Molecular and functional characterisation of a putative elovl4 gene and its expression in response to dietary fatty acid profile in Atlantic bluefin tuna (Thunnus thynnus)

Elongation of very long-chain fatty acid 4 (Elovl4) proteins are involved in the biosynthesis of very long-chain (> C24) fatty acids and in many teleost fish species they are key enzymes in the pathway for the production of docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Therefore, Elovl4 may be particularly important in Atlantic bluefin tuna (ABT; Thunnus thynnus) characterised by having high DHA to EPA ratios. The present study cloned and characterised both the function and expression of an elovl4 cDNA from ABT. The Elovl4 had an open reading frame of 915 base pairs encoding a putative protein of 304 amino acids. Alignment and phylogenetic analyses indicated that the Elovl4 isoform identified in the present study was an Elovl4b. Functional characterisation demonstrated that the Elovl4b enzyme had elongase activity towards all the polyunsaturated fatty acid (PUFA) substrates assayed. The ABT Elovl4b contributed to DHA biosynthesis by elongation of EPA and DPA to 24:5n-3, the latter being desaturated to 24:6n-3 by the action of fads2 (Δ6 desaturase). Additionally, the ABT Elovl4b has a role in the biosynthesis of very long-chain PUFA up to C34, compounds of key structural roles in neural tissues such as eye and brain, which had high levels of elovl4b transcripts. Surprisingly, while the relative expression of fads2, required for the production of DHA from EPA, was increased in liver of ABT fed a diet with reduced levels of EPA and DHA, expression of elovl4b was reduced. Results indicated that ABT has enzymes necessary for endogenous production of DHA from EPA and demonstrate that Elovl4b can effectively compensate for absence of Elovl2.Versión del edito

Functional Desaturase Fads1 (Δ5) and Fads2 (Δ6) Orthologues Evolved before the Origin of Jawed Vertebrates

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales

Genes for de novo biosynthesis of omega-3 polyunsaturated fatty acids are widespread in animals

Marine ecosystems are responsible for virtually all production of omega-3 (ω3) long-chain polyunsaturated fatty acids (PUFA), which are essential nutrients for vertebrates. Current consensus is that marine microbes account for this production, given their possession of key enzymes including methyl-end (or "ωx") desaturases. ωx desaturases have also been described in a small number of invertebrate animals, but their precise distribution has not been systematically explored. This study identifies 121 ωx desaturase sequences from 80 species within the Cnidaria, Rotifera, Mollusca, Annelida, and Arthropoda. Horizontal gene transfer has contributed to this hitherto unknown widespread distribution. Functional characterization of animal ωx desaturases provides evidence that multiple invertebrates have the ability to produce ω3 PUFA de novo and further biosynthesize ω3 long-chain PUFA. This finding represents a fundamental revision in our understanding of ω3 long-chain PUFA production in global food webs, by revealing that numerous widespread and abundant invertebrates have the endogenous capacity to make significant contributions beyond that coming from marine microbes. Copyright © 2018 The Authors, some rights reserved.Acknowledgments: We thank A. Magurran and J. Napier for comments on the manuscript and R. Ruivo for drawings in Figs. 1 and 3. Funding: This work received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland) funded by the Scottish Funding Council (grant reference HR09011), and their support is gratefully acknowledged. Access to the Institute of Aquaculture laboratories was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 262336 (AQUAEXCEL), Transnational Access Project Number 0095/06/03/13

Assessment of stress and nutritional biomarkers in cultured Octopus

The common octopus (Octopus vulgaris) is a promising species for aquaculture diversification, but massive mortality during the first life-cycle stages (paralarvae) is the main bottleneck for its commercial production in captivity. The aim of this study was to assess stress and nutritional condition biomarkers (HSP70, ROS enzymes and lipid peroxidation) (RNA/DNA, RNA/protein, protein/DNA and protein) in O. vulgaris paralarvae from different geographical origins and fed with Artemia enriched with marine phospholipids or microalgae (control group). To this end paralarvae were cultured for 30 days, in three different centres in Spain (Tarragona-Mediterranean area, Tenerife-Central Atlantic area and Vigo-North Atlantic area), under the same protocol, and fed on Artemia enriched with marine phospholipids (LC60) (Marine Lecithin LC 60®, PhosphoTech Laboratoires) or microalgae (control group). Dry weight and most biomarkers analysed in hatchlings showed significant differences related to their origin (centre). Fifteen day old paralarvae presented significant differences in specific growth rate (SGR) associated with their dietary regime, and also showed differences in biomarkers associated both with their geographical origin and dietary regime. The results suggest that the SGR of paralarvae were positively influenced by LC60, promoting growth and in agreement with the results of nutritional condition biomarkers (nucleic acids ratios). The antioxidant defences against oxidative damage were also boosted in the LC60 paralarvae group, possibly as a result of the elevated content in highly polyunsaturated fatty acids. In addition, the partial correlations found between biomarkers varied according to diet. However, no positive effect of LC60 on survival was observed. The high variability found among geographical origins, despite the use of the same rearing protocol, highlights the need to clarify the sources of such variability. Statement of relevance: Stress status varies among geographical origins and diets.En prensa2,04

