Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.     

Purification and characterization of an antibacterial protein in the skin secretion of rockfish Sebastes schlegeli

An antibacterial protein in the skin secretion of rockfish (Sebastes schlegeli) was purified by lectin affinity chromatography on Con A-Sepharose and gel filtration on TSKgel G3000SW. The antibacterial protein featured the high molecular mass and selective action against Gram-negative bacteria. The molecular mass of the protein was estimated to be approximately 150 kDa in gel filtration and approximately 75 kDa by SDS-PAGE, suggesting that it is dimeric. The antibacterial principle was an acidic glycoprotein with pI 4.5, 3.4% reducing sugar and 2.8% amino sugar. Its sugar chains had N-type (high mannose-type) oligosaccharide and sialic acid components. It inhibited strongly the growth of Aeromonas salmonicida, Photobacterium damselae and Shewanella putrefaciens with a minimum inhibitory concentration (MIC) of approximately 3 microg/ml, and moderately the growth of Vibrio parahaemolyticus and A. hydrophila with a MIC of 12.5 microg/ml and 25 microg/ml, respectively. The values of the minimum bactericidal concentration were almost equivalent to those of MIC. The potent sensitivity against virulent pathogens such as A. hydrophila, A. salmonicida and P. damselae may contribute considerably to the innate host defense mechanism to combat microbes on the mucosal surfaces of the rockfish.     

Efficacy of protein A-HRP in an immunological study of black rockfish (Sebastes schlegeli Higendorf) humoral immune responses

The efficacy of protein A-horse radish peroxidase (HRP), as compared to that of mouse polyclonal antibody raised against purified Ig, in detection of black rockfish (Sebastes schlegeli Higendorf) immunoglobulin (Ig) was examined. Protein A affinity chromatography successfully purified Ig from black rockfish serum; the purified-Ig could be visualised as two protein bands (MW 70 and 25kDa) following resolution with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. In SDS-PAGE immunoblot profiles of the purified-Ig, the mouse polyclonal antibody recognised both the light chain and heavy chains of rockfish Ig, whereas protein A-HRP immunostained only the heavy chain of rockfish Ig. These results suggest that protein A-HRP may be used to detect rockfish antibody-antigen complexes in immunoassays. In a 2-DE immunoblot assay for exploring antigenic profiles of Lactococcus garvieae KG9408, protein A-HRP successfully detected specific antibodies to antigenic proteins of L. garvieae in the rockfish Ig. In addition, enzyme linked immunosorbent assay (ELISA) showed a high correlation between the results obtained for positivity of L. garvieae when protein A-HRP and the mouse polyclonal antibody-was used to analyse samples from 25 diseased rockfish. These results collectively indicate that protein A-HRP has a high affinity for Ig, and may be useful for new investigations into the humoral immune responses of rockfish.     

Isolation and characterization of microsatellite markers for the Korean rockfish, Sebastes schlegeli

The Korean rockfish (Sebastes schlegeli) is an important commercial fish that is widely used in aquaculture. We isolated and characterized 18 polymorphic microsatellite loci from the Korean rockfish using a (GT)(13)-enriched genomic library. Polymorphism was assessed in 48 individuals from a single population collected from the northern coastal waters of the Yellow Sea. The observed and expected heterozygosities ranged from 0.0244 to 0.7660 (mean 0.4194) and 0.0244 to 0.8758 (mean 0.5002), respectively. Polymorphism at these loci indicated from two to 15 alleles (mean 5.7); 14 of 18 loci conformed to Hardy-Weinberg equilibrium. These markers should be useful for management and conservation studies of this species.     

Identification of antibacterial mechanism of L-amino acid oxidase derived from Trichoderma harzianum ETS 323

Although L-amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th-LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6-8 and less stable at both pH 4-5.5 and pH 9. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40 °C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50 °C. The results obtained by confocal microscopy and flow cytometry indicate that Th-LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs located at the N-terminus. The findings of increased exogenous H(2) O(2) production and reactive oxidative species accumulation in Th-LAAO-treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H(2)O(2) production are involved in the mechanism responsible for the antibacterial activity of Th-LAAO.     

Identification and expression analysis of an interferon stimulated gene 15 (ISG15) from black rockfish, Sebastes schlegeli

The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by IFNs, viral infection and double-stranded RNA (poly I:C). The ISG15 homolog cDNA was isolated from the black rockfish poly I:C stimulated leukocyte cDNA library. The black rockfish ISG15 homolog was found to consist of 1070bp encoding 160 amino acid residues. Compared with other known ISG15 peptide sequences, the most conserved regions of the black rockfish ISG15 peptide were found to be the tandem ubiquitin-like domains and a C-terminal LRLRGG conjugating motif, characteristic of mammalian and non-mammalian ISG15 proteins. A phylogenetic analysis based on the deduced amino acid sequence revealed a homologous relationship between the ISG15 sequence of black rockfish and that of Atlantic salmon, Atlantic cod, crucian carp and rainbow trout. The expression of the black rockfish ISG15 molecule was induced in the peripheral blood leukocytes (PBLs) from 1 to 12h following poly I:C stimulation, with a peak at 6h post-stimulation. The black rockfish gene was predominantly expressed in the PBLs and the spleen.     

