Detection Of Sex Pheromones Research Articles

Diet acts on sexual behavior development in a male moth.

In many animals, drastic changes are observed during sexual maturation characterized by the reproductive system development concomitantly to the sexual behavior ontogenesis. These modifications are under the control of internal and external factors such as food. Sexual maturation requires considerable energetic investment, and diet has been shown to affect reproductive activities in many taxonomic groups, especially in insects and vertebrates. By contrast, diet effects on sexual behavior development remain largely unexplored. To elucidate this aspect, we used the male moth Agrotis ipsilon which undergoes sexual maturation occurring between the third and the fifth day postemergence. During this period, males are sensitive to female sex pheromones and a stereotypical sexual behavior characterized by female-oriented flight takes place. In our study, we compared (1) sex pheromone detection by electroantennography recordings and (2) behavioral response in wind tunnel assays between males fed with different diets found in nature. Compared to standard sucrose diet, males fed with sucrose, fructose, and glucose supplemented with sodium (a mineral element necessary for the locomotor activity in several moths) did not respond better to female sex pheromones but clearly exhibited an earlier behavioral response. Thus, such a diet accelerates the development of sex pheromone-mediated oriented flight, probably by facilitating the central processing of sex pheromone information in male A. ipsilon moths. Our results provide new information on the influence of nutritional intake on the ontogenesis of male sexual behavior in animals.

Functional characterization of sensory neuron membrane protein 1a involved in sex pheromone detection of Apolygus lucorum (Hemiptera: Miridae)

The mirid bug Apolygus lucorum (Hemiptera: Miridae) is a polyphagous pest that affects a wide range of host plants. Its control remains challenging mainly due to its rapid reproduction, necessitating an understanding of sex pheromone communication. The recognition of sex pheromones is vital for courtship and mating behaviors, and is mediated by various chemosensory-associated proteins. Among these, sensory neuron membrane protein (SNMP), a CD36-related protein, is suggested to play crucial roles in detecting sex pheromones. In this study, we employed transcriptomic and genomic data from A. lucorum and phylogenetic approaches, and identified four putative SNMP genes (AlucSNMP1a, AlucSNMP1b, AlucSNMP2a, and AlucSNMP2b) with full open reading frames. Expression analysis revealed the ubiquitous presence of AlucSNMP transcripts in multiple tissues, with only AlucSNMP1a exhibiting male-biased expression in the antennae, suggesting its potential role in male chemosensation. Functional analysis using the Xenopus oocyte expression system, coupled with two-electrode voltage clamp recording, demonstrated that the co-expression of AlucSNMP1a with specific pheromone receptors (PRs) and the co-receptor (Orco) significantly enhanced electrophysiological responses to sex pheromones compared to the co-expression of PRs and Orco alone. Moreover, the results indicated that the presence of AlucSNMP1a not only affected the responsiveness to sex pheromones but also influenced the kinetics (activation and inactivation) of the induced signals. In contrast, the co-expression of AlucSNMP1b with AlucPR/Orco complexes had no impact on the inward currents induced by two pheromone compounds. An examination of the selective pressures on SNMP1 genes across 20 species indicated strong purifying selection, implying potential functional conservation in various insects. These findings highlight the crucial role of AlucSNMP1a in the response to sex pheromones.

Exploring the proteins and metabolites associated with male antennae responses to female exposure of Antheraea pernyi (Lepidoptera: Saturniidae) moths.

