Tissue Expression Patterns And Ligand-binding Abilities Of Five Chemosensory Proteins In Chrysopa Pallens(Rambur)

During the long-term evolutionary process,insects evolved a highly sensitive olfactory system to detect volatile compounds in the environments,and accordingly trigger physiological or behavioral responses such as finding food,mating,and locating oviposition sites.In the process of olfaction,chemosensory proteins(CSPs)and odorant binding proteins(OBPs)play important roles in the olfactory perception by transporting hydrophobic chemical volatiles across the sensillum lymph to odorant receptors(ORs).The lacewing Chrysopa pallens(Rambur)is an important natural predator of small soft-bodied arthropods,such as aphids,mites coccids,and thrips in the cotton and other agricultural system.To study the olfactory mechanism in C.pallens will undoubtedly facilitate the development and application of more effective predator behavioral manipulation strategies.However,research on the C.pallens olfactory gene functional study is rare.In this study,focusing on 5 CSP genes,the tissue expression patterns were firstly determined,andthen the physiological functions were explored by fluorescence competitive binding experiments,electroantennogram(EAG)recording assay and Y-tube olfactormeter.The main results are as follows:1.Gene cloning,sequence analysis and expression patterns of CpalCSPsBy transcriptome data analysis 5full-length cDNAs of candidate CSPsfrom C.pallens were previously obtained.In the present sutudy,they were verified byPCRexperiments.These 5 genes were named asCpalCSP 3,CpalCSP 4,CpalCSP14,CpalCSP15,CpalCSP16,respectively.Sequence analysis showed that the five genes possessed the typical characteristics of CSPs with 4 conserved cysteine residues in a common cysteine sequence motif of C-X6-C-X18-C-X2-C.Phylogenetic trees showed that the five CSPs were classified into two groups.Further RT-qPCRstudy showed that CpalCSP3,CpalCSP4,CpalCSP14,CpalCSP15,CpalCSP16 were highly expressed in the heads or antennae of both sexes.CpalCSP3was highly expressed in the head,CpalCSP4,CpalCSP14,CpalCSP16werehighly or specifically expressed in theantennae,CpalCSP155was highly expressed in theantennaeand head.The relative expression level of CpalCSP14 in the antennae was significantly higher than the other four genes.These tissue expression profiles suggest thatthe 5 CSP genes play roles in the olfaction or gustation,and CpalCSP14 isthe most importantamong the 5 genes.2.Prokaryotic expression and ligand-binding affinity analysis of CpalCSPsRecombinant proteinsof CpalCSPs wereobtained byprokaryotic expression and purified through affinity chromatography.The binding affinities of the five CpalCSPs were tested with 85 odorant compounds by fluorescence competitive binding experiment.Among the tested compounds,nerolidol,(+)-cedrol and beta-ionone had high binding affinities to CpalCSP3 with Ki values ranged from 8.79 to 9.79p,M.In addition,phlorizin dihydrate,dodecyl aldehyde,geranyl acetate and(+/-)-alpha-pinene also showed high binding affinities with Ki values from 11.54 to 19.06?M.CaplCSP14 could bind with high affinity(Ki<20?M)to various chemicals including aldehydes,esters andterpenoids,whichcis-3-hexenyl hexanoate and phenethyl acetate showingthe strongest bindings(Ki=0.74-1.12?M).However,the CaplCSP4,CaplCSP15,CaplCSP16 had low binding affinities to all the 85 odorant compounds.It suggests that CpalCSP3 and CaplCSP14 play an important role in the C.pallens olfaction bybinding and transporting thespecificodorant compounds.3.Electrophysiological andBehavioral responses of C.pallens to semiochemicalsThe electrophysiological responsesof C.pallens to the 23 ligandsof higher binding affinity to CpalCSPs were studied.The results showed that decanal,hexanal,phenylacetaldehyde,beta-ionone,nerolidol,ethyl benzoate,dodecyl aldehyde and geraniol elicited high relative EAG responses in males(EAG>1),whiledecanal,hexanal,phenylacetaldehyde,cis-3-hexenyl hexanoate,dodecyl aldehyde,ethyl benzoate,beta-ionone,phenethyl alcohol,benzyl alcohol andcis-3-hexenalelicited high relative EAG responses in females(EAG>1)."Y"tube olfactory test showed that decanal,cis-3-hexenyl hexanoate,(+)-alpha-pineneweresignificantly attractive toC.pallens,however,thecis-3-hexenyl acetate wasremarkablly repellent to C.pallens.These active semiochemicals can be used to develop the attractants or repellents of the C.pallens,thus improve the effectivenessofthe C.pallensin the biological control.