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There has been grave concern in stock enhancement programs regarding the effects of stocking practices on resource augmentation of ensuing generations through reproduction of released hatchery stocks. Evaluation of whether released seedlings successfully produce offspring in the natural environment is a first step toward a better understanding of such inter-generation stocking effects. Against this background, we examined the reproductive success of released hatchery stocks of a Pacific abalone species, Haliotis discus hannai, on the basis of microsatellite-based parentage analysis. Adults of hatchery-raised abalone were released from 2014 to 2016 into the Toyoura area of Funka Bay (Hokkaido Island, Japan), where natural gene flow from other habitats was expected to rarely occur. After the reproductive season of H. discus hannai in Funka Bay in 2016, we collected 2016-year-class wild-born juveniles from Toyoura and determined parentages between the juveniles and released individuals. The results of this analysis revealed that approximately 10% of the juveniles were descended from the released hatchery stocks.

Additional file 1: Fig. S1. Kin relationships among individuals. Small rectangles on the outer edge refer to individual fish. Pairs of individuals with first and second degree relationship are connected with dark and light lines, respectively.

Female sea urchins store the major yolk protein (MYP) in ovarian nutritive phagocytes before vitellogenesis. Using immunological procedures, we detected MYP in the testicular nutritive phagocytes of Pseudocentrotus depressus, the red sea urchin, and then compared the distribution of MYP between sexes during gametogenesis. MYP was purified from unfertilized eggs by ion exchange chromatography (Q Sepharose) and gel filtration (Superdex 200), and an antiserum (anti-MYP) was raised against MYP. Immunoblot analysis demonstrated that immature testes, as well as ovaries, contained a large quantity of MYP. Immunohistochemistry showed that MYP was distributed in the nutritive phagocytes occupying the follicular lumen in both males and females. In both sexes, as gametogenesis proceeded, the nutritive phagocytes degenerated and the gonadal lumen filled with gametes. MYP accumulated in ripe ova as a yolk protein in the mature ovary. In contrast, MYP was not detected in mature testes, because stored spermatozoa did not react with anti-MYP. We conclude that in male P. depressus, MYP is stored in the testicular nutritive phagocytes and utilized as the nutrient source for spermatogenesis.

The food availability of chum salmon fry is one of the principal factors affecting survival in their coastal residence period. The aim of this study was to better understand the quality and quantity of available food resources and the feeding habits of salmon fry in nearshore waters. We examined stomach contents of salmon fry and the zooplankton community structure of the water column during salmon fry’s coastal residence period (April and May) over four years in Yamada Bay, a coastal embayment of the Pacific Ocean in northern Japan. The mean water temperatures during study period were low in 2014 and 2013 (6.7 and 8.3 °C, respectively),and high in 2016 and 2015 (11.1 and 9.4 °C, respectively). Neocalanus plumchrus and Themisto japonica, typical cold-water zooplankton dominated the stomach contents of chum fry (except for 2016), however, they were not necessarily the most dominant in the water column, suggesting that chum fry selectively preyed upon these cold-water zooplankters. On the other hand, chum fry preyed primarily on decapod larvae in 2016 the warmest water year. Condition factor of chum fry were higher in cold-water years than those of warm-water years. Because these two cold-water zooplankton species are typically abundant in the Oyashio Current, the magnitude of Oyashio inflow may affect prey availability for chum salmon fry and their nutritional status. The relationship between the food environment at the coastal residence period of chum fry and the adult return rate after three years was also discussed.

The first studies that identified leptin and its receptor (LepR) in mammals were based on mutant animals that displayed dramatic changes in body-weight and regulation of energy homeostasis. Subsequent studies have shown that a deficiency of leptin or LepR in homoeothermic mammals results in hyperphagia, obesity, infertility and a number of other abnormalities. The physiological roles of leptin-mediated signaling in ectothermic teleosts are still being explored. Here, we produced medaka with homozygous LepR gene mutation using the targeting induced local lesions in a genome method. This knockout mutant had a point mutation of cysteine for stop codon at the 357th amino acid just before the leptin-binding domain. The evidence for loss of function of leptin-mediated signaling in the mutant is based on a lack of response to feeding in the expression of key appetite-related neuropeptides in the diencephalon. The mutant lepr−/− medaka expressed constant up-regulated levels of mRNA for the orexigenic neuropeptide Ya and agouti-related protein and a suppressed level of anorexigenic proopiomelanocortin 1 in the diencephalon independent of feeding, which suggests that the mutant did not possess functional LepR. Phenotypes of the LepR-mutant medaka were analyzed in order to understand the effects on food intake, growth, and fat accumulation in the tissues. The food intake of the mutant medaka was higher in post-juveniles and adult stages than that of wild-type (WT) fish. The hyperphagia led to a high growth rate at the post-juvenile stage, but did not to significant alterations in final adult body size. There was no additional deposition of fat in the liver and muscle in the post-juvenile and adult mutants, or in the blood plasma in the adult mutant. However, adult LepR mutants possessed large deposits of visceral fat, unlike in the WT fish, in which there were none. Our analysis confirms that LepR in medaka exert a powerful influence on the control on food intake. Further analyses using the mutant will contribute to a better understanding of the role of leptin in fish. This is the first study to produce fish with leptin receptor deficiency.

