Environmental salinity and dietary lipid nutrition strategy: Effects on flesh quality of the marine euryhaline crab Scylla paramamosain.

The quality of crustaceans' flesh has direct impact on consumers' purchase choices, with water environment and dietary nutrition being effective ways to regulate flesh quality. The aim of present study was to investigate the impacts of water salinity (low, 4 and medium, 23) and dietary lipid source (fish oil and soybean oil) on nutritional values, texture, taste and odor of flesh of mud crab. While water salinity had no significant influence on nutritional values of crab flesh, crabs fed soybean oil displayed significantly lower contents of amino acids and n-3 PUFAs in muscle. However, crabs reared at low salinity showed reduced flesh hardness, chewiness and gumminess likely related to altered myofiber structure, that impacted muscle texture. Furthermore, low salinity and dietary soybean oil weakened umami taste and aroma characteristics of crab flesh associated with decreased contents of free amino acids, flavor nucleotides, inorganic ions and odor active compounds in flesh.

Influence of Dietary Lipids and Environmental Salinity on the n-3 Long-Chain Polyunsaturated Fatty Acids Biosynthesis Capacity of the Marine Teleost Solea senegalensis.

Fish vary in their ability to biosynthesise long-chain polyunsaturated fatty acids (LC-PUFA) depending upon the complement and function of key enzymes commonly known as fatty acyl desaturases and elongases. It has been reported in Solea senegalensis the existence of a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid (DHA) from eicosapentaenoic acid (EPA), which can be modulated by the diet. The present study aims to evaluate the combined effects of the partial replacement of fish oil (FO) with vegetable oils and reduced environmental salinity in the fatty acid composition of relevant body compartments (muscle, hepatocytes and enterocytes), the enzymatic activity over α-linolenic acid (ALA) to form n-3 LC-PUFA through the incubation of isolated hepatocytes and enterocytes with [1-14C] 18:3 n-3, and the regulation of the S. senegalensis fads2 and elovl5 in the liver and intestine. The presence of radiolabelled products, including 18:4n-3, 20:4n-3 and EPA, provided compelling evidence that a complete pathway enabling the biosynthesis of EPA from ALA, establishing S. senegalensis, has at least one Fads2 with ∆6 activity. Dietary composition prevailed over salinity in regulating the expression of fads2, while salinity did so over dietary composition for elovl5. FO replacement enhanced the proportion of DHA in S. senegalensis muscle and the combination with 20 ppt salinity increased the amount of n-3 LC-PUFA in hepatocytes.

fat-1 transgenic zebrafish are protected from abnormal lipid deposition induced by high-vegetable oil feeding.

High dietary concentration of vegetable oil, particularly those rich in n-6 polyunsaturated fatty acids (PUFAs), can induce negative physiological effects including excessive lipid deposition in teleost fish. Omega-3 desaturase (Fat-1) of Caenorhabditis elegans is able to convert n-6 PUFAs to n-3 PUFAs and thus induces a low n-6/n-3 PUFAs ratio alleviating lipid deposition. In this study, we investigated the effects of dietary n-6 PUFAs on lipid metabolism of fat-1 transgenic zebrafish (Tg:fat-1), to explore the role of fat-1 in fish lipid metabolism. We first generated Tg:fat-1 zebrafish and assayed the effects of a low-fat diet (LFD) and a high-fat diet (HFD) prepared from soybean oil. Wild type zebrafish (WT) fed with HFD (HFD-WT) exhibited increased obesity and lipid deposition, especially in the abdominal cavity and liver. These defects were absent from HFD-Tg:fat-1. For each diet group, Tg:fat-1 exhibited significantly decreased levels of almost all hepatic lipid classes compared with WT. Expression levels of lipid synthesis-related genes and lipid deposition-related genes were markedly lower in the liver of HFD-Tg:fat-1 compared with HFD-WT. In contrast, the steatolysis-related genes significantly upregulated in HFD-Tg:fat-1. Then expression profiles of mitochondrial energy metabolism-related genes and ATP contents in the livers from LFD-WT, LFD-Tg:fat-1, HFD-WT, and HFD-Tg:fat-1 were determined. Our findings suggest that fat-1 protects fish from abnormal lipid deposition induced by high-vegetable oil feeding, through endogenously converting n-6 PUFAs to n-3 PUFAs. KEY POINTS: • fat-1 transgenic zebrafish (Tg:fat-1) can endogenously convert n-6 PUFAs to n-3 PUFAs. • Tg:fat-1 avoid serious abnormal lipid deposition induced by high-vegetable oil feeding. • fat-1 transgenosis effectively improved lipid metabolism and mitochondrial energy metabolism in zebrafish.

Dietary choline supplementation attenuated high-fat diet-induced inflammation through regulation of lipid metabolism and suppression of NFκB activation in juvenile black seabream (Acanthopagrus schlegelii).

