Effects on the survival rates, hematological parameters, and neurotransmitters in olive flounders, Paralichthys olivaceus, reared in bio-floc and seawater by Streptococcus iniae challenge.

Bacterial infections cause huge losses to aquaculture globally, and increased antibiotic resistance means that alternative methods of reducing mortality from bacterial diseases are required. We compared the resistance of Juvenile olive flounders, Paralichthys olivaceus, to Streptococcus iniae between those reared in biofloc and seawater conditions for ten months. Experimental fish were challenged with S. iniae at concentrations of 0, 3.36 × 106, 3.36 × 107, 3.36 × 108, and 3.36 × 109 colony forming units (CFU)/g fish for 96 h to evaluate the difference in S. iniae susceptibility of flounders reared in biofloc and seawater. The 96 h lethal concentration 50% (LC50) of fish injected with S. iniae was 2.41 × 109 CFU/g fish in biofloc and 1.51 × 108 CFU/g fish in seawater. Hematological parameters such as hemoglobin and hematocrit significantly decreased when fish were challenged by S. iniae. Plasma components such as calcium, glucose, cholesterol, total protein, GOT, GPT, and ALP were significantly altered by S. iniae infection and acetylcholinesterase activity was significantly inhibited. These results indicate that S. iniae infection affects the survival rates, hematological parameters, and neurotransmitter levels of flounders reared in biofloc and seawater, and that S. iniae susceptibility was higher in flounders reared in seawater than those reared in biofloc.

Toxic effects of waterborne ammonia exposure on hematological parameters, oxidative stress and stress indicators of juvenile hybrid grouper, Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀.

Juvenile hybrid grouper, Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀ (mean weight: 26.5 ±â€¯2.8 g, mean length: 11.8 ±â€¯1.3 cm) were exposed to different, sub-lethal levels of waterborne ammonia (0, 1, 2, 4, and 8 mg NH4+/L) for 2 weeks. We assessed the hematological parameters, antioxidant enzymes, and stress responses of juvenile hybrid grouper after 1 week and after 2 weeks. Hematological parameters such as hemoglobin and hematocrit levels, were significantly decreased by ammonia exposure. Plasma components such as the magnesium and total protein contents, and the glutamic oxaloacetic transaminase and glutamic pyruvic transaminase activities were significantly altered by ammonia exposure; however, no changes in the magnesium levels were detected. Antioxidant responses, such as superoxide dismutase and glutathione S-transferase activities, were also significantly affected by ammonia exposure. Stress indicator levels, i.e., plasma cholesterol and heat shock protein 70 levels, were significantly increased by ammonia exposure. The results of this study indicated that ammonia exposure has toxic effects on juvenile hybrid grouper and affects their hematological parameters, antioxidant enzymes, and stress responses.

Molecular aspects, genomic arrangement and immune responsive mRNA expression profiles of two CXC chemokine receptor homologs (CXCR1 and CXCR2) from rock bream, Oplegnathus fasciatus.

The CXCR1 and CXCR2 are the prototypical receptors and are the only known receptors for mammalian ELR+ (Glu-Leu-Arg) CXC chemokines, including CXCL8 (interleukin 8). These receptors transduce the ELR+ chemokine signals and operate the downstream signaling pathways in inflammation and innate immunity. In this study, we report the identification and characterization of CXCR1 and CXCR2 genes from rock bream fish (OfCXCR1 and OfCXCR2) at the molecular level. The cDNA and genomic DNA sequences of the OfCXCR1 and OfCXCR2 were identified from a transcriptome library and a custom-constructed BAC library, respectively. Both OfCXCR genes consisted of two exons, separated by an intron. The 5'-flanking regions of OfCXCR genes possessed multiple putative transcription factor binding sites related to immune response. The coding sequences of OfCXCR1 and OfCXCR2 encoded putative peptides of 355 and 360 amino acids (aa), respectively. The deduced aa sequences of OfCXCR1 and OfCXCR2 comprised of a G-protein coupled receptors (GPCR) family 1 profile with a GPCR signature and a DRY motif. In addition, seven conserved transmembrane regions were predicted in both OfCXCRs. While our multiple alignment study revealed the functionally significant conserved elements of the OfCXCR1 and OfCXCR2, phylogeny analyses further confirmed their position in teleost sub clade, in which they manifested an evolutionary relatedness with other fish counterparts. Based on comparative analyses, teleost CXC chemokine receptors appear to be distinct from their non-fish orthologs in terms of evolution (both CXCR1 and CXCR2) and genomic organization (CXCR2). Quantitative real-time PCR (qPCR) detected the transcripts of OfCXCR1 and OfCXCR2 in eleven examined tissues, with higher levels in head kidney, kidney and spleen highlighting their crucial importance in immunity. In vitro stimulation of peripheral blood leukocytes (PBLs) with concanavalin A (Con A) resulted in modulation of OfCXCR2 transcription, but not that of OfCXCR1. In addition, the magnitude of the OfCXCR1 and OfCXCR2 transcripts in head kidney and spleen was differentially increased after the in vivo administration of immune stimulants, LPS and poly I:C and in the infection models injected with rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. These lines of evidence suggest that these receptors may play an important role(s) in immune responsive signaling during pathogenesis of rock bream.