We developed tetranucleotide-repeat microsatellite markers for the masu salmon (Oncorhynchus masou) complex. 454 pyrosequencing was used to discover repeat motifs, and seven polymorphic microsatellite-primer sets were identified. The number of alleles detected at each locus ranged from four to 24 and the expected heterozygosity varied from 0.57 to 0.92. Cross-subspecies amplification for O. m. masou, O. m. ishikawae and O. m. subsp. was successful. These microsatellites can be utilized in studies of genetic structure, genetic diversity, and intra- and inter-subspecific hybridization, making a contribution to conservation and management of the Oncorhynchus masou complex.

We found that tandem repeat (TR) arrays were present in the ark shell Scapharca broughtonii mitochondrial genome, and we demonstrated the efficiency of using this TR region as a tool for the detection of genetic differentiation. In samples collected from seven populations off the Japanese, Chinese, Russian and Korean coasts, five TR types with length ranging from 481 to 601 bp (base pairs) were found, as were 18 haplotypes (n = 155). All the Japanese samples from SD, IK, AM and KG, which represented four geographically distinct populations, had the same type of tandem repeat array, 481-TR. All the samples from China and all but one sample from Russia had the same TR type, 511-TR; these finding may be strong evidence that the ark shell populations in China and Russia are very similar. Based on a neighbor-joining dendrogram of haplotypes, the Korean population was divided into two groups as follows: samples with haplotype H481-3 and samples with haplotypes H601-1, H601-2, H601-3 or H541-1. Based on the haplotype diversity and nucleotide diversity, the Chinese, Russian and Korean populations showed a higher diversity than Japanese populations (P

The present study analyzed the rate of occurrence of deformities at completion of yolk resorption after Japanese eel larvae were exposed to low-salinity (29 psu) or low-water-temperature (20 °C) conditions (after they had been reared under suitable conditions: 34 psu, 25 °C) at various points between 0 and 8 days post-fertilization (dpf) in order to determine the period of sensitivity of the larvae to low-salinity or low-temperature conditions. No significant differences were observed between the rate of deformed larvae obtained in the groups reared under suitable conditions and the corresponding rates in groups that were exposed to low-salinity or low-temperature conditions at 4 dpf or later, but the frequency of normal larvae showed a downward trend in experimental groups that were subjected to low-salinity or low-temperature conditions at 3 dpf or earlier. Occurrence rates of pericardial edema were significantly higher in experimental groups that were exposed to low-salinity or low-temperature conditions at 2 dpf or earlier. Therefore, it was concluded that the rearing conditions for Japanese eel larvae should be maintained at 34 psu salinity and 25 °C until at least 4 dpf in order to reduce the occurrence of larval deformity at completion of yolk resorption.

In recent years, massive sequencing approaches have allowed us to determine genomic structures of various organisms rapidly, raising novel applicability of the high-throughput sequence data obtained to various fields of biological studies. We present here a pipeline to search for microsatellite DNA and design PCR primers encompassing the microsatellites on genomic sequence data produced by 454 pyrosequencing. We tested this pipeline, called ‘Auto-primer’, on several fish genomic sequences and obtained many and various candidates for microsatellite DNA loci useful for detecting intraspecies genetic variability. This in silico search for microsatellite DNA is superior to conventional cloning methods, since any sequence patterns of repeat unit can be screened.

Muscles play important roles in feeding, respiration and swimming during not only adult stage but also larval stage in fish. However, there is no information of the muscle development in larval greater amberjack, Seriola dumerili (Risso). Here, we investigated muscle development in the greater amberjack, focusing primarily on the cranial muscles from 0 days post hatch (dph) to 12 dph using a modified whole-mount immunohistochemical staining method. We found that the muscles required for feeding develop by 3 dph, when the larvae begin to feed. Subsequently, muscle composition in the dorsal branchial arches changes to the adult form between 5 and 8 dph. At 8 dph, all the muscles required for feeding and respiration appear, whereas the dorsal, pelvic and caudal fin muscles required for swimming develop later. This report provides fundamental information on larval greater amberjack muscle development, which will enable the detection of abnormal larval muscles and improve larval rearing techniques by modifying environmental conditions.

Fugu (Takifugu rubripes) has contributed as an ideal model organism for understanding the structure and evolution of vertebrate genomes, but also has potential as a good model organism for developmental biology because of the availability of the genome information. However, there is no comprehensive report describing the developmental stages, which is fundamental data for developmental biology. Here we describe a series of stages of the embryonic development of fugu during the first 8 days after fertilization, i.e. from fertilization to hatching. We define seven periods of embryogenesis – the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. Stages subdividing these periods are defined based on morphological characteristics. In addition, as a model experiment of gene expression analysis using this staging series, we performed in situ hybridization of aldh1a2, aldh1a3 and cyp26a1 that play regulatory roles in retinoic acid (RA) metabolism essential for embryogenesis. This report provides fundamental information on fugu embryogenesis, which is anticipated to facilitate the use of fugu as a model organism for developmental studies.

