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Pheromone-binding proteins (PBPs) have been extensively investigated in lepidopteran moths, but their evolution and function in hemipteran species remain unclear. Our previous study demonstrated that an odorant-binding protein, OBP4, of the mirid bug Adelphocoris lineolatus functions as a candidate hemipteran PBP but clustered with lepidopteran antennae-binding proteins (ABPs) rather than in the PBP/general odorant-binding protein (GOBP) clade. In this study, we hypothesized that origin and function of PBPs in hemipteran bugs may differ from those of lepidopteran moths. To test this hypothesis, we first constructed a phylogenetic tree using insect OBPs from sister hemipteran and holometabolous lineages, and the results indicated that neither OBP4 nor other types of candidate PBPs of mirid bugs clustered with the lepidopteran PBP/GOBP clade. Then, a fluorescence competitive binding assay was employed to determine binding affinities of recombinant OBP4 protein to host plant volatiles, with functional groups different from A. lineolatus sex pheromone components. The results revealed that OBP4 highly bound the female adult attractant 3-hexanone and 15 other mirid bug biologically active plant volatiles. Finally, we examined cellular expression profiles of OBP4 in putative antennal sensilla that are related to female A. lineolatus host plant location. The fluorescence in situ hybridization and immunocytochemical labeling assay showed that the OBP4 gene was highly expressed in the multiporous olfactory sensilla medium-long sensilla basiconica rather than in the short sensilla basiconica or uniporous sensilla chaetica. These results, together with those of our previous studies, indicate that OBP4 not only functions in recognition of bug-produced sex pheromones in males, but is probably involved in detection of host plant volatiles in both A. lineolatus sexes. Our findings support the hypothesis that the origin and function of PBPs in hemipteran bugs differ from those of well-known PBPs in lepidopteran moths, which provides a novel perspective on evolutionary mechanisms of sex pheromone communication across insect orders.

Different types of insect feeding can activate distinct plant resistance mechanisms and trigger the generation of specific volatile compounds. Transcriptomic changes and the genomic basis underlying plant defense in response to chewing herbivores or phloem-feeding insects have been relatively well investigated, while better insight into molecular mechanisms underlying the plant defense response, in particular, volatile emissions triggered by sap-feeding insects such as the green plant bug Apolygus lucorum is needed. Here, we monitored transcriptomic and volatile metabolomic changes in cotton over time during A. lucorum infestation. RNA-seq analysis showed that 1614 transcripts were differentially expressed (log2|Ratio| ≥ 1; q ≤ 0.05) in cotton leaves infested by A. lucorum. All differentially expressed genes (DEGs) in jasmonic acid (JA; 48 genes) and salicylic acid (SA; 5 genes) pathways were upregulated, highlighting a central role of JA in A. lucorum-induced signaling without attenuating the SA pathway. Moreover, all DEGs (30 genes) involved in herbivore-induced volatile biosynthesis were upregulated. Consistently, A. lucorum-induced cotton volatile blends and synthetic methyl salicylate significantly attracted the parasitoid Peristenus spretus. The present data indicated that cotton plants after A. lucorum infestation undergo rapid, extensive transcriptome reprogramming mediated by complex signaling networks in which the JA and SA pathways act synergistically, leading to a specific volatile profile involved in an indirect plant defense.

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.

Background (E)-4,8-dimethylnona-1,3,7-triene (DMNT), one of the homoterpenes, is thought to contribute to plant indirect defense against insect herbivores. DMNT-enriched plants have great application potential to regulate insect behavior in the 'push & pull' strategy of pest management. However, de novo biosynthesis of DMNT in plants without a homoterpene metabolic pathway in their wild type is still not achieved, and the role of DMNT played in these plants and their interacted insects remains unclear. Results Cytochrome P450s and terpene synthases involved in homoterpenes biosynthesis in cotton plants were employed to generate DMNT-releasing tobacco plants. Single GhTPS14 transgenic Nicotiana tabacum only emitted (E)-nerolidol, the precursor of DMNT. Transgenic tobaccos expressing single GhCYP82Ls were unable to produce DMNT or TMTT, while DMNT was detected when exogenous (E)-nerolidol was added. Compared to wild-type plants, only co-expression of GhCYP82Ls and GhTPS14 in transgenic tobaccos triggered the constitutive release of single-component DMNT. Furthermore, DMNT-emitting transgenic tobacco plants, whether infested with Helicoverpa armigera larvae or not, significantly incited orientation behavior of parasitoid wasps Microplitis mediator. Conclusion Wild type N. tabacum plants have no DMNT metabolic pathway. DMNT could be de novo biosynthesized via co-expression of GhCYP82Ls and GhTPS14. What is more, the parasitoid wasp M. mediator could be recruited by DMNT-releasing transgenic tobaccos, especially by H. armigera-infested transgenic tobaccos, suggesting the potential roles of engineered N. tabacum in regulating the behavioral preference of M. mediator.

BACKGROUND: The Apolygus lucorum is one of the most destructive insect pests in China with a wide range of host plants. Interaction of A. lucorum with surrounding environment heavily relies on chemical communication. Deorphanization of receptors involved in odors detection elevates our understanding of the olfactory system of this pest and may help to develop a chemical ecology‐based control strategy. RESULTS: AlucOR80, an odorant receptor (OR) in A. lucorum was newly cloned. Gene expression analysis showed that this receptor was mainly expressed in the antennae and head of both sexes but with a male bias. The Xenopus oocytes heterologous expression system coupled with the two‐electrode voltage‐clamp (TEVC) recording revealed that AlucOR80 was tuned to 21 selected compounds. Furthermore, electroantennogram (EAG) tests confirmed that all 21 ligands of AlucOR80 were electrophysiologically active in antennae of both sexes. Behavioral trials in a three‐cage olfactometer indicated that 16 compounds were behaviorally active, amongst which, 12 components were attractants and four components were repellents for adults of both sexes. Butyl butyrate and Dimethyl disulfide (DMDS) were the strongest attractive and repellant compounds, respectively. Importantly, we found the repellency of 1, 8‐Cineole, S‐(−)‐cis‐Verbenol and (1S)–(1)‐beta‐Pinene against adults of A. lucorum. CONCLUSION: Although AlucOR80 is a general OR, may play important role in the olfactory perception of A. lucorum. Screening of AlucOR80 ligands by behavioral assay provided valuable insights by which olfactory‐based management approaches could be developed by utilizing the behaviorally active components as attractants or repellents. © 2019 Society of Chemical Industry

