Pheromone-binding Proteins Genes Research Articles

Molecular and Functional Characterization of Pheromone Binding Protein 2 from Cyrtotrachelus buqueti (Coleoptera: Curculionidae)

Pheromone-binding proteins (PBPs) play important roles in binding and transporting sex pheromones. However, the PBP genes identified in coleopteran insects and their information sensing mechanism are largely unknown. Cyrtotrachelus buqueti (Coleoptera: Curculionidae) is a major insect pest of bamboo plantations. In this study, a novel PBP gene, CbuqPBP2, from C. buqueti was functionally characterized. CbuqPBP2 was more abundantly expressed in the antennae of both sexes than other body parts, and its expression level was significantly male-biased. Fluorescence competitive binding assays showed that CbuqPBP2 exhibited the strongest binding affinity to dibutyl phthalate (Ki = 6.32 μM), followed by styrene (Ki = 11.37 μM), among twelve C. buqueti volatiles. CbuqPBP2, on the other hand, showed high binding affinity to linalool (Ki = 10.55), the main volatile of host plant Neosinocalamus affinis. Furthermore, molecular docking also demonstrated the strong binding ability of CbuqPBP2 to dibutyl phthalate, styrene, and linalool, with binding energy values of −5.7, −6.6, and −6.0 kcal/mol, respectively, and hydrophobic interactions were the prevailing forces. The knockdown of CbuqPBP2 expression via RNA interference significantly reduced the electroantennography (EAG) responses of male adults to dibutyl phthalate and styrene. In conclusion, these results will be conducive to understanding the olfactory mechanisms of C. buqueti and promoting the development of novel strategies for controlling this insect pest.

Pheromone-binding proteins based phylogenetics and phylogeography of Maruca spp. from Asia, Africa, Oceania, and South America.

Variations in the functional response of legume pod borer (Maruca vitrata) populations to sex pheromone blends were observed in Asia and Africa. Hence, this study was carried out to understand the differences in pheromone‐binding proteins (PBPs) among Maruca populations in Asia, Africa, Oceania, and South America. A de novo transcriptome assembly was adopted to sequence the entire transcribed mRNAs in M. vitrata from Taiwan. The raw‐sequence data were assembled using homologous genes from related organisms in GenBank to detect M. vitrata PBPs (MvitPBPs). Sections of the cDNA of MvitPBP of different length were used to design primers to amplify the full‐length cDNA of PBPs. All three PBP sequences comprised three exons interspersed by two introns. In total, 92 MvitPBP1 haplotypes, 77 MvitPBP2 haplotypes, and 64 MvitPBP3 haplotypes were identified in 105, 98, and 68 Maruca individuals, respectively. High pairwise F ST values (0.41–0.73) and phylogenetic analyses distinguished the putative Maruca species in South America from those occurring in rest of the world, and possibly two putative subspecies in Asia and Africa. The haplotype networks and Automatic Barcode Gap Discovery analyses also confirmed these results. The negative Tajima's D and Fu's F S values showed the recent demographic expansion of Maruca populations. Thus, this study confirmed the presence of different Maruca species and/or subspecies in different continents based on the diversity within PBP genes. Additional sampling and studies are suggested for Oceania and South America. The genetic differences among Maruca populations should be carefully considered while using sex pheromone lures and bio‐control agents.

Conservation and lineage-specific rearrangements in the GOBP/PBP gene complex of distantly related ditrysian Lepidoptera

