Plant volatile compound methyl benzoate is highly effective against Spodoptera frugiperda and safe to non-target organisms as an eco-friendly botanical-insecticide.

Recent studies have indicated that the plant volatile methyl benzoate (MB) exhibits significant insecticidal bioactivity against several common insects. However, the potential environmental hazards of MB and its safety to non-target organisms is poorly understood. In the present study, these characteristics were investigated through laboratory experiments and field investigations. The results revealed that MB was highly toxic to the agricultural pest, fall armyworm Spodoptera frugiperda. Compared with the commercial pesticide lambda-cyhalothrin, the toxicities of MB against S. frugiperda larvae and adults were comparable and 3.41 times higher, respectively. Behavioral bioassays showed that the percentage repellency of MB to S. frugiperda larvae was 56.72 %, and MB induced 69.40 % oviposition deterrence rate in S. frugiperda female adults. Furthermore, in terms of median lethal concentration (LC50) and median lethal doses (LD50), MB exhibited non-toxic effects on non-target animals with 3-d LC50 of > 1 % to natural predators (Coccinella septempunctata and Harmonia axyridis), 3-d LD50 of 467.86 µg/bee to the bumblebee Bombus terrestris, 14-d LC50 of 971.09 mg/kg to the earthworm Eisenia fetida, and 4-d LC50 of 47.30 mg/L to the zebrafish Brachydanio rerio. The accumulation of MB in the soil and earthworms was found to be extremely limited. Our comparative study clearly demonstrated that MB is effective as a selective botanical pesticide against S. frugiperda and it is safe to use in the tested environment, with no toxic effects on non-target animals and natural predators.

Functional Characterization of a Candidate Sex Pheromone Receptor AlinOR33 Involved in the Chemoreception of Adelphocoris lineolatus.

Sex pheromones are deemed to play a significant role in sexual communication of most insects. Although many sex pheromone components in mirid bugs have been identified, the roles of odorant receptors in sex pheromone perception in Adelphocoris spp. (Hemiptera: Miridae) remain unknown so far. Here, AlinOR33, a candidate sex pheromone receptor in Adelphocoris lineolatus was functionally characterized. Phylogenetic analysis showed that AlinOR33 clustered with the sex pheromone receptor AlucOR4 fromApolygus lucorum. Quantitative real-time PCR measurement revealed that the expression of AlinOR33 increased gradually from nymph to adult stage and reached its peak in the antennae of 3-day-old mated male bugs. The subsequent in situ hybridization demonstrated that AlinOR33 was mainly expressed in sensilla trichoid on the antennae of A. lineolatus. In the two-electrode voltage clamp recordings, AlinOR33/AlinOrco was specifically tuned to four sex pheromone components including butyl butyrate, hexyl hexanoate, trans-2-hexenyl butyrate and hexyl butyrate, and especially most sensitive to the major component trans-2-hexenyl butyrate. After dsAlinOR33 injection, the electroantennogram responses of males to four sex pheromone components were reduced significantly (∼50%). Compared to control bugs, dsAlinOR33-injected male bugs almost lost behavioral preference for trans-2-hexenyl butyrate. Furthermore, the wingbeat frequency of dsAlinOR33-injected male bugs notably declined. Therefore, we conclude that as a candidate sex pheromone receptor, AlinOR33 plays essential roles in the sexual behavior of A. lineolatus.

Coordinative mediation of the response to alarm pheromones by three odorant binding proteins in the green peach aphid Myzus persicae.

