Ocular-side lateralization of adult-type chromatophore precursors: development of pigment asymmetry in metamorphosing flounder larvae.

The adult-type chromatophores of flounder differentiate at metamorphosis in the skin of ocular side to establish asymmetric pigmentation. In young larva and before metamorphosis, adult-type melanophores that migrate to the ocular side during metamorphosis reside at the base of the dorsal fin as latent precursors. However, the migration route taken by these precursor cells and the mechanisms by which lateralization and asymmetric pigmentation develop on the ocular side are unknown. To further investigate this migration and lateralization, we used in situ hybridization with gch2 probe, a marker for melanoblasts and xanthoblasts (precursors of adult type chromatophores), to examine the distribution of chromatophore precursors in metamorphosing larvae. The gch2-positive precursors were present in the myoseptum as well as in the skin. This finding indicated that these precursors migrated from the dorsal part of the fin to the skin via the myoseptum. Additionally, there were much fewer gch2-positive cells in the myoseptum of the blind side than in the skin and myoseptum of the ocular side, and this finding indicated either that migration of the precursor cells into the myoseptum of blind side was inhibited or that the precursors were eliminated from the myoseptum of the blind side. Therefore, we propose that the signals responsible for development of asymmetric pigmentation in flounder reside not only in the skin but on a larger scale and in multiple tissues throughout the lateral half of the trunk.

Metamorphic pitx2 expression in the left habenula correlated with lateralization of eye-sidedness in flounder.

The bilateral symmetry of flounder larvae changes through the process of morphogenesis to produce external asymmetry at metamorphosis. The process is characterized by the lateral migration of one eye and pigmentation at the ocular side. Migration of the left or right eye to produce either dextral or sinistral forms, respectively, is usually fixed within a species. Here we propose a mechanism for the mediation of lateralization by the nodal-lefty-pitx2 (NLP) pathway in flounders, in which pitx2, the final left-right determinant of the NLP pathway, is re-expressed in the left habenula at pre-metamorphosis. After the initiation of left-sided pitx2 re-expression, the eye commences migration, when the habenulae shift their position on the ventral diencephalon rightwards in sinistral flounder (Paralichthys olivaceus) and leftwards in dextral flounder (Verasper variegatus). In addition, the right habenula increases in size relative to the left habenula in both species. Loss of pitx2 re-expression induces randomization of eye-sidedness, manifesting as normal, reversed or bilateral symmetry, with laterality of the structural asymmetry of habenulae being entirely inverted in reversed flounders compared with normal ones. Thus, flounder pitx2 appears to be re-expressed in the left habenula at metamorphosis to direct eye-sidedness by lateralizing the morphological asymmetry of the habenulae.

Adult-type pigment cells, which color the ocular sides of flounders at metamorphosis, localize as precursor cells at the proximal parts of the dorsal and anal fins in early larvae.

Flounders form left-right asymmetry in body coloration during metamorphosis through differentiation of adult-type melanophores and xanthophores on the ocular side. As the first step in investigating the formation of flounder body coloration asymmetry, in this study, we aimed to determine where the precursors of adult-type chromatophores distribute in larvae before metamorphosis. In Paralichthys olivaceus and Verasper variegatus, GTP cyclohydrolase 2 (gch2), a common marker of melanoblasts and xanthoblasts, was found to be transiently expressed in cells located along the bilateral skeletal muscles at the basal parts of the dorsal and anal fins of premetamorphic larvae. When V. variegatus larvae were fed with a strain of Artemia collected in Brazil, this gch2 expression was abolished and the differentiation of adult-type melanophores was completely inhibited, while the density of larval melanophores was not affected. In a cell trace test in which the cells at the basal part of the dorsal fin were labeled with DiI at the premetamorphic stage, adult-type melanophores labeled with DiI were found in the skin on the ocular side after metamorphosis. These data suggest that, in flounder larvae, adult-type melanophores are distributed at the basal parts of the dorsal and anal fins as unpigmented precursor cells.

Embryogenesis and expression profiles of charon and nodal-pathway genes in sinistral (Paralichthys olivaceus) and dextral (Verasper variegatus) flounders.

Although it is well known that flounder form external asymmetry by migration of one eye at metamorphosis, the control system that forms this asymmetry is unknown. To help elucidate this mechanism, we here describe the embryogenesis and expression profiles of the Nodal-pathway genes in the Japanese flounder, Paralichthys olivaceus. We also perform a comparative study of the laterality of the expression of these genes in sinistral (P. olivaceus) and dextral (Verasper variegatus) flounders. In P. olivaceus, Kupffer's vesicle forms at the 2-somite stage, after which left-sided expression of spaw starts at the 8-somite stage. Left-sided expression of pitx2 occurs in the gut field at the 15-somite to high-pec stages, in the heart field at the 21-somite stage, and in the dorsal diencephalon at the 27-somite to high-pec stages. In response to left-sided pitx2 expression, the heart, gut, and diencephalon begin asymmetric organogenesis at the pharyngula (heart) and the long-pec (gut and diencephalon) stages, whereas the eyes do not show signs of asymmetry at these stages. In both sinistral and dextral flounders, the Nodal-pathway genes are expressed at the left side of the dorsal diencephalon and left lateral-plate mesoderm. Considering these data together with our previous finding that reversal of eye laterality occurs to some extent in the P. olivaceus mutant reversed, in which embryonic pitx2 expression is randomized, we propose that although the Nodal pathway seems to function to fix eye laterality, embryonic expression of these genes does not act as a direct positional cue for eye laterality.

