Functional Research Of DcCHS And Its Application In Control Of Diaphorina Citri

Diaphorina citri Kuwayama(Hemiptera: Liviidae),is an important insect pest on citrus flush shoots and the main vector of huanglongbing(HLB).HLB is one of the most devasting citrus diseases,causing huge economic losses to the global citrus industry.Up to now,there is no effective agent or method to cure huanglongbing once it is established.Vector control of D.citri to cut off the transmission route of pathogens has been a critical part of the global strategy to manage HLB.Reducing the role of D.citri as a vector mainly depends on chemical insecticides,whereas,the frequent and longterm aplication of such chemicals has caused many negative effects such as pesticide residues,environmental pollution,as well as the selection for resistance.Therefore,it is necessary to explore new and environment-friendly scientific prevention and control strategies.Chitin widely occurs in arthropods and nematodes,but not in mammals.Therefore,the design of novel insecticides targeting genes in the chitin metabolic pathway has become an important research topic.Chitin synthase(CHS),a member of the glycosyltransferase superfamily,is the last rate-limiting enzyme during chitin synthesis,which can transfer the activated sugar groups of uridine diphosphate(UDP)to different sugar receptors.In this study,the molecular characteristics and functions of DcCHS were researched using D.citri as research material.These results will provide theoretical basis for the molecular control of D.citri using CHS.The main research results are as follows:1.Identification of DcCHS and spatiotemporal expression analysisSequences of Nilaparvata lugens CHS(Nl CHS)and Acyrthosiphon pisum CHS(Ap CHS)were used as queries in searching genome database of D.citri to obtain the nucleotide sequence of DcCHS.The open reading frame(ORF)of DcCHS was 3180 bp in length,encoding a protein of1059 amino acids with a predicted molecular mass of 129.9 k Da,and isoelectric point of 5.02.Genetic structure analysis revealed that DcCHS consisted of 19 exons and 18 introns.The structural domain analysis showed that DcCHS contained 14 transmembrane helical regions,two low-complexity regions,and one coiled helical region.Phylogenetic analysis revealed that DcCHS was close to its counterparts from hemipteran A.pisum,Toxoptera citricida,and Apis glycine.DcCHS was highly expressed during fifth-instar nymph stage and in the cuticle layer basing on quantitative real-time PCR(q PCR).Fluorescence in situ hybridization(FISH)further demonstrated that DcCHS was mainly distributed in the epidermis of D.citri.2.Effect of diflubenzuron on DcCHSIn order to further analyze the effect of DcCHS on the growth and development of D.citri,the fifth-instar nymphs of D.citri were treated with diflubenzuron(DFB)using a leaf-dip bioassay.The results showed that the expression level of DcCHS in the DFB group was significantly higherthan that in the control group.In addition,the cumulative mortality rate of the treatment group at48 h was 22.22%,which was 11.11% higher than that of the control group.After eclosion,the DFB-treated adults exhibited body shrinkage and deformed wings phenomenon.3.RNAi of DcCHS and its effect on epidermal structureTo further investigate the function of DcCHS,RNA interference(RNAi)was carried out on the fifth-instar nymphs of D.citri through membrane feeding.Along with transmission electron microscopy(TEM)was conducted to analyze the effect of silenceing of DcCHS on the epidermal structure of D.citri.The results showed that,the mortality rate of D.citri increased by 17% and the molting rate decreased by 33.4% after silenceing of DcCHS.The D.citri after moulting showed an abnormal wing phenotype.Furthermore,TEM observation showed that knockdown of DcCHS expression suppressed the formation of the epidermis of D.citri.These results suggest that DcCHS may play an important role in the growth and development of D.citri.