| The parasitoid wasp Microplitis mediator(Haliday)(Hymenoptera: Braconidae)is a generalist endoparasitoid that is reported to attack approximately 40 different lepidopteran larvae,including Helicoverpa armigera(Hübner),Mythimna separata(Walker)and several other moth species.M.mediator serves as important natural agent,effectively control major crop pests in the agricultural ecosystem.In China,it has been successfully mass-reared in the laboratory and used as biological control agent.The success of parasitic wasps in suppressing pest populations depends on their ability to locate hosts in the complex chemical environment.Like most of the insects,parasitic wasps can also find their hosts in the foraging environments and reproduction occurs through a series of behavioral activities,regulated mainly by semiochemicals.To study the chemsensory mechanism of M.mediator,which can help us through the regulation of the chemsensory behavior of parasitoids to improve the parasitic efficiency of M.mediator,and making M.mediator play a greater role in biological control of crop pests.In present study,we compare the structure characteristic of antennal chemsensilla in both sexes of M.mediator.By transcriptome sequencing of male and female antennae,we identified amount of chemosensory receptor genes involved in the chemosensery of M.mediator.We further analyzed the expression profile of the olfactory receptors(IRs and ORs)and odorant binding protein(OBPs)in the antenna of M.mediator.The main results are as follows:The antennae of both sexes of M.mediator consist of scape,pedicel and flagellum.The flagellum is composed of 16 antennomeres and exhibited strong sexual dimorphism in the length of each antennomere,which were longer in males than in females.Scanning electron microscopy studies reveal that eight different types of sensilla were identified on the antennae: s.trichodea,s.chaetica,s.placodea,s.basiconica type Ⅰ,s.basiconica type Ⅱ,s.basiconica type Ⅲ,s.coeloconica and s.campaniform.S.placodea and s.basiconica type Ⅱ with multiple wall pores and numerous dendrites to be olfactory sensilla.S.basiconica type Ⅰ and type Ⅲ with terminal pore to be gustatory sensilla.The s.basiconica type Ⅲ described in our study are absent in male antennae of M.mediator.Using Life Technologies Ion Proton ? seqensing platform and bioinformatics analysis,we identified60 candidate odorant receptors(Mmed ORs),six candidate ionotropic receptors(Mmed IRs)andtwogustatory receptors(Mmed GRs)from male and female antennae transcriptom of M.mediator.The full-length sequences of 40 OR sequences,six putative IR genes and two candidate GRscloned as full-length coding sequences by using the rapid amplification of c DNA ends PCR(RACE-PCR)method.We also conducted reverse transcription PCR(RT-PCR)combined with real-time quantitative PCR(q RT-PCR)forinvestigating the expression profiles of these chemosensory receptor genes in olfactory and non-olfactory tissues.RT-PCR results showed that the transcripts of Mmed OR24,Mmed OR26,Mmed OR28,Mmed OR29,Mmed OR31,Mmed OR32,Mmed OR34,Mmed OR36-OR43,Mmed OR45,Mmed OR46,Mmed OR48,Mmed OR49,Mmed OR50,Mmed OR52 and Mmed OR53 were only expressed in male and female antennae.The ORs with antennal-specific or dominant expression profiles may play crucial roles in olfactory chemoreception of wasps.Compared with ORs,IRs and GRs showed a ubiquitous expression pattern.By comparing expression levels in female antennae,male antennae and body tissues,we found that 25 OR genes(Mmed OR15,16,18,20,21,22,23,24,25,29,30,31,33,34,35,36,37,41,43,45,47,48,49,50,52)were more highly expressed in femaleantennae,which may be involved in the perception of volatile semiochemicals during host location process.Two ORs,OR19 and OR26,were more highly expressed in maleantennae,which may be involved in the perception ofsex pheromone.Mmed GR64 f was highly expressed in the female antennae,suggesting it may participate in sugar detection.Six IR