Abstract
Cytochrome P450 monooxygenases (P450s) are widely distributed multifunctional enzymes that play crucial roles in insecticide detoxification or activation. In this study, to ascertain the molecular mechanisms of P450s in the detoxification of Chinese honeybees, Apis cerana cerana Fabricius (A. c. cerana), we isolated and characterized four new P450 genes (Acc301A1, Acc303A1, Acc306A1, and Acc315A1). The open reading frames of the four genes are 1263 to 1608 bp in length and encode four predicted polypeptides of 499 to 517 amino acids in length. Real-time quantitative PCR (RT-qPCR) results showed that expression of all four genes was observed in all developmental stages. In addition, Western blot assays further indicated the RT-qPCR results that showed that the four genes were induced by pesticide (thiamethoxam, deltamethrin, dichlorovos, and paraquat) treatments. Furthermore, we also used double-stranded RNA-mediated RNA interference to investigate the functions of Acc301A1, Acc303A1,and Acc306A1 in the antioxidant defense of honeybees. RNA interference targeting Acc301A1, Acc303A1, and Acc306A1 significantly increased the mortality rate of A. c. cerana upon pesticide treatment. These results provide important evidence about the role of the four P450 genes involved in detoxification.
Highlights
Organisms have a variety of antioxidant enzymes and detoxifying enzymes, including glutathione S-transferases (GSTs), carboxylesterases, and cytochrome P450 monooxygenases (P450s), that reduce the potential damage of environmental pollutants (Li et al, 2007)
To analyze gene expression at different stages, samples were randomly collected from larvae (L1–L5, from the first to fifth instars), pupae (Pp: prepupae, white-eyed pupae (Pw): whiteeyed pupae, Pb: brown-eyed pupae, and Pd: dark-eyed pupae), and newly emerged adults (NE) (Michelette and Soares, 1993)
The four genes belong to the Mit P450 and CYP2 clades
Summary
Organisms have a variety of antioxidant enzymes and detoxifying enzymes, including glutathione S-transferases (GSTs), carboxylesterases, and cytochrome P450 monooxygenases (P450s), that reduce the potential damage of environmental pollutants (Li et al, 2007). The CYP3 clades, those of the CYP6 and CYP9 families, have frequently been shown to play a role in the detoxification and the metabolic resistance to insecticides across a range of insect species (Liu et al, 2010; Poupardin et al, 2010; Cifuentes et al, 2012; Musasia et al, 2013). Studies provided evidence that the CYP4G11 gene plays an important role in protecting honeybees from insecticide (phoxim, paraquat, decamethrin) damage (Shi et al, 2013). It is crucial to identify the functions of P450s and to explore stress response mechanisms at the gene expression regulation level in A. c. Our results provide preliminary insight into the changes in the gene transcription of Acc301A1, Acc303A1, Acc306A1, and Acc315A1 and their responses and resistance to four insecticides (dichlorovos, thiamethoxam, deltamethrin, and paraquat). Our results should be useful to facilitate further studies on the roles of P450 genes in pesticide resistance in insects
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