Assessment of genetic diversity in Coho salmon (Oncorhynchus kisutch) populations with no family records using ddRAD-seq.

OBJECTIVE: Selective breeding for desirable traits is becoming popular in aquaculture. In Miyagi prefecture, Japan, a selectively bred population of Coho salmon (Oncorhynchus kisutch) has been established with the original, randomly breeding population maintained separately. Since they have been bred without family records, the genetic diversity within these populations remains unknown. In this study, we estimated the genetic diversity and key quantitative genetic parameters such as heritability and genomic breeding value for body size traits by means of genomic best linear unbiased prediction to assess the genetic health of these populations. RESULTS: Ninety-nine and 83 females from the selective and random groups, respectively, were genotyped at 2350 putative SNPs by means of double digest restriction associated DNA sequencing. The genetic diversity in the selectively bred group was low, as were the estimated heritability and prediction accuracy for length and weight (h2 = 0.26-0.28; accuracy = 0.34), compared to the randomly bred group (h2 = 0.50-0.60; accuracy = 0.51-0.54). Although the tested sample size was small, these results suggest that further selection is difficult for the selectively bred population, while there is some potential for the randomly bred group, especially with the aid of genomic information.

Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food intake and stage specific effects on growth and fat allocation.

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.

Tetra-repeat microsatellite markers for the masu salmon (Oncorhynchus masou masou) and its application in cross-subspecies amplification.

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.

Complete mitochondrial DNA sequence of the ark shell Scapharca broughtonii: an ultra-large metazoan mitochondrial genome.

The complete mitochondrial (mt) genome of the ark shell Scapharca broughtonii was determined using long PCR and a genome walking sequencing strategy with genus-specific primers. The S. broughtonii mt genome (GenBank accession number AB729113) contained 12 protein-coding genes (the atp8 gene is missing, as in most bivalves), 2 ribosomal RNA genes, and 42 transfer tRNA genes, in a length of 46,985 nucleotides for the size of mtDNA with only one copy of the heteroplasmic tandem repeat (HTR) unit. Moreover the S. broughtonii mt genome shows size variation; these genomes ranged in size from about 47 kb to about 50 kb because of variation in the number of repeat sequences in the non-coding region. The mt-genome of S. broughtonii is, to date, the longest reported metazoan mtDNA sequence. Sequence duplication in non-coding region and the formation of HTR arrays were two of the factors responsible for the ultra-large size of this mt genome. All the tRNA genes were found within the S. broughtonii mt genome, unlike the other bivalves usually lacking one or more tRNA genes. Twelve additional specimens were used to analyze the patterns of tandem repeat arrays by PCR amplification and agarose electrophoresis. Each of the 12 specimens displayed extensive heteroplasmy and had 8-10 length variants. The motifs of the HTR arrays are about 353-362 bp and the number of repeats ranges from 1 to 11.

Distribution of pepsinogen- and ghrelin-producing cells in the digestive tract of Japanese eel (Anguilla japonica) during metamorphosis and the adult stage.

Pepsinogen is the precursor form of the gastric-specific digestive enzyme, pepsin. Ghrelin is a representative gastric hormone with multiple functions in vertebrates, including the regulation of growth hormone release, stimulation of food intake and gastrointestinal motility function. We investigated chronological changes in the distribution of pepsinogen-expressing cells by in situ hybridization and ghrelin-immunoreactive cells by immunohistochemistry in the Japanese eel (Anguilla japonica) during metamorphosis from the leptocephalus sage to the elver stage. The ghrelin-producing cells first appeared in the gastric cecum and pyloric portion of the stomach in the late phase of metamorphosing leptocephali, whereas the pepsinogen-producing cells were first detected in the early phase of the glass-eel stage. These suggest that endocrine cells differentiated earlier than exocrine cells in the eel stomach. Accompanying eel development, the distribution of ghrelin-producing cells spread to the esophagus and other regions of the stomach, but not to the intestine. These results may be related to the changes in dietary habits during metamorphosis in the Japanese eel.

Embryogenic staging of fugu, Takifugu rubripes, and expression profiles of aldh1a2, aldh1a3 and cyp26a1.

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.

Multiple cocaine- and amphetamine-regulated transcript (CART) genes in medaka, Oryzias latipes: cloning, tissue distribution and effect of starvation.

