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.     

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.     

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.     

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.     

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.     

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.     

Immunochemical identification and partial characterization of female-specific serum proteins in white-edged rockfish,Sebastes taczanowskii

Synopsis Female-specific serum proteins (FSSPs) in white-edged rockfish,Sebastes taczanowskii, were identified and partially characterized by immunochemical procedures. Two FSSPs, which clearly reacted with antiserum against egg proteins, were confirmed in the serum of mature females, and estrogen treatment induced similar FSSPs in the serum of mature males. Hence, the FSSPs were considered to be vitellogenin. The vitellogenin concentration in female fish was high during the vitellogenic period and low during gestation, parturition and the recovery period, indicating that vitellogenin is used only for yolk formation in the oocytes and not as a direct nutritional source for developing embryos during gestation. On the other hand, an FSSP (FS3), which was considered not to be vitellogenin, was also identified in the sera of mature females and males after estradiol-17β administration by using an antiserum (a-FS3) that removed the components of the male serum and egg extracts from the anti-mature female serum antiserum. Moreover, immunohistochemical observation with a-FS3 illustrated that FS3 was a major constituent of the ovarian fluid but not of vitellogenic oocytes. The cross-reactivities of these FSSPs among seven viviparous rockfishes demonstrated that vitellogenin existed in the sera of all rockfishes studied belonging to the generaSebastes andSebastiscus, whereas FS3 was not present in several species ofSebastes.     

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.     

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。     

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.     

Molecular characterization, expression, and functional analysis of two thioredoxins in the black rockfish (Sebastes schlegelii)

Thioredoxins (TRxs) are a family of small evolutionarily conserved proteins that are essential for the maintenance of cellular homeostasis. Two TRx homologue cDNAs were isolated from a black rockfish concanavalin A (Con A)/phorbol myristate acetate (PMA)-stimulated leucocyte cDNA library and named BrTPx1-1 and BrTPx1-2. As compared with other known TRx peptide sequences, the most conserved regions of both BrTRx1-1 and BrTRx1-2 peptides were found to be the redox-active site Trp-Cys-X-X-Cys (WCXXC). The TRx present in most species is a TRx1-2 protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in the larger 13 kDa BrTRx1-1 protein, a Cys-Pro-Pro-Cys (CPPC) active site was identified. Here, we report the identification of a new member of the TRx protein family from the teleost black rockfish, which defines a new subclass of 13-kDa TRx1-1 proteins. Phylogenetic analysis indicated that both BrTRx1-1 and BrTRx1-2 were grouped with other vertebrate TRx1 peptides. BrTRx1-1 expression was strongly induced in peripheral blood leucocytes (PBLs) 12-24 h following Con A/PMA stimulation, with peak expression at 24 h post-stimulation. BrTRx1-2 was induced in PBLs after stimulation with lipopolysaccharide (LPS), Con A/PMA, or poly I:C at 24 h. The BrTRx1-1 gene was predominantly expressed in the liver and gills, while BrTRx1-2 was expressed in PBLs and gills. After treatment with a high concentration (10 μg/mL) of rBrTRx1-1 or rBrTRx1-2, kidney leucocytes exhibited increased cell proliferation and viability under oxidative stress.     

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.     

Mariculture of kurosoi,Sebastes schlegeli

Synopsis The technology of collecting developing larvae from female kurosoiSebastes schlegeli, and raising the larvae to juveniles (100 mm total length (TL)) to be released into the oopen sea, is presented. Gravid females 40–46 cm TL were captured in May–June 1977–1980 and held in the laboratory until parturition. Fecundity of fish in this size range was 100 000–184 000. Larvae were sequentially fed rotifers,Artemia nauplii, and young sand lance,Ammodytes personatus, until reaching 25 mm; this required 35 days and yielded a survival rate of 50%. Thereafter, the fish were reared in separate size groups to avoid cannibalism. Minced or chopped sand lance and commercial food were provided until the final size of 100 mm was attained. The growth of juvenile kurosoi from 25 to 100 mm required 85 days, with a survival rate of 90%. The effect of released cultured fish on the local stock is being determined from information on the recapture of tagged fish.     

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.     

Molecular cloning and characterization of a short type peptidoglycan recognition protein (CfPGRP-S1) cDNA from Zhikong scallop Chlamys farreri

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and plays a crucial role in the innate immune responses as a pattern recognition receptor (PRR). The cDNA of a short type PGRP was cloned from scallop Chlamys farreri (named CfPGRP-S1) by homology cloning with degenerate primers, and confirmed by virtual Northern blots. The full length of CfPGRP-S1 cDNA was 1073 bp in length, including a 5' untranslated region (UTR) of 59 bp, a 3' UTR of 255 bp, and an open reading frame (ORF) of 759 bp encoding a polypeptide of 252 amino acids with an estimated molecular mass of 27.88 kDa and a predicted isoelectric point of 8.69. BLAST analysis revealed that CfPGRP-S1 shared high identities with other known PGRPs. A conserved PGRP domain and three zinc-binding sites were present at its C-terminus. The temporal expression of CfPGRP-S1 gene in healthy, Vibrio anguillarum-challenged and Micrococcus lysodeikticus-challenged scallops was measured by RT-PCR analysis. The expression of CfPGRP-S1 was upregulated initially in the first 12 h or 24 h either by M. lysodeikticus or V. anguillarum challenge and reached the maximum level at 24 h or 36 h, then dropped progressively, and recovered to the original level as the stimulation decreased at 72 h. There was no significant difference between V. anguillarum and M. lysodeikticus challenge. The results indicated that the CfPGRP-S1 was a constitutive and inducible acute-phase protein which was involved in the immune response against bacterial infection.     

Morphogenesis in hatchery-reared larvae of the black rockfish,Sebastes schlegeli,and its relationship to the development of swimming and feeding functions

The developmental sequence of morphological characteristics related to swimming and feeding functions was investigated in hatchery-reared larvae and juveniles ofSebastes schlegeli, a viviparous scorpaenid. The fish were extruded at an early larval stage, when the mean body size was 6.23 mm TL. Fin-ray rudiments became visible at 9.0 mm TL in the dorsal and anal fins, at 8.0 mm TL in the pectoral and pelvic fins and 6.0 mm TL (size at extrusion) in the caudal fin. Completion of segmentation of soft rays in the dorsal and anal fins was attained by 14 mm TL and in all fins by 17 mm TL. Branching of soft rays in the respective fins started and was completed considerably later than the completion of segmentation, as well as ossification of the fin-supports. Morphological transformation from larva to juvenile was apparently completed by about 17 mm TL. Although the completion of basic juvenile structures was attained by transformation at that body size, succeeding morphological changes occurred between 17 mm and 32 mm TL. Newly-extruded larvae possessed one or two teeth on the lower pharyngeal and pharyngobranchials 3 and 4, but lacked premaxillary, dentary, palatine and prevomer teeth. The fish attained full development of gill rakers and gill teeth by 15 mm TL, the upper and lower pharyngeal teeth subsequently developing into a toothplate. Development of the premaxillary, dentary and palatine teeth was completed at about 30 mm TL, by which time loop formation of the digestive canal and the number of pyloric caeca had attained the adult condition. The developmental sequence of swimming and feeding functions during larval and early juvenile periods appeared to proceed from primitive functions to advanced or complex ones, from the ability to produce propulsive force to that of swimming with high maneuverability and from development of the irreducible minimum function of passing food into the stomach to the ability to actively capture prey via passive food acquisition with the gill rakers and gill teeth. The relationship of morphological development to the behavior and feeding activity of artificially-produced hatchlings is also discussed.     

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.