Study On Resistance And Mechanism Of Asian Citrus Psyllid,Diaphorina Citri To Imidacloprid

The Asian citrus psyllid,Diaphorina citri,is a vector of citrus Huanglongbing(HLB),a destructive disease for citrus.Because there is no available cure to treat diseased trees,management of HLB has focused primarily on the use of insecticides to control the vector to further reduce the spread of pathogens.However,the inappropriately applications of conventional insecticides to control D.citri have led to high level of resistance.Up to now,few studies have been attributed to the resistance mechanisms in D.citri.Therefore,it is necessary to fully understand the molecular mechanism of insecticide resistance in D.citri.At the beginning,we conducted the monitoring of resistance to nine insecticides in D.citri collected from differents citrus orchards in Guangdong and compared the activities of three major detoxification enzymes.Then,we collected the resistant population after resistance monitoring of D.citri sample from the field around Guangdong.Secondly,based on transcriptome data and known insect resistance gene,the detoxification genes related to insecticide resistance were identified.And thirty detoxification genes were successfully cloned from D.citri.In addition,Quantitative real-time PCR(q RT-PCR)was performed to detect the overexpression patterns of detoxification genes in resistance and susceptible population of D.citri.And further functional verification of differentially expressed genes by RNA interference(RNAi).Finally,the expression profiles of six detoxification genes under different insecticides stress were analyzed by q RT-PCR and their potential physiological function were also verified.These results may contribute to comprehensively understand the detoxification metabolic mechanism in D.citri,and these data may also support the design of management strategies to control the resistance development in the field.The main results are as follows:(1)The relative resistance level of D.citri sampled from the four populations(Zengcheng,Huizhou,Qingyuan and Conghua)to nine different insecticides were investigated by a modified leaf-dip bioassay method.The bioassay results showed that the highest level of resistance for D.citri was found in imidacloprid with a resistance ratio of 15.12 in the Zengcheng population compared with the laboratory susceptible population.In addition,the highest level of resistance for D.citri was also found in dinotefuran with a resistance ratio of 6.16 in the Zengcheng population.However,the Qingyuan population had the highest resistance to bifenthrin and thiamethoxam,and the resistance ratios were 4.16 and 6.04,respectively.The Huizhou population had the highest resistance to chlorpyriphos and lambda-cyhalothrin,and the resistance ratios were 6.47 and 4.78,respectively.There was no resistance to clothianidin,acetamiprid or chlorfenapyr in all the four populations.The bioassay was continued in the next year by the diagnostic dose(LC₉₅).As a result,it was found that the resistance characteristics of D.citri to the nine different insecticides were not changed,and the resistance to imidacloprid was enhanced.(2)The synergism effects of PBO,TPP and DEM with imidacloprid were evaluated in Zengcheng-resistant and laboratory susceptible population of D.citri to measure the involvement of general esterase,mixed function oxidases and GST detoxifying enzymes in resistance mechanisms,respectively.The toxicity of imidacloprid was nearly four times greater in the presence of PBO than in the absence of PBO in the Zengcheng population.Meanwhile,a significant synergism was observed for TPP in the Zengcheng population(2.46-fold)but not in the susceptible population.Therefore,the results showed that the resistance to imidacloprid was mainly caused by mixed function oxidases and general esterase detoxification.To further examine whether these detoxification enzymes were involved in insecticide resistance,we compared the general esterase,P450 monooxygenase and GST activities of the adults between field and susceptible populations of the D.citri.The results showed that mean GST activity was significantly higher in the population from Qingyuan than in the susceptible population(1.61-fold).When?-naphthol was used as substrate,the general esterase activity of the Zengcheng and Huizhou populations was 3.18-and 2.71-fold higher than that of the susceptible population,respectively.However,the amount of general esterase was highest in the Huizhou and Qingyuan populations when?