Effects of Large-Scale Releases on the Genetic Structure of Red Sea Bream (Pagrus major, Temminck et Schlegel) Populations in Japan.

Large-scale hatchery releases are carried out for many marine fish species worldwide; nevertheless, the long-term effects of this practice on the genetic structure of natural populations remains unclear. The lack of knowledge is especially evident when independent stock enhancement programs are conducted simultaneously on the same species at different geographical locations, as occurs with red sea bream (Pagrus major, Temminck et Schlegel) in Japan. In this study, we examined the putative effects of intensive offspring releases on the genetic structure of red sea bream populations along the Japanese archipelago by genotyping 848 fish at fifteen microsatellite loci. Our results suggests weak but consistent patterns of genetic divergence (F(ST) = 0.002, p < 0.001). Red sea bream in Japan appeared spatially structured with several patches of distinct allelic composition, which corresponded to areas receiving an important influx of fish of hatchery origin, either released intentionally or from unintentional escapees from aquaculture operations. In addition to impacts upon local populations inhabiting semi-enclosed embayments, large-scale releases (either intentionally or from unintentional escapes) appeared also to have perturbed genetic structure in open areas. Hence, results of the present study suggest that independent large-scale marine stock enhancement programs conducted simultaneously on one species at different geographical locations may compromise native genetic structure and lead to patchy patterns in population genetic structure.

Inference of potential genetic risks associated with large-scale releases of red sea bream in Kanagawa prefecture, Japan based on nuclear and mitochondrial DNA analysis.

Since 1978, millions of hatchery-reared red sea bream (Pagrus major) juveniles have been released in Sagami Bay and Tokyo Bay in Kanagawa Prefecture, Japan. The stock enhancement program has contributed to total catch; however, no information regarding the genetic interactions with wild counterparts is available. Here, we combined 15 microsatellite loci and mitochondrial D-loop sequencing to characterize the genetic resources of red sea bream in Sagami Bay and Tokyo Bay and to elucidate the potential harmful genetic effects associated with fish releases. Both types of markers evidenced higher levels of genetic diversity in wild samples (SB and TB) compared with offspring before stocking (H07 and H08) as well as a hatchery-released sample recaptured in Sagami Bay (HR). Microsatellite F (ST) estimates and Bayesian clustering analysis found significant genetic differences among samples (F (ST) = 0.013-0.054), except for the two wild samples (F (ST) = 0.002) and HR vs. H07 (F (ST) = 0.007). On the other hand, mitochondrial-based Ф (ST) suggested haplotypic similarity between SB, H07, and HR. The low effective number of females contributing to the offspring over multiple generations may be responsible for the lack of haplotypic differentiation. Moreover, the putative hatchery origin to three fish (8 %) without deformity in the inter-nostril epidermis was inferred for the first time. Our results showed the usefulness of combining nuclear and mitochondrial markers to elucidate genetic interactions between hatchery-released and wild red sea bream and warned about potential harmful genetic effects should interbreeding takes place.

Production of symmetrical flatfish by controlling the timing of thyroid hormone treatment in spotted halibut Verasper variegatus.

The thyroid hormone plays an essential role in the metamorphosis in flatfish, during which external asymmetry (for example, eye relocation and pigmentation) is established. However, no information is available on the expression mechanisms of metamorphic asymmetry. Since the presence of malformed juveniles (having either ocular or blind side characteristics on both sides) was known in spotted halibut (Verasper variegatus), the effect of the timing of thyroid hormone treatment was investigated. When thyroxine (T4, 10 or 30 ng/ml) was administered to the hypothyroid larvae (continuously receiving 30 microg/ml thiourea) from 25 days after hatching (DAH), the occurrence of one type of symmetrical juvenile (symmetrical pseudoalbino having blind side characteristics on both sides) increased to more than three times, and the occurrence was significantly greater than that observed in the control group. In the fish in which T4 treatment was initiated prior to 15 DAH or after 60 DAH, the occurrence of another type of symmetrical juvenile (symmetrical ambicolorate having ocular side characteristics on both sides) became more than two times, and its occurrence was significantly greater than that observed in the control group. These results suggest that both sides of the larval body independently possess the potential to become either the ocular or the blind side of the juvenile body and that the timing of thyroid hormone increase is the determining factor of "which side to become." Based on previous studies, since thyroid hormone is expected to induce blind side characteristics, the differential responsiveness to thyroid hormone-longer in the left side (blind side in normal juvenile) than the right-is strongly suggested as the central mechanism for metamorphic asymmetry in spotted halibut.

Two species of parasitic copepods (Caligidae) new to Japan.

Two species of parasitic copepods (Siphonostomatoida: Caligidae), Caligus sclerotinosus Roubal, Armitage & Rohde, 1983, parasitic on red seabream Pagrus major (Temminck & Schlegel), and Lepeophtheirus longiventralis Yü & Wu, 1932, parasitic on spotted halibut Verasper variegatus (Temminck & Schlegel), are redescribed based on material found on their respective hosts cultured in Japan. Both species are new to Japan. Preliminary observation on the occurrence of L. longiventralis indicates that the larval development takes place on the body surface of the host and only the post-mated female migrates into the host's gill-cavities. Whilst the occurrence of L. longiventralis in Japan can be considered as due to natural causes, the occurrence of C. sclerotinosus is likely due to anthropogenic activities.