Oil from transgenic Camelina sativa as a source of EPA and DHA in feed for European sea bass (Dicentrarchus labrax L.).

Aquaculture, the fastest growing food production sector cannot continue to rely on finite stocks of marine fish as the primary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n-3), for feeds. A four-month feeding trial was conducted to investigate the impact of a de novo oil, with high levels of EPA and DHA, obtained from transgenic Camelina sativa on growth performance, tissue fatty acid profiles, and expression of lipid metabolism genes when used as a replacement for fish oil in feed for European seabass (Dicentrachus labrax). Triplicate groups of 50 juvenile fish (initial weight 16.7 ± 0.92 g) per tank were fed for 4 months with one of three isolipidic and isoproteic experimental diets consisting of a standard diet containing a commercial blend of fish oil and rapeseed oil (CFO), a diet containing transgenic Camelina oil (TCO), or a blend of fish oil and rapeseed oil with enhanced levels of EPA and DHA (EFO) formulated to match the n-3 LC-PUFA profile of the TCO feed. Final weight of fish fed the GM-derived oil was not different to fish fed either CFO or EFO. Slight lower growth performance of fish fed TCO at the beginning of the trial was related to transient reduced feed intake, possibly caused by glucosinolates in the raw Camelina sativa oil. The GM-derived oil improved the nutritional quality of the fish fillet by enhancing total n-3 PUFA levels compared to the fish fed the other two feeds, and maintained flesh EPA and DHA at the same levels as in fish fed the diets containing fish oil. The metabolic response in liver and intestine was generally relatively mild although diets TCO and EFO seemed to trigger a metabolic response consisting of an up-regulation of both ß-oxidation (cpt1a) and fatty acid transport (fabp1), possibly reflecting higher levels of LC-PUFA. Overall, the present study indicated that an oil of terrestrial origin, Camelina sativa, when engineered to contain high levels of EPA and DHA can replace fish oil in feeds for European seabass with no detrimental impact on growth or feed efficiency, while also maintaining or increasing tissue n-3 LC-PUFA contents.

Growth and digestive enzyme activities of rohu labeo rohita fed diets containing macrophytes and almond oil-cake.

The impact of plant-based diets on the digestive physiology of rohu Labeo rohita fingerlings (10.66 ±â€¯0.53 g) was evaluated. A diet with all protein supplied by fishmeal was included as a control (F). Four test diets containing 300 g/kg protein were formulated using the following plant ingredients and fishmeal in a 1:1 blend: almond oil-cake Terminalia catappa (FTC), duckweed Lemna minor (FLM), water fern Salvania molesta (FSM) and combination of these three ingredients (FTCLMSM). The final body weight and specific growth rate were significantly higher in rohu fed diet FLM compared to the other treatments. Significantly lower feed conversion ratio in rohu fed diet FLM showed that diet was utilized efficiently in this feeding regime compared to the other diets. The composition of diets also influenced the digestive enzyme activities of the fish. Thus, amylase, trypsin and chymotrypsin activities were significantly higher in rohu fed diet FLM compared to the rohu fed the other diets. Protease activity was significantly higher in rohu fed diets FTC and F and lipase activity was significantly higher in rohu fed diet FTC compared to the rohu fed the other diets. The inclusion of raw duckweed in feed replaced 300 g/kg of dietary fishmeal without affecting growth.

Assessment of a land-locked Atlantic salmon (Salmo salar L.) population as a potential genetic resource with a focus on long-chain polyunsaturated fatty acid biosynthesis.