-NA was used as substrate.The general esterase activity was more than 3-fold higher than that in the susceptible population.The P450 monooxygenase activity was 2.09-,1.60-and1.56-fold higher in Zengcheng,Huizhou and Qingyuan populations than in the susceptible population.Both the results of synergism effects and detoxifying enzyme activity analysis showed that general esterase and cytochrome P450 monooxygenase might contribute to the resistance of D.citri to insecticide,and glutathione S-transferase may play a minor role in the resistance of adult D.citri to insecticides,especially for imidacloprid.(3)Based on known insect resistance genes,we obtained 16 P450s,8 GSTs and 6carboxylesterase(Car Es)gene transcript by searching annotation detoxification genes from the D.citri transcriptome data.The c DNA fragments of 16 P450s,8 GSTs and 6Car Es were successfully cloned using RT-PCR technique from the susceptible population.And the Zengcheng population were selected by exposure to imidacloprid.After nine generations of continuous selection,the population showed 52.19-fold resistance to imidacloprid by the leaf-dip method in D.citri,regarding as resistance population.The transcriptional level of detoxification genes were compared between the imidacloprid-resistant and susceptible population using quantitative real-time PCR(q RT-PCR).The results showed that the expression of 10 P450 genes,6 GST genes and4 Car E genes were significantly greater in the imidacloprid-resistant population in comparison to the susceptible population among all of the tested detoxification genes.Relative expressions of CYP4g15,CYP303A1,CYP4C62,CYP6BD5,GSTS1 and EST-6 were moderately high(>5-fold increase)in the imidacloprid-resistant population.Notably,higher expression levels of the six selected genes in the imidacloprid-resistant population suggested that these genes may play a major role in imidacloprid metabolism associated with resistance.(4)To determine the function of CYP4g15,CYP303A1,CYP4C62,CYP6BD5,GSTS1 and EST-6,RNAi experiments were performed by the topical feeding application and then performed an imidacloprid bioassay to assess the contribution of these genes to imidacloprid resistance.The results showed that the expression levels of CYP4g15,CYP303A1,CYP4C62,CYP6BD5,GSTS1 and EST-6 were greatly decreased(by 71.5%,50.9%,46.7%,60.6%,72.1%and 56.6%,respectively)compared to those treated with RNase-free water and ds RNA-gfp when the ds RNA concentration was 100?g m L^-1.And the RNAi-mediated silencing of the six target genes increased the susceptibility of D.citri to imidacloprid.Therefore,these data suggested that the six overexpressed detoxification genes in imidacloprid-resistant population were implicated in imidacloprid resistance in D.citri.(5)The objective of this experiment was to examine and confirm which insecticides could effectively induce the expression of the six D.citri detoxification genes.Eight commonly used insecticides,which field populations of D.citri showed varying levels of resistance,were selected for study.For the neonicotinoid insecticide,there were varying levels of significant induction of all six detoxification genes.The expressions of CYP4C62 and GSTS1 genes were the most significantly upregulated in adults in almost all of the different treatment concentrations of the four neonicotinoid insecticides relative to controls.The expression of CYP4g15 gene was also the most significantly upregulated in adults treated with the thiamethoxam.For the pyrethroid insecticide,the lambda-cyhalothrin and bifenthrin with different concentrations were selected to research the induction of expression of the six detoxification genes in D.citri.The results showed that the expression of the six detoxification genes was significantly upregulated in D.citri treated with two insecticides,and the most affected genes were CYP4C62 and GSTS1(for CYP4C62,13.61-fold for lambda-cyhalothrin treatment at 0.5 mg L^-1 and an 8.69-fold for bifenthrin treatment at 1 mg L^-1).For the chlorpyrifos and chlorfenapyr,the CYP4C62 and GSTS1 genes were significantly higher in adults exposed to all concentrations of chlorpyrifos than in the control.However,expression of CYP4g15 and CYP303A1 was most affected when the concentrations of chlorfenapyr treatment were 2 and 5 mg L^-1,respectively.The expression levels of CYP4g15 and CYP303A1 were greatly increased(by 13.52-fold and 13.49-fold,respectively)compared with controls.In order to evaluate the roles of up-regulated detoxification genes after inducting,the functional analysis via RNAi of up-regulated detoxification genes were performed by the topical feeding application.The results suggested that CYP4C62 gene was involved in the resistance of D.citri to clothianidin,thiamethoxam,acetamiprid and chlorpyrifos;The GSTS1 gene was involved in the resistance of D.citri to clothianidin and thiamethoxam;The EST-6 gene was also involved in the resistance of D.citri to dinotefuran;And the CYP4g15 gene was involved in the resistance of D.citri to chlorfenapyr.