Leptin (Lep) is a key factor for the energy homeostasis in mammals, but the available data of its role in teleosts are not conclusive. There are large sequence differences among mammalian and teleost Lep, both at the gene and protein level. Therefore, in order to characterize Lep function in fish, the use of species-specific Lep is crucial. In this study, the cDNA sequence of salmon leptin a1 (lepa1) was used to establish a production protocol for recombinant salmon LepA1 (rsLepA1) in Escherichia coli, that enabled a final yield of 1.7 mg pure protein L⁻¹ culture. The effects of 20-day administration of rsLepA1 on growth and brain neuroendocrine peptide gene expression [npy, cart, agrp (-1 and -2), pomc (-a1, -a2, -a2s, and -b)] were studied in juvenile, immature Atlantic salmon (96.5±2.1g) fed a commercial diet to satiation. Intraperitoneal osmotic pumps were used to deliver rsLepA1 at four different concentrations (calculated pumping rates were 0, 0.1, 1.0 and 10 ng g⁻¹ h⁻¹). In the highest dosage group (10 ng g⁻¹ h⁻¹), the growth rate was significantly reduced, and pomc-a1 gene expression was higher than in controls. The results support the lipostatic hypothesis and suggest that sLepA1 reduces growth in Atlantic salmon by affecting food intake through the central pro-opiomelanocortin pathway.

Anadromous (sea-migrating) Arctic charr (Salvelinus alpinus) display pronounced seasonal variations in food intake and growth and is an interesting model for studying mechanisms of appetite regulation. In this study cDNAs encoding for ghrelin (GHRL) and leptin (LEP) in Arctic charr were cloned, after which stomach GHRL and liver LEP mRNA expressions were examined by qPCR during a seasonal feeding cycle of semi-wild anadromous Arctic charr. The fish were captured as they returned from summer feeding in seawater and transferred to an indoor tank where they were fed in excess until October the year after. Growth rate was low in late winter, increased in late spring and reached a peak during summer, and then declined during autumn, when the fish became sexually mature. The changes in growth rate were associated with corresponding changes in the proportion of fish that had been eating at each sampling date, and whole body lipid status. Stomach GHRL mRNA expression was high in late winter, decreased to a nadir in mid-summer and increased again to a high level in early autumn. Liver LEP mRNA remained low during winter, spring and early summer, after which there was a gradual, 7-fold increase until October. The seasonal changes in ghrelin and leptin support a role of these hormones in the long-term regulation of energy homeostasis in the anadromous Arctic charr. It cannot be excluded, however, that the increase in liver leptin expression during autumn is related to sexual maturation.

The present study reports the complete coding sequences for two paralogues for leptin (sLepA1 and sLepA2) and leptin receptor (sLepR) in Atlantic salmon. The deduced 171-amino acid (aa) sequence of sLepA1 and 175 aa sequence for sLepA2 shows 71.6% identity to each other and clusters phylogenetically with teleost Lep type A, with 22.4% and 24.1% identity to human Lep. Both sLep proteins are predicted to consist of four helixes showing strong conservation of tertiary structure with other vertebrates. The highest mRNA levels for sLepA1 in fed fish (satiation ration=100%) were observed in the brain, white muscle, liver, and ovaries. In most tissues sLepA2 generally had a lower expression than sLepA1 except for the gastrointestinal tract (stomach and mid-gut) and kidney. Only one leptin receptor ortholog was identified and it shares 24.2% aa sequence similarity with human LepR, with stretches of highest sequence similarity corresponding to domains considered important for LepR signaling. The sLepR was abundantly expressed in the ovary, and was also high in the brain, pituitary, eye, gill, skin, visceral adipose tissue, belly flap, red muscle, kidney, and testis. Fish reared on a rationed feeding regime (60% of satiation) for 10 months grew less than control (100%) and tended to have a lower sLepA1 mRNA expression in the fat-depositing tissues visceral adipose tissue (p0.05) and white muscle (n.s.). sLepA2 mRNA levels was very low in these tissues and feeding regime tended to affect its expression in an opposite manner. Expression in liver differed from that of the other tissues with a higher sLepA2 mRNA in the feed-rationed group (p0.01). Plasma levels of sLep did not differ between fish fed restricted and full feeding regimes. No difference in brain sLepR mRNA levels was observed between fish fed reduced and full feeding regimes. This study in part supports that sLepA1 is involved in signaling the energy status in fat-depositing tissues in line with the mammalian model, whereas sLepA2 may possibly play important roles in the digestive tract and liver. At present, data on Lep in teleosts are too scarce to allow generalization about how the Lep system is influenced by tissue-specific energy status and, in turn, may regulate functions related to feed intake, growth, and adiposity in fish. In tetraploid species like Atlantic salmon, different Lep paralogues seems to serve different physiological roles.

A pivotal role of conservation genetics is to establish an appropriate strategy to maintain the genetic diversity of animals with dwindling natural resources. Tackling this issue requires polymorphic genetic markers such as microsatellites. We developed 11 microsatellite markers for a marine bivalve, the ark shell (Scapharca broughtonii), whose natural resources have considerably declined in recent years. We applied the novel markers coupled with a previously reported one to multiplex polymerase chain reaction trials, successfully allocating them into four multiplex panels. Of the 11 markers, nine met Hardy–Weinberg equilibrium (P > 0.05) with a certain degree of polymorphisms in a natural population, suggesting the potential of the markers in its application to the conservation genetics for the ark shell.