The neuropeptide cocaine- and amphetamine-regulated transcript (CART) is important in the regulation of food intake in mammals and fish. The tissue distributions of six CART cDNAs (cart ch3, ch4, ch6, ch9, ch11, and ch22) from medaka, Oryzias latipes, were cloned and the effect of starvation on their expression was examined. As in other species, medaka cart ch3, ch4, ch6, ch9, and ch22 consisted of three exons, while medaka cart ch11 contained four. The six cysteine residues at the C-terminal end of the CART motif and three-dimensional structure were well conserved in all medaka CART peptides. Tissue distribution analysis revealed that cart ch3, ch4, ch6, ch11, and ch22 were primarily expressed in the brain, but that the highest rates of cart ch9 expression occurred in the skin, suggesting different functions among the homologous genes. Although CART ch3 mRNA levels decreased in response to 17 days starvation, these levels were restored by re-feeding. However, the finding that the five other CART mRNAs did not respond to starvation suggests that only CART ch3 has an anorexigenic function in medaka.

Leptin reduces Atlantic salmon growth through the central pro-opiomelanocortin pathway.

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.

Ontogeny and distribution of cholecystokinin-immuno reactive cells in the digestive tract of sharpsnout sea bream, Diplodus puntazzo (Cetti, 1777), during larval development.

The appearance and regional distribution of cholecystokinin-immuno reactive cells (CCK-IR) in the developing gut of larval Diplodus puntazzo were studied by means of immunohistochemistry, with the aim of understanding the role of this peptide hormone in the acquisition of digestive capacity. Immunohistochemical reaction showed CCK-IR cells from 10 days after hatching (DAH), near the pyloric sphincter and past the first bend in the midgut, as well as in the hindgut. At 25 DAH CCK-IR cells were scattered throughout the midgut, as well as in the hindgut. Since gastric glands appeared at 30 DAH, CCK-IR cells were most abundant in the anterior midgut, near and including the pyloric caeca, and just afore the ileo-rectal sphincter in the posterior midgut, as well as in the hindgut. In older larvae (39 DAH), CCK-IR cells were mainly distributed in the anterior midgut, including the pyloric caeca, as well as in the hindgut. No CCK-IR cells were detected in the foregut at any stage. The distribution pattern of CCK-IR cells differed from other species which also possess a rotated gut as D. puntazzo. In fact, although cells were abundant in regions where the ingested food is retained, so that they can be stimulated to modulating the release of digestive enzymes, a large number of cells occurred also in the hindgut.

Leptin and leptin receptor genes in Atlantic salmon: Cloning, phylogeny, tissue distribution and expression correlated to long-term feeding status.

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 (p<0.05) 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 (p<0.01). 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.

Muscle development in the Japanese flounder, Paralichthys olivaceus, with special reference to some of the larval-specific muscles.

We investigated muscle development in the Japanese flounder Paralichthys olivaceus, focusing primarily on the cranial muscles, using a whole mount immunohistochemical staining method. It is well established that during the very early stages of morphogenesis, until 4 days post hatching (dph), muscles required for feeding develop. Later, between 8 and 16 dph, the muscle composition in the dorsal branchial arches changes to the adult form. We discovered the presence of larval-specific muscles in this ontogenetic period, termed the larval branchial levators 2 and 3, located in the dorsal branchial arches. The larval branchial levators 2 and 3 disappear during the course of development, whereas the others remain as levator internus 1 and levator posterior, which have also been described in adult fish. In place of these regressed muscles, the levatores externi and levator internus 2 develop and regulate the branchial arches. In addition, we found that the levator posterior, which is thought to represent the fifth levator externus, and the levatores externi exhibit different origins. We also found that at least a part of the caudal fin musculature develops from the trunk myotome.

Leptin and ghrelin in anadromous Arctic charr: cloning and change in expressions during a seasonal feeding cycle.

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.

Genomic characterization of multiple leptin genes and a leptin receptor gene in the Japanese medaka, Oryzias latipes.

