Studies On Metabolic Resistance Mechanisms Of Chilo Suppressalis(Walker) To Chlorantraniliprole

The striped rice stem borer,Chilo suppressalis Walker(Lepidoptera:Pyralidae),is considered one of the most serious pests harmed on rice and some other grain crops,and has caused huge economic losses throughout Asia.Since 1970s,accompanied with the reform of rice farming system in China,especially for the widespread use of hybrid rice,which promotes the growth of C.suppressalis population leading to a worsening damage effect.The control of C.suppressalis is mainly depended on chemical insecticides.However,the abused application of chemical insecticides have make the C.sup.pressalis developed heavily resistance to various insecticides,which greatly reduced the effectiveness in controlling C.suppressalis.Chlorantraniliprole,a novel diamide insecticide targets the ryanodine receptor(RyR)with broad spectrum,highly efficiency,low toxicity and low residue,has been extensively used for control of lepidoptera insects.However,high level resistance in Plutella xylostella and C.suppressalis has been reported in southern and center east China within a decade of application.Nevertheless,chlorantraniliprole is still an important pesticide used for control of C.suppressalis and other lepidoptera pests worldwide.It is urgent to have an in-depth understanding of resistance mechanisms to make good use of this kind of novel insecticide and prolong its service life.Resistance of C.suppressalis to chloroacetamide has been reported in previous studies and the ryanodine receptor mutation corresponding to chloroacetamide resistance in P.xylostella has also been found,but the identified mechanisms did not mate well the resistance ratio,which means there should be metabolic resistance mechanism involved.Hence,in the present study,the three principal detoxification enzymes were studied by testing the synergism effects of three enzyme inhibitors on chlorantraniliprole and detoxifying enzyme activity.The Secondary detoxification enzymes and membrane-transporter proteins related to pesticide resistance were also studied.Searching from the genome of C.suppressalis,UDP-glycosyltransferase superfamily genes and ATP-binding cassette transporter(ABC transporter)superfamily genes were obtained and identified by bioinformatic approaches.Their potential functions on chlorantraniliprole resistance were evaluated by comparative analysis of these genes(cytochrome P450,Esterase,UDP-glycosyltransferase and ABC transporter)between the susceptible strain and resistance strain and their role were further confirmed by the RNAi for several genes.20 genes related to chlorantraniliprole resistance were identified preliminary.The role of UDP-glycosyltransferase in chlorantraniliprole resistance was confirmed.The molecular mechanism of the metabolic resistance of C.suppressalis to chlorantraniliprole was ascertained in this study to provide a theoretical basis for the resistance management strategies.The results were summarized as follows:1.The relationship between P450,Esterase and Glutathione-S-transferase and C.suppressalis resistance to chlorantraniliproleIn order to clarify the contribution of three main detoxification enzymes to chlorantraniliprole resistance,C.suppressalis with high resistance to chlorantraniliprole were collected in the field(YY)and selected by chlorantraniliprole in the laboratory(YYR).In addition,the synergistic effect of different synergists and activities of three detoxification enzymes in different strains were measured.After selected by chlorantraniliprole,the resistance ratio(RR)of the 1st and 4th instar larvae of the field population increased from 55.03 and 51.24 times to 91.80 and 74.06 times,respectively.The higher resistance ratio strain YYR to chlorantraniliprole was then obtained.The synergism studies showed the resistance was suppressed by P450 inhibitor PBO(3.57-fold),esterase inhibitor TPP(1.89-fold)and glutathione-S-transferase inhibitor DEM(1.58-fold)in the selected chlorantraniliprole resistant strain YYR(RER 83.87-fold),while the synergism ratio of three inhibitor PBO,TPP and DEM in susceptible strain were 1.81.1.59 and 1.29,respectively.Results showed that only PBO had significant synergistic effect in the resistant strain.The activities of cytochrome P450,esterase and glutathione-S-transferase in resistant strain YYR were 3.87-fold,2.19-fold and 1.47-fold higher than that of susceptible strain S,respectively.Both synergism test and detoxifying enzyme activity analysis showed that cytochrome P450 and esterase might contribute to the resistance of C.suppressalis to chlorantraniliprole,while the effect of glutathione S-transferase was not significant.2.The relative expression of P450 genes in chlorantraniliprole resistant strain and susceptible strain of C.suppressalisIn order to clarify the difference of transcript level of different P450 genes in chlorantraniliprole resistant and susceptible strains of C.suppressalis and their contribution to chlorantraniliprole resistance,the relative expression level of 69 cytochrome P450 genes in chlorantraniliprole resistant and susceptible strains of C.suppressalis were detected by real-time quantitative PCR.The results showed that CsCYP9A68,CsCYP6CV5,CsCYP6CT1,CsCYP6AB45,CsCYP6,CsCYP18A1,CsCYP321F3,CsCYP324A12,CsCYP4AUll and CsCYP341A15 was significantly over-expressed(relative expression ratio>3 and p<0.05)in chlorantraniliprole resistant strain,with 74.88(p=0.0189),10.58(p=0.0144),9.67(P=0.0001),8.08(p=0.0015),7.09(p=0.0269),6.79(p=0.0094),6.63(0.0008),4.48(p=0.0264),4.17(p=0.0068)and 3.41(p=0.0177)times of the expression level compared to the susceptible strain,respectively.The results suggested that the ten genes might play important roles in resistance of C.suppressalis to chlorantraniliprole.There were ten resistance related cytochrome P450 genes were found expressed mainly in the larval midgut(CsCYP6CV5,CsCYP324A12 and CsCYP321F3),fat body(CsCYP9A68)and head(CsCYP18A1,CsCYP6AB45,CsCYP6CTl,CsCYP6,CsCYP4AUll