Sensory neuron membrane proteins (SNMPs), homologs of the human fatty acid transport protein CD36 family, are observed to play a significant role in chemoreception, especially in detecting sex pheromone in Drosophila and some lepidopteran species. In the current study, two full‐length SNMP transcripts, MmedSNMP1 and MmedSNMP2, were identified in the parasitoid Microplitis mediator (Hymenoptera: Braconidae). Quantitative real‐time polymerase chain reaction analysis showed that the expression of MmedSNMP1 was significantly higher in antennae than in other tissues of both sexes. In addition, the MmedSNMP1 transcript was increased dramatically in newly emerged adults and there were no significant differences between adults with or without mating and parasitic experiences. However, compared with MmedSNMP1, the expression of MmedSNMP2 was widely found in various tissues, significantly increased at half‐pigmented pupae stage and remained at a relatively constant level during the following developmental stages. It was found that MmedSNMP1 contained eight exons and seven introns, which was highly conserved compared with other insect species. In situ hybridization assay demonstrated that MmedSNMP1 transcript was distributed widely in antennal flagella. Among selected chemosensory genes (odorant binding protein, odorant receptor, and ionotropic receptor genes), MmedSNMP1 only partially overlapped with MmedORco in olfactory sensory neurons of antennae. Subsequent immunolocalization results further indicated that MmedSNMP1 was mainly expressed in sensilla placodea of antennae and possibly involved in perceiving plant volatiles and sex pheromones. These findings lay a foundation for further investigating the roles of SNMPs in the chemosensation of parasitoids.

The alfalfa plant bug Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae), a serious pest of several agricultural crops, is extremely attracted by flowering plants to perform season host switching. Generally, chemoreception of A. lineolatus plays a crucial role in detecting food sources, locating mates and oviposition sites. In addition, odorant carrier proteins are believed to participate in the initial chemical communication and perception in insects. In this work, we identified 31 putative odorant carrier protein genes from the antennal transcriptomes of A. lineolatus, including 17 new odorant binding proteins (OBPs), 10 new chemosensory proteins (CSPs) and four Niemann-Pick type C2 proteins (NPC2s). Phylogenetic analysis demonstrated that the OBP and CSP genes from four mirid bug species (A. lineolatus, Adelphocoris suturalis, Apolygus lucorum and Lygus lineolaris) had a relatively close evolutionary relationship. Regular patterns and key conserved motifs of OBPs and CSPs in Hemiptera insects are also identified by using Multiple EM for Motif Elicitation (MEME) tool. Tissue expression profiles analysis showed that nine of the 17 OBPs, 10 CSPs and two of the four NPC2s were primarily or uniquely expressed in the adult antennae suggesting their putative roles in chemoreception. Our study provides the first insight into the roles of odorant carrier proteins in chemoreception of A. lineolatus, which will help to facilitate biological functions of odorant carrier proteins and develop novel insect behavioral regulation strategy in future.

Summary Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up‐regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)‐limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)‐limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)‐limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.

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.

Apolygus lucorum (Hemiptera: Miridae), one of the main insect pests, causes severe damage in cotton and many other economic crops. As is well-known, legs play important roles in the chemoreception of insects. In this study, the putative chemosensory proteins in legs of A. lucorum involved in close or contact chemical communication of adult bugs were investigated using RNA transcriptome sequencing and qPCR methods. Transcriptome data of forelegs, middle legs and hind legs of adult bugs demonstrated that 20 odorant binding protein (OBP) genes, eight chemosensory protein (CSP) genes, one odorant receptor (OR) gene, one ionotropic receptor (IR) gene and one sensory neuron membrane protein (SNMP) gene were identified in legs of A. lucorum. Compared to the previous antennae transcriptome data, five CSPs, IR21a and SNMP2a were newly identified in legs. Results of qPCR analysis indicated that all these putative chemosensory genes were ubiquitously expressed in forelegs, middle legs and hind legs of bugs. Furthermore, four types of sensilla on legs of A. lucorum including sensilla trichodea (subtypes: long straight sensilla trichodea, Str1; long curved sensilla trichodea, Str2), sensilla chaetica (subtypes: sensilla chaetica 1, Sch1; sensilla chaetica 2, Sch2; and sensilla chaetica 3, Sch3), sensilla basiconca (subtypes: medium-long sensilla basiconca, Sba1; short sensilla basiconca, Sba2) and Bohm bristles (BB) were found using scanning electron microscopy. Additionally, the largest number of sensilla was observed on hind legs, while the forelegs had the smallest number of sensilla. Our data provide valuable insights into understanding the chemoreception of legs in A. 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.

Ionotropic receptors (IRs), as a member of the conserved chemoreceptor families in the peripheral nervous system , play a critical role in the chemoreception of Drosophila . However, little is known about IRs in Hymenoptera insects. Here, we comprehensively characterized the gene structure, topological map and chemosensory roles of antennal IRs (MmedIRs) in the hymenopteran parasitoid wasp Microplitis mediator . We found that the IRs were conserved across various insect species. In the in situ hybridization assays, most IRs showed female antennae biased features, and there was no co-expression of the IRs and the olfactory receptor co-receptor (ORco). Moreover, three IR co-expressed complexes, IR75u-IR8a, IR64a1-IR8a and IR64a2-IR8a, were detected. Two genes with high similarity, IR64a1 and IR64a2, were located in distinct neurons but projected to the same sensillum . In two-electrode voltage-clamp recordings, IR64a1 was widely tuned to the chemicals from habitat cues released from host plants over long distances, whereas IR64a2 responded to a narrow range host cues and plant odors with low-volatility. Notably, IR64a2 was able to perceive Z 9-14: Ald, a vital sex pheromone component that is released from Helicoverpa armigera , which is the preferred host of M. mediator. Furthermore, most ligands of IR64a1 and IR64a2 can trigger electrophysiological responses in female wasps . We propose that IR64a1 and IR64a2 collaboratively perceive habitat and host cues to assist parasitoids in efficiently seeking hosts.

The alfalfa plant bug Adelphocoris lineolatus, an economically important pest, has representative behavioral characteristics with host plants transfer. Olfactory system is essential for insects to perceive ever-changing chemical signals in the external environment, and chemosensory genes play crucial roles in signals reception and transduction. In this work, we compared the differences in chemosensory genes expression before and after host plants transfer by constructing 12 antennal transcriptomes of male and female bugs, respectively. The results showed that the expression levels of most chemosensory genes in A. lineolatus changed to adapt to the transformation of the hosts plant. More remarkable, female bugs had more up-regulated chemosensory genes than males. Differentially expressed genes (DEGs) analysis revealed three odorant binding proteins (OBPs), three chemosensory proteins (CSPs), eight odorant receptors (ORs) and one ionotropic receptor (IR) showed significant differences when the host plant transferred. There were complex characteristics of up- and down- regulated genes in male and female adults, among which OBP19 showed higher expression in females exposing to the new host plant alfalfa, suggesting this OBP may be associated with the localization of the oviposition site. The OR54 and OR82 were up-regulated in both genders, indicating their possible roles in recognizing some alfalfa-specific volatiles. These findings will provide valuable insights in biological functions of chemosensory genes in A. lineolatus and facilitate the development of new targets for novel strategies to control the alfalfa plant bug and other herbivores.

