Michael V. Bell

Lipid nutrition of marine fish during early development: current status and future directions

Abstract Research on the dietary requirements of marine fish larvae has evolved from considerations of optimal dietary levels of n −3 HUFA to considerations of optimal dietary ratios of the two principal HUFAs, 22:6 n −3 and 20:5 n −3, and more recently to considerations of optimal dietary levels and ratios of all three dietary essential fatty acids, 22:6 n −3, 20:5 n −3 and 20:4 n −6. Our present understanding of the requirements and optimal dietary balance of 22:6 n −3, 20:5 n −3 and 20:4 n −6 is reviewed. Limitations of enriching live feed are considered, particularly from the point of view of achieving an optimal balance between levels of phospholipids and triacylglycerols in enriched live feeds that generate an optimal blend of essential fatty acids and energy-yielding fatty acids. It is concluded that the ideal marine fish larval diet is one containing circa 10% of the dry weight as n −3 HUFA-rich, marine phospholipids with less than 5% triacylglycerols, as exemplified by the lipid compositions of marine fish egg yolk, marine fish larvae themselves and their natural zooplankton prey. Such diets provide 22:6 n −3, 20:5 n −3 and 20:4 n −6 in the desired levels and ratios and simultaneously satisfy known requirements for phospholipids, inositol and choline. Approaches to developing marine fish larval diets more closely resembling this “gold standard” diet are considered.

The role of polyunsaturated fatty acids in fish

The physical properties of polyunsaturated and saturated fatty acids are compared in relation to melting points and fluidity. The role of polyunsaturated fatty acids on membrane fluidity and membrane bound enzyme activity is discussed. The influence of the environment, particularly temperature, on poikilothermic animals is considered in relation to membrane fatty acid composition and metabolism. The metabolic role of polyunsaturated fatty acids of the (n-3) series and their interaction with arachidonate metabolism is discussed.

Molecular species composition of the major diacyl glycerophospholipids from muscle, liver, retina and brain of cod (Gadus morhua)

The molecular species composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) from white muscle, liver, retina and brain of cod (Gadus morhua) were determined by high-performance liquid chromatography of the respective 1,2-diacylglycerol 3,5-dinitrobenzoyl derivatives. A minimum of 69 diacyl species was identified. In muscle and liver saturated fatty acid/polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid/PUFA molecular species were predominant, particularly 16∶0/20∶5 and 16∶0/22∶6 in PC, 16∶0/22∶6 and 18∶1/22∶6 in PE and 18∶0/22∶6 and 18∶1/22∶6 in PS. Didocosahexaenoyl species were major components of PC, PE and PS from retina, comprising 29.3, 71.8 and 59.7% of the respective totals. Didocosahexaenoyl species were also abundant in PE and PS from brain, accounting for 13.8 and 24.0% of the totals, respectively. DiPUFA species were important in muscle, totalling 21.2% in PC and 38.3% in PE. PC from all tissues had the largest amounts of species containing only saturated or monounsaturated fatty acids, accounting for 59.8% of PC from brain, including 12.8% of 18∶1/24∶1 plus 24∶1/18∶1.

Lipid conversions during enrichment of Artemia

Abstract Artemia nauplii were enriched for 24 h with radiolabelled fatty acid ethyl esters and then starved for a subsequent period of 24 h. Analyses of the distribution of radioactivity in lipids from samples taken at the end of the enrichment period and after the subsequent starvation showed that the ethyl esters were readily converted into other lipid classes, mainly triacylglycerols, during assimilation by the nauplii. The proportions of radioactivity recovered in free fatty acids and phospholipids increased during the starvation period indicating the mobilisation of fatty acids from triacylglycerols for use in catabolism and in the formation of biomembrane lipids. The distribution pattern of radioactivity from [U– 14 C ]22:6n−3 in the fatty acids of the nauplii demonstrates that Artemia are capable of retroconverting 22:6n−3 to 20:5n−3.

