Characterization and functional analysis of UDP-glycosyltransferases reveal their contribution to phytochemical flavone tolerance in Spodoptera litura.

Efficient detoxification is the key factor for phytophagous insect to adapt to phytochemicals. However, the role of uridine diphosphate (UDP)-glycosyltransferases (UGTs) in insect anti-defense to phytochemical flavone is largely unknown. In this study, 52 UGT genes were identified in Spodoptera litura and they presented evident gene duplication. UGT played a crucial part in larval tolerance to flavone because the enzyme activity and transcriptional level of 77 % UGT members were remarkably upregulated by flavone administration and suppression of UGT enzyme activity and gene expressions significantly increased larval susceptibility to flavone. Bacteria coexpressing UGTs had high survival rates under flavone treatment and flavone was dramatically metabolized by UGT recombinant cells, which indicated the involvement of UGTs in flavone detoxification. What's more, ecdysone pathway was activated by flavone. Topical application of 20-hydroxyecdysone highly upregulated UGT enzyme activity and more than half of UGT expressions. The effects were opposite when ecdysone receptor (EcR) and ultraspiracle (USP)-mediated ecdysone signaling pathway was inhibited. Furtherly, promoter reporter assays of 5 UGT genes showed that their transcription activities were notably increased by cotransfection with EcR and USP. In consequence, this study suggested that UGTs were involved in flavone detoxification and their transcriptional expressions were regulated by ecdysone pathway.

Role of the epsilon glutathione S-transferases in xanthotoxin tolerance in Spodoptera litura.

Spodoptera litura, a polyphagous lepidopteran pest, demonstrates a remarkable capacity to adapt to varying host plants by efficiently detoxifying phytochemicals. However, the underlying mechanism for this adaptation is not well understood. Herein, twenty eplison glutathione S-transferase genes (GSTes) were characterized and their roles in phytochemical tolerance were analyzed in S. litura. Most of the GSTe genes were mainly expressed in the larval midgut and fat body. Exposure to the phytochemicals, especially xanthotoxin, induced the expression of most GSTe genes. Molecular docking analysis revealed that xanthotoxin could form stable bonds with six xanthotoxin-responsive GSTes, with binding free energies ranging from -36.44 to -68.83 kcal mol-1. Knockdown of these six GSTe genes increased the larval susceptibility to xanthotoxin. Furthermore, xanthotoxin exposure significantly upregulated the expression of two transcription factor genes CncC and MafK. Silencing of either CncC or MafK reduced the expression of GSTe16, which exhibited the largest change in response to xanthotoxin. Additionally, analysis of the promoter sequence of GSTe16 revealed the presence of seven CncC/Maf binding sites. Luciferase reporter assays showed that CncC and MafK enhanced the expression of GSTe16, leading to the increased xanthotoxin tolerance in S. litura. These findings provide insight into the functions and transcriptional regulatory mechanisms of GSTes, thereby enhancing our understanding of the role of GSTs in the adaptation of lepidopteran pests to phytochemicals.

CYP321A Subfamily P450s Contribute to the Detoxification of Phytochemicals and Pyrethroids in Spodoptera litura.

Although the induction of cytochrome P450 monooxygenases involved in insect detoxification has been well documented, the underlying regulatory mechanisms remain obscure. In Spodoptera litura, CYP321A subfamily members were effectively induced by exposure to flavone, xanthotoxin, curcumin, and λ-cyhalothrin, while knockdown of the CYP321A genes increased larval susceptibility to these xenobiotics. Homology modeling and molecular docking analyses showed that these four xenobiotics could stably bind to the CYP321A enzymes. Furthermore, two transcription factor genes, CncC and MafK, were significantly induced by the xenobiotics. Knockdown of CncC or MafK reduced the expression of four CYP321A genes and increased larval susceptibility to the xenobiotics. Dual-luciferase reporter assays showed that cotransfection of reporter plasmids carrying the CYP321A promoter with CncC and/or MafK-expressing constructs significantly magnified the promoter activity. These results indicate that the induction of CYP321A subfamily members conferring larval detoxification capability to xenobiotics is mediated by the activation of CncC and MafK.

Nuclear receptors potentially regulate phytochemical detoxification in Spodoptera litura.

Phytochemicals are a class of potential pesticides for pest control. Our previous studies have demonstrated that the development of Spodoptera litura is suppressed by two phytochemicals, flavone and xanthotoxin. Generally, phytochemical is metabolized by insect detoxification enzyme systems. Nuclear receptor (NR) is the ligand-activated transcription factor that involved in the regulation of detoxification gene expressions. To explore how NR responds to phytochemical to mediate detoxification gene expression, in the present study, 19 NRs were firstly identified in S. litura genome. The transcriptional levels of most NRs were significantly induced in the midgut of S. litura larvae after exposure to flavone and xanthotoxin. RNAi-mediated knockdown of FTZF1, EcR, Dsf, and HR3 remarkably reduced the larval tolerance to flavone or xanthotoxin. In addition, many crucial detoxification genes were downregulated by dsNR administrations, which might be responsible for the high sensitivity of S. litura to phytochemicals. Molecular docking indicated that phytochemicals as the potential ligands had high affinity to bind to NRs. This study suggested that NR potentially regulated the transcriptional expression of detoxification genes in response to phytochemical stresses, which partially elucidated the mechanism of extensive host adaptation in S. litura and provided the theoretical evidences for the development of NR-targeted insecticides.