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.

Ionotropic receptors (IRs) mainly detect the acids and amines having great importance in many insect species, representing an ancient olfactory receptor family in insects. In the present work, we performed RNAseq of Microplitis mediator antennae and identified seventeen IRs. Full-length MmedIRs were cloned and sequenced. Phylogenetic analysis of the Hymenoptera IRs revealed that ten MmedIR genes encoded "antennal IRs" and seven encoded "divergent IRs". Among the IR25a orthologous groups, two genes, MmedIR25a.1 and MmedIR25a.2, were found in M. mediator. Gene structure analysis of MmedIR25a revealed a tandem duplication of IR25a in M. mediator. The tissue distribution and development specific expression of the MmedIR genes suggested that these genes showed a broad expression profile. Quantitative gene expression analysis showed that most of the genes are highly enriched in adult antennae, indicating the candidate chemosensory function of this family in parasitic wasps. Using immunocytochemistry, we confirmed that one co-receptor, MmedIR8a, was expressed in the olfactory sensory neurons. Our data will supply fundamental information for functional analysis of the IRs in parasitoid wasp chemoreception.

Pheromone binding proteins (PBPs) are well studied in lepidopteran moths and are considered to be crucial in detection of sex pheromones as well as some green leaf volatiles. In contrast, evidence that PBPs interact with sex pheromones of hemipteran species is not available. The mirid plant bug, Adelphocoris lineolatus (Goeze), is a notorious hemipteran pest that uses two butyrate esters, trans-2-hexenyl butyrate (E2HB) and hexyl butyrate (HB), and one hexenoic aldehyde trans-4-oxo-2-hexenal (E4O2H), as sex pheromones. In the present study, we report on an odorant binding protein, AlinOBP4, with particular focus on its potential physiological roles in the detection of A. lineolatus sex pheromone components. Phylogenetic analyses indicated that AlinOBP4 and two mirid orthologs clustered in a general phylogenetic clade with the lepidopteran ABX OBPs, the fly LUSH and the OBP83a/b subfamily. Cellular localization by fluorescence in situ hybridization and immunolabeling further demonstrated that AlinOBP4 was strongly expressed in the multiporous sensilla trichodea (str) and middle long sensilla basiconica (mlsba) of male A. lineolatus adults, suggesting a key role associated with sex pheromone and odorant detection. A ligand binding assay revealed that recombinant AlinOBP4 protein highly bound not only to the sex pheromone components E4O2H but also to some host plant volatiles. These findings together with the evidence of insect PBPs available in the literature support the view that AlinOBP4 is involved in sex pheromone detection in male A. lineolatus and provide foundational information for further elucidating the molecular mechanisms of chemosensory based mating behavior in hemipteran mirid bugs.

Pheromone-binding proteins (PBPs) are considered to play critical roles in sex pheromone detection. Lepidopteran moths can be divided into two taxa, those that use Type-I sex pheromones, such as C10-C18 unsaturated aldehydes, alcohols and acetates, and those that use Type-II pheromones, which are C17-C23 polyunsaturated hydrocarbons and their epoxide derivatives. To date, nearly all the characterized PBPs have been reported in moths with Type-I sex pheromones, and the physiological functions of PBPs in moths that use Type-II sex pheromones remains unclear. In the present study we functionally examine EoblPBP1 in Ectropis obliqua Prout, an important geometrid moth pest that uses Type-II sex pheromones. The phylogenetic analysis of the sequence indicated that EoblPBP1 clustered together with ScerPBP1, a geometrid PBP for detecting Type-II sex pheromones. Scanning electron microscopy showed that E. obliqua moths of both sexes mainly had six types of antennal sensilla, including two types of sensilla trichodea, Str-I and Str-II, sensilla basiconica (Sba), sensilla styloconica (Sst), sensilla chaetica (Sch) and sensilla auricillica (Sau). Of these, Str-I was confirmed to be male moth-specific and had five different subtypes. Fluorescence in situ hybridization revealed that EoblPBP1 was primarily expressed at the base of Str-I. A comparative binding assay showed that recombinant EoblPBP1 bound three sex pheromone components of E. obliqua, demonstrating its involvement in the detection of Type-II sex pheromones. Besides, EoblPBP1 also highly bound unsaturated acetates pheromones and the green leaf volatiles. These results indicate that PBP1 is associated with detecting Type-II sex pheromones in E. obliqua but cannot differentiate Type-II sex pheromones from Type-I sex pheromones or green leaf volatiles. Our findings provide a foundation for further study on molecular basis of Type-II sex pheromone recognition in lepidopteran moths.

The phytophagous mirid bugs of Apolygus lucorum, Lygus pratensis as well as three Adelphocoris spp., including Adelphocoris lineolatus, A. suturalis, and A. fasciaticollis are major pests of multiple agricultural crops in China, which have distinct geographical distribution and occurrence ranges. Like many insect species, these bugs heavily rely on olfactory cues to search preferred host plants, thereby investigation on functional co-evolution and divergence of olfactory genes seems to be necessary and is of great interest. In the odorant detection pathway, olfactory receptor co-receptor (Orco) plays critical role in the perception of odors. In this study, we identified the full-length cDNA sequences encoding three putative Orcos (AsutOrco, AfasOrco, and LpraOrco) in bug species of A. suturalis, A. fasciaticollis, and L. pratensis based on homology cloning method. Next, sequence alignment, membrane topology and gene structure analysis showed that these three Orco orthologs together with previously reported AlinOrco and AlucOrco shared high amino acid identities and similar topology structure, but had different gene structure especially at the length and insertion sites of introns. Furthermore, the evolutional estimation on the ratios of non-synonymous to synonymous (Ka/Ks) revealed that Orco genes were under strong purifying selection, but the degrees of variation were significant different between genera. The results of quantitative real-time PCR experiments showed that these five Orco genes had a similar antennae-biased tissue expression pattern. Taking these data together, it is thought that Orco genes in the mirid species could share conserved olfaction roles but had different evolution rates. These findings would lay a foundation to further investigate the molecular mechanisms of evolutionary interactions between mirid bugs and their host plants, which might in turn contribute to the development of pest management strategy for mirid bugs.

