Dastarcus Helophoroides P450 Participates In The Molecular Mechanism Of Cypermethrin Stress Response

Dastarcus helophoroides is an insect of the Coleoptera family as an important natural enemy,it has been widely used in the biological control of longhorn beetles.Cytochrome P450is one of the three major detoxification enzymes of insects,and it plays an important role in the process of insects’detoxification and metabolism of various chemical pesticides.With the extensive use of chemical pesticides,pesticides have a greater impact on natural enemy insects while killing pests.There are many related reports on the effects of pesticides on pests and the detoxification and metabolic mechanism of pests on pesticides.However,there are no related reports on the response mechanism of the natural enemy of pests,the Dastarcus helophoroides to pesticide stress.This article first explored the virulence of the commonly used insecticide cypermethrin to Eriophorum cypermethrin,and then used transcriptome sequencing technology to screen out the P450 genes that were highly expressed under cypermethrin treatment,and then used real-time fluorescence quantitative technology to detect the difference.After treatment with cypermethrin concentration,the expression of P450 genes was different.Finally,RNAi technology was used to verify the function of the related P450 genes,which laid the foundation for the subsequent exploration of the detoxification and metabolism process of cypermethrin.The main test results are as follows:1.Using the dipping method,the knockdown concentration KC₅₀ of cypermethrin on the Dastarcus helophoroides is 0.19 mg/L,and the KC₃₀ is 0.08 mg/L.After being knocked down,the chest and feet of the Dastarcus helophoroides twitched and could not crawl or feed normally.Cypermethrin is well tolerated,and death occurs only under treatment with higher concentration of cypermethrin.2.Use cypermethrin KC₃₀ concentration and acetone immersion to treat the Dastarcus helophoroides,extract the totalRNA 24h after treatment,construct a cDNA library,use Illumina technology to sequence to obtain the corresponding transcriptome data.Using|log₂^{（Fold Change）}|>0 as the standard,a total of 90 differentially expressed P450 genes were screened out,of which 57 genes had increased expression levels,the highest differential multiple was 39.4 times,and 33 genes had decreased expression levels.The highest drop was7.5 times.3.The 12 differentially expressed P450 genes were screened out from the transcriptome data,and the real-time fluorescence quantitative technology was used to detect the genetic effects of the cypermethrin KC₁₀ and KC₃₀ concentration treatments at 1h,4h,8h,12h,24h,and 48h.The results showed that the expression levels of these 12 genes increased in each time period after being stressed by pesticides.The most obvious increase in gene expression levels within 8h-24h was:pesticide treatment 8h The expression level of base Dh 1216 was 9times that of the control group.At 12 h,the expression level of gene Dh 775 was 9 times that of the control group.At 24 h,the expression level of gene Dh 1094 was 5 times that of the control group.4.UsingRNAi technology,the dsRNA of different injection amounts and different P450genes was injected into the body of the velvet armour.The results showed that when the P450gene of the velvet armour was interfered with a single interference,the expression of the gene Dh 775 decreased most significantly,down by 77.0%.The expression level of gene Dh 1094decreased the least,down by 47.3%;when two genes or three genes were mixed interference,within a certain injection volume,the gene expression would decrease with the increase of dsRNA injection volume.After the interference gene Dh 1094,the sensitivity of L.lindula to cypermethrin changed most obviously.When treated with KC₅₀ concentration for 24 hours,the knockdown rate rose to 96.7%.After the single interference gene Dh 775 and Dh 1216,the knockdown rate rose to respectively 68.2%,73.7%.In mixed interference,if the interference gene is the same,the sensitivity of Cypermethrin to Cypermethrin will increase with the increase of the injection volume.Therefore,it is inferred that the three P450 genes of Cypermethrin:Dh 1216,Dh 775,and Dh 1094 are related to their detoxification and metabolism functions of cypermethrin.