n-octyl acrylate is a candidate sex pheromone component involved in courtship in parasitoid wasp Microplitis mediator.

Sex pheromones are considered to play critical roles in partner communication of most parasitic Hymenoptera. However, the identification of sex pheromone components remains limited to a few families of parasitoid wasps. In this study, we functionally characterized a candidate sex pheromone component in Microplitis mediator (Hymenoptera: Braconidae), a solitary parasitoid of Noctuidae insects. We found that the body surface extract from female wasps could significantly stimulate courtship behavior of males. Gas chromatography-electroantennographic detection (GC-EAD) analysis revealed that a candidate semiochemical from extract triggered significant electrophysiological response of antennae of males. By performing gas chromatography-mass spectrometer (GC-MS) measurement, GC-EAD active compound was identified as n-octyl acrylate, a candidate sex pheromone component in female M. mediator. In electroantennogram (EAG) tests, antennae of male wasps showed significantly higher electrophysiological responses to n-octyl acrylate than those of females. Y-tube olfactometer assays indicated that male wasps significantly chose n-octyl acrylate compared with the control. Furthermore, male wasps showed a remarkable preference for n-octyl acrylate in a simulated field condition behavioral trial; simultaneously, n-octyl acrylate standard could also trigger significant courtship behavior in males. We propose that n-octyl acrylate, as a candidate vital sex pheromone component, could be utilized to design behavioral regulators of M. mediator to implement the protection and utilization of natural enemies.

A female-biased odorant receptor tuned to the lepidopteran sex pheromone in parasitoid Microplitis mediator guiding habitat of host insects.

INTRODUCTION: The parasitoid wasp Microplitis mediator is an important natural enemy of the turnip moth Agrotis segetum and other Noctuidae pests. In our field observation, it was fortuitously discovered that sex pheromone traps used for A. segetum also attract female wasps, verified by a simulated field condition dual-choice laboratory assay. Therefore, it was hypothesized that olfactory recognition could be crucial in this process. In this regard, a female-biased odorant receptor of the wasp, MmedOR49, attracted our attention. OBJECTIVES: To unravel the significance of the female-biased MmedOR49 regulating host pheromone recognition. METHODS: Expression analysis (fluorescence in situ hybridization; quantitative realtime PCR), in vitro (two-electrode voltage-clamp recordings) and in vivo (RNAi combined with behavioral assessments) functional studies, and bioinformatics (structural modeling and molecular docking) were carried out to investigate the characteristics of MmedOR49. RESULTS: MmedOR49 expression was detected in the antennae of females by FISH. Quantification indicated that the expression level of MmedOR49 increased significantly after adult emergence. In vitro functional study revealed that MmedOR49 was specifically tuned to cis-5-decenyl acetate (Z5-10:Ac), the major sex pheromone component of A. segetum. Molecular docking showed that Z5-10:Ac strongly bound to the key amino acid residues His 80, Ile 81, and Arg 84 of MmedOR49 through hydrogen bonding. Behavioral assays indicated that female wasps were significantly attracted by Z5-10:Ac in a three-cage olfactometer. RNAi targeting further confirmed that MmedOR49 was necessary to recognize Z5-10:Ac, as female wasps lost their original behavioral responses to Z5-10:Ac after down-regulation of the MmedOR49 transcript. CONCLUSION: Although M. mediator is a larval endoparasitoid, female wasps have a behavioral preference for a sex pheromone component of lepidopteran hosts. In this behavior, for female M. mediator, MmedOR49 plays an important role in guiding the habitat of host insects. These data provide a potential target for enhancing natural enemy utilization and pest control.

Characterization and target gene analysis of microRNAs in the antennae of the parasitoid wasp Microplitis mediator.

MicroRNAs (miRNAs), a class of non-coding single-strand RNA molecules encoded by endogenous genes, are about 21-24 nucleotides long and are involved in the post-transcriptional regulation of gene expression in plants and animals. Generally, the types and quantities of miRNAs in the different tissues of an organism are diverse, and these divergences may be related to their specific functions. Here we have identified 296 known miRNAs and 46 novel miRNAs in the antennae of the parasitoid wasp Microplitis mediator by high-throughput sequencing. Thirty-three miRNAs were predicted to target olfactory-associated genes, including odorant binding proteins (OBPs), chemosensory proteins, odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors. Among these, 17 miRNAs were significantly highly expressed in the antennae, four miRNAs were highly expressed both in the antennae and head or wings, while the remaining 12 miRNAs were mainly expressed in the head, thorax, abdomen, legs and wings. Notably, miR-9a-5p and miR-2525-3p were highly expressed in male antennae, whereas miR-1000-5p and novel-miR-13 were enriched in female antennae. The 17 miRNAs highly expressed in antennae are likely to be associated with olfaction, and were predicted to target one OBP (targeted by miR-3751-3p), one IR (targeted by miR-7-5p) and 14 ORs (targeted by 15 miRNAs including miR-6-3p, miR-9a-5p, miR-9b-5p, miR-29-5p, miR-71-5p, miR-275-3p, miR-1000-5p, miR-1000-3p, miR-2525-3p, miR-6012-3p, miR-9719-3p, novel-miR-10, novel-miR-13, novel-miR-14 and novel-miR-28). These candidate olfactory-associated miRNAs are all likely to be involved in chemoreception through the regulation of chemosensory gene expression in the antennae of M. mediator.

