Guo Ying-Ao, Si Feng-Ling, Han Bao-Zhu, Qiao Liang, Chen Bin
Chongqing Key Laboratory of Vector Control and Utilization, Institute of Entomology and Molecular Biology, College of Life Sciences, Chongqing Normal University, Chongqing 401331, PR China.
Chongqing Key Laboratory of Vector Control and Utilization, Institute of Entomology and Molecular Biology, College of Life Sciences, Chongqing Normal University, Chongqing 401331, PR China.
Acta Trop. 2024 Dec;260:107413. doi: 10.1016/j.actatropica.2024.107413. Epub 2024 Sep 27.
Cytochrome P450 monooxygenases (P450s), a multifunctional protein superfamily, are one of three major classes of detoxification enzymes. However, the diversity and functions of P450 genes from pyrethroid-resistant populations of Anopheles sinensis have not been fully explored. In this study, P450 genes associated with pyrethroid resistance were systematically screened using RNA-seq in three field pyrethroid-resistant populations (AH-FR, CQ-FR, YN-FR) and one laboratory resistant strain (WX-LR) at developmental stages, tissues, and post blood-meal in comparison to the laboratory susceptible strain (WX-LS) in An. sinensis. Importantly, the expression of significantly upregulated P450s was verified using RT-qPCR, and the function of selected P450s in pyrethroid detoxification was determined with RNA interference using four laboratory pyrethroid-resistant strains (WX-LR, AH-LR, CQ-LR, YN-LR). Sixteen P450 genes were significantly upregulated in at least one field-resistant population, and 44 were significantly upregulated in different developmental stages, tissues or post blood-meal. A total of 19 P450s were selected to verify their association with pyrethroid resistance, and four of them (AsCYP6P3v1, AsCYP6P3v2, AsCYP9J10, and AsCYP9K1) demonstrated significant upregulation in laboratory pyrethroid-resistant strains using RT-qPCR. Knockdown of these four genes all significantly reduced pyrethroid resistance and increased the mortality by 57.19% (AsCYP6P3v1 and AsCYP6P3v2 knockdown group), 38.39% (AsCYP9K1 knockdown group) and 48.87% (AsCYP9J10 knockdown group) in An. sinensis by RNAi, which determined the pyrethroid detoxification function of these four genes. This study revealed the diversity of P450 genes and provided functional evidence for four P450s in pyrethroid detoxification in An. sinensis for the first time, which increases our understanding of the pyrethroid resistance mechanism, and is of potential value for pyrethroid resistance detection and surveillance.
细胞色素P450单加氧酶（P450s）是一个多功能蛋白质超家族，是三大解毒酶类之一。然而，中华按蚊拟除虫菊酯抗性种群中P450基因的多样性和功能尚未得到充分研究。在本研究中，与实验室敏感品系（WX-LS）相比，利用RNA测序在三个田间拟除虫菊酯抗性种群（AH-FR、CQ-FR、YN-FR）和一个实验室抗性品系（WX-LR）的发育阶段、组织和血餐后，系统筛选了与拟除虫菊酯抗性相关的P450基因。重要的是，使用RT-qPCR验证了显著上调的P450s的表达，并使用RNA干扰在四个实验室拟除虫菊酯抗性品系（WX-LR、AH-LR、CQ-LR、YN-LR）中确定了所选P450s在拟除虫菊酯解毒中的功能。16个P450基因在至少一个田间抗性种群中显著上调，44个在不同发育阶段、组织或血餐后显著上调。总共选择了19个P450s来验证它们与拟除虫菊酯抗性的关联，其中四个（AsCYP6P3v1、AsCYP6P3v2、AsCYP9J10和AsCYP9K1）通过RT-qPCR在实验室拟除虫菊酯抗性品系中显示出显著上调。敲低这四个基因均显著降低了拟除虫菊酯抗性，并通过RNA干扰使中华按蚊的死亡率提高了57.19%（AsCYP6P3v1和AsCYP6P3v2敲低组）、38.39%（AsCYP9K1敲低组）和48.87%（AsCYP9J10敲低组），这确定了这四个基因的拟除虫菊酯解毒功能。本研究首次揭示了中华按蚊P450基因的多样性，并为四个P450s在拟除虫菊酯解毒中的功能提供了证据，这增加了我们对拟除虫菊酯抗性机制的理解，对拟除虫菊酯抗性检测和监测具有潜在价值。
