Classic Odorant Binding Proteins Research Articles

Molecular Characterization of Odorant-Binding Protein Genes Associated with Host-Seeking Behavior in Oides leucomelaena.

The identification of odorant-binding proteins (OBPs) involved in host location by Oides leucomelaena (O. leucomelaena Weise, 1922, Coleoptera, Galerucinae) is significant for its biological control. Tools in the NCBI database were used to compare and analyze the transcriptome sequences of O. leucomelaena with OBP and other chemosensory-related proteins of other Coleoptera insects. Subsequently, MEGA7 was utilized for OBP sequence alignment and the construction of a phylogenetic tree, combined with expression profiling to screen for candidate antennae-specific OBPs. In addition, fumigation experiments with star anise volatiles were conducted to assess the antennae specificity of the candidate OBPs. Finally, molecular docking was employed to speculate on the binding potential of antennae-specific OBPs with star anise volatiles. The study identified 42 candidate OBPs, 8 chemosensory proteins and 27 receptors. OleuOBP3, OleuOBP5, and OleuOBP6 were identified as classic OBP family members specific to the antennae, which was confirmed by volatile fumigation experiments. Molecular docking ultimately clarified that OleuOBP3, OleuOBP5, and OleuOBP6 all exhibit a high affinity for β-caryophyllene among the star anise volatiles. We successfully obtained three antennae-specific OBPs from O. leucomelaena and determined their high-affinity volatiles, providing a theoretical basis for the development of attractants in subsequent stages.

Transcriptome analysis used to identify and characterize odorant binding proteins in Agasicles hygrophila (Coleoptera: Chryspmelidae).

The transcriptomes of Agasicles hygrophila eggs and first instar larvae were analyzed to explore the olfactory mechanism of larval behavior. The analysis resulted in 135,359 unigenes and the identification of 38 odorant-binding proteins (OBPs), including 23 Minus-C OBPs, 8 Plus-C OBPs, and 7 Classic OBPs. Further analysis of differentially expressed genes (DEGs) revealed 10 DEG OBPs, with 5 (AhygOBP5, AhygOBP9, AhygOBP12, AhygOBP15 and AhygOBP36) up-regulated in first instar larvae. Verification of expression patterns of these 5 AhygOBPs using qPCR showed that AhygOBP9 and AhygOBP36 were mainly expressed in the adult stage with gradually increasing expression in the larval stage. AhygOBP5, AhygOBP12, and AhygOBP15 were not expressed in eggs and pupae, and their expression in larvae and adults showed no clear pattern. These 5 AhygOBPs may play an olfactory role in larval behavior, providing a basis for further investigation of their specific functions and clarifying the olfactory mechanism of A. hygrophila.

Expression, affinity, and binding mode analysis of antennal-binding protein X in the variegated cutworm Peridroma saucia (Hübner)

The variegated cutworm Peridroma saucia (Hübner) is a worldwide pest that causes serious damage to many crops. Odorant-binding proteins (OBPs) are small soluble proteins involved in the first step of odorant reception. In moths, antennal-binding protein Xs (ABPXs) represent a main subfamily of classic OBPs. However, their functions remain unclear. Here, we cloned the ABPX gene from the antennae of P. saucia. RT-qPCR and western-blot analyses showed that PsauABPX is antenna-predominant and male-biased. Further temporal expression investigation indicated that the expression of PsauABPX started 1 day before eclosion and reached the highest 3 days after eclosion. Next, fluorescence binding assays revealed that recombinant PsauABPX had high binding affinities with P. saucia female sex pheromone components Z11–16: Ac and Z9–14: Ac. Then, molecular docking, molecular dynamics simulation, and site-directed mutagenesis were employed to identify key amino acid residues involved in the binding of PsauABPX to Z11–16: Ac and Z9–14: Ac. The results demonstrated that Val-32, Gln-107 and Tyr-114 are essential for the binding to both sex pheromones. This study not only give us insight into the function and binding mechanism of ABPXs in moths, but could also be used to explore novel strategies to control P. saucia.

