Chlorfenapyr metabolism by mosquito P450s associated with pyrethroid resistance identifies potential activation markers

Chlorfenapyr is a pro-insecticide increasingly used in combination with pyrethroids such as a-cypermethrin or deltamethrin in insecticide treated bednets (ITNs) to control malaria transmitted by pyrethroid-resistant mosquito populations. Chlorfenapyr requires P450 activation to produce tralopyril and other bioactive metabolites. Pyrethroid resistance is often associated with elevated levels of chemoprotective P450s with broad substrate specificity, which could influence chlorfenapyr activity. Here, we have investigated chlorfenapyr metabolism by a panel of eight P450s commonly associated with pyrethroid resistance in An. gambiae and Ae. aegypti, the major vectors of malaria and arboviruses. Chlorfenapyr was activated to tralopyril by An. gambiae CYP6P3, CYP9J5, CYP9K1 and Ae. aegypti, CYP9J32. The K-cat/K-M value of 0.66 mu M-1 min(-1) for CYP9K1 was, 6.7 fold higher than CYP6P3 and CYP9J32 (both 0.1 mu M-1 min(-1)) and 22-fold higher than CYP9J5 (0.03 mu M-1 min(-1)). Further investigation of the effect of -cypermethrin equivalent to the ratios used with chlorfenapyr in bed nets (similar to 1:2 molar ratio) resulted in a reduction in chlorfenapyr metabolism by CYP6P3 and CYP6K1 of 76.8% and 56.8% respectively. This research provides valuable insights into the metabolism of chlorfenapyr by mosquito P450s and highlights the need for continued investigation into effective vector control strategies.