Department of Entomology, Texas A&M University Minnie Bell Heep Center, TAMU 2475 370 Olsen Blvd College Station, College Station, TX, 77843, USA.
Department of Poultry Science, Texas A&M University, College Station, TX, 77843, USA.
BMC Genomics. 2023 Mar 29;24(1):160. doi: 10.1186/s12864-023-09241-4.
Metabolic detoxification is one of the major mechanisms contributing to the development of resistance in mosquitoes, including the southern house mosquito, Culex quinquefasciatus. The three major detoxification supergene families, cytochrome P450s, glutathione S-transferases and general esterases, have been demonstrated to play an important role in metabolic resistance. In this study, we performed differential gene expression analysis based on high-throughput transcriptome sequencing on samples from four experimental groups to give insight into key genes involved in metabolic resistance to malathion in Cx. quinquefasciatus. We conducted a whole transcriptome analysis of field captured wild Cx. quinquefasciatus from Harris County (WI), Texas and a malathion susceptible laboratory-maintained Sebring colony (CO) to investigate metabolic insecticide resistance. Field captured mosquitoes were also phenotypically classified into the malathion resistant and malathion susceptible groups following a mortality response measure conducted using a Centers for Disease Control and Prevention (CDC) bottle assay. The live (MR) and dead (MS) specimens from the bottle assay, along with an unselected WI sample and a CO sample were processed for total RNA extraction and subjected to whole-transcriptome sequencing.
We demonstrated that the genes coding for detoxification enzymes, particularly cytochrome P450s, were highly up-regulated in the MR group compared to the MS group with similar up-regulation observed in the WI group compared to the CO group. A total of 1,438 genes were differentially expressed in comparison between MR and MS group, including 614 up-regulated genes and 824 down-regulated genes. Additionally, 1,871 genes were differentially expressed in comparison between WI and CO group, including 1,083 up-regulated genes and 788 down-regulated genes. Further analysis on differentially expressed genes from three major detoxification supergene families in both comparisons resulted in 16 detoxification genes as candidates potentially associated with metabolic resistance to malathion. Knockdown of CYP325BC1 and CYP9M12 using RNA interference on the laboratory-maintained Sebring strain significantly increased the mortality of Cx. quinquefasciatus after exposure to malathion.
We generated substantial transcriptomic evidence on metabolic detoxification of malathion in Cx. quinquefasciatus. We also validated the functional roles of two candidate P450 genes identified through DGE analysis. Our results are the first to demonstrate that knockdown of CYP325BC1 and CYP9M12 both significantly increased malathion susceptibility in Cx. quinquefasciatus, indicating involvement of these two genes in metabolic resistance to malathion.
代谢解毒是导致蚊子产生抗药性的主要机制之一，包括南方家蚊（Culex quinquefasciatus）。已证实三大解毒超级基因家族，细胞色素 P450s、谷胱甘肽 S-转移酶和普通酯酶，在代谢抗性中发挥重要作用。在这项研究中，我们基于来自四个实验组的高通量转录组测序样本进行差异基因表达分析，深入了解代谢性马拉硫磷抗性相关的关键基因。我们对来自德克萨斯州哈里斯县（WI）的野外捕获的野生南方家蚊（Cx. quinquefasciatus）和易感马拉硫磷的实验室 Sebring 种群（CO）进行了全转录组分析，以研究代谢性杀虫剂抗性。野外捕获的蚊子还根据疾病控制和预防中心（CDC）瓶测法进行的死亡率反应测量，表型分类为马拉硫磷抗性和马拉硫磷敏感组。从瓶测法的活（MR）和死（MS）标本，以及未选择的 WI 样本和 CO 样本中提取总 RNA 并进行全转录组测序。
我们证明，解毒酶编码基因，特别是细胞色素 P450s，在 MR 组中与 MS 组相比高度上调，在 WI 组中与 CO 组相比也观察到类似的上调。MR 与 MS 组之间的比较共鉴定出 1438 个差异表达基因，包括 614 个上调基因和 824 个下调基因。此外，WI 与 CO 组之间的比较共鉴定出 1871 个差异表达基因，包括 1083 个上调基因和 788 个下调基因。对两个比较中三个主要解毒超级基因家族的差异表达基因进一步分析，确定了 16 个解毒基因作为可能与马拉硫磷代谢抗性相关的候选基因。使用 RNAi 对实验室维持的 Sebring 株系进行 CYP325BC1 和 CYP9M12 的敲低，显著增加了南方家蚊暴露于马拉硫磷后的死亡率。
我们提供了南方家蚊代谢解毒马拉硫磷的大量转录组证据。我们还通过 DGE 分析验证了两个候选 P450 基因的功能作用。我们的结果首次表明，CYP325BC1 和 CYP9M12 的敲低均显著增加了南方家蚊对马拉硫磷的敏感性，表明这两个基因参与了马拉硫磷的代谢抗性。