Detection of sex pheromones of insects relies on the antennae. The female pheromone signal transmission in the male antennae ultimately initiates the courtship and mating behaviors of males. To investigate the proteins and metabolites involved in this neural transduction, integrative proteomics and metabolomics analysis including tandem mass tag (TMT) proteomic quantification and liquid chromatography tandem mass spectrometry (LC/MS)-based metabolomics was adopted for comparing proteomic and metabolic changes between the antennae of male moths following stimulation by females and the non-stimulated males of Antheraea pernyi (Guérin-Méneville, Lepidoptera: Saturniidae) in this study. A total of 92 differentially expressed proteins (DEPs) containing 52 upregulated and 40 downregulated proteins and 545 differentially expressed metabolites (DEMs) including 218 upregulated and 327 downregulated metabolites were identified from the antennae of female-stimulated male moths based on the proteome and metabolome data, respectively. Bioinformatics analysis was performed for the 45 DEPs and 160 DEMs, including Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encylopaedia of Genes and Genomes (KEGG) enrichment analysis and Human Metabolome Database (HMDB) annotation. A number of DEPs and DEMs related to neural transmission of female pheromone signals in the male antennae of A. pernyi were screened, including tyrosine hydroxylase, cryptochrome-1, tachykinin, arylalkylamine N-acetyltransferase, cadherin-23, glutathione S-transferase delta 3, tyramine, tryptamine, n-oleoyl dopamine, n-stearoyl dopamine, and n-stearoyl tyrosine. The altered expression levels of those proteins or metabolites were speculated involved in regulating the neuron activity for enhanced transmission of neural impulses and continuous perception, reception, and transduction of female pheromone signals. Our findings yielded novel insights into the potential mechanisms in the antennae of male A. pernyi responding to female attraction.

Olfactory Gene Families in Scopula subpunctaria and Candidates for Type-II Sex Pheromone Detection

Scopula subpunctaria, an abundant pest in tea gardens, produce type-II sex pheromone components, which are critical for its communicative and reproductive abilities; however, genes encoding the proteins involved in the detection of type-II sex pheromone components have rarely been documented in moths. In the present study, we sequenced the transcriptomes of the male and female S. subpunctaria antennae. A total of 150 candidate olfaction genes, comprising 58 odorant receptors (SsubORs), 26 ionotropic receptors (SsubIRs), 24 chemosensory proteins (SsubCSPs), 40 odorant-binding proteins (SsubOBPs), and 2 sensory neuron membrane proteins (SsubSNMPs) were identified in S. subpunctaria. Phylogenetic analysis, qPCR, and mRNA abundance analysis results suggested that SsubOR46 may be the Orco (non-traditional odorant receptor, a subfamily of ORs) of S. subpunctaria. SsubOR9, SsubOR53, and SsubOR55 belonged to the pheromone receptor (PR) clades which have a higher expression in male antennae. Interestingly, SsubOR44 was uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP25, SsubOBP27, and SsubOBP28 were clustered into the moth pheromone-binding protein (PBP) sub-family, and they were uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP19, a member of the GOBP2 group, was the most abundant OBP in the antennae. These findings indicate that these olfactory genes, comprising five candidate PRs, three candidate PBPs, and one candidate GOBP2, may be involved in type II sex pheromone detection. As well as these genes, most of the remaining SsubORs, and all of the SsubIRs, showed a considerably higher expression in the female antennae than in the male antennae. Many of these, including SsubOR40, SsubOR42, SsubOR43, and SsubIR26, were more abundant in female antennae. These olfactory and ionotropic receptors may be related to the detection of host plant volatiles. The results of this present study provide a basis for exploring the olfaction mechanisms in S. subpunctaria, with a focus on the genes involved in type II sex pheromones. The evolutionary analyses in our study provide new insights into the differentiation and evolution of lepidopteran PRs.

Antennal transcriptome analysis of olfactory genes and tissue expression profiling of odorant binding proteins in Semanotus bifasciatus (cerambycidae: coleoptera)

