No abstract

The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.

BackgroundRNA interference (RNAi) is an effective tool to examine the function of individual genes. Carboxylesterases (CarE, EC 3.1.1.1) are known to play significant roles in the metabolism of xenobiotic compounds in many insect species. Previous studies in our laboratory found that CarE expression was up-regulated in Aphis gossypii (Glover) (Hemiptera: Aphididae) adults of both omethoate and malathion resistant strains, indicating the potential involvement of CarE in organophosphorus (OP) insecticide resistance. Functional analysis (RNAi) is therefore warranted to investigate the role of CarE in A. gossypii to OPs resistance.Result CarE expression in omethoate resistant individuals of Aphis gossypii was dramatically suppressed following ingestion of dsRNA-CarE. The highest knockdown efficiency (33%) was observed at 72 h after feeding when dsRNA-CarE concentration was 100 ng/µL. The CarE activities from the CarE knockdown aphids were consistent with the correspondingly significant reduction in CarE expression. The CarE activity in the individuals of control aphids was concentrated in the range of 650–900 mOD/per/min, while in the individuals of dsRNA-CarE-fed aphids, the CarE activity was concentrated in the range of 500–800 mOD/per/min. In vitro inhibition experiments also demonstrated that total CarE activity in the CarE knockdown aphids decreased significantly as compared to control aphids. Bioassay results of aphids fed dsRNA-CarE indicated that suppression of CarE expression increased susceptibility to omethoate in individuals of the resistant aphid strains.ConclusionThe results of this study not only suggest that ingestion of dsRNA through artificial diet could be exploited for functional genomic studies in cotton aphids, but also indicate that CarE can be considered as a major target of organophosphorus insecticide (OPs) resistance in A. gossypii. Further, our results suggest that the CarE would be a propitious target for OPs resistant aphid control, and insect-resistant transgenic plants may be obtained through plant RNAi-mediated silencing of insect CarE expression.

Cytochrome P450 monooxygenases play a critical role in insecticide resistance by allowing resistant insects to metabolize insecticides. Previous studies revealed that two P450 genes, CYP9M10 and CYP6AA7, are not only up-regulated but also induced in resistant Culex mosquitoes. In this study, CYP9M10 and CYP6AA7 were separately co-expressed with cytochrome P450 reductase (CPR) in insect Spodoptera frugiperda (Sf9) cells using a baculovirus-mediated expression system and the enzymatic activity and metabolic ability of CYP9M10/CPR and CYP6AA7/CPR to permethrin and its metabolites, including 3-phenoxybenzoic alcohol (PBOH) and 3-phenoxybenzaldehyde (PBCHO), characterized. PBOH and PBCHO, both of which are toxic to Culex mosquito larvae, can be further metabolized by CYP9M10/CPR and CYP6AA7/CPR, with the ultimate metabolite identified here as PBCOOH, which is considerably less toxic to mosquito larvae. A cell-based MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cytotoxicity assay revealed that Sf9 cells expressing CYP9M10/CPR or CYP6AA7/CPR increased the cell line’s tolerance to permethrin, PBOH, and PBCHO. This study confirms the important role played by CYP9M10 and CYP6AA7 in the detoxification of permethrin and its metabolites PBOH and PBCHO.