Masato Aritaki

Pedigree analysis of recaptured fish in the stock enhancement program of spotted halibut Verasper variegatus

Spotted halibut Verasper variegatus hatchery juveniles produced in 2002 were genotyped using three microsatellite DNA markers (msDNA) and then released into natural waters. Subsequently, recaptured individuals were examined using msDNA. In order to evaluate the effectiveness of the stock enhancement program, from the genetic point of view, a pairwise FST test was implemented to estimate the genetic divergence between the wild captive broodstock, the hatchery offspring and the recaptured samples. The analysis showed significant differentiation between the broodstock and recaptured samples. Pedigree determination using msDNA was used to calculate the effective population size of the recaptured stock, which was found to be very low (Ne≈8). Equal family survivability was observed between the two recaptured stocks, but not between the released and recaptured stock. The number of identified families was higher and more equalized in the hatchery offspring compared to the recaptured samples, where the number of families declined. This fact was caused by an unequal family survivability just before or just after release. Separately, the number of contributing parents to the hatchery offspring was lower than the broodstock census number. Consequently, these two facts caused the genetic divergence of the recaptured stock from the broodstock.

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.

Genetic population evaluation of two closely related flatfish species, the rare barfin flounder and spotted halibut, along the Japanese coast

Barfin flounder and spotted halibut have been selected as target species for stock enhancement in Japan. Understanding the genetic condition of the wild stock is a principal requirement in any stock enhancement program. The genetic variability of barfin flounder and spotted halibut, and the population structure of spotted halibut were evaluated using microsatellite DNA markers (msDNA) and the control region of the mitocondrial DNA (mtDNA). Barfin flounder and spotted halibut showed high genetic variability at the msDNA level. Barfin flounder A was 16.7 and He was 0.860; spotted halibut An ranged from 7.7 to 10.2 and He ranged from 0.710 to 0.774. At the mtDNA level, high haplotype (h=0.922) and low nucleotide (π=0.002) diversities were observed for barfin flounder; however, low haplotype and nucleotide diversities (h=0.603–0.620 and π=0.001–0.002), and very low haplotype and nucleotide diversities (h=0.193 and π=0.0003) were observed for spotted halibut in the north and south locations, respectively. Slight genetic differentiation among spotted halibut sampling locations was observed from the msDNA. MtDNA analyses showed genetic differentiation between north and south locations, but not within them. The designation of north-specific and south-specific management units in the future stock enhancement activities of spotted halibut is recommended.

Dual appearance of xanthophores, and ontogenetic changes in other pigment cells during early development of Japanese flounder Paralichthys olivaceus

Flatfishes display a left–right asymmetry that is unique in the animal kingdom. In order to clarify the mechanisms of the asymmetrical development of pigment cells, changes in pigment cell densities were examined in Japanese flounder Paralichthys olivaceus. During development from symmetrical larvae to asymmetrical juveniles, pigment cell densities were monitored on the skin on both the left side (ocular side in juvenile; eventually has two eyes) and the right side (blind side in juvenile; eventually has no eyes). A symmetrical and constant decrease was observed in leucophores and larval type melanophores. A mostly symmetrical (slightly delayed on the blind side) and constant increase in iridophores from metamorphosis was observed. Adult-type melanophores appeared and then increased only after metamorphosis on the ocular side. However, the pattern of xanthophores was complicated: they first existed symmetrically and decreased symmetrically until metamorphosis, and they later increased only on the ocular side. The dual appearance of the xanthophores, as well as the differences between their depths and sizes on the ocular and blind sides, may suggest the presence of two types of xanthophores—just as melanophores are well known to exhibit two types. The ontogenetic study of pigment cells described here is likely to help to elucidate the process of abnormal pigmentation in flatfishes.

Embryogenesis and Expression Profiles of charon and Nodal-Pathway Genes in Sinistral (Paralichthys olivaceus) and Dextral (Verasper variegatus) Flounders

Abstract 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, Kupffers 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.

Genetic structure in species with shallow evolutionary lineages: a case study of the rare flatfish Verasper variegatus

We examined the genetic population divergence of the spotted halibut Verasper variegatus. A previous report suggested two conservation units for this species along the Japanese Pacific coast. Extending the coverage of the genomes (29 microsatellites and three mitochondrial DNA segments) revealed hitherto-undetected genetic population boundaries. We screened population samples from the major habitats along the Japanese coast and the Yellow Sea coast (East Asian Continent). Significant genetic differentiation was found in every comparison between the habitats. In most cases, the nuclear and mitochondrial population divergences were incongruent, most likely caused by differences between the two genomes in the effects of genetic drift after recent population isolation and bottleneck events. We discuss the ecological and evolutionary mechanisms of the genetic structure as well as the units of conservation. The present study illustrates the merits of wider coverage of genomes in genetic population analysis especially for species with a shallow population history.

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.

Microsatellite markers for a rare species of right-eye flounder Verasper variegatus (Pleuronectiformes, Pleuronectidae)

In the context of stock enhancement for aquatic animals, the risk of negative genetic impacts of releasing hatchery-produced seedlings upon the genetic diversity of indigenous recipient populations becomes a growing concern. This issue is particularly relevant to threatened or rare species such as a member of right-eye flounder, the spotted halibut Verasper variegatus. We present here 29 microsatellite markers for spotted halibut, expecting that these markers could serve as a molecular tool of choice to address a wide range of conservation issues in this species.

Pseudoalbinism and ambicoloration in hatchery-reared pleuronectids as malformations of asymmetrical formation

Morphological abnormalities in eye location and/or body coloration are commonly observed in hatchery-reared juveniles of many pleuronectid species, and have become one of the most serious problems in juvenile production for stock enhancement. In this study, these morphological abnormalities of six pleuronectid species—barfin flounder Verasper moseri, slime flounder Microstomus achne, stone flounder Platichthys bicoloratus, starry flounder Pl. stellatus, cresthead flounder Pseudopleuronectes schrenki, and marbled sole Ps. yokohamae—were successfully classified into four morphological types [normal, two types of pseudoalbinism (normal-eye position or top-eye position), and ambicoloration], following the same classification scheme proposed for other two pleuronectids. Based on our results, the characteristics of each morphological type are quite similar among species. It is confirmed that the normal type has the same ocular side and blind side characteristics as those of wild fish, not only in eye position and body coloration, but also in scales or dentition. The pseudoalbino types have the blind side characteristics of wild fish on both sides, with top-eye type in all characteristics, and normal-eye type in all other characteristics other than eye position. The ambicoloration type has all the ocular side characteristics of wild fish on both sides. Therefore, the pseudoalbinism and ambicoloration of hatchery-reared juveniles of pleuronectid species can be considered malformations of asymmetrical formation. Since this process normally occurs during metamorphosis, the term “metamorphosis-related malformation” is proposed for the abnormal formation of juveniles in pleuronectid species.

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.