Minus-C subfamily has diverged from Classic odorant-binding proteins in honeybees

Odorant-binding proteins (OBPs) in insects bind to volatile chemical cues that are important in regulating insect behavior. It is hypothesized that OBPs bind with specificity to certain volatiles and may help in transport and delivery to odorant receptors (ORs), and may help in buffering the olfactory response and aid the insect in various behaviors. Honeybees are eusocial insects that perceive olfactory cues and strongly rely on them to perform complex olfactory behaviors. Here, we have identified and annotated odorant-binding proteins and few chemosensory proteins from the genome of the dwarf honey bee, Apis florea, using an exhaustive homology-based bioinformatic pipeline and analyzed the evolutionary relationships between the OBP subfamilies. Our study confirms that the Minus-C subfamily in honey bees has diverged from the Classic subfamily of odorant-binding proteins.

Identification and Expression Analysis of Odorant-Binding and Chemosensory Protein Genes in Virus Vector Nephotettix cincticeps.

The insect odorant binding proteins (OBPs) and chemosensory proteins (CSPs) are involved in the perception and discrimination of insects to host odor cues. Nephotettix cincticeps, one of the destructive pests of rice plants, not only directly damages hosts by sucking, but also indirectly transmits plant viruses in the field. Previous study found that two rice volatiles ((E)-β-caryophyllene and 2-heptanol) induced by rice dwarf virus (RDV) mediated the olfactory behavior of N. cincticeps, which may promote virus dispersal. However, the OBPs and CSPs in N. cincticeps are still unknown. In this study, to identify the OBP and CSP genes in N. cincticeps, transcriptomic analyses were performed. In total, 46,623 unigenes were obtained. Twenty putative OBP and 13 CSP genes were discovered and identified. Phylogenetic analyses revealed that five putative OBPs belonged to the plus-C OBP family, and the other classic OBPs and CSPs were distributed among other orthologous groups. A total of 12 OBP and 10 CSP genes were detected, and nine OBP and three CSP genes were highly expressed in N. cincticeps antennae compared with other tissues. This study, for the first time, provides a valuable resource to well understand the molecular mechanism of N. cincticeps in the perception and discrimination of the two volatiles induced by RDV infection.

Ligand-binding specificities of four odorant-binding proteins in Conogethes punctiferalis.

Odorant-binding proteins (OBPs) play essential roles in lepidopteran insects' perception of host volatiles by binding and transporting hydrophobic ligands. The yellow peach moth (YPM), Conogethes punctiferalis (Guenée), is a serious agricultural pest, with broad host range and cryptic feeding habits. However, few studies about YPM perceiving pheromones and host plant odorants have been reported. In this study, four OBP genes (CpunOBP8, CpunOBP9, CpunABP, and CpunGOBP2) were cloned from the antennae of YPM. The recombinant proteins were expressed and purified by prokaryotic expression system, with their binding affinities to 26 ligands being tested. Four CpunOBPs all had six conserved cysteine residues, which were typical structural characteristics of classical OBPs. The fluorescence competitive binding assay indicated that CpunOBP8 and CpunABP could not only exhibit high binding affinities to female sex pheromones, but also to host plant odorants. For example, CpunOBP8 bound strongly with cis-10-hexadecenal, hexadecanal, and so forth, whereas CpunABP bound with cis-10-hexadecenal, camphene, and 3-carene. Comparatively, CpunOBP9 and CpunGOBP2 could only bind with host plant odorants, with CpunOBP9 binding strongly to 3-methyl-1-butanol, hexyl acetate, and so forth, while CpunGOBP2 displaying the widest binding spectra and correlating with 3-carene, pentyl acetate, and so forth. The results indicated that on the one hand, each of the four CpunOBPs had its specific binding spectra when binding and transporting olfactory ligands; on the other hand, the same ligand might be bound to more than one CpunOBPs, which would provide information for the potential application of semiochemicals in controlling YPM.

Spatial Expression Analysis of Odorant Binding Proteins in Both Sexes of the Aphid Parasitoid Aphidius gifuensis and Their Ligand Binding Properties.

