Genome-wide association studies unveil major genetic loci driving insecticide resistance in Anopheles funestus in four eco-geographical settings across Cameroon

Abstract Insecticide resistance is jeopardising malaria control efforts in Africa. Deciphering the evolutionary dynamics of mosquito populations country-wide is essential for designing effective and sustainable national and subnational tailored strategies to accelerate malaria elimination efforts. Here, we employed genome-wide association studies through pooled template sequencing to compare four eco-geographically different populations of the major vector, Anopheles funestus, across a South North transect in Cameroon, aiming to identify genomic signatures of adaptive responses to insecticides. Our analysis revealed limited population structure within Northern and Central regions ( F ST <0.02), suggesting extensive gene flow, while populations from the Littoral/Coastal region exhibited more distinct genetic patterns ( F ST >0.049). Greater genetic differentiation was observed at known resistance-associated loci, resistance-to-pyrethroids 1 (rp1) (2R chromosome) and CYP9 (X chromosome), with varying signatures of positive selection across populations. Allelic variation between variants underscores the pervasive impact of selection pressures, with rp1 variants more prevalent in Central and Northern populations ( F ST >0.3), and the CYP9 associated variants more pronounced in the Littoral/Coastal region ( F ST =0.29). Evidence of selective sweeps was supported by negative Tajima’s D and reduced genetic diversity in all populations, particularly in Central (Elende) and Northern (Tibati) regions. Genomic variant analysis identified novel missense mutations and signatures of complex genomic alterations such as duplications, deletions, transposable element (TE) insertions, and chromosomal inversions, all associated with selective sweeps. A 4.3 kb TE insertion was fixed in all populations with Njombe Littoral/Coastal population, showing higher frequency of CYP9K1 (G454A), a known resistance allele and TE upstream compared to elsewhere. Our study uncovered regional variations in insecticide resistance candidate variants, emphasizing the need for a streamlined DNA-based diagnostic assay for genomic surveillance across Africa. These findings will contribute to the development of tailored resistance management strategies crucial for addressing the dynamic challenges of malaria control in Cameroon. Author Summary Despite the widespread use of vector control tools to combat malaria in Cameroon, the disease burden remains high, particularly affecting children and pregnant women. This persistent burden is linked to intense resistance in malaria vectors, mainly driven by the overexpression of metabolic insecticide resistance genes. The evolutionary response of mosquito populations to both control interventions and agricultural environmental stimuli across Cameroon is not well understood. Understanding these dynamics is crucial for developing effective and sustainable strategies for malaria elimination country-wide. Here, we performed a genome-wide survey of Anopheles funestus across four eco-geographic regions in Cameroon, revealing limited population structure between the northern and southern regions. For the first time in Cameroon, we observed the emergence and widespread of two known resistance-related loci, rp1 and CYP9 loci. Additionally, we identified both known and novel replacement polymorphisms, along with complex signatures of genomic alterations such as large insertions and duplications, linked to selective sweeps. Notably, a 4.3kb structural variant was completely fixed in all regions, while the CYP9K1 resistant allele (A454A) was fixed only in the littoral/coastal region but remained under selection elsewhere highlighting the importance of designing a tailored resistance management strategies crucial for addressing the dynamic challenges of malaria control in Cameroon.