Identification Of Cytochrome P450 Genes And The Contribution Of CYP380C6 To Spirotetramat Resistance In Aphis Gossypii(Glover)

Cotton aphid(Aphis gossypii Glover)is a worldwide agricultural pest.Because of its short generation duration,strong fecundity and unreasonable use of insecticides in the field,the insecticide resistance of cotton aphid has developed rapidly and the resistant background is quite complex in China.Insect cytochrome P450 enzymes play a very important role in insect metabolism of plant secondary substance and insecticides,so that insects can adapt to different host plants and develop insecticide resistance.This study aimed to screen the P450 gene associated with the resistance of cotton aphid to spirotetramat,and preliminarily verify their functionality by RNA interference(RNAi).The spirotetramat susceptible(SS)and resistant strains(SR)were established by the laboratory selection and were used in the present study.Through the analysis of the synergism effect of PBO,cytochrome P450 was related to the resistance development of cotton aphid to spirotetramat.Thirty five P450 genes were identified by analyzing the transcriptome of cotton aphid.According to the phylogenetic tree,these 35 genes belong to the CYP2,CYP3,CYP4 and Mito clades.The Mrna expression levels of thirty-three cytochrome P450 genes in were determined in SS and SR strain using RT-qPCR,the expression of CYP6CY4,CYP6CY14,CYP6CY18,CYP6DC1 and CYP49A1 were significantly up-regulated in resistant third instar nymphs.The transcriptional levels of P450 CYP380C6,CYP353B1 and CYP307A1 were significantly up-regulated in resistant apterous adults.Combining with the transcript abundance of each gene,CYP380C6 was chosen for the next experiment.CYP380C6 belongs to the CYP4 clade,and the CYP4 clade is one of the major families associated with resistance.Therefore,CYP380C6 was chosen for the RNAi experiment.For the dsRNA feeding experiments,dsRNA was added to the artificial diet at a150 ng/?L concentration.An artificial diet containing dsRNA-ECFP was used as a control.One hundred spirotetramat-resistant A.gossypii were transferred onto the artificial diet for rearing.To analyze the efficiency of the dsRNA knockdown of P450 expression,the aphids were fed an artificial diet containing dsRNA(150 ng/?L)for 72 h and then collected for RT-qPCR.The successful RNAi reduced the expression levels of CYP380C6 to 0.49-and 0.26-fold in dsRNA-CYP380C6 treated(150 ng/?L)nymphs and adult aphids after 72 h,respectively,compared with control expression levels.Eighty resistant apterous adults were transferred to artificial diets containing spirotetramat(2500 mg/L)mixed with dsRNA-CYP380C6(150 ng/?L).Eighty resistant nymphs were transferred to artificial diets containing spirotetramat(50 mg/L)mixed with dsRNA-CYP380C6(150 ng/?L).DsRNA-ECFP was used as the control.Mortality significantly increased,from 65.84% in the control to 84.67%in dsRNA-CYP380C6 fed nymphs,respectively,in the presence of 50 mg/L spirotetramat.In adult aphids,the mortality dramatically increased,from 55.46 % in the control to 93.59% in the dsRNA-CYP380C6 fed adult aphids,under the 2500mg/L spirotetramat treatment.Suppress the transcripts of CYP380C6 by RNA interference effectively increased the sensitivity of resistant aphids to spirotetramat.The P450 genes related to spirotetramat resistance was screened out and its function was preliminary verified.It provides a theoretical basis for further research,and provides a possible target for the creation of new pesticides.