Parentage assignment in hatchery population of brown sole <Emphasis Type="Italic">Pleuronectes herzensteini</Emphasis> by microsatellite DNA markers

ABSTRACT:   Five loci ( Phz2 , Phz6 , Phz7 , Phz12 , and Phz14 ) of microsatellite DNA markers developed in a previous study for parentage assignment in the hatchery population generated by mating among 61 broodstock fish (35 females and 26 males) in a spawning tank, were selected. After natural spawning in the same tank, larvae collected at three different times were categorized into early phase (EP), middle phase (MP), and late phase (LP) groups. In the parental broodstock, the mean number of alleles per locus was 21.8 and expected heterozygosity ( H _E ) was 0.813. In the progeny, the mean number of alleles per locus decreased to 11.6 (EP), 14.4 (MP), and 6.4 (LP) and H _E to 0.796 (EP), 0.833 (MP), and 0.681 (LP). Parental assignment determined eight dams and six sires as major parents for the EP group. In the MP group, 13 dams and ten sires genetically contributed to spawning, but only three dams and two sires were involved in LP group progeny. In the hatchery population produced from a limited number of parental fish such as the LP group, genetic variability was apparently decreased.     

Survey of genetic variation at three microsatellite loci in captive populations of endangered Japanese minnow <Emphasis Type="Italic">Aphyocypris chinensis</Emphasis> with implications for reduction of inbreeding

The Japanese population of the cyprinid minnow Aphyocypris chinensis is nearing extinction in the wild. The genetic diversity of three microsatellite loci in five captive populations was investigated, and an effective breeding strategy to reduce inbreeding from pairwise relatedness (R _xy ) between each captive line is discussed. The average number of alleles ranged 2.33–4.67 and the average heterozygosity ranged 0.283–0.602. The pairwise relatedness observed in most combinations showed a significant decrease between the populations. It is suggested that exchange of individuals between different breeding lines should effectively stop inbreeding. Studies show that the effective population size (N _e ) estimated from the number of parental individuals was 8.54 in one captive population, which is insufficient to maintain genetic diversity. It is recommended that more parental individuals should be used, and to exchange fish in a rotating mating mode between institutions participating in captive breeding of A. chinensis.     

Effects of dietary thiamin on the physiological status of the grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

This study was conducted to evaluate the effects of dietary thiamin on the physiological status of the juvenile grouper, Epinephelus coioides. Graded levels of thiamin (0.08, 0.50, 2.12, 3.15, 4.63, 12.37 mg thiamin kg⁻¹ diet) were fed to grouper juveniles (mean weight: 16.97 ± 0.14 g) for 10 weeks. Although fish fed the thiamin-deficient (TD) diet showed no obvious symptoms of thiamin deficiency or increased mortality, those fed the lowest doses of thiamin (0.08 and 0.50 mg thiamin kg⁻¹ diet) had significantly decreased transketolase activity in the liver. In addition, the level of liver thiobarbituric acid reactive substances in fish fed the TD diet was 33–67% higher than that in fish with the thiamin-supplemented diet. There were no significant differences in superoxide dismutase activity between the different groups of fish.     

Parentage assignment of stocked black sea bream <Emphasis Type="Italic">Acanthopagrus schlegelii</Emphasis> in Hiroshima Bay using microsatellite DNA markers

The genetic contribution of 51 broodstock, comprising 29 females and 22 males, reared at Hiroshima City Marine Products Promotion Center for the production of stocked black sea bream was monitored during two consecutive years using seven microsatellite DNA loci. The high discrimination ability of these markers was reflected in the polymorphic identification content (PIC=0.831), the exclusion probability (Q≈1), and the low probability of identity index (I=3.635⁻¹⁰). The total number of breeders contributing to the mating process was estimated at 32 (62.7%) in 2000 and 30 (58.8%) in 2001. On pedigree reconstruction, 69.3% of the offspring were successfully assigned to a single broodstock pair. Loss of alleles accounted for 16.9% during seed production; nevertheless, 90.9% of males and 69.0% of females participated in the mating process. Based on microsatellite genetic tagging, 58.9% of the fish sampled during the two months after release were identified as hatchery stock, presenting no significant differences from wild conspecifics in either fork length or body weight.     

