Changes in both trans- and cis-regulatory elements mediate insecticide resistance in a lepidopteron pest, Spodoptera exigua.

The evolution of insect resistance to insecticides is frequently associated with overexpression of one or more cytochrome P450 enzyme genes. Although overexpression of CYP450 genes is a well-known mechanism of insecticide resistance, the underlying regulatory mechanisms are poorly understood. Here we uncovered the mechanisms of overexpression of the P450 gene, CYP321A8 in a major pest insect, Spodoptera exigua that is resistant to multiple insecticides. CYP321A8 confers resistance to organophosphate (chlorpyrifos) and pyrethroid (cypermethrin and deltamethrin) insecticides in this insect. Constitutive upregulation of transcription factors CncC/Maf are partially responsible for upregulated expression of CYP321A8 in the resistant strain. Reporter gene assays and site-directed mutagenesis analyses demonstrated that CncC/Maf enhanced the expression of CYP321A8 by binding to specific sites in the promoter. Additional cis-regulatory elements resulting from a mutation in the CYP321A8 promoter in the resistant strain facilitates the binding of the orphan nuclear receptor, Knirps, and enhances the promoter activity. These results demonstrate that two independent mechanisms; overexpression of transcription factors and mutations in the promoter region resulting in a new cis-regulatory element that facilitates binding of the orphan nuclear receptor are involved in overexpression of CYP321A8 in insecticide-resistant S. exigua.

Insecticides induce the co-expression of glutathione S-transferases through ROS/CncC pathway in Spodoptera exigua.

Glutathione S-transferases (GSTs) are a family of multifunctional enzymes that are involved in detoxification of electrophilic toxic compounds. Although the co-induced expression of GST genes by insecticides in insects has been documented in recent years, the underlying regulatory mechanisms are not understood. In this study, a total of thirty-one cytosolic S. exigua GSTs (SeGSTs) was cloned and identified. The bioinformatics and gene expression patterns were also analyzed. Out of them, SeGSTe9, SeGSTs6, SeGSTe1, SeGSTe6, SeGSTe8, SeGSTe14, and SeGSTd1 were significantly co-expressed following exposure to three insecticides (lambda-cyhalothrin, chlorpyrifos and chlorantraniliprole). The analysis of upstream sequences revealed that all of these seven SeGSTs harbored CncC/Maf binding site. The luciferase reporter assay showed that the pGL3-SeGST promoter construct exhibited a significant increase in luciferase activities after exposure to insecticides, and mutation of CncC/Maf binding site diminish the induction effect. These data indicate that CncC/Maf pathway regulates the co-expression of GST genes in response to different insecticides in S. exigua. Insecticides significantly enhanced the ROS content and treatment with the ROS inhibitor N-acetylcysteine (NAC) decreased the insecticide-induced luciferase activities of the PGL3-GSTe6 promoter construct, but not the CncC-mutated construct. These results indicate that ROS mediates GST gene expression after exposure to insecticides through CncC/Maf pathway. Overall, these data show that insecticides induce the co-expression of glutathione S-transferases through the ROS/CncC pathway in S. exigua.

[Chlorophyll content and net photosynthetic rate of Machilus pauhoi and M. leptophylla].

By using SPAD-502 chlorophyll meter, LI-6400 portable photosynthesis system, and spectrophotometer, the leaf SPAD value, net photosynthetic rate (Pn), and chlorophyll (a + b) content (Ct) of 3-year-old Machilus pauhoi and M. leptophylla seedlings were measured, and the relationships of SPAD value with Pn and Ct were analyzed. The M. pauhoi seedlings were grown from the seeds originated from Suichuan County of Jiangxi Province and Jian'ou County of Fujian Province, named as MPS and MPJ, respectively; while the M. leptophylla seedlings were grown from the seeds originated from Shangyou County of Jiangxi Province, named as MLG. There were significant differences in the mean chlorophyll content of MPS, MPJ, and MLG. The SPAD value and the contents of chlorophyll (a + b) (Ct), chlorophyll a (Ca) and chlorophyll b (Cb) were in the order of MPS < MLG < MPJ, with the mean SPAD value being 43.80, 45.12, and 50.67 and the Ct value being 1.944, 2.831, and 3.447 mg c g(-1), respectively. The chlorophyll content was influenced by the maturing degree of mesophyll tissues of M. pauhoi and M. leptophylla, being lower in current-year leaves than in 2-year-old leaves. The Ct of same age leaves at different crown layers of MPS and MPJ and of MLG was in the order of upper layer < middle layer < lower layer and of upper layer < lower layer < middle layer, respectively, and the SPAD value of the same lamina at different positions was in the order of apex < middle < base. SPAD value had a significant positive linear correlation with Ct, and a statistically not significant positive correlation with Pn.