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Reverse chemical ecology based on insect functional odorant binding proteins has been extensively studied to screen behaviourally active compounds, whereas chemosensory proteins (CSPs), which are reportedly involved in olfactory chemical reception and could serve as molecular targets remain unclear. In the present study, two behaviourally active compounds for Cnaphalocrocis medinalis, a serious pest of rice in Asia, were successfully screened via an antenna-biased CSP, CmedCSP33. Fluorescence competitive binding assays showed that CmedCSP33 could bind to seven out of 32 rice volatiles. Fluorescence quenching experiments revealed that CmedCSP33 forms a stable complex with nerolidol and β-ionone, and circular dichroism (CD) spectra demonstrated that these two compounds cause conformational changes in CmedCSP33. Furthermore, H-tube olfactometer bioassays showed that C. medinalis displayed prominent attractant responses to nerolidol and prominent repellent responses to β-ionone. Additionally, binding assays and CD spectra at different pH values implied that extensive conformational changes may be a general feature of CSPs for triggering the subsequent chemical transduction. Overall, our findings provide evidence for the involvement of CSPs in olfactory perception, and a protocol for effectively screening behaviourally active compounds.

Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play essential roles in insect chemosensory recognition. Here, we identified nine OBPs and nine CSPs from the Myzus persicae transcriptome and genome. Genomic structure analysis showed that the number and length of the introns are much higher, and this appears to be a unique feature of aphid OBP genes. Three M. persicae OBP genes (OBP3/7/8) as well as CSP1/4/6, CSP2/9 and CSP5/8 are tandem arrayed in the genome. Phylogenetic analyses of five different aphid species suggest that aphid OBPs and CSPs are conserved in single copy across all aphids (with occasional losses), indicating that each OBP and CSP class evolved from a single gene in the common ancestor of aphids without subsequent duplication. Motif pattern analysis revealed that aphid OBP and CSP motifs are highly conserved, and this could suggest the conserved functions of aphid OBPs and CSPs. Three OBPs (MperOBP6/7/10) are expressed antennae specifically, and five OBPs (MperOBP2/4/5/8/9) are expressed antennae enriched, consistent with their putative olfactory roles. M. persicae CSPs showed much broader expression profiles in nonsensory organs than OBPs. None of the nine MperCSPs were found to be antennae specific, but five of them (MperCSP1/2/4/5/6) showed higher expression levels in the legs than in other tissues. MperCSP10 mainly expressed in the antennae and legs. The broad and diverse expression patterns of M. persicae CSPs suggest their multifunctions in olfactory perception, development and other processes.

Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play essential roles in insect chemosensory recognition. Here, we identified nine OBPs and nine CSPs from the Myzus persicae transcriptome and genome. Genomic structure analysis showed that the number and length of the introns are much higher, and this appears to be a unique feature of aphid OBP genes. Three M. persicae OBP genes (OBP3/7/8) as well as CSP1/4/6, CSP2/9 and CSP5/8 are tandem arrayed in the genome. Phylogenetic analyses of five different aphid species suggest that aphid OBPs and CSPs are conserved in single copy across all aphids (with occasional losses), indicating that each OBP and CSP class evolved from a single gene in the common ancestor of aphids without subsequent duplication. Motif pattern analysis revealed that aphid OBP and CSP motifs are highly conserved, and this could suggest the conserved functions of aphid OBPs and CSPs. Three OBPs (MperOBP6/7/10) are expressed antennae specifically, and five OBPs (MperOBP2/4/5/8/9) are expressed antennae enriched, consistent with their putative olfactory roles. M. persicae CSPs showed much broader expression profiles in nonsensory organs than OBPs. None of the nine MperCSPs were found to be antennae specific, but five of them (MperCSP1/2/4/5/6) showed higher expression levels in the legs than in other tissues. MperCSP10 mainly expressed in the antennae and legs. The broad and diverse expression patterns of M. persicae CSPs suggest their multifunctions in olfactory perception, development and other processes.