The occurrence of morphological deformities under different incubation salinities (24, 30, 33, 36 and 42 practical salinity units [psu]) was examined in Japanese eel larvae. Fertilized eggs hatched at all salinity treatments. The survival rate until yolk resorption decreased at higher than 36 psu. The proportions of deformed larvae reared at 36 psu were significantly lower than at other treatments. Pericardial edema and abnormal lower jaw increased at lower than 33 psu and spinal curvature occurred at high salinity (42 psu). In contrast, the incubation salinity did not significantly affect the relative frequency of abnormal neurocranium. These results imply that the optimal salinities for rearing Japanese eel eggs and embryos are 34–35 psu from the viewpoints of survival and deformity. Taken together with previous study [Kurokawa, T., Okamoto, T., Gen, K., Uji, S., Murashita, K., Unuma, T., Nomura, K., Matsubara, H., Kim, H.S., Ohta, H., Tanaka, H., in press. Influence of Water Temperature on Morphological Deformities in Cultured Larvae of Japanese Eel, Anguilla japonica , at Completion of Yolk Resorption. J. World Aqua. Soc.], the rearing of eel eggs and embryos at 25 °C and 34–35 psu probably decreases the mortality and deformity rate of eel larvae at yolk resorption stage.

The present study was conducted to establish a homologous radioimmunoassay (RIA) for quantifying plasma leptin (Lep) levels in salmonid species, and to study Lep levels in relation to nutritional status. A part of the Lep peptide, a 14 amino acid long sequence, identical between a Salmo and an Oncorhynchus species was synthesised. Polyclonal antibodies were raised in rabbit against this antigen and both were subsequently used in the development of a RIA protocol for assessing plasma Lep levels. The limit of detection of the assay was 0.3 nM, and intra- and interassay coefficient of variation (CV) were 8.4% and 13%, respectively. Apart from Atlantic salmon and rainbow trout, the assay exhibits measuring parallelism for a range of fish species, including arctic char, Atlantic cod and turbot, suggesting that the established RIA is useful for quantifying Lep levels in several fish species. The RIA indicates that Lep is found in salmonid plasma at levels of 0.5-5 nM, which is comparable with other peptide hormones, and well within the measuring range of the RIA. A study of fed and fasted rainbow trout showed elevated plasma Lep levels during fasting. In addition there was no correlation between Lep levels and condition factor. These data suggest that the relation between circulating Lep levels and energy status differs from that in mammals. While Lep is linked to energy balance, it may not act as an adiposity signal in salmonids, possibly pointing to functional divergence among ectothermic and endothermic vertebrates.

Key peptide hormones involved in the control of appetite in vertebrates were identified, their genes characterized and their regulation studied in Atlantic salmon: two agouti-related proteins (AgRP), cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY). The AgRP-1 and AgRP-2 genes encode prepro-proteins of 142- and 117-amino acids, respectively. The deduced AgRP-2 protein has 10 cysteine residues in the C-terminal polycysteine domain, while the AgRP-1 lacks the 6th and 7th cysteine residues observed in other species. AgRP-1 was principally expressed in the pituitary and skin, while AgRP-2 was highly expressed in the mid-gut, red muscle and gonads. The CART gene, encoding 118-amino acids, was strongly expressed in the brain and eye. In addition to salmon CART, we identified three to six variants of the CART gene in lower vertebrates by mining available databases. The salmon NPY gene, encoding 100-amino acids, was mainly expressed in the brain and eye. AgRP-1 and CART mRNA levels in the brain decreased after 6 days of fasting while AgRP-2 and NPY showed no significant change, suggesting that AgRP-1 and CART are involved in feeding regulation in Atlantic salmon. The identification of multiple variants of these appetite-regulating genes emphasizes the importance to further investigate the complex regulation of these genes.

Studies on the cellulose utilization by animals have been conducted in keeping with the recent developments in molecular biology. In mollusks, endogenous cellulases have been reported from blue mussel, abalone, and freshwater snail. We previously reported the possibility of cellulose assimilation by Corbicula japonica , a representative bivalve dominant in brackish water environments in Japan, and the cloning of its endogenous cellulase (β-1,4-glucanase) gene (Sakamoto, K., Touhata, K., Yamashita, M., Kasai, A. and Toyohara, H., 2007. Cellulose digestion by common Japanese freshwater clam Corbicula japonica . Fish. Sci. 73, 675–683). However, the gene of β-glucosidase, another enzyme essential for the complete cellulose decomposition to glucose units, has not yet been isolated from the mollusk. Therefore, we attempted the molecular cloning of endogenous β-glucosidase from C. japonica and succeeded in the isolation of a cDNA with a 2832-bp open reading frame (ORF) encoding 943 amino acid residues ( CjCel1A ). CjCEL1A has 2 repeated GHF-1(Glycosyl Hydrolase family 1)-like domains and showed high similarity with known insect β-glucosidases and mammalian lactase-phlorizin-hydrolases. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCEL1A is likely to be produced in the secretory cells in the digestive gland, suggesting that CjCEL1A is a digestive β-glucosidase of C. japonica and is not derived from symbionts.

