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In Japanese flounder aquaculture, hypermelanosis occurs widely on the blind side. Rearing flounders in a net-lined tank was recently reported to prevent hypermelanosis. To effectively apply this method to larger tanks for aquaculture farming, the net setting method was examined. Juvenile flounders without darkened areas on the blind side [total length (TL) 13 cm] were selected, and reared for 6 months (TL 32 cm). In the control tank without a net, the median value of darkened area ratio (darkened area to blind side area) was 46%. By only covering the tank bottom with slack net, the darkened area ratio was suppressed to 8%, less than 1/5 of that in the control tank. At the end of experiment, bottom coverage ratios of 0%, 10%, 30%, 50%, and 100% revealed a negative correlation between bottom net coverage and darkened area ratio. In this experiment, the darkening area in the tank with 50% bottom net coverage decreased to 1/3 of the control. Although the occurrence of hypermelanosis differs depending on the production lot, these results are expected to serve as a reference for selecting the suitable net size to meet the level of clearness of the blind side.

In artificially reared flatfish, especially the Japanese flounder Paralichthys olivaceus, pigmented skin (hypermelanosis) frequently appears on the fish’s blind side after normal metamorphosis. As no practical prevention method has yet been proposed, we examined the effectiveness of a loose net placed inside the rearing tank that covers the bottom and walls like a pouch. When juveniles (standard length [SL] 6 cm) were transferred to the net-lined tank (mesh size 4 mm) before the first appearance of hypermelanosis, the pigmented area after 2 months covered about 0.5% of the blind side; this is about 1/40th of the area covered by pigment in fish reared in an ordinary tank (20%). Although the initial appearance of pigmentation in the axilla area (the area covered by the pectoral fin) was not suppressed, utilization of a larger mesh size (12 mm) decreased the expansion of pigmentation in this area. Juveniles reared in the net-lined tank were about 5–15% smaller (SL) than those reared in the ordinary tank, but their body depth:SL ratio was closer to that of wild-caught juveniles. From the results of this study, we propose that net-lined rearing tanks with larger-sized mesh are a practical method of preventing hypermelanosis in Japanese flounder aquaculture systems.

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.

Body coloration in flatfish is one of the most distinctive asymmetries in the animal kingdom, although the fundamental molecular mechanism of the pigmentation is unclear. In the dorso-ventral coloration (countershading) of other teleost fishes, ventral-specific expression of agouti signaling protein 1 (ASIP1), an endogenous antagonist of melanocortin 1 receptor (MC1R), has been reported to play a pivotal role. Contribution of ASIP1 is also suggested in the asymmetrical pigmentation of flatfish. In order to confirm the contribution of ASIP1 and further examine receptor function in the body coloration of Japanese flounder, expression levels of asip1, mc1r, melanocortin 5 receptor (mc5r), and melanin-concentrating hormone receptor 2 (mchr2) were measured in the normally pigmented area of the left side, the normally non-pigmented area of the right side, and the abnormally pigmented (exhibiting hypermelanosis) area of the right side. Measurement was also carried out under conditions of hypermelanosis stimulated by cortisol and during the transition from non-pigmentation to pigmentation in areas of hypermelanosis. Contrary to our expectations, no difference was detected in asip1 expression between pigmented and non-pigmented areas. There was also no difference between normal and hormonally stimulated pigmented conditions in areas of hypermelanosis or during the transition process. Instead, the expression levels of mc1r, mc5r, and mchr2 were consistently higher in pigmented areas, and were especially increased under hormonally stimulated conditions. In addition, expressions of these receptor genes increased prior to pigmentation in areas of future hypermelanosis. Our results suggest that MC1Rand MC5R, but not necessarily ASIP1, contribute to pigmentation and hypermelanosis in Japanese flounder. We propose a yet unknown molecular mechanism for asymmetrical pigmentation in flatfish that is distinct from that of countershading in other vertebrates.

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.

A correction to this paper has been published: https://doi.org/10.1007/s12562-021-01504-x

After metamorphosis, both eyes of the Japanese flounder Paralichthys olivaceus are located on its left side, with only the ocular side becoming pigmented. Staining, or hypermelanosis, occurs on the blind side at 2–3 months after metamorphosis, thereby lowering the market price of the fish. To understand the pigmentation expansion process, we performed scale transplantation between the blind and ocular sides of an individual. About 40 % of transplanted scales were successfully engrafted, regardless of donor or recipient site. When blind-side scales were transplanted to the ocular side, they became pigmented after 2 weeks, while no change was observed when the scales were transplanted to the blind side. Ocular-side scales did not lose pigment, regardless of the recipient site. However, after removal of transplanted ocular-side scales, pigmented scales regenerated after 3 weeks, even at blind-side sites. Identical results were obtained when the stained area on the blind side was used as the recipient location. When an ocular-side scale with skin tissue was inserted under blind-side scales, the scales immediately above the transplanted area became pigmented, whereas ocular-side scales stripped of tissue did not induce pigmentation. These results strongly suggest the presence of an ocular-side characteristic inducer in pigmented scale tissues.

