Monitoring of the genetic variability of the hatchery and recaptured fish in the stock enhancement program of the rare species barfin flounder <Emphasis Type="Italic">Verasper moseri</Emphasis>

ABSTRACT:   In relation to the stock enhancement program for barfin flounder, hatchery juveniles produced in 2001 were genotyped using microsatellite DNA markers (msDNA) and then released to natural waters. Subsequently, recaptured individuals, designated as 'tentative recaptured', were examined using msDNA. In order to evaluate the effectiveness of the stock enhancement program, pairwise F _ST and genic differentiation tests were used to estimate the genetic divergence between the wild samples, the hatchery broodstock, and the tentative recaptured samples. Analysis showed significant differentiation among these three groups. Pedigree determination by msDNA was used to establish the origin of the tentative recaptured individuals, in order to elucidate whether they were hatchery produced, 'real recaptured', or wild specimens. Wild individuals were not found. The effective population size of the real recaptured stock was very low ( N _e = 16.6). Equal family survivability was observed between the released and real recaptured stocks, indicating that the genetic variability of the released stock was maintained in the natural environment. Future broodstock management, breeding designs, and family contribution equalization of the offspring to be released will be required in this rare species to avoid unintended genetic differentiation between the wild population and the hatchery broodstock.     

Pedigree analysis of recaptured fish in the stock enhancement program of spotted halibut Verasper variegatus

ABSTRACT:   Spotted halibut Verasper variegatus hatchery juveniles produced in 2002 were genotyped using three microsatellite DNA markers (msDNA) and then released into natural waters. Subsequently, recaptured individuals were examined using msDNA. In order to evaluate the effectiveness of the stock enhancement program, from the genetic point of view, a pairwise F _ST test was implemented to estimate the genetic divergence between the wild captive broodstock, the hatchery offspring and the recaptured samples. The analysis showed significant differentiation between the broodstock and recaptured samples. Pedigree determination using msDNA was used to calculate the effective population size of the recaptured stock, which was found to be very low ( N _e ≈ 8). Equal family survivability was observed between the two recaptured stocks, but not between the released and recaptured stock. The number of identified families was higher and more equalized in the hatchery offspring compared to the recaptured samples, where the number of families declined. This fact was caused by an unequal family survivability just before or just after release. Separately, the number of contributing parents to the hatchery offspring was lower than the broodstock census number. Consequently, these two facts caused the genetic divergence of the recaptured stock from the broodstock.     

Population structure of Japanese spiny lobster Panulirus japonicus inferred by nucleotide sequence analysis of mitochondrial COI gene

ABSTRACT:   The mitochondrial DNA sequence for the COI region was determined for 73 Panulirus japonicus individuals collected at three sample locations in Japan. Fifty-one haplotypes, including 69 polymorphic sites without insertion and deletion, were detected, of which three dominant haplotypes were shared among the three sample locations. The nucleotide and haplotype diversities at the three sample locations were in the ranges of 0.009–0.010 and 0.959–0.990, respectively. No significant population subdivision was detected among the sample locations based on the F _st value and net nucleotide substitution rate, and analysis of the molecular variance model ( amova ). There was no characteristic geographic distribution pattern for these haplotypes. These results indicate there is no genetic differentiation between P. japonicus populations in Japan, and support the hypothesis that benthic individuals of P. japonicus are sustained from a population of a common pool of long-period phyllosoma larvae through long-distance larval transport within the Kuroshio Subgyre.     