Dietary inclusion of mealworm (Tenebrio molitor) meal as an alternative protein source in practical diets for juvenile rockfish (Sebastes schlegeli)

An 8‐week feeding trial was designed to evaluate the potential of yellow mealworm (Tenebrio molitor) as a locally available nutrient‐rich feedstuff for juvenile rockfish (Sebastes schlegeli). Experimental diets containing elevated levels of mealworm meal (WM) supplemented with synthetic methionine were formulated to be isonitrogenous, isolipidic and isoenergetic to a WM‐free fishmeal (FM) based control diet (designated as WM0, WM8, WM16, WM24 and WM32, respectively). To determine the necessity of dietary methionine supplementation at the highest inclusion of WM, a diet was prepared to contain 32% WM without methionine supplementation (WM32‐AA). Triplicate groups of rockfish juveniles (Mean ± S.E.; 3.11 ± 0.01 g) were fed one of the experimental diets to apparent satiation twice daily for 8 weeks. Fish growth performance in terms of weight gain and specific growth rate increased with increasing dietary inclusion of WM from 0 to 16% and then tended to decrease with further increase in dietary WM levels to 32%. Protein retention (PR) values followed the same trend as growth rates with the highest values found in fish offered WM16 diet. Although fish fed WM32‐AA diet showed significantly lower growth rate and PR values compared to those fed WM16 diet, their performance was still comparable to that of the WM‐free control group. Plasma triglyceride level was negatively affected by dietary WM inclusion and the lowest values were observed in the WM32‐AA group. Whole‐body and fillet proximate and essential amino acid compositions were not altered by dietary treatment and these values were comparable to those of the WM0 group. These findings suggested that WM might prove to be a promising alternative to FM in practical diets for juvenile rockfish and could be used at an inclusion level of up to 32% without having any adverse consequences for the health and performance of the fish. Although the diet containing 32% WM seemed to support a performance similar to that of the control diet, the recommended dietary inclusion level was no more than 16% of the diet dry matter.     

Development and application of monoclonal antibodies against IgM of black rockfish Sebastes schlegeli

Monoclonal antibodies (mAbs) against black rockfish Sebastes schlegeli serum immunoglobulin M (IgM) were developed, which showed a specific reaction with the heavy chain of S. schlegeli IgM in Western blotting and with surface IgM positive (sIgM⁺) lymphocytes in indirect immunofluorescence. mAb 2A6 was employed to investigate the antibody and sIgM⁺ lymphocyte responses of S. schlegeli injected with inactivated Edwardsiella tarda, by ELISA and flow cytometry. Compared with controls, the level of specific antibodies and the percentage of sIgM⁺ lymphocytes both increased in the immunized fish and simultaneously reached their peaks at day 35 after immunization.     

The complete mitochondrial genome of the rockfish Sebastes schlegeli (Scorpaeniformes, Scorpaenidae)

We isolated rockfish Sebastes schlegeli mitochondrial DNA by long-polymerase chain reaction (Long-PCR) with conserved primers, and sequenced it by primer walking using flanking sequences as sequencing primers. S. schlegeli mitochondrial DNA consists of 16,526 bp and its structural organization is conserved in comparison with other fish. Using mitochondrial control region sequences, we compared related species from the genus Sebastes (Scorpaeniformes, Scorpaenidae), showing the similarity of S. schlegeli isolated from Korean and Japanese waters. In this paper, we report the basic characteristics of the S. schlegeli mitochondrial genome including structural organization, base composition of rRNAs and the tRNAs and protein-encoding genes, and characteristics of mitochondrial tRNAs. These findings are applicable to aquaculture and to molecular phylogenetics in the genus Sebastes.     

Molecular cloning and characterization of peptidoglycan recognition proteins from the rockfish,Sebastes schlegeli

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are structurally conserved through evolution in both invertebrate and vertebrate animals. Here we report the identification and characterization of two long forms of PGRP (SsPGRP-L1 and SsPGRP-L2) from the rockfish, Sebastes schlegeli. The deduced amino acid sequences of SsPGRP-L1 and SsPGRP-L2, 466 and 482 residues respectively, contain the conserved PGRP domain and the four Zn²⁺-binding amino acid residues required for amidase activity. In addition to peptidoglycan-lytic amidase activity, recombinant SsPGRPs have broad-spectrum antimicrobial activity like zebrafish PGRPs. RT-PCR analysis of total RNA shows that the expression patterns of SsPGRP-L1 and SsPGRP-L2 genes are different, though they are widely expressed in the tissues that come in contact with bacteria. Overall, these data suggest that rockfish PGRPs are involved in the innate host defense of S. schlegeli against bacterial infections.     

