<i>An</i>. <i>funestus</i> and flies bioassays results.

Cross-resistance to insecticides in multiple resistant malaria vectors is hampering resistance management. Understanding its underlying molecular basis is critical to implementation of suitable insecticide-based interventions. Here, we established that the tandemly duplicated cytochrome P450s, CYP6P9a/b are driving carbamate and pyrethroid cross-resistance in Southern African populations of the major malaria vector Anopheles funestus. Transcriptome sequencing revealed that cytochrome P450s are the most over-expressed genes in bendiocarb and permethrin-resistant An. funestus. The CYP6P9a and CYP6P9b genes are overexpressed in resistant An. funestus from Southern Africa (Malawi) versus susceptible An. funestus (Fold change (FC) is 53.4 and 17 respectively), while the CYP6P4a and CYP6P4b genes are overexpressed in resistant An. funestus in Ghana, West Africa, (FC is 41.1 and 17.2 respectively). Other up-regulated genes in resistant An. funestus include several additional cytochrome P450s (e.g. CYP9J5, CYP6P2, CYP6P5), glutathione-S transferases, ATP-binding cassette transporters, digestive enzymes, microRNA and transcription factors (FC<7). Targeted enrichment sequencing strongly linked a known major pyrethroid resistance locus (rp1) to carbamate resistance centering around CYP6P9a/b. In bendiocarb resistant An. funestus, this locus exhibits a reduced nucleotide diversity, significant p-values when comparing allele frequencies, and the most non-synonymous substitutions. Recombinant enzyme metabolism assays showed that both CYP6P9a/b metabolize carbamates. Transgenic expression of CYP6P9a/b in Drosophila melanogaster revealed that flies expressing both genes were significantly more resistant to carbamates than controls. Furthermore, a strong correlation was observed between carbamate resistance and CYP6P9a genotypes with homozygote resistant An. funestus (CYP6P9a and the 6.5kb enhancer structural variant) exhibiting a greater ability to withstand bendiocarb/propoxur exposure than homozygote CYP6P9a_susceptible (e.g Odds ratio = 20.8, P<0.0001 for bendiocarb) and heterozygotes (OR = 9.7, P<0.0001). Double homozygote resistant genotype (RR/RR) were even more able to survive than any other genotype combination showing an additive effect. This study highlights the risk that pyrethroid resistance escalation poses to the efficacy of other classes of insecticides. Available metabolic resistance DNA-based diagnostic assays should be used by control programs to monitor cross-resistance between insecticides before implementing new interventions.多重抗性疟疾媒介对杀虫剂产生的交互抗性，正掣肘抗性治理工作的推进。解析其潜在的分子机制，对于制定适配的基于杀虫剂的防控策略至关重要。本研究证实，串联重复的细胞色素P450（cytochrome P450）家族成员CYP6P9a/b，是驱动南非主要疟疾媒介富氏按蚊（Anopheles funestus）种群产生氨基甲酸酯类与拟除虫菊酯类交互抗性的核心因子。 转录组测序（transcriptome sequencing）结果显示，在抗残杀威（bendiocarb）与抗氯菊酯（permethrin）的富氏按蚊中，细胞色素P450是表达上调最显著的基因家族。相较于敏感株富氏按蚊，南非（马拉维）地区的抗性富氏按蚊中，CYP6P9a与CYP6P9b基因的表达量显著上调，其表达倍数（Fold Change, FC）分别为53.4和17；而在西非加纳的抗性富氏按蚊种群中，CYP6P4a与CYP6P4b基因的表达上调倍数分别为41.1和17.2。抗性富氏按蚊中其他上调的基因还包括多种其他细胞色素P450（如CYP9J5、CYP6P2、CYP6P5）、谷胱甘肽S-转移酶（glutathione-S transferases）、ATP结合盒转运蛋白（ATP-binding cassette transporters）、消化酶、微小RNA（microRNA）以及转录因子，其表达上调倍数均小于7。靶向富集测序（Targeted enrichment sequencing）结果证实，已知的主要拟除虫菊酯抗性位点rp1与以CYP6P9a/b为核心的氨基甲酸酯类抗性显著相关。在抗残杀威的富氏按蚊中，该位点的核苷酸多样性（nucleotide diversity）降低，等位基因频率（allele frequencies）比较的p值具有统计学显著性，且存在最多的非同义替换（non-synonymous substitutions）位点。重组酶代谢实验（Recombinant enzyme metabolism assays）证实，CYP6P9a与CYP6P9b均可代谢氨基甲酸酯类杀虫剂。在黑腹果蝇（Drosophila melanogaster）中进行CYP6P9a/b的转基因表达实验显示，相较于对照组，携带这两个基因的果蝇对氨基甲酸酯类杀虫剂的抗性显著提升。此外，氨基甲酸酯类抗性与CYP6P9a基因型之间存在显著相关性：纯合抗性型富氏按蚊（携带CYP6P9a基因及6.5kb增强子结构变异）相较于纯合CYP6P9a敏感型个体（例如残杀威暴露下的优势比[Odds Ratio, OR]=20.8，P<0.0001）以及杂合型个体（OR=9.7，P<0.0001），对残杀威与恶虫威（propoxur）暴露的耐受能力显著更强。双重纯合抗性基因型（RR/RR）的存活能力更是优于其他所有基因型组合，呈现出加性效应。 本研究揭示了拟除虫菊酯抗性升级对其他类别杀虫剂防控效果构成的潜在风险。疟疾防控项目应在推行新的干预措施前，采用现有的基于DNA检测的代谢抗性诊断技术，监测不同杀虫剂间的交互抗性情况。