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 β-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 chemosensory system is essential for insects to detect exogenous compounds, and odorant binding proteins (OBPs) play crucial roles in odorant binding and transduction. In the alfalfa plant bug Adelphocoris lineolatus, an important pest of multiple crops, our understanding of the physiological roles of antenna-biased OBPs has increased dramatically, whereas OBPs related to gustation have remained mostly unexplored. In this study, we employed RNA sequencing and RACE PCR methods to identify putative OBPs from the adult forelegs of both sexes. Eight candidate OBPs were identified, and three OBPs (AlinOBP15, 16, and 17) were novel. Full-length sequence alignment and phylogenetic analyses suggested that these three candidate OBPs had characteristics typical of the insect OBP family. AlinOBP16 and 17 displayed six highly conserved cysteines, placing them in the classic OBP subfamily, whereas AlinOBP15 resembled AlinOBP14 and clustered with the Plus-C clade. Quantitative real-time PCR (qRT-PCR) revealed distinct and significant tissue- and sex-biased expression patterns. AlinOBP15 was highly expressed in female heads, and AlinOBP16 and 17 were strongly expressed in female antennae. In particular, AlinOBP11, the most abundant OBP gene in our foreleg transcriptome dataset, was predominately expressed in adult legs. Furthermore, four types of sensilla hairs were observed on the forelegs of adult A. lineolatus, including sensilla trichodea, setae, and two types of sensilla chaetica (Sch1 and Sch2). Anti-AlinOBP11 antiserum strongly labeled the outer sensillum lymph of Sch2, implying that it has important gustatory functions in A. lineolatus. Our current findings provide evidence that OBPs can be functionally expressed in the tarsal gustatory sensilla of hemipteran mirid species, broadening our understanding of OBP chemosensory function in insects and facilitating the discovery of new functional targets for the regulation of insect host-searching behaviors.

Olfactory receptors (OR) are believed to fulfil an indispensable role in insects host-seeking, mating and ovipositing. We obtained 4785 high-quality expressed sequencing tags (EST) from the antennal cDNA library of the parasitic wasp Microplitis mediator, a natural enemy of crop pests. After assembling, 786 contigs and 2130 singletons were generated. Using BLAST searches, a number of olfactory-related genes were identified, including ESTs encoding for 25 ORs. 14 full-length OR genes were cloned and their expression profiles in the wasp olfactory organs were quantified by real-time qRT-PCR. The results indicated a diverse distribution between the tissues and genders, yet the majority of OR genes are highly expressed in antennae. Three OR genes (Or2, Or12 and Or13) are highly expressed in female antennae, eight OR genes (ORco, Or3, Or4, Or5, Or7, Or8, Or9 and Or11) are highly expressed in male antennae. This is the first report on tissue-specific expression of wasp OR genes. Our study provides a foundational knowledge to explore and understand the molecular basis of odorant reception in this parasitic wasp and for the study of trophic interactions of natural enemy.

Insect odorant binding proteins (OBPs), one of the most important groups of odor carriers, are believed to play essential roles in chemoreception. In the present study, we focused on AfasOBP11 in Adelphocoris fasciaticollis. Expression profiles showed that AfasOBP11 was mainly expressed in the mouthparts of A. fasciaticollis. Additionally, two types of sensilla, sensilla trichodeum and sensilla basiconicum, were found on the mouthparts of bugs. Moreover, anti-AfasOBP11 antiserum strongly labeled the sensilla basiconica. In fluorescence binding assays, recombinant AfasOBP11 displayed much stronger binding abilities to non-volatile secondary metabolite compounds than to volatile odors, suggesting a role of AfasOBP11 in taste sensing. To further investigate the biological functions of AfasOBP11, the feeding behavior of wild-type, dsGFP-injected and dsAfasOBP11-injected bugs was evaluated by performing electrical penetration graph (EPG) tests. After RNA interference of target AfasOBP11, A. fasciaticollis bugs spent a longer time and pierced more frequently on the artificial diet containing 2.0% gossypol, indicating that RNAi treated bugs reduced sensitivity to gossypol. Our findings suggest that AfasOBP11 may play a vital role in chemoreception of A. fasciaticollis, especially in gustatory perception.

Highly developed chemoreception allows insects to detect foods, find mates, and escape natural enemies. We described the structures and distributions of antennal chemosensilla in Adelphocoris fasciaticollis Reuter by scanning electron microscopy and transmission electron microscopy. Seven major types of antennal sensilla were identified in adults of both sexes. Types 1 and 2 are sensilla chaetica and have thick cuticular walls with conspicuous grooves at their surfaces. Types 3 and 4 are multiporous sensilla trichodea with 1–3 dendrites located at the sensillum lymph, indicating a putative olfactory function. Types 5 and 6 are typical sensilla basiconica but share different characteristics in both external morphology and internal ultrastructure, and may be involved in the perception of host-associated odorants. The last sensilla were Bohm bristles. In addition to the morphological characterization, electrophysiological responses of antennal chemosensilla to 51 semiochemicals were investigated based on electroantennogram (EAG) recordings. Results revealed that different chemical stimuli elicited significantly different dose-dependent EAG responses, in which potential sex pheromone components and green leaf volatiles showed relatively higher EAG responses, but neither monoterpenes nor sesquiterpenes can elicit favorable EAG values. The results provided direct morphological and electrophysiological evidence that the adult antennae of A. fasciaticollis could function in searching for mates and host plants.

Aphids are major agricultural pests that cause significant yield losses in crop plants each year. (E)-β-farnesene (EβF) is the main or only component of an alarm pheromone involved in chemical communication within aphid species and particularly in the avoidance of predation. EβF also occurs in the essential oil of some plant species, and is catalyzed by EβF synthase. By using oligonucleotide primers designed from the known sequence of an EβF synthase gene from black peppermint (Mentha × piperita), two cDNA sequences, MaβFS1 and MaβFS2, were isolated from Asian peppermint (Mentha asiatica). Expression pattern analysis showed that the MaβFS1 gene exhibited higher expression in flowers than in roots, stems and leaves at the transcriptional level. Overexpression of MaβFS1 in tobacco plants resulted in emission of pure EβF ranging from 2.62 to 4.85 ng d− 1 g− 1 of fresh tissue. Tritrophic interactions involving peach aphids (Myzus persicae), and predatory lacewing (Chrysopa septempunctata) larvae demonstrated that transgenic tobacco expressing MaβFS1 had lower aphid infestation. This result suggested that the EβF synthase gene from Asian peppermint could be a good candidate for genetic engineering of agriculturally important crop plants.

