Functional Identification Of CYP9A Subfamily P450 Genes In Insecticide Detoxification Of Spodoptera Exigua (Hübner)

The beet armyworm,Spodoptera exigua（Hübner）,is a polyphagous insect of Noctuidae（Lepidoptera）and has more than 170 host plants.It is widely distributed around the world and has developed high levels of resistance against many kinds of insecticide.Cytochrome P450 monooxygenase of insects plays an important role in the adaptation to host and resistance to chemical insecticides.Preliminary studies showed that the CYP9A genes were amplified in S.exigua,the CYP9A members are clustered on the chromosome.The expression of multiple members of this subfamily can be up-regulated by xenobiotics or constitutively up-regulated in insecticide-resistant strains.Several P450s have been reported to relate to the detoxification of chemical insecticides or plant alleochemicals,but their specific detoxification function remain unknown.This study used CRISPR/Cas9-mediated gene cluster knockout in vivo combined with bioassay to identify the CYP9A P450s in S.exigua are related to its detoxification of fenvalerate.Then by in vitro functional expression,those specific P450s of 9A subfamily involved in detoxification were identified.This study provides direct evidence for P450-mediated insecticide detoxification and resistance evolution in S.exigua,and provides an important theoretical basis for the research on the functional evolution of insect P450 genes and the chemical defense mediated by them.The main results are as follows:1.Construction of the homozygous CYP9A gene cluster knockout strain of S.exiguaThe genome data of S.exigua showed that 9 out of 10 CYP9A P450s form a head-to-tail gene cluster on chromosome 17.In this study,a～130kb genomic fragment with CYP9A cluster in Wuhan susceptible strain WHS was knocked out using the CRISPR/Cas9genome editing technology,targeting the beginning and end of the CYP9A gene cluster in S.exigua.A set of primers were designed to verify the genotype according to the sequence of each P450 in the CYP9A cluster.The homozygous knockout strain WHS-d9A was obtained by single pair crossing and molecular screening after three generations（G₁to G₃）,providing important material for subsequent research on the function of CYP9A genes in S.exigua.2.Effects on insecticide sensitivity of CYP9A gene cluster knockout in S.exiguaThe sensitivity variation between the background strain WHS and the CYP9A knockout strain WHS-d9A to 10 insecticides was identified by bioassay.Compared to the WHS,the WHS-d9A displayed 9.3-and 3.9-fold enhancement on sensitivities to fenvalerate and esfenvalerate respectively.However,there was no significant difference in other two pyrethroid insecticides,etofenprox and bifenthrin,and the structurally different insecticides,pyridalyl,emamectin benzoate,chlorantraniliprole,spinosad,chlorfenapyr,chlorpyrifos and indoxacarb.The study indicates that one or more CYP9A P450s in CYP9A cluster of S.exigua are involved in the detoxification of fenvalerate and esfenvalerate.3.In vitro expression of S.exigua CYP9A genes and metabolism of esfenvalerateIn this study,10 CYP9A P450s of S.exigua were functionally expressed in vitro using the Bac-to-Bac baculovirus expression system and their metabolic capabilities to esfenvalerate were tested.The results showed that 8 P450（CYP9A10,CYP9A40,CYP9A11,CYP9A27,CYP9A9,CYP9A186,CYP9A30,CYP9A107 and CYP9A10）were capable of metabolizing esfenvalerate except CYP9A187 and CYP9A98.The metabolic rate of CYP9A40（0.62±0.0025 pmol 4’-OH product/min/pmol P450）is one order of magnitude higher than the other 7 P450s（between 0.0039±0.0004 and 0.029±0.0019pmol 4’-OH product/min/pmol P450）.In addition,the by-product 2’-hydroxy-esfenvalerate was detected in all samples.Particularly in the metabolites of CYP9A107,the amount of68%is 2’-hydroxy-esfenvalerate.This study confirmed that 8 S.exigua CYP9A P450s could metabolize esfenvalerate,suggesting that up-regulation of these P450 genes may contribute to fenvalerate resistance of S.exigua,and these P450s are potential resistance genes.Among them,CYP9A40 has a significantly higher metabolic capacity,which makes it likely to be a major gene in the mediation of fenvalerate resistance in the field.In general,the amplification of CYP9A subfamily of S.exigua and functional redundancy of the CYP9A P450s paralogs in insecticide detoxification is a big challenge to the pest control.These results provide new insights and ideas for further study of P450 detoxification and insecticide resistance of S.exigua.