We previously reported that the progression of staining-type hypermelanosis spontaneously ceased at a specific time and area in Japanese flounder Paralichthys olivaceus. To examine whether time is a limiting factor in the spontaneous cessation of staining, we experimentally controlled the initiation and duration of staining by manipulating the bottom substrate condition in the fish tanks. At 151 days post hatching (DPH; 11 weeks), spontaneous cessation of staining was observed in fish reared in tanks without a sandy substrate. However, staining resumed (or was initiated) in tanks where sand was removed from 11 weeks, indicating a strong but temporary effect of bottom sand and the absence of time limitation in the staining progression by 151 DPH. Extended duration of the inhibitory period of hypermelanosis expansion (9 weeks or more) aided in only a 20 % reduction of the final staining area because of the increased rate of staining expansion. The bottom sandy substrate decreased the visibility of the staining area in individuals, but this was observed only before the completion of the staining expansion. These findings are discussed in relation to possible presence of area limitation of future staining, as well as the fundamental nature of staining.

Barfin flounder larvae exhibit unique black coloration, as well as left-right asymmetry in juvenile stage as in other flatfish. In this study, we first assessed the changes in melanophores with development and then investigated their responsiveness to melanin-concentrating hormone (MCH) during metamorphosis. Larval-type melanophores appeared on both sides of the body before metamorphosis, whereas adult-type melanophores appeared only on the ocular side after metamorphosis. Even in the individuals of this species displaying black coloration, the density of larval-type melanophores was similar to that in transparent larvae of other species. However, unlike in transparent larvae, larval-type melanophores completely dispersed in the black larvae of this species. Therefore, the black coloration during larval stages was mainly due to dispersion, and not the density, of larval-type melanophores. In vitro MCH treatment revealed, for the first time, the responsiveness of melanophores in larval stages. On the ocular side, larval-type melanophores aggregated against MCH during larval stages, while, in the larvae at later metamorphic stages and in juveniles, larval-type melanophores did not aggregate, although aggregation of adult-type melanophores was noted. In contrast, on the blind side, the responsiveness of larval-type melanophores to MCH was consistently present from larval to juvenile stages. The metamorphic transition of MCH responsiveness from larval- to adult-type melanophores only on the ocular side suggests the larval (therefore, immature) nature of the blind side skin. We propose that the inhibited development, and thus the retention of the larval-type skin leads to the formation of the blind side characteristics and is the central mechanism for the flatfish asymmetry.

Abnormal flounder coloration frequently occurs in flounder hatcheries and diminishes the commercial value of the fish. To understand hypermelanosis, the progression of staining-type hypermelanosis in normally metamorphosed juveniles and ocular-side pigmentation in pseudoalbino juveniles were examined in the Japanese flounder Paralichthys olivaceus. Sixty-five days post hatching, juveniles (total length, 6 cm) were individually identified by color-marker implantation, and the darkened area of the body surface was examined for 10 weeks by image analysis of digital photographs of the fish taken from above or below the transparent tank. Staining was observed to mainly begin at the upper and lower bases of the tail fin, expanded anteriorly along the peripheral part of the trunk, and ceased after 2 months. The individuals in which staining occurred earlier expressed severe staining and small body size by the end of the experiment. Further, pigmentation of the ocular side in pseudoalbino juveniles ceased after 2 months, but the order of pigmentation was different from that on the blind side. In this case, darkening began from the posterior, but expanded from the center to the periphery of the trunk. Even at the end of the experiment, ctenoid scales were exclusively found within the darkened area, together with cycloid scales.

Staining-type hypermelanosis, defined as blind-side melanosis occurring after completion of metamorphosis, reduces commercial value in hatchery-produced flatfishes. Detailed characterization was performed on the stained area of juvenile Japanese flounder Paralichthys olivaceus to physiologically understand this phenomenon. From 80 to 120 days after hatching, juveniles were reared in sandy and sandless tanks. By classifying the staining degree into 7 levels, about 2 times higher occurrence of middle-level staining was reconfirmed in sandless tank (about 80 %) than in sandy tank (about 40 %). In the stained area, we found 3 types of chromatophores (melanophore, xanthophore, and iridophore) and ctenoid scales, which would be typically observed on the normal ocular side. Detailed examination on the melanophores revealed further similarity between the stained area and the normal ocular side, in terms of the distribution at 2 layers (shallower and deeper than scale), and the densities in both layers (about 1000 cells/mm2 above scale and 200 cells/mm2 beneath scale). These results strongly suggest that the staining is a status change in the body surface conditions from the blind side to that on the ocular side, and not a simple darkening caused by disordered proliferation of melanophores on the blind side.

