Cytochrome P450 Involved In The Resistance Of Buprofezin And The Cross-resistance Of Buprofezin In The Brown Planthopper,Nilarpavata Lugens(St(?)l)

The brown planthopper(BPH),a monophagous rice pest,could cause disaster economic loss on rice due to its sucking,egg-laying and spreading viral disease.Control of BPH in the field is still relied on chemical insecticides,which have caused BPH had developed resistance to various insecticides.Buprofezin,an insect growth regulator,N.lugens has developed resistance to buprofezin in 2006,and high resistance since 2013.The resistance mechanism of BPH to buprofezin kept unclear.As a metabolic enzyme,which participate metabolic mechanism of insecticide resistance,Cytochrome P450 might mediate the detoxification of buprofezin.In this paper,to verify if Cytochrome P450 participate the resistance to buprofezin and the Cross-resistance to other insecticides in buprofezin resistant N.lugens,the gene cloning,q-PCR,transgenic Drosophila,RNAi and bioassay techniques were adopted to explore the function of 4 P450 gene.1.Four P450 genes participate the buprofezin resistance in N.lugensIn prior research,we found CYP6ER1,CYP6CW1,CYP6CS1,and CYP439A1 genes significantly high expressed in mRNA level compared buprofezin resistant and susceptible strain in N.lugens.To explore whether these four P450 genes participate the buprofezin resistance,four transgenic Drosophila strains with CYP6ER1,CYP6CW1,CYP6CS1,and CYP439A1 of N.lugens were constructed respectively using the Drosophila GAL4/UAS binary expression system.The results showed CYP6ER1,CYP6CW1 could cause a significant increase on the tolerance of Drosophila to buprofezin,but CYP6CS1 and CYP439A1 could not.Then silencing of CYP6ER1 and CYP6CW1 gene in N.lugens also significantly restored its sensitivity to buprofezin in 2017YC field population.the above results showed that CYP6ER1 and CYP6CW1 gene involved in the resistance of the brown planthopper to buprofezin.Whether there are other mechanisms need to study further.2.Changes on CYP6ER1 and CYP6CW1 genes in different resistance stages of buprofezinCYP6ER1 and CYP6CW1 genes were verified involving in the resistance of the brown planthopper to buprofezin.In order to know the changes of CYP6ER1 and CYP6CW1 genes with the resistance increase of buprofezin in the brown planthopper,we selected the Bup-Rl strain(RR=524.7)with buprofezin technical compound for every generations and obtained a higher resistance strain(BUP-R2,RR>5000),the difference of mRNA levels of CYP6ER1 and CYP6CW1 genes in Bup-R2 and susceptible strain(Bup-S)was detected by q-PCR.There was no significant difference in the mRNA levels of the two P450 genes in the BUP-R2 strain compared with the BUP-S strain.Subsequently,by gene cloning and q-PCR,the four phenotypes of vA,vB,vC and vD of CYP6ER1 gene were found in BUP-R2 and BUP-S strains,and the mRNA level of vA phenotype in both strains is much higher than the other three phenotypes;it may be involved in BUP-R2 strain is a vA phenotype of the CYP6ER1 gene by combing the results of previous laboratory studies.Therefore,our results are as follows:with the increase of resistance to buprofezin in brown planthopper,the overexpression level of CYP6ER1 and CYP6CW1 gene is gradually weakened.Although the CYP6ER1 gene has different duplication,it has little contribution to the resistance of buprofezin.Perhaps in the later stages,the resistance mechanism of brown planthopper to buprofezin is mediated by target mutations.Our results provide new ideas for the study of the mechanism of the resistance of the brown planthopper to the buprofezin.3.Cross-resistance of buprofezin to other insecticides in buprofezin resistant strain.Rice-seedling dipping method was used to study cross-resistance between buprofezin and imidacloprid,thiamethoxam,dinotefuran,nitenpyram,cycloxaprid,acetamiprid,thiacloprid,clothianidin,sulfoxaflor,isoprocarb,chlorpyrifos,pymetrozine and triflumezopyrim.The results showed that there was a moderate level of cross-resistance to thiamethoxam(17.1-fold),isoprocarb(12.5-fold)and acetamiprid(24.9-fold),a low level of cross-risistance to imidacloprid(10.0-fold),pymetrozine(7.7-fold)and dinotefuran(5.0-fold)in Bup-R2 strain,which resistance ratio was more than 5000 times to buprofezin.And no cross-resistance to nitenpyram,thiacloprid,clothianidin,triflumezopyrim,chlorpyrifos and sulfoxaflor was observed in this strain.In view of the current suspension of imidacloprid and thiamethoxam for controlling of the brown planthopper in the field,and the three rice planthoppers in the field developed moderate resistance to chlorpyrifos and clothianidin,nitenpyram,sulfoxaflor and cycloxaprid could be rotated insecticides for the control of the brown planthopper,and triflumezopyrim and pymetrozine could be alternate insecticides to delay the development of resistance in N.lugens.