BackgroundInsect olfactory proteins can transmit chemical signals in the environment that serve as the basis for foraging, mate searching, predator avoidance and oviposition selection. Semanotus bifasciatus is an important destructive borer pest, but its olfactory mechanism is not clear. We identified the chemosensory genes of S. bifasciatus in China, then we conducted a phylogenetic analysis of the olfactory genes of S. bifasciatus and other species. And the expression profiles of odorant binding proteins (OBPs) genes in different tissues and different genders of S. bifasciatus were determined by quantitative real-time PCR for the first time.ResultsA total of 32 OBPs, 8 chemosensory proteins (CSPs), 71 odorant receptors (ORs), 34 gustatory receptors (GRs), 18 ionotropic receptors (IRs), and 3 sensory neuron membrane proteins (SNMPs) were identified. In the tissue expression analysis of OBP genes, 7 OBPs were higher expressed in antennae, among them, SbifOBP2, SbifOBP3, SbifOBP6, SbifOBP7 and SbifOBP20 were female-biased expression, while SbifOBP1 was male-biased expression and SbifOBP22 was no-biased expression in antennae. In addition, the expressed levels of SbifOBP4, SbifOBP12, SbifOBP15, SbifOBP27 and SbifOBP29 were very poor in the antennae, and SbifOBP4 and SbifOBP29 was abundant in the head or legs, and both of them were male-biased expression. While SbifOBP15 was highly expressed only at the end of the abdomen with its expression level in females three times than males. Other OBPs were expressed not only in antennae but also in various tissues.ConclusionWe identified 166 olfactory genes from S. bifasciatus, and classified these genes into groups and predicted their functions by phylogenetic analysis. The majority of OBPs were antenna-biased expressed, which are involved in odor recognition, sex pheromone detection, and/or host plant volatile detection. However, also some OBPs were detected biased expression in the head, legs or end of the abdomen, indicating that they may function in the different physiological processes in S. bifasciatus.

Sex Pheromone Receptors of Lepidopteran Insects

The sex pheromone receptors (SPRs) of Lepidopteran insects play important roles in chemical communication. In the sex pheromone detection processes, sex pheromone molecule (SPM), SPR, co-receptor (Orco), pheromone binding protein (PBP), sensory neuron membrane protein (SNMP), and pheromone degradation enzyme (PDE) play individual and cooperative roles. Commonly known as butterfly and moth, the Lepidopteran insects are widely distributed throughout the world, most of which are pests. Comprehensive knowledge of the SPRs of Lepidopteran insects would help the development of sex lure technology and the sex communication pathway research. In this review, we summarized SPR/Orco information from 10 families of Lepidopteran insects from corresponding studies. According to the research progress in the literature, we speculated the evolution of SPRs/Orcos and phylogenetically analyzed the Lepidopteran SPRs and Orcos with the neighbor-joining tree and further concluded the relationship between the cluster of SPRs and their ligands; we analyzed the predicted structural features of SPRs and gave our prediction results of SPRs and Orcos with Consensus Constrained TOPology Prediction (CCTOP) and SwissModel; we summarized the functional characterization of Lepidopteran SPRs and SPR-ligand interaction and then described the progress in the sex pheromone signaling pathways and metabotropic ion channel. Further studies are needed to work out the cryo-electron microscopy (EM) structure of SPR and the SPR-ligand docking pattern in a biophysical perspective, which will directly facilitate the understanding of sex pheromone signal transduction pathways and provide guidance in the sex lure technology in field pest control.

Sex pheromone receptors of the light brown apple moth, Epiphyas postvittana, support a second major pheromone receptor clade within the Lepidoptera

Sex pheromones facilitate species-specific sex communication within the Lepidoptera. They are detected by specialised pheromone receptors (PRs), most of which to date fall into a single monophyletic receptor lineage (frequently referred to as “the PR clade”) within the odorant receptor (OR) family. Here we investigated PRs of the invasive horticultural pest, Epiphyas postvittana, commonly known as the light brown apple moth. Ten candidate PRs were selected, based on their male-biased expression in antennae or their relationship to the PR clade, for functional assessment in both HEK293 cells and Xenopus oocytes. Of these, six ORs responded to compounds that include components of the E. postvittana (‘Epos’) sex pheromone blend or compounds that antagonise sex pheromone attraction. In phylogenies, four of the characterised receptors (EposOR1, 6, 7 and 45) fall within the PR clade and two other male-biased receptors (EposOR30 and 34) group together well outside the PR clade. This new clade of pheromone receptors includes the receptor for (E)-11-tetradecenyl acetate (EposOR30), which is the main component of the sex pheromone blend for this species. Interestingly, receptors of the two clades do not segregate by preference for compounds associated with behavioural response (agonist or antagonist), isomer type (E or Z) or functional group (alcohol or acetate), with examples of each scattered across both clades. Phylogenetic comparison with PRs from other species supports the existence of a second major clade of lepidopteran ORs including, EposOR30 and 34, that has been co-opted into sex pheromone detection in the Lepidoptera. This second clade of sex pheromone receptors has an origin that likely predates the split between the major lepidopteran families.

