Target-Site Mutation Genotyping of Insecticide-Resistant Female Anopheles Mosquitoes in Ota

Malaria control techniques in sub-Saharan Africa significantly depend on insecticide-based measures such as insecticide-treated nets (ITNs) and indoor residual spraying (IRS). However, the emergence of resistance in Anopheles mosquitoes, driven by target-site mutations, undermines their effectiveness and threatens elimination efforts. Key resistance markers include knockdown resistance mutations ( Kdr ) in the voltage-gated sodium channel ( VGSC ) gene and the G119S mutation in the Ace-1 gene, both of which reduce insecticide efficacy. This study investigated the morphological and molecular composition of Anopheles populations, their insecticide resistance status, and the prevalence of Kdr and Ace-1 mutations, as well as assessed genetic diversity, gene flow, and population structure across breeding sites in Ota, Ogun State, Nigeria. Using Coetzee’s key, a total of 478 adult female mosquitoes were obtained and morphologically identified, with molecular assays confirming a uniform species composition of Anopheles gambiae (390 bp) across all sites. Susceptibility assays revealed strong pyrethroid resistance, with permethrin mortality rates of 15–25%, far below the WHO threshold of 90%. However, bendiocarb produced 98–100% mortality, indicating high susceptibility. Genotypic analysis revealed a predominance of the Kdr-W (L1014F) allele, particularly in Nestle and Iju, where homozygous resistant individuals were more frequent. The Kdr -E (L1014S) allele was rare and largely confined to homozygous susceptible genotypes, suggesting it is not yet established. Deviations from the Hardy-Weinberg equilibrium were detected in Nestle populations, while sequence alignment confirmed L1014F mutations in Atan, Iju, and Chelsea. Population genetic analyses showed no strong subdivision (χ² = 0.7885, Fst = -1.3586, Gst = -0.0395, Kst = -0.13760), but high haplotype diversity (Hd = 0.94–1.00), moderate nucleotide diversity (π = 0.27–0.31), and substantial gene flow (Nm ≈ 8.24), supported by PCA and phylogenetic clustering. This study reveals widespread pyrethroid resistance but sustained carbamate susceptibility, providing the first genetic dataset from Ota to guide surveillance and resistance management strategies.