Co-expression of CYP9M10 and NADPH cytochrome P450-reductase in baculovirus expression system: Revealing the role of CYP9M10 in permethrin resistance in Culex quinquefasciatus

Cytochrome P450 monooxygenases are known to play a major role in insecticide resistance allowing resistant insects to metabolize insecticides at a higher rate. Our previous studies revealed that CYP9M10 gene was not only up-regulated in Culex quinquefasciatus  resistant mosquitoes but also induced by permethrin in resistant mosquitoes. In the current study, using baculovirus expression system, we co-expressed CYP9M10 and cytochrome P450 reductase (CPR) in insect Spodoptera frugiperda (SF9) cells,  characterized enzymatic activity and metabolic ability of CYP9M10 to permethrin, and conducted cytotoxicity assays. The highest catalytic activity toward 7-ethoxycoumarin for CYP9M10 was obtained at combination of 0.1 mM 5-aminolaevulinic acid (5-ALA) and 0.02 mM Fe³⁺ or 0.5ug/ml hemin combinated with 0.1mM 5-ALA. Permethrin cytotoxicity assays by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were examined in Sf9 insect cells, with and without expression of CYP9M10. Results revealed that CYP9M10 could play a role in detoxifying permethrin in the insect cells. In vitro metabolic assay using GC-MS further revealed that co-expressed 9M10/CPR could metabolize insecticide permethrin. Our results not only showed the advantage of using baculovirus insect expression system to study P450 proteins but also strongly demonstrated the role of CYP9M10 in metabolism of insecticide permethrin, the molecular basis of resistance development in mosquitoes.