The role of cholecystokinin and peptide YY in feed intake in Atlantic halibut (Hippoglossus hippoglossus) larvae.

Control of appetite and feed intake in fish larvae are still largely unexplored. Two of the key players in controlling vertebrate's feed intake are cholecystokinin (CCK) and peptide YY (PYY). Here we investigated the mRNA expression of pyy, cck and cck receptors (cckr) in the brain (head) and gut of Atlantic halibut larvae in response to three consecutive meals. We used Artemia nauplii cysts that are commonly ingested by halibut larvae when present as inert feed, and three water-soluble extracts as attractants to stimulate appetite. Cyst intake was not affected by the use of attractants and overall ingestion rate was low. Differences in mRNA expression of cck and pyy were observed between the halibut larvae that had eaten and those that had not despite readily available feed (cysts), supporting that mechanisms for control of feed intake are at least partly functional. All genes analysed were present in the brain and gut, however the different expression profiles between paralogues suggest potential divergent functions. In the gut, cck2 and pyyb mRNA expression was significantly higher in the larvae that ate cysts compared to larvae that decided to not eat, indicating that these genes play a satiety function in the halibut larvae similar to the general vertebrate scheme. However, cck2, cck2r1, and pyy mRNA expression in the brain were lower in the fed-filled larvae group compared to larvae before eating, which contrasts with the presumable anorectic function of these genes. Further research is required to fully evaluate how PYY and CCK affect the feeding biology in halibut larvae, contributing to formulate inert diets that can stimulate appetite and feed intake.

Cholecystokinin receptors in Atlantic salmon: molecular cloning, gene expression, and structural basis.

The peptide hormone cholecystokinin (CCK) exerts a wide range of digestive and CNS-related physiological signaling via CCK receptors in brain and gut. There is very limited information available on these receptors in Atlantic salmon. The aim of this study was to characterize CCK receptors in gut and brain of salmon. We have identified and cloned one CCK-1 receptor and duplicates of CCK-2 receptor in salmon. The phylogenetic analysis indicates the existence of one common ancestor gene for all CCK receptors. CCK-1R mRNA is highly expressed in pancreas followed by midgut, hindgut, gallbladder, and stomach indicating an involvement in pancreatic regulation and gallbladder contractions. CCK-2R1/gastrin mRNA is expressed at high levels in midgut and at relatively low levels in stomach, gallbladder, and pancreas. We postulate CCK-2R1/gastrin receptor to have gastrin-related functions because of its distribution and abundance in gastro-intestinal (GI) tissues. CCK-2R2 is relatively abundant in brain but has low expression levels in gut tissues supporting the hypothesis for involvement in the gut-brain signaling. Major functional motifs and ligand interaction sites in salmon are conserved with that of mammals. This information will be instrumental for comparative studies and further targeting receptor activation and selectivity of biological responses of CCK in salmon.

Characterization of small, mononuclear blood cells from salmon having high phagocytic capacity and ability to differentiate into dendritic like cells.

Phagocytes are the principal component of the innate immune system, playing a key role in the clearance of foreign particles that include potential pathogens. In vertebrates, both neutrophils and mononuclear cells like monocytes, macrophages and dendritic cells are all professional phagocytes. In teleosts, B-lymphocytes also have potent phagocytic ability. We have isolated a population of small (<5 µm), mononuclear blood cells from Atlantic salmon (Salmo salar L.) not previously characterized. In order to identify them, we have performed morphological, gene expression, flow cytometry, cytochemical, ultrastructural and functional analyses. Interestingly, they highly express the gene encoding CD83, the most characteristic cell surface marker for dendritic cells in mammals, and MHC class II limited to professional antigen presenting cells. They did not express genes nor did they have cell markers for B-cells, T-cells, monocytes/macrophages or neutrophils as shown by qRT-PCR, flow cytometry and immunoblotting. A remarkable feature of these cells is their potent phagocytic capacity. Their oxygen-independent killing mechanism, as shown by intense acid phosphatase staining, is supported by lack of respiratory burst and myeloperoxidase activity and the acid phosphatase's sensitivity to tartrate. They show a high level of morphological plasticity, as, upon stimulation with mitogens, they change morphology and obtain branching protrusions similarly to dendritic cells. We suggest, based on our findings, that the small, round cells described here are progenitor cells with potential to differentiate into dendritic like cells, although we can not exclude the possibility that they represent a novel cell type.

