| The evolution of insecticide resistance by insects threatens human welfare through its impact on crop protection and disease transmission.The introduction of new insecticides has resulted in a rapid increase in resistance,with many species now resisting several classes of insecticide.Understanding the molecular basis of resistance mechanisms,including discovering the function of resistance-related genes,is critical to designing novel resistance management strategies,and developing new insecticidal compounds.Rice is a staple food to more than 50%of the global population.The small brown planthopper,Laodelphax striatellus Fallen(Hemiptera:Delphacidae),is a serious pest in some rice growing areas,caused a huge decrease in rice production every year by sucking plant sap,as well as transmitting plant virus diseases.In the present work,we studied the mechanism of insecticide resistance,cross-resistance,multiple resistance and related gene function.This research provides us with vital information for insecticide resistance prediction and management for L.striatellus.1.Molecular mechanisms of ethiprole resistance in L.striatellusIn this study the ethiprole resistance mechanism was investigated in a field population that is highly resistant to ethiprole.This population was used to establish a laboratory population that was subjected to further selection to produce a resistant strain.Target genes were cloned,and comparisons were made between the resistant and the susceptible strains.The role of detoxification enzymes was examined,and the relative expression levels of 71 detoxification enzyme genes were tested using quantitative real time PCR.The laboratory selection enhanced the resistance from 107-fold to 180-fold.The reported Rdl-type target site mutation(A283N)seldom occurred in the resistant strain(1/4)and is unlikely to represent the major mechanism underlying the observed resistance.Of the 3 kinds of important detoxification enzymes,only P450 monooxygenase was found to be associated with ethiprole resistance.Moreover,2 genes,CYP4DE1 and CYP6CW3v2,were found to be overexpressed in the resistant strain(4.5 and 12.9 times).Furthermore,gene-silencing via a dsRNA feeding test was employed,and the results showed that the mRNA levels of CYP4DE1 and CYP6CW3v2 were reduced in the resistant strain(by 50%and 70%),whereas ethiprole susceptibility was increased(mortality increased from 35%and 35%to 78%and 81%).These results suggest that CYP4DE1 and CYP6CW3v2 play an important role in ethiprole resistance in L.striatellus.2.Cytochrome P450 associated with imidacloprid resistance in L.striatellusImidacloprid has been used as the principal insecticide for controlling rice planthoppers worldwide.Resistance to imidacloprid has been reported in a field population of L.striatellus.Thus,for good resistance management,the molecular mechanisms for imidacloprid resistance were studied.An imidacloprid-resistant strain was produced by selecting a field population with imidacloprid for 24 generations(RR increased by 13.99 times),synergism and enzyme activity assays showed that cytochrome P450 monooxygenase might associate with the imidacloprid resistance.Then,the mRNA expression levels of 57 P450 genes were compared between resistant and susceptible strains.Four CYP genes,CYP4C71v2,CYP4C72,CYP6AY3v2 and CYP353Dlv2,were found to be significantly over-expressed in the resistant strain(1.67,7.96,8.94 and 1.82 times,respectively).Furthermore,with four different resistant strains(Sus,Chlor-R,Delt-R and field strains)as original,imidacloprid resistance were selected for two generations,and then the expression levels of 10 identified detoxification genes were compared.Only the expression level of CYP353Dlv2 was found significantly increased,as compared to the original(2.89,1.82,2.88 and 6.75 times,respectively),which implies a strong correlation between CYP353Dlv2 expression levels and imidacloprid treatments.Additionally,experiments proved that the gene-silencing RNAi via a dsRNA feeding could depress the expression of CYP353Dlv2(70%,53%,56%and 66%expression,respectively),and significantly enhance the sensitivity of L.striatellus to imidacloprid,with the same dose mortality for the 4 strains increased from 30.9-31.4%to 61.7%,from 32.4?37.8%to 72.5%,from about 42.8%to 69.6%,and from 33.3-36.6%to 59.0%.Our study revealed the constitutive overexpression of four CYP genes associated with imidacloprid resistance in long term selection,whereas CYP353Dlv2 in short term selection in L.striatellus.3.Cross-resistance among common insecticides and its possible mechanism in L.striatellusThis study was conducted to assess the cross-resistance among common insecticides and its mechanism in L.striatellus.The selections with chlorpyrifos,deltamethrin and imidacloprid were conducted and both field population and a mixed resistant strain were employed as original to evaluate the cross-resistance.The mechanism was analyzed by comparing the expression levels of detoxification enzyme genes in the susceptible and the selected resistant strains.The