Long-chain (≥C20) Polyunsaturated Fatty Acids Research Articles

Proximate and fatty acids composition in the muscle of wild and farmed sardine (Sardinella brasiliensis)

Wild sardines contain a high level (2560 mg/100 g n-3 LC-PUFA of wet mass) of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), namely, PUFA with 20 and 22 carbon atoms, however, in captivity their fatty acid composition can be affected by food and farming conditions. Therefore, this study evaluated the proximate composition and fatty acid profile of sardines cultivated in the four seasons of the year as well as compared to those captured in the wild in the spring. In both analyses, farmed sardines differed significantly from wild specimens. Total lipid content was significantly higher, especially in winter, with 14.52 % and 2.37 % for farmed and wild sardines, respectively. The fatty acid profile had a prevalence of n-3 LC-PUFA in wild fish (17.6 %) and n-6 PUFA ​​in farmed fish (12.2 %). LC-PUFA showed higher relative levels in wild animals, 16.4 % against 1.2 % for farmed sardines in winter. In absolute terms, the amount of EPA (eicosapentaenoicacid) and DHA (docosahexaenoicacid) in farmed sardine was similar to the wild specimens in spring 4000 mg/100 g and 3000 mg/100 g, respectively.

Dietary LC-PUFA and environmental salinity modulate the fatty acid biosynthesis capacity of the euryhaline teleost thicklip grey mullet (Chelon labrosus).

The capacity to biosynthesise long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) depends upon the complement and function of key enzymes commonly known as fatty acyl desaturases and elongases. The presence of a Δ5/Δ6 desaturase enabling the biosynthesis of docosahexaenoic acid (22:6n-3, DHA) through the "Sprecher pathway" has been reported in Chelon labrosus. Research in other teleosts have demonstrated that LC-PUFA biosynthesis can be modulated by diet and ambient salinity. The present study aimed to assess the combined effects of partial dietary replacement of fish oil (FO) by vegetable oil (VO) and reduced ambient salinity (35ppt vs 20ppt) on the fatty acid composition of muscle, enterocytes and hepatocytes of C. labrosus juveniles. Moreover, the enzymatic activity over radiolabelled [1-14C] 18:3n-3 (α-linolenic acid, ALA) and [1-14C] 20:5n-3 (eicosapentaenoic acid, EPA) to biosynthesise n-3 LC-PUFA in hepatocytes and enterocytes, and the gene regulation of the C. labrosus fatty acid desaturase-2 (fads2) and elongation of very long chain fatty acids protein 5 (elovl5) in liver and intestine was also investigated. Recovery of radiolabelled products including stearidonic acid (18:4n-3, SDA), 20:5n-3, tetracosahexaenoic acid (24:6n-3, THA) and 22:6n-3 in all treatments except FO35-fish, provided compelling evidence that a complete pathway enabling the biosynthesis of EPA and DHA from ALA is present and active in C. labrosus. Low salinity conditions upregulated fads2 in hepatocytes and elovl5 in both cell types, regardless of dietary composition. Interestingly, FO20-fish showed the highest amount of n-3 LC-PUFA in muscle, while no differences in VO-fish reared at both salinities were found. These results demonstrate a compensatory capacity of C. labrosus to biosynthesise n-3 LC-PUFA under reduced dietary supply, and emphasise the potential of low salinity conditions to stimulate this pathway in euryhaline fish.

Functional characterization reveals a diverse array of metazoan fatty acid biosynthesis genes.

