Genetic population evaluation of two closely related flatfish species, the rare barfin flounder and spotted halibut, along the Japanese coast

ABSTRACT:   Barfin flounder and spotted halibut have been selected as target species for stock enhancement in Japan. Understanding the genetic condition of the wild stock is a principal requirement in any stock enhancement program. The genetic variability of barfin flounder and spotted halibut, and the population structure of spotted halibut were evaluated using microsatellite DNA markers (msDNA) and the control region of the mitocondrial DNA (mtDNA). Barfin flounder and spotted halibut showed high genetic variability at the msDNA level. Barfin flounder A was 16.7 and H _e was 0.860; spotted halibut A _n ranged from 7.7 to 10.2 and H _e ranged from 0.710 to 0.774. At the mtDNA level, high haplotype ( h  = 0.922) and low nucleotide (π = 0.002) diversities were observed for barfin flounder; however, low haplotype and nucleotide diversities ( h  = 0.603–0.620 and π = 0.001–0.002), and very low haplotype and nucleotide diversities ( h  = 0.193 and π = 0.0003) were observed for spotted halibut in the north and south locations, respectively. Slight genetic differentiation among spotted halibut sampling locations was observed from the msDNA. MtDNA analyses showed genetic differentiation between north and south locations, but not within them. The designation of north-specific and south-specific management units in the future stock enhancement activities of spotted halibut is recommended.     

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.     

Annual dynamics of marine birnavirus (MABV) in cultured Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and sea water

ABSTRACT:   Seasonal changes in the infection state of marine birnavirus (MABV) in Japanese flounder Paralichthys olivaceus and in rearing sea water are described. Sea water and 10–11 healthy fish were sampled monthly from April 2002 to February 2003. The MABV genome was detected throughout the year in > 80% of the fish examined at each sampling. The virus was isolated from the liver, kidney, and spleen, but not from the brain. The detection rate in each organ increased from April to October, and then decreased. Detection of virus antigen by the indirect fluorescent antibody technique also showed that the virus was present from spring to autumn (June–September) in the liver, kidney, and spleen, but not the brain. Sequence analysis of the MABV genome at the VP2–NS region revealed two specific mutations compared to the standard yellowtail strain (Y-6). It is suggested that the infection state of MABV in Japanese flounder changes to a latent or persistent infection after autumn. MABV was detected in sea water between September and February, suggesting that virus particles in the environment are relatively higher during cool seasons.     

Maternal and genetic effects on individual variation during early development in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   To investigate the changes in maternal effects during early development in Japanese flounder Paralichthys olivaceus , four-by-four factorial mating by artificial fertilization was performed to produce 16 families. Larvae and juveniles of each family were reared under common environmental conditions until completion of metamorphosis. The magnitude of maternal effects was estimated as the differences between sire and dam variance. Maternal effects were highest at hatching, and then disappeared at 30 days after hatching. During early development, larval size at hatching was largely affected by egg size, but this effect decreased as larvae grew. Estimated mean heritability during early development was nearly constant (0.14 ± 0.06 for total length and 0.10 ± 0.05 for body depth). Therefore, it is suggested that early development in Japanese flounder is affected by both maternal effect and genetic factors.     

Feeding habits of stone flounder <Emphasis Type="Italic">Platichthys bicoloratus</Emphasis> larvae in Mutsu Bay,Japan