Fortification of <Emphasis Type="Italic">niboshi</Emphasis> with histidine prepared from <Emphasis Type="Italic">katsuobushi</Emphasis> protein digest

ABSTRACT:   Free and total histidine contents in the muscle of various fishes and invertebrates caught in the Seto Inland Sea were estimated. These contents were higher in the muscles from skipjack, yellowfin tuna, yellowtail and mackerel than in those from lizard fish, sea bream and hairtail. Invertebrate muscles, except squid, contained low free and total histidine. Boiled and dried anchovy ( niboshi ) contained lower free histidine than that of dried anchovy (suboshi). To prepare free histidine-rich extracts, the waste residue of soup stock from smoke-dried and shaved skipjack ( katsuobushi ) was digested with proteases. The extract of the enzyme digest contained high free histidine and was used to fortify niboshi with free histidine. If taken, the fortified niboshi may suppress food intake, and prevent obesity.     

Chemical composition of dried eyeballs from <Emphasis Type="Italic">Euphausia superba</Emphasis> and <Emphasis Type="Italic">Euphausia pacifica</Emphasis>

ABSTRACT:   Dried krill eyeballs were obtained from Euphausia superba and Euphausia pacifica by using a patented industry method and their chemical compositions were examined. Crude protein content was 77.7% and 80.8% of the dry matter of E. superba and E. pacifica , respectively. The dominating amino acids in both krill were glutamic acid, aspartic acid, glycine, arginine, leucine and tyrosine. Crude fat content was 10.9% and 5.4% of dry matter of E. superba and E. pacifica , respectively. The main lipid class of the extracted lipids was phospholipids at 88.5% in E. superba and 96.4% in E. pacifica . The dominating fatty acids in both krill were 22:6 ( n- 3), 20:5 ( n- 3), 16:0 and 18:1 ( n- 9). Astaxanthin (3, 3'-dihydroxy-β, β-carotene-4, 4'-dione) content of E. superba and E. pacifica was 566 mg/100 g and 252 mg/100 g of dry matter, respectively. Retinol of E. superba and E. pacifica was 153.0 mg/100 g (510 000 IU/100 g) and 57.6 mg/100 g (192 000 IU/100 g) of dry matter, respectively. The lipophilic extract of E. superba by using n -hexane contained 1923 mg/100 g of astaxanthin at approximately four times higher than the dried eyeballs.     

Suspension feeding and growth of ark shell <Emphasis Type="Italic">Anadara granosa</Emphasis>: comparison with ubiquitous species <Emphasis Type="Italic">Scapharca subcrenata</Emphasis>

The ark shell Anadara granosa is a species peculiar to the Ariake Sound. To determine why distribution of A. granosa in Japan is largely confined to this area, we examined feeding and growth of A. granosa as functions of environmental and biological variables. The results were compared with those of another ark shell Scapharca subcrenata, which is ubiquitous in Japan. Feeding experiments indicated that A. granosa is eurythermal and euryhaline, as is S. subcrenata, but is adapted to temperature slightly higher than S. subcrenata. Weight-specific clearance rate (CR) of A. granosa as a function of soft-body dry-weight (w) followed the power function of w (CR=2.7×w ^−0.37), with coefficient and exponent very close to those for S. subcrenata. Growth rate of A. granosa increased linearly with daily ration, similar to S. subcrenata. Thus, feeding and growth characteristics of A. granosa were comparable to those of S. subcrenata and no ‘peculiarities’ of the former were detected. Therefore, the factors that make A. granosa a species restricted to the sound are probably not directly related to feeding or growth characteristics.     