General odorant binding proteins (GOBPs) and pheromone binding proteins (PBPs) form a monophyletic subfamily of insect odorant binding proteins (OBPs) specific for Lepidoptera, butterflies and moths. The GOBP/PBP genes include six subgroups (GOBP1–2, PBP-A–D) previously reported to form a complex arrayed in a conserved order in representative moths (superfamily Bombycoidea) and butterflies (Nymphalidae). Although our knowledge of lepidopteran genomes has increased greatly recently, the structure of the GOBP/PBP complex has been studied only for species that represent limited lineages of the highly diverged Ditrysia. To understand the evolution of this functionally important gene complex, we determined 69–149 kb genomic sequences that include GOBP2 and five PBP genes in three Ostrinia moths (Pyraloidea), O. nubilalis, O. furnacalis, and O. latipennis, using bacterial artificial chromosome (BAC) and fosmid clones. The structure of the GOBP2/PBP gene cluster was well conserved despite the different sex pheromone composition utilized by the three moths. Five expressed PBP genes in Ostrinia moths were the result of two duplications of PBP-A genes. Surprisingly, an allele containing a fusion gene between tandemly arrayed PBP-A genes was observed in O. nubilalis. We also revealed duplication and intra-chromosomal translocation of the GOBP1 gene in P. xylostella by fluorescence in situ hybridization (FISH) analysis. Additionally, we compared the structure of the GOBP/PBP gene complex of seventeen species covering six superfamilies and twelve families of the lepidopteran clade, Ditrysia, and found the gene order was basically conserved despite the frequent occurrence of lineage-specific gains, losses, inversions and translocations of these genes, compared with their neighboring genes. Our findings support the hypothesis that the structure of the GOBP/PBP gene complex was already established in the common ancestor of Ditrysia.

CRISPR/Cas9-mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis.

Pheromone‐binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP‐single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target‐gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1‐sgRNA injected eggs and 19.5% for PBP3‐sgRNA injected eggs. Second, by an in‐crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA‐injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.

RNAi-Induced Electrophysiological and Behavioral Changes Reveal two Pheromone Binding Proteins of Helicoverpa armigera Involved in the Perception of the Main Sex Pheromone Component Z11-16:Ald.

Pheromone binding proteins (PBPs) are thought to play key roles in insect sex pheromone recognition; however, there is little in vivo evidence to support this viewpoint in comparison to abundant biochemical data in vitro. In the present study, two noctuid PBP genes HarmPBP1 and HarmPBP2 of the serious agricultural pest, Helicoverpa armigera were selected to be knocked down by RNA interference, and then the changes in electrophysiological and behavioral responses of male mutants to their major sex pheromone component (Z)-11-hexadecenal (Z11-16:Ald) were recorded. There were no significant electrophysiological or behavioral changes of tested male moths in response to Z11-16:Ald when either single PBP gene was knocked down. However, decreased sensitivity of male moths in response to Z11-16:Ald was observed when both HarmPBP1 and HarmPBP2 genes were silenced. These results reveal that both HarmPBP1 and HarmPBP2 are required for the recognition of the main sex pheromone component Z11-16:Ald in H. armigera. Furthermore, these findings may help clarify physiological roles of moth PBPs in the sex pheromone recognition pathway, which in turn could facilitate pest control by exploring sex pheromone blocking agents.

Molecular Characterization, Expression Patterns and Binding Properties of Two Pheromone-Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck)

Insect pheromone-binding proteins (PBPs) play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors (ORs). However, the PBPs of the oriental fruit moth, Grapholita molesta, an important destructive pest of stone fruits worldwide, are not well characterized. In this study, two new putative PBP genes, GmolPBP2 and GmolPBP3, were identified from G. molesta antennae. The deduced amino-acid sequences of these two putative PBP genes are characteristic of the odorant binding protein family, containing six conserved cysteine residues. The genomic DNA sequence of each gene contained two introns. However, the lengths and positions of the introns differed. RT-PCR analyses revealed that the two GmolPBP genes are only expressed in the antennae of female and male moths. Quantitative real-time PCR indicated that the transcription levels of GmolPBP2 are far greater than those of GmolPBP3 in both female and male antennae. GmolPBP3 showed higher transcription levels in female antennae than in male antennae, while GmolPBP2 showed similar transcription levels in both female and male antennae. The transcript levels of both genes were significantly different in premating and post-coitum individuals, implying that mating affects the process of sex pheromone reception. To better understand the functions, two GmolPBPs were expressed in Escherichia coli, and the ligand binding assays were conducted. Results showed that GmolPBP2 has strong binding affinities to two sex pheromone components, E8–12:Ac and Z8–12:Ac, as well as weaker binding affinities to Z8-12:OH and 12:OH. GmolPBP2 also bound some ordinary odor molecules. However, the affinity of GmolPBP3 to both sex pheromones and ordinary odor molecules was very weak. These results show that GmolPBP2 plays the main role in pheromone discrimination and recognition in the oriental fruit moth.

