Functional Analysis Of Chemosensory Protein Genes In Oriental Armyworm Mythimna Separata

Chemosensation plays key roles for insect locating hosts and looking for mates.It has been shown that several proteins including chemosensory proteins(CSPs)involve in insect chemosensory process,though the exact physiological functions of CSPs are not clear.The oriental armyworm Mythimna separata is an overwhelming,migratory and polyphagous pest of numerous crops worldwide.In order to illustrate the functions of CSPs in M.separata,expression patterns,binding affinities,and molecular docking of several CSPs were studied.And also,functions of CSPs were investigated by RNAi techniques.The main findings and results are given below.Expression profiling of chemosensory protein genesSix CSP genes(MsepCSP5,MsepCSP8,MsepCSPll,MsepCSP14,MsepCSP15 and MsepCSP16)were cloned from M.separata.The expression profiles of CSP genes were analyzed by RT-qPCR.The results showed that MsepCSPs are widely distributed not only in developmental stages,but also in several body parts of M.separata.The relative expressions of six MsepCSPs in larvae were higher than those in pupae,while temporal analysis of female and male adults showed diverse expression patterns for all tested genes.The expressions of MsepCSP5,MsepCSP8 and MsepCSP16 were highest in 3 days old female moths,while the highest expressions of MsepCSPll and MsepCSP14 were in 1 day and 3 day old male moths,respectively.Both female and male moths exhibited the highest expression of MsepCSP15 at 1 and 5 days old adults,respectively.Further analysis on body parts revealed that MsepCSP5,MsepCSP8 and MsepCSP14 were highly expressed in antennae than those in the other tissues.MsepCSPll,MsepCSP15 and MsepCSP16 were highly expressed in wings,legs and abdomen,respectively.The present study demonstrated that MsepCSPs from M.separata may attribute to miscellaneous functions.Functional analysis of MsepCSP5Full-length cDNA of MsepCSP5 was cloned and verified by sequencing.MsepCSP5 recombinant protein was expressed in Escherichia coli,and purified successfully with Nickel ion affinity chromatography.Binding characteristics of MsepCSP5 with 35 candidate volatiles were tested with fluorescence competitive-binding assay at pH 7.4 and pH 5.0,and the highly bound(Ki<10 μM)compounds were validated followed by three dimensional structure,molecular docking and olfactometer bioassays.The results showed that MsepCSP5 exhibited higher binding affinities with tested compounds at pH 5 than pH 7.4,such as 2-heptanol,3-pentanol,trans-2-hexen-l-ol,1-octene-3-ol,methyl benzoate,(R)-(+)-a-pinene,(-)-limonene,(+)-3-carene and 2-undecanone.Moreover,three dimensional structure and molecular docking showed that amino acid residues of MsepCSP5 attributed to binding of the volatile compounds.Most of those compounds with high binding affinities could elicit strong behavioral responses of M.separata,such as 2-heptanol,3-pentanol,trans-2-hexen-1-ol,1-octene-3-ol,methyl benzoate and ethyl acetate.Furthermore,reduction in the MsepCSP5 transcripts abundance was achieved by dsMsepCSP5 injection,which affected the behavioral responses of M.separata to the representative semiochemicals.The injection of dsMsepCSP5 caused non-significant preference of M.separata to 3-pentanol and 1-octene-3-ol.The present study suggests that MsepCSP5 may involve in chemoreception and other functions in M.separata.Functional analysis of MsepCSP8The full-length MsepCSP8 gene was cloned from M.separata.MsepCSP8 recombinant protein was expressed in E.coli and purified successfully using affinity chromatography.The competitive binding assay confirmed that 20 of 56 ligands were highly bound to MsepCSP8 at pH 7.4 compared to pH 5.0.Ligand binding mechanism of highly bound(Ki<10 μM)compounds such as cyclohexanol,1-penten-3-ol,hexanal,octanal,trans-2-hexenal,2-tridecanone,terpinolene,a-terpinene,(-)-terpinen-4-ol was further confirmed by three dimensional structure,molecular docking and olfactometer bioassays.Three dimensional structure and molecular docking displayed that amino acid residues of MsepCSP8 are plausible to be involved in binding of the tested volatiles.The behavioral response experiments showed that M.separata elicited significant responses to five volatiles(cyclohexanol,1-penten-3-ol,hexanal,terpinolene and a-terpinene)from the compounds displaying high binding affinity for the MsepCSP8.RNAi results demonstrated that expression level of MsepCSP8 was decreased significantly until 3rd day post eclosion which showed non-significant preference of M.separata to the tested volatiles(hexanal and terpinolene).Present study demonstrates that MsepCSP8 is likely to contribute in mediating responses of M.separata adults to plant volatiles.Functional analysis of MsepCSP14In this study,we cloned the full-length MsepCSP14 gene from M.separata,expressed in E.coli and subsequently purified.Ligand binding experiments were conducted for 21 ligands at pH 7.4 and pH 5.0.The results displayed strong binding of MsepCSP14(Ki<10 μM)with linalool,farnesene,(-)-limonene,(-)-terpinen-4-ol,nonadecane,R-(+)-limonene,dodecyl aldehyde,β-lonone,nerolidol at pH 7.4.Three dimensional modeling and molecular docking revealed that amino acid in binding pocket MsepCSP14 are important for recognition and binding of hydrophobic ligands.Olfactometer bioassays showed that M.separata displayed attractiveness towards farnesene and nerolidol while repellency towards linalool and R-(+)-limonene.The present study revealed that MsepCSP14 might involve in the chemoreception of M.separata Overall,our results demonstrated that CSPs we have tested are widely distributed in all tissues of M.separata indicating their broad functions in chemodetection.Strong bindings of several tested ligands to MsepCSPs propose their contribution in mediating responses of M.separata to plant volatiles.RNAi experiments also showed aptness of CSP genes attribution in chemodetection.Therefore,molecular mechanism insights into CSPs can potentially assist for developing new approaches for insect pest management.