Long-chain (≥C20 ) polyunsaturated fatty acids (LC-PUFAs) are physiologically important fatty acids for most animals, including humans. Although most LC-PUFA production occurs in aquatic primary producers such as microalgae, recent research indicates the ability of certain groups of (mainly marine) invertebrates for endogenous LC-PUFA biosynthesis and/or bioconversion from dietary precursors. The genetic pathways for and mechanisms behind LC-PUFA biosynthesis remain unknown in many invertebrates to date, especially in non-model species. However, the numerous genomic and transcriptomic resources currently available can contribute to our knowledge of the LC-PUFA biosynthetic capabilities of metazoans. Within our previously generated transcriptome of the benthic harpacticoid copepod Platychelipus littoralis, we detected expression of one methyl-end desaturase, one front-end desaturase, and seven elongases, key enzymes responsible for LC-PUFA biosynthesis. To demonstrate their functionality, we characterized eight of them using heterologous expression in yeast. The P. littoralis methyl-end desaturase has Δ15/17/19 desaturation activity, enabling biosynthesis of α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid (DHA) from 18:2 n-6, 20:4 n-6 and 22:5 n-6, respectively. Its front-end desaturase has Δ4 desaturation activity from 22:5 n-3 to DHA, implying that P. littoralis has multiple pathways to produce this physiologically important fatty acid. All studied P. littoralis elongases possess varying degrees of elongation activity for saturated and unsaturated fatty acids, producing aliphatic hydrocarbon chains with lengths of up to 30 carbons. Our investigation revealed a functionally diverse range of fatty acid biosynthesis genes in copepods, which highlights the need to scrutinize the role that primary consumers could perform in providing essential nutrients to upper trophic levels.

Long-chain polyunsaturated fatty acid biosynthesis in a land-crab with advanced terrestrial adaptations: Molecular cloning and functional characterization of two fatty acyl elongases.

Depending on the presence and activities of the front-end fatty acyl desaturases and elongation of very long-chain fatty acid (Elovl) enzymes, animals have different capacities for long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis. Successful land colonisation in brachyuran crabs requires a shift towards terrestrial food chain with limited LC-PUFA availability. We cloned and functionally characterised two elovl genes from the purple land crab Gecarcoidea lalandii. The two Elovl contained all the necessary motifs of a typical polyunsaturated fatty acids (PUFA) Elovl and phylogenetically clustered in the Elovl1 and Elovl6 clades, respectively. The G. lalandii Elovl1 elongated saturated fatty acids, with low activities towards C20 and C22 PUFA substrates. Moreover, the G. lalandii Elovl6 was particularly active in the elongation of C18 PUFA, although it also recognised monounsaturated fatty acids as substrates for elongation. Collectively, the herein characterised G. lalandii elovl paralogues fulfil all the elongation steps involved in the LC-PUFA biosynthetic pathways. Tissue distribution of the G. lalandii elovl genes, along with the FA composition analyses, suggest the hepatopancreas and gill as key metabolic sites for fatty acid elongation. However, current data suggest that G. lalandii is unable to rely solely on biosynthesis to fulfil LC-PUFA requirements, since front-end desaturase appears to be absent in this species and other decapods.

Effects of diet and temperature on the fatty acid composition of the gammarid Gammarus locusta fed alternative terrestrial feeds

The fast and remarkable growth of global aquaculture in recent years has created new challenges, such as guaranteeing a sustainable supply of raw materials used for aquafeed formulation. Gammarids are low-trophic crustaceans with an increasing interest in aquaculture due to their high nutritional profiles and their capacity to grow under high-density conditions. Moreover, gammarids have the ability to thrive on a wide range of sidestreams while accumulating relatively high levels of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA). In the present study, juveniles of the marine gammarid Gammarus locusta were cultured at four different temperatures (5°C, 10°C, 15°C, and 20°C) for 21 days and fed three diets, including the seaweed Fucus sp. as control, and carrot leaves and coconut flesh representing two agri-food industry sidestreams. Our results indicate that both the survival and biomass of G. locusta were highly affected by diet, with coconut showing the lowest growth performance. The temperature had no effect on biomass, although high temperature (20°C) resulted in a decrease in survival. The effects of temperature on the gammarid fatty acids were not evident, with diet being the main modulator of the profiles. Furthermore, the results also reveal that the Fucus sp. diet was associated with relatively high percentages of n-3 and n-6 LC-PUFA. Interestingly, essential LC-PUFA such as eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) acids were detected in gammarids fed on either Fucus sp. or any of the sidestreams irrespectively of their presence in the diets. These results suggest an ability of G. locusta for LC-PUFA biosynthesis (trophic upgrading) and/or retention, making this species a promising candidate for the production of high-value ingredients for aquafeeds.

Insulin activates LC-PUFA biosynthesis of hepatocytes by regulating the PI3K/Akt/mTOR/Srebp1 pathway in teleost Siganus canaliculatus.

