Genetic mapping of resistance: A QTL and associated polymorphism conferring resistance to alpha-cypermethrin in Anopheles funestus

The heavy reliance on pyrethroid-based interventions has accelerated the spread of resistance malaria vectors including Anopheles funestus, jeopardising control efforts. The efficacy of Insecticide-based interventions, especially insecticide-treated nets (ITNs), the cornerstone of malaria control and management, is threatened by the widespread resistance complicating malaria control. Alpha-cypermethrin, a type II pyrethroid, is increasingly utilised in various ITN formulations, including those combined with piperonyl butoxide (PBO) and chlorfenapyr-based Interceptor(R) G2 (IG2) nets, to enhance effectiveness against resistant mosquito populations. Therefore, understanding the molecular basis of resistance is essential to monitor and track resistance trends for an effective malaria control program. In this study, we identified a 1.4 Mb QTL on the telomeric end of the left arm of chromosome 2, conferring resistance to -cypermethrin (rap1 QTL). Different crossing schemes and sequencing approaches were explored to determine the most effective strategy. Individual-based QTL mapping performed on segregating individuals from an isofemale family identified a QTL at the F7 generation. Higher recombination density relative to the physical genome in the F7 isofemale family, with a recombination every 240 kb, facilitated the detection of a QTL compared to the F2 family (335 kb/cM). Additionally, we exploited bulk segregate analysis (BSA) between susceptible and resistant phenotypes from the F7 isofemale family and an F7 mixed cross-family to perform cost-effective and rapid QTL-mapping discovery. The strongest signal in both independent BSA analyses overlaps with the rap1 QTL, further supporting its role in -cypermethrin resistance. The known resistant alleles of the cytochrome P450 CYP6P9a and 6.5-kb structural variant within the rap1 QTL strongly correlate with survival to -cypermethrin. In this study, we validated that previously developed DNA-based assays, originally designed to monitor permethrin resistance, are effective for tracking resistance to -cypermethrin as well. Additionally, we identified candidate variants that can serve as reliable markers for monitoring -cypermethrin resistance. Author SummaryIn the study we used genetic crosses between resistant and susceptible Anopleles funestus colonies to identify quantitative trait loci (QTL) associated with the resistance to -cypermethrin. We have identified a QTL on the left arm of chromosome 2 conferring resistance to -cypermethrin (rap1 QTL). Different crossing schemes and sequencing approaches were explored to determine the most effective and cost-efficient strategy to map candidate loci associated with resistance. In this study we assessed the efficiency and the cost-effectiveness of Bulk segregant analysis (BSA) mapping to detect candidate loci associated with resistance. BSA analysis enabled the detection of polymorphism in candidate regions that could serve as potential SNP-based marker to track resistance to -cypermethrin. Previously developed SNP-based markers to track resistance to permethrin in known resistant alleles show a strongly correlation with survival to -cypermethrin, suggesting shared mechanisms my underly resistance to both type I (permethrin) and type II (-cypermethrin) pyrethroids.