Masculinization of Convict Cichlid (<Emphasis Type="Italic">Cichlasoma nigrofasciatum</Emphasis>) by immersion in <Emphasis Type="Italic">Tribulus terrestris</Emphasis> extract

We have examined the effects of Tribulus terrestris (TT), a non-toxic herb, on sex reversal in the Convict Cichlid Cichlasoma nigrofasciatum with the aim of introducing a new environmentally friendly method for masculinization in C. nigrofasciatum. TT is a natural plant product that elevates the testosterone levels in humans and animals. Different concentrations (0.0, 0.10, 0.20 and 0.30 g/l) of TT extract were tested for their effect on sex reversal in C. nigrofasciatum by immersing newly hatched offspring once weekly for 2 months in TT extract. Of the dosages used in the present study, 0.30 g/l TT was the most effective in terms of masculinization, resulting in a maximum male ratio of 87.23% (P < 0.001). Sex ratios of 79 and 85% at 0.10 and 0.20 g/l TT, respectively, were also significantly different from the expected 1:1 ratio (P < 0.001). Histological examinations revealed that the testes of fish treated with TT extract contained all stages of spermatogenesis, clearly demonstrating that the administration of TT extract to C. nigrofasciatum stimulated spermatogenesis. Total survival rates in all treatments and the control were uniformly high, ranging from 88.57 to 90% (P > 0.05). We concluded that TT had no negative effect on the survival rate of C. nigrofasciatum. In addition, all groups of TT-treated fish exhibited successful growth acceleration compared to the control group, but only the 0.30 g/l TT treatment significantly improved the growth rate of C. nigrofasciatum. (P < 0.01). Sex reversal in C. nigrofasciatum demonstrated that TT-treated 0-day-old larvae showed successful sex reversal, spermatogenesis and a better growth rate than untreated progenies.     

Sterols from bivalves <Emphasis Type="Italic">Calyptogena soyoae</Emphasis> and <Emphasis Type="Italic">Bathymodiolus septemdierum</Emphasis> living in deep sea

From the two species of bivalves, Calyptogena soyoae around a cold seep and Bathymodiolus septemdierum near hydrothermal vents in the sea, sterols were isolated using high-pressure liquid chromatography analysis of the lipid fraction guided by characteristic ¹H NMR signals of the sterol skeleton. The minor sterol composition of C. soyoae included 24-methylenecycloartanal, cycloeucalenol, and obutusifoliol, which are known phytosterols. From B. Septemdierum, lathosterol and cholesterol as main sterols together with more diverse sterols were obtained. The difference between these species and their sterol contents is most likely because of feeding modes and metabolism of nutrients from their habitat.     

<Emphasis Type="Italic">Myxobolus nagaraensis</Emphasis> n. sp. (Myxozoa: Myxosporea) causes abdominal distension of freshwater goby <Emphasis Type="Italic">Rhinogobius</Emphasis> sp. OR type from the Nagara River

ABSTRACT:   A new myxosporean parasite was found in the body cavity and caudal peduncle of the freshwater goby Rhinogobius sp. Orange type (OR) collected from the Nagara River, Gifu Prefecture, Japan. Infected fish exhibited substantial swelling of the abdomen caused by large parasitic cysts approximately 10 mm in size, formed in the visceral cavity. The cyst was a compacted aggregate of several smaller cysts, similar to a bunch of grapes in appearance. Histological examination showed that plasmodia developed within the renal capsule, and finally occupied the visceral cavity. Spores were ovoid with an attenuated anterior end. Sutural ridges were conspicuous with several folds on the edge. Average spore size was 11.9 (10.5–13.5) μm long, 9.0 (8.0–10.0) μm wide, and 6.5 (6.0–7.0) μm thick. Two equal polar capsules were 5.5 (4.5–6 0) μm long and 3.0 (2.5–4.0) μm wide. Partial small subunit ribosomal DNA sequences of the myxosporean were distinct from those of other myxozoan species in GENBANK. A new species name, Myxobolus nagaraensis , is proposed for this parasite.     