The natural food for Atlantic salmon (Salmo salar) in freshwater has relatively lower levels of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) than found in prey for post-smolt salmon in seawater. Land-locked salmon such as the Gullspång population feed exclusively on freshwater type lipids during its entire life cycle, a successful adaptation derived from divergent evolution. Studying land-locked populations may provide insights into the molecular and genetic control mechanisms that determine and regulate n-3 LC-PUFA biosynthesis and retention in Atlantic salmon. A two factorial study was performed comparing land-locked and farmed salmon parr fed diets formulated with fish or rapeseed oil for 8 weeks. The land-locked parr had higher capacity to synthesise n-3 LC-PUFA as indicated by higher expression and activity of desaturase and elongase enzymes. The data suggested that the land-locked salmon had reduced sensitivity to dietary fatty acid composition and that dietary docosahexaenoic acid (DHA) did not appear to suppress expression of LC-PUFA biosynthetic genes or activity of the biosynthesis pathway, probably an evolutionary adaptation to a natural diet lower in DHA. Increased biosynthetic activity did not translate to enhanced n-3 LC-PUFA contents in the flesh and diet was the only factor affecting this parameter. Additionally, high lipogenic and glycolytic potentials were found in land-locked salmon, together with decreased lipolysis which in turn could indicate increased use of carbohydrates as an energy source and a sparing of lipid.

Early nutritional programming affects liver transcriptome in diploid and triploid Atlantic salmon, Salmo salar.

BACKGROUND: To ensure sustainability of aquaculture, plant-based ingredients are being used in feeds to replace marine-derived products. However, plants contain secondary metabolites which can affect food intake and nutrient utilisation of fish. The application of nutritional stimuli during early development can induce long-term changes in animal physiology. Recently, we successfully used this approach to improve the utilisation of plant-based diets in diploid and triploid Atlantic salmon. In the present study we explored the molecular mechanisms occurring in the liver of salmon when challenged with a plant-based diet in order to determine the metabolic processes affected, and the effect of ploidy. RESULTS: Microarray analysis revealed that nutritional history had a major impact on the expression of genes. Key pathways of intermediary metabolism were up-regulated, including oxidative phosphorylation, pyruvate metabolism, TCA cycle, glycolysis and fatty acid metabolism. Other differentially expressed pathways affected by diet included protein processing in endoplasmic reticulum, RNA transport, endocytosis and purine metabolism. The interaction between diet and ploidy also had an effect on the hepatic transcriptome of salmon. The biological pathways with the highest number of genes affected by this interaction were related to gene transcription and translation, and cell processes such as proliferation, differentiation, communication and membrane trafficking. CONCLUSIONS: The present study revealed that nutritional programming induced changes in a large number of metabolic processes in Atlantic salmon, which may be associated with the improved fish performance and nutrient utilisation demonstrated previously. In addition, differences between diploid and triploid salmon were found, supporting recent data that indicate nutritional requirements of triploid salmon may differ from those of their diploid counterparts.

Microbial and genetically engineered oils as replacements for fish oil in aquaculture feeds.

As the global population grows more of our fish and seafood are being farmed. Fish are the main dietary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, but these cannot be produced in sufficient quantities as are now required for human health. Farmed fish have traditionally been fed a diet consisting of fishmeal and fish oil, rich in n-3 LC-PUFA. However, the increase in global aquaculture production has resulted in these finite and limited marine ingredients being replaced with sustainable alternatives of terrestrial origin that are devoid of n-3 LC-PUFA. Consequently, the nutritional value of the final product has been partially compromised with EPA and DHA levels both falling. Recent calls from the salmon industry for new sources of n-3 LC-PUFA have received significant commercial interest. Thus, this review explores the technologies being applied to produce de novo n-3 LC-PUFA sources, namely microalgae and genetically engineered oilseed crops, and how they may be used in aquafeeds to ensure that farmed fish remain a healthy component of the human diet.

Molecular mechanism of dietary phospholipid requirement of Atlantic salmon, Salmo salar, fry.

