Putative Odorant-binding Proteins Research Articles

Interplay of hormones and metabolite excretion with fern pattern prove saliva as a potent indicator of male reproductive status in Kangayam breed cattle.

Kangayam cattle are one of the drought breeds in India with distinct attributes. Agricultural transformation has led to a decline in many pure-breed indigenous cattle, including the Kangayam breed. Hence, a study on the reproductive physiology of male Kangayam breed cattle is necessary to disentangle problems in the area of livestock improvement. In this study, we investigated the relationship between serum hormones and bio-constituents and ascertained the potential of saliva as an indicator of the reproductive status of Kangayam cattle (Bos indicus). The present study confirms that cholesterol was higher in intact males and lower in prepubertal and castrated males. Testosterone levels were also higher in intact males than in castrated or prepubertal males. Hence, it can be inferred that high cholesterol levels contribute to active derivatization of testosterone in intact males. In contrast, reduced cholesterol availability leads to decreased testosterone synthesis in castrated and prepubertal males. Furthermore, it is reasonable to speculate that testosterone could have influenced salivary fern patterns in intact males, and thus, fern-like crystallization in the saliva was apparent. The unique salivary compounds identified through GC-MS across various reproductive statuses of Kangayam males may advertise their physiological status to conspecifics. In addition, the presence of odorant-binding protein(OBP) in saliva further supports its role in olfactory communication. This study attested to a posssible interlink between gonadal status and serum biochemical profiles. The salivary fern pattern revealed in this study can be used as a predictive tool, and the presence of putative volatiles and OBP adds evidence to the role of saliva in chemical communication.

Genome-Wide Identification of the Genes of the Odorant-Binding Protein Family Reveal Their Role in the Olfactory Response of the Tomato Leaf Miner (Tuta absoluta) to a Repellent Plant

The remarkable biological and evolutionary adaptations of insects to plants are largely attributed to the powerful chemosensory systems of insects. The tomato leaf miner (Tuta absoluta) is a destructive invasive pest with a global distribution that poses a serious threat to the production of nightshade crops, especially tomatoes. Functional plants can attract or repel insect pests by releasing volatiles that interact with the olfactory system of insects, thereby reducing the damage of insect pests to target crops. However, there is limited research on the interaction between T. absoluta olfactory genes and functional plants. In this study, 97 members of the putative odorant-binding protein (OBP) family have been identified in the whole genome of T. absoluta. Phylogenetic analysis involving various Lepidopteran and Dipteran species, including D. melanogaster, revealed that OBP gene families present conserved clustering patterns. Furthermore, the Plus-C subfamily of OBP showed extremely significant expansion. Moreover, the expression levels of the OBP genes varied significantly between different developmental stages; that is, the highest number of OBP genes were expressed in the adult stage, followed by the larval stage, and fewer genes were expressed in high abundance in the egg stage. On the other hand, through a Y-tube olfactometer, we identified a functional plant—Plectranthus tomentosa—that significantly repels adult and larval T. absoluta. Finally, we screened the OBP genes in response to tomato and P. tomentosa volatiles at the genomic level of T. absoluta using RT-qPCR. These results laid a good foundation for controlling T. absoluta with functional plants and further studying olfactory genes.

Identification and expression analyses of the olfactory-related genes in different tissues' transcriptome of a predacious soldier beetle, Podabrus annulatus (Coleoptera, Cantharidae).

We sequenced and analyzed the transcriptomes from different tissues of the soldier beetle, Podabrus annulatus (Coleoptera: Cantharidae), and obtained 75.74 Gb clean reads which were assembled into 95,274 unigenes. Among these transcripts, 25,484 unigenes of highly quality were annotated. Based on annotation and tBLASTn results, we identified a total of 101 candidate olfactory-related genes for the first time, including 11 putative odorant-binding proteins (OBPs), 6 chemosensory proteins (CSP), 50 olfactory receptors (ORs), 25 gustatory receptors (GRs), 6 ionotropic receptors (IRs), and 3 sensory neuron membrane proteins (SNMPs). BLASTX best-hit results indicated that these chemosensory genes were most identical to their respective orthologs from Photinus pyralis. Phylogenetic analyses also revealed that the ORs, GRs, and IRs of Podabrus annulatus are closely related to those of Photinus pyralis. The fragment per kilobase per million mapped fragments (FPKM) values showed that the PannOBP2, PannOBP3, and PannOBP10 were predominantly expressed in the antennae, PannOBP1 in the abdomen-thorax, while others were not identified to be tissue-specific. These olfactory-related differentially expressed genes (DEGs) demonstrated different roles in the olfactory system of Podabrus annulatus. This study establishes the groundwork for future research into the molecular mechanism of olfactory recognition in Podabrus annulatus.

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.

Genome-Wide Analysis of Odorant-Binding Proteins and Chemosensory Proteins in the Bean bug Riptortus pedestris.

Insects have sensitive olfactory systems to interact with environment and respond to the change in host plant conditions. Key genes in the system can be potential targets for developing new and efficient pest behaviour control methods. Riptortus pedestris is an important soybean pest in East Asia and has caused serious damage to the soybean plants in Huang-Huai-Hai region of China. However, the current treatment of pests is dominated by chemical insecticides and lacks efficient sustainable prevention and control technologies. In this study, we identified 49 putative odorant-binding proteins (OBPs) (43 were new genes) and 25 chemosensory proteins (CSPs) (17 were new genes) in R. pedestris genome. These OBP and CSP genes are clustered in highly conserved groups from other hemipteran species in phylogenetic trees. Most RpedOBPs displayed antennal-biased expression. Among the 49 RpedOBPs, 33 were significantly highly expressed in the antennae, including three male-biased and nine female-biased. While many RpedCSPs were detected both in the antennae and in non-antennal tissues, only 11 RpedCSPs displayed antennal-biased expression, in which four RpedCSPs were male-biased and five RpedCSPs were female-biased. Some OBP and CSP genes showed sex-biased expression profiles. Our results not only provide a foundation for future exploration of the functions of RpedOBPs and RpedCSPs but also aid in developing environmentally friendly insecticides in the future.

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights.

Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi.

Olfactory Sensilla and Olfactory Genes in the Parasitoid Wasp Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae).

Simple SummaryParasitic wasps are the major natural enemies of many organisms, and therefore they are broadly used in the biological control of numerous agricultural and horticultural pests. For example, Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) is a tiny natural egg parasitoid of various agricultural pest insects, including Plutella xylostella, Helicoverpa armigera, Spodoptera frugiperda and Ectomyelois ceratoniae. However, how T. pretiosum seek and localise host insect eggs is still not clear. The olfactory system is critical in guiding insect behaviours, including mating, feeding and oviposition, which play pivotal roles in the interactions between parasitoid wasps and their hosts. This project aimed to investigate T. pretiosum major olfactory tissue (antennae) and the olfactory genes, including odorant binding proteins (OBPs) and odorant receptors (ORs). T. pretiosum adult antennae were examined under scanning electron microscopy, and four types of olfactory sensilla were observed. Using T. pretiosum genome, 22 OBPs and 105 ORs were identified, which were further compared with olfactory genes of other Hymenoptera insect species. The expression patterns of OBPs between T. pretiosum male and female adults were examined to identify female- or male-specific OBPs. This study enriches our knowledge of T. pretiosum olfactory system and will help better use it in the integrated pest management (IPM) for many insect pest species.Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) is a tiny natural egg parasitoid of several agricultural pest insects, which has been widely used in the biological control for Plutella xylostella, Helicoverpa armigera, Spodoptera frugiperda and Ectomyelois ceratoniae. However, limited studies have been conducted on T. pretiosum olfactory system, which is critical in regulating insect behaviours. In this study, T. pretiosum adult antennae were investigated under ascanning electron microscopy (SEM). Four types of olfactory sensilla were observed, including chaetica sensilla (CS), trichoid sensilla (TS), faleate sensilla (FS) and placoid sensilla (PS). Using T. pretiosum genome, 22 putative odorant binding proteins (OBPs) and 105 odorant receptors (ORs) were identified, which were further compared with olfactory genes of Apis mellifera, Nasonia vitripennis and Diachasma alloeum. The expression patterns of OBPs between T. pretiosum male and female adults were examined by quantitative real time PCR (qRT-PCR) approaches. Three female-specific OBPs (TpreOBP19, TpreOBP15 and TpreOBP3) were identified, which may play crucial roles in T. pretiosum host-seeking and oviposition behaviours. This study enriches our knowledge of T. pretiosum olfactory genes and improves our understanding of its olfactory system.