Genetic evaluation of interindividual relatedness for broodstock management of the rare species barfin flounder Verasper moseri using microsatellite DNA markers

ABSTRACT:   The barfin flounder has been recently declared to be rare, as the number of wild individuals in Japan has greatly reduced since 1975. The Japanese National Center for Stock Enhancement started the stocking program for this species on 1987. In the present study, microsatellite DNA loci were applied in order to determine the pedigree of the hatchery-produced juveniles for stock enhancement. Additionally, using six polymorphic microsatellite markers, the accuracy to assign dyads to categories of kinship was estimated using the relatedness estimator ( R _XY), similarity indexes (individual genetic identity [GI] and proportion of shared alleles [ P _S]), identification of kin groups using unweighted pair group method with arithmetic mean (UPGMA) clustering based on interindividual genetic distances (1- R _XY, 1- P _S, and 1-GI), a likelihood ratio approach, and partition methods to separate individuals into cohorts. The results were compared with the pedigree relationship previously obtained from parentage analysis. Estimation of the kinship relationship between individuals could be better inferred by means of UPGMA dendrogram clustering based on 1-GI or 1- P _S as interindividual genetic distances, or using computer software to perform partitioning of set of individuals.     

条斑星鲽和圆斑星鲽线粒体12S rRNA与16S rRNA基因序列及其系统分析

采用PCR产物直接测序法分别测定了条斑星鲽和圆斑星鲽线粒体基因组序列,并对12SrRNA和16S rRNA基因核苷酸全序列进行分析,条斑星鲽和圆斑星鲽线粒体12S rRNA的核苷酸序列长度分别为948 bp和949 bp,16S rRNA均为1716 bp。试验结果表明,由12S rRNA和16S rRNA基因所构建的两个系统树的结构基本一致,21种鱼主要分为3个大的分支:鲤形目、鲶形目聚为1支,鲑形目独立聚为1支,鲈形目和鲽形目聚为1大支。鲆鲽类与鲈形目的鲹科、鲷科鱼类聚类在一支,且与鲹科的日本竹荚鱼、大西洋竹荚鱼构成姊妹群,支持鲆、鲽类是从鲈形目分化出来的观点,而同属于鲽形目的鳎科鱼类塞内加尔鳎在12S rRNA树中成为一个独立的分支,在16S rRNA树中聚到鲈形目和鲽形目的分支中,并且与鲈形目关系更近些,鳎类是1个特殊类群,其分类地位有待进一步探讨。线粒体基因组序列已提交到GenBank中,序列号为:DQ403797和EF025506。     

Genetic population structure of <Emphasis Type="Italic">Nibea albiflora</Emphasis> in Yellow Sea and East China Sea

ABSTRACT:   The population genetic structure and level of gene flow of Nibea albiflora from the Yellow Sea and the East China Sea were examined with a 479-bp segment of a mtDNA control region. In total, 65 samples were collected from three locations and 37 haplotypes were obtained. Mean haplotype diversity and nucleotide diversity for the three populations ranged from 0.9130 ± 0.0308 (Zhoushan) to 0.9926 ± 0.0230 (Xiamen), and from 0.0073 ± 0.0043 (Qingdao) to 0.0099 ± 0.0057 (Xiamen). Analysis of molecular variance and pairwise F _ST revealed little genetic structure between the Yellow Sea and the East China Sea in N. albiflora . But based on the exact test of differentiation, the null hypothesis that N. albiflora within the Yellow Sea and the East China Sea constitutes a panmictic mtDNA gene pool was rejected. This might be caused by the broad spawning areas but not by the Yangtze River outflow. Mismatch distribution revealed that N. albiflora has undergone population expansion, possibly before the last 85 000–170 000 years. The existence of high gene flow between stocks in the studied area was supported by our results. Annual migrations, larval drift in the ocean currents, and recent range expansion could be the reasons for little genetic structure in the studied area.     

Occurrence and distribution of settling and newly settled spotted halibut Verasper variegatus and Japanese flounder Paralichthys olivaceus in shallow nursery grounds around Shimabara Peninsula, western Japan

