Risk Assessment Of Resistance To Clothianidin And Its Metabolic Resistance Mechanism In White-backed Planthoppe

The white-backed planthopper,Sogatella furcifera（Horváth）is a migratory rice pest,which is very destructive to the growth of rice.At present,the main measure to control S.furcifera is chemical control,but due to the unreasonable use of pesticides for a long time,S.furcifera has developed different degrees of resistance to different kinds of pesticides.Clothianidin,as the second generation of neonicotinoid insecticide,is widely used to control S.furcifera on rice.In order to evaluate the resistance risk of S.furcifera to clothianidin and delay the development of resistance of S.furcifera to clothianidin,scientific and effective pest control and resistance management strategies were formulated.In the present study,the resistance levels of field populations of S.furcifera in Guizhou Province to clothianidin and other commonly used insecticides were monitored.In addition,the clothianidin-resistance strain was obtained from the continuous screening of a field population with clothianidin in the laboratory using the rice seedling impregnation method,the realistic heritability and the cross-resistance spectrum of resistance of clothianidin-resistance strains were studied.The sublethal effects of clothianidin on S.furcifera were evaluated via the age-stage,two-sex life table procedure.The effects of clothianidin on the activity of P450 enzyme and the expression of P450 gene in S.furcifera,and the up-regulated expressions of CYP4CE3 and CYP6FJ3 in the resistance of S.furcifera to clothianidin metabolism were studied.The main research results are as follows:1.Monitoring the insecticide resistance of field population of S.furcifera in GuizhouThe resistance levels of field populations of S.furcifera to clothianidin and 12 other commonly used insecticides were monitored in 5 regions of Guizhou Province from 2021 to 2022by rice seedling impregnation method.The results showed that all field populations of S.furcifera had high level of resistance to chlorpyrifos（Resistance Ratio,RR=205.5-456.7-fold）,and the resistance levels of the other populations except for Anlong population increased significantly.S.furcifera had moderate to high levels of resistance to etofenprox（RR=19.9-106.6-fold）and buprofezin（RR=64.6-148.3-fold）,and moderate levels of resistance to acetamiprid（RR=19.4-53.1-fold）.S.furcifera had low to moderate levels of resistance to imidacloprid（RR=5.3-20.5-fold）,dinotefuran（RR=7.5-11.7-fold）,isoprocarb（RR=5.5-13.7-fold）,thiamethoxam（RR=4.6-11.7-fold）and sulfoxaflor（RR=4.0-11.2-fold）.In addition,the resistance levels of S.furcifera to nitenpyram（RR=2.3-5.9-fold）and clothianidin（RR=2.9-9.5-fold）remained susceptible to low levels.S.furcifera remained susceptible to cycloxaprid（RR=0.5-2.3-fold）,triflumezopyrim（RR=0.9-1.8-fold）.Therefore,chlorpyrifos,etofenprox and buprofezin should be used in limited quantities,and the insecticides with higher sensitivity such as clothianidin,nitenpyram,cycloxaprid and trifluoropyrimidine can be used alternately in the field population control of S.furcifera in Guizhou.2.Resistance risk assessment and cross-resistance of S.furcifer to clothianidinA field population of S.furcifera collected from Huishui County,Guizhou Province in 2020was used as the starting population.The clothianidin-resistant strain（CL-R）was obtained,and the field population was continuously screened with clothianidin for 24 generations in the laboratory using the rice seedling impregnation method.The resistance ratio of clothianidin-resistant strain（CL-R）was 10.2-fold（moderate resistance level）,and the LC₅₀of the first screened strain（G₀）was increased from 1.52 mg/L to 15.59 mg/L.The realistic heritability（h²）of resistance was 0.0980when the selection pressure was 80%,it was necessary to screen continuously for 21 generations to predict the 10-fold increase of the resistance ratio of S.furcifera to clothianidin,indicating that S.furcifera had certain resistance risk to clothianidin.The results of cross-resistance test showed that when CL-R developed 10.2-fold resistance to clothianidin,it showed low level cross-resistance to isoprocarb（CR=6.0-fold）and sulfoxaflor（CR=5.9-fold）,and more than 3-fold cross-resistance to imidacloprid（CR=4.7-fold）,nitenpyram（CR=4.3-fold）,thiamethoxam（CR=4.2-fold）,dinotefuran（CR=3.7-fold）and trifluoropyrimidine（CR=3.2-fold）.However,there was no significant cross-resistance to chlorpyrifos（CR=2.3-fold）,cycloxaprid（CR=2.1-fold）,buprofezin（CR=2.0-fold）and acetamiprid（CR=1.1-fold）.The above results indicated that single and continuous use of clothianidin as well as its mixing and alternate use with isoprocarb and sulfoxaflor should be avoided for controlling S.furcifera in the field,thus slowing the development of resistance to clothianidin and cross-resistance to other insecticides in S.furcifera.3.Sublethal effect of clothianidin on S.furciferaThe sublethal effects of clothianidin on the biological traits and population parameters of S.furcifera were evaluated via the age-stage,two-sex life table procedure.The results showed that the female adult longevity,fecundity and hatchability of F₀generation were significantly decreased after LC₁₀and（or）LC₃₀of clothianidin exposure compared to the control.Transgenerational effects showed that the pre-adult period,female adult longevity,total longevity,oviposition days（Od）,fecundity and mean generation time（T）of F₁generation were significantly decreased in the LC₁₀and LC₃₀groups compared to the control.Moreover,the development times of the third-and fifth-instar nymphs,total preoviposition period（TPOP）and doubling time（DT）were significantly shorter in the LC₁₀group than in the control and LC₃₀groups.Furthermore,the intrinsic rate of increase（r_i）and finite rate of increase（λ）values of the LC₁₀group were significantly higher than those of the control group.However,there was no significant difference in the male adult longevity,adult preoviposition period（APOP）and net reproductive rate（R₀）between the treated groups and the control.The above results showed that the sublethal concentration of clothianidin had a certain stimulating effect on the development rate of nymphae of S.furcifera,but it had a negative effect on its longevity and fecundity.4.Mechanism of metabolic resistance of S.furcifer to clothianidin mediated by cytochrome P450 oxidaseThe results of P450 enzyme activities,RT-q PCR,RNAi and biological activity assay showed that the treatment of clothianidin LC₁₀and LC₃₀could significantly enhance the P450 enzyme activity of susceptible strains of S.furcifera,and a total of 18 P450 genes were significantly induced to express,among which CYP4CE3 and CYP6FJ3 were significantly induced to increase the expression level.The CYP4CE3(22.4 and 28.8-fold for LC₁₀and LC₃₀treatments,respectively)and CYP6FJ3(13.4 and 22.8-fold for LC₁₀and LC₃₀treatments,respectively)showed the highest m RNA expression level in the both of LC₁₀and LC₃₀treatment groups compared with control.After successfully interfering with the expression of the above two P450 genes,the detoxification ability of S.furcifera to clothianidin decreased significantly.In addition,the activity of P450 in the resistant strain was significantly higher than that in the sensitive strain（1.67-fold）,and the expression levels of CYP4CE3 and CYP6FJ3 in CL-R were increased 4.88-fold and 3.96-fold respectively than that in the sensitive strain.When the expression levels of CYP4CE3 and CYP6FJ3 were inhibited,the sensitivity of insect resistance test to clothianidin increased significantly.These results indicated that P450 multifunctional oxidase and its key genes CYP4CE3 and CYP6FJ3 were involved in the detoxification process of clothianidin by S.furcifera and the evolution of resistance.