To clarify the feeding strategy of pelagic larvae of stone flounder in Mutsu Bay, the dietary composition and pray size was investigated from February to April during 1989–1999. Diets were compared with the numerical and volumetric compositions and frequency of occurrence of each prey species. Mensuration formulae were applied to estimate individual prey volume in the diet, while the length of planktonic species was measured from net and water samples. Prey shapes were assumed as sphere, cylinder, ellipsoid, pyramid, two elliptical cones, or a combination of ellipsoid and cylinder. Prey-size range increased as the larvae grew. Preflexion larvae fed mainly on copepod nauplii. Flexion and postflexion larvae ingested primarily appendicularians, with a suggestion that these larvae might depend on some parts of the microbial food web. Low frequencies of flexion and postflexion larvae with empty guts (1.7 and 1.4%, respectively) might be derived from feeding on slow-swimming appendicularians. From a size comparison between ‘house’-like organ length and trunk length of the appendicularian Oikopleura sp., almost all house-like organs with trunks in the larval diet were nonexpanded ‘house rudiments’, not expanded ‘houses’. Thus, stone flounder larvae may not chew the houses, but swallow the house rudiments with trunks.     

Changes in growth,higher salinity tolerance and behavioral traits in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> after repeated cross-breeding

ABSTRACT:     To improve stock enhancement strategies, the influences of repeated cross-breeding on several traits were studied in hatchery-reared juveniles of the Japanese flounder. Ten families (seven caught in the wild and three from cross-bred brood fish) were produced by artificial fertilization. Ninety-four days after hatching, we investigated settlement, burying in the sand and escape behavioral traits, and then measured tolerance of high salinity and six morphological traits. Juveniles from the repeatedly cross-bred brood fish showed significantly higher growth and tolerance than those from the wild-caught brood fish. Among the behavioral traits, a significantly lower settling speed and incidence of burying in the sand were observed in the juveniles from the repeatedly cross-bred families. Heritability ( h ²) of growth traits was more than 0.2 in both origins, while that of other traits was close to zero. These results suggest that repeated cross-breeding of Japanese flounder under hatchery conditions improves growth performance and leads to the loss of certain behavioral traits that are considered essential to survival in wild environments.     

Evaluation of cottonseed and soybean meal as partial replacement for fishmeal in diets for juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

This study was conducted to investigate the effect of dietary supplementation of cottonseed and soybean meal (CS) on growth performance, feed utilization, and gossypol accumulation in juvenile (mean body weight 11 g) Japanese flounder Paralichthys olivaceus. Five isonitrogenous and isocaloric experimental diets (designated CS0, CS10, CS20, CS30, and CS40) containing 0, 10, 20, 30, and 40% CS mixture (1∶1 w/w) were formulated. After 9 weeks of feeding trial, fish fed all diets did not show significant differences in growth performances, feed utilization, and whole body compositions. Hemoglobin content gradually decreased (P<0.05) with increments of CS inclusion levels. There was a positive linear relationship between dietary gossypol and total liver gossypol contents. Free radical scavenging activity in the experimental diets gradually increased with increment of dietary CS inclusion. This finding indicates that the mixture of cottonseed and soybean meal with methionine and lysine supplementation could replace up to 40% of fishmeal protein in diets. However, 20% fishmeal protein replacement by dietary inclusion of cottonseed (9.4%) and soybean (8.7%) meal might be the optimum and safe level for the commercial use of CS in juvenile Japanese flounder.     

Reproductive cycle,age, and body size at maturity and fecundity of female willowy flounder <Emphasis Type="Italic">Tanakius kitaharai</Emphasis>

Annual reproductive cycle, age and body size at maturity, and potential and relative fecundity were examined in female willowy flounder in the North Pacific off Japan. Vitellogenesis became active from September, followed by the beginning of spawning in December. The spawning season continued until May with its peak in January. Estimated maturation rate was 50% at a standard length of 16 cm, 30% in 2-year-olds, and almost 100% in ≥3-year-old fish. Potential fecundity increased with age, while relative fecundity decreased in older fish. The results clarify some aspects of the detailed reproductive biology of female willowy flounder and emphasize the importance of age-composition data in estimating reproductive potential within a population.     

