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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Undulated flooring in the rearing tank decreases hypermelanosis in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

In the aquaculture of the Japanese flounder Paralichthys olivaceus, hypermelanosis, a malpigmentation condition in which the scales of a significant area on the blind side express the characteristics of those on the ocular side, remains a major concern. Since introducing sand into the rearing tank effectively suppresses hypermelanosis, the inhibitory effects of various characteristics of the surface of the tank floor were investigated. Although an inhibitory effect was observed in both tanks with a sand-image floor and a sand-pasted floor, the strongest effect was found in tanks with a dimpled floor. In addition, covering the inner surface of the tank with net also inhibited hypermelanosis. Using a commercially available corrugated plate, the inhibition of hypermelanosis was confirmed with a noted increase in this effect when combined with light coloration. Juveniles tended to situate themselves in the valley portions of the corrugated plate, suggesting that floor contact with the blind side may contribute to hypermelanosis inhibition. Further observations on the floor contact area with various floor configurations suggested that hypermelanosis is locally suppressed in the areas with floor contact. Therefore, suppression by an undulated (both dimpled and corrugated) floor is likely due to an increase in floor contact area with the blind side.     

Pathological characteristics of olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> experimentally infected with <Emphasis Type="Italic">Streptococcus parauberis</Emphasis>

Pathological characteristics of olive flounder Paralichthys olivaceus experimentally infected with Streptococcus parauberis were studied. Various stressful conditions, aeration and netting stress in particular, led to induced mortality by S. parauberis. Netting stress-induced mortality was positively correlated to bacterial dose and stressful conditions. Inflammation of the heart and pericarditis was the major pathological change observed in olive flounder experimentally infected with S. parauberis. During the infected period, the number of bacteria in the infected olive flounder was recorded over time. S. parauberis remained in all fish organs tested, especially in the heart and brain.     

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.     

Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.     

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.     

Efficiency of stagnant water larviculture using disinfected amictic eggs of the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

There is a need to develop low-cost methods for larviculture that emphasize disease prevention. We evaluated stagnant water larviculture in flounder Paralichthys olivaceus using rotifer amictic eggs treated with glutaraldehyde. Growth and survival of the larvae were similar to in larvae reared by standard running water larviculture with daily feeding of rotifers (control). In the experimental group, the n-3 highly unsaturated fatty acid content of rotifers in the rearing water was higher than in the enriched rotifers fed to the control group. We also observed a similar pattern for the fatty acid composition of flounder larvae. The viable bacterial counts associated with rotifers in stagnant water were lower than those associated with enriched rotifers, and there were fewer bacterial genera. However, the viable bacterial count was the same or higher in the experimental rearing water compared with that in the control group. The α-Proteobacteria and Cytophaga–Flavobacterium–Bacteroidetes group dominated the bacterial community structure after larval hatching. To our knowledge, this is the first time that stagnant water larviculture, using only disinfected amictic eggs, has been successfully demonstrated on a mass production scale. This method appears to offer benefits including improved nutrition, more favorable bacterial communities, and lower cost.