The occurrence of morphological deformities under different rearing water temperatures (18, 20, 22, 24, 26, 28, and 30 C) was examined in Japanese eel larvae. The rates of hatching and survival until yolk resorption at 22–26 C were higher than those at other water temperatures. Fertilized eggs never hatched at 18 and 30 C. The rates of occurrence of abnormal larvae reared at the water temperatures 24–28 C were lower than those at 20 or 22 C. Pericardial edema and lower jaw deformities occurred most frequently at lower temperatures (20 and 22 C). In contrast, the incubation temperature did not significantly affect the relative frequency of some neurocranial deformities and of spinal curvature. These results imply that the optimal temperatures for rearing Japanese eel eggs and embryos are 24–26 C from the viewpoints of survival and deformity.

Cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptides are key regulators of pancreatic enzyme secretion in vertebrates. CCK stimulates enzyme secretion whereas peptide Y (PY), a NPY-related peptide, plays an antagonistic role to that of CCK. In fish, very little is known about how different nutrients affect the synthesis of CCK and PY in the digestive tract, and the mechanism by which CCK and PY actually regulate digestive enzyme secretion is not well understood. In order to determine how different nutrients stimulate the synthesis of CCK and PY in yellowtail (Seriola quinqueradiata), CCK and PY mRNA levels in the digestive tract were measured after oral administration of a single bolus of either phosphate-buffered saline (PBS: control), starch (carbohydrate), casein (protein), oleic acid (fatty acid) or tri-olein (triglyceride). In addition, in order to confirm the synthesis and secretion of digestive enzymes, the mRNA levels and enzymatic activities of three digestive enzymes (lipase, trypsin and amylase) were also analyzed. Casein, oleic acid and tri-olein increased the synthesis of lipase, trypsin and amylase, while starch and PBS did not affect the activity of any of these enzymes. CCK mRNA levels rose, while PY mRNA levels were reduced in fish administered casein, oleic acid and tri-olein. These results suggest that in yellowtail, CCK and PY maintain antagonistic control of pancreatic enzyme secretion after intake of protein and/or fat.

Leptin is a key factor for the regulation of food intake and energy homeostasis in mammals, but information regarding its role in teleosts is still limited. There are large differences between mammalian and teleost leptin at both gene and protein levels, and in order to characterize the function of leptin in fish, preparation of species-specific leptin is therefore a key step. In this study, full-length cDNA coding for rainbow trout leptin was identified. In spite of low amino acid sequence similarity with other animals, leptin is highly conserved between trout and salmon (98.7%). Based on the cDNA, we produced pure recombinant trout leptin (rt-leptin) in E. coli , with a final yield of 20 mg/L culture medium. We then examined the effects of intraperitoneal (IP) injection of rt-leptin on feeding behavior and gene expression of hypothalamic NPY and POMCs (POMC A1, A2 and B) in a short-term (8 h) experiment. The rt-leptin suppressed food intake and led to transient reduction of NPY mRNA levels, while the expression of POMCs A1 and A2, was elevated compared with vehicle-injected controls. These results for rainbow trout are the first that describe a physiological role of leptin using a species-specific orthologue in teleosts, and they suggest that leptin suppresses food intake mediated by hypothalamic regulation. This anorexic effect is similar to that observed in mammals and frogs and supports that the neuroendocrine pathways that control feeding by leptin are ancient and have been conserved through evolution.

Leukemia inhibitory factor (LIF) maintains embryonic stem cells in an undifferentiated state. To establish stable cultures of zebrafish embryonic stem cells, recombinant zebrafish LIF (zLIF) is needed because the LIF sequence varies greatly between species. In the current study, we identified the zebrafish (Danio rerio) and pufferfish (Tetraodon nigroviridis) orthologs of lif from genomic databases, and we isolated a cDNA encoding zLIF. Synteny analysis and comparison of sequences identified zebrafish and Tetraodon orthologs of human LIF. The cDNA for zLIF encoded a predicted 215-amino acid protein with a putative 32-amino acid signal peptide, two disulfide bonds, and two N-linked glycosylation sites. We found that transcription of zlif starts at the hatching period during embryogenesis and is present in the brain, visceral organs, bone, and skin.

We identified cDNA coding for a homologue to mammalian leptin in puffer, Takifugu rubripes, using genomic synteny around the human leptin gene. In addition to significant sequence homologies, the puffer leptin (pLEP) displays characteristic structural features in common with mammalian leptin. The pLEP mRNA was expressed mostly in the liver that contained abundant lipids. In addition, homologues to pLEP were found in the databanks for three fish species (salmon, medaka, and Tetraodon) and two amphibians (salamander and Xenopus). The phylogenetic analysis shows rapid rates of molecular divergence among leptins from different vertebrate classes, but not between mammals and avians.

To investigate the feeding and digestive physiology of early eel larvae, the development of cholecystokinin (CCK) and peptide YY (PYY) in the larval intestine, the feeding speed and pancreatic trypsin secretion in response to feeding were observed. CCK and PYY mRNA started to be expressed in the larval intestine at 8 days post-hatching (dph). This indicates that the pancreatic enzyme secretion is probably controlled by the CCK and PYY endocrine systems from the first feeding. Using fluorescein isothyiocyanate isomer (FITC)-labeled freeze-dried shark egg powder (Aquaran) as a diet, 10-dph eel larvae were estimated to feed at a rate of 0.04 μg Aquaran/min. The tryptic activity in the intestines of 11-dph larvae increased approximately 4.2-fold at 1 h of feeding, whereas that in the pancreas remained constant. These results indicate that the digestive tract of these larvae recognized Aquaran as a nutrient, and that the exocrine pancreas secreted digestive enzymes into the intestine and began to synthesize digestive enzymes in response to feeding.

