Transcriptomic signatures of high‑intensity pyrethroid resistance in Anopheles gambiae from a Cameroonian agricultural hotspot

Pyrethroid resistance continues to undermine malaria vector control across Africa, particularly in agricultural hotspots where insecticide pressure is high. However, the molecular mechanisms enabling Anopheles gambiae to survive extreme pyrethroid doses remain poorly understood. This study investigates transcriptomic responses, allele‑frequency shifts, and selection signatures associated with high‑ permethrin resistance intensity in An. gambiae from Mangoum, Cameroon. RNA‑seq analyses revealed strong overexpression of detoxification genes in both unexposed and permethrin‑exposed field mosquitoes. The UDP‑glycosyltransferase UGT308G1 showed the highest expression levels (FC = 105.1 in C–S; 50.8 in R–S). Within the P450 family, members of the CYP6Z cluster were markedly overexpressed, notably CYP6Z3 (FC = 68.8 in C–S; 41.4 in R–S), CYP6Z2 (FC = 29.0; 17.2), and CYP6Z1 (FC = 12.3; 6.0). Additional detoxification genes such as CYP9K1 (FC = 11.3; 4.7), CYP6M2 (FC = 8.5; 3.8), and the cuticle‑associated CYP4G17 (FC = 2.9; 2.5) were strongly upregulated. Increasing permethrin doses (1 × , 5 × , 10 ×) induced further upregulation of oxidative‑stress, mitochondrial, and translational pathways. Gene‑level population‑genetic metrics revealed strong selective sweeps at VGSC and across the rp1 region. High‑frequency nonsynonymous variants in CYP6AA1 (Ser395Thr), CYP6AA2 (Asn327Asp), CYP6P1 (Ile168Val and Leu374Met), and the validated marker Glu205Asp-CYP6P3 were nearly fixed in field populations and absent in lab susceptible strain, consistent with long‑term selection and hitchhiking. Additional metabolic variants in CYP12F2, UGT49A3, UGT308A2, and ABCA2 occurred at moderate to high frequencies and distinguished resistant from susceptible genetic backgrounds. RNAi‑mediated silencing of CYP6Z1, CYP6Z2, CYP6Z3, and CYP6Z4 significantly increased mortality under permethrin and alpha‑cypermethrin 1X, 5X and 10X exposure, confirming the functional involvement of the CYP6Z family in resistance escalation. High pyrethroid resistance intensity in An. gambiae arises from strong target‑site selection coupled with extensive metabolic adaptation. The convergence of transcriptomic signals, allele‑frequency patterns, and RNAi assays highlights the CYP6Z genes cluster, together with key P450, UGT, and ABC variants, as central components of a polygenic resistance architecture. These findings underscore the need to integrate metabolic markers into resistance surveillance to detect and manage escalating pyrethroid resistance.