Whole body proteome response to a dietary lysine imbalance in zebrafish Danio rerio.

Lysine (Lys) is an indispensable amino acid (AA) and is generally the first limiting AA in most vegetable proteins used in fish feeds. Lys availability may thus limit protein synthesis and accretion, and growth of fish. Metabolic effects of dietary Lys imbalance were examined by 2D-proteomics using zebrafish as model. The Control diet (Lys: 2.47 g kg(-1)) was based on zebrafish carcass AA profiles previously obtained. Two other experimental diets were deficient in Lys [Lys(-); 1.34 g kg(-1)] and Lys added in excess [Lys(+); 4.63 g kg(-1)]. Fish growth was monitored from 33 to 49 days post-fertilization and the whole body proteome screened by means of two-dimension gel electrophoresis and mass spectrometry. Growth rate was negatively affected in group Lys(-). Comparative proteomic analysis showed 45 spots differentially expressed among groups. Twenty-nine of these proteins were identified revealing proteins involved in muscle growth, energy and lipid metabolism, eye lens differentiation, chaperone activity and apoptosis. Lys deficiency is accompanied by a down-regulation of muscle proteins and up-regulation of proteins affected by fasting, energy deficit, growth arrest and apoptosis. Excess Lys was accompanied by an up-regulation of proteins related to glycolysis, steroidogenesis and sexual maturation.

The combined impact of plant-derived dietary ingredients and acute stress on the intestinal arachidonic acid cascade in Atlantic salmon (Salmo salar).

A study was conducted to assess the effect of substituting high levels of dietary fish oil (FO) and fishmeal (FM) for vegetable oil (VO) and plant protein (PP) on the intestinal arachidonic acid (AA) cascade in the carnivorous fish species Atlantic salmon. Four diets were fed to salmon over a period of 12 months, including a control FMFO diet, with varying replacements of plant-derived ingredients: 80 % PP and 35 % VO; 40 % PP and 70 % VO; 80 % PP and 70 %VO. Subsequently, fish were examined pre- (0 h) and post- (1 h) acute stress for blood parameters and intestinal bioactive lipidic mediators of inflammation (prostaglandins). Plasma cortisol responses were greatest in the FMFO group, while 80 % PP and 70 % VO fish exhibited increased plasma chloride concentrations. The n-3:n-6 PUFA ratio in intestinal glycerophospholipids from 70 % VO groups significantly decreased in both proximal and distal regions due to elevated levels of 18 : 2n-6 and the elongation/desaturation products 20 : 2n-6 and 20 : 3n-6. Increases in n-6 PUFA were not concomitant with increased AA, although the AA:EPA ratio did vary significantly. The 40 % PP and 70 % VO diet produced the highest intestinal AA:EPA ratio proximally, which coincided with a trend in elevated levels of PGF2alpha, PGE2 and 6-keto-PGF1alpha in response to stress. PGE2 predominated over PGF2alpha and 6-keto-PGF1alpha (stable metabolite of PGI2) with comparable concentrations in both intestinal regions. Cyclo-oxygenase-2 (COX-2) mRNA expression was an order of magnitude higher in distal intestine, compared with proximal, and was significantly up-regulated following stress. Furthermore, the 80 % PP and 70 % VO diet significantly amplified proximal COX-2 induction post-stress. Results demonstrate that high replacements with plant-derived dietary ingredients can enhance COX-2 induction and synthesis of pro-inflammatory eicosanoids in the intestine of salmon in response to acute physiological stress.

Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhua L.): cloning, tissue expression and comparative aspects.