The occurrence and distribution of settling and settled spotted halibut Verasper variegatus were compared with those of Japanese flounder Paralichthys olivaceus in shallow habitats around Shimabara Peninsula, western Japan, to elucidate the settlement and recruitment characteristics of spotted halibut, a rare pleuronectid flatfish in Japan. Push-net sampling conducted at nine sites in March and April 2003 collected spotted halibut from four nurseries located at eastern areas of Shimabara Peninsula in Ariake Bay; Japanese flounder were sampled from eight nurseries. Comparison of microhabitat distributions in a tidal-flat nursery during February?CApril in 2003?C2007 revealed that metamorphosing larvae and juveniles of spotted halibut, which showed the drastic body coloration (black to brown) and behavioral changes (frequent swimming to settlement), were distributed mainly in the lower edge of the intertidal area, whereas Japanese flounder inhabited both intertidal and subtidal areas. Metamorphosing larvae of spotted halibut (12.6?C16.5?mm standard length; n?=?24) were collected in March when the highest densities were observed, whereas metamorphosing larvae of Japanese flounder (10.0?C15.9?mm, n?=?55) were observed during March?CApril. Our results suggest that spotted halibut has a narrower time/space window for recruitment to the specific nurseries compared with Japanese flounder.     

Genetic variation of hatchery and wild stocks of the pearl oyster <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis> (Dunker, 1872), assessed by mitochondrial DNA analysis

In order to provide baseline information for the genetic resources, genetic variation in wild and cultured Pinctada fucata martensii from southern Korea and Japan was studied using nucleotide sequence analysis of 379 base pairs (bp) in the mitochondrial cytochrome oxidase subunit I gene (COI). The study included three hatchery stocks from Korea (Tongyeong) and Japan (Mie and Tsushima) and one wild stock from Korea (Geoje). A total of 3 haplotypes were identified in hatchery stocks of 78 individuals, of which 63 individuals shared 1 haplotype. Overall, nucleotide diversity (π) was low, ranging from 0.000 to 0.002, and haplotype diversity (h) ranged from 0.000 to 0.541. Considerably low haplotype and nucleotide diversities in hatchery stock indicated that low effective population size and consecutive selective breeding of P. fucata martensii could be responsible for the reduction in genetic variation. The wild stock exhibited low haplotype diversity (0.507 ± 0.039) with two shared haplotypes. The results of the present study with first record of wild pearl oyster in Korea support the possibility that the transplanted pearl oyster for overwintering experiments could have survived in winter. In order to enhance and/or maintain genetic diversity in the hatchery stock, further research should be directed toward genetic monitoring and evaluation of the hatchery and wild pearl oysters.     

Habitat utilization,feeding, and growth of wild spotted halibut <Emphasis Type="Italic">Verasper variegatus</Emphasis> in a shallow brackish lagoon: Matsukawa-ura,northeastern Japan

Habitat utilization, feeding, and growth of a rare pleuronectid flatfish, spotted halibut Verasper variegatus, were examined in a brackish lagoon in northeastern Japan: Matsukawa-ura. The distribution and date–length data of spotted halibut collected mainly from beam-trawl samplings during 1983–2008 indicated that age-0 juveniles [n = 25, 6.0–18.0 cm total length (TL)] and older spotted halibut (n = 71, 13.8–43.0 cm TL) inhabited almost the entire northern part of Matsukawa-ura. Comparative distribution surveys of spotted halibut, stone flounder, and marbled flounder during 1985–1989 revealed similar distribution patterns of these flatfishes; the highest densities were found around the central part of Matsukawa-ura, although significantly lower abundance was detected for spotted halibut. Linear growth equations of age-0 juveniles demonstrated that spotted halibut were able to achieve high growth after June, probably because abundant prey (e.g., mysids and gammarids) and suitable physical conditions (warmer water temperature and lower salinity) were present. The main prey items shifted from various crustaceans including mysids, caridean shrimps, and anomurans to brachyura Hemigrapsus spp. (≥20 cm TL). Our study shows that spotted halibut use the shallow brackish lagoon as an important nursery for juveniles, and also as a feeding ground for young and adults.     