In China, Aphidius gifuensis is one of the most common endoparasitoids of the green peach aphid Myzus persicae and grain aphid Sitobion miscanthi in the field. Insect odorant-binding proteins (OBPs) play vital roles in odor perception during feeding, host searching, mating and oviposition. In addition, some OBPs are involved in other physiological processes such as gustation and reproduction. In the present study, a comparative antennal transcriptomic analysis was applied between male and female A. gifuensis. The spatial expression patterns among antennae, heads, thoraxes, abdomens and legs of OBPs in both sexes were further profiled. Fifteen AgifOBPs were predicted, and 14 of them were identified by gene cloning, including 12 classic OBPs and 2 min-C OBPs. As expected, all OBPs were mainly expressed at high levels in antennae, heads or legs which are sensory organs and tissues. Finally, ligand binding properties of 2 OBPs (AgifOBP7 and AgifOBP9) were further evaluated. Female leg specifically expressed AgifOBP9 displays a broad and high binding property to aphid alarm pheromones, plant green volatiles and aphid sex pheromones (Ki < 10 μΜ). However, female leg specifically expressed AgifOBP7 displays poor affinity for all tested ligands except CAU-II-11 ((E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methoxybenzoate), a reported (E)-β-farnesene (EBF) analog with an exceptionally high binding affinity (Ki = 1.07 ± 0.08 μΜ). In summary, we reported the spatial expression pattern of the OBP repertoire in A. gifuensis, and further studied the binding properties of OBP7 and OBP9, which are mainly expressed in female legs, laying the foundation for the dissection of the contribution of OBPs to chemosensation in A. gifuensis.

Binding Properties of Odorant-Binding Protein 4 of Tirathaba rufivena to Areca catechu Volatiles.

Odorant-binding proteins (OBPs) play a key role in the olfactory system and are essential for mating and oviposition host selection. Tirathaba rufivena, a serious lepidopterous insect pest of the palm area in recent years, has threatened cultivations of Areca catechu in Hainan. Female-biased odorant-binding protein 4 of T. rufivena (TrufOBP4) expression was hypothesized to participate in the process of oviposition host recognition and localization. In this study, we cloned and analyzed the cDNA sequence of TrufOBP4. The predicted mature protein TrufOBP4 is a small, soluble, secretory protein and belongs to a classic OBP subfamily. Fluorescence binding assay results showed that TrufOBP4 had high binding abilities with the host plant volatiles, octyl methoxycinnamate, dibutyl phthalate, myristic acid and palmitic acid. These four components tend to dock in the same binding pocket based on the molecular docking result. The interactions and contributions of key amino acid residues were also characterized. This research provides evidence that TrufOBP4 might participate in the chemoreception of volatile compounds from inflorescences of A. catechu and can contribute to the integrated management of T. rufivena.

Identification and expression of odorant binding proteins in the egg-parasitoid Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae, Telenominae)

Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae) is an egg-parasitoid of the southern green stink bug, Nezara viridula (Linneaus) (Hemiptera, Pentatomidae). Many behaviors associated with female T. basalis host-finding and acceptance are mediated by chemosensory pathways, for which olfactory, gustatory and ionotropic receptors have been previously identified. Odorant binding proteins (OBPs) are small, globular proteins, one of the functions of which is the transport of odorant ligands through the aqueous lymph of chemosensory sensilla to these receptors. We identified 18 classical OBP sequences in the T. basalis genome and transcriptomes sharing an average 26.8% pairwise identity. Gene tree analyses suggest very limited lineage-specific expansion and identify potential orthologs among other scelionids and Hymenoptera. Transcriptome mapping and qPCR comparison of expression levels in antennae and bodies of both sexes determine that at least five TbOBPs are preferentially expressed in the female antennae. These are, therefore, prime candidates for further study to determine their role in detecting host-produced semiochemicals.