Odorant binding proteins (OBPs) play an essential role for insect chemosensation in insect peripheral nervous systems of antennae. Each antennal sensilla contains more than one OBP at high concentrations but the interactions and cooperation between co-localized OBPs are rarely reported. In present study, we cloned, expressed and purified eight OBPs of the green peach aphid Myzus persicae. The effects of knocking down the expression of these OBP genes by RNAi on the electrophysiological and behavioural responses of M. persicae to the aphid alarm pheromone, (E)-ß-farnesene (EßF) were investigated. The results showed that the aphids could still be repelled by EßF when the expression of each of three OBP genes was individually knocked down. However, the simultaneous knockdown of MperOBP3/7/9 expression significantly reduced the electrophysiological response and the repellent behaviours of M. persicae to EßF than the single OBP gene knockdown (P < 0.05). Rather than a normal saturation binding curve of individual OBP, the binding curve of MperOBP3/7/9 is bell-shaped with a higher affinity for the fluorescent probe N-phenyl-1-naphthylamine (1-NPN). The competitive binding assays confirmed that MperOBP3, MperOBP7, MperOBP9 and MperOBP3/7/9 mixture exhibited a stronger binding affinity for EßF, than for sex pheromones and plant volatiles with a dissociation constant of 2.5 µM, 1.1 µM, 3.9 µM and 1.0 µM, respectively. The competitive binding curve of MperOBP3/7/9 mixture to EßF is shallow without bottom plateau, suggesting a conformational change and a rapid dissociation after the displacement of all 1-NPN (in vivo after the saturation binding of all OBPs by EßF). The interaction between OBPs and formation of a heterogeneous unit may facilitate the delivery EßF to the OR at electrophysiological and behavioural levels during insect odorant signal transduction thus mediate M. persicae response to the alarm pheromone EßF.

Transcriptional identification of differentially expressed genes associated with division of labor in Apis cerana cerana.

Workers of Apis cerana cerana undergo an in-hive nursing to outdoor foraging transition, but the genes underlying this age-related transition remain largely unknown. Here, we sequenced the head transcriptomes of its 7-day-old normal nurses, 18- and 22-day-old normal foragers, 7-day-old precocious foragers and 22-day-old over-aged nurses to unravel the genes associated with this transition. Mapping of the sequence reads to Apis mellifera genome showed that the three types of foragers had a greater percentage of reads from annotated exons and intergenic regions, whereas the two types of nurses had a greater percentage of reads from introns. Pair- and group-wise comparisons of the five transcriptomes revealed 59 uniquely expressed genes (18 in nurses and 41 in foragers) and 14 nurse- and 15 forager-upregulated genes. The uniquely expressed genes are usually low-abundance long noncoding RNAs, transcription factors, transcription coactivators, RNA-binding proteins, kinases or phosphatases that are involved in signaling and/or regulation, whereas the nurse- or forager-upregulated genes are often high-abundance downstream genes that directly perform the tasks of nurses or foragers. Taken together, these results suggest that the nurse-forager transition is coordinated by a social signal-triggered epigenetic shift from introns to exons/intergenic regions and the resulting transcriptional shift between the nurse- and forager-associated genes.

Broadly Tuned Odorant Receptor AlinOR59 Involved in Chemoreception of Floral Scent in Adelphocoris lineolatus.

Plant volatiles such as floral scent compounds play a crucial role in mediating insect host locating, mate search, and oviposition sites selection. The alfalfa plant bug, Adelphocoris lineolatus (Goeze), is a seriously polyphagous herbivore of alfalfa and cotton that has an obvious preference for flowering host plants. In this study, we focused on the role of an odorant receptor AlinOR59 in the perception of plant volatiles in A. lineolatus. In situ hybridization showed that AlinOR59 was coexpressed with the coreceptor AlinORco in the ORNs cell located in the long curved sensilla trichodea on antennae of both genders. The Xenopus oocytes expression coupled with two-electrode voltage clamp recordings demonstrated that AlinOR59 responded to 15 plant volatiles. In electroantennogram assays, all of the above 15 compounds could excite electrophysiological responses in the antennae of adult bugs. Furthermore, an important floral scent compound, methyl salicylate, was utilized to evaluate the behavioral responses of A. lineolatus. It was found that adult bugs of both genders were significantly attracted to methyl salicylate. Taken together, our findings suggest that AlinOR59 plays a crucial role in the perception of floral scents in A. lineolatus and could be used as a potential target to design novel olfactory regulators for the management of bugs.

Bioactivities of synthetic salicylate-substituted carboxyl (E)-ß-Farnesene derivatives as ecofriendly agrochemicals and their binding mechanism with potential targets in aphid olfactory system.

