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Bemisia tabaci Gennadius biotypes B and Q are two of the most important worldwide agricultural insect pests. Genomic sequences of Type-2 B. tabaci chemosensory protein (BtabCSP2) were cloned and sequenced in B and Q biotypes, revealing key biotype-specific variations in the intron sequence. A Q260 sequence was found specifically in Q-BtabCSP2 and Cucumis melo LN692399, suggesting ancestral horizontal transfer of gene between the insect and the plant through bacteria. A cleaved amplified polymorphic sequences (CAPS) method was then developed to differentiate B and Q based on the sequence variation in exon of BtabCSP2 gene. The performances of CSP2-based CAPS for whitefly recognition were assessed using B. tabaci field collections from Shandong Province (P.R. China). Our SacII based CAPS method led to the same result compared to mitochondrial cytochrome oxidase-based CAPS method in the field collections. We therefore propose an explanation for CSP origin and a new rapid simple molecular method based on genomic DNA and chemosensory gene to differentiate accurately the B and Q whiteflies of the Bemisia complex around the world.

Chemosensory proteins (CSP) afford insects the ability to recognize complicated environmental cues and are involved in diverse physiological processes. The planthoppers Laodelphax striatellus and Sogatella furcifera are serious specialist pests of rice throughout eastern Asia. In this study, we identified 21 putative CSP genes (namely LsCSP1–12 and SfCSP1–9) in the two planthoppers L. striatellus and S. furcifera, and profiled their transcript accumulation in different developmental stages and body parts by real-time quantitative PCR (qPCR). All CSPs had the characteristic typical features of the CSP family, with the exception of LsCSP10 and SfCSP2, which lacked the predicted signal peptide sequence. Among these putative CSP genes, only two (LsCSP9 and SfCSP5) were more highly expressed in the antennae, indicating that they might be involved in olfaction. On the contrary, eleven genes (LsCSP2, 3, 4, 6, 8, 12; SfCSP1, 4, 6, 7, 8) and one gene (SfCSP9) were more highly expressed in the legs and heads (without antennae), respectively. The remainders of the genes were ubiquitously expressed in the heads (without antennae) and the bodies (without the legs and heads). The broad expression patterns in different body parts indicate that the CSPs of these two delphacid species may be involved in diverse functions besides chemosensation. These findings increase the understanding of the chemosensory protein system of Hemiptera species.

Tsetse fly exhibit species-specific olfactory uniqueness potentially underpinned by differences in their chemosensory protein repertoire. We assessed 1) expansions of chemosensory protein orthologs in Glossina morsitans morsitans, Glossina pallidipes, Glossina austeni, Glossina palpalis gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpis tsetse fly species using Café analysis (to identify species-specific expansions) and 2) differential expressions of the orthologs and associated proteins in male G. m. morsitans antennae and head tissues using RNA-Seq approaches (to establish associated functional molecular pathways). We established accelerated and significant (P90% of chemosensory transcripts) included cilium-associated mechanoreceptors, chemo-sensation, neuronal controlled growth/differentiation and regeneration/responses to stress. The expanded and tsetse fly species specific orthologs includes those associated with known tsetse fly responsive ligands (4-methyl phenol, 4-propyl phenol, acetic acid, butanol and carbon dioxide) and potential tsetse fly species-specific responsive ligands (2-oxopentanoic acid, phenylacetaldehyde, hydroxycinnamic acid, 2-heptanone, caffeine, geosmin, DEET and (cVA) pheromone). Some of the orthologs can potentially modulate several tsetse fly species-specific behavioral (male-male courtship, hunger/host seeking, cool avoidance, hygrosensory and feeding) phenotypes. The putative tsetse fly specific chemosensory gene orthologs and their respective ligands provide candidate gene targets and kairomones for respective downstream functional genomic and field evaluations that can effectively expand toolbox of species-specific tsetse fly attractants, repellents and other tsetse fly behavioral modulators., Author summary Tsetse flies are insect vectors of sleeping sickness in humans and nagana in livestock in sub-Sahara Africa. Tsetse flies identify their hosts (preferred and non-preferred) by detecting and processing odor cues emitted by the hosts in their environment. Tsetse flies use chemosensory proteins and associated pathways in their antennae to identify these cues. In this study, we identified expansions of these chemosensory protein in six tsetse fly species (Glossina morsitans morsitans, Glossina pallidipes, Glossina austeni, Glossina palpalis gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpis) with different known hosts. We also identified potential ligands to these proteins based on fruit fly (Drosophila melanogaster) orthologs. With G. m. morsitans as an example, we identified the proteins and associated molecular pathways preferentially expressed in tsetse fly antennae. These proteins may be responsible for the tsetse fly species-specific host discrimination, with the ligands eliciting species-specific behavioral responses in the flies. The expressed orthologs may be functionally important in odor detection in tsetse fly and lay down useful groundwork for downstream functional genomics R&D for more effective tsetse fly species-specific odor attractants and repellents for routine tsetse fly control operations.

The chemosensory gene families of insects encode proteins that are crucial for host location, mate finding, oviposition, and avoidance behaviors. The insect peripheral chemosensory system comprises odorant receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), odorant binding proteins (OBPs), chemosensory proteins (CSPs), and sensory neuron membrane proteins (SNMPs). These protein families have been identified from a large number of insect species, however, they still remain unidentified from several taxa that could provide important clues to their evolution. These taxa include older lepidopteran lineages and the sister order of Lepidoptera, Trichoptera (caddisflies). Studies of these insects should improve evolutionary analyses of insect chemoreception, and in particular shed light on the origin of certain lepidopteran protein subfamilies. These include the pheromone receptors (PRs) in the "PR clade", the pheromone binding proteins (PBPs), general odorant binding proteins (GOBPs), and certain presumably Lepidoptera-specific IR subfamilies. Hence, we analyzed antennal transcriptomes from Rhyacophila nubila (Trichoptera), Eriocrania semipurpurella, and Lampronia capitella (representing two old lepidopteran lineages). We report 37 ORs, 17 IRs, 9 GRs, 30 OBPs, 7 CSPs, and 2 SNMPs in R. nubila; 37 ORs, 17 IRs, 3 GRs, 23 OBPs, 14 CSPs, and 2 SNMPs in E. semipurpurella; and 53 ORs, 20 IRs, 5 GRs, 29 OBPs, 17 CSPs, and 3 SNMPs in L. capitella. We identified IR members of the "Lepidoptera-specific" subfamilies IR1 and IR87a also in R. nubila, demonstrating that these IRs also occur in Trichoptera. Members of the GOBP subfamily were only found in the two lepidopterans. ORs grouping within the PR clade, as well as PBPs, were only found in L. capitella, a species that in contrast to R. nubila and E. semipurpurella uses a so-called Type I pheromone similar to the pheromones of most species of the derived Lepidoptera (Ditrysia). Thus, in addition to providing increased coverage for evolutionary analyses of chemoreception in insects, our findings suggest that certain subfamilies of chemosensory genes have evolved in parallel with the transition of sex pheromone types in Lepidoptera. In addition, other chemoreceptor subfamilies show a broader taxonomic occurrence than hitherto acknowledged.

