Roderick Nigel Finn

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.

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.

Physiological energetics of developing embryos and yolk-sac larvae of Atlantic cod (Gadus morhua). I. Respiration and nitrogen metabolism

This paper provides the basis for a general model of catabolic metabolism for developing embryos and yolk-sac larvae of Atlantic cod (Gadus morhua L.). Yolk-dependent routine rates of oxygen consumption, ammonia excretion, and accumulation of ammonium ions were related to quantitative changes in contents of glucose, glycogen, lactate, free amino acids, proteins and lipid classes (lipid classes published separately) in order to determine the rate and sequence of catabolic substrate oxidation that occurs with development at 6.0°C, 34.5‰ S. The stoichiometric relation of the oxygen consumption and total ammonia production to substrate utilisation indicated that during the first 2 to 3 d of development, glycogen was the sole substrate of oxidative metabolism. After formation of the syncytium, free amino acids (75%) together with polar lipids (13%, mainly phosphatidyl choline) and neutral lipids (9%, mainly triacylglycerol) comprised the metabolic fuels of embryonic development. Following hatch (Day 16 post fertilisation), the fuels were free amino acids (32%), polar lipids (20%, mainly phosphatidyl choline), neutral lipids (17%, mainly triacylglycerol) and proteins (31%). Thus, the catabolic metabolism of endogenously feeding Atlantic cod larvae was predominantly fuelled by amino acids (67%) and lipids (32%), while glycogen only accounted for 1% of the total enthalpy dissipated. It is proposed that the above sequence of catabolic substrate oxidation is also generally applicable to other cold-water fishes which spawn eggs that do not contain oil glubules.

Respiration, nitrogen and energy metabolism of developing yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.)

Abstract The present investigation quantifies the partitioning of yolk enthalpy (C) into production of biomass (P), dissipation due to metabolism (R) and losses associated with excretion of nitrogenous end products (E) during the endogenous nutrition of developing yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). Further, the sequence of catabolic substrate oxidation and the mass-specific metabolic rate in relation to development have been established in order to estimate the nutritional and enthalpy requirements of first-feeding larvae. The larvae grew exponentially (G = 6% · day−1, 1%·°day−1) and maintained positive energy balance until day 33 post-hatch (200°day). This point coincided with maximum tissue mass, protein content, standard length, myotome height and respiratory exertion of the larvae. During this phase, the larvae conserved 59–61% of yolk enthalpy for biomass deposition, and 35% was dissipated in metabolism, with only 4–6% being lost via nitrogenous excretion, Thereafter, the larvae experienced negative energy balance, and the excess metabolism was supported by proteolytic and lipolytic recruitment of somatic tissue. This resulted in an increased (14%) excretory component. For the first 2 weeks following hatch, amino acids from the rapidly declining free pool constituted the sole metabolic fuel. Free amino acids were also simultaneously polymerized into protein. Thereafter, protein-bound amino acids became the preferred substrate, joined initially by polar lipids (mainly phosphatidyl choline) and then by neutral lipids (mainly triacylglycerol). During the period of positive energy balance, free amino acids constituted 31%, proteinic amino acids 44%, polar lipids 21% and neutral lipids 3% of the substrates used to fuel metabolism. At the time of maximum tissue mass, the yolk-sac larvae showed a significantly higher rate of oxygen consumption in light compared with in darkness. Within the size range of 200–800 μg · ind−1, the rate of oxygen consumption scaled isometrically with dry body mass throughout development, such that the mass-specific aerobic metabolic rate at 7°C was maintained at 72 ± 13 μmol · g−1 · hr−1 in light, and 62 ± 9 μmol · g−1 · hr−1 in darkness. Using the aerobic metabolic rate of the light-adapted larvae, we estimate that first-feeding larvae of Atlantic halibut (at 7°C) demand an exogenous enthalpy at a rate of 110 J · g−1 · hr−1, (2.6 kJ · g−1 · day−1) in order to sustain an instantaneous growth rate of 6% · day−1.

Vertebrate Yolk Complexes and the Functional Implications of Phosvitins and Other Subdomains in Vitellogenins

Abstract In nonplacental or nontrophotenic vertebrates, early development depends on the maternal provision of egg yolk, which is mainly derived from large multidomain vitellogenin (Vtg) precursors. To reveal the molecular nature of the protein pools in vertebrate oocytes, published data on the N-termini of yolk proteins has been mapped to the deduced primary structures of their parent Vtgs. The available evidence shows that the primary cleavage sites of Vtgs are conserved, whereas the cleavage products exist as multidomain variants in the yolk protein pool. The serine-rich phosvitin (Pv) domains are linearly related to the molecular masses of the lipovitellin heavy chain. The 3-D localization of Pv maps to the outer edges of the Vtg monomer, where it is proposed to form amphipathic structures that loop up over the lipid pocket. At this locus, it is proposed that Pv stabilizes the nascent Vtg while it receives its lipid cargo, thereby facilitating the hepatic loading and locking of lipid within the Vtg (C-sheet)-(A-sheet)-(LvL) cavity, and enhances its solubility following secretion to the circulating plasma. The C-terminal regions of Vtgs are homologous to human von Willebrand factor type D domains (Vwfd), which are conserved cysteine-rich molecules with homologous regions that are prevalent in Vtgs, lipophorins, mucins, integrins, and zonadhesins. Unlike human VWFD, lower vertebrate Vwfds do not contain RGD motifs, which are associated with extracellular matrix binding. Although its function in Vtg is unknown, the lubricant properties associated with mucins and the cell adhesion properties associated with integrins and zonadhesins implicate Vwfd in the genesis of hemostatic platelet aggregation. Similarly, the proteolytic inhibitory properties associated with the binding of factor VIII in humans suggest that Vwfd stabilizes Vtg during passage in the systemic circulation.