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.

Identification and characterization of olfactory genes in the parasitoid wasp Diadegma semiclausum (Hellén) (Hymenoptera: Ichneumonidae).

Diadegma semiclausum is an important parasitoid wasp and widely used in the biological control of the diamondback moth, Plutella xylostella, one of the most destructive pests of cruciferous plants. Insect olfactory system is critical in guiding behaviors including feeding, mating, and oviposition, in which odorant binding proteins (OBPs) and odorant receptors (ORs) are two key components. However, limited attention has been paid to D. semiclausum olfactory genes. In this study, a transcriptome sequencing was performed on the RNA samples extracted from D. semiclausum male and female adult antennae. A total of 17 putative OBP and 67 OR genes were annotated and further compared to OBPs and ORs from P. xylostella, and other hemipteran parasitoid species. The expression patterns of D. semiclausum OBPs between male and female antennae were examined using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Six OBPs (DsemOBP 6, 7, 8, 9, 10, and 14) demonstrated significantly higher expression levels in females than in males, which may assist in female D. semiclausum host-seeking and oviposition behaviors. This study advances our understanding of the olfactory system of D. semiclausum at the molecular level and paves the way for future functional studies aiming at increasing the efficacy to control P. xylostella by using D. semiclausum.

Identification of chemosensory genes by antennal transcriptome analysis and expression profiles of odorant-binding proteins in parasitoid wasp Aulacocentrum confusum

The endoparasitoid wasp, Aulacocentrum confusum (Hymenoptera: Braconidae), is a preponderant natural enemy of the larvae of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae), which is a destructive pest of mulberry trees. We first constructed the antennal transcriptome database of A. confusum. In total, we obtained 48,262,304 clean reads from the dataset and assembled 24,324 unigenes. A total of 12,690 (52.17%) unigenes indicated significant similarity (E-value < 10−5) compared to known protein sequences of other species from the NCBI non-redundant protein database. Gene ontology (GO) and cluster of orthologous groups (COG) analyses were used to determine the functional categories of these genes. A total of 84 putative chemosensory genes were identified from the antennal transcriptome of A. confusum, including 11 putative odorant-binding protein (OBP) genes, six chemosensory protein (CSP) genes, 44 olfactory receptor (OR) genes (including one olfactory co-receptor, Orco), 19 ionotropic receptor (IR) genes, and four sensory neuron membrane protein (SNMP) genes. Results of qPCR assays indicated that among of 11 AconOBPs, nine AconOBP genes were significantly expressed in the antennae of A. confusum adults. AconOBP8 was significantly expressed in the abdomen and AconOBP10 was highly expressed in the thorax. These findings can build a basis for further study on the processes of chemosensory perception in A. confusum at the molecular level.

A hypothesis on the capacity of plant odorant-binding proteins to bind volatile isoprenoids based on in silico evidences.

Volatile organic compounds (VOCs) from 'emitting' plants inform the 'receiving' (listening) plants of impending stresses or simply of their presence. However, the receptors that allow receivers to detect the volatile cue are elusive. Most likely, plants (as animals) have odorant-binding proteins (OBPs), and in fact, a few OBPs are known to bind 'stress-induced' plant VOCs. We investigated whether these and other putative OBPs may bind volatile constitutive and stress-induced isoprenoids, the most emitted plant VOCs, with well-established roles in plant communication and defense. Molecular docking simulation experiments suggest that structural features of a few plant proteins screened in databases could allow VOC binding. In particular, our results show that monoterpenes may bind the same plant proteins that were described to bind other stress-induced VOCs, while the constitutive hemiterpene isoprene is unlikely to bind any investigated putative OBP and may not have an info-chemical role. We conclude that, as for animal, there may be plant OBPs that bind multiple VOCs. Plant OBPs may play an important role in allowing plants to eavesdrop messages by neighboring plants, triggering defensive responses and communication with other organisms.