Effects of fish size and water temperature on the acute toxicity of boron to Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:     The acute toxicities of boron were examined for Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major in terms of fish size and water temperature. Japanese flounder of 0.1–70.0 g and red sea bream of 0.6–20.3 g were exposed to different concentrations of boron for 96 h at 20°C under semi-static conditions. In both fish species, the median lethal concentration (LC₅₀) for 96 h of boron increased linearly with increasing fish size, ranging from 108 to 252 mg B/L for the flounder, and from 97 to 172 mg B/L for the sea bream. The effect of water temperature on acute toxicity was examined for Japanese flounder of 0.6 and 1.5 g at 10, 15, 20 and 25°C, and for red sea bream of 0.6 and 2.4 g at 12, 15, 20 and 25°C. The toxicity of boron for the flounder increased linearly with increasing water temperature. The 96 h LC₅₀ values ranged from 299 to 108 mg B/L for the 0.6 g flounder and from 350 to 113 mg B/L for the 1.5 g flounder. A similar trend was shown for the 2.4 g red sea bream; however, the relationship for the 0.6 g red sea bream was not significant.     

基于线粒体NADH脱氢酶亚基2标记的东南太平洋不同表型间茎柔鱼群体遗传学分析

为了解东南太平洋茎柔鱼(Dosidicus gigas)大、中、小3种表型群体间的遗传分化和结构,利用线粒体NADH脱氢酶亚基2(ND2)基因,对东南太平洋大、中、小3种表型群,共90尾性成熟茎柔鱼样本进行群体遗传学研究.ND2基因序列测序结果显示3个表型群总的单倍型多样性指数(Hd)为0.818,核苷酸多样性指数(P...     

Fishery characteristics of barfin flounder Verasper moseri in southern Tohoku,the major spawning ground,after the start of large-scale stock enhancement in Hokkaido,Japan

Commercial landings of a rare pleuronectid flatfish, barfin flounder Verasper moseri, recovered drastically after large-scale stock enhancement in Hokkaido conducted since 2006. This study investigated commercial landings, fishing grounds, size distributions, and sex and age compositions of barfin flounder during 2007–2011 in southern Tohoku, their major spawning ground, which is over 700 km south of Hokkaido. Landings, mostly comprising stocked fish, increased drastically in southern Tohoku: from 2.0 tons in 2007 to 20.8 tons in 2010. Over 98 % of them were landed during January–April, with the peak period during February–March. Fishing logbook data of offshore bottom-trawl vessels during 2007–2010 revealed the upper continental slope off southernmost Tohoku as the main fishing ground (35°40′–36°50′N, annual weighted mean depth 267–299 m). Two size modes in the landings consisted of males (ca. 40 cm) and females (ca. 60 cm). The main age at fishery recruitment in southern Tohoku was younger for males (age 2+) than for females (ages 3+ and 4+), the result of which was that the female percentage was lowest at 1.5 % in 2009 and highest at 7.9 % in 2011. Our results showed clearly how the effectiveness of large-scale stock enhancement in Hokkaido extended to southern Tohoku in association with spawning migration of stocked barfin flounder.     

Comparison of visual acuity and visual axis of three flatfish species with different ecotypes

ABSTRACT:   The visual acuity, visual axis and visual accommodation of pointhead flounder, slime flounder, and red halibut were determined to obtain basic knowledge for developing appropriate fishing gear and fishing methods for sustainable fisheries. Each of these species has a different ecotype in terms of habitat, depth and prey species. Thus, it was hypothesized that they may differ in terms of visual acuity, visual axis and visual accommodation. Few studies have compared these characters in flatfishes from different ecotypes. We used histological methods to determine visual acuity (i.e. cone cell density) and visual axis (i.e. cone cell distribution) in each of these species. The maximum visual acuity was 0.127 in pointhead flounder (total length, TL 344 mm), 0.092 in slime flounder (TL 372 mm) and 0.109 in red halibut (TL 336 mm). Based on the cone cell distribution in the retina, the visual axis was upward and forward in pointhead flounder, forward and downward in slime flounder, and downward in red halibut. Finally, the mean angle of lens movement was −2° in pointhead flounder, −13° in slime flounder and −32° in red halibut. This measurement of lens movement indicated that the average near-point distance was 0.87 × TL in pointhead flounder, 0.65 × TL in slime flounder and 1.02 × TL in red halibut. At similar TL (336–355 mm), the visual acuity of these species differs depending on the direction in which they are looking.     

