Characterization and identification of cytochrome P450 monooxygenases involved in insecticide detoxification in Aedes albopictus mosquito larvae

The Asian tiger mosquito, Aedes albopictus (Skuse), is an important vector of dengue fever viruses as well as a potential vector for eastern equine encephalitis and West Nile viruses. Insecticide resistance due to detoxification catalyzed by cytochrome P450 monooxygenases (P450s) continues to be a major problem in mosquito control. These enzymes are often induced by xenobiotics found in the habitats of the mosquito larvae, resulting in increased detoxification rates. The effects of xenobiotics such as pesticides and tire-leachate compounds on insecticide toxicity and P450 expression in the larvae were examined.;Larvae pre-exposed to pesticides or tire-leachate compounds had significantly increased tolerance to the insecticide carbaryl. Pretreatment of the larvae with pentachlorophenol (PCP) or benzothiazole (BZT) almost eliminated the in vivo toxicity of carbaryl. Exposure to BZT also decreased the toxicity of rotenone and temephos up to five-fold compared to controls. A P450 inhibitor, piperonyl butoxide, reversed these in vivo effects, suggesting that P450s were involved. Microsomes from larvae treated with PCP or BZT had significantly increased catalytic activities in vitro when measured in incubations with three known P450 substrates. This was consistent with sharply increased intensity of at least four SDS-PAGE bands of microsomal proteins with molecular weights in the same range as P450s.;A microsomal protein (approximately 58 kD) from BZT-treated larvae was selected, trypsin-digested, and analyzed by mass spectrometry. Peptide mapping of the resulting tryptic digest by comparing it to a protein mass database identified the protein as a P450 possibly of the CYP4 family. Internal amino acid sequences of two HPLC-fractionated peptide fragments of the protein also had similarities with P450s of the CYP4 family. This indicates that the microsomal protein induced by BZT is a CYP4 P450.;This study provides important evidence that compounds present in the natural habitats of the mosquito larvae can induce P450s, which detoxify insecticides and thus cause insecticide tolerance. These findings will enable us to generate a molecular probe for P450-encoding genes in the mosquito. This probe will be used to monitor tolerance to insecticides detoxified by P450s in small samples from wild mosquito populations.