Our previous studies showed that in barfin flounder, α-melanocyte-stimulating hormone (α-MSH) stimulates pigment dispersion in xanthophores, while it shows negligible effects in melanophores. The present study was undertaken to evaluate whether these results are limited to barfin flounder by using Japanese flounder. Three subtypes of proopiomelanocortin gene encoding melanocortins (MCs) were expressed in the Japanese flounder pituitary, one of which was also expressed in the skin. Expression of melanocortin 5 receptor gene (Mc5r) was observed in isolated xanthophores, while that of Mc1r and Mc5r was found in melanophores. In the xanthophores of Japanese flounder skin, α-MSH as well as desacetyl (Des-Ac)-α-MSH and diacetyl (Di-Ac)-α-MSH exhibited dose-dependent pigment-dispersing activities, indicating that the signals of α-MSH-related peptides were mediated by MC5R. On the other hand, α-MSH did not stimulate pigment dispersion in melanophores, while Des-Ac-α-MSH and Di-Ac-α-MSH did, thus indicating that the expression of two different types of Mcr is related to the decrease in α-MSH activity. Thus, the molecular repertoire in MC system observed in Japanese flounder is similar to that in barfin flounder. Moreover, the relationship between the pigment-dispersing activities of α-MSH-related peptides and the expression of Mcr subtypes in xanthophores and melanophores were also similar between Japanese flounder and barfin flounder. Consequently, we hypothesize that inhibition of α-MSH activity could be due to the formation of heterodimers comprising MC1R and MC5R, often observed in G-protein-coupled receptors.

Morphological abnormalities in eye location and/or body coloration are commonly observed in hatchery-reared juveniles of many pleuronectid species, and have become one of the most serious problems in juvenile production for stock enhancement. In this study, these morphological abnormalities of six pleuronectid species—barfin flounder Verasper moseri, slime flounder Microstomus achne, stone flounder Platichthys bicoloratus, starry flounder Pl. stellatus, cresthead flounder Pseudopleuronectes schrenki, and marbled sole Ps. yokohamae—were successfully classified into four morphological types [normal, two types of pseudoalbinism (normal-eye position or top-eye position), and ambicoloration], following the same classification scheme proposed for other two pleuronectids. Based on our results, the characteristics of each morphological type are quite similar among species. It is confirmed that the normal type has the same ocular side and blind side characteristics as those of wild fish, not only in eye position and body coloration, but also in scales or dentition. The pseudoalbino types have the blind side characteristics of wild fish on both sides, with top-eye type in all characteristics, and normal-eye type in all other characteristics other than eye position. The ambicoloration type has all the ocular side characteristics of wild fish on both sides. Therefore, the pseudoalbinism and ambicoloration of hatchery-reared juveniles of pleuronectid species can be considered malformations of asymmetrical formation. Since this process normally occurs during metamorphosis, the term “metamorphosis-related malformation” is proposed for the abnormal formation of juveniles in pleuronectid species.

Cortisol, the main glucocorticoid in fish, increases during flatfish metamorphosis and peaks before the surge of thyroxine. A large body of evidence indicates the essential role of thyroxine in flatfish metamorphosis, whereas information on cortisol is limited. We administered cortisol to spotted halibut Verasper variegatus larvae in order to examine the effect on pigmentation during metamorphosis. Administration of 10 μg cortisol per mL of water from before the onset of metamorphosis (stage E) to metamorphic climax (stage G) induced the development of adult type pigment cells on the blind side of the metamorphosed juveniles and increased the occurrence of ambicolored juveniles. When 10 μg/mL cortisol was administered during stage D, stages E-F, stage G or stage H, only the administration during stages E-F induced the development of adult type pigment cells on the blind side. In addition, the expression of the gene dopachrome tautomerase (dct), a marker of melanoblasts, was enhanced at Stage E by cortisol administration. These results clearly indicated, for the first time, the enhancement of pigmentation by exogenous high-dose cortisol. Since endogenous cortisol is secreted in response to various kinds of stress in rearing conditions, these results indicate a possible influence of stress conditions in the occurrence of ambicoloration in flatfish.

The mechanism underlying malformation during flatfish metamorphosis is not clear. Here, the relationship between growth before metamorphosis and the occurrence of malformation after metamorphosis was examined in individuals reared in a single tank. Otoliths of starry flounder Platichthys stellatus larvae were labeled with alizarin complexone before metamorphosis [12 days after hatching (DAH)]. The positive linear regression between the lapillus otolith diameter and body length at 12 DAH enabled the estimation of individual larval size after completion of metamorphosis. At 12 DAH, the average body length of the typical pseudoalbino juveniles was significantly greater (5.37 mm) than that of the normal juveniles (5.13 mm) (P < 0.01, n = 100). In addition, of the larvae that were estimated to measure less than 4.8 mm at 12 DAH, approximately 80% metamorphosed into normal juveniles, whereas more than 60% of the larvae that measured more than 5.4 mm became pseudoalbino juveniles. These results indicate that the larvae that grow rapidly during the first 12 days are more likely to become pseudoalbino individuals after metamorphosis.