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n-3), to eicosapentaenoic acid, 20:5(n-3), in a cell line from the turbot, Scophthalmus maximus

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C18 to C20 polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n-3), and its elongation product, 20:4(n-3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 microM), U-14C (1 microM) or deuterated (d5; 25 microM) fatty acids. Stearidonic acid, 18:4(n-3), was metabolised to 20:5(n-3) in both cells lines, but more so in AS than in TF cells. Delta5 desaturation was more active in TF cells than in AS cells, whereas C18 to C20 elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n-3)) were produced by both cell lines, although there was significant production of 22:5(n-3) in both cultures, especially when 20:4(n-3) was supplemented. We conclude that limited elongation of C18 to C20 fatty acids rather than limited fatty acyl Delta5 desaturation accounts for the limited rate of conversion of 18:3(n-3) to 20:5(n-3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C18 to C20 PUFA by the turbot and the Atlantic salmon in vivo.

Fatty acid compositions of gonadal material and diets of the sea urchin, Psammechinus miliaris: trophic and nutritional implications

The fatty acid compositions of gonadal material was examined for the sea urchin Psammechinus miliaris (Gmelin) held in aquaria and fed either salmon feed pellets or the macroalga, Laminaria saccharina for 18 months. Gonadal material was also examined from P. miliaris collected from four field sites, including commercial scallop lines encrusted with the mussel, Mytilus edulis, sea cages stocked with Atlantic salmon Salmo salar and two intertidal sea-loch sites, characterised by either a fine mud or a macroalgal substratum. The fatty acid compositions of known and potential dietary material was examined. The proportions of certain fatty acids in the gonads of P. miliaris were significantly affected by diet type and location. Docosahexaenoic acid (DHA) 22:6 n-3 was significantly higher in the gonads of the sea urchins fed salmon feed in aquaria and collected from the salmon cages and scallop lines than in the gonads of the sea urchins fed L. saccharina in aquaria and collected from the intertidal sea loch sites. The salmon feed and the mussel tissue also contained a high proportion of this fatty acid. Stearidonic acid 18:4 n-3 and arachidonic acid 20:4 n-6, however, were found in significantly higher proportions than DHA in the gonads of the sea urchins fed L. saccharina and collected from the two intertidal sea-loch sites. L. saccharina was also found to contain high proportions of stearidonic and arachidonic acid. The gonads of the sea urchins collected from the intertidal site, characterised by a mud substratum, and from the scallop lines were found to contain a lower 18:1 n-9/18:1 n-7 ratio and a higher proportion of branched and odd-chained fatty acids, signifying a high dietary bacterial input, than the sea urchins held in the aquaria and collected from the salmon cage. 20:2 and 22:2 non-methylene-interrupted dienoic fatty acids (NMIDs) were found in P. miliaris fed diets lacking these fatty acids suggesting de novo biosynthesis. These results, therefore, suggest that the proportions/ratios of certain fatty acids in the gonads of P. miliaris could be used to give an indication of the predominant diet type of this species in the wild.

Changes in the fatty acid composition of phospholipids from turbot (Scophthalmus maximus) in relation to dietary polyunsaturated fatty acid deficiencies

Young turbot (1-20 g) were maintained for not less than 14 weeks on three diets: (1) a control diet containing normal amounts of polyunsaturated fatty acids (PUFA); (2) a diet totally deficient in PUFA; (3) a diet deficient in the (n-6) series of PUFA but containing (n-3) PUFA. At 14 weeks the fatty acid compositions of the phospholipids from liver, gut, gills and muscle were analysed. Large changes in the amounts of PUFA in the phospholipids were found. Fish maintained on the totally PUFA deficient diet 2 had retained arachidonic acid, 20:4(n-6), and docosahexaenoic acid, 22:6(n-3), at the expense of eicosapentaenoic acid, 20:5(n-3). Fish maintained on the (n-6) PUFA-deficient diet (3) contained decreased amounts of 20:4(n-6) and 22:6(n-3) while retaining 20:5(n-3). In all cases phosphatidylinositol had the lowest n-3/n-6 ratios. These results are discussed in terms of PUFA function.

