Ivar Rønnestad

Fish larval nutrition: a review of recent advances in the roles of amino acids

Marine pelagic fish eggs from various latitudes contain up to 50% of the total amino acid pool as free amino acids (FAAs). The FAA pool is established during final oocyte maturation and seems to derive from the hydrolysis of a yolk protein. During yolk resorption, the FAA pool is depleted and reaches low levels at first feeding. The FAA are predominantly used as metabolic fuel, but they are also utilized for body protein synthesis. Amino acids are also important catabolic substrates after the onset of first feeding and may account for 60% or higher of the energy dissipation. Since growth is primarily an increase in body muscle mass by protein synthesis and accretion and fish larvae have very high growth rates, they have a high dietary requirement for amino acids. Fish larvae that develop stomachs late in development have a low proteolytic and absorptive capacities of the digestive systems at first feeding. In vivo studies have shown higher absorption of FAA than peptides and protein bound amino acids from the larval gut in the early stages of marine fish larvae. In the ocean, marine fish larvae obtain a large supply of FAA by consuming plankton after first feeding. The FAA composition of live feed used in aquaculture may to some extent be manipulated within rearing conditions and species and strain selection. While microdiets are a promising feed for larval fish, no satisfactory techniques have at present been developed that allows delivery of high contents of FAA. New techniques using liposomes have the potential to alleviate this problem.

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.

The supply of amino acids during early feeding stages of marine fish larvae: a review of recent findings

In marine fish larvae, the sum of protein deposition, turnover and catabolism necessary for their rapid growth dictates a high amino acid (AA) requirement. Once the yolk is exhausted, the digestive tract becomes the vital organ that ensures a steady supply of dietary AA to the growing larval tissues. In this paper, we discuss the demand and availability of AA (free and polymerised pools) in relation to larval digestive capacity. The sources of AA from compound and live diets are described, and the early regulatory roles of cholecystokinin (CCK) and a retrograde peristaltic activity are highlighted.

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.

Amino acid requirements of fish larvae and post-larvae: new tools and recent findings

This paper reviews methodologies and recent findings in the study of the amino acid (AA) metabolism of fish larvae and post-larvae, in order to better understand the AA requirements. The larval indispensable AA (IAA) profile can be used as index of the IAA requirements. When turbot larvae and live food IAA profiles are compared, the profile of the latter seems to be deficient in some IAA. However, the larval IAA profile is only a rough indicator of AA requirements. A more precise estimate of the ideal dietary IAA profile implies the knowledge of the relative bioavailabilities of the individual AA, in particular, eventual differential rates of absorption and catabolism. Metabolic budgets (including unabsorbed AA, AA oxidation and AA retention) can be estimated using an in vivo method based on controlled tube-feeding of AA mixes containing a 14C-labelled AA. Results with fasted post-larval Senegal sole (Solea senegalensis) and fasted herring (Clupea harengus) larvae show a high retention of labelled doses of IAA (>60%) in the body, compared to catabolism as measured by liberated 14CO2 ( 40%) and a lower retention (<57%). So, from the onset of exogenous feeding, fish larvae have high catabolic losses of AA, but use DAA preferentially to IAA as energy substrates. A new method combining the use of 13C-labelled live food and 13C-NMR spectroscopy can be used to study simultaneously the relative bioavailability of several individual AA in fish larvae. In larval gilthead seabream (Sparus aurata) fed on rotifers, relative bioavailabilities (a combined measure of absorption efficiency and rate of catabolism) vary between AA being high for aspartate, glutamate and lysine and low for threonine. These estimates of relative biovailability of individual AA together with the IAA profiles of the larval seabream indicate that rotifers are deficient in threonine and leucine for larval seabream, threonine being the first limiting AA for protein synthesis. In order to define ideal IAA profiles for larval fish, further studies are needed on the factors affecting the relative bioavailability of IAA, such as species, age, developmental stage, temperature and the dietary nitrogen molecular form(s). Estimates of relative bioavailability of individual AA together with the IAA profile of the larval protein allow to determine the ideal dietary IAA profile for a given species.

The importance of free amino acids to the energy metabolism of eggs and larvae of turbot (Scophthalmus maximus)

A study was undertaken to establish the role of free amino acids (FAA) in aerobic energy dissipation in embryos of turbot (Scophthalmus maximus) which contain an oil globule in the egg. Laboratory-reared developing eggs and larvae (15°C, 34‰ salinity) were measured for oxygen uptake, ammonia excretion, contents of FAA, protein, and ammonium, and volumes of yolksac and oil globule. Newly spawned eggs from different batches contained 55 to 90 nmol egg−1 of FAA. Resorption of FAA occurred in parallel with the consumption of yolk. Resorption of the oil globule, however, occurred predominantly after hatching and mainly after yolk resorption. The combined data suggest that approximately 70% of the FAA are utilized as an energy substrate, while the rest are polymerized into body proteins. FAA become a significant energy substrate in the early egg stage and account for 100% of the aerobic energy dissipation 2 d after Fertilization then decrease to ca. 60% at the time of hatching. Lipids derived from the oil globule seem to be the main fuel after hatching and account for ca. 90% of the energy dissipation at the onset of first-feeding. Thus, the energetics of fish embryos which contain an oil globule seems to be different from those that depend exclusively on the nutritional reserves of the yolk.

In vivo studies of digestion and nutrient assimilation in marine fish larvae

Abstract This paper describes a method for quantifying the functionality of the digestive system in fish larvae. The system described can provide data for the gut absorption, oxidation and retention (assimilation) of nutrients. A tube-feeding setup (originally described in Aquaculture 116 (1993) (341–352) using 14 C-labelled dietary nutrients formed the basis of a new incubation system. This permitted unabsorbed nutrients evacuated from the gut to be distinguished from molecules originating from catabolism of the absorbed nutrient, both of which are present in the incubation water. The system is based on the release, transfer and entrapment of metabolically produced 14 C–CO 2 through manipulation of the water pH. The efficiency of the trap has been validated and tested, and provides 100.0±1.3% (S.D.) recovery. The usefulness of the method is demonstrated in a study in which Atlantic halibut post-larvae (46 days post first feeding) were fed a 14 C-labelled protein diet. These data show that this protein has a digestibility of 42% for halibut post-larvae. If oxidation had not been measured through the use of the CO 2 trap, digestibility would have been greatly underestimated (at about 25%).

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.

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.

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.