Cry toxins produced by Bacillus thuringiensis (Bt) are effective biological insecticides against certain insect species. However, there are potential risks of the evolved resistance of insects to Cry toxin owing to decreased binding of toxins to target sites in the brush border membranes of the larva midgut. The Cry toxins with different binding sites in the larval midgut have been considered to be a good combination to deploy in delaying resistance evolution. Bioassay results demonstrated that the toxicity of different Cry toxins ranked differently for each species. The toxicity ranking was Cry1Ac>Cry1Ab>Cry2Ab for Helicoverpa armigera, Cry1B>Cry1C>Cry2Ab for Spodoptera exigua, and Cry2Ab>Cry1B> Cry1C for S. litura. Only Cry2Ab was toxic to Agrotis ipsilon. Binding experiments were performed with 125I-Cry1Ab, 125I-Cry1Ac, 125I-Cry1B, 125I-Cry1C, 125I-Cry2Ab and the brush border membranes vesicles (BBMV) from H. armigera, S. exigua, S. litura and A. ipsilon. The binding of Cry1Ab and Cry1Ac was shown to be saturable by incubating with increasing concentrations of H. armigera BBMV (Kd=(45.00±2.01) nmol L−1 and (12.80±0.18) nmol L−1, respectively; Bmax=(54.95±1.79) ng and (55.44±0.91) ng, separately). The binding of Cry1B was shown to be saturable by incubating with increasing concentrations of S. exigua BBMV (Kd=(23.26±1.66) nmol L−1; Bmax=(65.37±1.87) ng). The binding of 125I-Cry toxins was shown to be non-saturable by incubating with increasing concentrations of S. litura and A. ipsilon BBMV. In contrast, Cry1B and Cry1C showed some combination with the BBMV of S. litura, and a certain amount of Cry2Ab could bind to the BBMV of A. ipsilon. These observations suggest that a future strategy could be devised for the focused combination of specific cry genes in transgenic crops to control target pests, widen the spectrum of insecticide effectiveness and postpone insect resistance evolution.

Cry toxins produced by Bacillus thuringiensis (Bt) are effective biological insecticides against certain insect species. In this study, bioassay results indicated that Cry1B and Cry1C were toxic to Spodoptera exigua . We also identified a cadherin-like gene in S. exigua that could enhance the toxicity of Cry1B and Cry1C. The cadherin-like gene identified from the larvae midgut tissue was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA of the gene consisted of 5 220 bp encoding 1 740 amino acid with a predicted molecular mass of 196 kD. BLAST search analysis showed that the predicted amino acid sequence had a high sequence identity to the published sequences of cadherin-like proteins from other Lepidoptera insects. Spatial expression of the cadherin-like gene detected by qRT-PCR analysis revealed that the cadherin-like gene was mainly present in the gut of 4th instar larvae and during different life stages. The results suggested that the commercial development of this synergist has the potential to enhance Cry1B and Cry1C toxicity against Lepidoptera insects.

Odorant receptors are thought to play critical roles in the perception of chemosensory stimuli by insects. The primary method to address the functions of odorant receptors in insects is to use in vitro binding assays between the receptors and potential chemical stimuli. We injected MmedOrco dsRNA into the abdominal cavity of a braconid wasp, Microplitis mediator, and assayed for expression of this gene 72 h after treatment (RNAi). Quantitative real-time PCR demonstrated that the level of mRNA expression in MmedOrco dsRNA-treated M. mediator was significantly reduced (>90%) when compared with water-treated controls. Furthermore, electroantennogram (EAG) responses of M. mediator to two chemical attractants, nonanal and farnesene, were also reduced significantly (~70%) in RNAi-treated M. mediator when compared to controls. RNAi-treated M. mediator also responded by walking/flying at a lower rate to both chemicals when compared with controls in a Y-tube olfactometer bioassay, which provides direct evidence that MmedOrco plays an important role in perception of nonanal and farnesene in M. mediator.

The full-length sequence of the odorant binding protein 5 gene, HarmOBP5, was obtained from an antennae cDNA library of cotton bollworm, Helicoverpa armigera (Hubner). The cDNA contains a 444 bp open reading frame, encoding a protein with 147 amino acids, namely HarmOBP5. HarmOBP5 was expressed in Escherichia coli and the recombinant protein was purified by affinity chromatography. SDS-PAGE and Western blot analysis demonstrated that the purified protein can be used for further investigation of its binding characteristics. Competitive binding assays with 113 odorant chemicals indicated that HarmOBP5 has strong affinity to some special plant volatiles, including (E)-s-farnesene, ethyl butyrate, ethyl heptanoate, and acetic acid 2-methylbutyl ester. Based on three-dimensional (3D) model of AaegOBP1 from Aedes aegypti, a 3D model of HarmOBP5 was predicted. The model revealed that some key binding residues in HarmOBP5 may play important roles in odorant perception of H. armigera. This study provides clues for better understanding physiological functions of OBPs in H. armigera and other insects.

Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines.

Microplitis mediator Haliday (Hymenoptera: Braconidae) is an important larval endoparasitoid of various lepidopteran pests, including Helicoverpa armigera (Hübner). In China, H. armigera is a key pest of cotton and is currently the focus of several biological control efforts that use M. mediator as principal natural enemy of this pest. To improve the success of biological control efforts, behavioral studies are needed that shed light on the interaction between M. mediator and H. armigera. In this study, we determined M. mediator response to volatile compounds from undamaged, mechanically injured, or H. armigera--damaged plants and identified attractive volatiles. In Y-tube olfactometer assays, we found that mechanically damaged plants and/or plants treated with H. armigera oral secretions did not attract wasps. However, volatiles from H. armigera-damaged plants elicited a strong attraction of both M. mediator sexes. Headspace extracts from H. armigera-damaged cotton were analyzed by coupled gas chromatography-electroantennographic detection (GC-EAD), and a total of seven different compounds were found to elicit electroantennogram (EAG) responses, including an unknown compound. Six different EAD-active volatiles were identified from caterpillar-damaged cotton plants, of which 3, 7-dimethyl-1, 3, 6-octatriene and (Z)-3-hexenyl acetate were the principal compounds. Olfactometer assays indicated that individual synthetic compounds of 3, 7-dimethyl-1, 3, 6-octatriene, (Z)-3-hexenyl acetate, and nonanal were attractive to M. mediator. Field cage studies showed that parasitism of H. armigera larvae by M. mediator was higher on cotton plants to which 3,7-dimethyl-1,3, 6-octatriene was applied. Our results show that the combination of terpenoids and green leaf volatiles may not only facilitate host, mate, or food location but may also increase H. armigera parasitism by M. mediator.