The present study aimed to investigate whether dietary choline can regulate lipid metabolism and suppress NFκB activation and, consequently, attenuate inflammation induced by a high-fat diet in black sea bream (Acanthopagrus schlegelii). An 8-week feeding trial was conducted on fish with an initial weight of 8·16 ± 0·01 g. Five diets were formulated: control, low-fat diet (11 %); HFD, high-fat diet (17 %); and HFD supplemented with graded levels of choline (3, 6 or 12 g/kg) termed HFD + C1, HFD + C2 and HFD + C3, respectively. Dietary choline decreased lipid content in whole body and tissues. Highest TAG and cholesterol concentrations in serum and liver were recorded in fish fed the HFD. Similarly, compared with fish fed the HFD, dietary choline reduced vacuolar fat drops and ameliorated HFD-induced pathological changes in liver. Expression of genes of lipolysis pathways were up-regulated, and genes of lipogenesis down-regulated, by dietary choline compared with fish fed the HFD. Expression of nfκb and pro-inflammatory cytokines in liver and intestine was suppressed by choline supplementation, whereas expression of anti-inflammatory cytokines was promoted in fish fed choline-supplemented diets. In fish that received lipopolysaccharide to stimulate inflammatory responses, the expression of nfκb and pro-inflammatory cytokines in liver, intestine and kidney were all down-regulated by dietary choline compared with the HFD. Overall, the present study indicated that dietary choline had a lipid-lowering effect, which could protect the liver by regulating intrahepatic lipid metabolism, reducing lipid droplet accumulation and suppressing NFκB activation, consequently attenuating HFD-induced inflammation in A. schlegelii.

The compositional and metabolic responses of gilthead seabream (Sparus aurata) to a gradient of dietary fish oil and associated n-3 long-chain PUFA content.

The replacement of fish oil (FO) with vegetable oil (VO) in feed formulations reduces the availability of n-3 long-chain PUFA (LC-PUFA) to marine fish such as gilthead seabream. The aim of this study was to examine compositional and physiological responses to a dietary gradient of n-3 LC-PUFA. Six iso-energetic and iso-nitrogenous diets (D1-D6) were fed to seabream, with the added oil being a blend of FO and VO to achieve a dietary gradient of n-3 LC-PUFA. Fish were sampled after 4 months feeding, to determine biochemical composition, tissue fatty acid concentrations and lipid metabolic gene expression. The results indicated a disturbance to lipid metabolism, with fat in the liver increased and fat deposits in the viscera reduced. Tissue fatty acid profiles were altered towards the fatty acid compositions of the diets. There was evidence of endogenous modification of dietary PUFA in the liver which correlated with the expression of fatty acid desaturase 2 (fads2). Expression of sterol regulatory element binding protein 1 (srebp1), fads2 and fatty acid synthase increased in the liver, whereas PPARα1 pathways appeared to be supressed by dietary VO in a concentration-dependent manner. The effects in lipogenic genes appear to become measurable in D1-D3, which agrees with the weight gain data suggesting that disturbances to energy metabolism and lipogenesis may be related to performance differences. These findings suggested that suppression of ß-oxidation and stimulation of srebp1-mediated lipogenesis may play a role in contributing toward steatosis in fish fed n-3 LC-PUFA deficient diets.

Molecular and functional characterisation of two elovl4 elongases involved in the biosynthesis of very long-chain (>C24) polyunsaturated fatty acids in black seabream Acanthopagrus schlegelii.

Elongation of very long-chain fatty acid (Elovl) 4 proteins are important fatty acyl elongases that participate in the biosynthesis of long-chain (C20-24) and very long-chain (˃C24) polyunsaturated fatty acids (LC-PUFA and VLC-PUFA, respectively) in teleost fish, especially in marine species. Moreover, knowledge of Elovl4 and other elongases such as Elovl2 has contributed to an advanced understanding of the LC-PUFA biosynthetic pathway in marine fish. In the present study, elovl4a and elovl4b were cloned from black seabream Acanthopagrus schlegelii and functionally characterised using recombinant expression in yeast. The elovl4a and elovl4b cDNA sequences included open reading frames (ORF) of 969 and 918 base pairs (bp), encoding proteins of 322 and 315 amino acids (aa), respectively. The functional characterisation of A. schlegelii Elovl4 proteins showed they were able to utilise all assayed C18-22 PUFA substrates except 22:6n-3. Moreover, it was particularly noteworthy that both A. schlegelii Elovl4a and Elovl4b proteins had the ability to elongate 20:5n-3 and 22:5n-3 to 24:5n-3, which can be potentially desaturated and ß-oxidised to 22:6n-3. Tissue transcript abundance analysis showed the highest expression of elovl4a and elovl4b in brain and eye, respectively, suggesting these tissues were major sites for VLC-PUFA biosynthesis in black seabream. The functions of the A. schlegelii Elovl4-like elongases, Elovl4a and Elovl4b, characterised in the present study, along with those of the Elovl5 and fatty acyl desaturase (Fads2) proteins of A. schlegelii characterised previously, provided evidence of the biosynthetic pathways of LC-PUFA and VLC-PUFA in this teleost species.