Evaluation of genetic diversity of eight grouper species <Emphasis Type="Italic">Epinephelus</Emphasis> spp. based on microsatellite variations

ABSTRACT:   Genetic diversity among eight species of grouper, Epinephelus bleekeri , E. coioides , E. malabaricus , E. ongus , E. akaara , E. maculatus , E. merra and E. fuscoguttatus , was studied using six microsatellite loci, Em-01 *, Em-03 *, Em-07 *, Em-08 *, Em-10 *, and CA-07 *, with the aim of exploring the feasibility of using microsatellite data for species identification. The results showed high levels of genetic differentiation among species ( F _ST  = 0.4403 and R _ST  = 0.4954). Species identification based on fixed allelic differences was possible between E. coioides , E. ongus , and E. fuscoguttatus at Em-01 * and between E. fuscoguttatus and E. ongus at Em-08 *. Private alleles were found in all species, except for E. ongus . Pairwise F _ST ranged 0.238–0.578 ( P  < 0.008 Bonferroni correction), and Nei's genetic distance ranged 0.433–2.710. Size homoplasy was observed at Em-03 * 157 allele, which was characterized by a T–C transition at the 119th nucleotide site of PCR products. The genetic assignment test unambiguously assigned each individual to the correct species. Thus, this test can be used for species identification of unknown individuals when the multilocus genotypes of the six microsatellite loci are available. The phylogenetic (neighbor-joining) tree, which was constructed based on the genetic distance matrix, separated the eight grouper species into two main groups.     

An appraisal of factors constraining the success of fish stock enhancement programmes

Stocking and introduction of fish have been widely applied in Yunnan Province, China, for many years to mitigate for declines in natural stocks and commercial catches, and to restore or create new fisheries. The present paper assesses the results of activities concerned with stock enhancement programmes to evaluate the factors which have contributed towards the success of these projects. Factors such as natural lake conditions, stocking density, the age and size of fish stocked, and the mechanisms of stocking are discussed. Stocking density was evaluated using the equations Y _p = [ B ( P / B ) U _f]/ K _f or PY_p = (P × U_f)/K_f, which were based on the ecological carrying capacity of the recipient lake and other criteria. The stocking density in most lakes in Yunnan was high, but the recapture rates and total yield did not reach the predicted levels. This may have been an effect of the size of the fingerlings released (finger-lings >10 cm have a greater chance of survival). Key management measures for stocking and the ecological impacts of overstocking grass carp, Ctenopharyngodon idella (Valenciennes), are also described.     

Genetic diversity of red sea bream Pagrus major in western Japan in relation to stock enhancement

SUMMARY: Stock enhancement is used in Japan as a tool to help the replenishment of wild populations of red sea bream Pagrus major . In this study, we analyzed the genetic diversity and composition of wild red sea bream at seven locations around Shikoku Island, South-west Japan, using three microsatellite loci. This analysis was done to test the hypothesis that: (i) red sea bream comprises a single Mendelian population along Japan; and (ii) stock enhancement programs around Shikoku Island are causing genetic differentiation among wild stocks. The results indicated that some locations from the Shikoku area were not significantly different from the rest of Japan, supporting the hypothesis of a single Mendelian population. Significant departures from Hardy–Weinberg equilibrium and significant pairwise F_ST among locations indicated genetic instability within this region. We suggest that the stock enhancement programs made in the region are the possible cause of this genetic instability. A management scheme for the hatcheries involved in the stock enhancement of red sea bream is presented.     

Methionine requirement of juvenile Japanese flounder Paralichthys olivaceus estimated by the oxidation of radioactive methionine

