Putative Odorant-binding Proteins Genes Research Articles

Identification and characterization of odorant binding proteins in the diamondback moth, Plutella xylostella.

Odorant binding proteins (OBPs) are a group of soluble proteins functioning as odorant carriers in insect antennae, mouth parts and other chemosensory organs. However, multiple insect OBPs have been detected in other tissues and various functions have been proposed. Therefore, a detailed expression profile including stages, tissues and sexes where OBPs are expressed will assist in building the links to their potential functions, enhancing the functional studies of insect OBPs. Here, we identified 39 putative OBP genes from its genome and transcriptome sequences of diamondback moth (DBM), Plutella xylostella. The expression patterns of identified PxylOBPs were further investigated from eggs, larvae, pupae, virgin adults, mated adults, larval midgut, larval heads, adult antennae, adult heads and adult tarsi. Moreover, P. xylostella larvae and adults with and without host plants for 5h were utilized to study the interactions between OBP expression and host plants. The results showed that most PxylOBPs were highly expressed in male and female adult antennae. The expression levels of certain PxyOBPs could be regulated by mating activities and feeding host plants. This study advances our knowledge of P. xylostella OBPs, which may help develop new strategies for more environmentally sustainable management of P. xylostella.

Key Amino Residues Determining Binding Activities of the Odorant Binding Protein AlucOBP22 to Two Host Plant Terpenoids of Apolygus lucorum.

Odorant binding proteins (OBPs) are considered to be highly expressed at antennae sensillum lymph and play crucial roles in detection of insect host plant volatiles. The polyphagous mirid bug Apolygus lucorum is one of a series of insect pests on many important agricultural crops that heavily rely on sophisticated olfaction to locate host plants. Previously, putative OBP genes and their tissue-related expression patterns in this pest species have been clarified. In this study, we characterized the ligand spectrum and the molecular binding mechanism of the antennae-biased AlucOBP22 to host plant volatiles of A. lucorum. Frist, the recombinant AlucOBP22 protein was constructed and purified, and its binding affinities to selected host plant volatiles were assessed. Two terpenoids, β-ionone and β-caryophyllene, could highly bind to AlucOBP22. Next, three-dimensional model prediction indicated that AlucOBP22 employed six α-helices to form a typical pocket for ligand accommodation. Molecular docking analysis suggested that both β-ionone and β-caryophyllene were located at the AlucOBP22 pocket with some hydrophobic amino acid residues close to the two chemicals, suggesting that hydrophobic interactions might be crucial for ligand-specific binding. Finally, site-directed mutagenesis combined with fluorescence binding assays revealed that mutants of five hydrophobic residues Leu5, Ile40, Met41, Val44, and Met45 displayed significantly decreased or completely abolished binding affinities to the two ligands. Our findings showed the specific binding characteristic of AlucOBP22 and suggested that hydrophobic residues and their hydrophobic interactions were involved in AlucOBP22 binding to terpenoids, which provided new insights into the molecular interaction mechanisms of hemipteran insect OBPs to host plant odors.

Identification and analysis of odorant-binding protein genes from the wolf spider Pardosa pseudoannulata (Araneae: Lycosidae) based on its transcriptome

Pardosa pseudoannulata, one of the dominant predators in the paddy ecosystem, is a potential resource in the biological control of rice pests. Olfaction is crucial for insect behaviors such as feeding, mating and foraging, and odorant-binding proteins (OBPs) play a vital role in smell sensing, so we suppose that OBPs may be also important in spider behaviors. In this study, we identified two putative OBP genes and obtained their complete cDNA sequences. PpseOBP1 is 842 bp long and encodes a 188 amino acids protein. PpseOBP2 is 996 bp long and encodes a 213 amino acids protein. By our predictions and analysis, both of the PpseOBPs share some similar physicochemical and structural characteristics with insect classic OBP genes, but they do not strictly observe the cysteine pattern of OBPs in insects, in which there are three amino acid residues between the second and the third cysteine residues but 27 amino acid residues in P. pseudoannulata. Phylogenetic analysis showed that two PpseOBPs clustered together at the edge of the tree, indicating their distant relationship with insects. Regarding insects, PpseOBPs were most closely related to the Periplaneta americana OBP1. Both PpseOBPs are mainly expressed in appendages (pedipalps and legs). Expression of PpseOBP1 was significantly higher in female appendages than in male appendages, while PpseOBP2 showed an extreme expression level in male pedipalps. This is the first report of molecular characterization and expression patterns of OBPs in Lycosidae and will establish a foundation for better understanding of molecular mechanisms of P. pseudoannulata olfaction and facilitate its function in biological control.