Lipid Composition of Oil Extracted from Wasted Norway Lobster (Nephrops norvegicus) Heads and Comparison with Oil Extracted from Antarctic Krill (Euphasia superba).

In the UK, the Norway lobster (Nephrops norvegicus) supports its most important shellfish fishery. Nephrops are sold either whole, or as "tails-only" for the scampi trade. In the "tailing" process, the "head" (cephalothorax) is discarded as waste. A smaller crustacean species, the Antarctic krill Euphasia superba, represents an economically valuable industry, as its extractable oil is sold as a human dietary supplement. The aim of this study was to determine the amount and composition of the oil contained in discarded Nephrops heads and to compare its composition to the oil extracted from krill. Differences due to Geographical variation and seasonal patterns in the amount and composition of lipid were also noted. Results indicated that Nephrops head waste samples collected from more southern locations in Scotland (Clyde Sea area) contained higher levels of oil when compared to samples collected from northern locations in Iceland. Moreover, seasonal differences within the Clyde Sea area in Scotland were also observed, with oil extracted from Nephrops head waste peaking at around 11.5% during the summer months when larger and more mature females were caught by trawl. At this time of the year, the valuable fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) accounted for around 23% of the total fatty acid content in oil extracted from Nephrops head waste. A seasonal effect on EPA content was found, with higher levels obtained in the summer, while no trend was found in DHA percentages. Finally, oil from Nephrops head waste contained a higher proportion of EPA and DHA than krill oil but these fatty acids were more abundantly linked to the neutral lipids rather to than polar lipids. The characterization of lipid that could be extracted from Nephrops head waste should be seen as a first step for the commercial use of a valuable resource currently wasted. This approach is extremely relevant given the current limited supply of EPA and DHA and changes in the Common Fisheries Policy.

Biosynthesis of very long-chain fatty acids (C>24) in Atlantic salmon: cloning, functional characterisation, and tissue distribution of an Elovl4 elongase.

The elongases of very long-chain fatty acids (Elovl) account for the rate-limiting condensation step of the elongation process in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovl family, Elovl4, has been regarded as a critical enzyme in vertebrates in the production of the so-called very long-chain fatty acids (VLC-FA), a group of compounds that has been scarcely explored in fish. Here we report on the cloning of a novel Elovl4-like elongase from Atlantic salmon (Salmo salar). The salmon Elovl4 cDNA codes for a putative protein containing 306 amino acids. Heterologous expression in yeast demonstrated that salmon Elovl4 efficiently elongated saturated FAs up to 36:0, with 24:0 and 26:0 appearing as preferred substrates. Additionally, salmon Elovl4 effectively converted C20 and C22 polyunsaturated fatty acids to elongated polyenoic products up to C36. Tissue distribution showed that Elovl4 mRNA transcripts are abundant in eye, brain and testes, suggesting that, as described in mammals, these tissues are important metabolic sites for the biosynthesis of VLC-FA. Our results are discussed in comparison with the functional analyses observed in Elovl4 proteins from other vertebrates, and also other Elovl proteins investigated previously in Atlantic salmon.

Vertebrate fatty acyl desaturase with Δ4 activity.

Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by Δ6 desaturation of 182n - 6 to produce 183n - 6 that is elongated to 203n - 6 followed by Δ5 desaturation. Synthesis of EPA from 183n - 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second Δ6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional Δ6/Δ5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 225n - 3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having Δ4 Fad activity. This is a unique report of a Fad with Δ4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 225n - 3 followed by Δ4 desaturation, as well as the more complicated pathway as described above.

Multiple genes for functional 6 fatty acyl desaturases (Fad) in Atlantic salmon (Salmo salar L.): gene and cDNA characterization, functional expression, tissue distribution and nutritional regulation.

Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct Delta5 and Delta6 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, Delta6fad_a and Delta5fad, corresponded to the previously cloned Delta6 and Delta5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Delta6fad_b and Delta6fad_c, had only Delta6 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 Delta6fad_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 are discussed in relation to gene structures and fish physiology.

Highly unsaturated fatty acid synthesis in Atlantic salmon: characterization of ELOVL5- and ELOVL2-like elongases.

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 complementary DNA (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-amino-acid (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 zebra fish, Acanthopterygian fish species lack elovl2 which is consistent with their negligible ability to biosynthesize 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 biosynthesize LC-PUFA and may provide a biotechnological tool to produce enhanced levels of LC-PUFA, particularly DHA, in transgenic organisms.