Genomic analysis of DDT resistance identifies importance of glutathione transferases in driving evolutionary response to vector control in the malaria vector Anopheles gambiae

Insecticide resistance in Anopheles gambiae poses a significant threat to malaria 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 in the late 1990s between a DDT-resistant ZAN/U strain (Originally from Zanzibar) and a susceptible FAST strain 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 for isolating glutathione S-transferase epsilon ( GSTe )-mediated resistance mechanisms that evolved during the DDT control program era. BSA analysis revealed a major quantitative trait locus on chromosome 3R spanning the GSTe gene cluster ( GSTe 1–8). BSA identified 20 significant amino acid substitutions distributed across all eight GSTe genes, in addition to variations in the promoter region of GSTe2 and GSTe3 , which were previously determined to be overexpressed in the ZAN/U strain. East African field-collected samples from the MalariaGEN Ag1000G project confirmed that many of these mutations are under long-term selection pressure in natural populations. Additionally, BSA analysis was performed on progeny from crosses established in early 2000s between a permethrin-resistant RSP-ST strain originating from Western Kenya and a susceptible strain, identifying expected target-site resistance at the vgsc locus. This work provides a characterisation of the GSTe cluster’s role in establishing the metabolic resistance foundation that continues to compromise vector control efforts today.