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.     

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.     

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.     

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.     

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.     

Cloning and characterization of novel microsatellite DNA markers for the grass rockfish,Sebastes rastrelliger,and cross‐species amplification in 10 related Sebastes spp

Here we report development and characterization of seven polymorphic loci derived from grass rockfish (Sebastes rastrelliger) genomic DNA phagemid libraries enriched for microsatellite motifs. Within grass rockfish, average allelic diversity was 11.3 alleles per locus and average heterozygosity was 0.73. The seven loci also were surveyed in 10 related species of Sebastes, where allelic diversity ranged from highly polymorphic to monomorphic. Deviations from Hardy–Weinberg were nonsignificant in all but one species/locus combination suggesting low occurrence of null alleles. Significant linkage disequilibrium was detected among three loci, but these events were limited to a single species in each case.     

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.     

Isolation and characterization of nine polymorphic microsatellite markers of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae)

Sebastiscus marmoratus is a typical sedentary fish and usually lives at the bottom of the coastal zone from Japan to the East China Sea. Next-generation sequencing platform was used to obtain microsatellite loci sequences of S. marmoratus, among which forty-eight perfect microsatellite repeats were suitable for primer design. Nine polymorphic microsatellite markers were exploited and characterized for it. The allele number of all polymorphic microsatellite markers ranged from 4 to 12. Expected and observed heterozygosity varied from 0.302 to 0.909 and from 0.190 to 0.625, respectively. In further studies, the genetic variation and population structure of this species would be investigated using these microsatellite loci.     

Isolation and characterization of polymorphic microsatellite markers for the endangered Korean freshwater fish Hemibarbus mylodon

Korean barbel, Hemibarbus mylodon, is an endangered freshwater species endemic to Korea. In order to undertake a conservation program for this species in Korea, it is essential to evaluate its genetic diversity and population structure. For this purpose, we developed 10 polymorphic microsatellite markers and examined their allelic variation using a total of 60 wild individuals collected from three different localities. The number of alleles per locus ranged from three to 26. The expected and observed heterozygosities per locus ranged from 0.19 to 0.94 and from 0.20 to 0.98, respectively. Significant departures from Hardy–Weinberg equilibrium expectations occurred in four loci. These markers will be useful for the assessment of genetic diversity for this species.     

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.     

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.     

Population genetic structure of wild and hatchery black rockfish Sebastes inermis in Korea, assessed using cross-species microsatellite markers

The population structure of the black rockfish, Sebastes inermis (Sebastidae), was estimated using 10 microsatellite loci developed for S. schlegeli on samples of 174 individuals collected from three wild and three hatchery populations in Korea. Reduced genetic variation was detected in hatchery strains [overall number of alleles (N(A)) = 8.07; allelic richness (A(R)) = 7.37; observed heterozygosity (H(O)) = 0.641] compared with the wild samples (overall N(A) = 8.43; A(R) = 7.83; H(O) = 0.670), but the difference was not significant. Genetic differentiation among the populations was significant (overall F(ST) = 0.0237, P < 0.05). Pairwise F(ST) tests, neighbor-joining tree, and principal component analyses showed significant genetic heterogeneity among the hatchery strains and between wild and hatchery strains, but not among the wild populations, indicating high levels of gene flow along the southern coast of Korea, even though the black rockfish is a benthic, non-migratory marine species. Genetic differentiation among the hatchery strains could reflect genetic drift due to intensive breeding practices. Thus, in the interests of optimal resource management, genetic variation should be monitored and inbreeding controlled within stocks in commercial breeding programs. Information on genetic population structure based on cross-species microsatellite markers can aid in the proper management of S. inermis populations.     

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.     

Isolation and characterization of microsatellite markers for the seagrass Cymodocea nodosa

In order to study the spatial patterns of genetic diversity of a clonal marine angiosperm, the seagrass Cymodocea nodosa, microsatellite markers were obtained by screening a genomic library enriched for the (CT) dinucleotide motif. Of 38 primer pairs defined, 15 amplified polymorphic microsatellites and are described. These loci identified a number of alleles ranging from two to seven, and showed expected heterozygosity ranging from 0.35 to 0.76, when a group of 40 individuals from Cadiz Bay in Spain was analysed. Additionally, we describe here the multiplexing conditions for 12 of these loci.     

Isolation and characterization of polymorphic microsatellite markers for Alternaria alternata

Alternaria alternata is of major significance as a food and feed contaminant and is able to produce a range of mycotoxins that may elicit adverse effects in both animals and humans. We describe the isolation and characterization of five microsatellite markers for studying A. alternata. Marker polymorphism was screened in 64 isolates of A. alternata. The number of alleles per locus ranged from eight to 24, and allelic diversity ranged from 0.425 to 0.882. These markers will be useful in the study of relationships and population genetics amongst isolates of A. alternata.     

Isolation and characterization of microsatellite markers from the budgerigar,Melopsittacus undulatus

The budgerigar, Melopsittacus undulatus, belongs to a group of birds that are among the most common captive‐held avian species that are also most threatened. Budgies are also a valuable resource as model species for many important vertebrate characteristics including auditory and ageing. Several microsatellite‐enriched genomic libraries were developed using standard protocols. Twelve of 20 primer pairs developed from microsatellite‐containing sequences were codominant and polymorphic when tested on 21 birds from three different states. The number of alleles detected ranged from two to four, with the expected and observed heterozygosities ranging from 0.09 to 0.61 and from 0.00 to 0.85, respectively. The genomic libraries and the primers described here will be useful in characterizing budgies.