The high sensitivity of the olfactory system is essential for feeding and oviposition in moth insects, and some chemosensory proteins (CSPs) are thought to play roles in this system by binding and carrying hydrophobic odorants across the aqueous sensillar lymph. In this study, to identify the olfactory CSPs from a repertoire of 21 CSP members in the notorious rice pest Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), tissue expression patterns were firstly examined by quantitative real-time polymerase chain reaction (qPCR). It showed that CSP2 was antennae specific and seven more CSPs (CSP1, 3, 4, 6, 15, 16, and 17) were antennae biased in expression, suggesting their olfactory roles; while other CSPs were multiple-tissue expressed and non-antennae biased, suggesting other functions for these genes. To further determine the ligand binding specificity, three putative olfactory genes (CSP1–3) were expressed in Escherichia coli cells, and binding affinity of these three recombinant CSP proteins were measured for 35 plant volatiles by the ligand binding assays. CSP1 and CSP2 exhibited high binding affinities (Ki ≤ 10.00 µM) for four (2-tridecanone, benzaldehyde, laurinaldehyde and 2-pentadecanone) and two (2-heptanol and (+)-cedrol) host plant volatiles, respectively; the three CSPs also showed moderate binding affinity (Ki = 10.01–20.00 µM) for 16 plant volatiles. Our study suggests that the three CSPs play essential roles in the perception of host plant volatiles, providing bases for the elucidation of olfactory mechanisms in this important pyralid pest.

Chemosensory proteins (CSPs) are soluble carrier proteins typically characterized by a six-helix bundle structure joined by two disulfide bridges and a conserved Cys spacing pattern (C1-X6-8 -C2-X16-21 -C3-X2 -C4). CSPs are functionally diverse with reported roles in chemosensation, immunity, development, and resistance. To expand our molecular understanding of CSP function in plant bugs, we used recently developed transcriptomic resources for Lygus lineolaris and Lygus hesperus to identify 17 and 14 CSP-like sequences, respectively. The Lygus CSPs are orthologous and share significant sequence identity with previously annotated CSPs. Three of the CSPs are predicted to deviate from the typical CSP structure with either five or seven helical segments rather than six. The seven helix CSP is further differentiated by an atypical C3-X3 -C4 Cys spacing motif. Reverse transcriptase PCR-based profiling of CSP transcript abundance in adult L. lineolaris tissues revealed broad expression for most of the CSPs with antenna specific expression limited to a subset of the CSPs. Comparative sequence analyses and homology modeling suggest that variations in the amino acids that comprise the Lygus CSP binding pockets affect the size and nature of the ligands accommodated.

Chemosensory proteins (CSPs) play important roles in chemosensation in insects, but their exact physiological functions remain elusive. In order to investigate the functions of CSPs in the oriental armyworm Mythimna separata, in the present study we explored expression patterns and binding characteristics of the CSP, MsepCSP8. The distinctive functions of MsepCSP8 were also validated by RNAi. The results showed that MsepCSP8 shares high sequence similarity with CSPs of other insect family members, including the characteristic four-cysteine signature motif. MsepCSP8 mRNA was specifically expressed in antennae of females at levels well above those in other tissues. Competitive binding assays confirmed that 20 out of 56 ligands bound more strongly to MsepCSP8 at pH 7.4 than at pH 5.0. Protein structure modeling and molecular docking analyses identified amino acid residues involved in binding volatile compounds, and behavioral response experiments showed that M. separata elicited significant responses to five volatiles from compounds displaying high binding affinity to MsepCSP8. MsepCSP8 transcript abundance was decreased by dsMsepCSP8 injection, which affected the behavioral responses of M. separata to representative semiochemicals. Our findings demonstrate that MsepCSP8 likely contributes to mediating responses of M. separata adults to plant volatiles.

Chemosensory proteins (CSPs) play important roles in insects' chemoreception, although their specific functional roles have not been fully elucidated. In this study, we conducted the developmental expression patterns and competitive binding assay as well as knock-down assay by RNA interference both in vitro and in vivo to reveal the function of NlugCSP10 from the brown planthopper (BPH), Nilaparvata lugens (Stal), a major pest in rice plants. The results showed that NlugCSP10 messenger RNA was significantly higher in males than in females and correlated to gender, development and wing forms. The fluorescence binding assays revealed that NlugCSP10 exhibited the highest binding affinity with cis-3-hexenyl acetate, eicosane, and (+)-β-pinene. Behavioral assay revealed that eicosane displayed attractant activity, while cis-3-hexenyl acetate, similar to (+)-β-pinene significantly repelled N. lugens adults. Silencing of NlugCSP10, which is responsible for cis-3-hexenyl acetate binding, significantly disrupted cis-3-hexenyl acetate communication. Overall, findings of the present study showed that NlugCSP10 could selectively interrelate with numerous volatiles emitted from host plants and these ligands could be designated to develop slow-release mediators that attract/repel N. lugens and subsequently improve the exploration of plans to control this insect pest.