Genetic identification of native populations of fluvial white-spotted charr <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis> in the upper Tone River drainage

ABSTRACT:   Stocking of exogenous, hatchery-reared white-spotted charr Salvelinus leucomaenis has been conducted throughout much of their range in Honshu Island, Japan, to increase angling opportunities. Although the native charr populations are thought to have declined because of hybridization with introduced fish, their distribution and genetic status have been uncertain. Fine population structures of charr in the upper Tone River drainage were examined using mitochondrial DNA and microsatellite analyses so as to clarify the presence of native populations. One common mtDNA haplotype was detected in all populations in the Ohashi River and Watarase River, and four and one tributary populations were monomorphic for such haplotypes, respectively. However, several haplotypes, considered to have originated from stocked hatchery fish, were observed in the stocked and the remaining populations. Judging from the genetic integrity over a fine geographic scale, the former were considered as indicative of native populations and the latter as admixtures with hatchery fish. Comparisons of genetic diversity, deviations from the Hardy–Weinberg equilibrium, principal component analysis, and relatedness estimations based on microsatellite DNA can also provide evidence for distinguishing native populations from those influenced by hatchery fish.     

Optimum temperature and salinity conditions for growth of green algae <Emphasis Type="Italic">Chlorella ellipsoidea</Emphasis> and <Emphasis Type="Italic">Nannochloris oculata</Emphasis>

ABSTRACT:   The effects of temperature and salinity on growth of green algae Chlorella ellipsoidea and Nannochloris oculata were determined to compare the optimum culture conditions. A four-temperature (15, 20, 25, and 30°C) × three-salinity (10, 20, and 30) factorial design with triplicates was applied. Specific growth rate (SGR), maximum density, and duration to reach maximum density of C. ellipsoidea were significantly affected by both temperature and salinity. The highest SGR was observed in C. ellipsoidea at 25°C and salinity 10, but the maximum density was very low. The highest maximum density was achieved in C. ellipsoidea at 15°C and 10. The slope constant of the linear relationship between semilogarithmic growth of C. ellipsoidea and day of culture was highest at 15°C and 10. The SGR and duration to reach maximum density of N. oculata were significantly affected by both temperature and salinity. However, maximum density of N. oculata was significantly affected by temperature, but not salinity. The highest maximum density was achieved in N. oculata at 25°C and 30, but SGR was significantly lower than that of N. oculata at 25°C and 10. The slope constant of the linear relationship between semilogarithmic growth of N. oculata and day of culture was highest at 25°C and 30. Based on these results, the condition of 15°C and salinity 10 seemed to be optimal for maximum density of C. ellipsoidea , and the condition of 25°C and 10 and 30 for SGR and maximum density for N. oculata , respectively.     

Genetic identification of <Emphasis Type="Italic">Conger myriaster</Emphasis> leptocephali in East China Sea

ABSTRACT:   A subsample of Conger leptocephali collected in the East China Sea (ECS) was genetically analyzed and most were found to be the larvae of Conger myriaster , the most commercially important marine eel species in East Asia. However, these leptocephali (22.3–74.0 mm total length [TL]) did not have a complete row of lateral pigmentation, like the leptocephali of this species are commonly known to have when they are collected over the continental shelf or in coastal waters as they recruit. These leptocephali were in the western edge of the Kuroshio Current and the mixing zone over the shelf in the northern part of the ECS, and two smaller leptocephali (22.3, 46.5 mm TL) were collected to the south of the Ryukyu Islands. This evidence of an ontogenetic change in the presence of lateral pigmentation of C. myriaster indicates that previous efforts to determine the spawning area and recruitment patterns based on only morphological examination of Conger leptocephali in the region must be reconsidered. New research efforts based on genetic identification methods are needed to understand the spawning ecology and recruitment of this important fishery species.     

