IR52a+ neurons in the subesophageal zone (SEZ), some of which are sexually dimorphic. Optogenetic activation of IR52a+ neurons in the wing activates second-order projections in the SEZ. Taken together, this study provides a molecular description of the chemosensory sensilla of a greatly understudied taste organ and defines a gene that regulates the sexual circuitry of the fly.

Functional characterization of pheromone receptors in the moth Athetis dissimilis (Lepidoptera: Noctuidae)

Sex pheromones are crucial for communication between females and males in moths, and pheromone receptors (PRs) play a key role in peripheral coding of sex pheromones. During the last decade, many PR candidates have been identified based on transcriptome sequencing and bioinformatic analysis, but their detailed functions remain mostly unknown. Here, focusing on four PR candidates of Athetis dissimilis (AdisOR1, AdisOR6, AdisOR11 and AdisOR14) identified in a previous study, we first cloned the full-length cDNAs and determined the tissue expression profiles by quantitative real-time PCR (qPCR). The results revealed that expression of three of these genes were male antennae-specific, while AdisOR11 was similar in expression between male and female antennae. Furthermore, the expression level of AdisOR1 was much higher than those of the other three genes. Then, functional analysis was conducted using Xenopus oocyte system. AdisOR1 responded strongly to the sex pheromone component Z9–14:OH and the potential pheromone component Z9,E12–14:OH, suggesting its important role in the sex pheromone perception; AdisOR14 showed specificity for Z9,E12–14:OH; while AdisOR6 and AdisOR11 did not respond to any of the pheromone components and analogs tested. Taken together, this study contributes to elucidate the molecular mechanism of sex pheromone reception and provides potential targets for development of OR based pest control techniques in A. dissimilis.

Modern studies of the role of the vomeronasal system in the perception of pheromones and their impact on social and sexual behavior

The vomeronasal system (VNS) provides regulation of a wide range of autonomic and affective functions, behavioral reactions in response to the specific chemical stimuli pheromones secreted by mammals, including humans. The results of experimental studies confirming the existence of VNS and explaining the basic mechanisms of its functioning are presented. The results of studies of healthy volunteers, explaining the effect of pheromones on a number of functions of the human body, are considered.

Ageing via perception costs of reproduction magnifies sexual selection.

Understanding what factors modulate sexual selection intensity is crucial to a wide variety of evolutionary processes. Recent studies show that perception of sex pheromones can severely impact male mortality when it is not followed by mating (perception costs of reproduction). Here, we examine the idea that this may magnify sexual selection by further decreasing the fitness of males with inherently low mating success, hence increasing the opportunity for sexual selection. We use mathematical modelling to show that even modest mortality perception costs can significantly increase variability in male reproductive success under a wide range of demographic conditions. We then conduct a series of assays suggesting that, in Drosophila melanogaster, failure to reproduce early in life may, via perception costs of reproduction, significantly reduces the subsequent fitness of males (ca 25%), due mostly to increased reproductive ageing. Altogether, our results strongly suggest that perception costs of reproduction can magnify sexual selection in a biologically significant way. Finally, we estimate that around 29% of available studies quantify sexual selection based on short-term fitness estimates that may fail to capture these effects (if they were present in their subject species), and suggest addressing the existence and impact of perception costs of reproduction across taxa should thus be a priority.

Differential Expression Analysis of Olfactory Genes Based on a Combination of Sequencing Platforms and Behavioral Investigations in Aphidius gifuensis

Aphidius gifuensis Ashmead is a dominant endoparasitoid of aphids, such as Myzus persicae and Sitobion avenae, and plays an important role in controlling aphids in various habitats, including tobacco plants and wheat in China. A. gifuensis has been successfully applied for the biological control of aphids, especially M. persicae, in green houses and fields in China. The corresponding parasites, as well as its mate-searching behaviors, are subjects of considerable interest. Previous A. gifuensis transcriptome studies have relied on short-read next-generation sequencing (NGS), and the vast majority of the resulting isotigs do not represent full-length cDNA. Here, we employed a combination of NGS and single-molecule real-time (SMRT) sequencing of virgin females (VFs), mated females (MFs), virgin males (VMs), and mated males (MMs) to comprehensively study the A. gifuensis transcriptome. Behavioral responses to the aphid alarm pheromone (E-β-farnesene, EBF) as well as to A. gifuensis of the opposite sex were also studied. VMs were found to be attracted by female wasps and MFs were repelled by male wasps, whereas MMs and VFs did not respond to the opposite sex. In addition, VFs, MFs, and MMs were attracted by EBF, while VMs did not respond. According to these results, we performed a personalized differential gene expression analysis of olfactory gene sets (66 odorant receptors, 25 inotropic receptors, 16 odorant-binding proteins, and 12 chemosensory proteins) in virgin and mated A. gifuensis of both sexes, and identified 13 candidate genes whose expression levels were highly consistent with behavioral test results, suggesting potential functions for these genes in pheromone perception.

Antennal Morphology and Localization of a Pheromone-Binding Protein of Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae).

In the sensory system of insects, olfactory sensilla constitute important functional elements for discriminating odors. Therefore, we used light microscopy and scanning electron microscopy to investigate the morphology and distribution of sensilla in the antennae of Lobesia botrana (Denis & Schiffermüller). In addition, we studied the expression of the gene encoding for pheromone-binding protein 1 (LbotPBP1) by in situ hybridization. Lobesia botrana antennae are filiform and are subdivided into three segments: scape, pedicel, and flagellum. The number of flagellum and their overall length were significantly higher and longer in males than in females. Six morphological types of sensilla (trichodea, chaetica, coeloconica, auricillica, basiconica, and styloconica) were identified on the antennae of both sexes. Trichodea sensilla were the most abundant on the antennae of L. botrana, and three subtypes, discerned by their lengths, were observed. However, sensilla trichodea subtype III was only present in male antennae. Moreover, LbotPBP1 expression was restricted to this type of sensilla, thus confirming its olfactory role, specifically under the context of sexual pheromone perception.