The molecular identification, odor binding characterization, and immunolocalization of odorant-binding proteins in Liriomyza trifolii

The Liriomyza trifolii is a highly invasive polyphagia pest. Understanding the physiological functions of odorant binding proteins (OBPs) in the chemical communication of L. trifolii can lead to effective pest management strategies. Seven full-length OBPs were identified by transcriptome screening of L. trifolii adults. Bioinformatics analyses classified the seven OBPs into two subfamilies (six classic OBPs, one minus-C OBP). The analysis of their expression in different development stages revealed that LtriOBP5 was highly expressed in the larval stage, LtriOBP4 in the pupa stage, and LtriOBP1, 2, 3, 6, 7 in the adult stage; the expression levels were higher in male adults than in females. The analysis of different tissues showed high expression of LtriOBP1, 3, 6, 7 in the antennae, which were selected for in vitro purification. To explore the ligand compounds of OBPs, fluorescence competitive binding experiments were performed. Immunofluorescence localization revealed that LtriOBP1, 3, 6, 7 showed strong binding abilities to plant volatiles and were located in the antennae, implying that LtriOBP1, 3, 6, 7 may play key roles in olfaction, such as host location. LtriOBP6 and LtriOBP7 had strong binding abilities to specific herbivore-induced plant volatiles, suggesting LtriOBP6 and LtriOBP7 may also play critical roles in chemoreception. This study provides preliminary exploration of the olfactory perception mechanism of L. trifolii, which can be used as a basis to design insect behavior regulators and develop highly effective insecticides using mixture of ligands and known pesticides.

Expression Profile and Ligand Screening of a Putative Odorant-Binding Protein, AcerOBP6, from the Asian Honeybee.

Simple SummaryThe olfactory sensillum, which is located in the antenna of insects, is the basic unit of the olfactory organ. Olfactory-related genes are expressed in the sensillum. It is believed that the process of olfaction recognition is mainly mediated by two gene families, odorant binding proteins (OBPs) and olfactory receptors (ORs). The honeybee possesses a large numbers of ORs, but few OBPs. Up to now, the function of OBPs in the honeybee has not yet been fully elucidated. In order to reveal the specific role of OBPs from Apis cerana cerana, we selected an OBP gene, AcerOBP6, which is highly expressed in the antennae of worker bees, acquired a purified protein via a prokaryotic expression system, and analyzed its function using bioinformatics, molecular biology, and electrophysiology. According to the result, AcerOBP6 was a protein with extensive binding affinity, and we speculated that its function was chiefly related to foraging. Overall, this research not only explains the essential role of OBPs in ligand binding, but also provides valuable resources to help researchers further understand the nature and mechanism of the olfactory system.Olfaction is essential in some behaviors of honeybee, such as nursing, foraging, attracting a mate, social communication, and kin recognition. OBPs (odorant binding proteins) play a key role in the first step of olfactory perception. Here, we focused on a classic OBP with a PBP-GOBP domain from the Asian honeybee, Apis cerana cerana. Beyond that, the mRNA expression profiles and the binding affinity of AcerOBP6 were researched. According to qRT-PCR analysis, AcerOBP6 transcripts were mainly expressed in the antennae of forager bees. In addition, we found that the expression level of AcerOBP6 was higher than that of AmelOBP6. The fluorescence competitive binding assay indicated that the AcerOBP6 protein had binding affinity with most of the tested odors, including queen pheromone, worker pheromone, and floral volatiles, among which the strongest one was linolenic acid (with a Ki value of 1.67). However, AcerOBP6 was not sensitive to the brood pheromones. A further study based on EAG assay revealed that the antennae had the strongest response to 2-heptanone. The EAG recording values of the selected ligands were all reduced after AcerOBP6 was silenced, with 8 of 14 declining significantly (p < 0.01) given that these odors could specifically bind to AcerOBP6. As revealed in our current study, AcerOBP6 might be a crucial protein involved in olfactory recognition for foraging. Overall, the research provides a foundation for exploring the olfactory mechanism of A. cerana cerana.

Comparative Genomics Provide Insights Into Function and Evolution of Odorant Binding Proteins in Cydia pomonella.

Insect olfaction is vital for foraging, mating, host-seeking, and avoidance of predators/pathogens. In insects, odorant binding proteins (OBPs) are involved in transporting hydrophobic odor molecules from the external environment to receptor neurons. The codling moth, Cydia pomonella, one of the most destructive insect fruit pests, causes enormous economic losses. However, little is known about the number, variety, gains and losses, and evolution of OBP genes in C. pomonella. Here we report the identification of 40 OBPs in C. pomonella, most (75%) of which are classic OBPs, using genomic and transcriptomic analyses. Two OBP genes were lost in C. pomonella relative to possible distant ancestor in Lepidoptera lineage based on an analysis of gene gains and losses. The phylogenetic tree and chromosome location showed that the expansion of OBP genes mainly resulted from tandem duplications, as the CpomGOBP2 gene was duplicated twice along with loss of CpomPBPB. Two positive selection sites of the CpomGOBP1 gene were identified while other OBP genes evolved under purifying selection. Our results provide fundamental knowledge of OBP genes allowing further study of their function in C. pomonella.

