Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm (Spodoptera litura) and RNA interference to evaluate its role in commonly used insecticides.

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, ß-cypermethrin and methomyl. The full-length complementary DNA sequence of CYP321B1 is 1814 bp long with an open reading frame of 1 488 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, ß-cypermethrin and methomyl with both lethal concentration at 15% (LC15 ) (50, 100 and 150 µg/mL, respectively) and 50%(LC50 ) dosages (100, 200 and 300 µg/mL, respectively). Addition of piperonyl butoxide (PBO) significantly increased the toxicity of chlorpyrifos, ß-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 ß-cypermethrin, respectively, at the LC50 dose levels. The results demonstrate that CYP321B1 might play an important role in chlorpyrifos and ß-cypermethrin detoxification in S. litura.

Expression analysis of two P450 monooxygenase genes of the tobacco cutworm moth (Spodoptera litura) at different developmental stages and in response to plant allelochemicals.

Cytochrome P450 monooxygenases (P450s) of insects are known to be involved in the metabolism or detoxification of plant allelochemicals and insecticides. Spodoptera litura (Lepidoptera, Noctuidae) is a polyphagous moth responsible for severe yield losses in many crops. In this study, two full-length P450 genes, CYP6B48 and CYP6B58, were cloned from S. litura. The cDNA sequences encode proteins with 503 and 504 amino acids, respectively. Phylogenetic analysis revealed that CYP6B48 and CYP6B58 belong to the CYP6B subfamily of P450s. Quantitative real-time PCR analyses showed that CYP6B48 and CYP6B58 were expressed only at larval stage, but not at pupal and adult stages. The highest levels of transcripts were found in the midguts and fat bodies of the larvae. No expression was detected in the ovary or hemolymph. Feeding with diets containing cinnamic acid, quercetin, or coumarin did not affect expression of CYP6B48. In contrast, diet supplemented with xanthotoxin dramatically increased the levels of CYP6B48 transcript in the midgut and fat bodies. Larvae fed with flavone had high levels of transcript of CYP6B48 in the midgut, whereas only slightly elevated levels were found in the fat bodies. Effects of the tested allelochemicals on CYP6B58 expression were minor. Hence, our findings show that S. litura responds to specific allelochemicals such as xanthotoxin with the accumulation of CYP6B48 transcripts, suggesting that specific signals in the food control the insect's ability to convert toxic allelochemicals to less harmful forms at the transcriptional level.