Identification and characterization of six peroxiredoxin transcripts from mud crab Scylla paramamosain: The first evidence of peroxiredoxin gene family in crustacean and their expression profiles under biotic and abiotic stresses.

The peroxiredoxins (Prxs) define a novel and evolutionarily conserved superfamily of peroxidases able to protect cells from oxidative damage by catalyzing the reduction of a wide range of cellular peroxides. Prxs have been identified in prokaryotes as well as in eukaryotes, however, the composition and number of Prxs family members vary in different species. In this study, six Prxs were firstly identified from the mud crab Scylla paramamosain by RT-PCR and RACE methods. Six SpPrxs can be subdivided into three classes: (a) three typical 2-Cys enzymes denominated as Prx1/2, 3, 4, (b) two atypical 2-Cys enzymes known as Prx5-1 and Prx5-2, and (c) a 1-Cys isoform named Prx6. The evolutionarily conserved signatures of peroxiredoxin catalytic center were identified in all six SpPrxs. Phylogenetic analysis revealed that SpPrx3, SpPrx4, SpPrx5s and SpPrx6 were clearly classified into Prx3-6 subclasses, respectively. Although SpPrx1/2 could not be grouped into any known Prx subclasses, SpPrx1/2 clustered together with other arthropods Prx1 or unclassified Prx and could be classified into the typical 2-Cys class. The comparative and evolutionary analysis of the Prx gene family in invertebrates and vertebrates were also conducted for the first time. Tissue-specific expression analysis revealed that these six SpPrxs were expressed in different transcription patterns while the highest expression levels were almost all in the hepatopancreas. Quantitative RT-PCR analysis exhibited that the gene expression profiles of six SpPrxs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and hypoosmotic salinity, suggesting that the SpPrxs might play different roles in response to various stresses. The recombinant proteins including the SpPrx1/2, SpPrx4, SpPrx5-1 and SpPrx6 were purified and the peroxidase activity assays indicated that all these proteins can reduce H2O2 in a typical DTT-dependent manner. To our knowledge, this is the first study about the comprehensive characterization of Prx gene family in Scylla paramamosain and even in crustaceans. These results would broaden the current knowledge of the whole Prx family as well as be helpful to understand and clarify the evolutionary pattern of Prx family in invertebrate and vertebrate taxa.

Molecular characterization of a cDNA encoding Na+/K+/2Cl- cotransporter in the gill of mud crab (Scylla paramamosain) during the molt cycle: Implication of its function in osmoregulation.

Although iono-regulatory processes are critical for survival of crustaceans during the molt cycle, the mechanisms involved are still not clear. The Na+/K+/2Cl- cotransporter (NKCC), a SLC12A family protein that transports Na+, K+ and 2Cl- into cells, is essential for cell ionic and osmotic regulation. To better understand the role of NKCC in the molt osmoregulation, we cloned and characterized a NKCC gene from the mud crab, Scylla paramamosain (designated as SpNKCC). The predicted SpNKCC protein is well conserved, and phylogenetic analysis revealed that this protein was clustered with crustacean NKCC. Expression of SpNKCC was detected in all the tissues examined but was highest in the posterior gills. Transmission electron microscopy revealed that posterior gills had a thick type of epithelium for ion regulation while the anterior gills possessed a thin phenotype related to gas exchange. During the molting cycle, hemolymph osmolality and ion concentrations (Na+ and Cl-) increased significantly over the postmolt period, remained stable in the intermolt and premolt stages and then decreased at ecdysis. Meanwhile, the expression of SpNKCC mRNA was significantly elevated (26.7 to 338.8-fold) at the ion re-establishing stages (postmolt) as compared with baseline molt level. This pattern was consistent with the coordinated regulation of Na+/K+-ATPase α-subunit (NKA α), carbonic anhydrase cytoplasmic (CAc) isoform and Na+/H+ exchanger (NHE) genes in the posterior gills. These data suggest that SpNKCC may be important in mediating branchial ion uptake during the molt cycle, especially at the postmolt stages.