A small number of male-biased candidate pheromone receptors are expressed in large subsets of the olfactory sensory neurons in the antennae of drones from the European honey bee Apis mellifera.

In the European honey bee (Apis mellifera), the olfactory system is essential for foraging and intraspecific communication via pheromones. Honey bees are equipped with a large repertoire of olfactory receptors belonging to the insect odorant receptor (OR) family. Previous studies have indicated that the transcription level of a few OR types including OR11, a receptor activated by the queen-released pheromone compound (2E)-9-oxodecenoic acid (9-ODA), is significantly higher in the antenna of males (drones) than in female workers. However, the number and distribution of antennal cells expressing male-biased ORs is elusive. Here, we analyzed antennal sections from bees by in situ hybridization for the expression of the male-biased receptors OR11, OR18, and OR170. Our results demonstrate that these receptors are expressed in only moderate numbers of cells in the antennae of females (workers and queens), whereas substantially higher cell numbers express these ORs in drones. Thus, the reported male-biased transcript levels are due to sex-specific differences in the number of antennal cells expressing these receptors. Detailed analyses for OR11 and OR18 in drone antennae revealed expression in two distinct subsets of olfactory sensory neurons (OSNs) that in total account for approximately 69% of the OR-positive cells. Such high percentages of OSNs expressing given receptors are reminiscent of male-biased ORs in moths that mediate the detection of female-released sex pheromone components. Collectively, our findings indicate remarkable similarities between male antennae of bees and moths and support the concept that male-biased ORs in bee drones serve the detection of female-emitted sex pheromones.

Sex-specific and caste-specific brain adaptations related to spatial orientation in Cataglyphis ants.

Cataglyphis desert ants are charismatic central place foragers. After long-ranging foraging trips, individual workers navigate back to their nest relying mostly on visual cues. The reproductive caste faces other orientation challenges, i.e. mate finding and colony foundation. Here we compare brain structures involved in spatial orientation of Cataglyphis nodus males, gynes, and foragers by quantifying relative neuropil volumes associated with two visual pathways, and numbers and volumes of antennal lobe (AL) olfactory glomeruli. Furthermore, we determined absolute numbers of synaptic complexes in visual and olfactory regions of the mushroom bodies (MB) and a major relay station of the sky-compass pathway to the central complex (CX). Both female castes possess enlarged brain centers for sensory integration, learning, and memory, reflected in voluminous MBs containing about twice the numbers of synaptic complexes compared with males. Overall, male brains are smaller compared with both female castes, but the relative volumes of the optic lobes and CX are enlarged indicating the importance of visual guidance during innate behaviors. Male ALs contain greatly enlarged glomeruli, presumably involved in sex-pheromone detection. Adaptations at both the neuropil and synaptic levels clearly reflect differences in sex-specific and caste-specific demands for sensory processing and behavioral plasticity underlying spatial orientation.

(Invited) Sensors for Agricultural Pests Management

Pests are undesired living organisms which cause major threat to plants, animals, humans and other organisms. It can directly feed on the crops and indirectly spread diseases and decrease the productivity of plants. Traditionally we have been using pesticides to get rid of them, but these pesticides are chemicals which tend to accumulate in the soil and water and pose detrimental effects to all the living creatures. To reduce the major loss in food production and use of chemical pesticides, we have come up with a few innovative approaches to control pests and also detect the toxic pesticides in an easy, affordable and eco-friendly way using sensors. We had developed sensors for effective use of biopesticides such as Helicoverpa armigera nuclear polyhedrosis virus (HaNPV), Spodoptera litura nuclear polyhedrosis virus (SlNPV) and Spilosoma obliqua nuclear polyhedrosis virus (SpilNPV). We had also designed a silicon dioxide-based microelectro-mechanical system (MEMS) sensors as tool for an early selective pest detection of female sex pheromones of Helicoverpa armigera and Bactrocera oleae. This robust device can also be used to selectively measure the pheromone at femtogram levels, and it is handy for early detection of pests. The device works well in both rainy and peak summer seasons. Using these systems farmers can take necessary measures in the pest attack region without using pesticides and would be able to support organic farming.

Anosmic flies: what Orco silencing does to olive fruit flies

BackgroundThe olive fruit fly (Bactrocera oleae) is the most destructive pest of the olive cultivation worldwide causing significant production losses and olive fruit impoverishment, as its larvae feed exclusively on the olive fruit. Reproductive and sexual behavior, as well as host-plant recognition of the fly, are highly dependent on its chemosensory system. Therefore, exploring the role of genes that play a critical role in olfaction, could reveal potential molecular targets that determine species-specific features on chemical communication and could be used to impair sexual behavior.ResultsIn this study we identified the gene that encodes the conserved olfactory co-receptor Orco (Odorant receptor co-receptor), which interacts with all divergent insect odorant receptors, and investigated how disruption of its expression affects chemoreception. We initially searched the expression profile of Bo-Orco in both sexes during sexual maturation, as well as pre- and post-mating communication by relative quantitative real time PCR (qRT-PCR) analysis suggesting that Bo-Orco was abundantly expressed in sexually mature adults.We further investigated the functional role of Bo-Orco in mating and oviposition behavior via transient gene silencing that was performed through in vivo dsRNA hemolymph injections in sexually mature flies 7 days after eclosion. Orco-knockdown phenotypes in both sexes showed reduced copulation rates in mating competitiveness tests, possibly through impaired olfactory-mediated detection of sex pheromone. In addition, oviposition was significantly inhibited in dsRNA-Orco injected females in a post-mating behavior test.ConclusionsOur results demonstrate that Orco plays a crucial role in the reproductive behavior of the olive fruit fly, since pre- and post-mating processes were affected. This is the first report in the olive fruit fly that links the chemosensory pathway with the mating behavior and the reproductive potential at a molecular basis, rendering this gene a potential target for the improvement of the olive fruit fly population control techniques.

First example of engineered β-cyclodextrinylated MEMS devices for volatile pheromone sensing of olive fruit pests

Olive oil is more preferred than other vegetable oils because of the increasing health concern among people throughout the world. The major hindrance in large-scale production of olive oil is olive fruit pests which cause serious economic damage to the olive orchards. This requires careful monitoring and timely application of suitable remedies before pest infestation. Herein we demonstrate efficacious utilization of covalently functionalized β-cyclodextrinylated MEMS devices for selective and sensitive detection of female sex pheromone of olive fruit pest, Bactocera oleae. Two of the MEMS devices, silicon dioxide surface-micromachined cantilever arrays and zinc oxide surface-microfabricated interdigitated circuits, have been used to selectively capture the major pheromone component, 1,7-dioxaspiro[5,5]undecane. The non-covalent capture of olive pheromones inside the β-cyclodextrin cavity leads to the reduction of resonant frequency of the cantilevers, whereas an increase in resistance has been found in case of zinc oxide derived MEMS devices. Sensitivity of the MEMS devices towards the olive pheromone was found to be directly correlated with the increasing availability of β-cyclodextrin moieties over the surface of the devices and thus the detection limit of the devices has been achieved to a value as low as 0.297 ppq of the olive pheromone when the devices were functionalized with one of the standardized protocols. Overall, the reversible usability and potential capability of the suitably functionalized MEMS devices to selectively detect the presence of female sex pheromone of olive fruit fly before the onset of pest infestation in an orchard makes the technology quite attractive for viable commercial application.

Essential role for SNMP1 in detection of sex pheromones in Helicoverpa armigera

The sensory neuron membrane protein, SNMP1, was initially discovered in moths and is associated with sex pheromone sensitive neurons, suggesting a role in the detection of these semiochemicals. Although DrosophilaSNMP1 has been reported to be involved in detecting of the sex pheromone cis-vaccenyl acetate (cVA), the role of this protein in moths in vivo is still largely unexplored. In this study we developed a SNMP1−/− homozygous mutant line of Helicoverpa armigera using CRISPR/Cas9. Wind-tunnel behavioral experiments showed that HarmSNMP1−/− males could not be attracted by sex pheromones (Z11-16:Ald/Z9-16:Ald = 97/3), while mating behavior obvervations revealed that the SNMP1 mutant males didn't react much to calling females and the rate of copulation was significantly decreased. The electrophysiological results indicated that HarmSNMP1 contributes to the detection of 16-carbon liner sex pheromones, (Z)-11-hexadecenal (Z11-16:Ald), (Z)-9-hexadecenal (Z9-16:Ald), (Z)-11-hexadecanol (Z11-16:OH) and 16-carbon acetate (Z)-11-hexadecenyl acetate (Z11-16:OAc), but is not required for detecting the 14-carbon sex pheromone component (Z)-9-tetradecenal (Z9-14:Ald) an analogue of Z11-16:Ald, (Z)-9-tetradecen-1-yl formate (Z9-14:OFor), which can activate the Z11-16:Ald-responsive neuron. Taken together, our studies indicated that HarmSNMP1 has an important role in the detection of long-chain sex pheromones, but is not essential for detecting shorter chain sex pheromone in vivo.

Sex pheromones and olfactory proteins in Antheraea moths: A. pernyi and A. polyphemus (Lepidoptera: Saturniidae).

Olfaction is essential for regulating the physiological and behavioral actions of insects with specific recognition of various odors. Antheraea moths (Lepidoptera: Saturniidae) possess relatively large bodies and antennae so that they are good subjects for exploring molecular aspects of insect olfaction. Current knowledge of the molecular aspects of Antheraea olfaction is focused on the Chinese tussah silkmoth A. pernyi Guérin-Méneville and another species A. polyphemus (Cramer) in their pheromones, odorant-binding proteins (OBPs), odorant receptors (ORs), odorant receptor coreceptors (ORCOs), sensory neuron membrane proteins (SNMPs), and odorant-degrading enzymes (ODEs). The first insect OBP, SNMP, and ODE were identified from A. polyphemus. This review summarizes the principal findings associated with the olfactory physiology and its molecular components in the two Antheraea species. Three types of olfactory neurons may have specific ORs for three respective sex-pheromone components, with the functional sensitivity and specificity mediated by three respective OBPs. SNMPs and ODEs are likely to play important roles in sex-pheromone detection, inactivation, and degradation. Identification and functional analysis of the olfactory molecules remain to be further performed in A. pernyi, A. polyphemus, and other Antheraea species.

Effects of DEHP on the ecdysteroid pathway, sexual behavior and offspring of the moth Spodoptera littoralis

Bis(2-ethylhexyl) phthalate (DEHP) is a widely produced plasticizer that is considered to act as an endocrine-disrupting chemical in vertebrates and invertebrates. Indeed, many studies have shown that DEHP alters hormonal levels, reproduction and behavior in vertebrates. Few studies have focused on the effects of DEHP on insects, although DEHP is found almost everywhere in their natural habitats, particularly in soils and plants. Here, we investigated the effects of DEHP on the sexual behavior and physiology of a pest insect, the noctuid moth Spodoptera littoralis. In this nocturnal species, olfaction is crucial for sexual behavior, and ecdysteroids at the antennal level have been shown to modulate sex pheromone detection by males. In the present study, larvae were fed food containing different DEHP concentrations, and DEHP concentrations were then measured in the adults (males and females). Hemolymphatic ecdysteroid concentrations, the antennal expression of genes involved in the ecdysteroid pathway (nuclear receptors EcR, USP, E75, and E78 and calmodulin) and sexual behavior were then investigated in adult males. The success and latency of mating as well as the hatching success were also studied in pairs consisting of one DEHP male and one uncontaminated female or one DEHP female and one uncontaminated male. We also studied the offspring produced from pairs involving contaminated females to test the transgenerational effect of DEHP. Our results showed the general downregulation of nuclear receptors and calmodulin gene expression associated with the higher concentrations of DEHP, suggesting peripheral olfactory disruption. We found some effects on male behavior but without an alteration of the mating rate. Effects on offspring mortality and developmental rates in the N + 1 generation were also found at the higher doses of DEHP. Taken together, the results of the study show for the first time that larval exposure to DEHP can induce delayed endocrine-disruptive effects in the adults of a terrestrial insect as well as effects on the next generation. To date, our study is also the first description of an impact of endocrine disrupter on olfaction in insects.