Elongase Reactions as Control Points in Long-Chain Polyunsaturated Fatty Acid Synthesis

Extent: 9p.Background: Δ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA. Methodology/Principal Findings: The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C20 and C22 polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3. Conclusions: The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.Melissa K. Gregory, Robert A. Gibson, Rebecca J. Cook-Johnson, Leslie G. Cleland and Michael J. Jame

Δ-6 desaturase substrate competition : dietary linoleic acid (18∶2n-6) has only trivial effects on α-linolenic acid (18∶3n-3) bioconversion in the teleost rainbow trout

It is generally accepted that, in vertebrates, omega-3 (n-3) and omega-6 (n-6) poly-unsaturated fatty acids (PUFA) compete for ?-6 desaturase enzyme in order to be bioconverted into long-chain PUFA (LC-PUFA). However, recent studies into teleost fatty acid metabolism suggest that these metabolic processes may not conform entirely to what has been previously observed in mammals and other animal models. Recent work on rainbow trout has led us to question specifically if linoleic acid (LA, 18:2n-6) and ?-linolenic acid (ALA, 18:3n-3) (?-6 desaturase substrates) are in direct competition for access to ?-6 desaturase. Two experimental diets were formulated with fixed levels of ALA, while LA levels were varied (high and low) to examine if increased availability of LA would result in decreased bioconversion of ALA to its LC-PUFA products through substrate competition. No significant difference in ALA metabolism towards n-3 LC-PUFA was exhibited between diets while significant differences were observed in LA metabolism towards n-6 LC-PUFA. These results are evidence for minor if any competition between substrates for ?-6 desaturase, suggesting that, paradoxically, the activity of ?-6 desaturase on n-3 and n-6 substrates is independent. These results call for a paradigm shift in the way we approach teleost fatty acid metabolism. The findings are also important with regard to diet formulation in the aquaculture industry as they indicate that there should be no concern for possible substrate competition between 18:3n-3 and 18:2n-6, when aiming at increased n-3 LC-PUFA bioconversion in vivo

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 towards 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 [1-14C]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

EFECTO DE LOS FOSFOLÍPIDOS MARINOS SOBRE BIOMARCADORES NUTRICIONALES Y DE ESTRÉS FISIOLÓGICO DE LAS PARALARVAS DEL PULPO COMÚN (OCTOPUS VULGARIS) BAJO CONDICIONES DE CULTIVO ESTANDARIZADAS EN TRES CENTROS

El efecto del enriquecimiento de la Artemia con fosfolípidos marinos (LC60), sobre el crecimiento, la supervivencia y los biomarcadores de condición nutricional (ARN/ADN) y de estrés fisiológico (proteínas de estrés (HSP70), enzimas antioxidantes y peroxidación lipídica) en paralarvas cultivadas de pulpo común (Octopus vulgaris) se evaluó bajo un protocolo estandarizado en tres centros. Las paralarvas recién eclosionadas mostraron diferencias significativas entre centros en peso seco y en la mayor parte de los biomarcadores analizados. Por el contario, en paralarvas cultivadas de 15 días, se encontraron diferencias significativas debidas a la dieta, presentando un incremento en la tasa de crecimiento específica (TCE) en el grupo alimentado con Artemia enriquecida con LC60 y mayores niveles de actividad GPX T y GR. Sin embargo, la dieta no tuvo efecto en la supervivencia de las paralarvas. Los biomarcadores de condición nutricional y de estrés fisiológicos presentaron diferencias entre los centros. Los resultados indican que el uso de fosfolípidos marinos como enriquecimiento de Artemia mejora el crecimiento de las paralarvas, si bien no se observa un efecto positivo de dicho tratamiento sobre la supervivencia de las mismas

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 and. 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