To clarify the environmental fate of the Cry1Ac protein from Bacillus thuringiensis subsp. kurstaki (Bt) contained in transgenic rice plant stubble after harvest, degradation was monitored under field conditions using an enzyme-linked immunosorbent assay. In stalks, Cry1Ac protein concentration decreased rapidly to 50% of the initial amount during the first month after harvest; subsequently, the degradation decreased gradually reaching 21.3% when the experiment was terminated after 7 mo. A similar degradation pattern of the Cry1Ac protein was observed in rice roots. However, when the temperature increased in April of the following spring, protein degradation resumed, and no protein could be detected by the end of the experiment. In addition, a laboratory experiment was conducted to study the persistence of Cry1Ac protein released from rice tissue in water and paddy soil. The protein released from leaves degraded rapidly in paddy soil under flooded conditions during the first 20 d and plateaued until the termination of this trial at 135 d, when 15.3% of the initial amount was still detectable. In water, the Cry1Ac protein degraded more slowly than in soil but never entered a relatively stable phase as in soil. The degradation rate of Cry1Ac protein was significantly faster in nonsterile water than in sterile water. These results indicate that the soil environment can increase the degradation of Bt protein contained in plant residues. Therefore, plowing a field immediately after harvest could be an effective method for decreasing the persistence of Bt protein in transgenic rice fields.

Apolygus lucorum (Hemiptera: Miridae) is an important insect pest of cotton and fruit trees in China. The adults prefer host plants at the flowering stage and their populations track flowering plants both spatially and temporally. In this study, we examine whether flower preference of its adults is mediated by plant volatiles and which volatile compositions play an important role in attracting them. In olfactometer tests with 18 key host species, the adults preferred flowering plants over non-flowering plants of each species. Coupled gas chromatography-electroantennography revealed the presence of seven electrophysiologically active compounds from flowering plants. Although the adults responded to all seven synthetic plant volatiles in electroantennography tests, only four (m-xylene, butyl acrylate, butyl propionate and butyl butyrate) elicited positive behavioral responses in Y-tube olfactometer bioassays. The adults were strongly attracted to these four active volatiles in multi-year laboratory and field trials. Our results suggest that these four fragrant volatiles, which are emitted in greater amounts once plants begin to flower, mediate A. lucorum’s preference to flowering host plants. We proved that the use of commonly occurring plant volatiles to recognize a large range of plant species can facilitate host selection and preference of polyphagous insect herbivore.

We determined whether plant volatiles help explain why Cotesia chilonis (a parasitoid of the target pest Chilo suppressalis) is less abundant in Bt than in non-Bt rice fields. Olfactometer studies revealed that C. chilonis females responded similarly to undamaged Bt and non-Bt rice plants. Parasitoids preferred rice plants damaged by 3rd-instar larvae of C. suppressalis, but did not differentiate between caterpillar-infested Bt and non-Bt plants. According to GC-MS analyses of rice plant volatiles, undamaged Bt and non-Bt rice plants emitted the same number of volatile compounds and there were no significant differences in the quantity of each volatile compound between the treatments. When plants were infested with and damaged by C. suppressalis larvae, both Bt and non-Bt rice plants emitted higher numbers and larger amounts of volatile compounds than undamaged plants, but there were no significant differences between Bt and non-Bt plants. These results demonstrate that the volatile-mediated interactions of rice plants with the parasitoid C. chilonis were not disrupted by the genetic engineering of the plants. We infer that parasitoid numbers are lower in Bt than in non-Bt fields because damage and volatile induction by C. suppressalis larvae are greatly reduced in Bt fields.

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.

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.

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.

Insects rely heavily on their sophisticated chemosensory systems to locate host plants and find conspecific mates. Although the molecular mechanisms of odorant recognition in many Lepidoptera species have been well explored, limited information has been reported on the geometrid moth Ectropis obliqua Prout, an economically important pest of tea plants. In the current study, we first attempted to identify and characterize the putative olfactory carrier proteins, including odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). By analyzing previously obtained transcriptomic data of third-instar larvae, five OBPs and 14 CSPs in E. obliqua were identified. Sequence alignment, conserved motif identification, and phylogenetic analysis suggested that candidate proteins have typical characteristics of the insect OBP or CSP family. The expression patterns regarding life stages and different tissues were determined by quantitative real-time PCR. The results revealed that four transcripts (OBP2, OBP4 and CSP8, CSP10) had larvae preferential expression profiles and nine candidate genes (PBP1, OBP1 and CSP2, CSP4, CSP5, CSP6, CSP7, CSP11, and CSP13) were adult-biased expressed. Further specific tissue expression profile evaluation showed that OBP1, OBP2, OBP4, and PBP1 were highly expressed at olfactory organs, implying their potential involvement in chemical cue detection, whereas CSPs were ubiquitously detected among all of the tested tissues and could be associated with multiple physiological functions. This study provided a foundation for understanding the physiological functions of OBPs and CSPs in E. obliqua and will help pave the way for the development of a new environmental friendly pest management strategy against the tea geometrid moth.

Insect chemosensory proteins (CSPs) are a family of small soluble proteins. To date, their physiological functions in insect olfaction remain largely controversial in comparison to odorant binding proteins (OBPs). In present study, we reported the antenna specific expression of three CSPs (AlinCSP4-6) from Adelphocoris lineolatus, their distinct chemosensillum distribution as well as ligand binding capability thus providing the evidence for the possible roles that they could play in semiochemical detection of the plant bug A. lineolatus. The results of qRT-PCR and western blot assay clearly showed that all of these three CSPs are highly expressed in the adult antennae, the olfactory organ of insects. Further cellular investigation of their immunolocalization revealed their dynamic protein expression profiles among different types of antennal sensilla. In a fluorescence competitive binding assay, the selective ligand binding was observed for AlinCSP4-6. In ad`dition, a cooperative interaction was observed between two co-expressed CSPs resulting in an increase of the binding affinities by a mixture of AlinCSP5 and AlinCSP6 to terpenoids which do not bind to individual CSPs. These findings in combination with our previous data for AlinCSP1-3 indicate a possible functional differentiation of CSPs in the A. lineolatus olfactory system.