We have isolated a novel gene, charon , that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer9s vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon9s function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm ( southpaw , cyclops , atv/lefty1 , lefty2 and pitx2 ) and diencephalon ( cyclops , atv/lefty1 and pitx2 ), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1 -mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer9s vesicle, play an important role in L/R patterning in zebrafish.

In teleosts, the embryonic fin fold consists of a peridermis, an underlying epidermis and a small number of mesenchymal cells. Beginning from such a simple structure, the fin skeletons, including the proximal and distal radials and lepidotrichia (finrays), develop in the dorsal fin fold at the larval stage. Their process of skeletogenesis and embryonic origin are unclear. Using flounder larvae, we report the differentiation process for chondrocytes and scleroblasts prior to fin skeletogenesis and the effects of retinoic acid (RA) on it. In early larvae, the mesenchymal cells grow between the epidermis and spinal cord to form a line of periodical condensations, which are proximal radial primordia, to produce chondrocytes. The prescleroblasts, which ossify the proximal radial cartilages, differentiate in the mesenchymal cells remaining between the cartilages. Then, mesenchymal condensations occur between the distal ends of the proximal radials, forming distal radial primordia, to produce chondrocytes. Simultaneously, condensations occur between the distal radial primordia and peridermis, which are lepidotrichia primordia, to produce prescleroblasts. Exogenous RA specifically inhibits the mesenchymal condensation prior to the proximal radial formation together with the down-regulation of sonic hedgehog (shh) and patched (pta) expression, resulting in the loss of proximal radials. Thus, it was indicated that differentiation of the precursor cells of radials and lepidotrichia begins in the proximal part of the fin fold and that the initial mesenchymal condensation prior to the proximal radial formation is highly susceptible to the effects of RA. Lepidotrichia formation does not occur where proximal radials are absent, indicating that lepidotrichia differentiation requires interaction with the radial cartilages. To examine the suggestion that neural crest cells contribute to the medial fin skeletons, we localized the HNK-1 positive cells in flounder embryos and slug and msxb-positive cells in pufferfish, Fugu rubripes, embryos. That the positive cells commonly arrive at the proximal part of the fin fold does not contradict the suggestion, but their final destiny as radial chondrocytes or lepidotrichia scleroblasts, should be further investigated.

Although pancreatic serine proteases have been cloned in teleosts, no sequence data are currently available on members of the carboxypeptidase (CP) family. Here, we cloned cDNAs coding for two preproCPAs, corresponding to mammalian preproCPA1 and preproCPA2, and one preproCPB from a pancreatic cDNA library of the Japanese flounder, Paralichthys olivaceus . The activation peptides of flounder proCPs completely retained the sequences for inhibition of enzymatic activity of proCPs just like mammalian proCPs. Of 306–309 amino acids in total, 95 amino acids are completely conserved between bovine CPA1 and CPB and flounder CPs. Notably, amino acid residues for Zn 2+ ligands, catalysis and substrate anchoring are completely conserved between flounder and bovine CPs. Three species of flounder preproCPs are all expressed in the pancreas of first feeding larvae.

To identify the teleost gastrin, CCK/gastrin family genes were isolated from puffer and flounder. The cDNA of puffer gastrin, CCK1 and CCK2 were 678, 752 and 533 bp, respectively. Puffer gastrin gene consists of three exons, in contrast, CCKs consist of four exons. Flounder gastrin mRNA (526 bp) was expressed in the intestine but not in the brain. It was developed synchronously with the stomach differentiation in the larval stage. The phylogenetic analysis shows that puffer and flounder gastrin classified into the vertebrate gastrin cluster and two types of CCK were probably produced by the genome duplication occurred in teleost phylogeny.

As food protein digestion is instrumental for promoting growth, the main protease in young marine fish larvae, trypsin, was studied in pre-leptocephalus larvae of Anguilla japonica . Tryptic enzyme activity was monitored until day 24 and rates of growth until day 36 after hatch in larval A. japonica derived from artificially matured parent fish. Tryptic activity increased with larval age and developmental stage until day 16, after which tryptic levels stabilized. In start-feeding larvae, tryptic activities increased after ingestion of an artificial diet, but elevated tryptic activities could also be found in larvae with guts void of prey, possibly due to intestinal retention of trypsin secreted in response to ingestion of a previous meal. Gut retention time for trypsin was estimated to be at least 15 h. The gut evacuation time for the artificial diet depended on the meal size and was in the range of 1–5.5 h. Rates of larval growth in length were ca. 5% day −1 during the yolk-sac stage, declining to ca. 1% day −1 in older larvae. Vision was highly important for initiation of feeding. Results provide the first quantitative information on aspects of protein digestion in eel larvae.

We cloned a 1401-bp cDNA encoding a novel pancreatic protein (PPSB) with two cysteine-rich somatomedin B-like domains from Japanese flounder (Paralichthys olivaceus). PPSB is predicted to be composed of 385 amino acids, including a signal sequence. The peptide sequence shares high homology with human placental protein 11 (PP11) and mouse T-cell specific protein (Tcl-30), which both contain a single somatomedin B-like domain. PPSB shares 47% and 44% identity with PP11 and Tcl-30, respectively. The unique point of PPSB is that an additional, somatomedin B-like domain is tandemly inserted. Unlike PP11 and Tcl-30, PPSB mRNA is specifically expressed by the exocrine pancreatic acinar cells, together with trypsinogen. Since PP11 has serine protease activity, it is predicted that its teleost homologue, PPSB, may function as a pancreatic digestive enzyme.

