Identification of carboxylesterase genes from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate the role in chlopyrifos resistance

Carboxylesterases (CarEs) are a well-documented family of enzymes that are ubiquitous in various animals, plants and microorganisms. The enzymes are involved in metabolism of specific hormones and detoxification of dietary and environmental xenobiotics in insects. The present study provides a genome-wide identification and functional characteristics of the CarE family in the diamondback moth, Plutella xylostella (L.), a highly insecticide-resistant and destructive pest worldwide. Genome-wide analysis identified a total of 65 putative CarEs from the P. xylostella genome, representing 7 of the 8 CarE subclasses in insects (α-esterase, β-esterase, juvenile hormone esterase, gliotactin, acetylcholinesterase, neurotactin and neuroligin).

Synergistic test of CarE inhibitor-triphenyl phosphate (TPP) showed that TPP decreased the resistance of the 3^rd-instar larvae of P. xylostella to chlorpyrifos by about 7.8 times. When treated with the lower doses (LC₃₀ and LC₅₀) of chlorpyrifos, the activity of CarE in the treated 3^rd-instar larvae of P. xylostellawas significantly higher than that in the control, suggesting that sublethal dose of chlorpyrifos could activate the CarE.

Gene expression profile study revealed that three genes (Pxae21, Pxae22 and Pxae31) from the α-esterase subclass were highly expressed in midgut of chlorpyrifos-resistant colony (CRC) as compared to other tissues and susceptible strain in qRT-PCR analysis. The qRT-PCR results also revealed that these genes could express at different developmental stages with predominant expression at the larval stage (the main feeding stage), suggesting their involvement with detoxification of plant defense compounds and insecticides.

RNAi knockdown of CarEs further confirmed their important roles in detoxification of insecticides because the chlorpyrifos susceptibility in dsRNA-injected P. xylostella (CRC) larvae was significantly increased with the rates of 84.6%, 79.2% and 58.3% for Pxae21, Pxae22 and Pxae31, respectively while the sensitivity of the 3^rd-instar instar larvae of P. xylostella to chlorpyrifos increased by 1.8 times, 1.8 times and 1.73 times, respectively. In summary, CarEs play important roles in metabolic detoxification of chlorpyrifos in P. xylostella. Three CarE genes, Pxae21, Pxae22 and Pxae31, are involved in insecticide resistance, and the variation of their expression levels can directly influence the susceptibility of P. xylostella to chlorpyrifos.