Mechanisms Of Detoxification And Metabolism To Indoxacarb Mediated By PxGSTO4 In Diamondback Moth,Plutella Xylostella (Lepidoptera:Plutellidae)

Diamondback moth?DBM?,Plutella xylostella?L.?is a worldwide the most important pest of cruciferous crops and its resistance has also become increasingly serious.Indoxacarb is a oxadiazine insecticide that has a significant effect on lepidopteran pests including P.xylostella and targets sodium ion channels.Since its launch in 1998,the field population of P.xylostella has produced high levels of resistance to indoxacarb due to its unreasonable use.Previous studies have found that point mutations in the sodium channel are associated with resistance to indoxacarb in P.xylostella,but whether the detoxification enzyme is involved in mediating metabolic resistance is unknown.Therefore,this study monitored the resistance of the field population of P.xylostella to the indoxacarb in four regions of Hubei in 2017-2018 and tested its detoxification enzyme activity.The software was used to analyze the pair-wise correlation coefficient comparison between LC₅₀ values of tested insecticides on field populations of P.xylostella,and pair-wise correlation between indoxcarb toxicities and enzyme activities.Based on this,the metabolic mechanism of the resistance to the indoxacarb in P.xylostella and its transcriptional regulation mechanism were further analyzed.The main results were briefly described as following:1.The resistance levels of P.xylostella field populations in the four regions of Wuxue,Xinzhou,Dangyang and Yunmeng in Hubei Province were observed in2017-2018.The results showed that the P.xylostella field populations have had different degrees of resistance to indoxacarb?Resistance Ratio=0.3-17.7?,P.xylostella collected in Xinzhou area in May 2017 and Dangyang area in October 2018 reached moderate level resistance.The resistance level of P.xylostella to the indoxacarb in Yunmeng and Dangyang areas increased year by year,while the Xinzhou and Wuxue areas were decreased.The software was used to analyze the pair-wise correlation coefficient comparison between LC₅₀ values of tested insecticides on field populations of P.xylostella,there is no cross-resistance between indoxacarb and chlorantraniliprole,emamectin benzoate,spinetoram and metaflumizone.The detoxification enzyme activity and correlation analysis showed that the three detoxification enzyme activities were significantly different among the field population of P.xylostella.Furthurmore,the pair-wise correlation analysis indicated significant positive correlation between the LC₅₀values of indoxacarb and detoxifying enzyme activity of glutathione S-transferase?r=0.975,P=0.025?,but carboxylesterase?r=0.535,P=0.456?and cytochrome P450monooxygenase?r=0.082,P=0.918?were not.Therefore,it is speculated that GSTs play an important role in the reduction of the sensitivity of P.xylostella to indoxacarb.2.Detoxification enzyme activities of carboxylesterase,glutathione S-transferase and cytochrome P450 monooxygenase in the susceptible population WH-SS and the resistant population GD-RR were detected.It was found that detoxification enzyme activities of the three detoxification enzymes were significantly higher than the sensitive population in resistant population GD-RR,which increased by 1.83,3.98 and 1.71 folds respectively.And during the selected process,glutathione S-transferase activity increased significantly in the fifth generation.The synergist test results showed that the synergistic ratios of triphenyl phosphate?TPP?,piperonyl butoxide?PBO?and diethyl maleate?DEM?to the field resistance population of P.xylostella were 1.53,2.50 and 3.32 folds respectively.In summary,GSTs may play a major role in the metabolism of indoxacarb.3.The expression levels of 22 GSTs mRNAs related to detoxification were detected.It was found that PxGSTO4 had the highest up-regulation in the field resistance population GD-RR compared with the sensitive population.After silencing PxGSTO4 by RNAi,the GSTs activity of P.xylostella was significantly reduced,the mortality to indoxacarb increased significantly.It is indicated that the overexpression of PxGSTO4 is related to the detoxification and metabolism to indoxcarb in P.xylostella.4.Previous studies showed that 13 transcription factors related to detoxification and metabolism were selected from P.xylostella.Then,the expression levels of 13 genes in susceptible and field resistant populations were compared by quantitative RT-PCR.The expression level of PxCRBL2 was significantly higher than susceptible population.In addition,knockdown of the PxCRBL2 by RNAi,the mortality of P.xylostella to indoxacarb increased significantly and the activity of GSTs was significantly decreased.By detecting changes in the expression of GSTs after silencing,it was found that the expression of PxGSTO4 gene was significantly down-regulated..These results indicated that the transcription factor PxCRBL2 mediates the detoxification metabolism of indoxacarb in P.xylostella by regulating the expression of PxGSTO4.