Despite the recent progress in the production of inert diets for fish larvae, feeding of most species of interest for aquaculture still relies on live feeds during the early life stages. Independently of their nutritional value, live feeds are easily detected and captured, due to their swimming movements in the water column, and highly digestible, given their lower nutrient concentration (water content>80%). The present paper reviews the main types of live feeds used in aquaculture, their advantages and pitfalls, with a special emphasis on their nutritional value and the extent to which this can be manipulated. The most commonly used live feeds in aquaculture are rotifers (Brachionus sp.) and brine shrimp (Artemia sp.), due to the existence of standardized cost‐effective protocols for their mass production. However, both rotifers and Artemia have nutritional deficiencies for marine species, particularly in essential n‐3 highly unsaturated fatty acids (HUFA, e.g., docosahexaenoic acid and eicosapentaenoic acid). Enrichment of these live feeds with HUFA‐rich lipid emulsions may lead to an excess dietary lipid and sub‐optimal dietary protein content for fish larvae. In addition, rotifers and Artemia are likely to have sub‐optimal dietary levels of some amino acids, vitamins and minerals, at least for some species. Several species of microalgae are also used in larviculture. These are used as feed for other live feeds, but mostly in the ‘green water’ technique in fish larval rearing, with putative beneficial effects on feeding behaviour, digestive function, nutritional value, water quality and microflora. Copepods and other natural zooplankton organisms have also been used as live feeds, normally with considerably better results in terms of larval survival rates, growth and quality, when compared with rotifers and Artemia. Nonetheless, technical difficulties in mass‐producing these organisms are still a constraint to their routine use. Improvements in inert microdiets will likely lead to a progressive substitution of live feeds. However, complete substitution is probably years away for most species, at least for the first days of feeding.

Running title: Characterisation of salmon ELOVL5-and ELOVL2 elongases Abbreviations: aa, amino acid; ARA, arachidonic acid (20:4n-6); DHA, docosahexaenoic acid (22:6n-3); EPA, eicosapentaenoic acid (20:5n-3); ER, endoplasmic reticulum; FA, fatty acid; FO, fish oil; HUFA, highly unsaturated fatty acids (carbon chain length ≥ C 20 with ≥ 3 double bonds); LO, linseed oil; ORF, open reading frame; PUFA, polyunsaturated fatty acids; Q-PCR, quantitative (real-time) polymerase chain reaction; RACE, rapid amplification of cDNA ends; RO, rapeseed oil; SO, soybean oil; UTR, untranslated region; VO, vegetable oil. 2Abstract Fish species vary in their capacity to biosynthesize the n-3 long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of LC-PUFA involves enzyme-mediated fatty acyl desaturation and elongation. Previously, a cDNA for an elongase, now termed elovl5a, had been cloned from Atlantic salmon. Here we report on the cloning of two new elongase cDNAs: a second elovl5b elongase, corresponding to a 294 aa protein, and an elovl2-like elongase, coding for a 287 aa protein, characterized for the first time in a nonmammalian vertebrate. Heterologous expression in yeast showed that the salmon Elovl5b elongated C18 and C20 PUFA, with low activity towards C22, while Elovl2 elongated C20 and C22 PUFA with lower activity towards C18 PUFA. All three transcripts showed predominant expression in the intestine and liver, followed by the brain. Elongase expression showed differential nutritional regulation. Levels of elovl5b and particularly of elovl2, but not of elovl5a, transcripts were significantly increased in liver of salmon fed vegetable oils (VO) compared to fish fed fish oil (FO). Intestinal expression showed a similar pattern.Phylogenetic comparisons indicate that, in contrast to salmon and zebrafish, Acanthopterygian fish species lack elovl2 which is consistent with their neglible ability to biosynthesise LC-PUFA and to adapt to VO dietary inclusion, compared to predominantly freshwater salmonids. Thus the presence of elovl2 in salmon explains the ability of this species to biosynthesise LC-HUFA and may provide a biotechnological tool to produce enhanced levels of LC-PUFA, particularly DHA, in transgenic organisms.3

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BackgroundExpansion of aquaculture is seriously limited by reductions in fish oil (FO) supply for aquafeeds. Terrestrial alternatives such as vegetable oils (VO) have been investigated and recently a strategy combining genetic selection with changes in diet formulations has been proposed to meet growing demands for aquaculture products. This study investigates the influence of genotype on transcriptomic responses to sustainable feeds in Atlantic salmon.ResultsA microarray analysis was performed to investigate the liver transcriptome of two family groups selected according to their estimated breeding values (EBVs) for flesh lipid content, 'Lean' or 'Fat', fed diets containing either FO or a VO blend. Diet principally affected metabolism genes, mainly of lipid and carbohydrate, followed by immune response genes. Genotype had a much lower impact on metabolism-related genes and affected mostly signalling pathways. Replacement of dietary FO by VO caused an up-regulation of long-chain polyunsaturated fatty acid biosynthesis, but there was a clear genotype effect as fatty acyl elongase (elovl2) was only up-regulated and desaturases (Δ5 fad and Δ6 fad) showed a higher magnitude of response in Lean fish, which was reflected in liver fatty acid composition. Fatty acid synthase (FAS) was also up-regulated by VO and the effect was independent of genotype. Genetic background of the fish clearly affected regulation of lipid metabolism, as PPARα and PPARβ were down-regulated by the VO diet only in Lean fish, while in Fat salmon SREBP-1 expression was up-regulated by VO. In addition, all three genes had a lower expression in the Lean family group than in the Fat, when fed VO. Differences in muscle adiposity between family groups may have been caused by higher levels of hepatic fatty acid and glycerophospholipid synthesis in the Fat fish, as indicated by the expression of FAS, 1-acyl-sn-glycerol-3-phosphate acyltransferase and lipid phosphate phosphohydrolase 2.ConclusionsThis study has identified metabolic pathways and key regulators that may respond differently to alternative plant-based feeds depending on genotype. Further studies are required but data suggest that it will be possible to identify families better adapted to alternative diet formulations that might be appropriate for future genetic selection programmes.

