Title Studies on the morphological abnormality of Pleuronectidae: focusing on the metamorphosis-related malformation in hatchery reared fish and its prevention
Authers Masato ARITAKI
Citation Bull. Fish. Res. Agen. No.37, 147-197, 2013
In Japan, pleuronectid species are important resources for coastal fisheries, and 12 species have been assigned for stock enhancement. However, juvenile production program of all pleuronectid species has encountered with frequent occurrence of morphological abnormalities such as abnormal pigmentation (pseudoalbinism on the ocular side and ambicoloration on the blind side), abnormal eye migration (incompletion or absence of eye migration, or both-eye migration to the top of head), and deficit and repetition of some parts of cranial bones, in relation to metamorphosis. The causes and mechanisms of these morphological abnormalities are totally not clarified other than some aspects in the Japanese flounder Paralichthys olivaceus and restricted pleuronectid species.
 Thus, objectives of this study are to obtain fundamental information concerning the occurrence of morphological abnormalities in hatchery reared pleuronectid species. In addition, by clarifying the causes and mechanisms, one possible way to prevent the abnormality is suggested.
 In chapterⅠ, in order to find a general trend of the morphological abnormalities, various asymmetrical characteristics of the juvenile body was examined in detail using eight pleuronectid species obtained from hatchery rearing.
 In chapterⅡ, the influence of feeding periods of Brazilian Artemia salina nauplii (BA) on the occurrence of pseudoalbinism was clarified in two pleuronectid species. The critical period for morphological abnormalities, and therefore for normal asymmetrical development, was determined from the occurrence of the pseudoalbinism.
 In chapterⅢ, three pleuronectid species, having differential growth and developmental speed, were reared at various temperatures. As the result, I found a clear relationship between the occurrence of morphological abnormalities and speed of larval growth and/or development specific for each species.
 In chapterⅣ, two pleuronectid species were experimentally reared at various temperatures and the tissue concentrations of thyroxine (T4) and 3, 5, 3′-triiodothyronine (T3) during metamorphosis were investigated. The result of this chapter suggested the relationship between thyroid hormone levels of metamorphosing larvae and occurrence of morphological abnormalities.
 Following the above mentioned research, I have discussed the possible mechanism and prevention method of morphological abnormalities in Pleuronectidae, and also described the subject for further studies in chapterⅤ.
Ⅰ. Morphological abnormality in hatchery-reared Pleuronectidae
 Morphological abnormalities are commonly observed in hatchery-reared juveniles of many pleuronectid species, at ratios much higher than those in wild fish. And therefore, the occurrence of abnormalities have become one of the most serious problems for juvenile production.
 In this chapter, the morphological abnormalities were examined on hatchery-reared juveniles of eight pleuronectid species (verfin flounder Verasper moseri, spotted halibut V. variegatus, slime flounder Microstomus achne, stone flounder Platichthys bicoloratus, starry flounder Pl. stellatus, brown sole Pseudopleuronectes herzensteini, cresthead flounder Ps. schrenki and marbled sole Ps. yokohamae), forcusing on asymmetrical characteristics (eye location, body color, upper jaw, pectoral fin, dentition, and scale) of each body side, comparing with the wild fish of each species.
 As the result, it was possible to classify all the juveniles into four morphological types (normal, two types of pseudoalbinism, and ambicoloration) in all 8 pleuronectid species examined. Normal fish had the same charactereristics as in wild fish for both body sides (left and right), after undergoing the asymmetrical metamorphosis. However, the morphological characteristics of both sides in pseudoalbino fish were similar to the blind side of the wild fish, and those in ambicolored fish was similar to the ocular side of the wild fish, suggesting the occurrence of symmetrical metamorphosis. Therefore morphological abnormalities of hatchery-reared juveniles in pleuronectid species were to be considered as “metamorphosisrelated malformation”.
Ⅱ. Critical timing for the metamorphosis-related malformation in Pleuronectidae
 In order to prevent metamorphosis-related malformation of pleuronectid juveniles, it is important to determine the critical developmental stage and/or timing for the malformation. In the Japanese flounder, it is known that BA feeding at a specific timing induces pseudoalbinism in juveniles at a ratio of 100%, and a close relationship was indicated between pseudoalbinism occurrence and the larval stage.
 In this chapter, the influence of timing of BA feeding was examined on the occurrence of pseudoalbinism in two pleuronectid species (spotted halibut and brown sole). BA was offered at six different feeing periods from early larval stage (first available to take artemia as diet) to juvenile stage.
