Insights from transcriptomic profiling identify the CYP6Z gene family as key drivers of pyrethroid resistance escalation in Anopheles gambiae from Cameroon

Malaria control efforts are stagnating, primarily due to increasing pyrethroid resistance in malaria vectors. Elucidating its molecular basis is essential for effective vector control. Here, via RNA-seq-based transcriptomic analyses, we elucidated the molecular basis of permethrin super-resistance in Anopheles gambiae from an agricultural setting in Cameroon with intense pesticide usage.The present study revealed significant overexpression of detoxification genes, including cytochrome P450, carboxylesterases, glutathione S-transferases, glycosyltransferases, and ABC-type xenobiotic transporter genes associated with insecticide resistance. Among the genes consistently overexpressed when surviving (R) and unexposed (C) mosquitoes were compared with the susceptible Kisumu strain (S), the glycosyltransferase UGT308G1 (AGAP007990) gene presented the greatest fold change [FC105.1 (C-S); 50.8 (R-S)]. Similarly, three CY6Z P450 genes were highly expressed [CYP6Z3 41 < FC < 63; CYP6Z2 17 < FC < 29; CYP6Z1 6 < FC < 12)]. Evidence of cuticular resistance was observed with the overexpression of several cuticular protein genes and related P450s, including CYP4G16/17. Signatures of selective sweeps were detected around the L1014F-kdr and E205D-CYP6P3 mutations. Moreover, the expression of key genes, including the protease, sensory appendage protein (SAP2), argininosuccinate lyase, P450 (CYP6Z, CYP6M2), and GST epsilon genes and cuticular proteins, was significantly upregulated in mosquitoes that survived at a 10-fold diagnostic concentration. RNA interference–induced knockdown supported the contribution of the CYP6Z gene family to resistance escalation through significant restoration of susceptibility when mosquitoes were exposed to increasing insecticide doses following gene silencing.Overall, this study provides valuable insights into the genetic mechanisms driving the aggravation of resistance in An. gambiae , highlighting the CYP6Z gene family as one of the key contributors.