This study examined the ontogenetic change in vertical distribution associated with algae and the thyroid hormone profiles of black rockfish (Sebastes schlegelii) under rearing conditions. Experimental observations on distribution were carried on black rockfish from the newly born larval stage to juveniles 75 days after birth (DAB). Position was categorized by vertical location [upper layer, mid layer and lower layer (LL)] and by association with artificial algae (floating algae, bottom algae and no association). Position of fish was observed four times a day, at 0400 (dawn), 1200 (noon), 1800 (dusk) and 2400 (midnight) hours. The majority of fish (>80%) reached transforming larval stage [17.8 ± 0.7 mm total length (TL)] at 25 DAB and juvenile stage (24.3 ± 0.6 mm TL) at 35 DAB. Newly born larvae were mainly distributed at surface and middle layers, the shift of vertical distribution to the LL occurred between 10 and 25 DAB, and over 98% of the individuals were distributed near the bottom after 35 DAB. Association behavior with algae was first observed from 20 to 30 DAB depending on the time of day. Percentage of fish associated with bottom algae increased from 25 to 40 DAB and reached 50–70% thereafter. Thyroid hormone (T4) showed two surges at 30 and 50 DAB. The first surge corresponded to the transforming larval stage and the occurrence of association behavior with algae. The results indicate that pelagic larvae settle to the bottom layer before metamorphosis, and association behavior with algae appears at the transforming larval stage. A high T4 concentration at 30 DAB indicates a close relationship with these morphological and behavioral developments.

Flatfishes display a left–right asymmetry that is unique in the animal kingdom. In order to clarify the mechanisms of the asymmetrical development of pigment cells, changes in pigment cell densities were examined in Japanese flounder Paralichthys olivaceus. During development from symmetrical larvae to asymmetrical juveniles, pigment cell densities were monitored on the skin on both the left side (ocular side in juvenile; eventually has two eyes) and the right side (blind side in juvenile; eventually has no eyes). A symmetrical and constant decrease was observed in leucophores and larval type melanophores. A mostly symmetrical (slightly delayed on the blind side) and constant increase in iridophores from metamorphosis was observed. Adult-type melanophores appeared and then increased only after metamorphosis on the ocular side. However, the pattern of xanthophores was complicated: they first existed symmetrically and decreased symmetrically until metamorphosis, and they later increased only on the ocular side. The dual appearance of the xanthophores, as well as the differences between their depths and sizes on the ocular and blind sides, may suggest the presence of two types of xanthophores—just as melanophores are well known to exhibit two types. The ontogenetic study of pigment cells described here is likely to help to elucidate the process of abnormal pigmentation in flatfishes.

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.

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.

In order to study the physiological characteristics of neoteny in teleosts, the development of the thyroid axis and digestive tract of the ice fish, Salangichthys microdon (Salmonoidei), were examined using wild caught samples in the Yura River and its estuary. In all developmental stages examined in this study, the digestive tract was straight, and no gastric gland was observed, even in adults. Since these features are only observed in larval stages of a related species, ayu, Plecoglossus altivelis (Salmonoidei), it is suggested that the ice fish retains larval features, not only in appearance but also in internal organs. Additionally, the thyroid axis exhibited a unique characteristic. Until the end of the juvenile stage, thyroid glands remained inactive, and no thyroid stimulating hormone (TSH) cells were observed. Activation of the thyroid gland and the first appearance of TSH cells occurred in adults. Since activation of the thyroid axis was generally known to occur by the time larval have transformed to juveniles in several teleosts, a delayed functioning of the thyroid axis was suggested as a specific characteristic, and probably a major factor contributing to neotenic development of the ice fish.

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.

水温がホシガレイ仔魚の発育と変態に関連した形態異常の出現に及ぼす影響を検討するため, 9℃～21℃の5段階の水温で飼育実験を行った。変態完了直後の稚魚の形態を眼位と左右の体色から正常, 白化, 両面有色魚の3タイプに区分した。正常魚は18℃区, 白化魚は12℃区, 両面有色魚は21℃区において最も高い出現率を示した。仔魚期における変態後の左右の形質が水温によって大きな影響を受けることから, 変態期の形態的左右分化は発育・成長や変態のタイミングと密接に関わる可能性が高いと判断された。

Morphological changes are described in Japanese flounder Paralichthys olivaceus larvae and juveniles with emphasis on growth during the period of metamorphosis. Ontogenetic changes in condition factor and lipid, glycogen and protein levels were also analysed to determine the utilization of stored energy. Fish grew from 6.6 to 20.3 mm L T (1.15 to 84.4mg in mass, M) during the period from 11 to 40 days after hatching (DAH) at 19.3°C. Per cent specific growth rate per day (% G day -1 ) for wet mass was lowest during the metamorphic phase (21-30 DAH) compared to pre- (11 -20 DAH) and post-metamorphic (31 40 DAH) phases. When L T and M were expressed as M = a L b T , the b value was highest during the pre-metamorphic phase and lowest during the metamorphic phase. These findings indicate that the developmental changes that occur during metamorphosis of Japanese flounder are closely related to the growth pattern. Moisture, lipid and glycogen contents were also at the lowest level during metamorphosis compared to pre- and post-metamorphosis, which suggest that Japanese flounder use their energy reserves to accomplish metamorphosis due to an apparent decline in feeding during this period.