A new non-classical fold of varroa odorant-binding proteins reveals a wide open internal cavity

Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites.

OBP14 (Odorant-Binding Protein) Sensing in Adelphocoris lineolatus Based on Peptide Nucleic Acid and Graphene Oxide.

Simple SummaryThe alfalfa plant bug, Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae), is a serious agricultural pest in China. Previous studies have shown that olfaction plays a crucial role in the survival and reproduction of herbivorous insects, such as A. lineolatus, and OBPs (odorant-binding proteins) are involved in the initial steps of olfaction. Due to the complex intracellular environment in insects, detection of the expression of OBP genes by conventional methods is limited in vitro. In this study, we developed a PNA-GO- (peptide nucleic acid and graphene oxide) based mRNA biosensor that allows detection of the expression of the AlinOBP14 gene in A. lineolatus. We demonstrate that AlinOBP14 is expressed in the adult head, which is consistent with previous research on the expression profiles of AlinOBP14 in A. lineolatus using quantitative real-time PCR. The results demonstrate that the method we developed is both feasible and reliable. In addition, this method exhibited good stability and specificity in detection of chemically synthesized targets in vitro as well as the expression of the target OBP gene in vivo.OBPs play a crucial role in the recognition of ligands and are involved in the initial steps of semiochemical perception. The diverse expression of OBP genes allows them to participate in different physiological functions in insects. In contrast to classic OBPs with typical olfactory roles in A. lineolatus, the physiological functions of Plus-C OBPs remain largely unknown. In addition, detection of the expression of insect OBP genes by conventional methods is difficult in vitro. Here, we focused on AlinOBP14, a Plus-C OBP from A. lineolatus, and we developed a PNA-GO-based mRNA biosensor to detect the expression of AlinOBP14. The results demonstrated that AlinOBP14 plays dual roles in A. lineolatus. The AlinOBP14 is expressed beneath the epidermis of the vertex and gena in heads of A. lineolatus, and it functions as a carrier for three terpenoids, while AlinOBP14 is also expressed in the peripheral antennal lobe and functions as a carrier for endogenous compounds such as precursors for juvenile hormone (JH) and JHⅢ. Our investigation provides a new method to detect the expression of OBP genes in insects, and the technique will facilitate the use of these genes as potential targets for novel insect behavioral regulation strategies against the pest.

Identification and functional characterization of odorant-binding proteins 69a and 76a of Drosophila suzukii

The fruit fly Drosophila suzukii is a fruit crop pest that causes a severe economic threat to soft summer fruit worldwide. The male sex pheromone, cis-vaccenyl acetate (cVA) has multiple functions in intra-species communication in Drosophila melanogaster, which is required in male to suppress male-male courtship. D. suzukii males do not produce cVA; however, the odorant receptor for cVA (Or67d) is still functional. The lack of cVA in D. suzukii casts the question of whether this pheromone might have been replaced by another compound similar to cVA that disrupts mating in D. suzukii. In order to address this question, we cloned two D. suzukii adult antenna-specific odorant-binding proteins (OBPs) DsOBP69a and DsOBP76a and aligned with their D. melanogaster orthologues. Moreover, we examined the binding properties of the newly identified recombinant proteins against 26 potential ligands including cVA, using the fluorescence-based ligand binding assay. The alignment showed that DsOBP69a and DsOBP76a, have six conserved cysteines and belong to the classic OBP family. Furthermore, our results revealed that cVA did not bind to DsOBP69a or DsOBP76a proteins. Interestingly, the floral odorant β-ionone and the bitter substance berberine chloride and coumarin displayed high binding ability. It is also worth noting that DsOBP69a and DsOBP76a have different affinities to (Z)-7-Tricosene that may reflect different functional roles. These findings suggest that DsOBP69a and DsOBP76a are potentially involved in olfaction and gustation of D. suzukii.

