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Hypermelanosis of the blind side of Japanese flounder frequently occurs in post-metamorphic juveniles reared in hatcheries. Due to the morphological similarity between darkened blind-side skin and normal ocular-side skin, hypermelanosis is regarded as a transition from normal blind-side skin to that of the ocular side. Hypermelanosis appears only in rearing tanks without sand on the bottom, and its incidence tends to increase under stressful conditions; however, the specific mechanisms responsible for its occurrence have not been identified to date. In teleosts, the hypothalamus-pituitary-interrenal axis (HPI axis) is activated under stress, which then accelerates cortisol secretion. Therefore, in this study, we orally administered cortisol to Japanese flounder to examine its effect on hypermelanosis. The results of our study indicated that cortisol promoted hypermelanosis in a dose-dependent manner. In addition, the effect of cortisol was canceled out in juveniles reared in tanks with bottom sand, suggesting that the well-documented inhibitory effect of bottom sand is stronger than the stimulatory effect of cortisol (i.e., stress) on hypermelanosis. However, administration of the cortisol antagonist RU486 failed to inhibit hypermelanosis, suggesting the contribution of other stimulatory factors to this process. Therefore, our findings strongly suggest that stress-induced cortisol release has the adverse effect of promoting hypermelanosis in juvenile flounders reared without bottom sand.

The aim of this work was to evaluate the effect of salinity changes on digestive enzyme activity and whole-body cortisol concentration in juveniles (46 DAH) of Japanese flounder. Two different salinity patterns were designed: a control group exposed to a constant flux of seawater ( S = 32) and a treatment group exposed to changes from full seawater ( S = 32) to low salinity water ( S = 10) each 6 h. The duration of the experiment was 2 days. Samples were taken every 6 h for enzyme activity and cortisol determination. Survival in both treatments was 92% at the end of the experiments. Cortisol levels were significantly higher ( P P > 0.05) between control and treatment groups. Trypsin activity decreased significantly ( P P P > 0.05) were found between treatment and control group. This work showed that juveniles of Japanese flounder could survive fluctuating exposure to low salinities ( S = 10) at least in short periods. The results suggest that juveniles of P. olivaceus may retain abilities to survive exposure to low salinities through shared phylogenetic relationship.

The aim of this work was to evaluate the effect of stocking density on growth, digestive enzyme activity and cortisol level in larvae (2–35 DAH) and juveniles (46–65 DAH) of Japanese flounder. The body weight and body length were recorded, enzyme activities (trypsin and lipase) and cortisol levels were examined from frozen samples anesthetized with 0.1% MS 222. In the first experiment using larvae, two different densities (1000 and 5000 individuals in triplicate 100 l tanks) were examined. The weight and total length of the low density group was significantly larger ( P P > 0.05) were found in cortisol concentrations before 30 DAH, while a significantly higher concentration ( P In the second experiment, juveniles of 46 DAH were kept in 100 l triplicate tanks at two different densities (200 and 2000 larvae in 100 l tanks, bottom area 0.22 m 2 ), and reared for 15 days. Bottom settled fish and swimming fish were separately sampled. On 61 DAH, the fish swimming were selectively captured in the high density tanks and 100 individuals were transferred to 30 l tanks and further reared for 4 days. The growth in total length and weight of swimming juvenile flounder was significantly lower ( P P P P > 0.05). In juveniles, specific activity of trypsin was significantly higher ( P P These results showed that the rearing density has a significant influence on Japanese flounder juveniles, and results in social hierarchy (bottom settled > swimming) at high densities, that further causes higher cortisol levels and lower growth rates in low rank individuals.

The effects of larval density, rearing media, and protein additions on fish larval survival were tested by rearing larvae in small containers. Yolk-sac larvae of Japanese flounder, Paralichthys olivaceus, were transferred 1 day after hatching to plastic dishes and cell-culture microplates by Pasture pipette at densities ranging from 0.3 to 30 individuals/ml and incubated at 16 °C without food. Dead larvae were removed and counted every 12 h. Three peaks of larval mortality were observed: the first peak appeared just after the transfer, the second corresponded to the completion of yolk absorption, and the third occurred 10–12 days after the transfer. Since about 50% of larvae survived until the third peak, it was clear that mortality at the second peak (completion of yolk absorption; equivalent to the first feeding) was not due to starvation. At the first mortality peak, better survival was unexpectedly recorded at higher larval densities (30 individuals/ml), suggesting that some material beneficial for survival is derived from the larvae. This idea was further supported by the improved survival in media previously used to rear larvae at high densities. The addition of bovine serum albumin (BSA) at 10 µg/ml effectively reduced mortality in microplates and in 30-l tanks. To reduce the larval mortality just after handling, addition of egg white at a dilution rate of 1/30,000 (vol/vol) or less is recommended as a practical alternative to BSA. Since the addition of polyethylene glycol 6000 (1 µg/ml) also effectively reduced mortality, reduction in mortality could not be attributed to a specific mechanism. The precise determination of the magnitude and timing of larval mortality was attained through the use of small containers, and a method to prevent mortality due to handling was identified in this study. These types of tests may allow the logical evaluation and establishment of better rearing procedures for seed production in aquaculture.

