No.68 (March,2005)
No.68 (March,2005)
A－382 Nozomi OKADA
Development of tissues involved in eye migration and role of thyroid hormone in metamorphosing Japanese flounder (Paralichthys olivaceus)
1.1 Asymmetrical development of bones and soft tissues during eye migration of metamorphosing Japanese flounder
The symmetrical body of flatfish larvae dramatically changes into an asymmetrical one after the metamorphosis. Eye migration results in the most significant asymmetrical development seen in any vertebrate. To understand the mechanisms involved in eye migration, bone and cartilage formation was observed during metamorphosis in laboratory-reared Japanese flounder using whole body samples and histological sections. Most of the hard tissues of the cranium (parasphenoid, trabecular cartilage, supraorbital canal, and supraorbital bar) exist symmetrically in the larval period before metamorphosis, and develop twisting in the same direction as the eye migrates. Increase of skin thickness beneath the eye was observed only on the blind side at the beginning of eye migration; this is the first definitive difference between the right and left sides of the body. The pseudomesial bar (Pb), a peculiar bone present only in flatfishes, develops from this thick skin, and grows dorsad. Novel sac-like structures were found, and named retrorbital vesicles (Rvs). The Rv of the blind side grew larger and faster than that of the ocular side when the right eye moved most drastically, whereas no difference was observed between the volume of right and left connective tissue in the head. Asymmetrical presence and growth of the Pb together with inflation of the Rv on the blind side may be responsible for right eye migration during metamorphosis of Japanese flounder.
1.2 Fine structure of soft and hard tissues involved in eye migration in metamorphosing Japanese flounder
The body of a Japanese flounder 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). 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 I 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 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 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 in right eye migration in the Japanese flounder becomes more significant.
2.1 Histological study of deformity in eye location in Japanese flounder
The internal structure of malformed Japanese flounder juveniles in eye location was histologically examined and deformed bones were identified as the tissue important for eye relocation. A deformed 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.
2.2 Insufficient ability to synthesize thyroid hormone in abnormal juveniles during seed production of the Japanese flounder
In flatfish hatcheries, various types of deformities occur during metamorphosis, at a relatively high ratio in some species. Since thyroid hormones (THs) are known to be essential for metamorphosis, the thyroid system was examined in a type of abnormal fish accounting for 4% of juveniles of the Japanese flounder observed in this study. From comparisons of external and internal structures between normal and abnormal fish, stasis during the metamorphosis was suggested as a fundamental reason for the abnormality, and malformed thyroid glands were found in the abnormal fish. In addition, serum thyroxine (T4) concentrations in the abnormal fish were reduced to less than 1/10 of that of normal. In order to examine the responsiveness of the abnormal fish to TH, T4 (0.1 ppm) and thiourea (TU, antithyroidal agent, 30 ppm) were administered. After 14 days of T4 treatment, all the abnormal characteristics disappeared, and fish recovered to normal, suggesting normal responsiveness to THs in peripheral tissues. In combination with the observation of thyroid follicles in T4 or TU treated fish, an insufficient ability of TH synthesis was suggested, and retardation of metamorphosis was induced by lowered TH levels in the abnormal fish in this specific case.
3.1 Bone development during metamorphosis of the Japanese flounder: differential responses to thyroid hormone
The larvae of flatfish change their body structure during metamorphosis, including dramatic translocation of one of the eyes from one side of the body to the other. Such metamorphic processes are in general promoted by THs. This study focuses on the response of individual tissues to hormones, and morphological characteristics were examined in hormone-deficient larvae of the Japanese flounder. Treatment of flounder larvae with an inhibitor of TH synthesis, TU (30 ppm) inhibited translocation of the right eye, shortening of dorsal fin rays, and body pigmentation, as previously reported. Treatment also inhibited elongation of the Pb, which is important for eye translocation, and formation of bones related to larval or juvenile characteristics, the actinost and the distal radial of the pectoral fin, and the pterygiophore of the anal fin. Bones other than these, which are similarly present in both larvae and juveniles, were unaffected. These results suggest the differential responsiveness of bones to TH deficiency during early developmental stages, which seems to play a significant role in the morphological changes from larvae to juveniles. To examine the period of hormone responsiveness, T4 treatment (0.1 ppb for 2 weeks) was started at two different daily ages for the fish receiving TU treatment. When T4 was given 2 weeks later than TU, the fish completely metamorphosed through the expected bone formation that had been suppressed by TU. However, when T4 treatment was started 4 weeks later than TU, body pigmentation did not occur, and the translocation of the right eye was not complete due to failure of Pb formation. It is suggested that individual tissues that change during metamorphosis have their own timing in response to THs.