BACKGROUND: The aphid alarm pheromone, (E)-ß-farnesene (EßF), is a natural product secreted from the aphid cornicle as a signal to warn companions of danger. Odorant binding proteins (OBPs) are the vital targets in insect signal transduction pathways. To improve bioactivity of EßF as more economic and stable aphid control agents, EßF derivatives containing an active substructure, salicylic acid moiety, were designed, synthesized, and evaluated for their bioactivities in a structure-function study under laboratory conditions. RESULTS: EßF derivatives, (E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methylbenzoate and (E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methoxybenzoate showed outstanding aphid-repellent activity at a dose of 5 µg against Acyrthosiphon pisum (repellency proportions of 67.3% and 71.2%, respectively) and Myzus persicae (repellency proportions of 80.0% and 74.4%, respectively) in laboratory. EßF and most of its derivatives bound strongly to ApisOBP9 with a higher affinity than those of the reported potential targets AphisOBP3 and ApisOBP7. The binding affinities to these three ApisOBPs were generally consistent with the in vivo aphid-repellent activity. A molecular docking study suggested that the hydrophobic effect was crucial for the interactions between the derivatives and the OBPs. CONCLUSION: New EßF derivatives containing salicylic acid moiety and their repellent activity, binding mechanism with three potential OBPs are presented. A new OBP, ApisOBP9, was characterized as a potential EßF and EßF derivatives binding protein for the first time. The hydrophobic nature of these analogues is responsible for their activity. Two analogues 3b and 3e with outstanding aphid-repellent activity could be new leads for aphid control agents.

Seroprevalence of Bartonella henselae and Identification of Risk Factors in China.

Serum samples were tested for Bartonella henselae IgG antibodies using indirect immunofluorescence assays. We then analyzed associated risk factors. Serum samples were considered positive when reactive at a dilution of more than 1:320. Differences between groups and risk factors associated with Bartonella exposure were statistically analyzed using Chi-square tests and the generalized linear model. 122 of 1,260 samples (9.68%) were positive for B. henselae infection. The infection rate ranged from 0% to 30.43% and differed significantly among age groups ( P < 0.01); infection rate in the 50-59 years group was significantly higher than that in other age groups. The seroprevalence of Bartonella varied significantly among sites within the four provinces, and the infection rate of field workers was significantly higher than that of urban workers.

Expressional and functional comparisons of two general odorant binding proteins in Agrotis ipsilon.

Insect general odorant binding proteins (GOBPs) have been long thought to bind and transport host plant volatiles to the olfactory receptors on the dendrite membrane of the olfactory neurons. Recent studies indicate that they can also bind female sex pheromones. In present study, two GOBP genes, AipsGOBP1 and AipsGOBP2 were cloned from the adult antennae of Agrotis ipsilon. Tissue expression profiles indicated that both of them are antennae-specific and more abundant in the female antennae than in the male antennae. Temporal expression profiles showed that both AipsGOBP1 and AipsGOBP2 began to express in antennae 3 days prior to adult emergence from pupae, and reached their highest expression level 3 and 4 days after adult emergence, respectively. Mating increased their expression in the female antennae but reduced their expression in the male antennae. In situ hybridization and immunolocalization demonstrated that both AipsGOBP1 and AipsGOBP2 are expressed and co-localized in sensilla basiconica and sensilla trichodea of both sexes. AipsGOBP2 exhibited a high binding affinity in vitro with the two major sex pheromone components Z7-12:Ac and Z9-14:Ac and the four plant volatiles cis-3-hexen-1-ol, oleic acid, dibutyl phthalate and ß-caryophyllene with Ki values less than 5 µM. AipsGOBP1, on the other hand, showed medium binding affinities with the five A. ipsilon sex pheromones and six plant volatiles. AipsGOBP2 also showed a broader ligand-binding spectrum and a greater ligand-binding affinity than AipsGOBP1 with the tested aldehyde and alcohol sex pheromones of Lepidoptera species. Taken together, our results indicate that AipsGOBP2 may play greater roles than AipsGOBP1 does in binding sex pheromones and host plant volatiles.

The terpene synthase gene family in Gossypium hirsutum harbors a linalool synthase GhTPS12 implicated in direct defence responses against herbivores.