 As the result, occurrence of pseudoalbinism was about 90-100% in the two pleuronectid species, when BA was given before flexion stage (E stage)(spotted halibut; 10mm TL, brown sole; 8mm TL) suggesting that expression (or non-expression) of pseudoalbinism have been determined before postflexion stage (F stage). Therefore, for the prevention of pseudoalbinism, and possibly other metamorphosis-related malformation too, the stages before postflexion is critically important in pleuronectid species.
Ⅲ. Relationship between the metamorphosis-related malformation and growth and/or development of pleuronectid larvae
 The Japanese flounder is a representative flatfish for juvenile production in Japan, and a body of information has been accumulated about the causes, mechanisms and prevention method of malformation. However, there is not enough information available for other flatfishes including pleuronectids. The objective of this chapter is to examine the effects of rearing temperatures on early larval development and occurrence of metamorphosis-related malformation in spotted halibut, slime flounder and brown sole.
 Newly hatched larvae were reared beyond the completion of metamorphosis at various temperatures (6-24°C). All juveniles were classified into four morphological categories as normal, pseudoalbinism (two types, regarding to normal or abnormal eye location) and ambicoloration, based on the body color (pigmentation) and eye location. The growth and developmental speed of the three species were relatively greater in brown sole, middle in spotted halibut and less in slime flounder, and accelerated concomitant with higher temperatures in all three species. The relationship between rearing temperatures and occurrence of the four morphological categories showed species specific tendencies consistent among the repeated experiments. The settlement ages that results in the highest ratio of normal juvenile were relatively young in brown sole, middle in spotted halibut and old in slime flounder. It is noticeable that these settlement ages were similar to those of the wild fish of each species.
 Results of the present study strongly suggest the importance to rear pleuronectid larvae at a similar developmental speed as in the wild fish, in order to let the larvae undergo normal metamorphosis, and produce maximum number of normal fish.
Ⅳ. Changes in tissue thyroid hormone levels in metamorphosing pleuronectid larvae reared at various temperatures
 In Japanese flounder and summer flounder Paralichthys dentatus, involvement of thyroid hormones in metamorphosis has been clarified. Because pseudoalbinism and ambicolorion of pleuronectid species were considered as “metamorphosis-related malformation”in chapterⅠ, it is speculated that thyroid hormones are also related to the occurrence of “metamorphosisrelated malformation”in pleuronectid species. In this chapter, I investigated the relationship between changes in tissue thyroid hormone levels and occurrence of normal, pseudoalbinism and ambicolortion by rearing the larvae of spotted halibut and blown sole at 9, 12, 15, 18, and 21℃.
 Tissue concentrations of T4 increased to maximum levels during metamorphic climax (stage G-H) at all the temperatures. No relationship was observed between tissue T3 levels and rearing temperatures. The peak T4 levels were significantly higher at lower temperatures, and appeared earlier when reared at higher temperatures. Since the occurrence of ambicolored fish and pseudoalbino fish was influenced by the rearing temperatures, it is possible that thyroid hormones are involved in the decision for normal formation or metamorphosis- related malformation of the juveniles in pleuronectid species, probably through the peak level or the timing of tissue T4 concentrations.
Ⅴ. The possible mechanism and prevention of metamorphosis-related malformation in Pleuronectidae
 In this sturdy, I have clarified that the morphological abnormality of Pleuronectidae was metamorphosis-related malformation, and that occurrence of the malformation was strongly influenced by the rearing temperature with specific patterns in each pleuronectid species. Furthermore, I have described that the occurrence of metamorphosis-related malformation was increased when the pleuronectid larvae were let to grow and/or develop at different speeds from wild fish. In addition, the extent and the timing of peak T4 levels were varied among the fish reared at different temperatures.
 These results strongly suggested the involvement of thyroid hormones, which controls metamorphosis of flounders, in the occurrence of metamorphosis-related malformation. Recently, I have obtained preliminary results in two pleuronectid species (spotted halibut and starry flounder) suggesting that the occurrence of metamorphosis-related malformation is directly decided by the timing of T4 increase. Therefore, the occurrence of normal juvenile and juveniles with metamorphosis-related malformation is probably decided by the growth and/or developmental speed of the larvae through the timing of T4 surge in pleuronectid species.
 As mentioned above, in order to prevent the occurrence of metamorphosis-related malformation, it is important to investigate the early life history of the target species in the sea, and to let the larvae grow and develop at a species specific speed similar to the wild fish.  It is necessary further to clarify the mechanism of metamorphosis-related malformation at a level of genetic embryology, in order to effectively develop the better method for reduced occurrence of the malformation.
URI http://www.fra.affrc.go.jp/bulletin/bull/bull37/37-04.pdf