Search

Pleuronectiform fish develop marked external asymmetry in eye location and skin color at metamorphosis. The bamboo sole, Heteromycteris japonica, also exhibits loss of the pectoral fins at metamorphosis. Because of its small body size, short generation time, and long spawning season, we focused on the bamboo sole as an experimental model to investigate metamorphic asymmetry and pectoral fin loss during development. In the present study, we utilized a small-scale culture system to evaluate bamboo sole larvae and larval development, and a microinjection system for fertilized eggs. The culture system described here uses an 18 L culture tank for rotifers (the first diet for larvae) and 5 L plastic beakers for larval culture. Under this system, most larvae completed metamorphosis, including one-eye migration and pigmentation of the ocular side, by 23 days postfertilization (dpf) at 25°C. Larvae at density of 120-150 per liter were grown from hatching to 23 dpf with a survival ratio of 60-75% per beaker. Pectoral fins, including coracoid and disk cartilage, formed but were completely lost in late metamorphosis without formation of proximal radials and fin rays. The microinjection system designed here is adequate for the bamboo sole and allows injection of 100 one-cell-stage embryos per day. We expect that the culture and microinjection systems described here will facilitate the use of the bamboo sole as an experimental model organism in developmental biology.

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.

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.