The internal structure of malformed Japanese flounder Paralichthys olivaceus juveniles in eye location was histologically examined and deformed bones were identified as the tissue important for eye relocation. A deformed pseudomesial bar (Pb) was the common feature for individuals with abnormal eye location (AEL individuals). Individuals with mild AEL had undeveloped Pb and those with serious AEL had no Pb or two Pb on both sides. When the Pb was absent, left–right asymmetry of the other hard tissues in the head region was disordered. For these reasons, Pb was considered important for eye relocation, and the possibility to define the left–right asymmetry of other tissues was suggested. The skin beneath the right eye and just outside the Pb is thickened in all normal individuals, while this skin was not thickened in most Pb-absent individuals, suggesting the possibility of the presence of upper control of Pb formation by the skin.

In order to clarify the mechanisms of neoteny in the ice goby (Leucopsarion petersii), we examined effects of thyroid hormone and thiourea (TU) treatments on their neotenic characteristics and the pituitary-thyroid axis. Adult ice goby were exposed to 3, 5, 3'-triiodothyronine (T3, 0.1 ppm), TU (inhibitor of thyroid hormone synthesis, 30 ppm), or the combination of the two for 2 weeks. Observations of whole body T3 levels, thyroid follicles and TSH immunoreactive cells in the pituitary suggests the presence of a functioning thyroidal system. However, all of the neotenic features did not disappear in T3-treated fish, suggesting the absence of T3 responsiveness in peripheral tissues. These results indicate the similarity between neoteny of the ice goby and obligatory-type neoteny of urodeles.

The body of a Japanese flounder (Paralichthys olivaceus) changes from a symmetrical to an asymmetrical form during metamorphosis. To obtain detailed information on the mechanisms of the migration of the right eye to the left side, soft and hard tissues in the head of larval flounders were examined using transmission electron microscopy (TEM). Retrorbital vesicles (Rvs) are pairs of sac-like structures under the eyes. It has been suggested that the asymmetrical development of Rvs, with the right (blind) one being bigger than the left, is the driving force behind eye migration. The present study revealed that the ultrastructure of the Rv sheath is quite similar to that of a lymphatic capillary. Thus, it is possible that the Rv is a part of the lymph system, and is probably related to the secondary vascular system in teleosts. If we assume that the Rv sheath has a high permeability to liquid, similar to lymphatic capillaries, it is not plausible that the active expansion of the Rv pushes the eyeball. On the other hand, the pseudomesial bar (Pb) is a bone that is unique to flounders and is present only on the right (blind) side. At the beginning of eye migration, an aggregation of fibroblast-like cells is observed in the dermis under the right eye, where the Pb will subsequently be formed. These cells have a well-developed rough endoplasmic reticulum (rER) and mitochondria, and are probably responsible for formation of the thick layers of collagen fibrils around them. Since it is unlikely that the active expansion of the Rv causes eye migration, the role played by the Pb and its rudiment becomes more significant in right eye migration in the Japanese flounder becomes more significant.

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.

Striped bonito Sarda orientalis larvae and juveniles were reared in a laboratory from hatching to 14 days post hatching, and the development of their digestive system was investigated histologically. On day 2 (first feeding day), the larval-type digestive system was differentiated. On day 3, a blind sac with densely distributed gastric glands developed and pyloric ceca appeared, suggesting that the digestive system was attaining adult-type structure. The yolk was completely absorbed on day 4. Bonito larvae preyed on rotifers and/or Artemia nauplii on day 2, but almost the whole gut was occupied by fish larvae from day 4. In the striped bonito, the adult-type digestive system was established right after first feeding, and this precocious development was well synchronized with the appearance of piscivory.

The appearance and distribution of cholecystokinin (CCK)-immunoreactive (IR) cells in the digestive tract of cultured developing larvae of bluefin tuna, Thunnus thynnus , were investigated by immunohistochemistry. The CCK-IR cells had an elongated triangular shape and a thin apex pointing towards the lumen of the gut. Very few weakly stained CCK-IR cells were found at hatching (0 days after hatching, DAH) and first feeding (3 DAH), while strongly stained CCK-IR cells were detected in all the larvae examined after 12 DAH. CCK-IR cells were scattered both in the pyloric caeca and the anterior intestine at 12 DAH, while the distribution was restricted to the pyloric caeca at 22 DAH. The relationships between the ontogeny of CCK and pancreatic enzymes are discussed.

