Chemosensory Genes In Macrocentrus Cingulum:Identification,Expression And Function

Macrocentrus cingulum Brischke(Hymenoptera:Braconidae)is deployed successfully as a biocontrol agent for corn pest insects from the genus Ostrinia in Europe and throughout Asia,including Japan,Korea and China.The odorants are recognized,bound and solubilize by odorant binding protein(OBP)is the initial biochemical recognition steps in olfaction which transport them across the sensillum lymph to specific receptors within the antennal sensilla to trigger behavioral responses.The aim of this study was to investigate the molecular mechanisms of olfaction in the M.cingulum,consequently,in an effort to expand our understanding of chemosensory pathways.We assembled antennal transcriptomes of M.cingulum by using next generation sequencing to identify the major chemosensory protein gene families.In total,129 olfactory protein genes(9 odorant binding proteins,8 chemosensory proteins,79 odorant receptors,20 gustatory receptors,and 13 ionotropic receptors)have been identified from the male and female antennal transcriptome.The sequences of all of these transcripts were confirmed by RT-PCR,and direct DNA sequencing.Expression profiles of gustatory receptors in olfactory and non-olfactory tissues were measured by qPCR.The sex-specific and sex-biased chemoreceptors expression patterns suggested that they may have important functions in sense detection which behaviorally relevant to odor molecules.This reported result provides a comprehensive resource of the foundation in semiochemicals driven behaviors at molecular level in this wasp.We examine the odorant binding effects on thermal stability of McinOBP2,McinOBP3 and their mutant form that lacks of third disulfide bonds.Real-time PCR experiments indicate that these two are expressed mainly in adult antennae,with expression levels differing by sex.Odorant binding affinities of aldehydes,terpenoids,and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes of the thermal stability of the proteins upon their affinities to odorants.The obtained results reveal higher affinity of trans-caryophelle,famesene and cis-3-Hexen-1-ol exhibits to both,wild-type and mutant McinOBP2 and McinOBP3.Although,conformational flexibility of the mutants and shape of binding cavity are make differences in odorant affinity between the wild-type and mutant.It suggested that lacking third disulfide bond in mutant proteins may have chance to incorrect folded structures which reduced the affinity to these odorants.The odorant binding protein McinOBP4 was characterized from the M.cingulum;it has been showed good binding affinity to corn green leaf volatiles.qPCR experiments indicated that the McinOBP4 was primarily expressed in wasp antennae,with transcripts level differing by the sex.From the fluorescence assay,McinOBP4 binds corn green leaf volatiles including terpenoides and aliphatic alcohols as well as aldehydes with good affinity.We have also conducted series of binding assay with first mutant(M1),which was lack of c-terminus 8 residues and second mutant(M2),Met119 was replaced by Leucine.In the acidic conditions,affinity 1-NPN to McinOBP4 and M1 were substantially decreased but increase in basic condition with no significant differences.The lack of C-terminus showed reduced affinity to terpenoides and aliphatic alcohols as well as aldehydes compounds of corn odorants.Finally,fluorescence assay revealed that the second mutant Met119 was significantly reduced binding a:ffinity to tested odorants.It indicates that Met119 was involved in binding these compounds through forming hydrogen bonds with the functional group of odorant compounds.These findings provide insight into the detailed chemosensory function of McinOBP4 and help to develop low release agents that attract of this wasp to improve eco-friendly pest management strategy.