RNA-Seq and differential gene expression analysis in Temora stylifera copepod females with contrasting non-feeding nauplii survival rates: an environmental transcriptomics study

BackgroundCopepods are fundamental components of pelagic food webs, but reports on how molecular responses link to reproductive success in natural populations are still scarce. We present a de novo transcriptome assembly and differential expression (DE) analysis in Temora stylifera females collected in the Gulf of Naples, Mediterranean Sea, where this copepod dominates the zooplankton community. High-Throughput RNA-Sequencing and DE analysis were performed from adult females collected on consecutive weeks (May 23rd and 30th 2017), because opposite naupliar survival rates were observed. We aimed at detecting key genes that may have influenced copepod reproductive potential in natural populations and whose expression was potentially affected by phytoplankton-derived oxylipins, lipoxygenase-derived products strongly impacting copepod naupliar survival.ResultsOn the two sampling dates, temperature, salinity, pH and oxygen remained stable, while variations in phytoplankton cell concentration, oxylipin concentration and oxylipin-per-diatom-cell production were observed. T. stylifera naupliar survival was 25% on May 23rd and 93% on May 30th. De novo assembly generated 268,665 transcripts (isoforms) and 120,749 unique ‘Trinity predicted genes’ (unigenes), of which 50% were functionally annotated. Out of the 331 transcript isoforms differentially expressed between the two sampling dates, 119 sequences were functionally annotated (58 up- and 61 down-regulated). Among predicted genes (unigenes), 144 sequences were differentially expressed and 31 (6 up-regulated and 25 down-regulated) were functionally annotated. Most of the significantly down-regulated unigenes and isoforms were A5 Putative Odorant Binding Protein (Obp). Other differentially expressed sequences (isoforms and unigenes) related to developmental metabolic processes, protein ubiquitination, response to stress, oxidation-reduction reactions and hydrolase activities. DE analysis was validated through Real Time-quantitative PCR of 9 unigenes and 3 isoforms.ConclusionsDifferential expression of sequences involved in signal detection and transduction, cell differentiation and development offered a functional interpretation to the maternally-mediated low naupliar survival rates observed in samples collected on May 23rd. Down-regulation of A5 Obp along with higher quantities of oxylipins-per-litre and oxylipins-per-diatom-cell observed on May 23rd could suggest oxylipin-mediated impairment of naupliar survival in natural populations of T. stylifera. Our results may help identify biomarker genes explaining variations in copepod reproductive responses at a molecular level.

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.

Identification of putative olfactory genes in newly hatched larvae of a Coleopteran ectoparasitoid Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) by transcriptome analysis

AbstractDastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is an effective ectoparasitoid of pine sawyer beetle Monochamus alternatus Hope (Coleoptera: Cerambycidae) which is the major median insect for transmission of pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Aphelenchida: Parasitaphelenchidae). A sensitive and acute olfactory system is extremely essential for newly hatched larva of D. helophoroides to forage for a host. Therefore, we established the transcriptome of newly hatched D. helophoroides larvae and characterized the expression profiles of olfactory genes. We identified 20 transcripts encoding putative odorant binding proteins (OBPs), 11 putative chemosensory proteins (CSPs), 9 putative odorant receptors (ORs), 11 putative ionotropic receptors (IRs), 7 putative gustatory receptors (GRs) and 4 putative sensory neuron membrane proteins (SNMPs) olfactory genes from the transcriptome of newly hatched D. helophoroides larvae. According to the phylogenetic analysis, 3 OBPs (DhelOBP6, DhelOBP14 and DhelOBP19), 1 CSP (DhelCSP11), 1 OR (DhelOrco1), 3 IRs (DhelIR3, DhelIR6 and DhelIR8), 3 GRs (DhelGR2, DhelGR4 and DhelGR5) and 2 SNMPs (DhelSNMP1 and DhelSNMP3) had high similarities with reported Coleopteran species. The highest expressed olfactory gene was DhelCSP3, followed by DhelOBP8 and DhelCSP2. The expression levels of the majority of OR, IR, GR and SNMP genes were low. These results indicate that CSPs and OBPs may play important roles in the olfactory recognition process in newly hatched D. helophoroides larvae. The findings from the present study are helpful in unraveling the profound mystery between D. helophoroides and M. alternatus, as well as improving the parasitism efficiency of D. helophoroides larvae.