Olfaction in antennae is essential for regulating insect behaviors such as host preference and oviposition site selection. To better understand the olfactory mechanisms in the cicada Subpsaltria yangi that has a very narrow host range far fewer than diets of most other cicadas, an antennal transcriptome was constructed in this study. We identified 10 unigenes encoding putative odorant-binding proteins (OBPs), 10 chemosensory proteins (CSPs), 8 sensory neuron membrane proteins (SNMPs), 7 gustatory receptors (GRs), 29 odorant receptors (ORs) and 13 ionotropic receptors (IRs). We found that the OBPs were separated into two groups, Classic OBPs and Plus-C OBPs, according to their motifs. Based on sequence alignment and phylogenetic analysis, we found five Orcos in OR family. However, no pheromone receptor was identified, which may be related to that cicadas are sound-producing insects and acoustic signals other than pheromones play most important role in sexual communication. We used qRT-PCR to examine the expression profile of ten OBPs genes in various organs, and found that they were mainly expressed in olfactory organs such as antennae and mouthparts. Our results make it possible for future research of the olfactory system of S. yangi at the molecular level, and provide important bases for elucidation of the molecular mechanisms and evolution of chemosensation in sap-sucking insects.

Bradysia odoriphaga is an agricultural pest insect affecting the production of Chinese chive and other liliaceous vegetables in China, and it is significantly attracted by sex pheromones and the volatiles derived from host plants. Despite verification of this chemosensory behavior, however, it is still unknown how B. odoriphaga recognizes these volatile compounds on the molecular level. Many of odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play crucial roles in olfactory perception. Here, we identified 49 OBP and 5 CSP genes from the antennae and body transcriptomes of female and male adults of B. odoriphaga, respectively. Sequence alignment and phylogenetic analysis among Dipteran OBPs and CSPs were analyzed. The sex- and tissue-specific expression profiles of 54 putative chemosensory genes among different tissues were investigated by quantitative real-time PCR (qRT-PCR). qRT-PCR analysis results suggested that 22 OBP and 3 CSP genes were enriched in the antennae, indicating they might be essential for detection of general odorants and pheromones. Among these antennae-enriched genes, nine OBPs (BodoOBP2/4/6/8/12/13/20/28/33) were enriched in the male antennae and may play crucial roles in the detection of sex pheromones. Moreover, some OBP and CSP genes were enriched in non-antennae tissues, such as in the legs (BodoOBP3/9/19/21/34/35/38/39/45 and BodoCSP1), wings (BodoOBP17/30/32/37/44), abdomens and thoraxes (BodoOBP29/36), and heads (BodoOBP14/23/31 and BodoCSP2), suggesting that these genes might be involved in olfactory, gustatory, or other physiological processes. Our findings provide a starting point to facilitate functional research of these chemosensory genes in B. odoriphaga at the molecular level.

The spread of the exotic insect pest sycamore lace bug Corythucha ciliata (Say) is increasing worldwide. The identification of behaviorally active compounds is crucial for reducing the current distribution of this pest. In this study, we identified and documented the expression profiles of genes encoding chemosensory proteins (CSPs) in the sycamore lace bug to identify CSPs that bind to the alarm pheromone geraniol. One CSP (CcilCSP2) that was highly expressed in nymph antennae was found to bind geraniol with high affinity. This finding was confirmed by fluorescence competitive binding assays. We further discovered one candidate chemical, phenyl benzoate, that bound to CcilCSP2 with even higher affinity than geraniol. Behavioral assays revealed that phenyl benzoate, similar to geraniol, significantly repelled sycamore lace bug nymphs but had no activity toward adults. This study has revealed a novel repellent compound involved in behavioral regulation. And, our findings will be beneficial for understanding the olfactory recognition mechanism of sycamore lace bug and developing a push-pull system to manage this pest in the future.

Chemosensory proteins (CSP) are soluble carrier proteins that may function in odorant reception in insects. CSPs have not been thoroughly studied at whole-genome level, despite the availability of insect genomes. Here, we identified/reidentified 283 CSP genes in the genomes of 22 mosquitoes. All 283 CSP genes possess a highly conserved OS-D domain. We comprehensively analyzed these CSP genes and determined their conserved domains, structure, genomic distribution, phylogeny, and evolutionary patterns. We found an average of seven CSP genes in each of 19 Anopheles genomes, 27 CSP genes in Cx. quinquefasciatus, 43 in Ae. aegypti, and 83 in Ae. albopictus. The Anopheles CSP genes had a simple genomic organization with a relatively consistent gene distribution, while most of the Culicinae CSP genes were distributed in clusters on the scaffolds. Our phylogenetic analysis clustered the CSPs into two major groups: CSP1-8 and CSE1-3. The CSP1-8 groups were all monophyletic with good bootstrap support. The CSE1-3 groups were an expansion of the CSP family of genes specific to the three Culicinae species. The Ka/Ks ratios indicated that the CSP genes had been subject to purifying selection with relatively slow evolution. Our results provide a comprehensive framework for the study of the CSP gene family in these 22 mosquito species, laying a foundation for future work on CSP function in the detection of chemical cues in the surrounding environment.

The diamondback moth was estimated to increase costs to the global agricultural economy as the global area increase of Brassica vegetable crops and oilseed rape. Sex pheromones traps are outstanding tools available in Integrated Pest Management for many years and provides an effective approach for DBM population monitoring and control. The ratio of two major sex pheromone compounds shows geographical variations. However, the limitation of our information in the DBM pheromone biosynthesis dampens our understanding of the ratio diversity of pheromone compounds. Here, we constructed a transcriptomic library from the DBM pheromone gland and identified genes putatively involved in the fatty acid biosynthesis, pheromones functional group transfer, and β-oxidation enzymes. In addition, odorant binding protein, chemosensory protein and pheromone binding protein genes encoded in the pheromone gland transcriptome, suggest that female DBM moths may receive odors or pheromone compounds via their pheromone gland and ovipositor system. Tissue expression profiles further revealed that two ALR, three DES and one FAR5 genes were pheromone gland tissue biased, while some chemoreception genes expressed extensively in PG, pupa, antenna and legs tissues. Finally, the candidate genes from large-scale transcriptome information may be useful for characterizing a presumed biosynthetic pathway of the DBM sex pheromone.

