Acta Entomologica Sinica ›› 2020, Vol. 63 ›› Issue (8): 961-972.

• RESEARCH PAPERS •

### Role of the cytochrome P450 gene AsCYP6Z2 in the maintenance of deltamethrin resistance in Anopheles sinensis (Diptera: Culicidae)

HAN Bao-Zhu, CHE Lin-Rong, CHEN Xiao-Jie, YAN Zhen-Tian, QIAO Liang*, CHEN Bin*

1.  (Institute of Entomology and Molecular Biology, Chongqing Normal University, Chongqing 401331, China)
• Online:2020-08-20 Published:2020-09-09

Abstract: 【Aim】 To investigate whether the cytochrome P450 gene AsCYP6Z2 is related to the pyrethroid resistance in Anopheles sinensis. 【Methods】 The coding region of the AsCYP6Z2 gene of  An. sinensis was cloned. The expression profiles of this gene in different developmental stages of female mosquitoes of the deltamethrinsusceptible strain (WX-LS) and resistant strain (YN-LR) of An. sinensis and its expression profiles in different segments of female pupae of WX-LS were detected by real-time quantitative PCR (qPCR). The posterior segment of female pupal abdomen was incubated with 25 mg/mL deltamethrin solution and acetone (control), respectively, and the consequent expression response of CYP6Z2 between the deltamethrin treatment group and the acetone control group was compared by qPCR. After injection with dsAsCYP6Z2 (the RNAi group) and dsEGFP (the control group) into the 10 h old pupa, the difference in the expression level of AsCYP6Z2 between the RNAi group and the control group was compared by qPCR, the knockout rate of female adults after exposure to 0.05% deltamethrin film in 1 h and the mortality of female adults after exposure to 0.05% deltamethrin film for 1 h and then recovered for 24 h in the RNAi group and the control group were determined by bioassay method. Furthermore, the amino acid residues interacting with deltamethrin were predicted by using molecular docking. 【Results】 The coding region of AsCYP6Z2 gene (GenBank accession no.: MT840336) was cloned, and its ORF is 1 251 bp in length, encoding 416 amino acids without signal peptide and transmembrane domain. Developmental expression profiles revealed that AsCYP6Z2 gene was highly expressed in the period from 30 h old pupa to 3 h old adult, and its expression level in the resistant strain was significantly higher than that in the susceptible strain. Tissue expression profiles revealed that AsCYP6Z2 was highly expressed in the posterior segments of abdomen, followed by in the anterior segments of abdomen, throax and head. At 12 h and 24 h after incubation of the posterior segments of female pupal abdomen with 25 mg/mL deltamethrin solution, the expression level of AsCYP6Z2 increased by 4-fold and 13-fold as compared with the control (incubated with acetone), respectively. After RNAi of AsCYP6Z2, the expression level of AsCYP6Z2 gene in the RNAi group decreased by about 80% compared with the control group (dsEGFP injection group). When the female adults were exposed to 0.05% deltamethrin film in 1 h, the individuals in the RNAi group showed significant knockdown phenomenon about 20 min in advance, and the knockdown rate was significantly higher than that in the control group. In addition, the mortality of individuals in the RNAi group after exposure to 0.05% deltamethrin film for 1 h and then recovered for 24 h increased by 17% compared to the control group, indicating that silencing the AsCYP6Z2 gene results in a significant increase in mosquito sensitivity to deltamethrin. The molecular docking between deltamethrin and the predicted AsCYP6Z2 protein showed that deltamethrin can enter the binding pocket of AsCYP6Z2 protein, forming a Pi-sulfide interaction with Cys-155 and generating more stable hydrogen bonds. The side chain can also form a stable hydrophobic interaction network with Ala-165, Val-72, Leu-76, Leu-82, Ala-24 and other amino acid residues of AsCYP6Z2. 【Conclusion】 The results indicate that the AsCYP6Z2 gene is involved in maintaining the deltamethrin resistance phenotype of An. sinensis, laying a foundation for further exploring the molecular mechanism of AsCYP6Z2 gene in the metabolism of pyrethroid insecticides.