Odorant-binding proteins and chemosensory proteins potentially involved in host plant recognition in the Asian citrus psyllid, Diaphorina citri.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are two families of small water-soluble proteins involved in odor detection and subsequent signal transmission. Determination of their binding mechanisms and specificity towards different odorants is important for developing OBPs/CSPs as targets in pest control management. We re-annotated genes encoding putative OBPs and CSPs in the Asian citrus psyllid (Diaphorina citri) draft genome using various bioinformatic tools. Genes encoding nine OBPs (seven Classic and two Plus-C) and 12 CSPs were identified, consistent with our previous transcriptomic results. Tissue-specific and developmental expression analyses suggested that genes encoding six OBPs and four CSPs were predominantly expressed in antennae, and displayed various expression patterns in different development stages, suggesting potential involvement in olfactory perception. Competitive fluorescence binding assays with 13 candidate ligands, including known host plant volatiles, sex pheromone components and repellents, showed that DcitOBP3 could bind to various odorants, whereas DcitOBP6, 8 and 9 bound specifically to host plant terpenoids. DcitCSP1 and 12 could also bind to certain terpenoids with high binding specificity. OBP- and CSP-encoding genes were systematically identified by annotating the draft D. citri genome and those potentially involved in odorant detection and signal transmission were identified by analyzing their tissue-expression profiles and odorant-binding affinities, particularly to the peripheral molecular perception of host plant terpenoids. The identified genes may provide potential targets for efficient pest control. © 2020 Society of Chemical Industry.

Candidate genes coding for odorant binding proteins and chemosensory proteins identified from dissected antennae and mouthparts of the southern green stink bug Nezara viridula

The southern green stink bug Nezara viridula (Hemiptera: Pentatomidae) is a highly polyphagous pest that can significantly impact many major crops worldwide. Insect odorant binding proteins (OBPs) and chemosensory proteins (CSPs) transport chemicals and play critical roles in chemoreception. Studies on N. viridula OBPs and CSPs should increase our overall understandings on chemosensory systems and chemical ecology of stink bugs, which may lead to improved pest control. In this study, we identified candidate genes encoding putative OBPs and CSPs in N. viridula by generating transcriptomes from dissected antennae and mouthparts. In total, the 42 unigenes were identified coding for OBPs (34 Classic OBPs and eight Plus-C OBPs) and 13 unigenes coding for CSPs. Expression profiles of OBP- and CSP -encoding genes were compared between antennae and mouthparts based on FKPM values. Candidates for antenna-predominant OBPs and CSPs were selected for real-time quantitative PCR analyses. Analyses of tissue expression profiles revealed that 17 OBP-encoding genes, and four CSP genes were primarily expressed in antennae, suggesting their putative roles in perception of volatiles. The sex-biased expression patterns of these antenna-predominant genes suggested that they may have important functions in reproduction of the insect. This is a systematic analysis on OBPs and CSPs in a stink bug, providing a comprehensive resource for future functional studies not only for N. viridula, but also for other stink bugs as well.

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 of putative odorant binding proteins in the peach fruit borer Carposina sasakii Matsumura (Lepidoptera: Carposinidae) by transcriptome analysis and their expression profile

