Studies On Chlorpyrifos Resistance Mechanisms In Nilaparvata Lugens(St(?)l)

The brown planthopper,Nilaparvata lugens(stal),is a major insect pest of rice crops,and its high frequency outbreak often leads to severe loss of rice production and quality.Chemical control has thus far been the primary strategy used to manage N.lugens.However,N.lugens has developed resistance towards many kinds of insecticides.Given its high levels resistance,imidacloprid application has been dramatically reduced and is not recommended for the wide use in controlling N.lugens in China since 2006,and usage of fipronil for pest control in rice crops is currently banned in China due to its high toxicity to non-target animal since 2009.Pymetrozine,an azomethine pyridine insecticide,has been commonly used for N.lugens control in China recently.Meanwhile,to provide more choices in available insecticides in N.lugens control and decrease the stress in resistance development to pymetrozine,insecticides belonging to organophosphate or carbamate classes have also been used in China in recent years,such as chlorpyrifos.Nevertheless,the field resistances have also been reported to pymetrozine and chlorpyrifos.Insecticide resistance-associated fitness cost is a key factor for the insecticide resistance management,which are frequently affected by certain environment factors.The influence mechanism of environment factors on fitness costs and the resistance mechanisms in the chlorpyrifos selected strain of N.lugens are still not clear.Present study developed a chlorpyrifos resistance strain through continuous chlorpyrifos selection.Using this resistant strain,we investigated biochemical and molecular mechanisms of clorpyrifos resistance and determined the effects of temperatures on fitness costs in the resistant strain.1.Point mutation in acetylcholinesterase 1 confered chlorpyrifos resistance in the brown planthopperA laboratory strain R9 was obtained through 9-generation selection,with 253.08-fold resistance to chlorpyrifos.S9 was a susceptible counterpart(resistance ratio=2.25-fold)from the same origin as R9,but without any selection with insecticides.PBO(piperonyl butoxide)and TPP(triphenyl phosphate)significantly synergized chlorpyrifos in R9,but the synergistic ratio(SR)was less than 3-fold,indicating other important mechanisms for the high resistance.The target-site insensitivity was supported by the key property differences of crude of acetylcholinesterases(AChEs)between R9 and S9.Compared to S9,three mutations(G119S,F331C and I332L)were detected in NlAChEl from individuals of R9 and field populations,but no mutation was detected in NlAChE2.G119S and F331C could decrease insecticide sensitivities in recombinant NlAChE1,while I332L took effect through increasing the influence of F331C on target insensitivity.F331C might be deleterious because of its obvious influence on the catalytic efficiency of NlAChE1,whereas I332L would decrease these adverse effects and maintain the normal function of NlAChE1.2.The roles of carboxylesterases(CarEs)and cytochrome P450 enzymes in chlorpyrifos resistance development in the brown planthopperIn order to explore the key genes of detoxifying enzymes within CarEs and P450s,A chlorpyrifos-resistance R16 strain of N.lugens was selected through another 7-generation chlorpyrifos exposure for R9,with the increased number of RR from 253.08 to 404.33.Meanwhile,a chlorpyrifos-sensitive counterpart S16 strain of N.lugens was obtained without any selection with insecticides,with the reduced number of RR from 2.25 to 1.50.Two synergistic agents,TPP and PBO,significantly synergized chlorpyrifos toxicity in R16,with a SR of 2.55-fold and 6.34-fold respectively,while DEM showed no any synergism,with a 1.11-fold SR.These dates confirmed CarEs and P450 enzymes play important roles in chlorpyrifos metabolic resistance mechanism.11 CarE genes and 38 P450 genes(belong to CYP 3 and CYP4 clan)of BPH were retrieved from NCBI gene databases.The primers were synthesized for further Real-time fluorescence quantification.Fold change of relative quantity of these genes in R16 were calculated,compared to these number in S16.The resulted indicated 4 genes for CarEs and 9 genes for P450s were overexpressed,among which N1XP₀22186544.1 gene,belonged to?-esterase for CarEs,ECYP6AY1,belonged to Clade 3 within 4 genes and CYP4C67,belonged to Clade 4 within 5 genes have the maximum fold change.Unsurprisingly,2 genes within CarEs and 4 genes belong to P450s were under-expressed in R16.RNA interference(RNAi)technique was used to eliminate specific gene product within CarEs in R16 and confirmed the detoxifying roles of three genes for the reduced toxicity of clorpyrifos in R16,among which N1XP₀22186544.1 gene maybe has the most important detoxifying function,indicating its possible roles in chlorpyrifos metabolic resistance mechanism.3.The effects of temperatures on fitness costs in the chlorpyrifos-resistant strain of the brown planthopperCompared to S9 population,the relative fitness was only 0.206 in R9 population under the laboratory condition(25? humidity 70-80%and 16 h light/8 h dark photoperiod).Temperatures varied.the resistance-associated fitness costs in R9 when compared to S9,with enhanced costs at high temperatures and reduced costs at low temperatures,such as 0.174 at 32? and 0.527 at 18?.The copulation rate and fecundity in R9 were two key factors for the reduced costs at low temperatures.Another finding was that R9 individuals needed much more time to recover from heat-shock than that of S9,but R9 and S9 individuals were similarly sensitive to cold-shock.The low fitness cost at low temperatures would increase the overwintering population,which might further increase risks of the rapid development and widespread distribution of chlorpyrifos resistance in N.lugens.