The metabolic resistance blueprint: Genomic dissection of DDT resistance in historical kdr-free East African 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 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 (GSTe1-8). 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. This work provides a characterisation of the GSTe clusters role in establishing the metabolic resistance foundation that continues to compromise vector control efforts today. 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.