3.2 Effect of stage-specific thyroidal stasis on the eye migration of Japanese flounder
Since TH is the main stimulator of eye migration in flounder metamorphosis, the timing of TH responsiveness was determined in the tissue responsible for eye migration in Japanese flounder. TU (30 ppm, an inhibitor of TH synthesis) was administered starting at different stages of metamorphosis, and the inhibitory effects on the tissue related to eye migration were histologically examined. When TU treatment was started before the E stage, eye migration was significantly inhibited due to lack of formation of the Pb, a bone important for eye migration. When treatment started after the F stage, the process was not significantly inhibited due to the minor inhibitory effect on Pb formation. This result indicates a specific requirement by stage F for TH in the Pb. These results suggest that the thyroidal status during the early phase of metamorphosis is extremely crucial for the Pb formation and therefore important for the completion of eye relocation in Japanese flounder, although the process of eye migration is gradual and continuous.
1.1 Asymmetrical development of bones and soft tissues during eye migration of metamorphosing Japanese flounder
The symmetrical body of flatfish larvae dramatically changes into an asymmetrical one after the metamorphosis. Eye migration results in the most significant asymmetrical development seen in any vertebrate. To understand the mechanisms involved in eye migration, bone and cartilage formation was observed during metamorphosis in laboratory-reared Japanese flounder using whole body samples and histological sections. Most of the hard tissues of the cranium (parasphenoid, trabecular cartilage, supraorbital canal, and supraorbital bar) exist symmetrically in the larval period before metamorphosis, and develop twisting in the same direction as the eye migrates. Increase of skin thickness beneath the eye was observed only on the blind side at the beginning of eye migration; this is the first definitive difference between the right and left sides of the body. The pseudomesial bar (Pb), a peculiar bone present only in flatfishes, develops from this thick skin, and grows dorsad. Novel sac-like structures were found, and named retrorbital vesicles (Rvs). The Rv of the blind side grew larger and faster than that of the ocular side when the right eye moved most drastically, whereas no difference was observed between the volume of right and left connective tissue in the head. Asymmetrical presence and growth of the Pb together with inflation of the Rv on the blind side may be responsible for right eye migration during metamorphosis of Japanese flounder.
1.2 Fine structure of soft and hard tissues involved in eye migration in metamorphosing Japanese flounder
The body of a Japanese flounder 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). 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 I 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 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 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 in right eye migration in the Japanese flounder becomes more significant.
2.1 Histological study of deformity in eye location in Japanese flounder
The internal structure of malformed Japanese flounder juveniles in eye location was histologically examined and deformed bones were identified as the tissue important for eye relocation. A deformed 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.
2.2 Insufficient ability to synthesize thyroid hormone in abnormal juveniles during seed production of the Japanese flounder
In flatfish hatcheries, various types of deformities occur during metamorphosis, at a relatively high ratio in some species. Since thyroid hormones (THs) are known to be essential for metamorphosis, the thyroid system was examined in a type of abnormal fish accounting for 4% of juveniles of the Japanese flounder observed in this study. From comparisons of external and internal structures between normal and abnormal fish, stasis during the metamorphosis was suggested as a fundamental reason for the abnormality, and malformed thyroid glands were found in the abnormal fish. In addition, serum thyroxine (T4) concentrations in the abnormal fish were reduced to less than 1/10 of that of normal. In order to examine the responsiveness of the abnormal fish to TH, T4 (0.1 ppm) and thiourea (TU, antithyroidal agent, 30 ppm) were administered. After 14 days of T4 treatment, all the abnormal characteristics disappeared, and fish recovered to normal, suggesting normal responsiveness to THs in peripheral tissues. In combination with the observation of thyroid follicles in T4 or TU treated fish, an insufficient ability of TH synthesis was suggested, and retardation of metamorphosis was induced by lowered TH levels in the abnormal fish in this specific case.