Analyses of structural dynamics revealed flexible binding mechanism for the Agrilus mali odorant binding protein 8 towards plant volatiles.

Volatiles from host plants are an important source of insect pest attractants and repellents. Insect odorant binding proteins (OBPs) have been widely characterized, but the molecular binding dynamics and underlying mechanisms are still not well understood. Thus, we characterized binding characteristics of AmalOBP8 from the apple buprestid beetle (Agrilus mali Matsumura), an unprecedented serious threat to rare apple germplasm resources and local ecosystems. Fluorescence studies demonstrated that the quenching mechanism was clearly static. AmalOBP8 was found to bind with both volatiles at single independent sites. Negative thermodynamic parameters suggested that binding interactions between AmalOBP8 and both volatiles could occur spontaneously. Hydrogen bonding was the key force in AmalOBP8's binding to geranyl formate, for which the amino acid residue Trp106 played a critical role in the binding pocket. Multiple Leu residues in AmalOBP8 created a strong hydrophobic environment, and formed the binding pocket for (Z)-3-hexenyl hexanoate. Compared to classic OBPs, in addition to lack of one disulfide bridge, AmalOBP8 had a small α-helix (α7) at the C-terminus, resulting in greater flexibility and adaptability for this protein to bind with different compound molecules. Key residues of AmalOBP8 in binding interactions with plant volatiles were clarified. AmalOPB8 had a large ligand binding spectrum and great flexibility in binding with plant volatiles, providing good molecular targets for screening insect attractants and repellents. Our results can promote understanding of insects' perception of various odorants, and establish a foundation for discovery of new pest control agents. © 2020 Society of Chemical Industry.

Olfactory Reception of Host Alarm Pheromone Component by the Odorant-Binding Proteins in the Samurai Wasp, Trissolcus japonicus (Hymenoptera: Scelionidae).

The samurai wasp, Trissolcus japonicus, is the predominant egg parasitoid of the brown marmorated stink bug, Halyomorpha halys, in its native range in China. (E)-2-Decenal is a major component of the alarm pheromone of H. halys, an important invasive insect pest with significant economic importance. T. japonicus can be strongly repelled by (E)-2-decenal, and thus its host location efficiency would be reduced in the field. Better understanding on the molecular basis of olfactory reception of this host alarm pheromone component by T. japonicus may provide opportunities to develop novel approaches to enhance biological control efficacy of the parasitoid against H. halys. We identified six Odorant Binding Proteins (OBPs) from T. japonicus by transcriptome sequencing, within which three classical OBPs were expressed in a heterologous expression system with E. coli, harvested, and then challenged with (E)-2-decenal in binding assay experiments. TjapOBP2 showed the highest binding ability to (E)-2-decenal, compared to TjapOBP1 and TjapOBP3. Our results unambiguously suggest that TjapOBP2 would play an important role in discriminating (E)-2-decenal and could be a possible target for further intervention in the parasitoid–host system.

Expression and functional characterization of odorant-binding protein genes in the endoparasitic wasp Cotesia vestalis.

Odorant-binding proteins (OBPs) are crucial in insect's olfactory perception, which participate in the initial step of odorant molecules transporting from the external environment to olfactory receptor neurons. To better understand the roles for OBPs in olfactory perception in Cotesia vestalis, a solitary larval endoparasitoid of diamondback moth, Plutella xylostella, we have comprehensively screened the genome of C. vestalis, and obtained 20 CvesOBPs, including 18 classic OBPs and two minus-C OBPs. Motif-pattern analysis indicates that the motifs of C. vestalis OBPs are highly conserved in Hymenoptera. The results of tissue expression analysis show that five OBPs (CvesOBP1/11/12/14/16) are highly expressed in male antennae, whereas six other OBP genes (CvesOBP7/8/13/17/18/19) are significantly transcriptionally enriched in female antennae. The results of RNA interference experiments for three most highly expressed OBP genes (CvesOBP17/18/19) in female antennae demonstrate that they are likely involved in parasitic processes of female wasps, as the wasps take a longer time to target the hosts when they are knocked down.

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

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

Identification 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.