Insulin is well known an important metabolic regulator in glucose and lipid metabolism. It has been proved to activate long-chain (≥ C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis in mammals, but little is known about such a role in fish. To explore the effects and molecular mechanisms of insulin in fish LC-PUFA biosynthesis, we treated the rabbitfish S. canaliculatus hepatocyte line (SCHL) cells with 65nM insulin for 12h, and the results showed that the mRNA levels of genes encoding the key enzymes and transcription factor involved in rabbitfish LC-PUFA biosynthesis such as Δ6Δ5 fads2, elovl5 and srebp1, as well as those of PI3K pathway genes including pdk1, akt2 and mtor increased significantly. Moreover, SCHL cells treated with different PI3K/Akt pathway inhibitors (LY294002, Wortmannin, AKTi-1/2) alone or combined with insulin decreased the mRNA levels of PI3K/Akt/mTOR downstream signaling genes, including Δ6Δ5 fads2, Δ4 fads2, elovl5, elovl4 and srebp1. While PI3K/Akt agonists (740 Y-P, IGF-1, SC-79) had the opposite results. The results of fatty acid composition analysis of hepatocytes showed that insulin stimulation increased the Δ6Δ5 Fads2-dependent PUFA desaturation indexes, while Elovl5-dependent PUFA elongation indexes had upward trends, and consequently LC-PUFA contents increased. Taken together, these results indicated that insulin activated LC-PUFA biosynthesis probably through PI3K/Akt/mTOR/Srebp1 pathway in S. canaliculatus hepatocytes.

Lack of ∆5 Desaturase Activity Impairs EPA and DHA Synthesis in Fish Cells from Red Sea Bream and Japanese Flounder.

Long-chain (≥ C20) polyunsaturated fatty acids (LC-PUFA), such as eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), are necessary for human health and are obtained from marine fish-derived oils. Marine fish are LC-PUFA-rich animals; however, many of them require LC-PUFA for growth. Therefore, it is suggested that they do not have sufficient ability to biosynthesize LC-PUFA. To evaluate in vivo LC-PUFA synthetic activity in fish cells, fish-derived cell lines from red sea bream (Pagrus major, PMS and PMF), Japanese flounder (Paralichthys olivaceus, HINAE), and zebrafish (Danio rerio, BRF41) were incubated with n-3 fatty acids labeled by radioisotopes or stable isotopes, and then, n-3 PUFA were analyzed by thin-layer chromatography or liquid chromatography-mass spectrometry. Labeled EPA and DHA were biosynthesized from labeled α-linolenic acid (18:3n-3) in BRF41, whereas they were not detected in PMS, PMF, or HINAE cells. We next cloned the fatty acid desaturase 2 (Fads2) cDNAs from PMF cells and zebrafish, expressed in budding yeasts, and then analyzed the substrate specificities of enzymes. As a result, we found that Fads2 from PMF cells was a ∆6/∆8 desaturase. Collectively, our study indicates that cell lines from red sea bream and Japanese flounder were not able to synthesize EPA or DHA by themselves, possibly due to the lack of ∆5 desaturase activity. Furthermore, this study provides a sensitive and reproducible non-radioactive method for evaluating LC-PUFA synthesis in fish cells using a stable isotope and liquid chromatography-mass spectrometry.

Expression of long-chain polyunsaturated fatty acids biosynthesis genes during the early life-cycle stages of the tropical gar Atractosteus tropicus.