The peach fruit borers Carposina sasakii Matsumura (Lepidoptera: Carposinidae) are serious fruit borers in East Asia. Odorants have been reported to elicit their olfactory responses, including sex pheromone and host plant volatiles. However, little is known about their molecular aspects of olfactory perception to these odorants. Odorant binding proteins (OBPs) are believed to play an important role in insect olfaction, which recognize and bind odorants, and transport them to odorant receptors located on olfactory neurons. We identified 12 putative OBPs (CsasOBP1-12) expressed in intact heads (with antennae and mouthparts) of C. sasakii adult males and females by transcriptome analysis, including 4 putative pheromone binding proteins (PBPs) (CsasOBP1-2, 7, 11) and 4 putative general odorant binding proteins (GOBPs) (CsasOBP6, 9–10, 12). A phylogenetic tree was constructed to characterize these OBPs. An analysis using fluorescence quantitative PCR showed that CsasOBP3, 5, 7, 9–12 have the highest expression level in the intact heads, suggesting that among them there may be the OBPs playing key roles in C. sasakii olfaction. In the 7 OBPs, CsasOBP5, 7, 11–12 have higher level of expressions in intact heads of C. sasakii adult males than females, while CsasOBP3, 9–10 are expressed equally in the two sexes. Moreover, CsasOBP2 is expressed dominantly in the intact heads and wings, with an equal level between them. CsasOBP8 expression is highest in the wings, while CsasOBP1, 4, 6 are expressed dominantly in the abdomens. Our study is helpful for understanding C. sasakii OBPs’ functions and C. sasakii olfaction from molecular view.

Antennal transcriptome analysis and expression profiles of odorant binding proteins in Clostera restitura

Clostera restitura Walker (Lepidoptera: Notodontidae) is one of the most destructive defoliators of poplars in China. We constructed an antennal transcriptome using Illumina Hiseq 2500™ sequencing and characterized the expression profiles of odorant binding proteins for better understanding of the olfactory receptive system and the role of putative olfactory proteins in C. restitura. A total of 165 transcripts were identified, including 43 transcripts encoding putative odorant-binding proteins (OBPs), 13 chemosensory proteins (CSPs), 78 odorant receptors (ORs), 15 ionotropic receptors (IRs), 13 gustatory receptors (GRs), and 3 sensory neuron membrane proteins (SNMPs). Furthermore, we systematically analyzed expression patterns of eight OBPs from different tissues of both C. restitura sexes by using reverse transcription PCR and quantitative real time PCR (RT-qPCR). The expression level of CresGOBP2 in female antennae was approximately two times higher than in males, and two pheromone binding proteins PBPs (CresPBP1 and -PBP3) and three OBPs (CresOBP9, −10, and −16) were more highly enriched in male antennae than in female antennae. CresOBP10 showed a remarkably high expression in legs compared to other studied insects. Our results suggested that these proteins might play a key role in foraging, seeking mates, and host recognition in C. restitura. Our findings provided a foundation for future studies on the molecular mechanisms controlling the olfactory system in C. restitura and potential novel targets for pest control strategies.

Molecular and Functional Characterization of Odorant Binding Protein 7 From the Oriental Fruit Moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae).

Odorant-binding proteins (OBPs) are widely and abundantly distributed in the insect sensillar lymph and are essential for insect olfactory processes. The OBPs can capture and transfer odor molecules across the sensillum lymph to odorant receptors and trigger the signal transduction pathway. In this study, a putative OBP gene, GmolOBP7, was cloned using specific-primers, based on the annotated unigene which forms the antennal transcriptome of Grapholita molesta. Real-time PCR (qRT-PCR) analysis revealed that GmolOBP7 was highly expressed in the wings of males and the antennae of both male and female adult moths, while low levels were expressed in other tissues. The recombinant GmolOBP7 (rGmolOBP7) was successfully expressed and purified via Ni-ion affinity chromatography. The results of binding assays revealed that rGmolOBP7 exhibited a high binding affinity to the minor sex pheromone 1-dodecanol containing Ki of 7.48 μM and had high binding capacities to the host-plant volatiles, such as pear ester, lauraldehyde and α-ocimene. RNA-interference experiments were performed to further assess the function of GmolOBP7. qRT-PCR showed that the levels of mRNA transcripts significantly declined in 1 and 2 day old male and female moths, treated with GmolOBP7 dsRNA, compared with non-injection controls. The EAG responses of dsRNA-injected males and females to pear ester, as well as the EAG responses of dsRNA-injected males to 1-dodecanol, were significantly reduced compared to the GFP-dsRNA-injected and non-injected controls. We therefore infer that GmolOBP7 has a dual function in the perception and recognition of the host-plant volatiles and sex pheromones.