<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.     

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.     

真鲷精子诱导漠斑牙鲆减数雌核发育

采用紫外线(UV)灭活的冷冻真鲷(Pagrosomus major)精子激发漠斑牙鲆(Paralichthys lethostigma)卵子发育.冷冻真鲷精子适宜的UV灭活剂量为72 mJ/cm2,随着处理时间延长,胚胎的孵化率呈现哈特维希效应,其中40 s灭活组胚胎孵化率达到最高[(29士1.9)％],单倍体率100％.利用冷休克法抑制卵子第二极体排出,成功获得雌核发育二倍体仔鱼.冷休克处理温度为0～2℃,处理起始时间3 min、持续时间45 min时,雌核发育二倍体胚胎的孵化率最高,达(9.4士0.71)％,获得批量雌核发育仔鱼.雌核发育仔鱼经流式细胞仪和染色体倍性鉴定,均为二倍体(2n=48),未发现单倍体和非整倍体现象.雌核发育二倍体组和单倍体组与正常二倍体组组孵化时间差异显著,分别历时64 h 59 min和55 h 49 min.雌核发育二倍体仔鱼与正常二倍体仔鱼形态特征差异不显著(P＞0.05),单倍体仔鱼明显畸形.研究表明,采用适宜的紫外线剂量灭活的冷冻真鲷精子可成功诱导漠斑牙鲆雌核发育,获得雌核发育二倍体后代,研究结果可为漠斑牙鲆全雌化苗种生产提供技术和理论依据.     

Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (<Emphasis Type="Italic">Pseudosciaena crocea</Emphasis>) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit 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. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1:100 with Ringer’s solution and UV irradiated with doses ranging from 0–150 J cm⁻². The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm⁻², after fertilization, motility was <10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm⁻². A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 20°C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures (2°C, 3°C or 4°C) and five shock durations (4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 ± 2.99%) were obtained at 3°C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.     

Improving growth in juvenile turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis> Rafinesque) by rearing fish in switched temperature regimes

The effect of thermal history (16 and 20°C) on growth of juvenile turbot, Scophthalmus maximus (initial mean weight 72.6 g, n = 157) was studied. Fish were divided into four groups, two groups remaining at constant temperature (C16, C20), while fish in the other groups were transferred from either 16 to 20°C (F16-20) or from 20 to 16°C (F20-16). Between 35 and 42 fish in each tank were individually tagged at the start of the experiment. The final mean weights were significantly higher in the F20-16 group (230 g) than in the C20 (213 g), F16-20 (211 g) and C16 (205 g) groups. The overall growth rate was highest in the F20-16 group (1.17% day⁻¹) but comparable in the three other groups (1.00–1.04% day⁻¹). Our findings indicate that, even at near-optimal temperature for a given size, the temperature history of the fish may influence future growth. Based on these indications, we conclude that as turbot grow larger, the temperature should be reduced to take advantage of the change in optimal temperature for growth with increasing fish size rather than rearing at constant temperatures.     

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.     

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.     

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.     

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.     

Repeatable immersion infection with <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis> reproducing clinical signs and moderate mortality

Pseudotuberculosis is a bacterial septicaemia caused by Photobacterium damselae subsp. piscicida in several marine fish species. Yellowtail Seriola quinqueradiata is the most sensitive fish species to this disease. The internal organs of naturally infected yellowtail exhibit whitish spots, tubercle-like tissue structures, consisting of bacterial accumulations. There have been many trials for experimental infection, however adequate method of infection that reproduces moderate mortality and primary clinical signs has not yet established. Present investigation evaluated an immersion infection method by using logarithmic culture-phase bacteria resulting in higher mortality than that using stationary culture-phase bacteria. Typical white spots on the spleen and kidney were also observed constantly in dead fish. Transmission electron microscopy and fluorescent antibody microscopy showed bacterial clusters not only in the spleen and kidney but also in the blood channels in the secondary gill filaments. These results were confirmed repeatedly by plural experiments. The use of logarithmic-phase bacteria in immersion infection is an appropriate technique to reproduce moderate mortality and primary clinical signs, which will be a reliable infection method also for the challenge test of pseudotuberculosis vaccine.     

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.