Genome wide population genetics and molecular surveillance of insecticide resistance in Anopheles stephensi mosquitoes from Awash Sebat Kilo in Ethiopia.

Since the detection of the Asian mosquito Anopheles stephensi in Dijbouti in 2012, it has spread throughout the Horn of Africa. This invasive vector continues to expand across the continent and is a significant threat to malaria control programs. Vector control methods, including insecticide-treated nets and indoor residual spraying, have substantially reduced the malaria burden. However, the increasing prevalence of mosquitoes resistant to insecticides, including An. stephensi populations, undermines ongoing malaria elimination efforts. Understanding population structure, gene flow between populations, and the distribution of insecticide resistance mutations is essential for guiding effective malaria control strategies. Here, we generated whole genome sequencing data for An. stephensi sourced from Awash Sebat Kilo, Ethiopia (n = 27) and compared with South Asian populations (n = 45; India and Pakistan) to assess genomic diversity, population structure, and uncovering insecticide resistance mutations. Population structure analysis using genome-wide single nucleotide polymorphisms (n = 15,533,476) revealed Ethiopian isolates clustering as a distinct ancestral group, separate from South Asian isolates. Three insecticide resistance-associated SNPs (gaba gene: A296S and V327I; vgsc L1014F) were detected. Evidence of ongoing selection was found in several loci, including genes previously associated with neonicotinoids, ivermectin, DDT, and pyrethroid resistance. This study represents the first whole genome population genetics study of invasive An. stephensi, revealing genomic differences from South Asian populations, which can be used for future assessments of vector population dispersal and detection of insecticide resistance mechanisms.