Different roles suggested by sex-biased expression and pheromone binding affinity among three pheromone binding proteins in the pink rice borer, Sesamia inferens (Walker) (Lepidoptera: Noctuidae)

Pheromone binding proteins (PBPs) are thought to bind and transport hydrophobic sex pheromone molecules across the aqueous sensillar lymph to specific pheromone receptors on the dendritic membrane of olfactory neurons. A maximum of 3 PBP genes have been consistently identified in noctuid species, and each of them shares high identity with its counterparts in other species within the family. The functionality differences of the 3 proteins are poorly understood. In the present study, 3 PBP cDNAs (SinfPBP1, 2, 3) were identified from the pink rice borer, Sesamia inferens, for the first time. The quantitative real-time PCR indicated that the 3 PBPs displayed similar temporal but very different sex related expression profiles. Expression of SinfPBP1 and SinfPBP2 were highly and moderately male biased, respectively, while SinfPBP3 was slightly female biased, as SinfPBPs were expressed at very different levels (PBP1>PBP2≫PBP3) in male antennae, but at similar levels in female antennae. Furthermore, the 3 SinfPBPs displayed different ligand binding profiles in fluorescence competitive binding assays. SinfPBP1 exhibited high and similar binding affinities to all 3 sex pheromone components (Ki=0.72–1.60μM), while SinfPBP2 showed selective binding to the alcohol and aldehyde components (Ki=0.78–1.71μM), and SinfPBP3 showed no obvious binding to the 3 sex pheromone components. The results suggest that SinfPBP1 plays a major role in the reception of female sex pheromones in S. inferens, while SinfPBP3 plays a least role (if any) and SinfPBP2 functions as a recognizer of alcohol and aldehyde components.

Analysis of a cDNA library from the antenna of Cnaphalocrocis medinalis and the expression pattern of olfactory genes

Chemoreception is a key feature in selection of host plants by insects. In this study, preliminary characterization and isolation of cDNA clones from Cnaphalocrocis medinalis antennal libraries identified eight olfactory genes, including two putative general odorant-binding proteins (GOBPs), three pheromone binding proteins (PBPs) and three chemosensory proteins (CSPs). The expression profiles of these eight genes in different tissues (antenna, head (without antennae), thorax, abdomen, leg and wing) were measured by real time qPCR. The results showed that GOBP and PBP genes in C. medinalis seemed to be antenna-specific, but differentially expressed in male and female antennae; while CSP genes were expressed ubiquitously during different developmental stages, but with an extremely elevated transcript level in antennae, legs and wings compared to head, thorax and abdomen. And also, the transcription levels of olfactory genes depended on the age, sex, and mating status of the adults. These findings support the hypothesis that OBPs and CSPs play dynamic roles during development of C. medinalis and are likely to be involved in broader physiological functions.

Functional differentiation of pheromone-binding proteins in the common cutworm Spodoptera litura