In the seed production of several marine teleosts, large numbers of dead larvae are frequently observed on the water surface around the time of first feeding. We tried to prevent such “surface death” by the addition of chicken egg white (EW) into the rearing water. In the first experiment, larvae of striped bonito, Sarda orientalis , at the first feeding stage (2 and 3 days after hatching) were transferred to plastic beakers using a 5-ml pipette at a density of 10 larvae/l, in the absence or presence of EW (80 μl/l). No diet was supplied, the water was not renewed throughout the experiment, and dead larvae were removed daily. Numbers of surviving larvae of the EW-treated group were significantly higher than those of the control group. In particular, survival at the first observation (next day after transfer) was less than 30% in the control, while 100% in the EW-treated group. In a second experiment, mortality at 2 h after transfer was compared with special attention to the location of the dead larvae. Significantly ( P Dules auriga , was also higher in the presence of EW, especially during a period when dead larvae were frequently found on the water surface in the control. Thus, the addition of EW to the rearing water is suggested to prevent surface death in a variety of marine fish larvae, and the possibility of applying the method in practical rearing is discussed.

Japanese flounder larvae were reared for 43 days in glass aquariums with and without sand in the bottom to study the influence of the substrate on the pigmentation pattern, pituitary growth and ACTH and MSH production. Similar results were obtained in terms of survival, growth and ocular side pigmentation in both groups. However, a clear difference was observed in blind side pigmentation and only 28% of the fish cultured with sand developed this abnormality, contrasting with 58% of those reared without sand. A clear increase in the pituitary volume was observed during larval development, being maximal at post metamorphosis. No differences in the pituitary volume could be found among pigmented, albino and ambicoloured juveniles, although those reared with sand had a larger pituitary than those reared without sand. ACTH and MSH producing cells were detected by immunohistochemistry. The proportion of the pituitary occupied by ACTH or MSH cells (ratio) was highest at early larval stages, and decreased with development to a minimum value at the climax of metamorphosis. No significant difference in the ACTH ratio was found between the two groups, although juveniles reared without sand showed a slightly higher ratio, probably reflecting the stress experienced by these fish. The MSH ratio was significantly higher in fish reared with sand. No significant differences were found in the ACTH and MSH ratios among pigmented, albino and ambicoloured fish. Fish produced with a sandy bottom showed a lower degree of hyperpigmentation and possessed larger pituitaries that had higher MSH ratios than those reared without sand.

In order to examine the putative involvement of adenocorticotropin (ACTH) and melanocyte-stimulating hormone (MSH), the two major hormones controlling melanophores, in albinism of flounders, production of the hormones was immunocytochemically examined in fish in which albinism had been induced by dietary arachidonic acid (ARA) supplementation. Larvae of Japanese flounder were fed live prey enriched with ARA from A, C, F and H stages (4, 13, 26, and 35 days after hatching, respectively). A high incidence of malpigmentation (80%, with 37–45% total albinos) was found in the fish fed ARA-supplemented diets prior to metamorphosis (Stage H, 35 days after hatching). The incidence of albinism was reduced compared to control treatment when ARA-supplemented diets were fed after metamorphosis. No significant differences were found among the groups fed the ARA-enriched live prey from A, C and F stages. No effect of ARA-supplemented diet was observed on the volumes of ACTH- and MSH-producing cells or total pituitary volume. However, it might be possible that although the total volume of ACTH- and MSH-producing cells was not significantly affected, the rate of hormone production or release by those cells might have been different among treatment groups of fish with differences in pigmentation. A direct effect of ARA and/or its metabolites on skin or on the differentiation of adult-type melanophores is also suggested.

Changes in phototaxis of chum (Oncorhynchus keta) and coho (O. kisutch) salmon were examined after treatment with thyroid hormones, using troughs (0.25 × 2 m long, 20 cm deep) with open (1200–2400 lux, 1 m long) and shaded (400–680 lux, 1 m long) water. In some experiments, half of the trough was covered to provide shelter ( 30 mg 150 l , immersion) or thiourea ( 15 g 150 l , immersion) for 56 days from early April. Coho smolts were treated with triiodothyronine (T3, 15 mg 150 l , immersion) for 4 days in July–August. T4-treated chum preferred open water by 80–90%. More than 78% of fry treated with thiourea and the control preferred to be in shade or to be in shelter. T3-treated coho preferred open water after 3 and 4 days, whereas the control fish stayed in shaded water. The preference of T3-treated fish for open water was observed 1 week after the end of treatment and the preference disappeared during the following week. These findings suggest that thyroid hormones play an important role in the phototaxis of salmonids in freshwater.