Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm (Spodoptera litura) and RNA interference to evaluate its role in commonly used insecticides.
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abstract
Insect cytochrome P450 monooxygenases (CYPs or P450s) play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura, has developed resistance to a wide range of insecticides. In the present study, a novel P450 gene, CYP321B1, was cloned from S. litura. The function of CYP321B1 was assessed using RNA interference (RNAi) and monitoring resistance levels for three commonly used insecticides, including chlorpyrifos, -cypermethrin and methomyl. The full-length complementary DNA sequence of CYP321B1 is 1814 bp long with an open reading frame of 1488 bp encoding 495 amino acid residues. Quantitative reverse-transcriptase polymerase chain reaction analyses during larval and pupal development indicated that CYP321B1 expression was highest in the midgut of fifth-instar larvae, followed by fat body and cuticle. The expression of CYP321B1 in the midgut was up-regulated by chlorpyrifos, -cypermethrin and methomyl with both lethal concentration at 15% (LC15 ) (50, 100 and 150 g/mL, respectively) and 50%(LC50 ) dosages (100, 200 and 300 g/mL, respectively). Addition of piperonyl butoxide (PBO) significantly increased the toxicity of chlorpyrifos, -cypermethrin and methomyl to S. litura, suggesting a marked synergism of the three insecticides with PBO and P450-mediated detoxification. RNAi-mediated silencing of CYP321B1 further increased mortality by 25.6% and 38.9% when the fifth-instar larvae were exposed to chlorpyrifos and -cypermethrin, respectively, at the LC50 dose levels. The results demonstrate that CYP321B1 might play an important role in chlorpyrifos and -cypermethrin detoxification in S. litura.
