Faculty of Science, Ain Shams University, Cairo, Egypt.
Faculty of Science, Ain Shams University, Cairo, Egypt; Egypt Japan University of Science Technology, Alexandria, Egypt.
Mol Cell Probes. 2020 Aug;52:101579. doi: 10.1016/j.mcp.2020.101579. Epub 2020 Apr 28.
Over the past decades, the extensive use of pyrethroids insecticides for vector control has resulted in the development of insecticide resistance. Cytochrome P450 has been recognized to play a critical role in the metabolic detoxification of insecticides. In the current study, Culex pipiens mosquitoes were collected from Giza Governorate in Egypt and tested for insecticide susceptibility against deltamethrin. First detection of Knockdown resistance gene (Kdr) mutations in field collected mosquitoes was performed. Activities of cytochrome oxidase P450 detoxification enzyme that synchronized with the resistance development, was assessed. Expression profiles of cytochrome P450s and their putative corresponding regulating miRNAs, which was previously reported in Cx. pipiens pallens were evaluated in pyrethroid resistant field-collected Cx. pipiens using RT-qPCR and stem-loop RT-qPCR, respectively. Specific stem-loop reverse transcription primers and forward primers were designed for miRNAs profiling. Our results elucidated the pyrethroid resistance development and revealed its relation to the metabolic and target site modification mechanisms with a first report of L1014F-kdr mutation detection. RT-qPCR results have showed an up-regulation in the expression of the studied P450 transcripts. Negative correlations were found between the expression of P450s and their regulatory miRNAs except for CYP9J35, where positive correlation was found with its corresponding miR-13. Interestingly, our data was the first to detect negative correlation between miR-285 and its putative CYP6Cp1 target gene. These findings highlighted the significance of identifying P450 gene along with regulatory miRNAs as a key mechanism implicated in pyrethroid resistance in field Culex vector population. The elucidation of this mechanism would shed light on the development of insecticide resistance and would help in shaping strategies to combat such vectors.
在过去的几十年中，广泛使用拟除虫菊酯杀虫剂来进行病媒控制，导致了昆虫抗药性的发展。细胞色素 P450 已被认为在杀虫剂的代谢解毒中发挥关键作用。在当前的研究中，从埃及吉萨省收集了库蚊，并对其对溴氰菊酯的杀虫剂敏感性进行了测试。首次检测到现场采集的蚊子中的击倒抗性基因 (Kdr) 突变。评估了与抗性发展同步的细胞色素氧化酶 P450 解毒酶的活性。先前在淡色库蚊中报道的细胞色素 P450 的表达谱及其假定的相应调节 miRNA，使用 RT-qPCR 和茎环 RT-qPCR 分别在抗药性现场采集的库蚊中进行了评估。为 miRNA 分析设计了特定的茎环反转录引物和正向引物。我们的研究结果阐明了拟除虫菊酯的抗性发展，并揭示了其与代谢和靶标修饰机制的关系，首次报道了 L1014F-kdr 突变的检测。RT-qPCR 结果显示，研究的 P450 转录物的表达上调。除了 CYP9J35 之外，还发现 P450 的表达与其调节 miRNA 之间存在负相关，而 CYP9J35 与相应的 miR-13 之间存在正相关。有趣的是，我们的数据首次检测到 miR-285 与其假定的 CYP6Cp1 靶基因之间存在负相关。这些发现强调了鉴定 P450 基因及其调节 miRNA 作为参与现场库蚊种群中拟除虫菊酯抗性的关键机制的重要性。阐明这一机制将有助于揭示昆虫抗药性的发展，并有助于制定对抗此类病媒的策略。