Herbivore-induced terpenes have been reported to function as ecological signals in plant-insect interactions. Here, we showed that insect-induced cotton volatile blends contained 16 terpenoid compounds with a relatively high level of linalool. The high diversity of terpene production is derived from a large terpene synthase (TPS) gene family. The TPS gene family of Gossypium hirsutum and Gossypium raimondii consist of 46 and 41 members, respectively. Twelve TPS genes (GhTPS4-15) could be isolated, and protein expression in Escherichia coli revealed catalytic activity for eight GhTPS. The upregulation of the majority of these eight genes additionally supports the function of these genes in herbivore-induced volatile biosynthesis. Furthermore, transgenic Nicotiana tabacum plants overexpressing GhTPS12 were generated, which produced relatively large amounts of (3S)-linalool. In choice tests, female adults of Helicoverpa armigera laid fewer eggs on transgenic plants compared with non-transformed controls. Meanwhile, Myzus persicae preferred feeding on wild-type leaves over leaves of transgenic plants. Our findings demonstrate that transcript accumulation of multiple TPS genes is mainly responsible for the production and diversity of herbivore-induced volatile terpenes in cotton. Also, these genes might play roles in plant defence, in particular, direct defence responses against herbivores.

Key site residues of pheromone-binding protein 1 involved in interacting with sex pheromone components of Helicoverpa armigera.

Pheromone binding proteins (PBPs) are widely distributed in insect antennae, and play important roles in the perception of sex pheromones. However, the detail mechanism of interaction between PBPs and odorants remains in a black box. Here, a predicted 3D structure of PBP1 of the serious agricultural pest, Helicoverpa armigera (HarmPBP1) was constructed, and the key residues that contribute to binding with the major sex pheromone components of this pest, (Z)-11- hexadecenal (Z11-16:Ald) and (Z)-9- hexadecenal (Z9-16:Ald), were predicted by molecular docking. The results of molecular simulation suggest that hydrophobic interactions are the main linkage between HarmPBP1 and the two aldehydes, and four residues in the binding pocket (Phe12, Phe36, Trp37, and Phe119) may participate in binding with these two ligands. Then site-directed mutagenesis and fluorescence binding assays were performed, and significant decrease of the binding ability to both Z11-16:Ald and Z9-16:Ald was observed in three mutants of HarmPBP1 (F12A, W37A, and F119A). These results revealed that Phe12, Trp37, and Phe119 are the key residues of HarmPBP1 in binding with the Z11-16:Ald and Z9-16:Ald. This study provides new insights into the interactions between pheromone and PBP, and may serve as a foundation for better understanding of the pheromone recognition in moths.

A simpler and more cost-effective peptide biosynthetic method using the truncated GST as carrier for epitope mapping.

There is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. Here, we describe improved biosynthetic peptide (BSP) method using a newly developed plasmid pXXGST-3 as vector, which has a viral E7 gene in the cloning sites of pXXGST-1. It is crucial to employ pXXGST-3 instead of pXXGST-1, since it makes use of the BSP method simpler and easier to perform, and more cost-effective for epitope mapping. These merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between BamH I and Sal I sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. The protocol involves the following core steps: (i) design of plus and minus strands of DNA fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed DNA fragments; (iii) development of r-clones using pXXGST-3 vector expressing each 8/18mer peptide fused with truncated GST188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on SDS-PAGE gel followed by sequencing of inserted DNA fragments for each verified r-clone; and (v) Western blotting with either monoclonal antibodies or polyclonal antibodies. This improved GST188-BSP method provides a powerful alternative tool for epitope mapping.

Aminopeptidase N5 (APN5) as a Putative Functional Receptor of Cry1Ac Toxin in the Larvae of Athetis lepigone.

Athetis lepigone was a new lepidopteran pest and caused severe damage to maize crops in China. We have detected that Cry1Ac protoxin and toxin were highly active against the larvae of A. lepigone. However, there is no report about the mode of action of Bt Cry1Ac toxin against this pest until now. A 110 kDa APN5 protein from BBMV of A. lepigone was identified as the binding receptor of Cry1Ac toxin using Ligand blotting. The Cry1Ac receptor APN5 was cloned from A. lepigone larval midgut mRNA and named as AlAPN5 (GenBank accession no.: KU950745). AlAPN5 had a GATEN motif and been classified to Class 5 APNs. 79.2% reduction in mortality was observed when A. lepigone larvae were injected with siRNA of the AlAPN5 gene and treated with Cry1Ac toxin. These data demonstrate that AlAPN5 is a putative functional receptor and maybe the only receptor of Cry1Ac in A. lepigone.

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.

Odorant-binding and chemosensory proteins identified in the antennal transcriptome of Adelphocoris suturalis Jakovlev.

