Evolutionary genetics of insecticide resistance in Culex quinquefasciatus

Culex quinquefasciatus mosquitoes play an important role in the transmission of vector-borne diseases of public health importance including lymphatic filariasis (LF) as well as many arboviruses. Insecticide-based approaches are one of the most important interventions to mitigate disease burden; nevertheless increased resistance of vectors to insecticides imposes a challenge for the sustainability and effectiveness of both current and future vector control interventions. Hence, understanding the dynamics and likely mechanisms underlying the evolution of resistance will be critical to effective decision-making in insecticide resistance management strategies. The present study was set out to investigate the genetic basis of insecticide resistance in C. quinquefasciatus from Uganda. Two objectives were developed, 1) to investigate patterns of insecticide resistance across the south of the country and how this might reflect local selection and genetic structure and 2) to investigate the basis of the molecular mechanisms underlying resistance to all four classes of insecticides recommended for vector control. The population genetic study compared and contrasted microsatellite markers and two resistance-associated loci (Vgsc-1014F and Ace1-119S). While no significant difference in genetic diversity across populations were detected by microsatellites, higher frequency of Vgsc-1014F compared to the Ace1-119S mutations was observed in all populations suggesting that the Ugandan Eastern – Southwest populations are under a heterogeneous selection pressure, which created a pattern of local adaptation in these populations. Additionally, the copy number (CN) assay developed in this study indicated the presence of CN variation in the voltage gated sodium channel (Vgsc) gene in about 10% of the individuals assayed from these populations. Genotypic/phenotypic association tests conducted on bendiocarb resistant-individuals suggested that this resistant phenotype was not underlying solely by the 119S target-site mutation in the Ace-1 gene. Indeed, synergist bioassays show an increase of mortality of around 25% in mosquitoes pre-exposed to either TTP or PBO, indicating a possible resistance mediated by detoxification enzymes. Using a novel whole-transcriptome microarray we profiled the bendiocarb-resistant phenotype and implicated two P450s (Cyp-Cx1 and Cyp6n23) with the highest up-regulation expression compared to a susceptible strain. Remarkably, the predicted Cyp-Cx1 is closely related to two P450s from the family Cyp6, which were already validated in vitro as insecticide metabolizers in A. gambiae and A. aegypti, which corroborates a likely association of metabolic resistance in the investigated bendiocarb-resistant phenotype. Taken together the results yielded by genomic and transcriptomic experiments provide evidence that Ugandan C. quinquefasciatus mosquitoes are under heterogeneous selection pressure imposed by insecticides from distinct classes, and that the evolution of insecticide resistance is mediated by at least two main genetic mechanisms; target-site mutations (Vgsc-1014F and Ace1-119S) as well as over-expression of detoxification enzymes.