The zebrafish genome encodes the largest vertebrate repertoire of functional aquaporins with dual paralogy and substrate specificities similar to mammals

BackgroundAquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown.ResultsThe screening and isolation of transcripts from the zebrafish (Danio rerio) genome revealed eighteen sequences structurally related to the four subfamilies of tetrapod aquaporins, i.e., aquaporins (AQP0, -1 and -4), water and glycerol transporters or aquaglyceroporins (Glps; AQP3 and AQP7-10), a water and urea transporter (AQP8), and two unorthodox aquaporins (AQP11 and -12). Phylogenetic analyses of nucleotide and deduced amino acid sequences demonstrated dual paralogy between teleost and human aquaporins. Three of the duplicated zebrafish isoforms have unlinked loci, two have linked loci, while DrAqp8 was found in triplicate across two chromosomes. Genomic sequencing, structural analysis, and maximum likelihood reconstruction, further revealed the presence of a putative pseudogene that displays hybrid exons similar to tetrapod AQP5 and -1. Ectopic expression of the cloned transcripts in Xenopus laevis oocytes demonstrated that zebrafish aquaporins and Glps transport water or water, glycerol and urea, respectively, whereas DrAqp11b and -12 were not functional in oocytes. Contrary to humans and some rodents, intrachromosomal duplicates of zebrafish AQP8 were water and urea permeable, while the genomic duplicate only transported water. All aquaporin transcripts were expressed in adult tissues and found to have divergent expression patterns. In some tissues, however, redundant expression of transcripts encoding two duplicated paralogs seems to occur.ConclusionThe zebrafish genome encodes the largest repertoire of functional vertebrate aquaporins with dual paralogy to human isoforms. Our data reveal an early and specific diversification of these integral membrane proteins at the root of the crown-clade of Teleostei. Despite the increase in gene copy number, zebrafish aquaporins mostly retain the substrate specificity characteristic of the tetrapod counterparts. Based upon the integration of phylogenetic, genomic and functional data we propose a new classification for the piscine aquaporin superfamily.

Piscine aquaporins: an overview of recent advances.

Aquaporins are a superfamily of integral membrane proteins that facilitate the rapid and yet highly selective flux of water and other small solutes across biological membranes. Since their discovery, they have been documented throughout the living biota, with the majority of research focusing on mammals and plants. Here, we review available data for piscine aquaporins, including Agnatha (jawless fish), Chondrichthyes (chimaeras, sharks, and rays), Dipnoi (lungfishes), and Teleostei (ray-finned bony fishes). Recent evidence suggests that the aquaporin superfamily has specifically expanded in the chordate lineage consequent to serial rounds of whole genome duplication, with teleost genomes harboring the largest number of paralogs. The selective retention and dichotomous clustering of most duplicated paralogs in Teleostei, with differential tissue expression profiles, implies that novel or specialized physiological functions may have evolved in this clade. The recently proposed new nomenclature of the piscine aquaporin superfamily is discussed in relation to the phylogenetic signal and genomic synteny, with the teleost aquaporin-8 paralogs used as a case study to illustrate disparities between the underlying codons, molecular phylogeny, and physical locus. Structural data indicate that piscine aquaporins display similar channel restriction residues found in the tetrapod counterparts, and hence their functional properties seem to be conserved. However, emerging evidence suggests that regulation of aquaporin function in teleosts may have diverged in some cases. Cell localization and experimental studies imply that the physiological roles of piscine aquaporins extend at least to osmoregulation, reproduction, and early development, although in most cases their specific functions remain to be elucidated.

The sequence of catabolic substrate oxidation and enthalpy balance of developing embryos and yolksac larvae of turbot (Scophthalmus maximus L.)