A head transcriptome provides insights into odorant binding proteins of the bamboo grasshopper.

The bamboo grasshopper Ceracris kiangsu is a famous bamboo pest in China. The identification of genes involved in olfactory behavior of C. kiangsu is necessary for better understanding the molecular basis and expression profiles of behavior ecology. However, necessary genomic and transcriptomic data are lacking in the species, limiting control efficiency. The primary objective of this study was to find and describe odorant binding proteins in the head of the bamboo grasshopper. We performed the paired-end sequencing on an Illumina Hiseq2000 following the vendor's recommended protocol. Functional annotation was performed by comparison with public databases. OBP genes were first identified using BLASTN and BLASTX results from our C. kiangsu datebase, which was established from the date of transcriptome sequencing. The gene-specific primers were used to conduct RT-PCR to detect the tissue distribution of OBPs using a SYBR Premix ExTaq kit following the manufacturer's instructions with a real-time thermal cycler. We obtained more than 133million clean reads derived from the C. Kiangsu heads using the next-generation sequencing, which were assembled into 260,822 unique sequences (average 814bp). We have detected eight putative odorant binding protein genes (OBPs) of C. kiangsu for the first time, and analyzed the expression profiles of the OBPs in different tissues (head, antenna, mouthpart, body and leg). Our results reveal that the eight OBPs display a clear divergence, strongly indicating that they possessed diverse functions, and thus provides comprehensive sequence analysis for elucidating the molecular basis of OBPs in C. kiangsu. In addition, we find that the relative expression levels of OBP1, OBP2 and OBP8 are significantly higher in the antennae as compared to the other OBP genes, suggesting that these three OBP genes play crucial roles in the locust's odorant discrimination. In general, this is the first study to characterize the complete head transcriptome of C. kiangsu using high-throughput sequencing. The study opens a window for functional characterization of the OBPs of C. kiangsu, with potential for new or refined applications of semiochemicals for control of this notorious pest.

Identification and sex expression profiling of odorant-binding protein genes in Trichogramma japonicum (Hymenoptera: Trichogrammatidae) using RNA-Seq

Trichogramma japonicum Ashmead (Hymenoptera: Trichogrammatidae) is a biological control agent that parasitizes the eggs of many lepidopteran insects. The control efficiency is closely related to the ability of the parasitoid to use chemical volatiles in searching for hosts, and the odorant binding proteins (OBPs) may serve as a first step in the perception of these chemical cues. Understanding the ability of Trichogramma to detect relevant host odorant cues might help to design better strategies to control target lepidopteran insect pests. In the present study, we assembled the transcriptome of T. japonicum using Illumina sequencing technology and identified 15 putative OBP genes. All the OBP genes have complete open reading frames and contain six conserved cysteines. In addition, sex-biased expression was found in eight OBP genes by using quantitative real-time PCR, which suggested different functions of these genes. Phylogenetic analyses revealed that Hymenoptera OBP genes were divided into Classic, Minus-C, and Double Minus-C subfamilies. All the identified OBP genes of T. japonicum belong to the Classic subfamily. These results provide an important foundation for a better understanding of the complex chemoreception system of T. japonicum.

Genome-wide identification and characterization of odorant-binding protein (OBP) genes in the malaria vector Anopheles sinensis (Diptera: Culicidae).

