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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.

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.

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.

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.

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.

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.

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 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.

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 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.