Long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), including eicosapentaenoic acid (EPA, 20:5n-3), arachidonic acid (ARA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3), are essential in multiple physiological processes, especially during early development of vertebrates. LC-PUFA biosynthesis is achieved by two key families of enzymes, fatty acyl desaturases (Fads) and elongation of very long-chain fatty acid (Elovl). The present study determined the expression patterns of genes encoding desaturases (fads1 and fads2) and elongases (elovl2 and elovl5) involved in the LC-PUFA biosynthesis during early life-stages of the tropical gar Atractosteus tropicus. We further analyzed the fatty acid profiles during early development of A. tropicus to evaluate the impact of Fads and Elovl enzymatic activities. Specific oligonucleotides were designed from A. tropicus transcriptome to perform qPCR (quantitative polymerase chain reaction) on embryonic and larval stages, along with several organs (intestine, white muscle, brain, liver, heart, mesenteric adipose, kidney, gill, swim bladder, stomach, and spleen) collected from juvenile specimens. Fatty acid content of feeds and embryonic and larval stages were analyzed. Results show that fads1, fads2, elovl2 and elovl5 expression was detected from embryonic stages with expression peaks from day 15 post hatching, which could be related to transcriptional and dietary factors. Moreover, fads1, fads2 and elovl2 showed a higher expression in intestine, while elovl5 showed a higher expression in liver, suggesting that the tropical gar activates its LC-PUFA biosynthetic machinery to produce ARA, EPA and DHA to satisfy physiological demands at crucial developmental milestones during early development.

Seasonal and site-specific variation in the nutritional quality of temperate seaweed assemblages: implications for grazing invertebrates and the commercial exploitation of seaweeds

In coastal ecosystems, seaweeds provide habitat and a food source for a variety of species including herbivores of commercial importance. In these systems seaweeds are the ultimate source of energy with any changes in the seaweeds invariably affecting species of higher trophic levels. Seaweeds are rich sources of nutritionally important compounds such as polyunsaturated fatty acids (PUFA) and are particularly rich in long-chain (≥ C20) PUFA (LC-PUFA). In southern Australia, the ‘Great Southern Reef’ has one of the most diverse assemblages of seaweeds in the world, which support highly productive fisheries and have been recognised as a promising resource of omega-3 LC-PUFA. Despite this, there is little information on the biochemical composition of most species and how it varies between sites and seasons. To address this knowledge gap, we undertook a survey to assess seasonal variability in the biochemical composition (fatty acids and nitrogen content) of abundant understory seaweeds across three sites in eastern Tasmania. The availability of nutritional compounds differed between sites and was primarily driven by differences in the biomass and the biochemical composition of the nutritious red seaweeds at each site. This variability may explain regional differences in the productivity of commercial fisheries. At the species level, seasonal changes in fatty acid composition were highly variable between species and sites, indicating that multiple environmental drivers influence fatty acid composition of seaweeds in this system. This finding suggests that commercial harvest of seaweeds from eastern Tasmania will need to consider species and site-specific variability in fatty acid composition.

Sp1 is Involved in Vertebrate LC-PUFA Biosynthesis by Upregulating the Expression of Liver Desaturase and Elongase Genes.

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n−6 to 18:3n−6, 18:2n−6 to 20:2n−6, and 18:3n−3 to 20:3n−3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

Methyl-end desaturases with ∆12 and ω3 regioselectivities enable the de novo PUFA biosynthesis in the cephalopod Octopus vulgaris

The interest in understanding the capacity of aquatic invertebrates to biosynthesise omega-3 (ω3) long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) has increased in recent years. Using the common octopus Octopus vulgaris as a model species, we previously characterised a ∆5 desaturase and two elongases (i.e. Elovl2/5 and Elovl4) involved in the biosynthesis of LC-PUFA in molluscs. The aim of this study was to characterise both molecularly and functionally, two methyl-end (or ωx) desaturases that have been long regarded to be absent in most animals. O. vulgaris possess two ωx desaturase genes encoding enzymes with ∆12 and ω3 regioselectivities enabling the de novo biosynthesis of the C18 PUFA 18:2ω6 (LA, linoleic acid) and 18:3ω3 (ALA, α-linolenic acid), generally regarded as dietary essential for animals. The O. vulgaris ∆12 desaturase (“ωx2”) mediates the conversion of 18:1ω9 (oleic acid) into LA, and subsequently, the ω3 desaturase (“ωx1”) catalyses the ∆15 desaturation from LA to ALA. Additionally, the O. vulgaris ω3 desaturase has ∆17 capacity towards a variety of C20 ω6 PUFA that are converted to their ω3 PUFA products. Particularly relevant was the affinity of the ω3 desaturase towards 20:4ω6 (ARA, arachidonic acid) to produce 20:5ω3 (EPA, eicosapentaenoic acid), as supported by yeast heterologous expression, and enzymatic activity exhibited in vivo when paralarvae were incubated in the presence of [1-14C]20:4ω6. These results confirmed that several routes enabling EPA biosynthesis are operative in O. vulgaris whereas ARA and docosahexaenoic acid (DHA, 22:6ω3) should be considered essential fatty acids since endogenous production appears to be limited.