Anopheles sinensis is a major malaria vector. Insect odorant-binding proteins (OBPs) may function in the reception of odorants in the olfactory system. The classification and characterization of the An. sinensis OBP genes have not been systematically studied. In this study, 64 putative OBP genes were identified at the whole-genome level of An. sinensis based on the comparison between OBP conserved motifs, PBP_GOBP, and phylogenetic analysis with An. gambiae OBPs. The characterization of An. sinensis OBPs, including the motif's conservation, gene structure, genomic organization and classification, were investigated. A new gene, AsOBP73, belonging to the Plus-C subfamily, was identified with the support of transcript and conservative motifs. These An. sinensis OBP genes were classified into three subfamilies with 37, 15 and 12 genes in the subfamily Classic, Atypical and Plus-C, respectively. The genomic organization of An. sinensis OBPs suggests a clustered distribution across nine different scaffolds. Eight genes (OBP23-28, OBP63-64) might originate from a single gene through a series of historic duplication events at least before divergence of Anopheles, Culex and Aedes. The microsynteny analyses indicate a very high synteny between An. sinensis and An. gambiae OBPs. OBP70 and OBP71 earlier classified under Plus-C in An. gambiae are recognized as belonging to the group Obp59a of the Classic subfamily, and OBP69 earlier classified under Plus-C has been moved to the Atypical subfamily in this study. The study established a basic information frame for further study of the OBP genes in insects as well as in An. sinensis.

Molecular Characterization and Expression Profiling of Odorant-Binding Proteins in Apolygus lucorum.

Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) is one of the most important agricultural pests, with broad host range and cryptic feeding habits in China. Chemosensory behavior plays an important role in many crucial stages in the life of A. lucorum, such as the detection of sex pheromone cues during mate pursuit and fragrant odorants during flowering host plant localization. Odorant-binding proteins (OBPs) are involved in the initial biochemical recognition steps in semiochemical perception. In the present study, a transcriptomics-based approach was used to identify potential OBPs in A. lucorum. In total, 38 putative OBP genes were identified, corresponding to 26 ‘classic’ OBPs and 12 ‘Plus-C’ OBPs. Phylogenetic analysis revealed that A. lucorum OBP proteins are more closely related to the OBP proteins of other mirid bugs as the same family OBP clustering together. Quantitative real-time PCR analysis for the first reported 23 AlucOBPs revealed that the expression level of 11 AlucOBP genes were significantly higher in antennae of both sexes than in other tissues. Three of them were male antennae-biased and six were female antennae-biased, suggesting their putative roles in the detection of female sex pheromones and host plant volatiles. In addition, three, four, two and one AlucOBPs had the highest degree of enrichment in the stylet, head, leg, and in abdomen tissues, respectively. Two other OBPs were ubiquitously expressed in the main tissues, including antennae, stylets, heads, legs and wings. Most orthologs had similar expression patterns, strongly indicating that these genes have the same function in olfaction and gustation.

Identification of putative odorant binding protein genes in Asecodes hispinarum, a parasitoid of coconut leaf beetle (Brontispa longissima) by antennal RNA-Seq analysis

Asecodes hispinarum (Hymenoptera: Eulophidae) is an endoparasitoid and an efficient biological control agent which attacks larvae of Brontispa longissima, a serious insect pest of Palmae plants in China. Odorant binding proteins (OBPs) are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. No previous study has reported on OBPs in A. hispinarum. In this study, we conducted the large-scale identification of OBP genes from the antennae of A. hispinarum by using transcriptome sequencing. Approximately 28.4 million total raw reads and about 27.3 million total clean reads were obtained, and then 46,363 unigenes were assembled. Of these unigenes, a total of 21,263 can be annotated in the NCBI non-redundant database. Among the annotated unigenes, 16,623 of them can be assigned to GO (Gene Ontology). Furthermore, we identified 8 putative OBP genes, and a phylogenetic tree analysis was performed to characterize the 8 OBP genes. In addition, the expression of the 8 OBP genes in different A. hispinarum body tissues was analyzed by real-time quantitative polymerase chain reaction (qRT-PCR). The results indicated that the 8 OBP genes were expressed accordingly to sexes and tissues, but all highly expressed in antennae. The finding of this study will lay the foundation for unraveling molecular mechanisms of A. hispinarum chemoperception.