Immunization of cultured juvenile rockfish Sebastes schlegeli against Microcotyle sebastis (Monogenea)

To determine whether immunization with Microcotyle sebastis antigen could induce protection against the parasite's establishment, naive juvenile rockfish were immunized by injection or immersion with whole worm antigen of M. sebastis. The infestation intensities of immunized groups following a challenge (2 wk after boosting) with 5000 M. sebastis eyed-eggs were significantly lower than those of control groups, when determined 7 wk postinfection. The fish in the groups boosted with M. sebastis antigen showed stronger protection than unboosted groups. The control group injected with FCA only showed a significantly smaller number of worms than the control group, which was immersed in PBS containing seawater. The results strongly suggest that both specific and nonspecific immune factors participate in the protection of rockfish against M. sebastis establishment.     

Purification and characterization of luteinizing hormone from pituitary glands of rockfish, Sebastes schlegeli

To acquire greater knowledge of the reproductive function of luteinizing hormone (LH) in the viviparous rockfish Sebastes schlegeli, LH from the pituitary glands of mature rockfish was isolated, purified, and localized and its biological activity was characterized. The molecular mass of purified LH was estimated to be approximately 33 kDa, similar to that of known LH. When rockfish LH was purified by reverse-phase high-performance liquid chromatography, its N-terminal amino acid sequences were found to coincide with those of predicted cDNA sequences of rockfish gonadotropin α (ssGTHα) and ssLHβ mature peptides. Immunocytochemical analysis using antisera against ssGTHα (molecular weight [MW], ~14.5 kDa) and ssLHβ (MW, ~18.5 kDa) indicated that the LH-producing cells are mainly distributed throughout the proximal pars distalis and along the periphery of the pars intermedia. Further, in vitro ovarian follicle analysis demonstrated that purified intact rockfish LH significantly enhances E(2) secretion in a dose-dependent manner. This is the first report on the purification and characterization of LH from a viviparous teleost, and these results will enable future research and increase our understanding of the mechanisms underlying the maturation of such fish.     

野生与养殖许氏平鲉消化酶活力的比较

用生物化学方法测定了野生和养殖许氏平鲉胃、肠和肝胰脏的蛋白酶、淀粉酶和脂肪酶活力,并对2组个体的上述消化酶活力分别进行了比较。结果表明:在37℃和最适pH条件下,除肝胰脏淀粉酶和脂肪酶外,养殖许氏平鲉各部位3种消化酶的活力全部高于野生许氏平鲉;野生与养殖许氏平鲉胃、肠3种消化酶活力均差异显著(P0.05),肝胰脏3种消化酶活力差异均不显著(P0.05);野生与养殖许氏平鲉消化酶活力的这种差异可能与野生许氏平鲉在自然环境中无法获得稳定充足的饵料有关。     

Multiplex PCR system applied for analysing microsatellite loci of Schlegel's black rockfish,Sebastes schlegeli

Two multiplex PCR amplifications were performed to analyse six microsatellite loci of Schlegel's black rockfish, Sebastes schlegeli, an important commercial fish in the northern part of Japan and an important species for the stock enhancement program in this area. We analysed 67 wild samples from Yamada Bay, Iwate Prefecture, Japan. The observed genotype frequencies agreed with the Hardy–Weinberg expectations at all loci, and the observed heterozygosities ranged from 0.072 to 0.897.     

Isolation and characterization of microsatellite markers for the heavily exploited rockfish Sebastes schlegeli, and cross-species amplification in four related Sebastes spp.

Here we report development and characterization of 14 polymorphic microsatellite loci from Sebastes schlegel. Polymorphism at these loci revealed from 3 to 23 alleles. The observed heterozygosity ranged from 0.34 to 1.00, and the expected heterozygosity ranged from 0.31 to 0.95. No linkage disequibrium was found. Two loci were significantly deviated from HWE (P < 0.01). The 14 loci were also surveyed in four other Sebastes species and 12 loci successfully amplified, where allelic diversity ranged from highly polymorphic to monomorphic. These results demonstrate these microsatellite markers can be used for the study of intra- and inter-specific genetic diversity.     

Depletion of praziquantel in plasma and muscle tissue of cultured rockfish Sebastes schlegeli after oral and bath treatment

Depletion of praziquantel in plasma and muscle tissue after oral and bath treatments was studied in cultured rockfish Sebastes schlegeli. In the oral treatment, a single dose of 400 mg praziquantel kg(-1) body weight was administered by intubation of the stomach. A bath treatment at 100 ppm of praziquantel for 4 min was also carried out. Plasma and muscle tissue samples were collected at 3, 6, 12, 24, 48, 72, 96, 120, 144 and 168 h post-treatment, and analyzed for praziquantel by reversed-phase HPLC using diazepam as the internal standard. Following oral treatment, praziquantel was detected in plasma and muscle tissue until 96 h after treatment. In plasma the praziquantel concentration was highest at the 9 h sampling time and declined sharply at the 48 h sampling point. The concentrations of praziquantel in the muscle tissue were lower than those in the plasma, and the highest value was found at the 9 h sampling time. Following bath treatment, praziquantel was found in plasma and muscle tissue until 72 and 24 h after treatment, respectively. In plasma the praziquantel concentration was highest at the 12 h sampling time and declined sharply thereafter. The concentrations of praziquantel in the muscle tissue were significantly lower than those in the plasma, and the concentrations declined consistently with time.