MiR-24 is involved in vertebrate LC-PUFA biosynthesis as demonstrated in marine teleost Siganus canaliculatus

Recently, microRNAs (miRNAs) have emerged as crucial regulators of lipid metabolism. However, the miRNA-mediated regulatory mechanism on long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis in vertebrates remains largely unknown. Here, we address a potentially important role of miRNA-24 (miR-24) in the regulation of LC-PUFA biosynthesis in rabbitfish Siganus canaliculatus. miR-24 showed significantly higher abundance in liver of rabbitfish reared in brackish water than in seawater for fish fed vegetable oil diets and in S. canaliculatus hepatocyte line (SCHL) cells incubated with alpha-linolenic acid (ALA) than the control group. Similar expression patterns were also observed on the expression of sterol regulatory element-binding protein-1 (srebp1) and LC-PUFA biosynthesis related genes. While opposite results were observed on the expression of insulin-induced gene 1 (insig1), an endoplasmic reticulum membrane protein blocking Srebp1 proteolytic activation. Luciferase reporter assays revealed rabbitfish insig1 as a target of miR-24. Knockdown of miR-24 in SCHL cells resulted in increased Insig1 protein, and subsequently reduced mature Srebp1 protein and expression of genes required for LC-PUFA biosynthesis, and these effects could be attenuated after additional insig1 knockdown. Opposite results were observed with overexpression of miR-24. Moreover, increasing endogenous insig1 by knockdown of miR-24 inhibited Srebp1 processing and consequently suppressed LC-PUFA biosynthesis in rabbitfish hepatocytes. These results indicate a potentially critical role for miR-24 in regulating LC-PUFA biosynthesis through the Insig1/Srebp1 pathway by targeting insig1. This is the first report of miR-24 involved in LC-PUFA biosynthesis and thus may provide knowledge on the regulatory mechanisms of LC-PUFA biosynthesis in vertebrates.

Sustainable alternatives to dietary fish oil in tropical fish aquaculture

AbstractMarine tropical carnivorous (MTC) fish are less tolerant to high dietary lipid compared with cold and temperate climate fish. However, insufficient n‐3 long‐chain (≥C20) polyunsaturated fatty acids (LC‐PUFA) compromises MTC fish health and growth which requires attention when choosing lipid sources in aquafeed formulations. Fish oil (FO) has been the major lipid source for feeding key MTC fish. We estimated that more than 200 thousand tonnes of FO were used to feed farmed MTC fish in 2016, representing nearly 25% of global FO production. Nevertheless, recent increases in FO prices and demand, and the foreseen inability of wild fisheries to produce sufficient FO in the future, have created a need for cheaper and more sustainable alternatives. Meta‐analysis showed that feeding vegetable oils (VO) to MTC fish can slightly reduce their growth, with a marked decrease in n‐3 LC‐PUFA content in the fish tissues corresponding with the increased replacement of FO and fishmeal in the diet. Rendered terrestrial animal fat or modulating the bioconversion of n‐3 LC‐PUFA precursors in VO can be utilised further in MTC fish nutrition with more research recommended on these approaches. Oils and oil‐rich meals from non‐food marine organisms are rich in n‐3 LC‐PUFA and represent an underutilised alternative to FO for tropical aquaculture. With oils rich in n‐3 LC‐PUFA produced from transgenic plants presently gaining regulatory approvals and becoming available as a renewable alternative to FO, aquaculture in the tropics and around the globe is ready to leap into a new phase of sustainable expansion.