The phospholipid (PL) requirement in fish is revealed by enhanced performance when larvae are provided PL-enriched diets. To elucidate the molecular mechanism underlying PL requirement in Atlantic salmon, Salmo salar, were fed a minimal PL diet and tissue samples from major lipid metabolic sites were dissected from fry and parr. In silico analysis and cloning techniques demonstrated that salmon possess a full set of enzymes for the endogenous production of PL. The gene expression data indicated that major PL biosynthetic genes of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylinositol (PtdIns) display lower expression in intestine during the early developmental stage (fry). This is consistent with the hypothesis that the intestine of salmon is immature at the early developmental stage with limited capacity for endogenous PL biosynthesis. The results also indicate that intact PtdCho, PtdEtn and PtdIns are required in the diet at this stage. PtdCho and sphingomyelin constitute the predominant PL in chylomicrons, involved in the transport of dietary lipids from the intestine to the rest of the body. As sphingomyelin can be produced from PtdCho in intestine of fry, our findings suggest that supplementation of dietary PtdCho alone during early developmental stages of Atlantic salmon would be sufficient to promote chylomicron formation. This would support efficient transport of dietary lipids, including PL precursors, from the intestine to the liver where biosynthesis of PtdEtn, PtdSer, and PtdIns is not compromised as in intestine facilitating efficient utilisation of dietary energy and the endogenous production of membrane PL for the rapidly growing and developing animal.

Evaluation of a high-EPA oil from transgenic Camelina sativa in feeds for Atlantic salmon (Salmo salar L.): Effects on tissue fatty acid composition, histology and gene expression.

Currently, one alternative for dietary fish oil (FO) in aquafeeds is vegetable oils (VO) that are devoid of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFAs). Entirely new sources of n-3 LC-PUFA such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids through de novo production are a potential solution to fill the gap between supply and demand of these important nutrients. Camelina sativa was metabolically engineered to produce a seed oil (ECO) with > 20% EPA and its potential to substitute for FO in Atlantic salmon feeds was tested. Fish were fed with one of the three experimental diets containing FO, wild-type camelina oil (WCO) or ECO as the sole lipid sources for 7 weeks. Inclusion of ECO did not affect any of the performance parameters studied and enhanced apparent digestibility of individual n-6 and n-3 PUFA compared to dietary WCO. High levels of EPA were maintained in brain, liver and intestine (pyloric caeca), and levels of DPA and DHA were increased in liver and intestine of fish fed ECO compared to fish fed WCO likely due to increased LC-PUFA biosynthesis based on up-regulation of the genes. Fish fed ECO showed slight lipid accumulation within hepatocytes similar to that with WCO, although not significantly different to fish fed FO. The regulation of a small number of genes could be attributed to the specific effect of ECO (311 features) with metabolism being the most affected category. The EPA oil from transgenic Camelina (ECO) could be used as a substitute for FO, however it is a hybrid oil containing both FO (EPA) and VO (18:2n-6) fatty acid signatures that resulted in similarly mixed metabolic and physiological responses.

Effects of dietary fatty acids on mitochondrial phospholipid compositions, oxidative status and mitochondrial gene expression of zebrafish at different ages.

Mitochondrial decay is generally associated with impairment in the organelle bioenergetics function and increased oxidative stress, and it appears that deterioration of mitochondrial inner membrane phospholipids (PL) and accumulation of mitochondrial DNA (mtDNA) mutations are among the main mechanisms involved in this process. In the present study, mitochondrial membrane PL compositions, oxidative status (TBARS content and SOD activity) and mtDNA gene expression of muscle and liver were analyzed in zebrafish fed two diets with lipid supplied either by rapeseed oil (RO) or a blend 60:40 of RO and DHA500 TG oil (DHA). Two feeding trials were performed using zebrafish from the same population of two ages (8 and 21 months). Dietary FA composition affected fish growth in 8-month-old animals, which could be related to an increase in stress promoted by diet composition. Lipid peroxidation was considerably higher in mitochondria of 8-month-old zebrafish fed the DHA diet than in animals fed the RO diet. This could indicate higher oxidative damage to mitochondrial lipids, very likely due to increased incorporation of DHA in PL of mitochondrial membranes. Lipids would be among the first molecules affected by mitochondrial reactive oxygen species, and lipid peroxidation could propagate oxidative reactions that would damage other molecules, including mtDNA. Mitochondrial lipid peroxidation and gene expression of 21-month-old fish showed lower responsiveness to diet composition than those of younger fish. Differences found in the effect of diet composition on mitochondrial lipids between the two age groups could be indicating age-related changes in the ability to maintain structural homeostasis of mitochondrial membranes.