Chemosensory protein (CSP) and gustatory receptor genes have been identified in all major arthropod groups. However, odorant binding proteins (OBP) and olfactory receptor genes are insect specific, suggesting that both gene families originated after the Hexapoda-Crustacea split (~470 million years ago). The seemingly parallel diversification of OBP and olfactory receptors has been suggested as coevolution between these genes after insect terrestrialization. To test this hypothesis we used the recently published transcriptomes of the jumping bristletail Lepismachilis y-signata and the firebrat Thermobia domestica to search for putative OBP and CSP sequences and analyzed their relationship to binding proteins of other insects and crustaceans. Our results suggest an evolution and expansion of OBPs as an adaptation to a terrestrial insect lifestyle, independently from the emergence of olfactory receptors.

More than half of the proteome from mandibular glands in caterpillars is represented by chemosensory proteins. Based on sequence similarity, these proteins are putative transporters of ligands to gustatory receptors in sensory organs of insects. We sought to determine whether these proteins are inducible by comparing, both qualitatively and quantitatively, the salivary (mandibular and labial) proteomes from caterpillars (Vanessa cardui) reared on different plants and artificial diet containing either bacteria or bacterial cell-walls. We included a treatment where the caterpillars were switched from feeding on artificial diet to plant material at some point in their development. Additionally, we evaluated the degree of overlap between the proteomes in the hemolymph-filled coelom and salivary glands of caterpillars reared on plant material. We found that the quality and quantity of the identified proteins differed clearly between hemolymph-filled coelome, labial and mandibular glands. Our results indicated that even after molting and two-day feeding on a new diet, protein production is affected by the previous food source used by the caterpillar. Candidate proteins involved in chemosensory perception by insects were detected: three chemosensory (CSPs) and two odorant-binding proteins (OBPs). Using the relative amounts of these proteins across tissues and treatments as criteria for their classification, we detected hemolymph- and mandibular gland-specific CSPs and observed that their levels were affected by caterpillar diet. Moreover, we could compare the protein and transcript levels across tissues and treatment for at least one CSP and one OBP. Therefore, we have identified specific isoforms for testing the role of CSPs and OBPs in plant and pathogen recognition. We detected catalase, immune-related protein and serine proteases and their inhibitors in high relative levels in the mandibular glands in comparison to the labial glands. These findings suggest that the mandibular glands of caterpillars may play an important role protecting the caterpillar from oxidative stress, pathogens and aiding in digestion. Contamination with hemolymph proteins during dissection of salivary glands from caterpillars may occur but it is not substantial since the proteomes from hemolymph, mandibular and labial glands were easily discriminated from each other by principal component analysis of proteomic data.

Chemosensory proteins (CSPs) are believed to be important in the detection of semiochemicals, and have been reported to be involved in development, molting, leg regeneration and phase transition. In the current study, six CSP genes were identified in the beet armyworm Spodoptera exigua , namely SexiCSP1-6, from the full length cDNA library. Multiple alignment and phylogenetic analysis indicated that they were very diverse as seen in CSPs of other insect species. Expression patterns determined by quantitative real-time PCR (qPCR) revealed that they were highly expressed in antenna. SexiCSP1-3, SexiCSP5 and SexiCSP6 were male antenna-biased, while SexiCSP4 was female-biased in the antenna. In addition to antenna, they were expressed at high levels in legs and wings, suggesting potential roles in contact chemoreception and non-olfactory roles. Compared with the expression in antenna, legs and wings, only SexiCSP1 showed relatively high expression in heads, thoraxes and abdomen. These results extended olfactory gene resource in the beet armyworm and provided basis for further gaining a better understanding of CSPs roles in this pest.

Insect chemosensory proteins (CSPs) are small, water-soluble proteins which can bind and transport hydrophobic odorants through sensillum lymph to activate odorant receptors, thus play important roles in the olfactory recognition. In the present study, by searching the transcriptome data sets, a total of 22 candidate CSP genes were identified from Cnaphalocrocis medinalis , a serious lepidopteran rice pest in Asia. CSP proteins encoded by these genes showed typical characteristics: an N-terminal signal peptide, four conserved cysteine residues and the pattern of cysteine spacing. The expression profiles of these putative CSP genes were investigated using real-time quantitative PCR. The results showed that five genes ( CmedCSP4 , CmedCSP8 , CmedCSP11 , CmedCSP18 and CmedCSP21 ) were expressed primarily in antennae, suggesting their involvement in olfactory processes; whilst other genes are mainly expressed in non-olfactory tissues, such as abdomen and legs, indicating a broader physiological function for these CSPs. The findings will lead to a better understanding of the molecular mechanisms of chemoreception in C. medinalis .

In this study, we constructed a high-quality cDNA library from the antennae of the Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). A total of 1,235 colonies with inserts greater than 0.7 kb were sequenced and analyzed. Homology searching coupled with bioinformatics analysis identified 15 and 7 cDNA sequences, respectively, encoding putative odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). A phylogenetic tree of CsupCSPs showed that each CsupCSP has orthologs in Manduca sexta and Bombyx mori with strong bootstrapping support. One CSP was either very specific or more related to the CSPs of another species than to conspecific CSP. The expression profiles of the OBPs and CSPs in different tissues were measured by real-time quantitative PCR. The results revealed that of the 11 OBP genes, the transcript levels of CsupOBP1, CsupOBP5, and CsupOBP7 were higher in both male and female antennae than those in other tissues. And CsupCSP7 was highly expressed in both male and female antennae. Based on these results, the possible physiological functions of CsupOBPs and CsupCSPs were discussed.

Chemoreception is a key feature in the selection of host plants by insects. In our current study, we identified two olfactory genes in Frankliniella occidentalis (Pergande), one encoding a putative pheromone binding protein (PBP) and the other one encoding a chemosensory protein (CSP), through an analysis of an antennal cDNA library. The expression profiles of both genes were examined in tissues of the antennae, abdomen, legs, and wings using quantitative reverse transcription and real-time polymerase chain reaction. We found that the PBP and CSP genes were expressed ubiquitously during various developmental stages of F. occidentalis. However, the levels of PBP and CSP transcripts in males were higher than those in females. The levels of PBP and CSP gene expression in adult F. occidentalis also varied with age. These findings support the hypothesis that odorant binding proteins and CSPs play dynamic roles in the development of F. occidentalis, and are likely to be involved in broader physiological processes.

In insects, flight and sophisticated olfactory systems go hand in hand and are essential to survival and evolutionary success. Females of many Lepidopteran species have secondarily lost their flight ability, which may lead to changes in the olfactory capabilities of both larval and adult stages. The gypsy moth, Lymantria dispar, an important forest pest worldwide, is currently undergoing a diversification process with three recognized subspecies: the Asian gypsy moth (AGM), Lymantria dispar asiatica; the Japanese gypsy moth (JGM), Lymantria dispar japonica; and the European gypsy moth (EGM), Lymantria dispar dispar. Females of EGM populations from North America have lost their flight capacity whereas the JGM and AGM females are flight capable, making this an ideal system to investigate the relationship between flight and olfaction. We used next-generation sequencing to obtain female antennal and larval head capsule transcriptomes in order to (i) investigate the differences in expression of olfaction-related genes among populations; (ii) identify the most similar protein sequences reported for other organisms through a BLAST search, and (iii) establish the phylogenetic relationships of these sequences with respect to other insect species. Using this approach, we identified 115 putative chemosensory genes belonging to five families of olfaction-related genes. A principal component analysis (PCA) revealed that the gene-expression patterns of female antennal transcriptomes from different subspecies were more similar to one another than to the larval head capsules of their respective subspecies supporting strong chemosensory differences between the two developmental stages. An analysis of the shared and exclusively expressed genes for three populations shows no evidence that loss of flight affects the number or type of genes being expressed. These results indicate either (a) that loss of flight does not impact the olfactory gene repertoire or (b) that the secondary loss of flight in American EGM populations may be too recent to have caused major changes in the genes being expressed. However, we found higher expression values for most olfaction-related genes in EGM females, suggesting that differences in transcription rates could be an adaptation of flightless females to their chemical environment. Differences in olfactory genes and their expression in the larvae appear to be unrelated to the flight ability of adult females and are likely adaptations to different ecological pressures., Frontiers in Ecology and Evolution, 5, ISSN:2296-701X

While it has been well characterized that chemosensory receptors in guts of mammals have great influence on food preference, much remains elusive in insects. Insect chemosensory proteins (CSPs) are soluble proteins that could deliver chemicals to olfactory and gustatory receptors. Recent studies have identified a number of CSPs expressed in midgut in Lepidoptera insects, which started to reveal their roles in chemical recognition and stimulating appetite in midgut. In this study, we examined expression patterns in midgut of 21 Spodoptera litura CSPs (SlitCSPs) characterized from a previously reported transcriptome, and three CSPs were identified to be expressed highly in midgut. The orthologous relationships between midgut expressed CSPs in S. litura and those in Bombyx mori and Plutella xylostella also suggest a conserved pattern of CSP expression in midgut. We further demonstrated that the expression of midgut-CSPs may change in response to different host plants, and SlitCSPs could bind typical chemicals from host plant in vitro. Overall, our results suggested midgut expressed SlitCSPs may have functional roles, likely contributing to specialization and adaption to different ecosystems. Better knowledge of this critical component of the chemsensation signaling pathways in midguts may improve our understanding of food preference processes in a new perspective.

The olfaction system of insects plays an important role in mediating various physiological behaviors, including locating hosts, avoiding predators, and recognizing mates and oviposition sites. Therefore, some key genes in the system present valuable opportunities as targets for developing novel green pesticides.Athetis lepigone, a noctuid moth can feed on more than 30 different host plants making it a serious polyphagous pest worldwide, and it has become one of the major maize pests in northern China since 2011. However, there are no reports on effective and environmentally friendly pesticides for the control of this pest. In this study, we identified 28 genes encoding putative odorant binding proteins (OBPs) and 20 chemosensory protein (CSPs) genes based on our previousA. lepigonetranscriptomic data. A tissue expression investigation and phylogenetic analysis were conducted in an effort to postulate the functions of these genes. Our results show that nearly half (46.4%) of theAlOBPsexhibited antennae-biased expression while many of theAlCSPswere highly abundant in non-antennal tissues. These results will aid in exploring the chemosensory mechanisms ofA. lepigoneand developing environmentally friendly pesticides against this pest in the future.

As a frequently used neonicotinoid insecticide, imidacloprid can impair the chemoreceptive behavior of honey bees even at sublethal doses, while the physiochemical mechanism has not been further revealed. Here, multiple fluorescence spectra, thermodynamic method, and molecular docking were used to study the interaction and the functional inhibition of imidacloprid to the recombinant CSP1 protein in Asian honey bee, Apis cerana. The results showed that the fluorescence intensity (λem = 332 nm) of CSP1 could be significantly quenched by imidacloprid in a dynamic mode. During the quenching process, ΔH > 0, ΔS > 0, indicating that the acting forces of imidacloprid with CSP1 are mainly hydrophobic interactions. Synchronous fluorescence showed that the fluorescence of CSP1 was mainly derived from tryptophan, and the hydrophobicity of tryptophan decreased with the increase of imidacloprid concentration. Molecular docking predicted the optimal pose and the amino acid composition of the binding process. Circular dichroism (CD) spectra showed that imidacloprid reduced the α-helix of CSP1 and caused the extension of the CSP1 peptide chain. In addition, the binding of CSP1 to floral scent β-ionone was inhibited by nearly 50% of the apparent association constant (KA) in the presence of 0.28–2.53 ng/bee of imidacloprid, and the inhibition rate of nearly 95% at 3.75 ng/bee of imidacloprid at sublethal dose level. This study initially revealed the molecular physiochemical mechanism that sublethal doses of neonicotinoid still interact and inhibit the physiological function of the honey bees' chemoreceptive system.

Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) of arthropods are thought to be involved in chemical recognition which regulates pivotal behaviors including host choice, copulation and reproduction. In insects, OBPs and CSPs located mainly in the antenna but they have not been systematically characterized yet in Bemisia tabaci which is a cryptic species complex and could damage more than 600 plant species. In this study, among the 106,893 transcripts in the head assembly, 8 OBPs and 13 CSPs were identified in B. tabaci MED based on head transcriptomes of adults. Phylogenetic analyses were conducted to investigate the relationships of B. tabaci OBPs and CSPs with those from several other important Hemipteran species, and the motif-patterns between Hemiptera OBPs and CSPs were also compared by MEME. The expression profiles of the OBP and CSP genes in different tissues of B. tabaci MED adults were analyzed by real-time qPCR. Seven out of the 8 OBPs found in B. tabaci MED were highly expressed in the head. Conversely, only 4 CSPs were enriched in the head, while the other nine CSPs were specifically expressed in other tissues. Our findings pave the way for future research on chemical recognition of B. tabaci at the molecular level.

The Yunnan pine and Simao pine caterpillar moths, Dendrolimus houi Lajonquière and Dendrolimus kikuchii Matsumura (Lepidoptera: Lasiocampidae), are two closely related and sympatric pests of coniferous forests in southwestern China, and olfactory communication systems of these two insects have received considerable attention because of their economic importance. However, there is little information on the molecular aspect of odor detection about these insects. Furthermore, although lepidopteran species have been widely used in studies of insect olfaction, few work made comparison between sister moths on the olfactory recognition mechanisms. In this study, next-generation sequencing of the antennal transcriptome of these two moths were performed to identify the major olfactory genes. After comparing the antennal transcriptome of these two moths, we found that they exhibit highly similar transcripts-associated GO terms. Chemosensory gene families were further analyzed in both species. We identified 23 putative odorant binding proteins (OBP), 17 chemosensory proteins (CSP), two sensory neuron membrane proteins (SNMP), 33 odorant receptors (OR), and 10 ionotropic receptors (IR) in D. houi; and 27 putative OBPs, 17 CSPs, two SNMPs, 33 ORs, and nine IRs in D. kikuchii. All these transcripts were full-length or almost full-length. The predicted protein sequences were compared with orthologs in other species of Lepidoptera and model insects, including Bombyx mori, Manduca sexta, Heliothis virescens, Danaus plexippus, Sesamia inferens, Cydia pomonella, and Drosophila melanogaster. The sequence homologies of the orthologous genes in D. houi and D. kikuchii are very high. Furthermore, the olfactory genes were classed according to their expression level, and the highly expressed genes are our target for further function investigation. Interestingly, many highly expressed genes are ortholog gene of D. houi and D. kikuchii. We also found that the Classic OBPs were further separated into three groups according to their motifs, which will help future functional researches. Surprisingly, no pheromone receptor was identified in the two Dendrolimus species, which may indicate a special pheromone identification mechanism in Dendrolimus. Our work allows for further functional studies of pheromones and host volatile recognition genes, and give novel candidate targets for pest management.

Insects have evolved many physiological and behavioral adaptations to recognize external complex chemicals. Olfaction plays an important role in perceiving volatile chemicals, utilizing them to locate host sites, conspecifics, and enemies. Chemosensory proteins (CSPs) are present in high concentrations within the sensory sensilla of insects and are endowed with a heterogeneous range of functions. However, direct evidence for the involvement of CSPs in olfactory function is still lacking. In this study, a fluorescence-based ligand binding assay using Bdor-CSP2 illustrated its ability to bind the majority of the selected ligands of different shapes and chemical structures that are ecologically significant, host plant volatiles of Bactrocera dorsalis. RNAi-mediated silencing coupled with electrophysiological tests showed lower electrophysiological responses to (3Z)-hex-3-en-1-ol, trans-2-hexenal, 6-methylhept-5-en-2-one, and 3-methylbutyl acetate in dsBdor CSP2 treated flies compared with the untreated controls. The reduced expression of Bdor-CSP2 by RNA interference was confirmed by semi-quantitative PCR, real-time quantitative PCR and Western blot, which suggested the RNAi-treatment was responsible for the observed reduction of antennal responses in EAG recordings. These data suggest that the expression of Bdor-CSP2 is necessary for the recognition of antennal responses to some plant host volatiles by B. dorsalis.

We sequenced the antenna transcriptome of the brown planthopper (BPH), Nilaparvata lugens (Stal), a global rice pest, and performed transcriptome analysis on BPH antenna. We obtained about 40million 90bp reads that were assembled into 75,874 unigenes with a mean size of 456bp. Among the antenna transcripts, 32,856 (43%) showed significant similarity (E-value

Chemosensory proteins (CSPs) are small soluble proteins endowed with heterogeneous functions. The information so far available for CSPs suggested these well-defined and conserved proteins were involved in diverse activities, including chemical communication, feeding, development, mating, immune regulation, as well as circadian rhythms. However, the detailed mechanisms of these physiological functions remain elusive. To explore the underlying mechanisms of CSPs and their interaction partners, a cDNA library from the head of Plutella xylostella was screened against CSP1 to identify proteins involved in the PxylCSP1-related physiological activities. Protein kinase C (PKC) was screened out as a putative interacting protein of PxylCSP1. The full length of PxylPKC cDNA was obtained, and the results of semi-quantitative real-time PCR and quantitative real-time PCR revealed that PxylPKC showed similar expression pattern as PxylCSP1. In vivo and in vitro interactions between PxylCSP1 and PxylPKC were further confirmed by co-immunoprecipitation and GST pull-down assays, respectively. These findings extended our knowledge on the mechanisms of CSP-regulated functions, and providing new target proteins to facilitate the design of novel intervention strategies against the pest.

Chemosensory proteins (CSPs) are a group of small soluble proteins found so far exclusively in arthropod species. These proteins act in chemical communication and perception. In this study, a gene encoding the Type 1 CSP (BtabCSP1) from the agricultural pest Bemisia tabaci (whitefly) was analyzed to understand sequence variation and expression specificity in different biotypes. Sequence analysis of BtabCSP1 showed significant differences between the two genetically characterized biotypes, B and Q. The B-biotype had a larger number of BtabCSP1 mutations than the Q-biotype. Similar to most other CSPs, BtabCSP1 was more expressed in the head than in the rest of the body. One-step RT-PCR and qPCR analysis on total messenger RNA showed that biotype-Q had higher BtabCSP1 expression levels than biotype-B. Females from a mixed field-population had high levels of BtabCSP1 expression. The interaction of BtabCSP1 with the insecticide thiamethoxam was investigated by analyzing the BtabCSP1 expression levels following exposure to the neonicotinoid, thiamethoxam, in a time/dose-response study. Insecticide exposure increased BtabCSP1 expression (up to tenfold) at 4 and 24 h following 50 or 100 g/ml treatments.

