Expression and functional analysis of cytochrome P450 genes in the wolf spider Pardosa pseudoannulata under cadmium stress.

ABSTRACT

Cytochrome P450 enzymes (CYPs), encoded by Halloween genes, mediate the biosynthesis of molting hormone, ecdysteroids, in arthropods. In this report, the effect of heavy metal cadmium (Cd) stress on the expression of cytochrome P450 genes in the wolf spider Pardosa pseudoannulata was analyzed. The results showed the expression levels of genes encoding for Cd transporters including ABC transporters, zinc transporters, calcium channel proteins and calcium binding proteins were inhibited or induced by Cd stress. In addition, the increase in metallothionein (MT) content and glutathione peroxidase (GPX) activity and decrease in total acetylcholine esterase (AChE) activity were also detected. Apparently, these detoxification methods did not completely protect the spider from the cytotoxicity of Cd stress. Increased mortality of P. pseudoannulata was observed when they were under Cd tress. In total 569 CYP genes belonging to 62 CYP subfamilies were obtained from P. pseudoannulata RNA-seq databases. BlaxtX analysis showed that 150, 161, 11, and 40 CYP genes were similar to the genes dib, phm, sad and shd, respectively, which are thought to catalyze the biosynthesis of ecdysteroids. Gene expression analysis suggested that 25 dib encoding genes, 27 phm encoding genes, 2 sad encoding genes, and 6 shd encoding genes were differentially expressed in TS2 vs. S2 comparison (Cd-treated 2nd instar spider vs. 2nd instar spider), respectively. There were 70 dib, 70 phm and 19 shd encoding genes either upregulated or downregulated, while 3 sad encoding genes were upregulated in TS5 vs. S5 (Cd-treated 5nd instar spider vs. 5nd instar spider). Genes related to heme binding and essential for activating the CYPs were also differentially expressed. Expression levels of cuticle related genes were significant differentially expressed, implying the changes in activities of chitin synthases and chitinase. Therefore we assume that unsuccessful molting process may occur on P. pseudoannulata due to influenced ecdysteroids levels, thus increasing mortality of spider.