Roles of selenoprotein antioxidant protection in zebrafish, Danio rerio, subjected to dietary oxidative stress.

In vertebrates, selenium (Se) is an essential micronutrient for vertebrates that is involved in antioxidant protection and thyroid hormone regulation among other roles and functions through its incorporation into proteins, the selenoproteins. Long-chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are essential nutrients for fish although high dietary levels may lead to increased oxidative stress due to the high degree of unsaturation. The present study investigated the effects of Se supplementation on zebrafish, Danio rerio, oxidative status together with selenoprotein expression profiles when subjected to a high-DHA diet. Fish were fed for 8 weeks with one of the four experimental diets, containing high or low DHA in combination with or without organic Se (7 mg/kg). Fish performance, Se content, fatty acid composition and TBARS of zebrafish were determined, as well as gene expression of selected selenoproteins in liver and muscle. The Se levels in whole fish reflected dietary content. High dietary DHA increased oxidative stress as indicated by reduced growth and high TBARS content, although Se supplementation reduced oxidation. The expression patterns of selenoproteins varied between liver and muscle with only deiodinase type II displaying a transcriptional response when high dietary Se was supplied. High dietary DHA decreased selenoprotein W expression in muscle and sps2 expression in liver regardless of the dietary Se content. These data suggest that oxidative stress protection associated with a high dietary intake of Se may not be solely mediated by transcriptional changes in teleost selenoprotein expression.

Effect of salinity on the biosynthesis of n-3 long-chain polyunsaturated fatty acids in silverside Chirostoma estor.

The genus Chirostoma (silversides) belongs to the family Atherinopsidae, which contains around 150 species, most of which are marine. However, Mexican silverside (Chirostoma estor) is one of the few representatives of freshwater atherinopsids and is only found in some lakes of the Mexican Central Plateau. However, studies have shown that C. estor has improved survival, growth, and development when cultured in water conditions with increased salinity. In addition, C. estor displays an unusual fatty acid composition for a freshwater fish with high docosahexaenoic acid (DHA)/ eicosapentaenoic acid (EPA) ratios. Freshwater and marine fish species display very different essential fatty acid metabolism and requirements, and so the present study investigated long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis to determine the capacity of C. estor for endogenous production of EPA and DHA, and the effect that salinity has on these pathways. Briefly, C. estor were maintained at three salinities (0, 5, and 15 ppt), and the metabolism of ¹4C-labeled 18:3n-3 was determined in isolated hepatocyte and enterocyte cells. The results showed that C. estor has the capacity for endogenous biosynthesis of LC-PUFA from 18-carbon fatty acid precursors, but that the pathway was essentially only active in saline conditions with virtually no activity in cells isolated from fish grown in freshwater. The activity of the LC-PUFA biosynthesis pathway was also higher in cells isolated from fish at 15 ppt compared with fish at 5 ppt. The activity was around fivefold higher in hepatocytes compared with enterocytes; although the majority of 18:3n-3 was converted to 18:4n-3 and 20:4n-3 in hepatocytes, the proportions of 18:3n-3 converted to EPA and DHA were higher in enterocytes. The data were consistent with the hypothesis that conversion of EPA to DHA could contribute, at least in part, to the generally high DHA/EPA ratios observed in the tissue lipids of C. estor.

Desaturases and elongases involved in long-chain polyunsaturated fatty acid biosynthesis in aquatic animals: From genes to functions.

