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.     

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.     

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.     

Thyroid hormone deficiency in abnormal larvae of the Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   In large-scale rearing of juveniles of the Japanese flounder Paralichthys olivaceus, a certain morphological abnormality occurred spontaneously in 4% of the fish. These fish showed a slight but clearly different appearance from any developmental stage of this species, and did not settle when all the other juveniles in the same tank completed metamorphosis and had settled. From comparisons of external and internal structures between the normal and the abnormal fish, the abnormality was attributed to unbalanced progress of metamorphosis, mainly due to metamorphic stasis. The thyroid of the abnormal fish was apparently activated morphologically. In addition, serum thyroxine (T4) concentrations in the abnormal fish were reduced to less than 1/10 of that of normal fish. After 14 days of T4 treatment (0.1 p.p.m) of the abnormal fish, all the abnormal characteristics disappeared, and the fish recovered to normal, suggesting normal responsiveness to thyroid hormones in peripheral tissues, whereas thiourea treatment (30 p.p.m., 14 days) further delayed metamorphosis. These results suggest that these abnormal fish were suffering from thyroid hormone deficiency, and were unable to secrete a sufficient amount of thyroid hormone to complete metamorphosis.     

Immunostimulatory effects of fermented vegetable product on the non-specific immunity of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

The stimulatory effect of fermented vegetable product (FVP) upon the phagocytic and superoxide generation of leukocytes was studied in the Japanese flounder Paralichthys olivaceus. The phagocytic activity of casein-induced, intraperitoneal leukocytes was investigated and quantified, that is the activity significantly increased (P<0.05 or <0.01) by the addition of FVP beyond 3 mg/kg body weight. Further analysis investigated the effect of FVP on superoxide generation in leukocytes. Established in vitro cytochrome c reduction assay was used to measure superoxide generation; reduced levels of FVP in assay samples had a profound effect on superoxide generation. FVP was also incorporated in commercial diets and fed to Japanese flounder for 4 weeks. The phagocytic activities and superoxide generation of peritoneal induced leukocytes were significantly higher (P<0.05, <0.01) in fish fed the FVP supplemented diet than fish fed the control diet. FVP feeding in fish had a significantly higher (P<0.05) activity of lysozyme than in the control fish.     

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.     

Optimum dietary phosphorus level of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> reared in the recirculating system

ABSTRACT:   An 8-week feeding trial was conducted to determine the optimum dietary phosphorus level of juvenile Japanese flounder Paralichthys olivaceus reared in the recirculating system. The basal diet containing 0.33% total phosphorus (TP) was supplemented with NaH₂PO₄·H₂O to provide 0.33, 0.51, 0.71, 0.94, 1.10 and 2.12% TP for the experimental diets. Fish averaging 2.0 ± 0.04 g (mean ± SD) were fed in triplicate groups with the experimental diets for 8 weeks. After 8 weeks, fish fed the 0.51% TP diet showed significantly higher weight gain (WG), feed efficiency (FE), specific growth rate (SGR), and protein efficiency ratio (PER) than those fed the 0.33% and 2.12% TP diets ( P  < 0.05). Meanwhile, no difference was observed in WG, FE, SGR and PER of fish fed the 0.51, 0.71, 0.94 and 1.10% TP diets. Fish fed the 0.33% TP diet showed phosphorus deficiency symptoms such as poor growth and a deformed head by the end of 8 weeks. Ash and phosphorus contents of bone increased with the increase of dietary phosphorus concentrations. anova test, the quadratic regression analysis, and the broken line analysis suggested that the optimum dietary phosphorus level could be between 0.45 and 0.51% TP for maximum WG in juvenile Japanese flounder.     

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.     