Odorant binding characteristics of three recombinant odorant binding proteins in Microplitis mediator (Hymenoptera: Braconidae).

Odorant binding proteins (OBPs) are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. In this study, three new putative OBP genes, MmedOBP8-10, were identified from a Microplitis mediator (Hymenoptera: Braconidae) antennal cDNA library. Quantitative real-time PCR (qRT-PCR) analysis revealed that all three of the OBP genes were expressed mainly in the antennae of adult wasps. The three OBPs were recombinantly expressed in Escherichia coli and purified by Ni ion affinity chromatography. Fluorescence competitive binding assays were performed using N-phenyl-naphthylamine as a fluorescent probe and 45 small organic compounds as competitors. These assays demonstrated that the three M. mediator OBPs can bind a broad range of odorant molecules with different binding affinities. They can bind the following ligands: nonane, farnesol, nerolidol, nonanal, β-ionone, acetic ether, and farnesene. In a Y-tube assay with these ligands as odor stimuli and paraffin oil as a control, all ligands, except nerolidol and acetic ether, were able to elicit behavioral responses in adult M. mediator. The wasps were significantly attracted to β-ionone, nonanal, and farnesene and repelled by nonane and farnesol. The results of this work provide insight into the chemosensory functions of the OBPs in M. mediator.

Identification and expression profile analysis of odorant-binding protein genes in Apolygus lucorum (Hemiptera: Miridae)

Insect odorant-binding proteins (OBPs) are one group of olfactory-related proteins and are believed to play essential roles in the physiological activity of insects. In this study, we constructed a cDNA library from the antennae of Apolygus lucorum (Meyer-Dur). Twelve novel putative OBP genes were identified from the antennal cDNA library, and the expression profiles of the 12 OBPs in different tissues (antennae, heads, thoraxes, abdomens, legs and wings) were analyzed by real-time quantitative PCR. The results revealed that AlucOBP2, AlucOBP7, AlucOBP8, AlucOBP11 and AlucOBP12 are highly expressed in antennae. Especially the transcript levels of AlucOBP7, AlucOBP8 and AlucOBP11 in male antennae were significantly higher than in female antennae, suggesting their potential olfactory functions in host seeking and mate finding of A. lucorum. However, AlucOBP1, AlucOBP5 and AlucOBP6 were highly expressed in the heads, whereas AlucOBP3 was specifically expressed only in the legs. In addition, AlucOBP4, AlucOBP9 and AlucOBP10 were expressed in almost all chemosensory tissues (antennae, heads, wings and legs). Our research is meaningful for understanding the molecular basis of the insect chemoreception requirements and provides valuable target genes to control the insect pests.

Genome analysis and expression patterns of odorant-binding proteins from the Southern House mosquito Culex pipiens quinquefasciatus.