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.

Molecular characterization and expression of sensory neuron membrane proteins in the parasitoid Microplitis mediator (Hymenoptera: Braconidae).

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.

Antennal ionotropic receptors IR64a1 and IR64a2 of the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidate) collaboratively perceive habitat and host cues.

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 Z9-14: 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.

Characterization of antennal chemosensilla and associated odorant binding as well as chemosensory proteins in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae).

Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) expressed in antennal chemosensilla are believed to be important in insect chemoreception. In the current study, we fully described the morphological characteristics of the antennal sensilla in parasitoid wasp Microplitis mediator and analyzed the expression patterns of OBPs and CSPs within the antennae. In M. mediator, eight types of sensilla were observed on the antennae. Sensilla basiconica type 2 and s. placodea with wall pores may be involved in olfactory perception, whereas s. basiconica type 1 and type 3 with tip pores may play gustatory functions. Among the 18 OBPs and 3 CSPs in M. mediator, 10 OBPs and 2 CSPs were exclusively or primarily expressed in the antennae. In situ hybridization experiments indicated that the 12 antennae-enriched OBPs and CSPs were mapped to five morphological classes of antennal sensilla, including s. basiconica (type 1-3), s. placodea and s. coeloconica. Within the antennae, most of OBP and CSP genes were expressed only in one type of sensilla indicating their differentiated roles in detection of special type of chemical molecules. Our data will lay a foundation to further study the physiological roles of OBPs and CSPs in antennae of parasitoid wasps.

Functional characterization of a Niemann-Pick type C2 protein in the parasitoid wasp Microplitis mediator.

Niemann-Pick type C2 (NPC2) is a type of small soluble protein involved in lipid metabolism and triglyceride accumulation in vertebrates and arthropods. Recent studies have determined that NPC2 also participates in chemical communication of arthropods. In this work, two novel NPC2 proteins (MmedNPC2a and MmedNPC2b) in Microplitis mediator were identified. Real-time quantitative PCR (qPCR) analysis revealed that MmedNPC2a was expressed higher in the antennae than in other tissues of adult wasps compared with MmedNPC2b. Subsequent immunolocalization results demonstrated that NPC2a was located in the lymph cavities of sensilla placodea. To further explore the binding characterization of recombinant MmedNPC2a to 54 candidate odor molecules, a fluorescence binding assay was performed. It was found MmedNPC2a could not bind with selected fatty acids, such as linoleic acid, palmitic acid, stearic acid and octadecenoic acid. However, seven cotton volatiles, 4-ethylbenzaldehyde, 3,4-dimethylbenzaldehyde, ß-ionone, linalool, m-xylene, benzaldehyde and trans-2-hexen-1-al showed certain binding abilities with MmedNPC2a. Moreover, the predicted 3D model of MmedNPC2a was composed of seven ß-sheets and three pairs of disulfide bridges. In this model, the key binding residues for oleic acid in CjapNPC2 of Camponotus japonicus, Lue68, Lys69, Lys70, Phe97, Thr103 and Phe127, are replaced with Phe85, Ser86, His87, Leu113, Tyr119 and Ile143 in MmedNPC2a, respectively. We proposed that MmedNPC2a in M. mediator may play roles in perception of plant volatiles.

Identification of odorant binding proteins and chemosensory proteins in Microplitis mediator as well as functional characterization of chemosensory protein 3.

Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play important roles in transporting semiochemicals through the sensillar lymph to olfactory receptors in insect antennae. In the present study, twenty OBPs and three CSPs were identified from the antennal transcriptome of Microplitis mediator. Ten OBPs (MmedOBP11-20) and two CSPs (MmedCSP2-3) were newly identified. The expression patterns of these new genes in olfactory and non-olfactory tissues were investigated by real-time quantitative PCR (qPCR) measurement. The results indicated that MmedOBP14, MmedOBP18, MmedCSP2 and MmedCSP3 were primarily expressed in antennae suggesting potential olfactory roles in M. mediator. However, other genes including MmedOBP11-13, 15-17, 19-20 appeared to be expressed at higher levels in body parts than in antennae. Focusing on the functional characterization of MmedCSP3, immunocytochemistry and fluorescent competitive binding assays were conducted indoors. It was found that MmedCSP3 was specifically located in the sensillum lymph of olfactory sensilla basiconca type 2. The recombinant MmedCSP3 could bind several types of host insects odors and plant volatiles. Interestingly, three sex pheromone components of Noctuidae insects, cis-11-hexadecenyl aldehyde (Z11-16: Ald), cis-11-hexadecanol (Z11-16: OH), and trans-11-tetradecenyl acetate (E11-14: Ac), showed high binding affinities (Ki = 17.24-18.77 µM). The MmedCSP3 may be involved in locating host insects. Our data provide a base for further investigating the physiological roles of OBPs and CSPs in M. mediator, and extend the function of MmedCSP3 in chemoreception of M. mediator.

Cloning and expression profile of ionotropic receptors in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae).

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.

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.

Molecular characterization and expression profiles of olfactory receptor genes in the parasitic wasp, Microplitis mediator (Hymenoptera: Braconidae).

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.