A novel full-length cDNA that encodes for the Atlantic cod (Gadus morhua L.) PepT1-type oligopeptide transporter has been cloned. This cDNA (named codPepT1) was 2,838 bp long, with an open reading frame of 2,190 bp encoding a putative protein of 729 amino acids. Comparison of the predicted Atlantic cod PepT1 protein with zebrafish, bird and mammalian orthologs allowed detection of many structural features that are highly conserved among all the vertebrate proteins analysed, including (1) a larger than expected area of hydrophobic amino acids in close proximity to the N terminus; (2) a single highly conserved cAMP/cGMP-dependent protein kinase phosphorylation motif; (3) a large N-glycosylation-rich region within the large extracellular loop; and (4) a conserved and previously undescribed stretch of 8-12 amino acid residues within the large extracellular loop. Expression analysis at the mRNA level indicated that Atlantic cod PepT1 is mainly expressed at intestinal level, but that it is also present in kidney and spleen. Analysis of its regional distribution along the intestinal tract of the fish revealed that PepT1 is ubiquitously expressed in all segments beyond the stomach, including the pyloric caeca, and through the whole midgut. Only in the last segment, which included the hindgut, was there a lower expression. Atlantic cod PepT1, the second teleost fish PepT1-type transporter documented to date, will contribute to the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful tool for the study of gut functional regionalization, as well as a marker for the analysis of temporal and spatial expression during ontogeny.

FABP3 and FABP10 in Atlantic salmon (Salmo salar L.)--general effects of dietary fatty acid composition and life cycle variations.

The increased use of dietary plant oil supplementation combined with high dietary lipid loads challenges the lipid transport systems of cultivated fish species. Fatty acid binding proteins (FABPs) have been thoroughly studied as intracellular fatty acid transporters in vertebrates, but no data have been reported in Atlantic salmon. In the present study, comparative characterizations were performed, and dietary influence of plant oil supplementation on FABP3 and FABP10 expression was studied for several tissues in two separate dietary trials. In trial I, groups (6 fish each) were fed diets for 42 weeks (body mass 142+/-1 to 1463+/-83 g) (mean+/-S.D.), containing graded levels of rapeseed oil substituting for fish oil using a linear regression design. In trial II, groups (3 fish each) were fed 100% fish oil or 100% plant oil for 22 months (0.160+/-0.052 to 2523+/-590 g) (mean+/-S.D.) and sampled at regular intervals. Liver and muscle tissues appeared to express several FABPs possibly linked to different metabolic functions. FABPs mRNA expression did not change with dietary inclusion of 75% rapeseed oil, whereas FABP3 protein expression seemed to be affected by dietary rapeseed oil inclusion. Significant changes in red muscle FABP3 mRNA expression correlate to significant changes in total beta-oxidation capacity during the energy consuming process of smoltification.

Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon.

BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, beta-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1AA and EF1AB) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1AB>EF1AA>beta-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1AB>EF1AA>S20>beta-actin>18S rRNA>GAPDH. CONCLUSION: Overall, this work suggests that the EF1AA and EF1AB genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon.

Dietary rapeseed oil affects the expression of genes involved in hepatic lipid metabolism in Atlantic salmon (Salmo salar L.).

Supplies of marine fish oils (FO) are limited, and sustainable production in aquaculture dictates that alternatives that do not compromise fish health and product quality, such as vegetable oils, must be found. Nutrigenomics will increase our understanding of how nutrition influences metabolic pathways and homeostatic control, and may be used to measure and validate subtle changes in organ-specific, metabolic gene expression signatures. We compared 2 groups of Atlantic salmon fed diets containing 100% FO or 75% rapeseed oil (RO) for 42 wk. A small-scale cDNA microarray was constructed to screen for changes in the expression of lipid metabolism genes in the liver resulting from this partial substitution of RO for FO. Delta5 fatty acid desaturase gene expression was significantly greater in fish fed 75% RO than in fish fed the control diet; this was confirmed by quantitative real time PCR analysis. In addition, several genes, among these mitochondrial proteins, peroxisome proliferator-activated receptor gamma, as well as other transcription factors, coactivators, and signal transducers, showed significant differential regulation. This partially validated microarray may be used for further gene expression profiling using other dietary comparisons, and for further characterization of selected genes.