The Roles Of Cytochrome P450 Monoxygenases In Resistance To Imidacloprid In Nilaparvata Lugens And Sogatella Furcifera

Planthoppers are notorious pests of rice crop throughout Asia,which caused serious economic losses.At present,chemical control is the major way to control planthoppers,which unavoidably lead to resistance to many kinds of insecticides,including organochlorines,organophosphates,carbamates,pyrethroids and neonicotinoids.Under the pressure of insecticide selection,insect could evolve resistance to each insecticide.With spread of insect and widely use of insecticides,insect could accelerate the resistance to new substituted insecticides,and the cross-resistance will become an increasingly serious problem.Molecular mechanism of insecticide resistance included the reduced target sensitivity,which consisted of point mutations and quantitative change,and the enchanced detoxification.Detoxification enzymes,including cytochrome P450 monooxygenases,glutathione S-transferases,and carboxylesterase,could enhance the ability to metabolize insecticides.Cytochrome P450 monooxygenases which exist in nearly all living organism,are one of the largest gene superfamilies.In insects,the physiological functions of P450 involved in biosynthesis and catabolism of endogenous compounds and metabolization of xenobiotic compounds,such as natural products and insecticides.As mix-function oxidases,P450s can metabolize almost all kinds of insecticides.Although point mutations or quantitative change in nicotinic acetylchoine receptors associated with resistance to imidacloprid have been reported in the laboratory-selected resistant strains of Nilaparvata lugens,the mechanisms were not currently prevalent in field populations.In contrast,both in laboratory-selected resistant strains and field populations,P450s played important roles in imidacloprid resistance.In this study,through continuous selection by imidacloprid,a resistant strain was obtained,and the over-expressed P450 genes in the resistant strain were identified.On the other hand,the P450 genes induced by the insecticide treatment were examined in a susceptible strain.The mechanism of P450s in metabolic resistance to neonicotinoid insecticides in planthoppsers and the cross-resistance between the different insecticides were studied using RNAi and functional expression of P450s.1.Expression induction of P450 genes by imidacloprid in Nilaparvata lugensFifty-five P450 genes were identified from genome database of Nilaparvata lugens.Among these P450 genes,only two genes had been confirmed to be related with imidacloprid resistance until now.In this study,the toxicity of imidacloprid against eight field populations of N.lugens was first tested,in which Huzhou and Wuxi populations showed the highest resisitance to imidacloprid,with resistance ratios of 71.83-and 82.26-fold,respectively.PBO significantly synergized imidacloprid in eight field populations,especially in Huzhou population with the synergistic ratio of 3.52-fold,which indicated that P450s played important roles in imidacl oprid resistance in N.lugens.Eighteen P450 genes(eight in Clade 3,eight in Clade 4 and two in Clade 2)in the susceptible strain were up-regulated by imidacloprid,among which eight genes(CYP4CE1,CYP417A1,CYP425B1,CYP6AX1,CYP6AY1,CYP6CS1,CYP6CW1 and CYP6ER1)from CYP3 and CYP4 Clade were increased toabove 2.0-fold,Three genes(CYP6CS1,CYP6CW1 and CYP6ER1)from CYP3 Clade were increased to above 4.0-fold.Eight P450 genes induced to above 2.0-fold were selected to determine the expression and induction levels in Huzhou population.The expression levels of seven P450genes were higher in Huzhou population than that in susceptible strain,with the biggest differences for CYP6CS1(9.8-fold),CYP6ER1(7.7-fold)and CYP6AY1(5.1-fold).The induction levels for all tested genes were bigger in Sus strain than that in Huzhou population except CYP425B1,which indicated that the relevant P450 genes were easilyinduced when their expressions were low in the relatively susceptiblepopulations,but the further induction became difficult when their expression levels were already at the high levels in resistant populations.2.Metabolic resistanceto imidacloprid relies on synergism of multiple P450 enzymesin Nilaparvata lugensAlthough point mutations or quantitative change in nicotinic acetylchoine receptors had been reported to be associated with imidacloprid resistance in the laboratory-selected resistant strains of Nilaparvata lugens,these mechanisms were not currently prevalent in field populations.However,both in laboratory-selected resistant strains and field populations,P450s played important roles in imidacloprid resistance through synergistic experiments.In this study,a N.lugens field population was collected from a paddy field,and was separated into two strains.One strain(G25)was obtained by the continuous selection by imidacloprid in laboratory for 25 generations,and the