Chlorpyrifos-and Fipronil-resistance Mediated By Carboxylesterases In The Diamondback Moth,Plutella Xylostella(Lepidoptera:Plutellidae)

Diamondback moth(DBM),Plutella xylostella,is a worldwide major pest of cruciferous vegetables.Carboxylesterases(CarEs)are one of the most important detoxification enzymes in insects.They can detoxify many kinds of insecticides in different pests.Based the DBM genome and transcriptome databases,we found 3 carboxylesterase genes that showed high midgut-specific expression in chlorpyrifos-resistant population(CRP)and 2 in fipronil-resistant population(FRP).Topical application was used to test the toxicity of chlorpyrifos and fipronil to a susceptible population(SP)and two resistant populations.The synergistic effect of triphenyl phosphate(TPP),a carboxylesterase inhibitor,was also tested on chlorpyrifos and fipronil.The LC30 and LC50 concentrations of two insecticides were used to treat their respective resistant populations,and the effect on the carboxylesterase activity after the treatment was determined.Stage-and tissue-specific expression patterns of Pxae21,Pxae22 and Pxae31 were profiled by qRT-PCR.Expression levels of candidate genes and the insecticide susceptibility of the 3rd instar larvae were also examined after RNA interference(RNAi).1.The toxicity and synergistic effect of TPP on P.xylostellaCompared with the susceptible population,the resistance ratio of CRP was 3638 times to chlorpyrifos and FRP 284.2-fold to fipronil.Our study also showed that the CarE inhibitor triphenyl phosphate was able to synergize the toxicity of chlorpyrifos(SR,synergize ratio 7.8)and fipronil(SR 6.0).2.Effect of low dose treatment on CarE specific activity in P.xylostellaAfter treatment with low doses(LC30 and LC50)of chlorpyrifos,the CarE activity in the treated 3rd larvae of P.xylostella was suppressed in a short time,but then increased higher than the control by 48h.The same phenomenon was found in the FRP.The CarE activity was remarkably high in the LC30 treatment(2.53-fold)and LC50 treatment(1.75-fold)for larvaes of P.xylostella at 24h.These results demonstrated that chlorpyrifos and fipronil could activate the CarE.3.Expression pattern analysis of CarEs in P.xylostellaThe expression levels of Pxae21,Pxae22 and Pxae31 in the CRP and FRP were all higher than in the SP,and they mainly expressed in the larvae stage.Tissue-specific expression profiling showed the three genes were primarily expressed in the midgut,while almost no expression was found in the head,cuticle and fatbody.4.Functional verification of CarEs candidate genes in P.xylostellaThe expression levels were significantly suppressed at 48h after RNAi of Pxae21,Pxae22 and Pxae31(about 83.9%,92.8%,67.5%respectively)in CRP compared with the control.Injection of dsPxae22 and dsPxae31 in FRP could also result in significant decrease of transcription after 36h when compared with the control(about 65%and 63%respectively).The results indicated an effective silencing after dsRNA injection of these genes in P.xylostella.