The Metabolic And Target Resistant Mechanism Of The Brown Planthopper(Nilaparvata Lugens St(?)l)to Etofenprox

Nilaparvata lugens Stal(brown planthopper,BPH),the primary insect pest of rice crops,is well known as a notorious pest throughout Asia because of its frequent outbreak.In addition to directly extracting nourishment from plants,oviposition and virus disease transmission by N.lugens result in more severe damage.Many insecticides including organophosphates,carbamates and neonicotinoids,have been used against N.lugens,nevertheless,N.lugens have developed high levels of resistance to many insecticides in most areas in China,resulting in severe yield reduction and significant economic loss.Etofenprox is a non-ester pyrethroid insecticide with an ether linkage instead of the ester linkage in traditional pyrethroid insecticides.Because of its excellent effects on rice planthoppers and low toxicities to fish and honeybee,etofenprox has been used frequently to control rice planthoppers in many countries and areas,such as Taiwan(China).With the forthcoming registration of etofenprox to control rice planthoppers in mainland China,it is significant to reveal the resistance mechanisms of etofenprox in advance,especially in N.lugens,the most important rice planthoppers in China.Resistance to most pyrethroid insecticides commonly arises by the enhanced detoxification by metabolic enzymes,target-site insensitivity,delayed cuticular penetration and behavioral change.Cytochrome P450 monooxygenases,a multigenic superfamily of enzymes,are the most important detoxification enzymes to biosynthesize endogenous compounds and detoxify xenobiotic compounds.The overexpression of P450 genes is a common metabolic mechanism for pyrethroids resistance.Based on the available N.lugens genome sequences,54 P450s have been annotated and many of which were known to play important roles in insecticide resistance.Voltage-gated sodium channels(VGSC)are responsible for the generation and propagation of nerve action potentials across the membranes of excitable cells and are the primary targets of various neurotoxins including pyrethroid insecticides.In this study,a N.lugens population developed etofenprox resistance to a high level through the successive selection by etofenprox for 16 generations in the laboratory(G16).To explore the potential P450 genes that might be associated with etofenprox resistance,mRNA levels of all P450 genes in G16 and its counterpart,the susceptible population US16 were determined and compared.In addition,we have identified three mutations(T276A,M946T and L1042F)in NlNav from the etofenprox resistant population.We isolated 41 full-length NlNav cDNA clones from the brown planthopper.Furthermore,we tested the gating and pharmacology properties of the eleven NlNav variants,which exhibited distinct channel gating and different sensitivities to etofenprox.And all three mutations reduced the sensitivity of NlNav1-1 to etofenprox,which may be the main reason of the resistance to etofenprox.1.Metabolic resistance to etofenprox relies on synergism of multiple P450 enzymes in N.lugensAn etofenprox resistance selection of N.lugens was performed in order to understand the resistance mechanisms.Through successive selection by etofenprox for 16 generations in the laboratory,an etofenprox-resistant strain(G16)with the resistance ratio(RR)of 422.3-fold was obtained.The resistance was partly synergised(2.68-fold)with the metabolic inhibitor PBO,suggesting a role for P450 monooxygenases.In this study,11 P450 genes were significantly up-regulated in G16,among which eight genes was above 2.0-fold higher than their expression in US16,a population with the same origin of G16 but without contacting any insecticide in the laboratory.The expression level of four genes(CYP6AY1,CYP6FU1 and CYP408A1 from Clade 3,and CYP425A1 from Clade 4)were above 4.0-fold when compared to that in US 16.RNA interference(RNAi)was performed to evaluate the importance of the selected P450s in etofenprox resistance.When CYP6FU1,CYP425A1 or CYP6AY1 was interfered,the susceptibility was significantly recovered in both G16 and US16,while the knockdown of CYP408A1 or CYP353D1 did not cause significant changes in etofenprox susceptibility.We supposed that CYP6FU1 was the most important P450 member for etofenprox resistance because of the highest expression level and the most noticeable effects on resistance ratios following RNAi.2.Functional and