Comparison of Thraustochytrids Aurantiochytrium sp., Schizochytrium sp., Thraustochytrium sp., and Ulkenia sp. for Production of Biodiesel, Long-Chain Omega-3 Oils, and Exopolysaccharide

Heterotrophic growth of thraustochytrids has potential in coproducing biodiesel for transportation, as well as producing a feedstock for omega-3 long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), especially docosahexaenoic acid (DHA) for use in nutraceuticals. In this study, we compared eight new endemic Australian thraustochytrid strains from the genera Aurantiochytrium, Schizochytrium, Thraustochytrium, and Ulkenia for the synthesis of exopolysaccharide (EPS), in addition to biodiesel and LC-PUFA. Aurantiochytrium sp. strains readily utilized glucose for biomass production, and increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased biomass yield by an average factor of 1.7. Ulkenia sp. strain TC 010 and Thraustochytrium sp. strain TC 033 did not utilize glucose, while Schizochytrium sp. strain TC 002 utilized less than half the glucose available by day 14, and Thraustochytrium sp. strain TC 004 utilized glucose at 4 % w/v but not 2 % w/v of the culture suggesting a threshold requirement between these values. Across all strains, increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased total fatty acid methyl ester content by an average factor of 1.9. Despite an increasing literature demonstrating the capacity of thraustochytrids for DHA synthesis, the production of EPS from these organisms is not well documented. A broad range of EPS yields was observed. The maximum yield of EPS was observed for Schizochytrium sp. strain TC 002 (299 mg/L). High biomass-producing strains that also have high lipid and high EPS yield may be better candidates for commercial production of biofuels and other coproducts.

Echium oil is better than rapeseed oil in improving the response of barramundi to a disease challenge

Pathogen infection stimulates the fatty acid (FA) metabolism and the production of pro-inflammatory derivatives of FA. Barramundi, Lates calcarifer, was fed on a diet rich in preformed long-chain (⩾C20) polyunsaturated fatty acids (LC-PUFA) from fish oil (FO), to compare with diets containing high levels of C18 precursors for LC-PUFA – stearidonic (SDA) and γ-linolenic acid (GLA) – from Echium plantagineum (EO), or rapeseed oil (RO) rich in α-linolenic acid (ALA), but a poor source of LC-PUFA and their precursors. After 6weeks, when growth rates were similar amongst the dietary treatments, a sub-lethal dose of Streptococcus iniae was administered to half of the fish, while the other half were maintained unchallenged and were pair-fed with the infected fish. Under a disease challenge situation, the tissue FA depots depleted at 3days post-infection (DPI) and were then restored to their previous concentrations at 7DPI. During the infection period, EO fish had a higher content of n3 and n6 PUFA in their tissues, higher n3:n6 PUFA ratio and reduced levels of the eicosanoids, TXB2 and 6-keto-PGF1α, in their plasma compared with RO fish. Fish fed on FO and EO had a longer lasting and enduring response in their FA and eicosanoid concentrations, following a week of bacterial infection, compared with those fed on RO. EO, containing SDA and GLA and with a comparatively higher n3:n6 PUFA ratio, proved more effective than RO in compensating for immunity stress.

Coping with sub-optimal water temperature: Modifications in fatty acid profile of barramundi as influenced by dietary lipid

Metabolic responses to sub-optimal temperature deplete lipid depots, remodel membrane lipid and alter the fatty acid profile in the whole body and tissues of ectothermic vertebrates including fish. The magnitude of these changes may depend on dietary history including oil sources with different fatty acid compositions. Barramundi, Lates calcarifer (Perciformes, Latidae), a tropical ectothermic fish, was fed on diets either rich in dietary long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from fish oil, rich in stearidonic and γ-linolenic acid (SDA and GLA, respectively) from Echium plantagineum, or rapeseed oil deficient in LC-PUFA. Following 5weeks at the optimum temperature of 30°C when growth rates were comparable amongst dietary treatments, water temperature was dropped to 20°C for 1week for half of the animals and maintained at 30°C for the other half. Decreased temperature increased the liver and skeletal muscle content of LC-PUFA in fish fed on echium oil compared with rapeseed oil, while dietary LC-PUFA depots in fish oil fed-fish depleted rapidly in the week of sub-optimal temperature. The lipid unsaturation index of cellular membrane in the liver and muscle increased under low temperature at the same rate regardless of dietary oil. Therefore, rapid exposure of an ectothermic vertebrate to a lower and sub-optimal temperature caused significant modulation in fatty acid composition. We propose that the tolerance of barramundi, a representative of tropical farmed fish, to sub-optimal temperature will be enhanced when fatty acid substrates closer to the LC-PUFA are available in their diet.