To survive, the entomopathogenic fungus Beauveria bassiana, which shows promise as a biocontrol agent for a variety of pests, including agricultural and forestry pests and vectors of human pathogens, must tailor gene expression to the particular pH of its environment. The pH response transcription factor gene BbPacC and its flanking sequence were cloned from this fungus. Quantitative reverse transcription (RT)-PCR revealed that it is highly induced by alkaline pH and salt stress, and the expression level achieved twice that of the housekeeping gene γ-actin. A microfluorometric assay indicated that the 1479 bp promoter region could activate the expression of enhanced green fluorescent protein (EGFP) under the same conditions. Truncation analysis showed that the 1479, 1274, 1040, 888 and 742 bp promoters have similar efficiencies in activating expression of β-glucuronidase (GUS). The GUS activities of corresponding transformants reached approximately 50 % that of those containing the strong constitutive promoter PtrpC. A truncation upstream at the –572 bp position (referenced to the translation start codon ATG), however, resulted in a significant loss of GUS activity. Both the upstream absences of the −502 and −387 bp positions caused almost complete loss of GUS activity. These results suggest that PPacC is an efficient, alkaline, and salt-inducible promoter, the core cis-elements are mainly located within the –742 to –502 bp region, and promoters equal to or longer than 742 bp may be feasible for regulating gene expression in response to an ambient pH or salt stress.

Semiochemicals such as sex pheromones and plant volatiles are crucial components of insect mating systems and host plant localization. In the olfactory signal transduction pathway, odorant-binding proteins (OBPs) are important elements that function in the first step of the pathway by carrying hydrophobic semiochemicals across the sensillum lymph to the olfactory receptors (ORs). In this study, we examined the binding affinities of semiochemicals to AlinOBP10, a putative OBP from the alfalfa plant bug, Adelphocoris lineolatus, that we demonstrate is expressed mainly in sensory organs. We then characterized the biological activities of the high affinity semiochemicals by measuring their electrophysiological activities in antennae and behavioral responses in the plant bug. AlinOBP10 displayed weak binding affinities to two major putative pheromone components, hexyl butyrate and (E)-2-hexenyl butyrate. In contrast, AlinOBP10 exhibited higher binding affinities to six host plant volatiles, namely myrcene, β-pinene, β-ionone, 3-hexanone, (E)-2-hexenal, and 1-hexanol. The biological activities of these six putative ligands were further studied in electroantennogram recordings and Y-tube olfactometer trials. The three compounds, (E)-2-hexenal, 1-hexanol, and 3-hexanone elicited strong electrophysiological responses, but elicited distinct behaviors. While 3-hexanone was attractive to female adults, (E)-2-hexenal and 1-hexanol were significant repellents. Although a weak electrophysiological response was elicited with β-pinene, it was a strong repellent. These results demonstrate that AlinOBP10 can interact with attractants, as well as repellents, with some specificity toward plant volatiles over sex pheromones.

Host selection by female moths is fundamental to the survival of their larvae. Detecting and perceiving the non-volatile chemicals of the plant surface involved in gustatory detection determine the host preference. In many lepidopteran species, tarsal chemosensilla are sensitive to non-volatile chemicals and responsible for taste detection. The tea geometrid Ectropis obliqua is one devastating chewing pest selectively feeding on limited plants, requiring the specialized sensors to forage certain host for oviposition. In present study, we revealed the distribution of chemosensilla in the ventral side of female fifth tarsomere in E. obliqua. To investigate its molecular mechanism of gustatory perception, we performed HiSeq 2500 sequencing of the male- and female- legs transcriptome and identified 24 candidate odorant binding proteins (OBPs), 21 chemosensory proteins (CSPs), 2 sensory neuron membrane proteins (SNMPs), 3 gustatory receptors (GRs) and 4 odorant receptors (ORs). Several leg-specific or enriched chemosensory genes were screened by tissue expression analysis, and clustered with functionally validated genes from other moths, suggesting the potential involvement in taste sensation or other physiological processes. The RPKM value analysis revealed that 9 EoblOBPs showed sex discrepancy in the leg expression, 8 being up-regulated in female and only 1 being over expressed in male. These female-biased EoblOBPs indicated an ecological adaption related with host-seeking and oviposition behaviors. Our work will provide basic knowledge for further studies on the molecular mechanism of gustatory perception, and enlighten a host-selection-based control strategy of insect pests.

The insect sensory neuron membrane proteins (SNMPs) SNMP1 and SNMP2 are transmembrane domain-containing proteins and are homologs of the vertebrate CD36 transmembrane proteins. It has been suggested that SNMPs play a significant role in insect chemoreception. Previous studies have demonstrated that SNMP1 is expressed in the pheromone-sensitive olfactory receptor neurons (ORNs), whereas SNMP2 is expressed in the supporting cells. In this study, we identified two full-length SNMP transcripts, AipsSNMP1 and AipsSNMP2, in the black cutworm moth Agrotis ipsilon (Hufnagel). The qRT-PCR results indicated that the AipsSNMP1 and AipsSNMP2 transcripts were expressed significantly higher in the antennae than in other tissues of both sexes. The expression of AipsSNMP1 and AipsSNMP2 in the antennae from different development stages of both sexes was investigated and was shown to begin to express in the pupae stage from 3 days before emergence and then increased dramatically at the day of the emergence, and the high expression levels were maintained during the following 4 days after the emergence in both sexes. The mating status had no effect on the expression levels of the AipsSNMP1 and AipsSNMP2 transcripts. Consistent with previous in situ hybridization studies in other Lepidoptera insects, our immunolocalization results at protein level demonstrated that both AipsSNMP1 and AipsSNMP2 were expressed in pheromone-sensitive sensilla trichodea but with a completely different expression profile. AipsSNMP1 is more uniformed and highly expressed along the membrane of the ORN dendrites, whereas AipsSNMP2 is widely distributed at the bottom of the sensilla trichodea and highly localized in the sensillum lymph. Our studies provide further detailed evidence for the involvement and general functional role of insect SNMPs in the detection of sex pheromones and general odorant molecules.

Insect chemosensory proteins (CSPs) have been proposed to capture and transport hydrophobic chemicals from air to olfactory receptors in the lymph of antennal chemosensilla. They may represent a new class of soluble carrier protein involved in insect chemoreception. However, their specific functional roles in insect chemoreception have not been fully elucidated. In this study, we report for the first time three novel CSP genes (AlinCSP1-3) of the alfalfa plant bug Adelphocoris lineolatus (Goeze) by screening the antennal cDNA library. The qRT-PCR examinations of the transcript levels revealed that all three genes (AlinCSP1-3) are mainly expressed in the antennae. Interestingly, these CSP genes AlinCSP1-3 are also highly expressed in the 5(th) instar nymphs, suggesting a proposed function of these CSP proteins (AlinCSP1-3) in the olfactory reception and in maintaining particular life activities into the adult stage. Using bacterial expression system, the three CSP proteins were expressed and purified. For the first time we characterized the types of sensilla in the antennae of the plant bug using scanning electron microscopy (SEM). Immunocytochemistry analysis indicated that the CSP proteins were expressed in the pheromone-sensitive sensilla trichodea and general odorant-sensitive sensilla basiconica, providing further evidence of their involvement in chemoreception. The antennal activity of 55 host-related semiochemicals and sex pheromone compounds in the host location and mate selection behavior of A. lineolatus was investigated using electroantennogram (EAG), and the binding affinities of these chemicals to the three CSPs (AlinCSP1-3) were measured using fluorescent binding assays. The results showed several host-related semiochemicals, (Z)-3-hexen-1-ol, (E)-2-hexen-1-al and valeraldehyde, have a high binding affinity with AlinCSP1-3 and can elicit significant high EAG responses of A. lineolatus antennae. Our studies indicate the three antennae-biased CSPs may mediate host recognition in the alfalfa plant bug A. lineolatus.