Pheromone-binding proteins (PBPs), a sub-family of odorant-binding proteins, are thought primarily to bind and transport the sex pheromones in moths. Considering multiple components of sex pheromone and multiple PBP genes exist in a single species, PBPs may contribute to the discrimination of different sex pheromone components. However, so far this discrimination is still unclear. Our previous ligand-binding assays showed that Spodoptera litura PBP1 (SlitPBP1) did not exhibit an obvious binding specificity among different sex pheromone components. In this study, binding specificity of the other two PBPs in S. litura (SlitPBP2 and SlitPBP3) was further investigated. As a result, SlitPBP2 was capable of binding all four sex pheromone components with similar affinities; whereas SlitPBP3 showed very weak binding affinities to them except Z9,E12–14:Ac. Similar results were also obtained from studied pheromone analogs, to which SlitPBP2 showed much stronger affinities than SlitPBP3. However, both SlitPBP2 and SlitPBP3 exhibited overall weaker affinities to sex pheromones and their analogs than SlitPBP1. In addition, quantitative real time PCR showed that three SlitPBP genes exhibited a very different sex-biased expression in adult antenna with male-biased for SlitPBP1 and SlitPBP2 while female-biased for SlitPBP3. Finally, ligand-binding assays indicated that the two SlitPBPs showed a similar pH-dependent conformational change as reported SlitPBP1, but these three SlitPBPs showed different behavior across a pH range or something similar. Taken together, our data suggest that in S. litura PBP1 and PBP2 may play critical roles in the perception of female sex pheromones, but do not show an obvious discriminative ability among different sex pheromone components; whereas PBP3 may have other functions.

Sex pheromone recognition and immunolocalization of three pheromone binding proteins in the black cutworm moth Agrotis ipsilon

Insect pheromone binding proteins (PBPs) are believed to solubilize and transport hydrophobic sex pheromones across sensillum lymph to membrane-associated pheromone receptors. To address the molecular mechanisms of PBPs in insect pheromone perception, we undertook a systemic study on the PBPs of the black cutworm Agrotis ipsilon at transcript as well as protein level from tissue distribution and cellular localization to pheromone binding affinity. We cloned three full-length PBP genes AipsPBP1-3 from A. ipsilon antennae, and demonstrated that AipsPBP1–3 transcripts were highly expressed in male antennae. The electron microscopic examinations revealed at least six types of olfactory sensilla on male and female antenna: trichodea, chaetica, basiconica, coeloconica, squamiformia and Böhm bristles. The immunocytochemistry results demonstrated that AipsPBP1-3 proteins were strongly expressed in the sensillum lymph of the trichoid sensilla of male moth. The binding assays showed that AipsPBP1 had high binding affinities with the major sex pheromone components Z7-12:Ac and Z9-14:Ac among five related chemicals and was clustered together with the long trichoid sensilla-expressing LdisPBPs of Lymantria dispar. AipsPBP2 showed high binding affinities also with Z11-16:Ac. AipsPBP3 displayed a high affinity only with Z11-16:Ac. Our studies provide further detail evidences for the involvement of moth PBPs in pheromone discrimination and selective recognition of specific components of the female sex pheromone blends.

Molecular Characterization and Expression Pattern of Two Pheromone-Binding Proteins from Spodoptera litura (Fabricius)

Pheromone perception is thought to be mediated by pheromone-binding proteins (PBPs) in the lymph surrounding the olfactory receptors. We cloned and characterized two PBP genes (SlitPBP1 and SlitPBP2) from the common cutworm, Spodoptera litura (F.; Lepidoptera: Noctuidae), which encode PBPs belonging to two different PBP groups. Western blot analysis of the crude antennal extracts with SexigPBP1 antibody revealed a single immunoreactive band (much stronger in male than in female) of approximately 16 kDa, in agreement with the calculated values for SlitPBPs. From genomic DNA, two introns and a similar exon/intron structural pattern were identified in each PBP genes, but the introns differed in length within and between PBP genes. The expression patterns of two SlitPBP genes, with respect to tissue distribution and sex, were further investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR. Although the two PBP genes were expressed only in the antennae of both sexes, reflecting the antennal specificity of PBPs, the transcription levels of PBP genes differed between the sexes and the genes. The transcription levels of SlitPBP1 and SlitPBP2 in females were only 2.1% and 7.0%, respectively, relative to those in males, and the levels of PBP2 compared with PBP1 were 31.4% and 95.3% in males and females, respectively. These differential expression levels might suggest different roles played by the two SlitPBPs in the perception of sex pheromone both in males and females.