Marine ecosystems are rich in "omega-3" long-chain (C20-24) polyunsaturated fatty acids (LC-PUFA). Their production has been historically accepted to derive mostly from marine microbes. This long-standing dogma has been challenged recently by the discovery that numerous invertebrates, mostly with an aquatic life-style, have the enzyme machinery necessary for the de novo biosynthesis of polyunsaturated fatty acids (PUFA) and, from them, LC-PUFA. The key breakthrough was the detection in these animals of enzymes called "methyl-end desaturases" enabling PUFA de novo biosynthesis. Moreover, other enzymes with pivotal roles in LC-PUFA biosynthesis, including front-end desaturases and elongation of very long- chain fatty acids proteins, have been characterised in several non-vertebrate animal phyla. This review provides a comprehensive overview of the complement and functions of these gene/protein families in aquatic animals, particularly invertebrates and fish. Therefore, we expand and re-define our previous revision of the LC-PUFA biosynthetic enzymes present in chordates to animals as a whole, discussing how key genomic events have determined the diversity and distribution of desaturase and elongase genes in different taxa. We conclude that both invertebrates and fish display active, but markedly different, LC-PUFA biosynthetic gene networks that result from a complex evolutionary path combined with functional diversification and plasticity.

Investigation of highly unsaturated fatty acid metabolism in the Asian sea bass, Lates calcarifer.

Lates calcarifer, commonly known as the Asian sea bass or barramundi, is an interesting species that has great aquaculture potential in Asia including Malaysia and also Australia. We have investigated essential fatty acid metabolism in this species, focusing on the endogenous highly unsaturated fatty acid (HUFA) synthesis pathway using both biochemical and molecular biological approaches. Fatty acyl desaturase (Fad) and elongase (Elovl) cDNAs were cloned and functional characterization identified them as ∆6 Fad and Elovl5 elongase enzymes, respectively. The ∆6 Fad was equally active toward 18:3n-3 and 18:2n-6, and Elovl5 exhibited elongation activity for C18-20 and C20-22 elongation and a trace of C22-24 activity. The tissue profile of gene expression for ∆6 fad and elovl5 genes, showed brain to have the highest expression of both genes compared to all other tissues. The results of tissue fatty acid analysis showed that the brain contained more docosahexaenoic acid (DHA, 22:6n-3) than flesh, liver and intestine. The HUFA synthesis activity in isolated hepatocytes and enterocytes using [1-(14)C]18:3n-3 as substrate was very low with the only desaturated product detected being 18:4n-3. These findings indicate that L. calcarifer display an essential fatty acid pattern similar to other marine fish in that they appear unable to synthesize HUFA from C18 substrates. High expression of ∆6 fad and elovl5 genes in brain may indicate a role for these enzymes in maintaining high DHA levels in neural tissues through conversion of 20:5n-3.

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray.

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr-smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)-salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community.

Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/SGP microarray.

Interferons (IFNs) are cytokines that have proinflammatory, antiviral, and immunomodulatory effects and play a central role during a host response to pathogens. The IFN family contains both type I and type II molecules. While there are a number of type I IFNs, there is only one type II IFN. Recently both type I and type II IFN genes have been cloned in salmonid fish and recombinant proteins produced showing IFN activity. We have stimulated an Atlantic salmon cell line (SHK-1) with both type I and type II recombinant salmonid IFNs and analyzed the transcriptional response by microarray analysis. Cells were exposed to recombinant IFNs for 6 or 24 h or left unexposed as controls. RNA was hybridized to an Atlantic salmon cDNA microarray (salmon 17K feature TRAITS/SGP array) in order to assess differential gene expression in response to IFN exposure. For IFN I and II, 47 and 72 genes were stimulated, respectively; most genes were stimulated by a single IFN type, but some were affected by both IFNs, indicating coregulation of the IFN response in fish. Real-time PCR analysis was employed to confirm the microarray results for selected differentially expressed genes in both a cell line and primary leukocyte cultures.