The contribution of the thyroid hormone to the metamorphosis of the coral trout Plectropomus leopardus was examined. Juveniles, aged 35 days, were exposed to thyroxine (T4, 0.1 p.p.m) and thiourea (TU; inhibitor of thyroid hormone synthesis, 30 p.p.m) for 13 days. All the fish in the T4-treated group had completed metamorphosis (second metamorphosis, pigmentation and resorption of fin spines) within three days after the beginning of the experiment (day 3), whereas it took 13 days for this to occur in the control fish. Moreover, the fish in the TU group had not completed metamorphosis even by day 13. Settling behavior was significantly stimulated by T4 treatment, as was the resorption of the dorsal and pelvic fin spines, the reduction of serration on the spines, and the transition of body color into an opaque reddish hue. Fish in the TU group had longer dorsal and pelvic spines, retarded pigmentation and an abnormal black coloration. These results suggest that thyroid hormone accelerates most changes during the second metamorphosis in the coral trout.

In 1999, spotted halibut Verasper variegatus were experimentally reared at 9, 12, 15, 18, and 21°C from eggs through early juveniles at the Miyako Station of Japan Sea-Farming Association in Iwate Prefecture. Tissue concentrations of thyroxine (T4) increased to maximum levels during metamorphic climax (H-stage) at all the temperatures. The peak T4 levels were higher at lower temperatures, whereas no relationship was observed between tissue 3,5,3′-triiodothyronine levels and rearing temperatures. However, epithelial cell height of thyroid follicles and the number of vacuoles in colloid were not different among rearing temperatures. Although the peak T4 levels were different, thyroid activity to produce T4 might not be so different at all temperatures. Therefore, much T4 observed at low temperatures was possibly due to the accumulation of the unused hormone in blood and tissue.

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.

The appearance and distribution of cholecystokinin (CCK)-producing cells were investigated in the digestive tract of developing larvae of cultured Atlantic halibut, Hippoglossus hippoglossus. The CCK-producing cells were detected immunohistochemically, by use of a primary antiserum against CCK cloned for the Japanese flounder, Paralichthys olivaceus. No CCK-immunoreactive (IR) cells were detected in first-feeding larvae (33 days after hatching, DAH). Forty-five DAH or 12 days after first feeding, there were a few scattered CCK-IR cells in the epithelium of the anterior midgut in about 30% of the examined larvae. All larvae older than 52 DAH had CCK-IR cells in the anterior midgut, particularly frequent in the most anterior region adjacent to the pyloric caeca. No CCK-IR cells were detected in the foregut, the hindgut, or the midgut posterior to the first curvature. The CCK-IR cells spanned the intestinal epithelium from the basal lamina to the lumen and were triangular in shape, with the nucleus in the basal part and a thin apex toward the lumen. The mechanisms controlling release of bile, pancreatic enzymes, and peristalsis during the 12 days between first feeding and the first detection of CCK-IR cells remain to be clarified.

The patterns of entry of thyroid hormones into live tilapia oocytes were examined by incubating ovarian follicles in L-15 medium containing 125 I-labeled thyroxine (T4) or 3,5,3′-triiodothyronine (T3). As judged from HPLC profiles, radioactivity in extracts of follicles immersed in T3 was identified to reside in T3, while most of the radioactivity in the extract of T4 immersed follicle was not associated with T4. Radioactivity of T3 immersed follicles reached a constant level after 18 h of incubation. Entry of T3 into the oocytes was non-saturable within the range of 0.5–5000 ng/ml of T3 in the incubation medium, suggesting the absence of specific mechanisms for T3 entry into the oocyte. Presence of female plasma at a level of 20% of incubation medium inhibited the T3 entry into the oocytes by approximately 80%. When follicles were back-transferred to medium without T3, only 15% of T3 in the oocyte disappeared within the following 24 h. From our results, we conclude that free T3 seems to enter oocytes freely across the membranes by diffusion, and that T3 in the oocytes may bind to some molecules in the oocyte. However, during egg formation in vivo, contribution of free T3 entry into the oocytes did not seem to be significant when considering the free T3 ratio in female plasma.

ホシガレイの仔稚魚期における消化系の発達および変態関連ホルモンの動態を調べた。消化系は開口時と変態開始期に大きく変化した。開口時には腸管が回転し, 胃・腸・直腸が区分された。着底が観察された変態開始期には胃腺と幽門垂が分化した。腸上皮細胞の高さは摂餌開始から増加し, 変態中期からは停滞した。甲状腺ホルモンとコルチゾルは卵中にも存在したが, ふ化数日後には低い値に減少した。コルチゾルは変態期直前にピークを示したのに対し, 甲状腺ホルモンのT4は変態最盛期にピークを示した。一方, T3は仔魚期を通じて低い値で推移した。

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.