pharmacological characterization of sodium channels from N.lugensTo evaluate the structure,function and toxin pharmacology of sodium channels from the brown planthopper,we isolated 41 full-length NlNav cDNA clones from the brown planthopper.A total of nine alternative exons were identified.Six of them were previously reported in sodium channel genes from other insect species and the other three of them were unique in NlNav.The 41 cDNA clones represent 24 distinct types of splicing variants.29 RNA editing sites were identified from 41 NlNav sodium channel clones,and all of them were unique to N1Nav.16 of them were A-to-I or U-to-C editing sites,however,none resulted in amino acid change.The electrophysiological properties and of these variants were examined using two-electrode voltage clamp.A broad range of voltage-dependent activation,steady-state fast inactivation was displayed among functional sodium channel clones.Analysis of the 11 functional NlNav variants not only revealed a wide range of activation and fast inactivation,but also a large range of slow inactivation and distinct pharmacological properties.Altogether,these results suggest that the functional diversity of sodium channels in the brown planthopper is mainly achieved by alternative splicing.3.The target resistant mechanism of N.lugens to entofenprox relies on the mutations of sodium channelStudies on the mechanism of knockdown resistance in the past two decades led to the identification of many sodium channel mutations that are associated with pyrethroids resistance in arthropod species.Three mutations(T276A,M946T and L1042F)were identified in etofenprox-resistant NlNav,among which T276A in segment 5 of domain I(IS5)was a novel mutation in NlNav,whereas L1042F and M946T were kdr and super-kdr mutation found in many insect resistant populations.To test if different mutation combinations of these mutations can decrease the sensitivity of NlNav,we generated 7 mutant channels.After in vitro transcription and injection,the wild type channel and mutant channels were separately expressed in oocytes.Using two-electrode voltage clamp,we found all three mutations decreased the sensitivity of NlNav1-1 to etofenprox,permethrin and deltamethrin,which may be the main reason of the population resistance.T276A substitution reduced the sensitivity of the NlNav1-1 channel to etofenprox,permethrin and deltamethrin by 12-fold,4-fold and 2-fold,respectively.Previously,modeling and functional analysis predicted a second pyrethroid receptor site(PyR2)formed by residues in IL45,IS5,and IIS6.Here,we reported the identification of a second mutation(T276A)in PyR2 that contributes to the low pyrethroid sensitivity of sodium channel in the insect resistant population other than L to F,which have been found in various arthropod pests and disease vectors.4.Investigating the effects of exon b in negatively regulating the brown planthopper sodium channel expression/function in vitroMammalian sodium channel isoforms are encoded by nine ? subunit genes,which exhibit distinct expression patterns and are expressed in various nerve cells,tissues and developmental stages.Different from mammals,most insect species carry only one sodium channel gene except for several aphid species.Indeed,alternative splice and RNA editing events have been demonstrated to produce a broad range of voltage-dependent activation,inactivation and distinct pharmacological properties of sodium channels.The 41 brown planthopper sodium channel cDNA clones represented 24 distinct types of splicing variants.When functionally expressed in Xenopus oocytes,the eleven NlNav variants lacking exon b(VSIYYFPT)generated sodium currents large enough for further functional characterization,whereas the variants possessing exon b produced little sodium currents,suggesting the role of exon b in negatively regulating sodium channel expression/function.The amino acid sequence of exon b was highly conserved among 20 insect and spider species and the genomic structure was nearly identical as an internally optional splice exon.However,two amino acids in exon b,tyrosine and phenylalanine(Y774 and F776),are completely consistent in these 20 species.Furthermore,we also determined that two critical amino acids were associated with current expression in Xenopus oocytes.We hypothesize that the size of amino acid residue side chains and phosphorylation of exon b contribute to the current expression level in vitro.