3.1 Bone development during metamorphosis of the Japanese flounder: differential responses to thyroid hormone
The larvae of flatfish change their body structure during metamorphosis, including dramatic translocation of one of the eyes from one side of the body to the other. Such metamorphic processes are in general promoted by THs. This study focuses on the response of individual tissues to hormones, and morphological characteristics were examined in hormone-deficient larvae of the Japanese flounder. Treatment of flounder larvae with an inhibitor of TH synthesis, TU (30 ppm) inhibited translocation of the right eye, shortening of dorsal fin rays, and body pigmentation, as previously reported. Treatment also inhibited elongation of the Pb, which is important for eye translocation, and formation of bones related to larval or juvenile characteristics, the actinost and the distal radial of the pectoral fin, and the pterygiophore of the anal fin. Bones other than these, which are similarly present in both larvae and juveniles, were unaffected. These results suggest the differential responsiveness of bones to TH deficiency during early developmental stages, which seems to play a significant role in the morphological changes from larvae to juveniles. To examine the period of hormone responsiveness, T4 treatment (0.1 ppb for 2 weeks) was started at two different daily ages for the fish receiving TU treatment. When T4 was given 2 weeks later than TU, the fish completely metamorphosed through the expected bone formation that had been suppressed by TU. However, when T4 treatment was started 4 weeks later than TU, body pigmentation did not occur, and the translocation of the right eye was not complete due to failure of Pb formation. It is suggested that individual tissues that change during metamorphosis have their own timing in response to THs.
3.2 Effect of stage-specific thyroidal stasis on the eye migration of Japanese flounder
Since TH is the main stimulator of eye migration in flounder metamorphosis, the timing of TH responsiveness was determined in the tissue responsible for eye migration in Japanese flounder. TU (30 ppm, an inhibitor of TH synthesis) was administered starting at different stages of metamorphosis, and the inhibitory effects on the tissue related to eye migration were histologically examined. When TU treatment was started before the E stage, eye migration was significantly inhibited due to lack of formation of the Pb, a bone important for eye migration. When treatment started after the F stage, the process was not significantly inhibited due to the minor inhibitory effect on Pb formation. This result indicates a specific requirement by stage F for TH in the Pb. These results suggest that the thyroidal status during the early phase of metamorphosis is extremely crucial for the Pb formation and therefore important for the completion of eye relocation in Japanese flounder, although the process of eye migration is gradual and continuous.
A－383 Are-Lang CHAN, Hideo YOSHIDA and Yasunori SAKURAI
Maturation, reproductive cycle，and characteristics of spawning season of saffron cod, Eleginus gracilis (Tilesius) in waters of Hokkaido, Japan
The histological reproducibility of saffron cod, Eleginus gracilis, examined in the waters eastern coast of Hokkaido Japan, by examining histological sections and another materials of samples that collected September 1983 to December 1986.
In the process of histological examination, maturity was divided into eight grades from chromatin-nucleolus stage (Ⅰ) to spent stage (Ⅷ) in ovaries, and from multiplication stage (Ⅰ) to spent stage (Ⅵ) in testis. Especially in ovaries, during the course of ovarian maturation, a portion of the oocytes became isolated from immature oocytes at the yolk vesicle stage(Ⅱ) and gradually developed into a group of yolky oocytes. When these oocytes reached the migratory stage (Ⅴ) they began to change into transparent mature eggs. Since these form of maturation, we defined them as typical “group-synchronous oocyte development” type.
Also, saffron cod was estimated to spawn within a very short term in January intensively, because almost all females reached spent stage(Ⅷ) from ripe stage(Ⅶ) in this period.