Pyridoxine deficiency of grouper, <Emphasis Type="Italic">Epinephelus coioides</Emphasis>: physiological and biochemical alteration

Pyridoxine is essential for animals. The purpose of this study was to investigate the pyridoxine deficiency symptoms and the fit dose of pyridoxine to keep normal physiological function in grouper, Epinephelus coioides. Graded levels of pyridoxine (0, 1, 2, 4, 8, 16, 32 mg pyridoxine kg⁻¹ diet) were fed to grouper juveniles (14.83±0.31 g) for 8 weeks, and then some physiological and biochemical parameters were measured. Pyridoxine deficiency symptoms such as anorexia, poor survival, convulsion, helically swimming and hyperirritability were observed in fish fed the pyridoxine-deficient diet, and these fish also showed low whole blood haemoglobin level, liver glutamic-pyruvic transaminase (GPT) and superoxide dismutase (SOD) activities. Slight symptoms of pyridoxine deficiency also appeared among fish fed the diet with very low dose (1 mg pyridoxine kg⁻¹ diet) of pyridoxine in 6 weeks. Broken-line regression analysis showed that the optimal pyridoxine levels for these fish to keep normal survival, whole blood haemoglobin level, liver GPT and SOD activities were approximately 1.75, 1.87, 2.22 and 2.05 mg kg⁻¹, respectively.     

Induction of (<Emphasis Type="Italic">Z</Emphasis>)-8-heptadecene in marine green alga <Emphasis Type="Italic">Bryopsis maxima</Emphasis> by mechanical wounding

ABSTRACT:   After mechanical wounding of the marine green alga Bryopsis maxima in a phosphate buffer, one main organic component was released to the solution. The organic compound was characterized by a molecular ion at 238  m/z by gas chromatography–mass spectrometry (GC-MS) and was deduced as 8-heptadecene by the fragment pattern. To identify the stereochemistry of 8-heptadecene, synthetic ( Z / E )-8-heptadecene was coinjected to the solution of wounded B. maxima . Comparing retention times and mass spectra with the synthetic sample, the target hydrocarbon obtained from wounded B. maxima was coincident with ( Z )-8-heptadecene.     

Effects of fish CYP inducers on difloxacin <Emphasis Type="Italic">N</Emphasis>-demethylation in kidney cell of Chinese idle (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

A drug–drug interaction occurs when the effect of one drug is altered by the presence of another drug which is generally associated with the induction of cytochrome P450s (CYPs) activity. Thus, unexpected treatment failures often happen resulting from inappropriate coadministration in fisheries. However, little information is available about CYP induction in fish. The reaction of difloxacin (DIF) biotransformation to sarafloxacin (SAR) belongs to N-demethylation catalyzed mainly by CYP(s). In order to supply useful information on CYP induction, the present study assessed the effects of fish-specific CYP inducers on DIF N-demethylation and enzyme kinetics in kidney cell of Chinese idle (CIK; grass carp (Ctenopharyngodon idellus)) by RP-HPLC. Results demonstrated that the amounts of SAR formation and enzymatic parameters Clint and Vmax were significantly increased due to β-naphthoflavone (BNF) pretreatment. Therefore, we suggest that CYP1A may be involved in DIF N-demethylation in CIK. This study provides instructive information to ensure treatment success via avoiding CYP induction in fisheries.     

Genetic variation in mitochondrial genes and intergenic spacer region in harmful algae <Emphasis Type="Italic">Chattonella</Emphasis> species

In this study, nuclear ribosomal RNA gene internal transcribed spacer regions and the cox2-cox1 fragment of the mitochondrial (mt) genome were sequenced in 24 strains of Chattonella spp. Variability in both regions showed that the mt genome sequences of Chattonella spp. have a higher evolutionary rate than the nuclear rRNA gene sequences. A maximum likelihood tree based on the mt sequence grouped the Japanese Chattonella strains into two groups (Groups A and B), although no correlation was observed amongst the phylogenetic groups, their morphologies, or the isolated areas. Groups A and B were clearly identified by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using Fokl, without the need for a sequencing experiment. The PCR-RFLP assay revealed that Chattonella cells obtained from sea water in Oita, Japan, in 2004 and 2005 belonged to Group B. This is the first report showing the genetic variation in Chattonella spp. using a PCR-RFLP identification protocol.