Identification And Functional Analysis Of Gustatory Receptor Genes Inbactrocera Dorsalis’ Forleg(Diptera:Tetriphitidae)

Many insects recognize distinct non-volatile chemicals including sugars, amino acids, orbitter compounds by taste and their extremely sensitive gustatory systems. After the insectstaste organs exposed to invironmental stimuli substances, gustatory receptors(Grs) in tasteneurons can encode this chemical signals, and convert the chemical signals into electricalsignals, then transmit it to the central nervous system through nerves axons in the form ofpulses.The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most importantagricultural pests in Asian and Pacific countries. Studying of the mechanism of B.dorsalisgustatory systems and the function of gustatory receptors, is contributed to understand thefunctions of insect taste perception systems for feeding, avoidance, courtship, mating andoviposition behaviors, and to help to design of a new control strategie by a particular tasteprotein target sites.We identified six gustatory receptor genesfrom the foreleg transcriptome database ofB.dorsalis and studied their tissue expression patterm. We studied the fuctions of threegustatory receptor genes by RNAi technology and behaviors analysis.The main results wereas follows:1）The sensibility of B.dorsalis to sugars and bitter compoudsInsect gustatory systems showed obviously behaviors responsing to attractive or aversivecompouds. In this study, the sensibility of adult B.dorsalis to sucrose, glucose, trehalose,fructose, maltose, arabinose, sorbitol, quinine, caffeine, denatonium and berberine weremeasured by Proboscis extension reflex test and two-choice preference assay. The resultshows that adult flies can recognize shese sugars or bitter compounds, effectively. Theresponse to sugar solutions are concentration gradient, on the other hand, bitter compoundscan inhibit feeding behaviors effectively in very low concentrations.2）Identification and sequences analysis of BdorGr gene in B.dorsalis’ forelegsWe identified six gustatory receptor genesfrom the foreleg transcriptome database ofB.dorsalis and named BdorGr5a, BdorGr28b, BdorGr32a, BdorGr68a, BdorG63a andBdorGr21a, then obtained their full-length cDNA sequences by RACE-PCR. And the homology of these amino acid sequence compare with DmelGr5a,DmelGr28b, DmelGr32a,DmelGr68a, DmelGr63a and DmelGr21awere56%,61%,53%,33%,67%and78%,respectively. Furdermore, the result of phylogenetic analysis betweenBdorGrs and severalinsect gustatory receptors also indicated their relationship.3）Tissue Expression Pattern of BdorGrs GenesTo study the tissue-specific expression and quantitative analysis of six BdorGrsGenes,we performed researchs on mRNA expression level for these six BdorGrsGenes in differenttissues of female and male adult flies by Real-time quantitative PCR methods. We found thatsix BdorGrsGenes were mainly expressed in proboscis and forelegs. The expression of levelof BdorGr32a and BdorGr68a in male forelegs was higher than female forelegs or othertissues, in addition, BdorGr63a and BdorGr21a were found expressed in antenne both infemale and male.4）RNAi BdorGr5a, BdorGr32a and BdorGr68a in B.dorsalisMicroinjection approach has been applied to inject dsRNA for BdorGr5a,BdorGr32aand BdorGr68a into the pupae abodomen cavity. The mRNA expression level ofBdorGr5a,BdorGr32a and BdorGr68a were reduced by about50%at24hr,48hr and72hrafter eclosion post-injection. Therefore, the dsRNA abodomen injection method was effectiveto be used for gustatory receptor genes RNAi study inB.dorsalis. Biological behavior analyzesshow that the flies of BdorGr5a dsRNA injection show lower sensitivity to trehalose,suggesting that BdorGr5a gene may involve in trehalose perception. However, we didn’tfound any difference betewwn BdorGr32a or BdorGr68a dsRNA injected groups and controlsin courtship and mating behavior analyses.