To reveal the ontogeny of pancreatic exocrine function in the early larval stage of eel, cDNAs encoding major pancreatic enzymes, trypsinogen, amylase and lipase were identified from the Japanese eel Anguilla japonica and their expression pattern in larvae was analyzed. The cloned eel trypsinogen precursor consisted of 224 amino acids and showed 82.2% identity to trypsinogen-2 of winter flounder Pleuronectes americanus. The eel amylase precursor consisted of 512 amino acids and showed 77% identity to winter flounder amylase. Eel pancreatic lipase was composed of 470 amino acids and had 58.3% of identity to human pancreatic lipase. In the eel larvae, mRNA expression of trypsinogen and amylase was first detected at 6 days post-hatching (d.p.h.), and the expression level increased between 7 and 8 d.p.h. In contrast, mRNA expression of lipase was first detected at 8 d.p.h. Eel larvae start to feed actively at 8 d.p.h. Thus, it was indicated that eel pancreas starts to synthesize digestive enzymes at 6 d.p.h. and acquires full function by the onset of exogenous feeding at 8 d.p.h.

The appearance and distribution of cholecystokinin (CCK)-immunoreactive (IR) cells in the digestive tract of cultured developing larvae of bluefin tuna, Thunnus thynnus , were investigated by immunohistochemistry. The CCK-IR cells had an elongated triangular shape and a thin apex pointing towards the lumen of the gut. Very few weakly stained CCK-IR cells were found at hatching (0 days after hatching, DAH) and first feeding (3 DAH), while strongly stained CCK-IR cells were detected in all the larvae examined after 12 DAH. CCK-IR cells were scattered both in the pyloric caeca and the anterior intestine at 12 DAH, while the distribution was restricted to the pyloric caeca at 22 DAH. The relationships between the ontogeny of CCK and pancreatic enzymes are discussed.

To elucidate the physiological role of the calcitonin gene-related peptide (CGRP) in teleosts, the expressions of calcitonin gene-related peptide receptor (CGRPR) and CGRP mRNA were examined using the reverse transcription–polymerase chain reaction method in the gills of flounder during adaptation from seawater to freshwater. Another related receptor mRNA, which is estimated to be a calcitonin receptor (calcitonin receptor-like receptor; CTRLR), was also monitored in the gills. The expressions of CGRPR mRNA in the gills decreased after transferring flounder from seawater to dilute seawater and became undetectable in freshwater conditions. In contrast, CTRLR mRNA expression in the gills did not change during acclimation. Interestingly, CGRP mRNA was not detected in gills when flounder were transferred from seawater to dilute seawater or freshwater. Therefore, CGRP secreted from other tissue, such as the intestine, may act on the gills and play a role in the seawater adaptation of flounder.

cDNAs encoding three neuropeptide Y (NPY)-related peptides were identified from the Japanese flounder. One peptide corresponds to NPY, and its mRNA was expressed in the brain and retina. The second peptide corresponds to peptide YY (PYY), and its mRNA was expressed in the brain but surprisingly not in the digestive organs. This suggests that PYY lacks function as a digestive hormone in flounder. The third peptide corresponds to peptide Y (PY), and its mRNA occupied the brain and the endocrine cells of the intestine and the pancreatic accessory islets, but not of the principal islet. This suggests that PY has a dual role as a neuropeptide and a digestive hormone. In flounder larvae, PY cells appear in the intestinal epithelia at 3 days posthatching (dph), when larvae start feeding, in contrast to the endocrine pancreas at 30 dph. Therefore, the endocrine system of PY, which probably regulates the pancreatic enzyme secretion, develops synchronously with the start of feeding in the intestine but about 1 month later in the endocrine pancreas in flounder.

To better understand the digestive physiology and phylogeny of the pancreatic serine proteases of teleosts, we cloned trypsin, chymotrypsin and elastase from flounder (Paralichthys olivaceus). Fifty phage plaques randomly chosen from a flounder pancreatic cDNA library were found to contain three species of trypsin, two species of chymotrypsin and four species of elastase. cDNAs of two species of carboxypeptidase A, one carboxypeptidase B and lipase were also obtained. In total, 23 out of 24 digestive enzyme cDNAs were those of proteolytic enzymes. Such a high ratio of proteolytic enzyme cDNA in the pancreas may reflect the carnivorous feeding habits of flounder. A phylogenetic comparison of the peptide sequences of flounder enzymes with those of other teleosts and mammals suggested that duplication of trypsin, chymotrypsin and elastase occurred before the divergence of the ray finned fish. It is also hypothesized that functional descendants of both duplicated genes of elastase exist in the teleosts and mammals, whereas only one of the genes of trypsin and chymotrypsin gave rise to the functional descendants in the teleosts but not in the mammals.