Olfactory-based behaviors in mosquitoes are mediated by odorant-binding proteins (OBPs). They form a multigenic family involved in the peripheral events in insect olfaction, specifically the transport of odorants to membrane-bound odorant receptors. OBPs contribute to the remarkable sensitivity of the insect's olfactory system and may be involved in the selective transport of odorants.We have employed a combination of bioinformatics and molecular approaches to identify and characterize members of the “classic” OBP family in the Southern House mosquito Culex pipiens quinquefasciatus ( = Cx. quinquefasciatus), a vector of pathogens causing several human diseases. By taking advantage of the recently released genome sequences, we have identified fifty-three putative Cx. quinquefasciatus OBP genes by Blast searches. As a first step towards their molecular characterization, expression patterns by RT-PCR revealed thirteen genes that were detected exclusively and abundantly in chemosensory tissues. No clear differences were observed in the transcripts levels of olfactory-specific OBPs between antennae of both sexes using semi-quantitative RT-PCR. Phylogenetic and comparative analysis revealed orthologous of Cx. quinquefasciatus OBPs in Anopheles gambiae and Aedes aegypti. The identification of fifty-three putative OBP genes in Cx. quinquefasciatus highlights the diversity of this family. Tissue-specificity study suggests the existence of different functional classes within the mosquito OBP family. Most genes were detected in chemosensory as well as non chemosensory tissues indicating that they might be encapsulins, but not necessarily olfactory proteins. On the other hand, thirteen “true” OBP genes were detected exclusively in olfactory tissues and might be involved specifically in the detection of “key” semiochemicals. Interestingly, in Cx. quinquefasciatus olfactory-specific OBPs belong exclusively to four distinct phylogenetic groups which are particularly well conserved among three mosquito species.

Identification and expression profiling of putative odorant-binding proteins in the malaria mosquitoes, Anopheles gambiae and A. arabiensis

Olfaction plays a major role in host-seeking behaviour of mosquitoes. An informatics-based genome-wide analysis of odorant-binding protein (OBP) homologues is undertaken, and 32 putative OBP genes in total in the whole genome sequences of Anopheles gambiae are identified. Tissue-specific expression patterns of all A. gambiae OBP candidates are determined by semi-quantitative Reverse Transcription (RT)-PCR using mosquito actin gene as internal expression control standard. The results showed that 20 OBP candidates had strong expression in mosquito olfactory tissues (female antennae), which indicate that OBPs may play an important role in regulating mosquito olfactory behaviours. Species-specific expression patterns of all putative anopheline OBPs are also studied in two of the most important malaria vectors in A. gambiae complex, i.e. A. gambiae and A. arabiensis, which found 12 of the putative OBP genes examined displayed species-differential expression patterns. The cumulative relative expression intensity of the OBPs in A. arabiensis antennae was higher than that in A. gambiae (the ratio is 1441.45:1314.12), which might be due to their different host preference behaviour. While A. gambiae is a highly anthropophilic mosquito, A. arabiensis is more opportunistic (varying from anthropophilic to zoophilic). So the latter should need more OBPs to support its host selection preference. Identification of mosquito OBPs and verification of their tissue- and species-specific expression patterns represent the first step towards further molecular analysis of mosquito olfactory mechanism, such as recombinant expression and ligand identification.

"Plus-C" odorant-binding protein genes in two Drosophila species and the malaria mosquito Anopheles gambiae.

Olfaction plays a crucial role in many aspects of insect behaviour, including host selection by agricultural pests and vectors of human disease. Insect odorant-binding proteins (OBPs) are thought to function as the first step in molecular recognition and the transport of semiochemicals. The whole genome sequence of the fruit fly Drosophila melanogaster has been completed and a large number of genes have been annotated as OBPs, based on the presence of six conserved cysteine residues and a conserved spacing between the cysteines. These proteins can be divided into three distinct subgroups; those with only one six-cysteine motif, those with two such motifs and those with one motif, three extra conserved cysteines and a conserved proline immediately after the sixth cysteine. This study concentrates on the last two subgroups, referred to as 'dimer' OBPs and 'Plus-C' OBPs, respectively. We determined the tissue-specific transcript levels of all of these OBP genes of D. melanogaster using semiquantitative RT-PCR. The results showed that the expression patterns can vary within a subgroup of genes and that this technique is valuable for assessing which of the putative OBP genes are likely to be involved in Drosophila olfaction. The publicly available genomes of another fruit fly Drosophila pseudoobscura, the malaria mosquito Anopheles gambiae and the yellow fever mosquito Aedes aegypti were searched by Blast against each Plus-C OBP and dimer OBP of D. melanogaster. Related genes were found in all of the other species and the relationships of these with the D. melanogaster genes and their possible biological functions are discussed.