Study On Sulfoxaflor Resistance And Its Mechanism In Nilaparvata Lugens(St?l)

The brown planthopper,Nilaparvata lugens?St?l?,is a major rice pest in China and in some other Asian countries.It causes great economic losses by serious damages every year.At present,the application of insecticides remains the principal method for N.lugens control.However,due to heavy and frequent application of insecticides,the species has evolved different levels of resistance to many of the major classes of insecticides.Sulfoxaflor is the initial commercial product from the new sulfoximine class of insect control agents and it's also the first pesticide on the market synchronously with the world in China's history.Sulfoxaflor exhibits a high degree of efficacy against a wide range of sap-feeding insects and has great potential in insecticide resistance management of N.lugens.In order to delay sulfoxaflor resistance development and prolong the application effective of this insecticide in the field,sulfoxaflor resistance monitoring and correlation analyses of insecticides in the field populations of N.lugens were studied.In the present study,the sublethal effects of sulfoxaflor on the N.lugens were evaluated via the age-stage,two-sex life table procedure.Additionally,a sulfoxaflor-resistant strain was obtained from a field population by successive selection with sulfoxaflor in the laboratory,and the risk of resistance development,the cross-resistance spectrum,inheritance of resistance,fitness costs and the metabolic mechanisms of sulfoxaflor resistance in N.lugens were investigated.The main results of this research are as follows:1.Sulfoxaflor resistance monitoring in the field populations of N.lugens in ChinaThe susceptibilities of fifty-two field populations of N.lugens to sulfoxaflor and nine other frequently used insecticides were measured in nine provinces of China from2013 to 2017 by using the rice-seedling dip method.The results showed that the LC₅₀values of field populations of N.lugens to sulfoxaflor varied from 1.63 to 15.36 mg/L,revealing that N.lugens has developed a low level of resistance?resistance ratio,RR=0.8-8.0-fold?to sulfoxaflor compared with the labortory susceptible strain(LC₅₀=1.93mg/L).Among fifty-two field populations,84.62%field populations remained susceptible and 15.38%field populations showed low level of resistance to sulfoxaflor.More importantly,the resistance levels of N.lugens to sulfoxaflor showed an increasing trend from 2013 to 2017.The susceptibility of field populations of N.lugens to sulfoxaflor was significantly correlated with the responses of N.lugens to imidacloprid,nitenpyram,dinotefuran,thiamethoxam,clothianidin and to buprofezin.This study reveals the current resistance levels of N.lugens to sulfoxaflor in China and implies a potential cross resistance pattern which need to be taken into account in the rational use of this new insecticide in N.lugens resistance management programs.2.Sublethal effects of sulfoxaflor on N.lugensIn the present investigation,the sublethal effects of sulfoxaflor on the N.lugens were evaluated via the age-stage,two-sex life table procedure.Our results demonstrated that the adult longevity and fecundity values of F₀ individuals were significantly lower in the LC₃₀ group than in the control group.Compared with those of the control group,the preadult period;female adult period;total preoviposition period?TPOP?;longevity;fecundity;hatchability;and development time of 1^st,3^rd,and 4^th instar nymphs of the F₁individuals of the LC₃₀ group were significantly reduced,whereas no significant differences were found between the LC₁₅ group and the control group.The development time of 5^th instar nymphs was significantly shorter in both the LC₁₅ and LC₃₀ groups than in the control group.However,the development time of 2^nd instar nymphs and the adult preoviposition period?APOP?were significantly longer in the LC₁₅ group than in the control group.In addition,the net reproductive rate?R₀?,mean generation time?T?and gross reproduction rate?GRR?were significantly lower in the LC₃₀ group than in the control group.However,the intrinsic rate of increase?r_i?and finite rate of increase???values were not influenced by low lethal concentrations of sulfoxaflor.These results indicated that the sublethal effects of sulfoxaflor reduce the survival and reproductive capability of N.lugens in successive generations.Our findings would be useful for assessing the overall effects of sulfoxaflor on N.lugens.3.Resistance selection and biochemistry mechanism of sulfoxaflor resistance in N.lugensA sulfoxaflor-resistant