Using targeted next-generation sequencing to characterize genetic differences associated with insecticide resistance in Culex quinquefasciatus populations from the southern U.S.

Resistance to insecticides can hamper the control of mosquitoes such as Culex quinquefasciatus, known to vector arboviruses such as West Nile virus and others. The strong selective pressure exerted on a mosquito population by the use of insecticides can result in heritable genetic changes associated with resistance. We sought to characterize genetic differences between insecticide resistant and susceptible Culex quinquefasciatus mosquitoes using targeted DNA sequencing. To that end, we developed a panel of 122 genes known or hypothesized to be involved in insecticide resistance, and used an Ion Torrent PGM sequencer to sequence 125 unrelated individuals from seven populations in the southern U.S. whose resistance phenotypes to permethrin and malathion were known from previous CDC bottle bioassay testing. Data analysis consisted of discovering SNPs (Single Nucleotide Polymorphism) and genes with evidence of copy number variants (CNVs) statistically associated with resistance. Ten of the seventeen genes found to be present in higher copy numbers were experimentally validated with real-time PCR. Of those, six, including the gene with the knock-down resistance (kdr) mutation, showed evidence of a ≥ 1.5 fold increase compared to control DNA. The SNP analysis revealed 228 unique SNPs that had significant p-values for both a Fisher’s Exact Test and the Cochran-Armitage Test for Trend. We calculated the population frequency for each of the 64 nonsynonymous SNPs in this group. Several genes not previously well characterized represent potential candidates for diagnostic assays when further validation is conducted.

杀虫剂抗药性会阻碍致倦库蚊（Culex quinquefasciatus）等蚊虫的防控，该蚊虫已知可传播西尼罗河病毒（West Nile virus）等虫媒病毒（arboviruses）。杀虫剂施用对蚊虫种群施加的强选择压力，可导致与抗药性相关的可遗传的遗传变异。本研究旨在通过靶向DNA测序（targeted DNA sequencing）技术，解析抗杀虫剂与易感型致倦库蚊之间的遗传差异。为此，本研究构建了包含122个已知或被推测参与杀虫剂抗药性过程的基因的检测面板，并使用离子激流PGM（Ion Torrent PGM）测序仪，对美国南部7个种群的125份无亲缘关系的蚊虫个体样本进行测序；这些样本对氯菊酯（permethrin）和马拉硫磷（malathion）的抗药性表型，已通过此前美国疾病控制与预防中心（CDC）的瓶式生物测定得到确认。数据分析环节包括筛选与抗药性存在统计学关联的单核苷酸多态性（Single Nucleotide Polymorphism，SNP）以及存在拷贝数变异（Copy Number Variant，CNV）证据的基因。在被检出拷贝数升高的17个基因中，有10个通过实时荧光定量PCR（real-time PCR）完成了实验验证。其中6个基因（包括携带击倒抗性（knock-down resistance，kdr）突变的基因）的拷贝数相较于对照DNA呈现≥1.5倍的升高。SNP分析共筛选出228个独特的单核苷酸多态性位点，这些位点在费希尔精确检验（Fisher’s Exact Test）与科克兰-阿米亚特趋势检验（Cochran-Armitage Test for Trend）中均得到了具有统计学意义的p值。本研究针对该组中的64个非同义单核苷酸多态性（nonsynonymous SNPs）位点，计算了其种群频率。若干此前未被充分表征的基因，在完成进一步验证后，有望成为诊断检测的潜在候选靶标。