Effect of dietary taurine and lipid contents on conjugated bile acid composition and growth performance of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   The effects of dietary taurine levels and lipid contents on the conjugated bile acid composition and growth performance of juvenile Japanese flounder Paralichthys olivaceus were investigated. Six types of diet (three different levels of taurine at two different levels of lipid) were fed to juveniles (average body weight, 0.04 g). Fishmeal that was washed with 70% ethanol to remove taurine was used as the sole protein source. Feeding experiments were carried out at 20°C for 6 weeks. At the end of the experiments, fish were weighed and analyzed for free amino acids in the body and the composition of the conjugated bile acids. The body weight and percent weight gain of the juveniles were improved by the dietary taurine supplementation. The taurine contents of the whole body and tissues increased with the increase of the dietary taurine contents. The conjugated bile acids in the gall bladder consisted of taurocholic acid and taurochenodeoxycholic acid, which increased with the increase of the dietary taurine level. Taurocholic acid accounted for more than 95% of the total conjugated bile acids. This indicates that taurine is the sole amino acid to conjugate bile acid in Japanese flounder.     

Genes expressed in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> spleen: analysis of genes involved in immune function

ABSTRACT:   Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and for profiling gene expression. A cDNA library developed from messenger RNA of the Japanese flounder, Paralichthys olivaceus spleen was constructed by directional cloning, in order to isolate functional genes involved in immunity in fish. A total of 1010 ESTs from the library were sequenced and compared with sequences in the GenBank database. Of the 1010 ESTs, 618 ESTs (61%) were identified as being homologous with known genes from many organisms by BLAST searches, whereas 392 (39%) appeared to be unknown and are likely to represent newly described genes. Of the identified genes, 105 (17%) encoded proteins associated with cell/organism defence and homeostasis. Of these 105 genes, 21 were identified for the first time in Japanese flounder. These included macrophage inflammatory protein (MIP)-3 α, granulin-A, novel immune-type receptor (NITR), interferon regulatory factor (IRF)-10, presenilin-like protein-2, and antizyme inhibitor. A comparison of ESTs derived from spleen, kidney, liver and leukocytes suggested that expression of immune-related genes in different tissues, organs, or cells are different.     

Effect of taurine supplemented practical diet on growth performance and taurine contents in whole body and tissues of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   The effect of dietary taurine on juvenile Japanese flounder was determined by feeding three taurine-supplemented experimental diets (TAU) and a commercial diet (CD) to evaluate a practical diet for juvenile Japanese flounder. Juvenile Japanese flounder were reared on the three experimental diets supplemented with taurine at 0, 0.5, 1.0% and CD. These diets were fed to juvenile Japanese flounder of an initial mean body weight of 0.2 g for 6 weeks at 20°C and the taurine contents of the whole body and tissues were analyzed. The final average body weight of juvenile Japanese flounder fed the 1.0% TAU was significantly higher than that of the other groups. Taurine contents in the whole body and tissues increased with the increase in dietary taurine level. These results indicate that juvenile Japanese flounder require at least 15 mg/g taurine in the diet, even though a combined mix of fish, krill and squid meal was the main protein source in the experimental diets.     

Production and characterization of monoclonal antibodies to leukocytes and serum immunoglobulin in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Two monoclonal antibodies (MAbs: JFW1 and JFW10) were produced against peripheral blood leukocytes (PBL) in Japanese flounder. Additionally, MAbs against flounder immunoglobulin (Ig; JFW20 and JFW21) were generated for the surface marker of Ig⁺ leukocytes using purified serum Ig as an antigen. MAb JFW1 recognized the surface marker of granulocytes and monocytes and MAb JFW10 specifically bound to the surface antigen of thrombocytes. Flow cytometric analysis of PBL incubated with JFW1, JFW10, JFW20 and JFW21 revealed that 2.5–7.4, 23.7–50.1, 25.2–26.1 and 5.2–8.3% of all leukocytes were positive for these markers. Analysis of head kidney leukocytes (HKL) showed that JFW1, JFW10, JFW20 and JFW21 bound to 30.5–36.3, 1.9–2.8, 6.4–8.3 and 1.9–3.0% of all leukocytes, respectively. Western blot analysis after SDS-PAGE showed that JFW10 recognizes a protein of 115 kDa from lysed PBL. JFW20 recognized the 70 and 74 kDa proteins of the heavy chain of Ig from serum. No band was observed for either JFW1 or JFW21. These antibodies will be useful for the identification and isolation of Japanese flounder leukocyte subpopulations and will facilitate immunological studies of flounder.     