In the present paper, we investigated the expression of fatty acyl desaturase and elongase genes in a marine teleost, cobia, a species of great interest due to its considerable aquaculture potential. A cDNA was cloned that, when expressed in yeast, was shown to result in desaturation of 18:3n-3 and 18:2n-6, indicating that it coded for a Δ6 desaturase enzyme. Very low desaturation of 20:4n-3 and 20:3n-6 indicated only trace Δ5 activity. Another cloned cDNA enabled elongation of 18:4n-3, 18:3n-6, 20:5n-3 and 20:4n-6 in the yeast expression system, indicating that it had C18-20 and C20-22 elongase activity. Sequence comparison and phylogenetic analysis confirmed that it was homologous to human ELOVL5 elongase. However, the cobia Elovl5 elongase also had low activity toward C24 HUFA. The cobia Δ6 desaturase had a preference for 18:3n-3, but the elongase was generally equally active with both n-3 and n-6 substrates. Expression of both genes was 1-2 orders of magnitude greater in brain than other tissues suggesting an important role, possibly to ensure sufficient docosahexaenoic acid (DHA, 22:6n-3) synthesis in neural tissues through elongation and desaturation of eicosapentaenoic acid (EPA; 20:5n-3).3

Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA;, that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesis EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long fatty acids (Elovl). Previously, two cDNAs encoding functionally distinct Δ5 and Δ6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, Δ6fad_a and Δ5fad, corresponded to the previously cloned Δ6 and Δ5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Δ6fad_b and Δ6fad_c, had only Δ6 activity, converting 47 and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for ∆6Fad_b and Δ6Fad_c, respectively. Both ∆6fad_a and ∆6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with ∆6fad_b also highly expressed in gill, whereas ∆6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the ∆6fad_a gene in liver and the ∆6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for ∆6fad_a in intestine, ∆6fad_b in liver, or ∆6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes were discussed in relation to gene structures and fish physiology.3

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Solea senegalensis is an unusual marine teleost with very low dietary requirement for longchain polyunsaturated fatty acids (LC-PUFA) during early development. Aquaculture is rapidly becoming the main source of health-beneficial fish products for human consumption. This, associated with limited supply of LC-PUFA-rich ingredients for fish feeds, render S. senegalensis a highly interesting species in which to study the LC-PUFA biosynthesis pathway. We have cloned and functionally character -3 series) and elovl5 with the potential to catalyze docosahexaenoic acid (DHA) biosynthesis from eicosapentaenoic acid (EPA). Changes in expression of both transcripts were determined during embryonic and early larval development, and transcriptional regulation in response to dietary n-3 LC-PUFA was assessed during larval and post-larval stages. There was a marked pattern of regulation during early ontogenesis, with transcripts showing peak expression coinciding with the start of exogenous feeding. Although elovl5 transcripts were present in fertilized DHA/EPA ratio (~ 11) to meet the high demands for early embryonic development. Fatty acid profile was significantly up-regulated in response to LC-PUFA-poor diets, which may suggest biological relevance of this pathway in reducing LC-PUFA dietary requirements in this species, compared to other marine teleosts. These results indicate that sole is capable of synthesizing DHA from EPA through a Sprecher-independent pathway. Suggested Reviewers: Xiaozhong Zheng xzheng@staffmail.ed.ac.uk Dr Zheng has done considerable work involving cloning and functional characterization of Fads and Elovls in fish and is therefore a very specific expert in this field.Johnathan A. Napier jon.napier@bbsrc.ac.uk Prof. Napier is an expert in Fad molecular biology and has considerable expertise in pathways of DHA biosynthesis.Chantal Cahu Chantal.Cahu@ifremer.fr Dr. Chantal Cahu is an expert in marine larvae nutrition including fatty acid metabolism. In addition, she has also been involved in molecular and genomic studies, including the cloning and Fad expression in fish.Gabriel Mourente gabriel.mourente@uca.es Dr. Gabriel Mourente is an expert in marine larvae nutrition including fatty acid metabolism. He has additionally performed several larval nutrition studies in S. senegalensis and has also worked specifically with enzymes of the pathway of LC-PUFA biosynthesis.Manuel Yúfera manuel.yufera@icman.csic.es Dr. Manuel Yúfera is an expert in marine larvae nutrition including fatty acid metabolism and has additionally performed several larval nutrition studies in S. senegalensis. Fatty acyl desaturase and elongase cDNAs corresponding to a 4fad (with some 5 activity for PUFA of the n-3 series) and elovl5 were cloned from a marine teleost (Solea senegalensis) and functionally characterized by heterologous expression in yeast. Historically, it was believed that the pathway for DHA synthesis would involve a 4 desaturase, as this would be the most direct route, but the presence of 4Fad enzymes cou...