Age validation and growth variability of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> off the Pacific coast of northern Japan

ABSTRACT:   This study examined age and growth of Japanese flounder Paralichthys olivaceus off the Pacific coast of northern Japan, and determined whether the growth patterns of male and female fish in northern (40–41°N) and southern (37–38°15'N) waters differ. In total 8095 specimens were collected between January 1999 and December 2005. Zonation consisting of opaque and translucent bands on the otolith was evident. Within each opaque band a thin and clear check (ring mark) was observed in all specimens examined. Monthly change in the frequency of appearance of a ring mark on the outer margin of the otolith indicates that ring marks form between July and August. The von Bertalanffy growth model showed a sexual dimorphism in growth, as females grew faster and reached a larger size than males. The growth patterns obtained by tracking the observed total length for monthly collections showed a rapid increase in total length between August and October. Spatial variation in the growth pattern of male and female fish between northern and southern waters was evident, as southern fish were significantly larger than northern counterparts during 1.25–3.00 years post hatch.     

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.     

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.     

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.     

圆斑星鲽线粒体基因组全序列结构及其进化

利用圆斑星鲽(Verasper variegatus Temminck et Schlegel)和相关鱼类的部分线粒体基因序列,设计出6对扩增引物,通过PCR扩增产物直接测序和引物行走(Primer walking)法测定圆斑星鲽线粒体基因组全序列,并对其进行结构与进化分析。圆斑星鲽线粒体基因组序列长17273 bp,其基因序列及构成都与其他硬骨鱼基本相同,包括37个基因(2个rRNA基因、22个tRNA基因和13个蛋白质编码基因)和2个非编码区(控制区和WANCY区)。在22个tRNA基因中,tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer(第2个)、tRNAGlu和tRNAPro的编码基因位于L链上,其余则位于H链上。在13个蛋白质编码基因中,除了COⅠ基因的起始密码子为GTG外,其余均以ATG为起始密码子。蛋白质编码基因包括具有完整TAA终止密码子的ND1,COⅠ,ATP8,ND4L,ND5,而其他蛋白编码基因则具有不完全终止密码子。在L链上,ND6是仅有的一个蛋白质编码基因。圆斑星蝶的控制区包含1个终止相关序列区(ETAS)、6个中央保守序列区(CSB-A、B、C、D、E、F)和3个保守序列区(CSB-1、2、3)以及长串联重复区(Tandemly repeat sequence)。用NJ法和MP法对5个目22种鱼mtDNA的13个蛋白质编码基因的氨基酸序列进行系统分析,结果显示,圆斑星蝶与石鲽关系最近,鲽形目的鲆、鲽类与鲈形目的科(Carangidae)、鲷科(Sparidae)鱼类亲缘关系较近,而同属于鲽形目的塞内加尔鳎(Solea senegalensis)没有和任何目聚在一起,成为一个独立的分支。圆斑星蝶的基因组全序列序列已提交到GenBank,登录号为DQ403797,控制区单元型序列的GenBank登录号为DQ834444~7。     

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.     

Influences of feeding frequency of extruded pellet and moist pellet on growth and body composition of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> in suboptimal water temperatures

This study was carried out to investigate the influences of feeding frequency of extruded pellet and moist pellet on growth and body composition of juvenile flounder (initial mean weight 6.3 g) in sub optimal water temperatures. A 2 (diets: extruded pellet and moist pellet)×2 (feeding frequencies: two and three times daily)×2 (water temperatures: 12 and 17°C) factorial design with three replications was used. After 60 days of feeding, the feeding frequency did not significantly affect growth performance of fish. Weight gain, daily feed intake, feed efficiency and protein efficiency ratio were significantly (P<0.05) higher for fish reared at the higher water temperature. At the same water temperature, weight gain, feed efficiency, and protein efficiency ratio were significantly (P<0.05) higher for fish fed the extruded pellet than moist pellet. The results of this study indicate that feeding frequency of two times daily is sufficient for optimal growth of juvenile flounder when reared in suboptimal water temperatures, and an increase in water temperature from 12 to 17°C improved growth and feed efficiency.     

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.