Abstract A model of catabolic metabolism during the endogenous developmental phase of turbot ( Scophthalmus maximus L.), a species whose pelagic eggs contain a single oil globule, is presented. Yolk-dependent routine rates of oxygen consumption and ammonia excretion and flux of ammonium ions were related stoichio-metrically to quantitative changes in the contents of free glucose, glycogen, lactate, free amino acids, proteins, and lipid classes with their associated fatty acids, in order to determine the rate and sequence of catabolic substrate oxidation that occurs with development. These data were further related to the changes in caloric contents of the eggs and larvae in order to derive an enthalpy balance equation for the period of yolk dependence. The stoichiometric relation of the oxygen consumption and ammonia production with the quantitative changes in substrates indicated that, following the first 18–19 hr of glycogen dependence, free amino acids (84%) together with a small amount of phosphatidyl choline (9%) and later wax esters (5%) comprised the metabolic fuels of embryonic development. Following hatch (day 4.4 post fertilisation), wax esters (33%) and triacylglycerols (25%) were initially catabolised with the remaining free amino acids (10%). Upon exhaustion of the free amino acids on day 6 post fertilisation, body proteins (32%) were recruited and catabolised together with wax esters and triacylglycerols. Thus, the catabolic metabolism of endogenously feeding turbot embryos and larvae were equally fuelled by amino acids (50%, with similar amounts being supplied from both the free and protein bound pools) and lipids (50%, mainly of neutral origin), while carbohydrates (predominantly glycogen) were only quantitatively important during the early cellular division stages. We argue that this pattern of catabolic substrate oxidation is also generally applicable to other marine fishes which spawn eggs containing oil globules. For an enthalpy balance equation of the form P = C + R + E, a turbot larva at 15°C, utilised 60% of yolk and oil globule enthalpy (C) for growth (P), 37% was dissipated due to metabolism (R) while only 3% was lost via excretion of nitrogenous end products (E).

The Maturational Disassembly and Differential Proteolysis of Paralogous Vitellogenins in a Marine Pelagophil Teleost: A Conserved Mechanism of Oocyte Hydration

Abstract A structural analysis of the differential proteolysis of vitellogenin (Vtg)-derived yolk proteins in the maturing oocytes of a marine teleost that spawns very large pelagic eggs is presented. Two full-length hepatic cDNAs (hhvtgAa and hhvtgAb) encoding paralogous vitellogenins (HhvtgAa and HhvtgAb) were cloned from nonestrogenized Atlantic halibut, and the N-termini of their subdomain structures were mapped to the oocyte and egg yolk proteins (Yps). The maturational oocyte Yp degradation products were further mapped to the free amino acid (FAA) pool in the ovulated egg. The deduced amino acid sequences conformed to the linear NH2-(LvH-Pv-LvL-beta′-CT)-COO− structure of complete teleost Vtgs. However, the Yps did not match the expected cleavage products of complete Vtgs. Specifically, the phosvitin subdomain of the HhvtgAa paralogue remains covalently attached to the lipovitellin light chain, while the phosvitin subdomain of the HhvtgAb paralogue remains covalently attached to a C-terminal fragment of the lipovitellin heavy chain (LvH). During oocyte hydration, the LvH of the HhvtgAa paralogue is disassembled and extensively degraded to FAA. In the HhvtgAb paralogue, the LvH is nicked in the C-sheet in a manner similar to that seen in lamprey and other teleosts. A small part of the C-teminal end of the LvH-Ab undergoes proteolysis to FAA, together with the phosvitin, beta′ component, and much (∼65%) of the lipovitellin light chain (LvL-Ab). The independently measured FAA pool in the ovulated egg corroborates that calculated from differential proteolysis of the Yps. Based on the 3:1 (HhvtgAb:HhvtgAa) Yp expression ratio, each paralogue contributes approximately equal amounts of FAA to the organic osmolyte pool of the hydrating oocyte during maturation.

Physiological energetics of developing embryos and yolk-sac larvae of Atlantic cod (Gadus morhua). II. Lipid metabolism and enthalpy balance

This paper presents quantitative data for the changes in the contents of total lipids, lipid classes and their associated fatty acids, together with the changes in caloric contents of developing eggs and yolk-sac larvae of Atlantic cod (Gadus morhua L.). During development between Day 0 to 28 post fertilisation, 32% of catabolic metabolism was fuelled by lipids. On a mass-specific basis, polar lipids (mainly phosphatidyl choline) contributed 60%, and neutral lipids (mainly triacyl-glycerol) contributed 40% to this catabolic component, with each class supplying similar amounts of fatty acids as fuel. The fatty acids supplied by phosphatidyl choline were catabolised non-selectively (i.e. in proportion to their presence in the egg), with about half of them being polyunsaturated. However, of the fatty acids esterified in triacylglycerol, the larvae showed an apparent oxidation preference for monoenes over polyunsaturates or saturates. Routine rates of oxygen consumption and ammonia production were related to the caloric contents of the eggs and larvae in order to derive an enthalpy balance equation (of the form P=C+R+E) for an Atlantic cod larva during its period of endogenous nutrition. For the interval of Day 0 to 25 post fertilisation (the period of yolk dependence), integration of the physiological and caloric data revealed that Atlantic cod larvae conserved 53% of yolk enthalpy (C) for growth (P), 42% was dissipated due to metabolism (R) while only 5% was lost via excretion (E).