The Development and Characterization of a Cell Culture System from Indian Mud Crabs Scylla serrata.

Commercially available culture media and supplements were tested for their potential to produce primary cell cultures from tissues of Indian mud crabs Scylla serrata. Eight commercially available culture media from Sigma-Aldrich (Leibovitz's L-15, Medium 199, Grace's Insect Medium, Minimal Essential Medium, Dulbecco's Modified Eagle Medium, TC-100 Insect Medium, IPL-41 Insect Medium, and Roswell Park Memorial Institute) were examined. Three different supplements (amino acid and sugar [AS], crab muscle extract [CME], and natural seawater [NSW]) were also examined. The hemocyte culture appeared to grow well for a maximum period of 21 d in 2 × L-15 medium supplemented with AS and 15% fetal bovine serum (FBS). Partial amplification and sequencing of the cytochrome oxidase subunit I (COI) gene confirmed that the primary hemocytes originated from Indian mud crabs. The effects of four metals on hemocyte viability were evaluated using the MTT assay. Of the four metals examined (arsenic, lead, cobalt, and nickel), cobalt and nickel were more toxic to the crab cells than the other metals. Both acridine orange/ethidium bromide and Hoechst staining showed the presence of apoptosis and necrosis in metal-treated groups, which suggests that metals in an aquatic environment induce death of the Indian mud crab's hemocytes. The hemocyte primary cell culture was also used to study the cytotoxicity effect of bacterial extracellular products from Vibrio harveyi and white spot syndrome virus. This study demonstrates that hemocyte primary cell culture can be used as a tool to study viral and bacterial pathogenesis and to assess the cytotoxicity of pollutants present in aquatic environments.

Intracellular Copper Zinc Superoxide dismutase (icCuZnSOD) from Asian seabass (Lates calcarifer): molecular cloning, characterization and gene expression with reference to Vibrio anguillarum infection.

Copper Zinc Superoxide dismutase (CuZnSOD) is the family of most important antioxidant metalloenzymes that protects tissues from damage by reactive oxygen species (ROS). In the present study, the intracellular copper zinc SOD from the Asian seabass Lates calcarifer (Lc-icCuZnSOD) was identified by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) technique. The full-length cDNA of Lc-icCuZnSOD consisted of 809 nucleotides with an open-reading frame of 465 bp encoding 154 amino acids and N-Glycosylation site (NVTA) within. The predicted molecular mass of the protein is 15.84 kDa with an estimated pI of 5.52. The deduced amino acid sequence of Lc-icCuZnSOD shared high degree of homology with known CuZnSODs from other species. CuZn binding sites (H47, H49, H64, and H121 for Cu(2+) and H72, H81, and ASP84 for Zn(2+)), two cysteine residues (aa 58 and 147) that form a disulfide bond, and CuZnSOD family signature sequences (GFHVHAFGDNT, aa 45-55 and GNAGGRLACGVI, aa 139-150) were highly conserved among fish species. Temporal and tissue specific expression of Lc-icCuZnSOD was significantly differentially altered in Asian seabass challenged with Vibrio anguillarum indicating possible role in antioxidant activities involved in the innate immune defense mechanisms.

Characterization of four lytic transducing bacteriophages of luminescent Vibrio harveyi isolated from shrimp (Penaeus monodon) hatcheries.

Four lytic bacteriophages designated as φVh1, φVh2, φVh3, and φVh4 were isolated from commercial shrimp hatcheries, possessing broad spectrum of infectivity against luminescent Vibrio harveyi isolates, considering their potential as biocontrol agent of luminescent bacterial disease in shrimp hatcheries, and were characterized by electron microscopy, genomic analysis, restriction enzyme analysis (REA), and pulsed-field gel electrophoresis (PFGE). Three phages φVh1, φVh2, and φVh4 had an icosahedral head of 60-115 nm size with a long, noncontractile tail of 130-329 × 1-17 nm, belonged to the family Siphoviridae. φVh3 had an icosahedral head (72 ± 5 nm) with a short tail (27 × 12 nm) and belonged to Podoviridae. REA with DraI and PFGE of genomic DNA digested with ScaI and XbaI and cluster analysis of their banding patterns indicated that φVh3 was distinct from the other three siphophages. PFGE-based genome mean size of the four bacteriophages φVh1, φVh2, φVh3, and φVh4 was estimated to be about 85, 58, 64, and 107 kb, respectively. These phages had the property of generalized transduction as demonstrated by transduction with plasmid pHSG 396 with frequencies ranging from 4.1 × 10(-7) to 2 × 10(-9) per plaque-forming unit, suggesting a potential ecological role in gene transfer among aquatic vibrios.