A mu class glutathione S-transferase from Manila clam Ruditapes philippinarum (RpGSTµ): cloning, mRNA expression, and conjugation assays.

Glutathione S-transferases (GSTs) are enzymes that catalyze xenobiotic metabolism in the phase II detoxification process. GSTs have a potential for use as indicators or biomarkers to assess the presence of organic and inorganic contaminants in aquatic environments. In this study, a full-length cDNA of a mu (µ) class GST (RpGSTµ) was identified from Manila clam (Ruditapes philippinarum) and biochemically characterized. The 1356 bp of the cDNA included an open reading frame of 651 bp encoding a polypeptide of 217 amino acid residues with a molecular mass of 25.04 kDa and an estimated pI of 6.34. Sequence analysis revealed that the RpGSTµ possessed several characteristic features of µ class GSTs, such as a thioredoxin-like N-terminal domain containing binding sites for glutathione (GSH), a C-terminal domain containing substrate binding sites, and a µ loop. The recombinant RpGSTµ (rRpGSTµ) protein exhibited GSH-conjugating catalytic activity towards several substrates, and significantly strong activity was detected against 4-nitrophenethyl bromide (5.77 ± 0.55) and 1-chloro-2,4-dinitrobenzene (CDNB, 3.19 ± 0.05). Kinetic analysis as a function of GSH and CDNB concentrations revealed relatively low Km values of 1.03 ± 0.46 mM and 0.56 ± 0.20 mM, respectively, thereby indicating a GSH-conjugation attributed with high rates. The optimum pH and temperature for the catalytic activity of the rRpGSTµ protein were 7.7 and 37°C, respectively. The effect of two inhibitors, Cibacron blue and hematin, on the activity of rRpGSTµ was evaluated and the IC50 values of 0.65 µM and 9 µM, respectively, were obtained. While RpGSTµ transcripts were highly expressed in gills and hemocytes, a significant elevation in mRNA levels was detected in these tissues after lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly I:C) and live bacterial (Vibrio tapetis) challenges. These findings collectively suggest that RpGSTµ functions as a potent detoxifier of xenobiotic toxicants present in the aquatic environment, and that its mRNA expression could be modulated by pathogenic stress signal(s).

Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties.

Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297 bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11 kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the 'Q-X-V-X-G' motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon in vivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response.

Fine structure of Longicollum pagrosomi (Acanthocephala: Pomphorhynchidae) and intestinal histopathology of the red sea bream, Pagrus major, infected with acanthocephalans.

The results described the structure of Longicollum pagrosomi and histopathological characters of the intestine of the red sea bream, Pagrus major, infected with acanthocephalans, using the light and electron microscopes. Among the six samples of P. major, L. pagrosomi was identified in the posterior intestine of five fish samples. Adult L. pagrosomi (total length, 8-27 mm) is divided into the presoma (proboscis, anterior neck, and posterior neck) and metasoma (trunk). The proboscis had vertically arranged hooks (40 µm in length), with ten hooks per row, and the septum was observed between the posterior neck and trunk. The tegument thickness of the proboscis was approximately 15 µm, and it was composed of thin, circular muscle fibers. The outer fibrous membrane was approximately 1 µm, and the connective tissue layer was approximately 35 µm in thickness in the anterior neck. The tegument of the posterior neck enclosed the cephalic ganglion and had longitudinal and vertical muscle fibers, and the tegument thickness was approximately 45 µm. The tegument of the body, which was approximately 1 mm in thickness, was composed primarily of muscle and collagen fibers, and the structure of the tegument was different, depending on the body region. The acanthocephalans had ovaries and oval-shaped eggs with an eggshell (77.5 × 17.1 µm), floating within the body cavity of the trunk. In the infected posterior intestine of P. major, the presoma and the anterior part of the metasoma of L. pagrosomi passed through the intestinal wall and infected the intestinal tissue, perforating the loose connective tissue. In the inflammatory connective tissue, collagen and muscle fibers were fragmented and revealed partial necrosis. Lipid drops and eosinophilic granular cells aggregated in the connective tissue of the tissue capsule. In the vicinity of the acanthocephalan, the mucosal epithelia contained hypertrophied nuclei, and the epithelial layer was collapsed. In an extreme case, the mucosal fold was degenerated because of pressure from the acanthocephalan.