In insects, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are primary peripheral olfactory proteins playing critical roles in odorant detection. In this study, we present the first identification of OBPs and CSPs from the transcriptome of grape phylloxera Daktulosphaira vitifoliae Fitch, an important pest that damages both roots and leaves of grapes. The OBPs contained six conserved cysteine residues and the CSPs contained four conserved cysteine residues in this insect. Phylogenetic analysis showed that most of the olfactory proteins were closely related to OBPs and CSPs from other aphids. However, DviOBP7 and DviCSP9 were different because they were classified into different independent branches, respectively. Real-time polymerase chain reaction (RT-PCR) was used to examine the tissue expression of these transcripts. DviOBP1, DviOBP6, and DviOBP7 were uniquely or primarily expressed in antennae and not in the body. DviOBP2 was more abundantly expressed in the body than in the antennae. The expression levels of OBPs and CSPs of phylloxera varied depending upon where they were expressed in different body tissues.

Anomala corpulenta is an insect pest that is widely distributed in China, and it can cause large economic losses in agriculture, horticulture, and forestry. It is difficult to control because most of the lifecycle is underground. Trapping adults would be an environmentally sustainable way to reduce damage caused by this insect. Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are considered to be important chemodetection proteins. In this study, we investigate the expression of odorant-binding protein genes and chemosensory protein genes in different tissues of the adult and other developmental stages. The antenna had the highest expression levels for OBPs and CSPs of all tissues, except for OBP7 which was most highly expressed in the head without antenna. Besides the antenna, the head without antenna had the next highest expression levels for all OBP and CSP genes. The expression of OBP2, OBP3, OBP14, and CSP in the adult was higher than in other stages, while, OBP7 was lower than in other stages. Before estimating the expression of OBPs and CSPs, seven candidate reference genes were tested for stability among the tissues of adult and other development stages. GAPDH was the best reference gene in different tissues of the adult, and RPL13a was the best reference gene in different development stages. Our results provide a foundation for further research on the molecular aspects of chemical communication of this insect.

The relationship between the diversity of chemosensory proteins (CSPs) and their limited gene repertoire is of great interest. We have previously demonstrated that mRNA editing is responsible for this diversity. In this report, we describe hypotheses we have developed from phylogenetic analyses of CSP genes and RNA variants of the silkworm moth, Bombyx mori. Our analysis is based on previously published DNA/RNA/peptide sequencing data of the different variants from different tissues ( Xuan et al., 2014 , Xuan et al., 2016 ). We find and describe conventional A-to-I RNA editing, and C-to-U/U-to-C editing events in different CSP variants and pinpoint loci we believe encode the ADAR and ADAT enzymes and variants thereof that are responsible for these events. We also describe several non-conventional editing events including G-to-I and C-to-I, suggesting the existence of a multitude of mutation mechanisms in insects as found in microbes, viruses and plants. In particular, our phylogenetic analysis indicates that some mRNA sequences are more closely related to edited mRNA than to the gene sequence and shows evidence of tissue-specific DNA-dependent (Dd) or RNA-dependent (Rd) mutations depending on the gene. These descriptive findings in the silkworm are useful for molecular evolution of Dd-and Rd-RNA polymerases in relation with a specific gene family, as well as evolutionary biology and adaptation of RNA, cell, tissue or organism to new environment.

Chemoreception plays an important role in insects for sensing information when searching for host and oviposition sites. An understanding of the chemosensory mechanism could aid in the development of new methods to effectively prevent damage from insects in agriculture. We have constructed a legs cDNA library for Apolygus lucorum and sequenced 1584 ESTs, from which we identified 669 unigenes. From this collection we identified one putative odorant binding protein (AlucOBP5) and three chemosensory proteins (AlucCSP2, AlucCSP3, AlucCSP4) genes. Using real-time PCR method, we assessed the expression of these genes in the head, thorax, abdomen, wing, antenna and mouthparts. Results indicate that the expression of these genes had tissue- and gender-specificity. AlucCSP2 and AlucCSP3 were specifically expressed in female wings. AlucCSP4 was expressed relatively highly in female wings but also expressed in other tissues. AlucOBP5 was expressed in female abdomen and male legs with high levels in the latter. Expression vectors for these proteins were constructed and expressed in BL21(DE3). The purified proteins were then tested for binding properties using bis-ANS as the fluorescent ligand. AlucOBP5 could bind strongly with phenyl acetaldehyde, 1-hexanol, 3-hexenal and β-ionone. AlucCSP2 and AlucCSP3 had low affinity with all general odorants. AlucCSP4 did not bind with any of the standards. All four proteins could bind with gossypol, meletin with high affinity and could also bind with rutin hydrate, although AlucCSP4 had weak binding capacity. AlucCSP3 and AlucCSP4 could bind weakly with catechin, while AlucCSP2 and AlucOBP5 could not.

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera (Hubner) was obtained from antennal cDNA libraries and expressed in Escherichia coli. The real time quantitative PCR (RT-qPCR) results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings. Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles. The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde, whereas methyl phenylacetate, 2-decanone, 1-pentanol, carvenol, isoborneol, nerolidol, 2-nonanone and ethyl heptanoate have relatively weak binding affinity. Moreover, the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33–38 (α1), 40–48 (α2), 62–72 (α3), 80–96 (α4), 98–108 (α5), and 116–119 (α6), two pairs of disulfide bridges Cys49-Cys55, Cys75-Cys78. The two amino acid residues, Ile94 and Trp101, may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.