Three experiments were carried out to clarify the effect of thyroid hormones on the pigmentation of larval Japanese flounder Paralichthys olivaceus. The first two experiments were conducted to investigate the critical concentration of thyroxine (T-4) which causes abnormal pigmentation, and the third was to determine the sensitive stage of larval development at which albinism is induced by exogenous T-4. The larvae treated with over 10 nM T-4 showed a significant increase of albinism in Experiments 1 (concentration: 0, 1, 10, 50 nM) and Experiment 2 (concentration: 0, 1, 5, 10 nM). in Experiment 3, seven groups were immersed in 10 nM T-4 at specific stages: between A and B, C and D, E and F, G and I, C and F and A and F. The groups of hormonal treatment at stages E and F (prometamorphosis) produced more than 90% albinism, indicating that differentiation of adult-type melanophores took place at this point. Another 4 groups of larvae were separately treated with levels of 10 nM T-4 at stages A and B, C and D, E and F and G to I. T-4 content of larvae increased corresponding to the developmental stage 180 ng/g-wet weight in A and B, 740 in C and D, and 1350 in E and F, but decreased to 130 in stage G to I (n=3, p

We tested the hypothesis that metabolism of thyroid hormones by the brain of Atlantic salmon (Salmo salar) changes when rheotactic behavior reverses during parr-smolt transformation (PST). We measured brain and liver thyroxine (T4) and 3,5,3'-triiodo-L-thyronine (T3) outer-ring deiodination (ORD) and inner-ring deiodination (IRD) activities and plasma T4and T3levels in Atlantic salmon held under natural photoperiod in fresh water at 10°C in the spring of 1993 and 1994. We also measured changes in T4, T3, and cortisol levels during the change in rheotactic behavior. Condition factor decreased while salinity tolerance improved from mid-March to late April. The turbidity-induced transition from upstream to downstream swimming occurred in mid to late April. The main changes in brain deiodination were reduced T3IRD (1993 study) and elevated T4ORD (1994 study). In both years, a high ratio of T4ORD/T3IRD activities in the brain indicated an increased potential for T3production in the brain during advanced PST. Liver deiodination profiles differed between years, but during advanced PST the low T4ORD activity and low T4ORD/T3IRD activity ratio suggested a low potential for hepatic, and hence systemic, T3production. However, plasma T4was increased in downstream swimmers at 1 d (1993) and 4 h (1994) after the turbidity increase. Since at this time brain deiodination pathways were poised towards T3production, the surge in plasma T4would likely increase local T3formation in brain. We conclude that during PST there is no major change in hepatic deiodination and hence probably no major change in systemic T3availability. But deiodination properties in brain during late PST indicate the potential for local T3formation. This may be significant when plasma T4increases at the time of downstream migration.

The entry and metabolism of 3H-cortisol in oocytes were investigated using isolated follicles of the tilapia (Oreochromis mossambicus) in order to examine the mechanisms of incorporation of maternal hormones into oocytes. The composition of 3H-labeled steroids in the oocyte was analyzed by high-performance liquid chromatography. A significant amount of cortisol was converted to cortisone and an unidentified molecule by the follicular layer. The contents of 3H-cortisol and 3H-cortisone in the oocyte reached an equilibrium level within 12 hr, whereas the content of the unidentified metabolite continued to increase for 36 hr. The total content of the incorporated cortisol and its metabolites was proportional to cortisol in the medium over the concentration range of 5 ng/ml to 5 microg/ml. The amounts of cortisone and the unidentified molecule increased proportionally when the concentration of cortisol in the medium was lower than 500 ng/ml, whereas they reached a plateau when the concentration of cortisol exceeded 500 ng/ml. Cortisol entry was reversible, because 90% of cortisol and cortisone in the oocyte was lost within 18 hr when the medium was changed to that without 3H-cortisol. On the other hand, 50% of the unidentified molecule was preserved at the end of the incubation. In conclusion, the entry of cortisol into the oocyte was considered to be nonspecific and due probably to simple diffusion. However, a considerable amount of cortisol (50-70%) was specifically converted to cortisone and another unidentified molecule during passage through the follicular layer.

Distributional changes in branchial chloride cells were examined in Japanese sea bass (Lateolabrax japonicus) juveniles transferred from seawater (SW) to fresh water (FW) during their migration season toward low salinity habitat in nature. Chloride cells were identified by immunocytochemistry with a specific antiserum for Na+,K+-ATPase. In fish reared in SW as controls, branchial chloride cells were localized exclusively in the filaments and absent in the lamellae. When sea bass were transferred from SW to FW, chloride cells emerged in gill lamellae, starting at the proximal part of the lamellae and thereafter spread over the lamellar epithelium. On 7th and 15th days after FW transfer, chloride cells were mostly found on the lamellae, whereas the number of filament chloride cells was decreased. These results suggest that, in Japanese sea bass juveniles, chloride cells in the gill lamellae are important in FW adaptation, and that lamellar chloride cells originated from the filaments and migrated to the lamellae during FW adaptation.

The metabolic aspects of ionic and osmotic regulation in fish are not well understood. The objective of this study was to examine changes in carbohydrate metabolism during seawater (SW) acclimation in the euryhaline tilapia (Oreochromis mossambicus). Hepatic activities of three key enzymes of the intermediary metabolism, phosphofructokinase, glycogen phosphorylase and glucose 6-phosphate dehydrogenase, together with glycogen content and plasma glucose concentration were measured at 0, 0.5, 1, 2, 3, 6, 12, 24, 48 and 96 h after the direct transfer of tilapia from fresh water (FW) to 70% SW. Plasma growth hormone, prolactin177 and prolactin188, Na+ and Cl− concentrations were also measured. Plasma Na+ and Cl− levels were highest at 12 h, but returned to FW levels at 24 h after transfer, suggesting the tilapia were able to osmoregulate within 24 h after transfer. Plasma glucose levels were significantly higher in 70% SW than in FW during the course of acclimation, especially in the early stages. Hepatic enzyme activities and glycogen content did not change significantly during the acclimation period. Our results suggest the possibility that glucose is an important energy source for osmoregulation during the acclimation to hyperosmotic environments in O. mossambicus.