A part of genomic DNA including the calcitonin gene-related peptide (CGRP) gene was cloned from flounder by the genome-walking method. The intron/exon boundary was predicted to occur exactly at the same position as in salmon. The 37-amino acid molecule coded by the region from the intron/exon boundary to the stop codon was preceded by a typical Lys-Arg cleavage signal and included a cleavage/amidation site common to the CGRP of other vertebrates. The predicted amino acid sequence of flounder CGRP had 78%, 78%, 78%, 81%, and 73–78% identity to that of salmon, cod, frog, chicken, and mammalian CGRPs, respectively. Among vertebrates, CGRP is more conserved than calcitonin (CT) because the identity of flounder CT to mammalian CTs is 31–50%. Expression analysis indicated that this hormone is synthesized in the brain, heart, intestine, testis, and ovary. Since we have previously shown that the CGRP receptor is expressed in these tissues, it is suggested that CGRP secreted from each tissue functions in a paracrine or autocrine manner.

The appearance and distribution of cholecystokinin (CCK)-producing cells were investigated in the digestive tract of developing larvae of cultured Atlantic halibut, Hippoglossus hippoglossus. The CCK-producing cells were detected immunohistochemically, by use of a primary antiserum against CCK cloned for the Japanese flounder, Paralichthys olivaceus. No CCK-immunoreactive (IR) cells were detected in first-feeding larvae (33 days after hatching, DAH). Forty-five DAH or 12 days after first feeding, there were a few scattered CCK-IR cells in the epithelium of the anterior midgut in about 30% of the examined larvae. All larvae older than 52 DAH had CCK-IR cells in the anterior midgut, particularly frequent in the most anterior region adjacent to the pyloric caeca. No CCK-IR cells were detected in the foregut, the hindgut, or the midgut posterior to the first curvature. The CCK-IR cells spanned the intestinal epithelium from the basal lamina to the lumen and were triangular in shape, with the nucleus in the basal part and a thin apex toward the lumen. The mechanisms controlling release of bile, pancreatic enzymes, and peristalsis during the 12 days between first feeding and the first detection of CCK-IR cells remain to be clarified.

Disulfiram causes undulation of the notochord and bending of pharyngeal cartilages in fish embryos. Using flounder embryos, this study aimed to elucidate which process of pharyngeal arch development was affected by the drug. Since disulfiram is known to block the synthesis of retinoic acid (RA) in vivo, we first examined whether the drug suppresses the expression of Hoxd-4 and shh, RA responsive genes related with the pharyngeal arch development. Disulfiram at a concentration which induces undulation of the notochord and bending of cartilage elements did not affect the expression of these genes. On the other hand, similar phenotypes of anomalies were found to be reproduced both in the notochord and pharyngeal cartilages by α, α′-dipyridyl which reduces the mechanical stability of collagen. Thus, we suppose that disulfiram causes anomalies by decreasing the mechanical stability of collagen and not by suppressing the expression of RA-responsive genes. Since disulfiram blocks ascorbate dehydrogenase...

To understand the developmental process of the endocrine system, which controls the pancreatic exocrine function in Japanese flounder, Paralichthys olivaceus, the expression patterns of cholecystokinin (CCK) and pancreatic polypeptide (PP) during the larval stage were analyzed by immunohistochemistry and in situ hybridization. Expression of CCK in the intestinal epithelia started at 2 days posthatching (dph), 1 day prior to the first feeding. Endocrine pancreatic cells containing insulin were present in the pancreas primodium at hatching, and these endocrine cells formed an islet at 3 dph and developed into the principle islet neighboring gallbladder at 20 dph. However, PP cells were not contained in the principle islet even after metamorphosis. PP cells were contained in the accessory islets differentiated around the proximal part of the pyloric appendages at 30 dph (early metamorphosis stage). Therefore, we infer that the stimulative regulation of pancreatic enzyme secretion by CCK starts to function at the first feeding, whereas the restrictive regulation by PP develops about 1 month later in flounder larvae. In addition, we observed that CCK immunoreactive cells appeared in the accessory islets at 30 dph, similar to PP cells, even though CCK mRNA expression could not be detected in cells from the islets. This indicates the possibility that a peptide that is cross-reacted with the CCK antibody, i.e., gastrin, is synthesized in the flounder islets.

The phenotypes of skeletal malformations induced in pharyngeal arches and pectoral fins of Japanese flounder larvae by retinoic acid (RA), disulfiram, 2,2′-dipyridyl and azetidine-2-carboxylic acid were characterized, RA controls gene expression essential for pharyngeal and pectoral fin development; disulfiram is an inhibitor of RA synthase, 2,2′-dipyridyl and azetidine-2-carboxylic acid are inhibitors of collagen synthesis. In larvae exposed to RA at shield stage for 1 h, the Meckel's cartilage did not form in mandible arch. Exposure to RA from hatching period shifted the growth direction of the pharyngeal cartilages posteriorly. Disulfiram did not affect the cartilage formation when given before hatching, even though it shortened the trunk. However, disulfiram exerted teratogenic effects when given after hatching time, inducing bending pharyngeal cartilages and S-shaped pectoral fin plate. 2,2′-Dipyridyl given from hatching also caused bending of pharyngeal cartilages and S-shaped pectoral fin plates. Azetidine-2-carboxylic acid reduced the size of cartilages, without causing remarkable malformation. Thus, it was demonstrated that both RA and inhibitor of its synthesis, and inhibitors of collagen synthesis exert specific teratogenic effects on both pharyngeal and pectoral skeletons of flounder larvae.