Biosynthesis of polyunsaturated fatty acids in aquatic ecosystems: General pathways and new directions

It is now well established that the long-chain, omega-3 (ω3 or n-3) polyunsaturated fatty acids (PUFA) are vitally important in human nutrition, reflecting their particular roles in critical physiological processes (see Chap. 14). In comparison to terrestrial ecosystems, marine or freshwater ecosystems are characterised by relatively high levels of long-chain n-3PUFA and, indeed, fish are the most important source of these vital nutrients in the human food basket. Virtually all PUFA originate from primary producers but can be modified as they pass up the food chain. This is generally termed trophic upgrading, and various aspects of these phenomena have been described in Chaps. 2, 6 and 7 (this volume). However, while qualitative aspects of essential fatty acid production and requirements in aquatic ecosystems are relatively well understood, in order to fully understand and model ecosystems, quantitative information is needed on synthesis and turnover rates of n-3PUFA at different trophic levels in the food web. The present chapter describes the biochemistry and molecular biology involved in the various pathways of PUFA biosynthesis and interconversions in aquatic ecosystems.

Biosynthesis and tissue deposition of docosahexaenoic acid (22∶6n−3) in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) weighing ca. 5 g and previously acclimated for 8 wk on a diet comprising vegetable oil (11%), fish meal (5%), and casein (48%) as the major constituents were fed a pulse of diet containing deuterated (D5) (17,17,18,18,18)-18∶3n−3 ethyl ester. The synthesis and tissue distribution of D5-22∶6n−3 was determined 3,7,14, 24, and 35 d after the pulse. The whole-body accumulation of D5-22∶6n−3 was linear over the first 7 d, corresponding to a rate of 0.54±0.12 μg D5-22∶6n−3/g fish/mg D5-18∶3n−3 eaten/d. Maximal accretion of D5-22∶6n−3 was 4.3±1.2 μg/g fish/mg of D5-18∶3n−3 eaten after 14 d. The amount of D5-22∶6n−3 peaked in liver at day 7, in brain and eyes at day 24, and plateaued after day 14 in visceral and eye socket adipose tissue and in the whole fish. The majority of D5-22∶6n−3 was found in the carcass (remaining tissues minus the above tissues analyzed separately) at all times. On a per milligram lipid basis, liver and eyes had the highest concentration of D5-22∶6n−3. The experimental diet also contained 21∶4n−6 ethyl ester as a marker to estimate the amount of food eaten by individual fish. From such estimates it was calculated that the great majority of the D5-tracer was catabolized, with the combined recovery of D5-18∶3n−3 plus D5-22∶6n−3 being 2.6%. The recovery of 21∶4n−6 was 57.6%. The concentration of 22∶6n−3 in the fish decreased during the 13-wk period, and the amount of 22∶6n−3 synthesized from 18∶3n−3 was only about 5% of that obtained directly from the fish meal in the diet.

Lipid composition during growth of motile and coccolith forms of Emiliania huxleyi

Abstract Flagellated and coccolith cell types of Emiliania huxleyi were grown axenically and the lipid class and fatty acid compositions of the main polar lipid classes examined throughout the growth cycle. The lipid compositions of the two cell types were similar with only slight differences noted, the coccolith form tending to have higher levels of neutral lipids than the flagellate. Methyl and ethyl ketones were present in both cell types. The proportion of phospholipids and glycolipids increased during the log-phase, while neutral lipids (free fatty acids, triacylglycerols, ketones and hydrocarbons) achieved their highest levels in the late stationary-phase. The polar lipids of both cell types were very rich in polyunsaturated fatty acids (PUFA). In sulphoquinovosylglyercol + phosphatidylethanolamine, 18:3 n -3 and 18:4 n -3 were the predominant fatty acids totalling 42.4–55.7%, while 18:5 n -3 (33.1–79.2%) was the main fatty acid in digalactosyldiacylglycerols. This fatty acid was also predominant in monogalactosydiacylglycerols (36.6–57.7%), with lesser amounts of 18:4 n -3 and 22:6 n -3. Phosphatidylcholine was dominated by two molecular species, 22:6 n -3/22:6 n -3 and 14:0/22:6 n -3, accounting for 25.7–33.7 and 42.0–58.3%, respectively. Changes in PUFA composition over the growth cycle were small. The C 18 PUFA associated with thylakoid glycolipids tended to increase during the log-phase and decreased in the late stationary-phase while 22:6 n -3 peaked in the late stationary-phase. The results are discussed in relation to the ecology of this alga.