Ontogenetic changes in whole-body immunoreactive cortisol concentrations (IRC) and aggressive behavior were examined in yellowtailSeriola quinqueradiata(Temminck & Schlegel; Carangidae). Baseline IRC significantly increased during the transition from larval to juvenile stage and was correlated with the onset of aggressive behavior. Handled fish (13.1 ± 2.6 ng/g tissue) showed an IRC level about three times higher than unhandled fish (4.7 ± 1.4 ng/g tissue), indicating that whole-body immunoreactive cortisol level may be an indicator of stress in juvenile yellowtails. Behaviorally subordinate fish (8.6 ± 1.6 ng/g tissue,n = 4) showed IRC levels significantly higher than dominant fish (0.6 ± 0.3 ng/g tissue, n = 4). Whole-body immunoreactive cortisol levels may thus reflect stress and social status in juvenile yellowtails, and the inverse relationship between social rank and IRC may result from agonistic interactions., General and Comparative Endocrinology, 109(2), pp.286-292; 1998

In Japanese flounder (Paralichthys olivaceus), metamorphic period involves not only transformation from larva to juvenile but also migration from offshore areas to estuaries. In the present study, the role of endocrine systems in low-salinity adaptation was examined during early development and metamorphosis of the flounder. Survival rate 48 hr after transfer to 1/8 SW was relatively high in yolk-sac larvae, decreased gradually to 0% at premetamorphosis, and increased to 100% at metamorphic climax. The ratio of prolactin (PRL)-immmunoreactive part to whole pituitary increased gradually during larval stages and reached a constant level during metamorphosis. When the larvae at premetamorphosis and metamorphic climax and the benthic juveniles were transferred from SW to 1/4 SW, PRL-immunoreactive part increased significantly 48 hr after the transfer at all stages examined. Whole-body concentration of cortisol was measured with a modified extraction method which is much robuster to lipid-rich sample than the ordinary method, but no significant difference was observed after the transfer. Whole-body concentrations of thyroid hormones decreased slightly but significantly at premetamorphosis and metamorphic climax. These results suggest possible involvement of PRL and thyroid hormones in low-salinity adaptation of the flounder during metamorphosis and inshore migration.

Cortisol is one of the central hormones in osmoregulation in fish, especially in seawater adaptation. A cDNA of 453 bp was cloned from liver mRNA of freshwater-reared tilapia (Oreochromis mossambicus), by reverse transcription polymerase chain reaction (RT-PCR) with primers designed for the hormone-binding domain of glucocorticoid receptors (GRs) in mammals and rainbow trout. The sequence of PCR product has 83% homology to the trout GR at the nucleotide level and 92% at the amino acid level. The PCR product of tilapia showed highest homology (74% at the amino acid level) to GR among human steroid hormone receptors, including mineralocorticoid receptor. The length of the receptor mRNA of tilapia was about 6.5 kb as determined by Northern blot hybridization. The mRNA concentration in the gills was relatively higher among various organs, the highest concentration being observed in blood cells. Signal intensity of the receptor message in the gills was stronger in fish reared in freshwater than in those reared in seawater or in concentrated (160%) seawater. During early development of tilapia, the highest concentration of receptor mRNA in the total RNA extracted from the whole egg was found just after fertilization, and its concentration decreased steadily toward hatching. The absolute amount of receptor mRNA per egg increased gradually before the initiation of cortisol production by the embryo. When embryos were transferred from fresh water to seawater 2 days before hatching, no difference was observed in the signal intensity of the receptor mRNA among embryos after 1, 2 (the day of hatching), 4, and 7 days.

Survival of the seed of goldstriped amberjack Seriola lalandi was consistently enhanced by intramuscular injection of triiodothyronine (T3) to the broodstock fish at a dose of 20 mg/kg body weight. Survival rates at the time of transfer to net cages (35 d after hatching) were 2.6–5.0% for the T3 treatment while only 0.3–2.1% for the control. Survival rates 36 mo after hatching was estimated to be 12 times higher in the T3-treated group (1.1%) than in the control (0.09%). T3 concentration in newly spawned eggs increased from control levels of 0.3–1.0 ng/g to 68–127 ng/g 2 d after injection and subsided with successive spawns. T3 concentration in the treated larvae was about 360 times higher than in the control upon hatching (29 np/p vs 0.08 ng/g), but became indistinguishable from the control 6 d after hatching. These results indicate injection of T3 to the broodstock of S. lalndi to be of practical value in aquaculture of the species.