Molecular Characterization of Two Pheromone Binding Proteins and Quantitative Analysis of their Expression in the Beet Armyworm, Spodoptera exigua Hübner

Pheromone binding proteins (PBP) play an important role in insect pheromone communication. However, the PBP for the beet armyworm, Spodoptera exigua Hübner (Lepidoptera: Noctuidae), an important agricultural pest worldwide, remains unaddressed. We report the cloning of two PBP genes, SexigPBP1 and SexigPBP2, from the antennal cDNA of S. exigua by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends-PCR (RACE-PCR). The deduced PBP amino acid sequences are characteristic of the odorant binding protein (OBP) family, although the two PBPs are only 44% identical. From an analysis of the genomic DNA, two introns and a similar intron/extron structural pattern were identified in each of the two PBP genes. RT-PCR analysis revealed that the two PBP genes are only expressed in antennae. Real-time PCR further indicated that the expression of SexigPBP1 is much higher than that of SexigPBP2, regardless of sex. However, the female expression levels for SexigPBP1 and SexigPBP2 are about 39% and 73%, respectively, relative to male levels. Finally, phylogenetic analysis suggested that PBPs from the Noctuidae are divided into three distinct groups based on the primary sequences.

Molecular characterization and evolution of pheromone binding protein genes in Agrotis moths

Pheromone-binding proteins (PBPs) are soluble transporter proteins that increase the capture and the solubilization of pheromone molecules in the lymph surrounding the olfactory receptors. A polymerase chain reaction-based method was used to identify PBP genes in Agrotis species for an evolutionary genomic study of noctuid moth PBPs. From genomic DNA we determined the structure of different PBP genes in the two closely related species, Agrotis ipsilon and A. segetum. In all, we clearly identified four genes ( Aips-1, Aips-2, Aseg-1 and Aseg-2) that represent two distinct PBP orthology groups. We found that the four genes have the same exon–intron structure and that they comprise three exons and two introns but differ in length mainly in the second intron. The three exons of Aseg-2 and Aips-2 have the same lengths but both intron 1 and intron 2 differ in length between the genes. In contrast, Aips-1 and Aseg-1 show dissimilarity only in the length of intron 2. Interestingly, introns 1 and 2 are inserted in the same positions in the Aips-1, Aips-2, Aseg-1 and Aseg-2 genes. These findings show that the Agrotis PBP genes have common ancestry and probably originate from gene duplication before the speciation of ipsilon and segetum. We found that expression of Aips-1/Aseg-1 and Aips-2/Aseg-2 is antennal-specific, but expression is not restricted to the male antennae.

050 Développement d’un simulateur de chirurgie de la cataracte

Pheromone-binding proteins (PBPs), a sub-family of odorant-binding proteins, are thought primarily to bind and transport the sex pheromones in moths. Considering multiple components of sex pheromone and multiple PBP genes exist in a single species, PBPs may contribute to the discrimination of different sex pheromone components. However, so far this discrimination is still unclear. Our previous ligand-binding assays showed that Spodoptera litura PBP1 (SlitPBP1) did not exhibit an obvious binding specificity among different sex pheromone components. In this study, binding specificity of the other two PBPs in S. litura (SlitPBP2 and SlitPBP3) was further investigated. As a result, SlitPBP2 was capable of binding all four sex pheromone components with similar affinities; whereas SlitPBP3 showed very weak binding affinities to them except Z9,E12–14:Ac. Similar results were also obtained from studied pheromone analogs, to which SlitPBP2 showed much stronger affinities than SlitPBP3. However, both SlitPBP2 and SlitPBP3 exhibited overall weaker affinities to sex pheromones and their analogs than SlitPBP1. In addition, quantitative real time PCR showed that three SlitPBP genes exhibited a very different sex-biased expression in adult antenna with male-biased for SlitPBP1 and SlitPBP2 while female-biased for SlitPBP3. Finally, ligand-binding assays indicated that the two SlitPBPs showed a similar pH-dependent conformational change as reported SlitPBP1, but these three SlitPBPs showed different behavior across a pH range or something similar. Taken together, our data suggest that in S. litura PBP1 and PBP2 may play critical roles in the perception of female sex pheromones, but do not show an obvious discriminative ability among different sex pheromone components; whereas PBP3 may have other functions.