results demonstrated that cross-resistance existed among the insecticides with different modes of action,chlorpyrifos,deltamethrin and imidacloprid.The Delt-R strain selected fron field population showed 264 times resistance to deltamethrin,and meanwhile 49 and 24 times to chlorpyrifos and imidacloprid.When the Mix strain was selected with deltamethrin for 7 generations,the deltamethrin resistance increased from 52 fold to 179 fold.Meanwhile,its resistance to chlorpyrifos increased from 32 to 118 fold,and to imidacloprid from 18 to 69 fold.Similar situation appeared in the strains selected with chlorpyrifos and imidacloprid.The detoxification enzyme genes CYP6AY3v2,CYP6FU1,CYP353Dlv2,and CYP439A1v3 overexpressed in the Deltamethrin-selected strain(1.8,2.0,1.8 and 4.5 folds,respectively);CYP6AY3v2,CYP306A2v2 and CYP353D1v2 were found to be up-regulated in the Chlorpyrifos-selected strain(1.8,1.9 and 4.0 folds);and CYP4C72,CYP6AY3v2 and CYP353D1v2 up-regulated in the Imidacloprid-selected strain(1.8,1.9 and 4.0 folds).The overexpression of CYP6AY3v2 and CYP353DIv2 present in all three resistant strains selected with different insecticides and was in concert with cross-resistance among these strains.These results suggest that CYP6AY3v2 and CYP353Dlv2 might be associated with the cross-resistance among chlorpyrifos,deltamethrin and imidacloprid in L.striatellus.4.Cytochrome P450 CYP353D1v2 functioning as metabolic enzyme degrading different insecticides in L.striatellusCytochrome P450s are associated with metabolizing a wide range compounds including insecticides.CYP353Dlv2 is associated with the resistance to different insecticides in L.striatellus.Thus,this study was conducted to express CYP353Dlv2 in Sf9 cell as recombinant protein,in order to assess its ability to metabolize imidacloprid and chlorpyrifos.The results indicate that CYP353Dlv2 was functionally expressed in insect cell Sf9.Moreover,the enzyme activity for degradation of imidacloprid and chlorpyrifos in vitro was tested by measuring substrate depletion and analyzing the formation of metabolites.NADPH-dependent depletion of imidacloprid and chlorpyrifos(eluting at 2.3 and 9.7 min respectively)and paralleled formation of an unknown metabolite(eluting at 13.6 and 1.4 min respectively),were observed after incubating the compounds separately with the P450 complex.Incubations carried out in the absence of a NADPH regenerating system showed no change in the control chromatogram of the parental imidacloprid and chlorpyrifos molecules.These results suggested that CYP353Dlv2 potentially metabolized imidacloprid,as well as chlorpyrifos.This result supports the role of CYP353Dlv2 in the reported deltamethrin,chlorpyrifos and imidacloprid resistance,and indicates that CYP353Dlv2 may have a specialized role in cross-resistance among these insecticides.In conclusion,CYP353Dlv2 has been expressed in Sf9 insect cells,and was proved to catalyse the degradation of imidacloprid and chlorpyrifos,which demonstrated that CYP353Dlv2 functions in cross-resistance among insecticides in L.striatellus.5.Multiple insecticides resistance and overexpression of multiple detoxification genes in the field populations of L.striatellus FallenInsecticide resistance is the most important phenomenon that threatens sustainable pest management.Thus,Knowledge of the resistance occurs in field is necessary for insecticides resistance management.This study was conducted to assess the resistance to Avermectins,chlorpyrifos,deltamethrin,imidacloprid and ethiprole in three field populations of L.striatellus,and its relation with the resistance related detoxification genes identified previously in laboratory strains.Topical application bioassay and quantitive RT-PCR were used.The results indicated that all the three population showed multiple resistance to mentioned insecticides,but the resistance level to different insecticides varied with populations.Jianhu population was high resistant to chlorpyrifos,deltamethrin,imidacloprid(302,114 and 53 fold,respectively),but low to avermectins and ethiprole(7 and 4 fold).Jining population showed high and moderate resistance to chlorpyrifos,ethiprole and imidacloprid(170,107 and 23 fold),and Wujiang population to chlorpyrifos,deltamethrin and ethiprole(164,29 and 14 fold).Among the 15 genes tested,13 resistance related genes found in laboratory strains all over-expressed in one or more field populations.For different populations,the number of over-expressed genes(11,8 and 11),the highest expressed gene(CYP439Alv3 in Jianhu,CYP6A3v2 in Jining and CYP314Alv2 in Wujiang)and their expression level were different(50.6,11.0 and 4.4).In conclusion,our study revealed that field populations of L.striatellus with different multiple resistances spectrum usually varied in their over-expression of detoxification genes,the resistance related genes identified previously could be functioned in field populations,and studies with laboratory selected strains could provide valuable information. |
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