Growth and amino acid oxidation studies were conducted to estimate methionine requirement of juvenile Japanese flounder, Paralichthys olivaceus , by using the purified diets containing 500 g kg^–1 crude protein from casein, gelatine and crystalline amino acids (CAA). Diets with six graded levels of methionine (5.3, 8.3, 11.3, 14.3, 17.3 and 20.3 g kg^–1 diet) were fed to triplicate groups of the juvenile (initial weight 2.8 ± 0.05 g) twice a day for 40 days. To prevent leaching losses, CAA were precoated using carboxymethylcellulose (CMC), and further diets were bound by CMC and κ-carrageenan. Based on broken-line analysis of percentage weight gain and feed conversion efficiency, the methionine requirements of Japanese flounder in the presence of 0.6 g kg^–1 of cystine were 14.9 and 14.4 g kg^–1 dry diet, respectively. After the growth study was finished, a direct estimate of methionine requirement was made by examining the influence of dietary methionine level on ¹⁴C-methionine oxidation by determining radioactive carbon dioxide, protein and nonprotein fractions of the whole body. The dose–response curve between expired radioactive CO₂ and dietary methionine levels showed that the optimum methionine level for the flounder was estimated to be within the range of 14.3–17.3 g kg^–1 of diet in high agreement with values obtained from the growth study.     

Genetic variation and population structure of the Pacific cod <Emphasis Type="Italic">Gadus macrocephalus</Emphasis> in Korean waters revealed by mtDNA and msDNA markers

To investigate the extent of genetic differentiation among wild populations of the Pacific cod Gadus macrocephalus, we have examined genetic polymorphism at five locations within Korean waters [Boryeong in the West Sea (WC-BR); Jinhae Bay in the South Sea (SC-JH); Jumunjin (EC-JM), Jukbyeon (EC-JB), and Bangeojin (EC-BJ) off the eastern coast of Korea] using mitochondrial DNA (mtDNA) control region and microsatellite DNA (msDNA) markers. Nucleotide sequence analysis of 584 bp in the variable portion of the 5′ end of the mtDNA control region revealed 27 variable nucleotide sites among 184 individuals, which defined eight, three, and 11 haplotypes in the western, southern, and eastern coast populations, respectively. The mtDNA analysis revealed a low variability but significant local differentiation among populations from these three areas within Korean waters. msDNA analysis also revealed moderate polymorphism in the wild populations, with a mean of 13.8–22.6 alleles per locus for the five msDNA markers and observed (and expected) heterozygosities of 0.755 (0.825) for the WC-BR, 0.793 (0.810) for the SC-JH, 0.920 (0.905) for the EC-BJ, 0.783 (0.865) for the EC-JB, and 0.804 (0.812) for the EC-JM populations. Analysis of msDNA loci indicated that Pacific cod sampled at the WC-BR, SC-JH, and EC-JB sites belong to genetically distinct populations. However, no significant difference was found between the Pacific cod population from SC-JH and that from EC-BJ. Consequently, three genetically distinct populations, namely, WC-BR, SC-JH and EC-BJ, and EC-JB, were identified using msDNA analysis. These results indicate that genetically distinct populations of Pacific cod are present in Korean coastal waters where spawning aggregations occur.     

Gonadal maturation and spawning migration of stocked female barfin flounder Verasper moseri off the Pacific coast of northern Japan

Commercial catches of barfin flounder Verasper moseri, an important target flatfish for stock enhancement programs in northern Japan, have recovered remarkably since the large-scale release of seedlings from Hokkaido in 2006. However, their reproductive ecology remains unclear. Our study of 2008–2012 investigated seasonal changes in the maturity of stocked female barfin flounder (n = 4,123) off the Pacific coast from Hokkaido to Tohoku (35.7°–43.4°N). Histological observation of ovaries revealed that fish undergoing vitellogenesis were observed mainly off Hokkaido and partially off northern Tohoku during August–January (40.2°–43.4°N). However, fully matured or spawning fish were not found near those areas. They were observed only in southern Tohoku. Ongoing spawning was observed off southernmost Tohoku (35.8°–37.5°N around 300 m depth) from early February through late April, when commercial landings had decreased drastically off Hokkaido. Spent fish were caught only rarely off southern Tohoku, but were observed frequently off Hokkaido during April–June. These results demonstrate that spawning grounds form at the upper continental slope off southernmost Tohoku during February–April. Relations between maturation traits and seasonal landings strongly suggest that female barfin flounder repeatedly migrate more than 700 km from feeding grounds off Hokkaido to spawning grounds off southern Tohoku.