Gene duplications can have a major role in adaptation, and gene families underlying chemosensation are particularly interesting due to their essential role in chemical recognition of mates, predators and food resources. Social insects add yet another dimension to the study of chemosensory genomics, as the key components of their social life rely on chemical communication. Still, chemosensory gene families are little studied in social insects. Here we annotated chemosensory protein (CSP) genes from seven ant genomes and studied their evolution. The number of functional CSP genes ranges from 11 to 21 depending on species, and the estimated rates of gene birth and death indicate high turnover of genes. Ant CSP genes include seven conservative orthologous groups present in all the ants, and a group of genes that has expanded independently in different ant lineages. Interestingly, the expanded group of genes has a differing mode of evolution from the orthologous groups. The expanded group shows rapid evolution as indicated by a high dN/dS (nonsynonymous to synonymous changes) ratio, several sites under positive selection and many pseudogenes, whereas the genes in the seven orthologous groups evolve slowly under purifying selection and include only one pseudogene. These results show that adaptive changes have played a role in ant CSP evolution. The expanded group of ant-specific genes is phylogenetically close to a conservative orthologous group CSP7, which includes genes known to be involved in ant nestmate recognition, raising an interesting possibility that the expanded CSPs function in ant chemical communication.

By mimicking biological olfaction, biosensors have been used for the detection of important ligands in complex environments. An olfactory biosensor based on chemosensory proteins (CSPs) was designed by immobilizing honeybee CSPs (Ac-ASP3) on the interdigitated golden electrodes. Its responses to ligands of pheromones and floral odors were recorded by impedance spectroscopy. The relative decrease of charge transfer resistance of the biosensor is proportional to the logarithm of ligand concentration from 10 −7 M to 10 −3 M. To explore the molecular recognition processes of the biosensor, the tertiary structure of the protein was modeled and the protein-ligand interactions were investigated by the molecular docking. Our docking results verified the validity of experiments and showed that the specific ligands could form hydrogen bonds with some of the conserved residues, such as Cys 60 and Gln 64 of Ac-ASP3. Furthermore, combining the molecular modeling with impedance detection, the accuracy, specificity and predictability of the ligands binding to the protein could be improved. Thus, CSPs will provide a promising approach for chemical molecular sensing at low concentrations.

Our knowledge of the molecular basis of odorant reception in insects has grown exponentially over the past decade. Odorant receptors (ORs) from moths, fruit flies, mosquitoes, and the honey bees have been deorphanized, odorant-degrading enzymes (ODEs) have been isolated, and the functions of odorant-binding proteins (OBPs) have been unveiled. OBPs contribute to the sensitivity of the olfactory system by transporting odorants through the sensillar lymph, but there are competing hypotheses on how they act at the end of the journey. A few ODEs that have been demonstrated to degrade odorants rapidly may act in signal inactivation alone or in combination with other molecular traps. Although ORs in Drosophila melanogaster respond to multiple odorants and seem to work in combinatorial code involving both periphery and antennal lobes, reception of sex pheromones by moth ORs suggests that their labeled lines rely heavily on selectivity at the periphery.

Research in the field of insect-host plant interactions has indicated that constituents of insect saliva play an important role in digestion and affect host chemical defense responses. However, most efforts have focused on studying the composition and function of regurgitant or saliva produced in the labial glands. Acknowledging the need for understanding the role of the mandibular glands in herbivory, we sought to make a qualitative and semi-quantitative comparison of soluble luminal protein fractions between mandibular and labial glands of Vanessa gonerilla butterfly larvae. Amylase and lysozyme were inspected as possible major enzymatic activities in the mandibular glands aiding in pre-digestion and antimicrobial defense. Although detected, neither of these enzymatic activities was prominent in the luminal protein preparation of a particular type of gland. Proteins isolated from the glands were identified by mass spectrometry and by searching an EST-library database generated for four other nymphalid butterfly species, in addition to the public NCBI database. The identified proteins were also quantified from the data using "Quanty", an in-house program. The proteomic analysis detected chemosensory proteins as the most abundant luminal proteins in the mandibular glands. In comparison to these proteins, the relative amounts of amylase and lysozyme were much lower in both gland types. Therefore, we speculate that the primary role of the mandibular glands in Lepidopteran larvae is chemoreception which may include the detection of microorganisms on plant surfaces, host plant recognition and communication with conspecifics.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) of aphids are thought to be responsible for the initial molecular interactions during olfaction that mediate detection of chemical signals. Analysis of the diversity of proteins involved comprises critical basic research work that will facilitate the development of sustainable pest control strategies. To help us better understand differences in the olfactory system between winged and wingless grain aphids, we constructed an antennal transcriptome from winged and wingless Sitobion avenae (Fabricius), one of the most serious pests of cereal fields worldwide. Among the 133,331 unigenes in the antennal assembly, 13 OBP and 5 CSP putative transcripts were identified with 6 OBP and 3 CSP sequences representing new S. avenae annotations. We used qPCR to examine the expression profile of these genes sets across S. avenae development and in various tissues. We found 7 SaveOBPs and 1 SaveCSP were specifically or significantly elevated in antennae compared with other tissues, and that some transcripts (SaveOBP8, SaveCSP2 and SaveCSP5) were abundantly expressed in the legs of winged or wingless aphids. The expression levels of the SaveOBPs and SaveCSPs varied depending on the developmental stage. Possible physiological functions of these genes are discussed. Further molecular and functional studies of these olfactory related genes will explore their potential as novel targets for controlling S. avenae.

How organisms adapt to new environments is of fundamental biological interest, but poorly understood at the genetic level. Chemosensory systems provide attractive models to address this problem, because they lie between external environmental signals and internal physiological responses. To investigate how selection has shaped the well-characterized chemosensory system of Drosophila melanogaster, we have analysed genome-wide data from five diverse populations. By couching population genomic analyses of chemosensory protein families within parallel analyses of other large families, we demonstrate that chemosensory proteins are not outliers for adaptive divergence between species. However, chemosensory families often display the strongest genome-wide signals of recent selection within D. melanogaster. We show that recent adaptation has operated almost exclusively on standing variation, and that patterns of adaptive mutations predict diverse effects on protein function. Finally, we provide evidence that chemosensory proteins have experienced relaxed constraint, and argue that this has been important for their rapid adaptation over short timescales., Fruit flies gain valuable information about their environment by sensing chemicals. Here, Arguello et al. show strong signals of recent selection on the chemosensory system of the fruit fly Drosophila melanogaster, consistent with the adaptation of populations to their local chemical environment.