Function Analysis Of Cytochrome P450 Monooxygenase And UGT Glycosyltransferase Genes Conferring Cyantraniliprole Resistance In Aphis Gossypii Glover

The cotton aphid,Aphis gossypii Glover(Hemiptera: Aphididae),is an important agricultural pest in the world,which has caused economic damage to cotton and other cash crops.The control of cotton aphids has been largely dependent on the application of chemical insecticides in China.Due to the long-term application of chemical pesticides,cotton aphids have evolved the high lever of resistance to many types of traditional insecticides.Cyantraniliprole is the second-generation ryanodine receptor activator insecticide.It has been used primarily for the control of sap-feeding insect pests due to its unique mechanism of action.In this study,the synergist experiment and the activity of detoxification enzymes were used to evaluate whether P450 and UGT genes can confer cyantraniliprole resistance in cotton aphids.The molecular mechanism of resistance to cyanodiamide in cotton aphids was further verify byRNAi and transgenic drosophila technology.The main results are as follow:Continuous selection with cyantraniliprole,the cyantraniliprole resistant cotton aphid strain(CyR)was established.The CyR strain has developed a medium level of resistance to cyantraniliprole with a 23.58-fold resistance ratio.Piperinyl butoxide(PBO),5-Nitrouracil(5-Nul)and Sulfinpyrazone(Sul)significantly increased the sensitivity of cyantraniliprole,with synergistic ratios of 3.40-fold,2.51-fold and 2.35-fold in the CyR strain,respectively.And PBO,5-Nul and Sul significantly increased the toxicity of α-cypermethrin,with synergistic ratios of 24.47-fold,7.94-fold and7.02-fold in the CyR strain,respectively.The activities of cytochrome P450 monooxygenase increased significantly(2.42-fold)in the CyR strain,compared with the SS strain,whereas GSTs enzyme activity and Car E enzyme with 2-Naphthyl acetate as substrate activity didn’t show the obvious change in the CyR strain,the Car E enzyme with 1-Naphthyl acetate activity was lower than that of SS strain.These indicate P450 monooxygenase is involved in the cyantraniliprole resistance of cotton aphids.The quantitative real-time PCR results revealed that the transcripts of CYP380C6,CYP6CY7,CYP6CY21,CYP6DA2,CYP4CJ1,UGT341A4,UGT344B4,UGT344D6,UGT344J2 and UGT344M2 increased significantly in the CyR strain compared with the susceptible strain(SS),with the 2.38-,2.60-,3.64-,3.15-,2.45-,4.97-,1.74-and2.79-fold,respectively.Compared with the body tissues,CYP6CY7 and UGT344B4 were significantly highly expressed in the midgut,with the CYP6CY7 and UGT344B4 increased to 3.05-fold and 8.22-fold,respectively.The functional roles of the CYP380C6,CYP6CY21,CYP6CY7,CYP6DC1 and CYP4CJ1 in cyantraniliprole resistance in cotton aphids were evaluated byRNAi.After treating by ds RNA-CYP380C6,ds RNA-CYP6CY21,ds RNA-CYP6CY7,ds RNA-CYP6DC1 and ds RNA-CYP4CJ1 the sensitivity of cotton aphids to cyantraniliprole increased and mortality dramatically increased by 18.07%,15.68%,15.27%,20.00% and 22.07%compared to that of the control.This indicates the overexpression of CYP380C6,CYP6CY21,CYP6CY7,CYP6DC1 and CYP4CJ1 may be involved in the resistance of cyantraniliprole in cotton aphids.An approach of transgenic Drosophila melanogaster successfully was used by us to constructed UAS-CYP380C6,UAS-CYP6CY7,UAS-CYP6CY21,UAS-CYP6DA2,UAS-CYP4CJ1,UAS-UGT344B4,UAS-UGT344M2,UAS-UGT344D6 and UAS-UGT341A4 transgenic lines.The transgenic lines crossed with the Esg-GAL4line(y w;Esg-Gal4 UAS-GFP/CyO)and stimulated the specific overexpression of P450 and UGT genes in the midgut tissue of the D.melanogaster.Bioassays showed that the midgut-specific overexpression of Esg>CYP380C6,Esg>CYP6CY7,Esg>CYP6CY21,Esg>CYP4CJ1,Esg>UGT341A4,Esg>UGT344B4 and Esg>UGT344M2 significantly increased resistance to cyantraniliprole.Compared with the control flies,the resistance ratio was 5.25-,3.88-,2.46-,1.55-,1.67-,2.55-and 3.30-fold,respectively.The overexpression of Esg>CYP380C6,Esg>CYP6CY7,Esg>CYP6CY21,Esg>UGT344B4 and Esg>UGT344M2 conferred α-cypermethrin cross-resistance.Compared with the control flies,the resistance ratio was 4.36-,3.71-,4.31-,4.71-and 5.06-fold,respectively.When transgenic lines crossed with the Act5-GAL4 line(y w;Act5C-Gal4/CyO),transgenic expression of the P450 and UGT genes in broad body tissues in D.melanogaster.Bioassays indicated that the overexpression of Act5>CYP380C6,Act5>CYP6CY21,Act5>CYP4CJ1,Act5>UGT344B4,Act5>UGT344M2 and Act5>UGT341A4 conferred cyantraniliprole resistance.Compared with the control flies,the resistance ratio was 4.38-,1.61-,2.42-,2.13-,3.57-and 3.10-fold,respectively.the overexpression of Act5>CYP380C6 、Act5>CYP6CY7、Act5>CYP6CY21、Act5>UGT341A4和Act5>UGT344M2 could be related to α-cypermethrin cross-resistance.Compared with the control flies,the resistance ratio was 2.41-,3.00-,2.13-,2.39-and 1.67-fold,respectively.Bioassays show the overexpression of P450 and UGT gene can confer cyantraniliprole resistance and α-cypermethrin cross-resistance in cotton aphids.In conclusion,these findings indicate that the overexpression of P450 and UGT genes are involved in the detoxification of cotton aphid against cyantraniliprole.These results are of great significance for comprehensive understanding the mechanism of cyantraniliprole resistance.