Detection of hepatitis a virus from oyster by nested PCR using efficient extraction and concentration method.

The molecular methods using polymerase chain reaction have been proposed as useful tools for the identification of viral pathogens in food and water. However, the PCR-based methods are highly dependent on the methods of virus concentration and nucleic acid purification due to the low sensitivity of PCR in the presence of PCR inhibitors. We developed TPTT [tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead] protocol in order to detect hepatitis A virus (HAV) inoculated in oyster digestive glands. The detection limit of HAV precipitated with zirconium hydroxide was 10(5) fold less sensitive in a nested PCR than that precipitated the HAV supernatant twice with PEG/NaCl (16% polyethylene glycol 6,000, 0.525 M NaCl) in a 1:2 (v/v) ratio, which provided an efficient detection of 0.0148 PFU/g from approximately 0.05 g of oyster homogenate. This method is efficient for potential use in the detection of HAV from shellfish and is more sensitive than most currently published tests.

Distribution of marine birnavirus in cultured olive flounder Paralichthys olivaceus in Korea.

Surveys of marine birnavirus (MABV) were undertaken in cultured olive flounder Paralichthys olivaceus from the south and west coastal areas and Jeju in Korea during the period January 1999 to April 2007. MABV was detected in all seasons from the fry, juveniles and adult fish from the areas examined. Evident cytopathic effects of the virus including rounding and cell lysis were observed in chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells, but not in fathead minnow (FHM) and epithelial papilloma of carp (EPC) cells. Nucleotide sequences of the VP2/NS junction region of the Korean isolates showed 97.8% ~ 100% similarity, and they belonged to the same genogroup. Cross neutralization tests with serotype-specific rabbit antisera against MABV strains exhibited a close antigenic relationships between strains, and were distinct from infectious pancreatic necrosis virus (IPNV) strains. Coinfection of MABV with bacteria (Streptococcus iniae, Vibrio spp.) and viruses (nervous necrosis virus, lymphocystis disease virus, viral hemorrhagic septicemia virus) was observed.

Genetic variation and geographic distribution of megalocytiviruses.

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae have caused mass mortalities in marine and freshwater fish in Asian countries. In this study, partial major capsid protein (MCP) gene of seven Japanese and six Korean megalocytiviruses was sequenced and compared with the known megalocytiviruses to evaluate genetic variation and geographic distribution of the viruses. Comparison of MCP gene nucleotide sequences revealed sequence identity of 92.8% or greater among these 48 isolates. A phylogenetic tree clearly revealed three clusters: genotype I including nine Japanese isolates, thirteen Korean isolates, one Chinese isolates, one Thailand isolate and one South China Sea isolate; genotype II including five freshwater fish isolates in Southeast Asian countries and Australia; and the remaining genotype III mainly consisted of flatfish isolate in Korea and China. This suggests that viruses belonging to the genotype I widely distribute among various fish species in many Asian countries. Conversely, the epidemic viruses belonged to genotype II and III are may be still locally spreading and constrained in their prevalence to the limited host fish species, i.e., genotype II viruses mainly distribute in Southeast Asian countries, whereas genotype III viruses distribute in flatfish species in Korea and China.

Occurrence of tetracycline resistance genes tet(M) and tet(S) in bacteria from marine aquaculture sites.

Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea. The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp. piscicida, and unidentified Gram-positive bacteria. The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 microg/ml of oxytetracycline and only four isolates had MICs less than 31.3 microg/ml. 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location. One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea. The tet(S) gene was detected in L. garvieae from yellowtail in Japan and in Vibrio sp. from seawater in Korea. This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes. These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment.