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

item WANG, RUI-LONG - South China Agricultural University
item ZHU-SALZMAN, KEYAN - Texas A&M University
item Baerson, Scott
item XIN, XIAO-WEI - South China Agricultural University
item LI, JUN - South China Agricultural University
item SU, YI-JUAN - South China Agricultural University
item ZENG, REN-SEN - South China Agricultural University

Submitted to: Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2016
Publication Date: 5/13/2016
Citation: Wang, R., Zhu-Salzman, K., Baerson, S.R., Xin, X., Li, J., Su, Y., Zeng, R. 2016. Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm moth (Spodoptera litura) and RNA interference to evaluate its role in commonly used insecticides. Insect Science. DOI 10.1111/1744-7917.12315.

Interpretive Summary: The Tobacco Cutworm (Spodoptera litura) represents one of the most economically significant herbivorous pests, and is responsible for damage to field crops such as cotton, tobacco and groundnut. Many field populations of S. litura have developed resistance to commonly used insecticides such as chlorpyrifos, ß-cypermethrin and methomyl, due to frequent applications and overuse. Evolved insecticide resistance in S. litura therefore represents a serious threat to both agriculture and natural ecosystems. Insects have developed the capacity to metabolize numerous inhibitory compounds, for example through the acquisition of genes encoding specialized metabolic enzymes such as cytochrome P450s. Cytochrome P450 enzymes often play a role in the emergence of insecticide resistance in field populations. This work focuses on the cytochrome P450 genes of S. litura, and we show in this study that a specific S. litura P450 gene is likely to be involved in the metabolism of two commonly-used insecticides. Severely inhibiting the expression of this P450 gene in vivo results in insects which are much more sensitive to inhibition from these compounds, indicating that the gene (designated CYP321B1), plays a role in their detoxification. Additionally, CYP321B1 expression levels increase significantly in insects exposed to these inseticides. Taken together, the results from this work strongly suggest a role for CYP321B1 in the detoxification of insecticides commonly used for the control of this pest.

Technical Abstract: BACKGROUND: 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 (Lepidoptera, Noctuidae) has been shown to be resistant to a wide range of insecticides. In this study, a novel P450 gene, CYP321B1, were 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. RESULTS: The full-length cDNA sequence of CYP321B1 is 1814 bp long with an ORF of 1488 bp. The encoded 495 amino acid protein has a predicted mass of 57.03 kDa and a theoretical PI value of 8.49. Reverse transcriptase-quantitative PCR (RT-qPCR) analyses during larval and pupal development indicated that CYP321B1 expression was highest in 5th-instar larvae. The highest expression levels were observed in the midgut of 5th-instar larvae, followed by fat body and cuticle. The expression of CYP321B1 in the midgut of S. litura was up-regulated by chlorpyrifos, ß-cypermethrin and methomyl with both LC15 (50, 100 and 150 µg/mL, respectively) and LC50 dosages (100, 200 and 300 µg/mL, respectively). RNAi-mediated silencing of CYP321B1 further increased mortality by 25.6% and 38.9% when the 5th-instar larvae were exposed to chlorpyrifos and ß-cypermethrin, respectively, at the LC50 dose levels. CONCLUSION: The results demonstrated that silencing of CYP321B1 increased the susceptibility of S. litura to chlorpyrifos and ß-cypermethrin, suggesting that CYP321B1 may play an important role in chlorpyrifos and ß-cypermethrin detoxification in S. litura.