strain?RR=183.63-fold?was obtained in the laboratory by successive selection with sulfoxaflor for 39 generations from a field population.Sulfoxaflor-resistant populations showed significant levels of cross-resistance to dinotefuran,nitenpyram,thiamethoxam,clothianidin,imidacloprid,and cycloxaprid.However,they exhibited only minor or no cross-resistance to isoprocarb,etofenprox,chlorpyrifos,triflumezopyrim,and buprofezin.Sulfoxaflor was synergized by the inhibitor piperonyl butoxide?PBO?and triphenyl phosphate?TPP?in SFX-SEL with2.69-fold and 1.48-fold relative synergistic ratios compared with UNSEL,respectively.The cytochrome P450 monooxygenase?P450?and esterase?EST?activities in SFX-SEL(G₃₉)were significantly higher than in UNSEL?3.50-and 1.85-fold respectively?.The results indicated that the enhanced detoxification of P450 and EST enzymes is associated with sulfoxaflor resistance,and the P450 may play a more important role.4.Inheritance and fitness costs of sulfoxaflor resistance in N.lugensTo determine the inheritance mode and fitness costs of sulfoxaflor resistance in N.lugens,reciprocal crosses and age-stage,two-sex life table were performed between the SFX-SEL and SS strains.Progenies of reciprocal crosses?F₁RS and F₁SR?showed similar dose-mortality responses(LC₅₀)to sulfoxaflor and also exhibited a similar degree of dominance?D?,-0.16 for F₁RS and-0.26 for F₁SR,and ranged between-1 and 0,indicating that sulfoxaflor resistance in N.lugens was inherited as autosomal and incompletely recessive.Significant differences between observed and expected mortalities of F₂ individuals suggested that sulfoxaflor resistance is associated with multiple genes.The resistant strain had a relative fitness of 0.75 with a substantially decreased female adult period,oviposition days,total fecundity,egg hatchability and female adult survival rate,and prolonged pre-adult period and total pre-oviposition period?TPOP?.The results indicated that development of the resistance may have a significant fitness cost for the resistant population.5.Transcriptome analysis of resistant and susceptible strains of N.lugensIn order to obtain the comprehensive transcriptome of resistant and susceptible strains of N.lugens,and excavate resistance genes contributied to sulfoxaflor resistance in N.lugens,the cDNA library was constructed using the RNA extracted from third-instar nymphs and generated by Illumina.A total of 372133456 clean reads were obtained.The results showed that a total of 466 genes were significantly upregulated more than 2.0-fold and 178 genes were significantly downregulated more than 2.0-fold in resistance strain compared with susceptible strain.The results of GO enrichment showed that the different expression genes were significantly enriched in“structural constituent of cuticle”,“generation of precursor metabolites and energy”,“nucleotide phosphorylation”,“ATP generation from ADP”,“purine nucleoside diphosphate metabolic process”and“pyridine-containing compound metabolic process”.Combined the results of biochemistry mechanism study,a total of twenty-seven carboxylesterase?CarE?genes and fifty-two cytochrome P450 monooxygenase?P450?genes were identified from our transcriptome database,and two CarE and three P450 genes were significantly upregulated in resistance strain compared with susceptible strain.The P450gene CYP6ER1 showed a remarkably high expression level with 18.70-fold change,which predicted that CYP6ER1 maybe the key resistance candidate gene of sulfoxaflor resistance in N.lugens.6.Identification and functional research of P450 genes involved in sulfoxaflor resistance in N.lugensCombined the results of transcriptome and biochemistry mechanism of sulfoxaflor resistance,the mRNA expression levels of 54 P450 genes in resistant and susceptible strains were analysed by qRT-PCR.The results showed that eight P450 genes?CYP15G1,CYP6CS1,CYP6CW1,CYP6ER1,CYP4C62,CYP4DE1,CYP417A1 and CYP419A1?were upregulated more than 2.0-fold in SFX-SEL compared with UNSEL,among which CYP6ER1 showed a remarkably high expression level with a 36.87-fold change.The expression levels of CYP6ER1 in SFX-SEL compared to UNSEL during the resistance selection showed a similar increasing trend with resistance development.RNAi reduced the expression of CYP6ER1 and significantly enhanced the susceptibility of SFX-SEL to sulfoxaflor.These results suggest that the enhanced detoxification of P450 enzymes caused by upregulation of several P450 genes is considered to be the metabolic resistance mechanism and CYP6ER1 might play an important role in sulfoxaflor resistance in N.lugens.