Muscle development in the bamboo sole Heteromycteris japonicus with special reference to larval branchial levators.

Muscle development in the bamboo sole Heteromycteris japonicus was investigated, focusing primarily on the cranial muscles, using an improved whole mount immunohistochemical staining method with potassium hydroxide, hydrogen peroxide and trypsin. Larvae of H. japonicus had branchial levators, but not all of them were retained in adults, a condition also seen in the Japanese flounder Paralichthys olivaceus. In particular, larval branchial levators II and III disappeared during development, while I and IV remained to become the levator internus I and levator posterior, which were well-defined muscles in adults. In place of the atrophied muscles, levatores externi and levator internus II developed and regulated the branchial arches. The results showed that the muscle composition in the dorsal branchial arches changed to the adult form before metamorphosis in H. japonicus, as seen in P. olivaceus, and this transformation may be common to all members of that group.

Isolation and characterization of 11 polymorphic microsatellite loci in the whitegirdled goby (Pterogobius zonoleucus) and cross-species amplification in the serpentine goby (P. elapoides).

Eleven polymorphic microsatellite markers were developed from a (CA)(n) -enrichment library of the whitegirdled goby (Pterogobius zonoleucus). Polymorphism at these loci ranged from 2 to 12 alleles, and observed and expected heterozygosities from 0.05 to 0.90 and from 0.05 and 0.86, respectively. All loci conformed to Hardy-Weinberg equilibrium, with no significant linkage disequilibrium between all locus pairs. Cross-species amplification tests were successful in P. elapoides, and most loci were polymorphic. These microsatellite markers will be useful in further population genetic studies of both species.

Asymmetrical development of bones and soft tissues during eye migration of metamorphosing Japanese flounder, Paralichthys olivaceus.

The symmetrical body of flatfish larvae dramatically changes into an asymmetrical form after 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, Paralichthys olivaceus, by 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 by twisting in the same direction as that in which the eye migrates. An increase in skin thickness beneath the eye was observed only on the blind side at the beginning of eye migration; this was the first definitive difference between the right and left sides of the body. The pseudomesial bar, a peculiar bone present only in flatfishes, developed from this thick skin and grew dorsad. Novel sac-like structures were found and named retrorbital vesicles. The retrorbital vesicle of the blind side grew larger and faster than that of the ocular side when the right eye moved most dramatically, whereas no difference was observed between the volume of right and left connective tissue in the head. The asymmetrical presence and growth of the pseudomesial bar together with inflation of the retrorbital vesicle on the blind side may be responsible for right eye migration during metamorphosis in the Japanese flounder.

Effect of Thyroid Hormones on the Stage-specific Pigmentation of the Japanese Flounder Paralichthys olivaceus.

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 (T4) which causes abnormal pigmentation, and the third was to determine the sensitive stage of larval development at which albinism is induced by exogenous T4. The larvae treated with over 10 nM T4 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 T4 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 T4 at stages A and B, C and D, E and F and G to I. T4 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<0.01). Changes in the body content of T4 were reflected in the groups with higher incidence of albinism. The present experiment has revealed the T4 sensitive period for the induction of albinism in Japanese flounder. The absence of normal coloration in the juveniles after T4 treatment clearly indicates the involvement of the thyroid in pigmentation at the early stages of development.

Developmental Sequence of Chloride Cells in the Body Skin and Gills of Japanese Flounder (Paralichthys olivaceus) Larvae.

The developmental sequence of chloride cells was examined in both the body skin and gills of Japanese flounder (Paralichthys olivaceus) larvae by whole-mount immunocytochemistry using an antiserum specific for Na(+),K(+)-ATPase. In premetamorphic larvae at 0 and 4 days after hatching (days 0 and 4), immunoreactive chloride cells were distributed only in the yolk-sac membrane and body skin. Premetamorphic larvae at days 8-18 possessed both cutaneous and branchial chloride cells. Large chloride cells in the skin of premetamorphic larvae often formed multicellular complexes, suggestive of their ion-secreting function. Cutaneous chloride cells decreased in size and density at the beginning of metamorphosis (days 21 and 24), and disappeared at the metamorphic climax (days 28 and 33). In contrast, branchial chloride cells first appeared at day 8, and increased during metamorphosis. These results indicate that the site for ion secretion in seawater may shift from cutaneous to branchial chloride cells during metamorphosis. The appearance of branchial chloride cells before the differentiation of gill lamellae suggests that the primary function of the gills during the early development is ion regulation rather than gas exchanges.

Dual appearances of pigment cells from in vitro cultured embryonic cells of Japanese flounder: an implication for a differentiation-associated clock.

The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin-EDTA in Dulbecco's modified Ca(2+)-, Mg(2+)-free phosphate buffered saline and then cultured in vitro using L-15-based fetal calf serum-supplemented growth medium at 20 degrees C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions. The first group of pigment cells, which is relatively larger in cell size (about 70 microns wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 microns) and overwhelmingly higher in cell density than the first, does so about 1 month after plating. The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis. Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

An association of melanophores appearing at metamorphosis as vehicles of asymmetric skin color formation with pigment anomalies developed under hatchery conditions in the Japanese flounder, Paralichthys olivaceus.

The mechanisms for asymmetric skin color formation in the Japanese flounder are studied with particular concerns to causes for pigment disorder (hypomelanosis) occurring under hatchery conditions. For an analysis of normal pigmentation, fish were raised with wild zooplanktons in an indoor hatchery, whilst for hypomelanosis, they were raised with Brazilian Artemia nauplii, a diet used in the hatcheries. Morphological observations, counting of melanophores, histochemical assay of DOPA-positive immature cells (melanoblasts), and radiometric estimation of tyrosinase activities in skins of developing larvae and juveniles indicate that 1) the structural plan for pigmentation in this species is bilaterally symmetric until metamorphosis, utilizing large-sized melanophores (hence larval melanophores) as main vehicles, and 2) an asymmetric coloration characteristic to metamorphosed juveniles is formed by an intensive development of smaller-sized melanophores (hence adult-type melanophores) appearing selectively in the ocular side at the later stages of metamorphosis and by an absence of it in the blind. These findings apparently indicate that 1) two types of melanophores occur in this species which differ with respect to morphological properties and developmental fate, and 2) selective differentiation of adult type melanophores in the ocular side of the body at or after metamorphosis is primarily responsible for an asymmetric coloration of its adult form. The similar assays on the fish fed with Artemia nauplii indicate that defective development of adult-type melanophores results in hypomelanosis in their ocular-sided skins, yielding a pigmentary pattern seen in the blind side of the metamorphosed juveniles with normal pigmentation.(ABSTRACT TRUNCATED AT 250 WORDS)