Impact of sustainable feeds on omega-3 long-chain fatty acid levels in farmed Atlantic salmon, 2006-2015.

As the global population and its demand for seafood increases more of our fish will come from aquaculture. Farmed Atlantic salmon are a global commodity and, as an oily fish, contain a rich source of the health promoting long-chain omega-3 fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Replacing the traditional finite marine ingredients, fishmeal and fish oil, in farmed salmon diets with sustainable alternatives of terrestrial origin, devoid of EPA and DHA, presents a significant challenge for the aquaculture industry. By comparing the fatty acid composition of over 3,000 Scottish Atlantic salmon farmed between 2006 and 2015, we find that terrestrial fatty acids have significantly increased alongside a decrease in EPA and DHA levels. Consequently, the nutritional value of the final product is compromised requiring double portion sizes, as compared to 2006, in order to satisfy recommended EPA + DHA intake levels endorsed by health advisory organisations. Nevertheless, farmed Scottish salmon still delivers more EPA + DHA than most other fish species and all terrestrial livestock. Our findings highlight the global shortfall of EPA and DHA and the implications this has for the human consumer and examines the potential of microalgae and genetically modified crops as future sources of these important fatty acids.

Replacement of Marine Fish Oil with de novo Omega-3 Oils from Transgenic Camelina sativa in Feeds for Gilthead Sea Bream (Sparus aurata L.).

Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) are essential components of the diet of all vertebrates. The major dietary source of n-3 LC-PUFA for humans has been fish and seafood but, paradoxically, farmed fish are also reliant on marine fisheries for fish meal and fish oil (FO), traditionally major ingredients of aquafeeds. Currently, the only sustainable alternatives to FO are vegetable oils, which are rich in C18 PUFA, but devoid of the eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) abundant in FO. Two new n-3 LC-PUFA sources obtained from genetically modified (GM) Camelina sativa containing either EPA alone (ECO) or EPA and DHA (DCO) were compared to FO and wild-type camelina oil (WCO) in juvenile sea bream. Neither ECO nor DCO had any detrimental effects on fish performance, although final weight of ECO-fed fish (117 g) was slightly lower than that of FO- and DCO-fed fish (130 and 127 g, respectively). Inclusion of the GM-derived oils enhanced the n-3 LC-PUFA content in fish tissues compared to WCO, although limited biosynthesis was observed indicating accumulation of dietary fatty acids. The expression of genes involved in several lipid metabolic processes, as well as fish health and immune response, in both liver and anterior intestine were altered in fish fed the GM-derived oils. This showed a similar pattern to that observed in WCO-fed fish reflecting the hybrid fatty acid profile of the new oils. Overall the data indicated that the GM-derived oils could be suitable alternatives to dietary FO in sea bream.

Incorporation of [1-14C]arachidonic and [1-14C]eicosapentaenoic acids into the phospholipids of peripheral blood neutrophils from the plaice, Pleuronectes platessa L.

The incorporation of [1-14C]arachidonic acid and [1-14C]eicosapentaenoic acids into phospholipids was studied in peripheral blood neutrophils from plaice, a marine fish whose lipids are rich in (n-3) polyunsaturated fatty acids. The incorporation of both labelled fatty acids into phospholipids was approximately equal when presented individually, and at equal concentration, to the cells. Arachidonic acid was relatively preferred when both acids were present in the incubations. When incorporation was expressed per unit mass of phospholipid class, the order of incorporation was PI greater than PC greater than PE greater than PS greater than sphingomyelin for both fatty acids. However, the specificity for incorporation into PI was significantly greater with arachidonic acid. Eicosapentaenoic acid was incorporated into PC to a greater extent than arachidonic acid. The results are discussed in relation to the possible roles of arachidonic acid and eicosapentaenoic acid in polyunsaturated fatty acid metabolism in plaice neutrophils.