Metabolic Resistance And Regulation Mechanism Of Nilaparvata Lugens (ST(?)L) Against Clothianidin

The brown planthopper（BPH）,Nilaparvata lugens（St（?）l）（Hemiptera:Delphacidae）is one of the most destructive rice pests in Asian countries and causes serious economic losses to rice production in China every year.At present,the control of brown planthopper still mainly relies on chemical insecticides,but the resistance of brown planthopper has become increasingly prominent due to the large use of insecticides,and has developed varying levels of resistance to a variety of insecticides.Neonicotinoid insecticides,including clothianidin,have been widely used to control brown planthoppers in rice fields,but the resistance of brown planthoppers to clothianidin has increased in the field.Therefore,it is of significance to clarify the evolution mechanism of brown planthopper resistance to clothianidin for designing high-quality resistance management strategies,extending the application period of the insecticide and ensuring the safe production of rice.Based on this,in this study,the resistance levels of brown planthopper to clothianidin in field populations from different geographical locations of China were determined.A clothianidin-resistant strain of brown planthopper was obtained in laboratory with successive selection,and the cross-resistance spectrum,resistance inheritance mode and fitness cost were invstigated.Furthermore,the metabolic resistance mechanism of brown planthopper to clothianidin was exploered by a series of physiological,biochemical and molecular biology techniques.The main research results are listed as follows:1.Monitoring of resistance to clothianidin in the field population of N.lugensDuring 2018-2020,the resistant levels of BPH field populations in 14 regions of 7provinces in China to clothianidin were monitored by rice seedling dip method.The results showed that the brown planthoppers in most areas had developed high levels resistance to clothianidin,of which 20 populations（76.92%）developed high levesl resistance wheras 6（23.08%）exhibited moderate levels of resistance.Comprehensive analysis of the resistance level of brown planthoppers to clothianidin from 2012 to 2020showed that the resistance of brown planthoppers to clothianidin in field populations in China exhibited a growing tendency.2.Biochemical mechanism and related biological characteristics of clothianidin resistance in N.lugensThe clothianidin-resistant strain CLR（RR=199.09）was obtained by continuous clothianidin treatment from an indoor susceptible strain CLS.Detection of cross-resistance showed that the CLR strain exsited cross-resistance to imidacloprid,thiamethoxam,nitenpyram,sulfoxaflor,dinotefuran,cycloxaprid and isoprocarb,respectively,but no cross-resistance was observed to chlorpyrifos,buprofezin and triflumezopyrim,respectively.Through the reciprocal cross between the CLR and the CLS strains,it was revealed that the resistance of N.lugens to clothianidin was inherited as an autosomal trait with incomplete dominance.There was a significant difference between the observed and the expected mortality of the F₂ generation obtained by self-cross and back-cross of the F₁generation,indicating that the resistance of BPH to clothianidin was controlled by multiple genes.The two-sex life table was used to compare the CLS and CLR strains having the same genetic background.The results showed that compared with CLS strain,the development duration of the fourth-instar nymphs and APOP of CLR were significantly prolonged,the fecundity of female adults was significantly decreased,and the relative fitness was 0.78,indicating that the resistance of N.lugens to clothianidin was accompanied by fitness cost.The synergist experiment showed that only PBO had synergistic effect on clothianidin in the CLR strain.The enzyme activities of detoxification enzymes in the clothianidin resistant and susceptible strains of BPH also showed that only the activity of cytochrome P450 significantly enhanced in the CLR strain,indicating that P450 played a key role in the resistance of BPH to clothianidin.3.Identification of clothianidin resistance related P450 genes in N.lugensTranscriptome sequencing of clothianidin resistant and susceptible strains showed that a total of 1878 transcripts were differentially expressed in the resistant and susceptible strains,of which 1169 transcripts were highly expressed in the CLR library and 709 transcripts were lowly expressed in the CLR library.The expression levels of fifty-four P450 genes of N.lugens were analyzed by quantitative real-time PCR.The results showed that CYP6ER1 was significantly overexpressed in the CLR strain,and the up-regulated fold was 14.25-fold,which was consistent with RNA-seq.Result of RNAi showed that when the expression of CYP6ER1 in the CLR strain was knocked down,the susceptibility of BPH to clothianidin was restored,and the results confirmed the role of CYP6ER1 in the development of N.lugens resistance to clothianidin.Further studies showed that CYP6ER1 in CLR encoded two polymorphic sequences,however,there is no significant difference in the susceptibility of transgenic Drosophila melanogaster overexpressing different variants,indicating that CYP6ER1 polymorphism in the CLR had no effect on clothianidin resistance.4.Transcriptional regulation mechanism of CYP6ER1 overexpression in the clothianidin-resistant strain of N.lugensTo explore the transcriptional regulation mechanism of CYP6ER1,six transient expression vectors of the CYP6ER1 promoter with different lengths were constructed and their fluorescence activity was detected.The results showed that-1388 bp～-1208 bp of the CYP6ER1 promoter was an important binding region of transcription factors.Based on online prediction,the expression levels of Nl AP-1,Nl BR-C,Nl Cnc C,Nl CEBP and Nl Maf K in the CLR and CLS were compared,and the transcription factor Nl AP-1 was significantly overexpressed in the CLR.After knockdown of Nl AP-1 in the CLR,susceptibility to clothianidin significantly restored and the expression of CYP6ER1 was significantly down-regulated.Further promoter activity experiments showed that Nl AP-1significantly enhanced the promoter activity of CYP6ER1,and both predicted binding sites could interact with Nl AP-1.The results elucidated the transcriptional regulatory mechanism of CYP6ER1 overexpression in the clothianidin-resistant strain of N.lugens.Our Studies provided a theoretical basis for revealing the molecular mechanism and regulatory mechanism of N.lugens resistance to clothianidin.