From the relationship between body length and fecundity, G. Eleginus was suggested higher production type in southern waters, and lower production type in northern waters.
The histological reproducibility of saffron cod, Eleginus gracilis, examined in the waters eastern coast of Hokkaido Japan, by examining histological sections and another materials of samples that collected September 1983 to December 1986.
In the process of histological examination, maturity was divided into eight grades from chromatin-nucleolus stage (Ⅰ) to spent stage (Ⅷ) in ovaries, and from multiplication stage (Ⅰ) to spent stage (Ⅵ) in testis. Especially in ovaries, during the course of ovarian maturation, a portion of the oocytes became isolated from immature oocytes at the yolk vesicle stage(Ⅱ) and gradually developed into a group of yolky oocytes. When these oocytes reached the migratory stage (Ⅴ) they began to change into transparent mature eggs. Since these form of maturation, we defined them as typical “group-synchronous oocyte development” type.
Also, saffron cod was estimated to spawn within a very short term in January intensively, because almost all females reached spent stage(Ⅷ) from ripe stage(Ⅶ) in this period.
From the relationship between body length and fecundity, G. Eleginus was suggested higher production type in southern waters, and lower production type in northern waters.
A－384 Setsuo SAITOH, Tatsunari MORI
Verification and identification of clonal lines induced by chromosome manipulation in Japanese flounder (Paralichthys olivaceus)
II. Verification and identification by microsatellite DNA polymorphism
Microsatellite DNA analysis was applied for confirmation and identification of clones of Japanese flounder, Paralichthys olivaceus. Twelve homozygous and five heterozygous clonal lines were examined by microsatellite DNA markers which have been developed for Japanese flounder. PCR Amplification was done using four of the primer sets designed according to each locus (Pol-2* , Pol-3*, Pol-4*, Pol-5*), and the fragments were analyzed using an DNA sequencer. The alleles present in DNA from each clonal line were identical to the same lines and were easily distinguished among the different lines. In some clonal fish, however, alleles were not detected even in the same clonal line.
II. Verification and identification by microsatellite DNA polymorphism
Microsatellite DNA analysis was applied for confirmation and identification of clones of Japanese flounder, Paralichthys olivaceus. Twelve homozygous and five heterozygous clonal lines were examined by microsatellite DNA markers which have been developed for Japanese flounder. PCR Amplification was done using four of the primer sets designed according to each locus (Pol-2* , Pol-3*, Pol-4*, Pol-5*), and the fragments were analyzed using an DNA sequencer. The alleles present in DNA from each clonal line were identical to the same lines and were easily distinguished among the different lines. In some clonal fish, however, alleles were not detected even in the same clonal line.
A－385Yasuko KONDA and Yuichi SAKAI
An experimental study to observe the gut contents of juvenile sea cucumber, Apostichopus japonicus
In this study, we developed a new method to observe the gut content of juvenile sea cucumber, Apostichopus japonicus. We applied Bleaching Method to observe diatoms taken in by the juvenile sea cucumber and compared with Dissecting Method. By Bleaching Method, we could handle large number of sea cucumbers without any loss or destruction of diatom frustule. From the observation of juvenile sea cucumbers ( size range : 288－791 μm body length ), reared with natural benthic diatoms cultivated in Shikabe, southeastern Hokkaido, it revealed that juvenile sea cucumbers were able to take in 8 μm to 128 μm length benthic diatoms.
In this study, we developed a new method to observe the gut content of juvenile sea cucumber, Apostichopus japonicus. We applied Bleaching Method to observe diatoms taken in by the juvenile sea cucumber and compared with Dissecting Method. By Bleaching Method, we could handle large number of sea cucumbers without any loss or destruction of diatom frustule. From the observation of juvenile sea cucumbers ( size range : 288－791 μm body length ), reared with natural benthic diatoms cultivated in Shikabe, southeastern Hokkaido, it revealed that juvenile sea cucumbers were able to take in 8 μm to 128 μm length benthic diatoms.