Chamnanya Saowanee, Kiddela Benyapa, Saingamsook Jassada, Nachaiwieng Woottichai, Lumjuan Nongkran, Somboon Pradya, Yanola Jintana
Center of Veterinary Medical Diagnostic and Animal Health Innovation, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, 50100, Thailand.
Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, 50200, Thailand.
Infect Dis Poverty. 2025 Jan 13;14(1):2. doi: 10.1186/s40249-024-01269-2.
The cytochrome P450s-mediated metabolic resistance and the target site insensitivity caused by the knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene were the main mechanisms conferring resistance to deltamethrin in Culex quinquefasciatus from Thailand. This study aimed to investigate the expression levels of cytochrome P450 genes and detect mutations of the vgsc gene in deltamethrin-resistant Cx. quinquefasciatus populations in Thailand.
Two field-collected strains of Cx. quinquefasciatus, Cq_SP and Cq_NiH, were selected with deltamethrin to generate the resistant strains Cq_SP-R and Cq_NiH-R, respectively. Bioassays were tested on larvae and adults of each strain according to WHO methods. Eight cytochrome P450 genes were analyzed for the expression level using quantitative real time-PCR. The cDNA of mosquitoes was amplified and sequenced for four fragments of vgsc gene. The kdr L1014F mutation and the haplotype of the CYP9M10 gene were detected in survivor and dead mosquitoes after exposure to the deltamethrin WHO test paper. Statistical analyses were performed using Fisher's exaction test.
Bioassay tests revealed a significantly higher resistance level in Cq_SP-R than in Cq_NiH-R strains in both larvae and adults. All eight cytochrome P450 genes were significantly overexpressed in larvae of Cq_NiH-R strain compared to the parent and susceptible Cq_Sus strains. The CYP6AA7 and CYP9J34 genes had the highest expression ratios, exceeding 24-fold in Cq_NiH-R larvae. In Cq_SP-R strain, the CYP4H34 and CYP9J34 genes were overexpressed in both stages. The kdr L1014F mutation was found in Cq_SP-R and its parent Cq_SP strains with a significantly higher mutant allele frequency in the survivor mosquitoes than in dead mosquitoes (P < 0.0001). The V240M and novel L925F mutations were found only in Cq_SP-R strain. Heterozygous genotype for the D-Cu( +)/Cu(-) of CYP9M10 gene was detected in Cq_NiH and Cq_NiH-R strains but other strains were mostly homozygous for the Cu(-)/Cu(-).
Overexpression of multiple cytochrome P450 genes alone has a relatively minor impact on resistance. The combined mechanisms of cytochrome P450- and kdr-mediated resistance result in significantly higher resistance to deltamethrin in Cx. quinquefasciatus. This study supports sustainable public health initiatives in Thailand to address the evolving challenges of insecticide resistance.
细胞色素P450介导的代谢抗性以及电压门控钠通道（vgsc）基因中的击倒抗性（kdr）突变引起的靶位点不敏感性是泰国致倦库蚊对溴氰菊酯产生抗性的主要机制。本研究旨在调查细胞色素P450基因的表达水平，并检测泰国溴氰菊酯抗性致倦库蚊群体中vgsc基因的突变。
选择两个野外采集的致倦库蚊品系Cq_SP和Cq_NiH，分别用溴氰菊酯进行筛选，以产生抗性品系Cq_SP-R和Cq_NiH-R。根据WHO方法对每个品系的幼虫和成虫进行生物测定。使用定量实时PCR分析八个细胞色素P450基因的表达水平。对蚊子的cDNA进行扩增，并对vgsc基因的四个片段进行测序。在暴露于溴氰菊酯WHO测试纸后的存活和死亡蚊子中检测kdr L1014F突变和CYP9M10基因的单倍型。使用Fisher精确检验进行统计分析。
生物测定试验表明，Cq_SP-R品系在幼虫和成虫中的抗性水平均显著高于Cq_NiH-R品系。与亲本和敏感的Cq_Sus品系相比，Cq_NiH-R品系幼虫中的所有八个细胞色素P450基因均显著过表达。CYP6AA7和CYP9J34基因的表达率最高，在Cq_NiH-R幼虫中超过24倍。在Cq_SP-R品系中，CYP4H34和CYP9J34基因在两个阶段均过表达。在Cq_SP-R及其亲本Cq_SP品系中发现了kdr L1014F突变，存活蚊子中的突变等位基因频率显著高于死亡蚊子（P < 0.0001）。V240M和新的L925F突变仅在Cq_SP-R品系中发现。在Cq_NiH和Cq_NiH-R品系中检测到CYP9M10基因的D-Cu( +)/Cu(-)杂合基因型，但其他品系大多为Cu(-)/Cu(-)纯合子。
单独多个细胞色素P450基因的过表达对抗性的影响相对较小。细胞色素P450和kdr介导的抗性联合机制导致致倦库蚊对溴氰菊酯的抗性显著更高。本研究支持泰国的可持续公共卫生倡议，以应对杀虫剂抗性不断演变的挑战。