The American cockroach (Periplaneta americana) is an urban pest with a precise chemosensory system that helps it achieve complex physiological behaviours, including locating food and mating. However, its chemosensory mechanisms have not been well studied. Here, we identified 71 putative odorant carrier protein genes in P. americana, including 57 new odorant-binding proteins (OBPs) and 11 chemosensory proteins (CSPs). To identify their physiological functions, we investigated their tissue expression patterns in antennae, mouthparts, legs, and the remainder of the body of both sexes, and determined that most of these genes were expressed in chemosensory organs. A phylogenetic tree showed that the putative pheromone-binding proteins of P. americana were in different clades from those of moths. Two genes, PameOBP24 and PameCSP7, were expressed equally in antennae of both sexes and highly expressed amongst the OBPs and CSPs. These genes were expressed in Escherichia coli and the resultant proteins were purified. The binding affinities of 74 common odorant compounds were tested with recombinant PameOBP24 and PameCSP7. Both proteins bound a variety of ligands. Our findings provide a foundation for future research into the chemosensory mechanisms of P. americana and help in identifying potential target genes for managing this pest.

Chemosensory proteins (CSPs) are a class of soluble proteins present in high concentrations in the sensilla of insect antennae. It has been proposed that they play an important role in insect olfaction by mediating interactions between odorants and odorant receptors. Here we report, for the first time, the presence of five CSP genes in the tsetse fly Glossina morsitans morsitans, a major vector transmitting nagana in livestock. Real-time quantitative reverse transcription PCR showed that three of the CSPs are expressed in antennae. One of them, GmmCSP2, is transcribed at a very high level and could be involved in olfaction. We also determined expression in the antennae of both males and females at different life stages and with different blood feeding regimes. The transcription of GmmCSP2 was lower in male antennae than in females, with a sharp increase in 10-week-old flies, 48 h after a bloodmeal. Thus there is a clear relationship between CSP gene transcription and host searching behaviour. Genome annotation and phylogenetic analyses comparing G. morsitans morsitans CSPs with those of other Diptera showed rapid evolution after speciation of mosquitoes.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are two families of small water-soluble proteins, abundant in the aqueous fluid surrounding olfactory receptor neurons in insect antennae. OBPs are involved in the first step of olfactory signal transduction, carrying airborne semiochemicals to the odorant receptors and can be classified into three groups: Classic OBPs, Plus-C OBPs and Atypical OBPs. Here, we identified and annotated genes encoding putative OBPs and CSPs in the pea aphid Acyrthosiphon pisum using bioinformatics. This identified genes encoding 13 Classic and two Plus-C OBPs and 13 CSPs. Homologous OBP sequences were also identified in nine other aphid species, allowing us to compare OBPs across several aphid and non-aphid species. We show that, although OBP sequences are divergent within a species and between different orders, there is a high similarity between orthologs within a range of aphid species. Furthermore, the phylogenetic relationships between OBP orthologs reflect the divergence of aphid evolution lineages. Our results support the 'birth-and-death' model as the major mechanism explaining aphid OBP sequence evolution, with the main force acting on the evolution being purifying selection.

Some chemosensory proteins (CSPs) are expressed in insect sensory appendages and are thought to be involved in chemical signalling by ants. We identified 14 unique CSP sequences in expressed sequence tag (EST) libraries of the red imported fire ant, Solenopsis invicta. One member of this group (Si-CSP1) is highly expressed in worker antennae, suggesting an olfactory function. A shotgun proteomic analysis of antennal proteins confirmed the high level of Si-CSP1 expression, and also showed expression of another CSP and two odorant-binding proteins (OBPs). We cloned and expressed the coding sequence for Si-CSP1. We used cyclodextrins as solubilizers to investigate ligand binding. Fire ant cuticular lipids strongly inhibited Si-CSP1 binding to the fluorescent dye N-phenyl-naphthylamine, suggesting cuticular substances are ligands for Si-CSP1. Analysis of the cuticular lipids showed that the endogenous ligands of Si-CSP1 are not cuticular hydrocarbons.

