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.     

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.     

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.     

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.     

Evaluation of different <Emphasis Type="Italic">Pyropia yezoensis</Emphasis> extracts as feed additives for growth and immunity of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We evaluated the effects of diets incorporating the red algae Pyropia yezoensis, prepared by several different extraction methods, on the growth of juvenile Japanese flounder Paralichthys olivaceus. We assessed growth performance, as well as the levels of amino acids, fatty acids, insulin-like growth factor I (IGF-I) and interleukins (ILs). Four experimental diets were developed based on different methods of processing P. yezoensis. A commercial feed, laver powder (P), high-pressure heat extraction of laver (HPHE) and acid hydrolysis extraction of laver were used as the experimental diets. Three experimental replicates were established for each diet (40 fish/group, body weight 123.7 ± 1.1 g), and the fish were fed for 6 weeks. We found no significant differences in weight gain, specific growth rate or feeding efficiency among the groups (P > 0.05); however, the fish fed HPHE had the greatest growth performance. Fish fed the laver extracts exhibited the highest protein efficiency ratio compared with the control and P groups. The experimental groups fed P. yezoensis extracts had significantly higher levels of IGF-I (P < 0.05) than those of the control group. High levels of IL-2 were found in the P and HPHE groups, IL-12 in the HPHE group, and IL-6 in all experimental groups. Therefore, these results suggest that a P. yezoensis extract improved the growth performance and immunity of Japanese flounder. In particular, the high-pressure heating process was a useful extraction method for preparing a P. yezoensis extract, which had beneficial effects as a dietary supplement in Japanese flounder.     

Proteolytic digestive enzyme response in Japanese flounder larvae <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> fed two types of rotifers <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> species complex

Two common live feeds, the Brachionus plicatilis species complex SS-type and L-type were used to assess whether there were any differences in protein hydrolysis and digestive trypsin activity in first feeding Japanese flounder. There were no significant differences in hydrolysis activity at 2, 3 and 7 days after hatching (DAH). At 5 DAH, hydrolysis activity was significantly higher in larvae fed SS-type (p?<?0.05) at 50 kDa in 1.5- and 3-h incubation whereas L-type treatment had not completely hydrolyzed the proteins after 3 h at the same molecular weight. Larvae fed SS-type had significantly higher (p?<?0.05) trypsin activity at 3, 5, 6, 7 DAH. Contribution of live prey to trypsin fraction in larvae showed significantly higher (p?<?0.05) fraction for SS-type at 5 DAH (2.18?±?0.44%) and 6 DAH (2.04?±?0.29%) and the effect of exogenous trypsin from live prey was relatively low when compared to the total trypsin activity in larvae. This study discusses the differences in ability to digest proteins in Japanese flounder when fed different rotifer morphotypes and highlights the adaptability of this species to alternative rotifer morphotypes during its early developmental stages.     

<Emphasis Type="Italic">Neoheterobothrium hirame</Emphasis> (Monogenea) alters the feeding behavior of juvenile olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We monitored feeding behavior and survival of starved juvenile olive flounder experimentally infected with the gill monogenean Neoheterobothrium hirame. Infected flounder increased amount of the time spent in the water column by 117% when trying to capture live mysids, Neomysis sp. They also showed different feeding patterns from those of uninfected fish and made fewer attacks towards prey during one feeding attempt. Although the average numbers of mysids captured by individuals were similar between infected and uninfected fish, heavily infected fish tended to catch less prey. These results indicate that N. hirame reduces the feeding efficiency of the host for capturing live prey and possibly makes them more vulnerable to predation during feeding. We could not detect any obvious difference in survival rates between uninfected, lightly and heavily infected fish during 3 months of starvation. There was no evidence that starvation makes fish more susceptible to N. hirame. The present study provides first experimental evidence that N. hirame affects feeding behavior of juvenile olive flounder and supports the idea that this parasite indirectly reducing the host’s survival and may be responsible for the recent reduction of the flounder population in Japan.     

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.     

Characterization of <Emphasis Type="Italic">pax3a</Emphasis> and <Emphasis Type="Italic">pax3b</Emphasis> genes in artificially induced polyploid and gynogenetic olive flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) during embryogenesis

Although chromosome set manipulation techniques including polyploidy induction and gynogentic induction in flatfish are becoming increasingly mature, there exists a poor understanding of their effects on embryonic development. PAX3 plays crucial roles during embryonic myogenesis and neurogenesis. In olive flounder (Paralichthys olivaceus), there are two duplicated pax3 genes (pax3a, pax3b), and both of them are expressed in the brain and muscle regions with some subtle regional differences. We utilized pax3a and pax3b as indicators to preliminarily investigate whether chromosome set manipulation affects embryonic neurogenesis and myogenesis using whole-mount in situ hybridization. In the polyploid induction groups, 94 % of embryos in the triploid induction group had normal pax3a/3b expression patterns; however, 45 % of embryos in the tetraploid induction group showed abnormal pax3a/3b expression patterns from the tailbud formation stage to the hatching stage. Therefore, the artificial induction of triploidy and tetraploidy had a small or a moderate effect on flounder embryonic myogenesis and neurogenesis, respectively. In the gynogenetic induction groups, 87 % of embryos in the meiogynogenetic diploid induction group showed normal pax3a/3b expression patterns. However, almost 100 % of embryos in the gynogenetic haploid induction group and 63 % of embryos in the mitogynogenetic diploid induction group showed abnormal pax3a/3b expression patterns. Therefore, the induction of gynogenetic haploidy and mitogynogenetic diploidy had large effects on flounder embryonic myogenesis and neurogenesis. In conclusion, the differential expression of pax3a and pax3b may provide new insights for consideration of fish chromosome set manipulation.     

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.     

Estimation of genetic parameters for growth traits of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> using an animal model

To estimate genetic parameters of growth traits in Japanese flounder Paralichthys olivaceus, full-sib and half-sib families were produced in three consecutive years at the Beidaihe Central Experiment Station in China. Each year 8–28 families were produced. The body weight, body length and body depth at 180, 240, and 360 days of age were measured for 5,224 individuals. Four animal models were used to examine the phenotypic variation of growth traits and were compared using the likelihood ratio test. The results showed that estimates for additive genetic effect heritabilities varied greatly depending on the model, trait and age. The maternal effect had a significant impact on phenotypic variation only for body depth at 180 days of age, which explained 49% of the phenotypic variance. The ratio of full-sib effect to phenotypic variation ranged from 0.09 to 0.22. Growth traits all exhibited low heritability (0.13–0.39), indicating that there is the potential for family selection breeding for these traits in Japanese flounder. Using the full model with the fixed, full-sib family, additive and maternal genetic effects, genetic correlations among the three traits for fish of the same age were estimated to be more than 0.80. Generally, the genetic correlations gradually increased as age increased.