The “n−3 LC-PUFA sparing effect” of modified dietary n−3 LC-PUFA content and DHA to EPA ratio in Atlantic salmon smolt

n−3 Long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) are used extensively by fish via β-oxidation when in dietary surplus. Therefore it is of interest to optimize n−3 LC-PUFA deposition in fish via a reduction in β-oxidation which may be induced by manipulation of dietary fatty acids. This study tested whether Atlantic salmon smolt fed a diet with a higher docosahexaenoic acid (DHA):eicosapentaenoic acid (EPA) ratio and a lower content of n−3 LC-PUFA to that of fish oil (FO) based diets would enhance deposition of n−3 LC-PUFA in fish tissues. Comparisons were made between fish fed: a FO diet, a blend of 50% rapeseed and 50% tuna oil diet (model oil, MO 1), a blend of 50% rapeseed, 25% tuna and 25% FO diet (MO 2), and a blend of 50% FO and 50% chicken fat diet (FO/CF). The dietary DHA:EPA ratio was in the order MO 1>MO 2>FO/CF~FO. Dietary n−3 LC-PUFA content was approximately 2-fold lower in fish fed the MO 1, MO 2 and FO/CF diets compared to the FO diet. There were comparable amounts of n−3 LC-PUFA in the muscle of FO, MO 1 and FO/CF fed fish. Our findings indicate that the right balance in both absolute and relative amounts of EPA and DHA can promote n−3 LC-PUFA retention.

Regiospecific Analyses of Triacylglycerols of Hoki (Macruronus novaezelandiae) and Greenshell™ Mussel (Perna canaliculus)

AbstractThe lipid profiles of the two most important New Zealand marine oil sources were investigated, with particular attention to the regioisomeric compositions of triacylglycerides (TAG), using 13C‐nuclear magnetic resonance analysis. Oils from hoki (Macruronus novaezelandiae) and Greenshell™ mussel (Perna canaliculus) (GSM) were analyzed for their lipid content, lipid class and fatty acid profile. The regiospecific distribution of long chain (C ≥ 20) polyunsaturated fatty acids (LC‐PUFA) between the sn‐1,3 and sn‐2 glycerol positions was calculated from 13C responses in the carbonyl region in the triacylglycerol fraction. Rendered hoki oil (RHO) produced from the viscera and filleting discards, had a similar lipid profile to that of hoki liver oil (HLO) confirming that the liver is the major source of oil in RHO. The regioisomeric distribution of fatty acids showed differences between the two oil sources. Docosahexaenoic acid (DHA) had a regioisomeric distributional preference to the sn‐2 position in TAG from all the oils (59.2% HLO, 54.3% RHO and 63.4% GSM). Eicosapentaenoic acid (EPA) had a more even distribution along the triacylglycerol backbone in hoki TAG (29.1% HLO, 33.6% RHO) while there was a slight sn‐2 positional preference in the GSM TAG (37.6%). This regioisomeric information is vital to distinguish LC‐PUFA‐rich marine oils from other marine sources for authentication purposes.

Metabolic engineering of omega-3 long-chain polyunsaturated fatty acids in plants using an acyl-CoA Δ6-desaturase with ω3-preference from the marine microalga Micromonas pusilla

Long-chain (≥C 20) polyunsaturated fatty acids (LC-PUFA) EPA and DHA (20:5 Δ5,8,11,14,17 and 22:6 Δ4,7,10,13,16,19) have well-documented health benefits against coronary heart disease, rheumatoid arthritis and other disorders. Currently, the predominant sources of these fatty acids are marine fish and algal oils, but research is being conducted to ensure that a sustainable, land-based production system can be developed. We here describe the metabolic engineering of an artificial pathway that produces 26% EPA in leaf triacylglycerol using a newly-identified Δ6-desaturase from the marine microalga Micromonas pusilla. We also demonstrate that this enzyme appears to function as an acyl-CoA desaturase that has preference for ω3 substrates both in planta and in yeast. Phylogenetic analysis indicates that this desaturase shares highly conserved motifs with previously described acyl-CoA Δ6-desaturases.