Study On The Expression Characteristics Of CYP450 Genes In Phortica Okadai Following Treatment With Beta-cypermethrin

Objective:This study aimed to explore the expression characteristics of cytochrome P450?CYP450?genes in Phatica okadai?P.okadai?from Zunyi area following treatment with beta-cypermethrin,which may establish a foundation for the study of the mechanism of vector insect metabolic resistance to pyrethroid insecticides.Methods:1.The establishment of laboratory animal model of P.okadai:In the surrounding area of Zunyi,P.okadai was trapped,and was raised in the laboratory after morphological identification.Then,stereomicroscopy and scanning electron microscopy were used for observing the morphological characteristics of the eggs,larvae,pupa,and adults of P.okadai.Furtherly,cytochrome c oxidase subunit 1?cox1?gene was applied for molecular identification of P.okadai.2.Toxicity determination of P.okadai following treatment with beta-cypermethrin:Referring to the test methods of fly resistance to insecticides-the bioassay methods for musca domestica?GB/T26350-2010 version?,we used the membrane method to carry out the toxicity determination of P.okadai?female flies without mating,3-5 days after eclosion,and no exposure to any insecticide?following treatment with beta-cypermethrin.3.Screening of the reference gene:Based on our previous transcriptome database,we selected five candidate reference genes used for real-time PCR,including 18S RNA,EF-1?,GAPDH,?-Actin and?-Tublin.Combined with the?Ct value analysis,GeNorm software analysis,NormFinder software analysis and BestKeeper analysis,the best reference was determined.4.Based on our previous transcriptome database,four CYP450 genes associated with metabolic resistance of pyrethroid insecticides were selected to analyze the expression difference in P.okadai following treatment with beta-cypermethrin(LC₂₀=0.061mg/L)at 0,1,2,4,8,12,24 h,and the?-Tublin gene was the internal reference for real-time PCR.5.Difference of CYP450 genes expression in P.okadai following treatment with beta-cypermethrin:the expression of CYP450 gene was detected by RNA sequencing in P.okadai at 0h and 1h exposure to cypermethrin,and the expression of candidate CYP450 genes were validated by real-time PCR.Results:1.There was no obvious difference in morphology between the in the Zunyi area and other parts of China.The cox1 gene sequence identity is 100%.Besides,there was no obvious difference in morphology between P.okadai and P.variegata.The sequence homology of the cox1 gene in P.okadai was 98%as compared to Italian P.variegata.2.The virulence regression calculation showed that the LC₂₀ of beta-cypermethrin against P.okadai was 0.061 mg/L.3.Under the stress of beta-cypermethrin,the?-Tublin gene was the best reference gene for real-time PCR among the five candidate reference genes.4.Real-time PCR results of CYP450 genes showed that the expression levels of four genes were significantly changed at 1 h exposure to beta-cypermethrin.The expression of cyp9f2 gene was up-regulated,and the expression of other three genes was down-regulated.5.RNA sequencing analysis showed that there were 103 differentially expressed CYP450 genes in P.okadai following treatment with beta-cypermethrin at 1 h.Based on the differentially expressed multiples|log₂^Ratio|?1 and q value<0.05,21 differentially expressed CYP450 genes,including 5 up-regulated genes and 16 down-regulated genes,were identified,which were validated by real-time PCR.Conclusion:1.The P.okadai in Zunyi area was morphologically consistent with the P.variegata,whereas the phylogenetic analysis based on the cox1 gene showed that there were some differences between them.2.Following treatment with beta-cypermethrin,the LC₂₀ of beta-cypermethrin against P.okadai was 0.061 mg/L,and the best reference gene was?-Tublin gene.3.The metabolism of insecticides in P.okadai may be associated with CYP450 genes,such as cyp28d1,cyp28d2,cyp4d2 and cyp49a1,within 1 h exposure to low-concentration beta-cypermethrin.