We comprehensively surveyed leptin (LEP) and leptin receptor (LEPR) genes in medaka, Oryzias latipes and identified two LEP (mLEP-A and mLEP-B) genes and one LEPR (mLEPR) gene. The gene arrangement around both mLEPs in medaka chromosomes 6 and 23 were well conserved with human chromosome 7q31 including LEP. This means that both mLEP-A and mLEP-B are orthologs of human LEP and paralogs derived from whole-genome duplication early in the teleost lineage. The expression of mLEP-A mRNA was relatively high in the liver, and mLEP-B was expressed in the brain and eye. The 3-D modeling of both mLEP-A and mLEP-B protein showed conservation of the four-helix structure that is characteristic in vertebrate leptin. Human LEPR and leptin receptor overlapping the transcript (LEPROT) genes are continuously located on chromosome 1p31. In contrast, medaka LEPR and LEPROT are located on chromosomes 4 and 17, respectively, but both genomic regions showed genomic synteny with the human genome around the LEPR on chromosome 1p31. This result could mean that the medaka chromosome regions around the LEPR and LEPROT are paralogous genomic regions derived from whole-genome duplication, and that the overlapping gene of LEPR and LEPROT was subsequently lost in the medaka genome.

A homologous salmonid leptin radioimmunoassay indicates elevated plasma leptin levels during fasting of rainbow trout.

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.

Molecular cloning of endogenous beta-glucosidase from common Japanese brackish water clam Corbicula japonica.

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 (beta-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 beta-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 beta-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 beta-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 beta-glucosidase of C. japonica and is not derived from symbionts.

Characterization, tissue distribution, and regulation of agouti-related protein (AgRP), cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) in Atlantic salmon (Salmo salar).

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.

Feeding restriction alters expression of some ATP related genes more sensitively than the RNA/DNA ratio in zebrafish, Danio rerio.

The growth rate of fish shows extensive plasticity in response to various environments. Metabolic responses of fish to excessive nutritional shortages such as starvation have been reported, but the effects of moderate nutrient shortage remain unclear. We examined expression levels of some genes related to ATP metabolism and to myogenesis, the RNA/DNA ratio, and the protein/DNA ratio of fish under different feeding conditions: a diet of 212-432% (frequent feeding, FR) or 32-82% (restricted feeding, RE) of initial body weight per week was supplied. The expression levels of nucleoside diphosphate kinase (NDK)-Z2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and myogenin genes of RE fish were higher than those of FR fish, although the RNA/DNA ratio and the protein/DNA ratio were unaffected by the feeding amount. Moreover, expression levels of NDK-Z2 and GAPDH were upregulated to a greater extent than those for myogenin and myostatin 1 under restricted feeding. Together, our results show that gene expression is more sensitive to nutrient conditions of fish than traditional indicators such as the RNA/DNA ratio. The ATP metabolic system is more sensitive to moderate nutrient shortages than the myogenic system.

Ghrelin, cholecystokinin, and peptide YY in Atlantic salmon (Salmo salar): molecular cloning and tissue expression.

Gastrointestinal (GI) peptide hormones, ghrelin (GHRL), cholecystokinin (CCK), and peptide YY (PYY) genes were identified in Atlantic salmon, Salmo salar. Full-length cDNAs encoding two isoforms of GHRL (GHRL-1 and GHRL-2), two isoforms of CCK (CCK-L and CCK-N) and peptide YY (PYY) cDNA were obtained. The GHRL-1 and GHRL-2 genes encoded proteins of 111- and 108-amino acids, respectively. Both types of GHRL were mainly expressed in the stomach, but also weakly expressed in the pyloric caeca, mid-gut, adipose tissue, and testis. The CCK-L and CCK-N genes encoded preproproteins of 132- and 140-amino acids, respectively. Both types of CCK were strongly expressed in the brain and comparatively weakly expressed in other tissues, including the digestive tract. In the digestive tract, CCK-L was mainly expressed in the pyloric caeca and hind-gut, while CCK-N was only expressed in the pyloric caeca. The PYY gene encoded for 97-amino acid residues and was mainly expressed in the brain and anterior part of the intestine, including the pyloric caeca. In an experiment, we demonstrated that 6 days starvation led to, increased GHRL-1 mRNA levels in the GI tract (stomach), while there no significant changes in expression levels for the other hormones in the GI tract. This suggests an orexigenic role for GHRL-1 in Atlantic salmon. These data contribute to elucidate the functional relationships among teleost gastrointestinal peptide hormones.

Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss).

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.

Nutrient control of release of pancreatic enzymes in yellowtail (Seriola quinqueradiata): involvement of CCK and PY in the regulatory loop.

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.