Adelphocoris suturalis Jakovlev (Hemiptera: Miridae) is an insect pest that causes severe agricultural damage to cotton and many other important crops. In insects, olfaction is very important throughout their lifetime. There are two groups of small soluble proteins, named odorant binding proteins (OBPs) and chemosensory proteins (CSPs), which are suggested to participate in the initial biochemical recognition steps of insect olfactory signal transduction. In this study, a total of 16 OBPs (12 classical OBPs and 4 plus-C OBPs) and 8 CSPs, were identified in the antennal transcriptome of A. suturalis. The sex- and tissue-specific profiles of these binding protein genes showed that 13 of the 16 OBP transcripts were highly expressed in the antennae of both sexes, and 4 OBPs (AsutOBP1, 4, 5 and 9) were expressed higher in the male antennae compared to the female antennae. Three CSPs (AsutCSP1, 4 and 5) were expressed specifically in the antennae of both sexes, and AsutCSP1 was expressed higher in the male antennae than in the female antennae. Our findings identify several novel OBP and CSP genes for further investigation of the olfactory system of A. suturalis at the molecular level.

Silencing of odorant binding protein gene AlinOBP4 by RNAi induces declining electrophysiological responses of Adelphocoris lineolatus to six semiochemicals.

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. Here, we injected AlinOBP4-siRNA into the conjunctivum between prothorax and mesothorax of the lucerne plant bug, Adelphocoris lineolatus and evaluated the silencing of AlinOBP4 by reverse transcription polymerase chain reaction (RT-PCR) analysis, quantitative real-time PCR (qPCR) test and electroantennogram (EAG) assay. The combination of RT-PCR and qPCR analyses revealed that the levels of messenger RNA transcript were significantly reduced ∼95% in AlinOBP4-siRNA-treated A. lineolatus males and ∼75% in RNAi-treated females within 48 hours. It was found that there are different EAG responses between male and female bugs when the AlinOBP4 gene was silenced by RNAi. The EAGs of A. lineolatus to two plant volatiles, tridecanal and hexyl alcohol, were reduced 9.09% and 79.45% in RNAi-treated males, 62.08% and 62.08% in RNAi-treated females compared to the controls, separately. Antennae of RNAi-treated bugs showed significantly lower electrophysiological responses to four sex pheromone analogs, butyl butanoate, 1-hexyl butyrate, (E)-2-hexenyl butyrate and hexyl hexanoate. The EAG recordings were reduced 35.43%, 35.24%, 39.96% and 78.47% in RNAi-treated males and 64.52%, 18.13%, 36.88% and 49.52% in RNAi-treated females, respectively. The results suggested that AlinOBP4 might play dual-roles in the identification of plant volatiles and sex pheromones. It was suspected that AlinOBP4 may have different functions in odor perception between male and female A. lineolatus.

Probiotic Diversity Enhances Rhizosphere Microbiome Function and Plant Disease Suppression.

Bacterial communities associated with plant roots play an important role in the suppression of soil-borne pathogens, and multispecies probiotic consortia may enhance disease suppression efficacy. Here we introduced defined Pseudomonas species consortia into naturally complex microbial communities and measured the importance of Pseudomonas community diversity for their survival and the suppression of the bacterial plant pathogen Ralstonia solanacearum in the tomato rhizosphere microbiome. The survival of introduced Pseudomonas consortia increased with increasing diversity. Further, high Pseudomonas diversity reduced pathogen density in the rhizosphere and decreased the disease incidence due to both intensified resource competition and interference with the pathogen. These results provide novel mechanistic insights into elevated pathogen suppression by diverse probiotic consortia in naturally diverse plant rhizospheres. Ecologically based community assembly rules could thus play a key role in engineering functionally reliable microbiome applications. IMPORTANCE: The increasing demand for food supply requires more-efficient control of plant diseases. The use of probiotics, i.e., naturally occurring bacterial antagonists and competitors that suppress pathogens, has recently reemerged as a promising alternative to agrochemical use. It is, however, still unclear how many and which strains we should choose for constructing effective probiotic consortia. Here we present a general ecological framework for assembling effective probiotic communities based on in vitro characterization of community functioning. Specifically, we show that increasing the diversity of probiotic consortia enhances community survival in the naturally diverse rhizosphere microbiome, leading to increased pathogen suppression via intensified resource competition and interference with the pathogen. We propose that these ecological guidelines can be put to the test in microbiome engineering more widely in the future.

