Anopheles gambiae bulk segregant analysis
Insecticide resistance in Anopheles gambiae threatens malaria vector control and eradication efforts across sub-Saharan Africa. While target-site resistance mechanisms are well-characterised, the evolutionary origins of metabolic resistance remain poorly understood. We employed bulk segregant analysis (BSA) using unique historical genetic crosses established between DDT-resistant ZAN/U strains (Zanzibar, late 1990s)  and susceptible FAST5 strains to map the genomic architecture of early metabolic resistance. Critically, the ZAN/U strain exhibited DDT resistance without kdr mutations, providing an ideal genetic background to isolate glutathione S-transferase epsilon (GSTe)-mediated resistance mechanisms that evolved during the initial DDT era. BSA analysis revealed a major quantitative trait locus on chromosome 3R spanning the GSTe gene cluster, with 22 significant amino acid substitutions distributed across all eight GSTe genes (GSTe1-8). Validation using East African field collected samples from the MalariaGEN Ag1000G project confirmed that many of these historically derived mutations are now present as naturally occurring variants under long-term selection pressure. Additionally, BSA analyses using permethrin-resistant crosses from the same period (late 1990s), originated from a cross between permethrin resistant RSP-ST strain and susceptible FAST5 strain, identified expected target-site resistance at the vgsc locus, contrasting with the metabolic resistance mechanisms underlying DDT resistance. These findings demonstrate that resistance mutations detected in contemporary field populations have deep evolutionary roots, originating in the early DDT era before being maintained through subsequent pyrethroid selection pressure. This work provides a comprehensive genomic characterisation of the GSTe cluster's role in establishing the metabolic resistance foundation that continues to compromise vector control efforts today.