Identification and Expression Profiling of Chemosensory Genes in Hermetia illucens via a Transcriptomic Analysis.

The black soldier fly, Hermetia illucens, is a cosmopolitan insect of the family Stratiomyidae (Diptera). Chemosensory genes encode proteins involved directly in the detection of odorants. In this study, we sequenced the antennal transcriptome of H. illucens adults to identify chemosensory genes. Putative unigenes encoding 27 odorant binding proteins (OBPs), five chemosensory proteins (CSPs), 70 odorant receptors (ORs), 25 ionotropic receptors (IRs), 10 gustatory receptors (GRs) and two sensory neuron membrane proteins (SNMPs) were identified. Tissue-specific expression profiles of the identified OBPs, CSPs and SNMPs were investigated using RT-PCR. Eight OBPs (HillOBP1-2, 9, 11-14, and 17), one CSP (HillCSP5) and one SNMP (HillSNMP1) were predominantly expressed in antennae. Further real-time quantitative PCR analyses revealed that the antennae-enriched unigenes also exhibited significant differences in expression between males and females. Among the sex-biased unigenes, six ORs showed female-biased expression, suggesting that these genes might participate in female-specific behaviors such as oviposition site searching. Sixteen ORs and two OBPs showed male-biased expression, indicating that they may play key roles in the detection of female sex pheromones. Our study is the first attempt to delineate the molecular basis of chemoreception in H. illucens. Our data provide useful information for comparative studies on the differentiation and evolution of Dipteran chemosensory gene families.

Sensilla localization and sex pheromone recognition of odorant binding protein OBP4 in the mirid plant bug Adelphocoris lineolatus (Goeze)

Pheromone binding proteins (PBPs) are well studied in lepidopteran moths and are considered to be crucial in detection of sex pheromones as well as some green leaf volatiles. In contrast, evidence that PBPs interact with sex pheromones of hemipteran species is not available. The mirid plant bug, Adelphocoris lineolatus (Goeze), is a notorious hemipteran pest that uses two butyrate esters, trans-2-hexenyl butyrate (E2HB) and hexyl butyrate (HB), and one hexenoic aldehyde trans-4-oxo-2-hexenal (E4O2H), as sex pheromones. In the present study, we report on an odorant binding protein, AlinOBP4, with particular focus on its potential physiological roles in the detection of A. lineolatus sex pheromone components. Phylogenetic analyses indicated that AlinOBP4 and two mirid orthologs clustered in a general phylogenetic clade with the lepidopteran ABX OBPs, the fly LUSH and the OBP83a/b subfamily. Cellular localization by fluorescence in situ hybridization and immunolabeling further demonstrated that AlinOBP4 was strongly expressed in the multiporous sensilla trichodea (str) and middle long sensilla basiconica (mlsba) of male A. lineolatus adults, suggesting a key role associated with sex pheromone and odorant detection. A ligand binding assay revealed that recombinant AlinOBP4 protein highly bound not only to the sex pheromone components E4O2H but also to some host plant volatiles. These findings together with the evidence of insect PBPs available in the literature support the view that AlinOBP4 is involved in sex pheromone detection in male A. lineolatus and provide foundational information for further elucidating the molecular mechanisms of chemosensory based mating behavior in hemipteran mirid bugs.

Silencing of OBP genes: Generation of loss-of-function mutants of PBP by genome editing.