counterpart(S25)was reared without contacting any insecticide.There was no significant synergistic effect of three synergists in S25.However,PBO and TPP significantly synergized imidacloprid in G25,with the synergistic ratio of 5.88-fold for PBO,which indicated that P450s played important roles in imidacloprid resistance.Compared to S25,the over-expression of twelve P450 genes(six in CYP4 Clade,five in CYP3 Clade,and one in Mito Clade)was observed in G25,with ratios above 5.0-fold for CYP6AY1,CYP6ER1,CYP6CS1,CYP6CW1,CYP4CE1 and CYP425B1.In G25,RNAi against CYP6AY1,CYP6ER1,CYP6CW1 and CYP4CE1 decreased the imidacloprid resistance.The recombinant P450s of CYP6AY1,CYP6ER1,CYP6CW1 and CYP4CE1 could metabolize imidacloprid efficiently,although the enzymatic activity was different among enzymes.In order to make clear of the roles of thsee four P450 genes in imidacloprid resistance at different resistance development stages,their expression levels in 25 successive selection generations were tested.The results indicated that CYP6ER1 was important at all stages,and others might only contribute to resistance at certain stages.The results indicated that,to completely reflect roles of P450s in insecticide resistance,their over-expression in resistant individuals,expression changes at the stages of resistance development,and catalytic activities against insecticides should be co-considered.3.Induction of P450 genes in Nilaparvata lugens and Sogatella furcifera by two neonicotinoid insecticidesNilaparvata lugens and Sogatella furcifera are two primary planthoppers on rice and often concur in rice paddy fields.Imidacloprid was used extensively to control these two planthopper species,and imidacloprid resistance was observed in both planthoppers.So,to develop the substituted insecticide for planthopper control is urgent.Cycloxaprid,a novel cis-neonicotinoid insecticide,shows high efficiency against to the imidacloprid-resistant and susceptible populations of planthoppers.It was suggested that cycloxaprid was hydrolyzed to nitromethyleneimidazole(NMI),which then showed its toxicity against insects.In this study,the induction of P450 genes was compared in S.furcifera following treatment by IMI and nitromethyleneimidazole(NMI),in two planthopper species by IMI at LD85 dose,and in N.lugens among three IMI doses(LD15,LD50 and LD85).When IMI and NMI at LD85 dose were applied to S.furcifera,the expression changes in most P450 genes were similar,including the up-regulation of nine genes and down-regulation of three genes.In terms of the expression changes in 12 homologous P450 genes between N.lugens and S.furcifera treated with IMI at LD85 dose,10 genes had very similar patterns,such as up-regulation in seven genes,down-regulation in one gene and no significant changes in two genes.When three different IMI doses were applied to N.luge,ns,the changes in P450 gene expression were much different,such as up-regulation in four genes at all doses and dose-dependent regulation of other genes.For example,CYP6AY1 could be induced by all IMI doses,while CYP6ER1 was only up-regulated by LD50 dose,although both genes were reported important in IMI resistance.In conclusion,P450 genes in two planthopper species showed similar regulation patterns in responding to IMI,and the two neonicotinoid insecticides had similar effects on P450 gene expression,although the regulation was often dose-dependent.4.No cross-resistance between imidacloprid and pymetrozine in the Nilaparvata lugens:status and mechanismsImidacloprid was widely used in N.lugens control because of its efficacy and relatively safe to mammals.However,imidacloprid was currently suspended to control N.lugens due to the high resistance,and consequently another insecticide,pymetrozine,has been widely used to control N.lugens in recent years.To investigate the possible cross-resistance between imidacloprid and pymetrozine is very important to avoid the adverse effects on the resistance development and insect pest control.Bioassays of N.lugens populations in two areas in five consecutive years showed that imidacloprid resistance decreased greatly,while pymetrozine resistance increased significantly.PBO could synergize both imidacloprid and pymetrozine in resistant populations,which indicated the importance of P450s in the resistance to two insecticides.Imidacloprid resistance was reported to be associated with two P450s,CYP6AY1 and CYP6ER1,which could metabolize imidacloprid efficiently.However,the recombinant proteins of these two P450s did not show any enzymatic activity to metabolize pymetrozine.The pymetrozine susceptibility did not change when CYP6AY1 and CYP6ER1 mRNA levels were reduced by RNAi,although which could obviously decrease imidacloprid resistance.In vivo and in vitro studies provided evidences to demonstrate that there was no cross-resistance between imidacloprid and pymetrozine in N.lugens,which was different from the findings in Bemisia tabaci.