Effects of water temperature on early development of Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

ABSTRACT:   To determine an optimal temperature range for efficient production of healthy eel larvae Anguilla japonica , the effect of water temperature on hatching, survival, and deformity rates was examined. The early ontogeny of morphological features in this species by incubating eggs at five different temperatures (19, 22, 25, 28 and 31°C) was examined. Hatching occurred at 24, 28, 46 and 58 h after fertilization when incubated at 28, 25, 22 and 19°C, respectively; no eggs hatched at 31°C. The growth rate of prefeeding larvae increased as water temperature was elevated and acquisition of feeding ability was also accelerated at higher temperature. Significantly high hatching rates (76–86%, P  < 0.05) and survival rates (61–86%, P  < 0.05) were observed at relatively high temperatures (22–28°C). The deformities were 'open lower jaw', 'pericardial edema', and 'notochordal bending', in which the mouth was deformed with a downward projecting lower jaw, the pericardial cavity was swollen, and the notochord was bent or twisted to various degrees, respectively. Open lower jaw and pericardial edema were especially predominant at 19°C, with rates of 68 and 92%, respectively, compared with 31 and 10% at 25 and 28°C, respectively. The occurrence of notochordal bending was not affected by temperature. The optimal temperature for incubation and rearing A. japonica eggs and prefeeding larvae is approximately 25–28°C.     

Effects of temperature changes on the endogenous rhythm of oxygen consumption in the Japanese flounder <Emphasis Type="Italic">paralichthys olivaceus</Emphasis>

ABSTRACT:   The effects of temperature changes on the endogenous rhythm of oxygen consumption in the Japanese flounder Paralichthys olivaceus which were acclimated to 3, 14 or 20°C in advance were investigated. The oxygen consumption rate (OCR) of individuals (total length >28 cm) that were acclimated to 3°C, maintained a relatively low value of 6.9 ± 2.3 mL (mean ± SD) O₂/kg ww (wet weight)/h till 5.8°C, which is considered a 'minimum' rate. Thereafter, the OCR gradually increased up to 18.7 mL O₂/kg ww/h at the temperature of 10.6°C. The OCR of individuals acclimated to 14°C stabilized with 124.2 ± 24.1 mL O₂/kg ww/h, when tested at the relatively constant temperature of 14.4 ± 0.17°C. Maximum entropy spectral analysis revealed that the peak OCR occurred at 23.9 h intervals, which could be a circadian rhythm. The mean OCR showed a peak at 06.00–08.00 hours. For the temperature above 25°C in which the fish were acclimated to 20°C, the rhythmic patterns of OCR were observed until the experimental temperature reached 26.4°C. For the temperature above 28.4°C, the metabolic activity rhythm of the OCR was dampened and then the OCR abruptly rose again. It is assumed that the oscillator of the endogenous rhythm lost the governing of normal metabolic activity in Japanese flounders above 28.4°C. These results suggest that the temperatures of around 5.8, 26.4 and 28.4°C are the 'lower limit temperature', 'upper incipient lethal temperature', and the 'critical thermal maximum', respectively, for the endogenous rhythm of oxygen consumption in the Japanese flounder.     

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.     

Quantitative genetic parameters for growth-related and morphometric traits of hatchery-produced Japanese flounder Paralichthys olivaceus in the wild

To understand quantitative genetic characteristics of hatchery‐produced Japanese flounder in the wild, heritability and genetic correlation of growth‐related and morphometric traits were examined in yearling released individuals at a coastal region in northeast Japan. Quantitative genetic parameters were estimated with restricted maximum likelihood following reconstruction of pedigree by a likelihood method using seven microsatellite loci. Estimates of heritability were 0.65 and 0.51 for growth‐related traits (body length and the proportion of body length to body depth) and 0.45–0.62 for morphometric traits (vertebral count and dorsal and anal fin ray counts). Genetic correlation was significantly positive (0.61) between dorsal and anal fin ray counts, indicating the possibility of pleiotropic genes or gametic phase disequilibrium for these traits. All the estimates of heritability and genetic correlation in the released individuals were close to those of hatchery‐reared juveniles, suggesting that yearling released individuals had similar quantitative genetic characteristics of growth and morphometric traits in the wild to hatchery‐reared juveniles.