Moths generally rely on pheromone communication for mate finding. The pheromone components of most moths are produced by a common pathway of fatty-acid biosynthesis coupled with species-specific modifications of the final products. Some genes involved in moth pheromone production have previously been described, whereas others remain to be characterized and thus the molecular mechanisms accounting for the production of species-specific blends are far from understood. The turnip moth, Agrotis segetum, has a multicomponent pheromone, consisting of at least four components derived from palmitic and stearic acid. Different populations produce and respond to different pheromone blends, which makes this species an excellent model for research on genes and molecular mechanisms involved in moth pheromone production. For this purpose, we performed an expressed sequence tag (EST) analysis of two cDNA libraries, one representing the female pheromone gland and the other representing the remainder of the insect body. Among 2285 ESTs analysed altogether, we identified a unigene set of 707 putative gene representatives. The comparative distribution of those in the two libraries showed the transcriptomes of the tissues to be clearly different. One third of the gene representatives were exclusively found in the pheromone gland. From sequence homology to public database information we assigned putative functional roles for a majority of the unigenes and then compared functional profiles of the two tissues. In the set of ESTs more abundant in the pheromone gland library, we found homologues of an acyl-CoA Delta11-desaturase, a G-protein subunit, a chemosensory protein as well as a juvenile hormone binding protein.

Chemosensory proteins (CSPs) are associated with insect sensory organs, including the sensillum lymph in some cases. However, they are also commonly expressed in nonsensory tissues that lack gustatory and olfactory neurones. We characterized the sex and development specific expression patterns of four CSP genes from the Eastern spruce budworm (ESB) using Northern blots. CfumAY426540.2 was detected at high levels in adult moths. Conversely, CfumAY426538 was expressed in all stages except adult moths, and was most abundant during late stages of the 6th instar. CfumAY701858 was expressed in all stages, while CfumAY426539 was detected less frequently, at specific developmental stages such as the 5th to 6th instar moult. During a natural moult, and a premature moult induced by the ecdysteroid agonist tebufenozide, CfumAY701858 and CfumAY426539 were up-regulated, while CfumAY426538 appeared to be down-regulated. Our results suggest that some members of the CSP gene family from the ESB may be involved in development, including moulting.

Two different classes of chemosensory proteins (CSPs) in Locusta migratoria have been identified on the basis of the molecular cloning of a series of different cDNAs from the antennae of this insect. Several CSP isoforms have been purified and biochemically characterized from antennal and wing extracts, some of them corresponding to expression products predicted for the identified cDNAs. In wings, the nature of the main endogenous ligand binding to these proteins was determined as oleoamide by a gas chromatography-mass spectrometric approach. One of these isoforms has been expressed in a bacterial system with high yield and used in a fluorescent binding assay. Competitive binding experiments have indicated the presence of long-chain compounds among the best ligands.

A small expressed sequence tag (EST) project generating 506 ESTs from 375 cDNAs was undertaken on the antennae of male Manduca sexta moths in an effort to discover olfactory receptor proteins. We encountered several clones that encode apparent transmembrane proteins; however, none is a clear candidate for an olfactory receptor. Instead we found a greater diversity of odourant binding proteins (OBPs) than previously known in moth antennae, raising the number known for M. sexta from three to seven. Together with evidence of seventeen members of the family from the Drosophila melanogaster genome project, our results suggest that insects may have many tens of OBPs expressed in subsets of the chemosensory sensilla on their antennae. These results support a model for insect olfaction in which OBPs selectively transport and present odourants to transmembrane olfactory receptors. We also found five members of a family of shorter proteins, named sensory appendage proteins (SAPs), that might also be involved in odourant transport. This small EST project also revealed several candidate odourant degrading enzymes including three P450 cytochromes, a glutathione S-transferase and a uridine diphosphate (UDP) glucosyltransferase. Several first insect homologues of proteins known from vertebrates, the nematode Caenorhabditis elegans, yeast and bacteria were encountered, and most have now also been detected by the large D. melanogaster EST project. Only thriteen entirely novel proteins were encountered, some of which are likely to be cuticle proteins.