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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 dietary inclusion of various sources of green tea on growth,body composition and blood chemistry of the juvenile olive flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

This study was performed to determine the effect of the dietary inclusion of various sources of green tea on growth, body composition and blood chemistry of the olive flounder. Twenty-five juvenile fish were distributed into each of 15 180 l flow-through tanks. Five experimental diets with triplicates were prepared: control, raw leaves, dry leaves, by-product and extract. The 5% various sources (raw leaves, dry leaves and by-product) of green tea were included in the experimental diets at the expense of 5% wheat flour. The extract was diluted with water and mixed with the ingredients to maintain 5% green tea at the same concentration as in the other diets. Weight gain and specific growth rate of fish fed the control diet and experimental diet containing extract were higher than those of fish fed the diets containing raw and dry leaves and by-product. Feed and protein efficiency ratio for fish fed the control diet and diet containing extract was higher than for fish fed the diets containing raw leaves and by-product. The level of low-density lipoprotein (LDL) cholesterol of fish fed the control diet was higher than that of fish fed the other diets containing various sources of green tea. The glutamic oxaloacetic transaminase (GPT) concentration of fish fed the diets containing raw leaves and extract was lower than that of fish fed the control diet. Crude lipid content of liver in fish fed the control diet was higher than that in fish fed the diets containing raw and dry leaves and by-product but was not significantly different from that of fish fed the diet containing extract. Dietary inclusion of extract was the most effective way to improve growth and feed utilization of the olive flounder among the various sources of green tea, and all sources of green tea were effective in lowering serum LDL cholesterol and GPT of the fish.     

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.