In the insect phylum, the relationships between individuals and their environment are often modulated by chemical communication. Odorant binding proteins (OBPs) are widely and robustly expressed in insect olfactory organs and play a key role in chemosensing and transporting hydrophobic odorants across the sensillum lymph to the olfactory receptor neuron. In this study, a novel OBP gene (AlinOBP1) in the lucerne plant bug, Adelphocoris lineolatus was identified, cloned and expressed. Real-time PCR results indicated that the expression level of AlinOBP1 gene differed in each developmental stage (from first instar to adult) and was predominantly expressed in the antennae of adults. The expression level of AlinOBP1 was 1.91 times higher in male antennae than in female antennae. The binding properties of AlinOBP1 with 114 odorants were measured using a fluorescence probe, N-phenyl-1-naphthylamine (1-NPN), with fluorescence competitive binding. The results revealed that AlinOBP1 exhibits high binding abilities with two major putative pheromone components, ethyl butyrate and trans-2-hexenyl butyrate. In addition, it was observed that six volatiles released from cotton, octanal, nonanal, decanal, 2-ethyl-1-hexanol, beta-caryophyllene and beta-ionone also bind to AlinOBP1. Immunocytochemistry analysis showed that AlinOBP1 was expressed in the sensillum lymph of sensilla trichodica and sensilla basiconca. Our results demonstrate that AlinOBP1 may function as a carrier in the chemoperception of the lucerne plant bug. (C) 2011 Wiley Periodicals, Inc.

The rice stem borer, Chilo suppressalis Walker is one of the most important insect pests on rice in Asia, north Africa and southern Europe. Transgenic Bt rice has been developed in the laboratory with good resistance to this pest and other Lepidopteran insects, which will provide a possible alternative tool for this pest control. The full-length cDNAs encoding an aminopeptidase N (CsAPN) and a cadherin (CsCad) were cloned from C. suppressalis. CsAPN showed common features of, and high identities to, other insect APNs in its deduced amino acid sequence. Although a full-length cDNA encoding cadherin-like protein has been reported in GenBank, the newly isolated cadherin here (CsCad) showed some differences in its amino acid sequence, especially at the 7th cadherin repeat region (CR7), which indicated the newly isolated CsCad might be another allele. CsAPN and CsCad were successfully expressed in insect Tn cells, and the blot analysis showed these two proteins could bind Bt toxin Cry1Ab. The results will provide valuable information for the studies of toxin mode of action and the possible toxin resistance mechanisms in this pest.

Polygalacturonase (PG) is an enzyme in the salivary glands of piercing-sucking mirid bugs (Hemiptera: Miridae) that plays a key role in plant feeding and injury. By constructing a full-length cDNA library, we cloned and characterized 14 PG genes from the salivary glands of Apolygus lucorum, a pestiferous mirid bug in cotton, fruit trees and other crops in China. BLAST search analysis showed that the amino acid sequences deduced from transcripts of the PG genes were closely related to PGs from other mirid bugs. Phylogenetic analysis showed that the PGs of mirid bugs had six main branches, PG1-PG6 (Genbank accession numbers: KF881899~KF881912). We investigated the mRNA expression patterns of the A. lucorum PG genes using real-time PCR. All 14 PGs were expressed significantly higher in the salivary glands than in other tissues (head, thorax, abdomen, leg and wing). For eggs and nymphs, the expression levels of these PGs were much higher in the 5th instar stage than in the egg, and 1st and 3rd instar stages. The PG expression levels in 1-day-old adults were very low, and increased in 5, 20 and 30-day-old adults. Additionally, PG expression levels were generally similar between males and females. The possible physiological functions of PGs in A. lucorum were discussed.

Antennal olfaction is extremely important for insect survival, mediating key behaviors such as host preference, mate choice, and oviposition site selection. Multiple antennal proteins are involved in olfactory signal transduction pathways. Of these, odorant receptors (ORs) and ionotropic receptors (IRs) confer specificity on olfactory sensory neuron responses. In this study, we identified the olfactory gene repertoire of the economically important agricultural pest moth, Helicoverpa armigera, by assembling the adult male and female antennal transcriptomes. Within the male and female antennal transcriptomes we identified a total of 47 OR candidate genes containing 6 pheromone receptor candidates. Additionally, 12 IR genes as well as 26 odorant-binding proteins and 12 chemosensory proteins were annotated. Our results allow a systematic functional analysis across much of conventional ORs repertoire and newly reported IRs mediating the key olfaction-mediated behaviors of H. armigera.

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.

In response to insect herbivory, plants emit elevated levels of volatile organic compounds for direct and indirect resistance. However, little is known about the molecular and genomic basis of defense response that insect herbivory trigger in cotton plants and how defense mechanisms are orchestrated in the context of other biological processes. Here we monitored the transcriptome changes and volatile characteristics of cotton plants in response to cotton bollworm (CBW; Helicoverpa armigera) larvae infestation. Analysis of samples revealed that 1,969 transcripts were differentially expressed (log2|Ratio| ≥ 2; q ≤ 0.05) after CBW infestation. Cluster analysis identified several distinct temporal patterns of transcriptome changes. Among CBW-induced genes, those associated with indirect defense and jasmonic acid pathway were clearly over-represented, indicating that these genes play important roles in CBW-induced defenses. The gas chromatography-mass spectrometry (GC-MS) analyses revealed that CBW infestation could induce cotton plants to release volatile compounds comprised lipoxygenase-derived green leaf volatiles and a number of terpenoid volatiles. Responding to CBW larvae infestation, cotton plants undergo drastic reprogramming of the transcriptome and the volatile profile. The present results increase our knowledge about insect herbivory-induced metabolic and biochemical processes in plants, which may help improve future studies on genes governing processes.