PoolSeq Genome‐Wide Association Studies and Microbial Signature Analyses Identify Novel Candidates Associated With Pyrethroid Resistance Evolution in <i>Anopheles funestus</i> in Cameroon

Intensification of insecticide resistance in malaria vectors is undermining efforts to sustain control strategies. The evolutionary features underlying such exacerbation in major vector such as Anopheles funestus are only partially understood. PoolSeq whole genome analysis of Anopheles funestus from Mibellon (Cameroon), (alive and dead at 1×, 5× and 10× concentrations), failed to identify hits associated with resistance escalation. However, stronger signals emerge at the rp1 and CYP9 loci when comparing these phenotypes to the susceptible reference strain FANG, with genomic analysis using F₃ crosses implicating these regions in resistance evolution. Temporal genomic between 2014 and control unexposed 2021 populations confirmed substantial genomic changes largely confined to these two regions with evidence of selective sweeps linked to the presence of multiple novel replacement polymorphisms and signatures of complex genomic evolution emerging from major cytochrome P450 genes within the CYP9 and rp1 regions at increasing allelic frequencies in field individuals and alive genetic crosses, indicating that those variants are potentially driving resistance evolution. Combined genotyping of the rp1-based 4.3 kb SV and CYP9K1 (G454A) in alive and dead genetic crosses underscores their significant contribution to super-resistant phenotype in Anopheles funestus population in Mibellon. On the other hand, microbial composition changes, notably Elizabethkingia anophelis was associated with resistance evolution, suggesting their potential role in shaping the resistance phenotype while Serratia marcescens and Asaia bongorensis correlate with susceptibility. Genetic events and microbial symbionts associated with resistance evolution offer promising avenues for developing molecular markers to manage insecticide resistance.