geneshad higher expression levels in antennae than that in body parts.The Mmed IR8 a,Mmed IR25 a.1,Mmed IR25 a.2,Mmed IR64 a and Mmed IR76 bshowed higher expression levels in female antennae than those in male antennae,indicating that IR-mediated signaling may play more important roles in host recognition behavior.We performed RNAseq(Illumina Hiseq 2500 platform)of M.mediator antennae and identified seventeen IRs,includingsix IRs previously reportedand 11 new IRs.Phylogenetic analysis of the Hymenoptera IRs revealed that ten Mmed IR genes encoded “antennal IRs” and seven encoded “divergent IRs”.Among the IR25 a orthologous groups,two genes,Mmed IR25 a.1 and Mmed IR25 a.2,were found in M.mediator.Gene structure analysis of Mmed IR25 a revealed a tandem duplication of IR25 a in M.mediator.The tissue distribution and development specific expression of the Mmed IR 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,Mmed IR8 a,was expressed in the olfactory sensory neurons.The staining ORNs obviously associated with the s.placodea on the antenna.Based on the sequence similarity to insect ORs,107 novel OR genes were identified from M.mediator Hi Seq 2500 antennal transcriptome data.Phylogenetic analysis of the set of OR genes from M.mediator and Microplitis demolitor revealed that Mmed OR genes can be classified into different subfamilies,and the majority Mmed ORs in each subfamily shared high sequence identities and clear orthologous relationships to M.demolitor ORs.Within “R” subfamily,six Mmed OR genes,Mmed OR98,124,125,126,131 and 155,shared a similar gene structure,each gene had seven exons and six introns.Comparison of exon/intron structure indicated that the lengths of corresponding exons are close to each other.The Mmed OR genes were arranged in clusters within the genome.The coding regions of the six Mmed OR genes were in the same transcriptional orientation and were separated by a short distance RACE-PCR experiments revealed that the OR genes within the cluster were transcribed as single m RNAs,and a bicistronic m RNA for two adjacent genes(Mmed OR124 and Mmed OR98)was also detected in female antennae by RT-PCR.In situ hybridization experiments indicated that each OR gene within the cluster was expressed in a different number of cells.Moreover,there was no co-expression of the two highly related OR genes,Mmed OR124 and Mmed OR98,which appeared to be individually expressed in a distinct population of neurons.The results demonstrated that Mmed OR98 and Mmed OR124 were mainly transcribed as single m RNAs,rather than a bicistronic m RNA,and were expressed in different ORNs in female antennaeBy using in situ hybridization,we investigated the expression profile of 18 OBP genes in the male and female antennae of M.mediator.A total of 11 OBP(Mmed OBP1-8,14,18 and 19)genes were detected expression in antennae.Seven genes(Mmed OBP11-13,15,16,17 and 20)were not detected signals in both sexes.The results indicated that these genes may be expressed at levels below the detection threshold of in situ hybridization assays.Analysesthe distribution pattern of OBP probeslabeled cells in the male and female antennae,10 OBP genes were expressed at the bases ofantennal sensilla.Mmed OBP2,Mmed OBP6 and Mmed OBP7 probes labled cells were restricted to thes.placodeafrom both sexs;Mmed OBP14 postive cells wereobserved under thes.placodeafrom male antennae;Mmed OBP18 and Mmed OBP1 postive cellspostive cells were located,respectively,at the bases of s.basiconica type Ⅰand type Ⅱ from both sexs;In male antennae,Mmed OBP3 probe labeled cells were located at the based of the s.basiconica type Ⅰ,while in female antenna,the labeled cells were located at the based of s.basiconica type Ⅰ and s.basiconica type Ⅲ;Mmed OBP4 and Mmed OBP5 postive cells wererestricted to thes.basiconica type Ⅲfrom female antennae;Mmed OBP8 postive cellswererestricted to thes.coeloconicafrom both sexs. |
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