and CsCYP324A1).In conclusion that over-expression of multiple P450s in the resistant strain,which further confirmed that the enhancement of P450 detoxification activity was one of the mechanism contribute to chlorantraniliprole resistance in C.suppressalis.These 14 over-expressed genes may be involved in the resistance of C.suppressalis to chlorantraniliprole,of which 10 genes which are significantly over-expressed in resistant strain is more likely to be resistance related gene,but further research is needed for specific confirmation.3.The relative expression of esterase genes in chlorantraniliprole resistant strain and susceptible strain of C.suppressalisIn order to clarify the difference of transcript level of different esterase genes in chlorantraniliprole resistant and susceptible strains of C.suppressalis and their contribution to chlorantraniliprole resistance,the relative expression levels of 51 esterase genes in chlorantraniliprole resistant and susceptible strains of C.suppressalis were detected by real-time quantitative PCR.The results showed that CsEST36,CsEST46 and CsEST7 was significantly over-expressed(relative expression ratio>4 and p<0.05)in chlorantraniliprole resistance strain,with 20.96(p=0.0409),4.83(p=0.04496)and 4.51(p=0.02538)times of the expression level compared to the susceptible strain,respectively.There were three resistance related esterase genes were found expressed mainly in the larval head,followed by midgut(CsEST7 and CsEST46)and integument(CsEST36).The results suggested that the three genes may play some roles in resistance of C.suppressalis to chlorantraniliprole.In this study,we found that 3 esterase genes were significantly over-expressed in the resistant strain,and further confirmed that the enhancement of esterase activity was one of the resistance mechanisms contribute to chlorantraniliprole resistance in C.suppressalis,but further research is needed for specific confirmation.4.Uridine diphosphate-glycosyltransferase genes in C.suppressalis and their contribution to chlorantraniliprole resistanceUDP-glycosyltransferases(UGT)represent major phase II metabolizing enzymes that are evolved in living organisms.It has been reported that UGT genes do contribute to insecticide resistance in some insects in recently.In order to identify the UGT of C.suppressalis and their contribution to chlorantraniliprole resistance.In this work,UGT genes in C.suppressalis were identified by a genome-wide bioinformatic approach and the relative expression level of them in chlorantraniliprole resistant and susceptible strains of C.suppressalis were detected by real-time quantitative PCR.Tissue-biased expression of CsUGTs were also detected by real-time quantitative PCR.Finally,the contribution of candidate genes to antagonism were tested by RNA interference.In this study,24 UGT genes were identified from the C.suppressalis genome and designated by UGT nomenclature committee.The results showed that CsUGT40AL1,CsUGT40AP1 and CsUGT33AG3 were significantly over-expressed in chlorantraniliprole resistant strain.These resistance related CsUGTs were found mainly expressed in the larval Malpighian tubules(Cs UGT40ALl and Cs UGT33AG3)and midgut(Cs UGT40APl).More importantly,the function of three CsUGTs were further confirmed by RNA interference,silence of CsUGT40ALl or CsUGT33AG3 made the insects more susceptible to chlorantraniliprole,and the susceptibility increased significantly higher in the resistant strain than in the susceptible one.In conclusion,this work identified 24 UGT genes in C.suppressalis and confirmed that two UGT genes,CsUGT40AL1 and CsUGT33AG3,were involved in the chlorantraniliprole resistance in C.suppressalis.The results not only proposed a new mechanism for chlorantraniliprole resistance in C.suppressalis and provide an example of UGT participate in the detoxification of insecticides in insect,but also provide a foundation for the research of the function of UGT gene in C.suppressalis5.ABC transport genes in C.suppressalis and their contribution to chlorantraniliprole resistanceABC transporters are important transport proteins involved in detoxification and metabolism.In order to clarify ABC transport genes in C.suppressalis and their contribution to chlorantraniliprole resistance,ABC transporter genes in C.suppressalis were identified by a genome-wide bioinformatic approach and their relative expression level in chlorantraniliprole resistant and susceptible strains were detected by real-time quantitative PCR.Tissue-biased expression of CsABCs were also detected by real-time quantitative PCR.Verapamil was used as the ABC transporter inhibitor to evaluate the toxicities of chlorantraniliprole in the resistant and susceptible strains in vivo.In this sudy,37 CsABCs were identified from the C suppressalis genome and designated by using Guidelines for Human Gene Nomenclature.The relative expression level of 37 CsABCs in chlorantraniliprole resistant and susceptible strains were detected by real-time quantitative PCR.Compared to the susceptible strain,5 ABC transporter genes was over-expressed in chlorantraniliprole resistance strain YYR and were found mainly expressed in the larval midgut.The synergism studies showed that verapamil increased the toxicity of chlorantraniliprole by a factor of 2.24 in YYR strain,but no significant synergism was observed in S strain(SR 1.98).In conclusion,this work identified 37 ABC transporter genes from C.suppressalis in the genome database by bioinformatic approaches.The involvement of ABC transporters in chlorantraniliprole resistance were further confirmed by using an ABC transporter inhibitor verapamil.5 ABC transporter genes,CsABCA3,CsABCA5,CsABCC1,CsABCC3 and CsABCD2,might involve in the chlorantraniliprole resistance in C.suppressalis.This result not only provide an example of ABC transporters participate in the detoxification of insecticides in insect,but also provide a foundation for the research of the function of ABC transporter genes in C.suppressalis...