Epitomics: IgG-epitome decoding of E6, E7 and L1 proteins from oncogenic human papillomavirus type 58.

To enable rational multi-epitope vaccine and diagnostic antigen design, it is imperative to delineate complete IgG-epitome of the protein. Here, we describe results of IgG-epitome decoding of three proteins from high-risk (HR-) oncogenic human papillomavirus type 58 (HPV58). To reveal their entire epitomes, employing peptide biosynthetic approach, 30 precise linear B-cell epitopes (BCEs) were mapped on E6, E7 and L1 proteins using rabbits antisera to the respective recombinant proteins. Using sequence alignment based on BCE minimal motif, the specificity and conservativeness of each mapped BCE were delineated mainly among known HR-HPVs, including finding 3 broadly antibody cross-reactive BCEs of L1 that each covers almost all HR-HPVs. Western blots revealed that 13 of the 18 BCEs within L1-epitome were recognized by murine antisera to HPV58 virus-like particles, suggesting that these are antibody accessible BCEs. Also, a highly conserved epitope (YGD/XTL) of E6 was found to exist only in known common HR-HPVs, which could be used as the first peptide reference marker for judging HR-HPVs. Altogether, this study provides systemic and exhaustive information on linear BCEs of HR-HPV58 that will facilitate development of novel multi-epitope diagnostic reagents/chips for testing viral antibodies and 'universal' preventive HPV peptide vaccine based on L1 conserved BCEs.

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 and comparative expression analysis of odorant binding protein genes in the tobacco cutworm Spodoptera litura.

Insect odorant binding proteins (OBPs) are thought to involve in insects' olfaction perception. In the present study, we identified 38 OBP genes from the antennal transcriptomes of Spodoptera litura. Tissue expression profiles analysis revealed that 17 of the 38 SlitOBP transcripts were uniquely or primarily expressed in the antennae of both sexes, suggesting their putative role in chemoreception. The RPKM value analysis revealed that seven OBPs (SlitPBP1-3, SlitGOBP1-2, SlitOBP3 and SlitOBP5) are highly abundant in male and female antennae. Most S. litura antennal unigenes had high homology with Lepidoptera insects, especially genes of the genus Spodoptera. Phylogenetic analysis of the Lepidoptera OBPs demonstrated that the OBP genes from the genus Spodoptera (S. litura, Spodoptera littoralis and Spodoptera exigua) had a relatively close evolutionary relationship. Some regular patterns and key conserved motifs of OBPs in genus Spodoptera are identified by MEME, and their putative roles in detecting odorants are discussed here. The motif-patterns between Lepidoptera OBPs and CSPs are also compared. The SlitOBPs identified here provide a starting point to facilitate functional studies of insect OBPs at the molecular level both in vivo and in vitro.

Identification and Expression Analysis of Putative Chemosensory Receptor Genes in Microplitis mediator by Antennal Transcriptome Screening.

Host-seeking, ovipositional behavior and mating of insects are controlled mainly by odor perception through sensory organs such as antennae. Antennal chemoreception is extremely important for insect survival. Several antennal chemosensory receptors are involved in mediating the odor detection in insects, especially the odorant receptors (ORs) and ionotropic receptors (IRs), to ensure the specificity of the olfactory sensory neuron responses. In the present study, we identified the chemosensory receptor gene repertoire of the parasitoid wasp Microplitis mediator, a generalist endoparasitoid that infests more than 40 types of Lepidopterous larvae and is widely distributed in the Palaearctic region. By transcriptome sequencing of male and female antennae we identified 60 candidate odorant receptors, six candidate ionotropic receptors and two gustatory receptors in M. mediator. The full-length sequences of these putative chemosensory receptor genes were obtained by using the rapid amplification of cDNA ends PCR (RACE-PCR) method. We also conducted reverse transcription PCR (RT-PCR) combined with real-time quantitative PCR (qPCR) for investigating the expression profiles of these chemosensory receptor genes in olfactory and non-olfactory tissues. The tissue- and sex-biased expression patterns may provide insights into the roles of the chemosensory receptor in M. mediator. Our findings support possible future study of the chemosensory behavior of M. mediator at the molecular level.