Pheromone binding proteins (PBPs) are small soluble proteins (about 15kDa) that play striking roles in the detection of sex pheromones in insects. Many studies including structural analysis, binding simulation, and in vitro assays have been performed to clarify the modes of action of PBPs. Although these studies have provided valuable contributions toward the understanding of which key amino acid components contribute to the correct folding of PBPs and their binding affinities to sex pheromones, the functional characteristics of PBPs in the natural environment is still obscure. Recent developments in genome editing have begun to enable the functional examination of PBPs in in vivo. Among insect PBPs, BmPBP1 is one of the most well-characterized, there being rich understanding of its structure, biochemical analysis, binding affinity, localization, and the relationship between the type of olfactory receptors and its expression. A recent study has shown that BmPBP1 contributes sensitivity, but not selectivity of sex pheromone detection in the silkmoth Bombyx mori. In this chapter, based on a current report of the functional characterization of BmPBP1 using genome editing, we provide one example of a useful analytical method to clarify the functional role of PBP in vivo.

Identification and Expression Analysis of Chemosensory Receptor Genes in Bradysia odoriphaga (Diptera: Sciaridae).

The chive midge, Bradysia odoriphaga, is a major insect pest affecting Chinese chive production in China. Its adult life stage is nonfeeding and has a short life span. Hence, the perception of chemical stimuli is important for its adult behavior and reproductive success. To better understand its chemosensory process at the molecular level, chemosensory receptor genes were identified based on transcriptomes of B. odoriphaga. In total, 101 chemosensory genes were identified from the antenna and body transcriptomes, including 71 odorant receptors (ORs), 18 ionotropic receptors (IRs), 5 gustatory receptors (GRs), and 7 sensory neuron membrane proteins (SNMPs). Phylogenetic analysis indicated that most of these genes have homologs among other Dipteran insects. A transcript abundance comparison based on FPKM values was conducted to analyze the sex- and tissue-specific expression profiles of these chemosensory genes. Moreover, quantitative real-time PCR of OR transcripts was performed on different tissues (female antennae, male antennae, heads, and legs) to verify the transcriptional expression levels of ORs in the transcriptomes. This analysis suggested that 44 ORs showed significantly higher expression in the female antennae, while 16 OR transcripts were most highly expressed in the male antennae and may play significant roles in sex pheromone detection. In addition, some IRs and GRs might be involved in CO2 and sugar detection and temperature sensing. In the present study, 101 chemosensory genes were identified, and their putative functions were predicted. This work could provide a basis to facilitate functional clarification of these chemosensory genes at the molecular level.

Identification and expression analysis of odorant binding proteins and chemosensory proteins from dissected antennae and mouthparts of the rice bug Leptocorisa acuta

The rice bug, Leptocorisa acuta (Tunberg) (Hemiptera: Alydidae), is a notorious pest in Asia, and it is significantly attracted by the volatiles derived from host plants. However, it remains unknown how L. acuta recognizes host volatile compounds at the molecular level. Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are thought to be responsible for the initial biochemical recognition during olfactory perception. Here, we followed the RNA Sequencing (RNA-Seq) approach to identify candidate genes encoding OBPs and CSPs from dissected antennae and mouthparts of L. acuta. In total, 26 unigenes were identified coding for OBPs (22 Classic OBPs and four Plus-C OBPs), and 17 unigenes coding for CSPs. Real-time quantitative PCR (RT-qPCR) revealed that 11 OBPs (LacuOBP1, 5, 6, 7, 9, 11, 13, 14, 17, 20 and 23) and nine CSPs (LacuCSP2, 3, 4, 5, 6, 8, 9, 10 and 12) were predominantly expressed in antennae, indicating that they might be essential for detection of general odorants and pheromones. Among these antennae-predominantly expressed genes, LacuOBP11 and LacuOBP13 showed male-biased expression and therefore may play crucial roles in the detection of sex pheromones. Seven LacuOBPs (LacuOBP4, 8, 10, 12, 21, 25 and 26) and two CSPs (LacuCSP7 and LacuOBP11) were predominantly expressed in mouthparts, suggesting that these genes might be involved in taste perception. Our work provides a starting point to facilitate functional study of these OBPs and CSPs in L. acuta at the molecular level in the future.