Sofia Morais

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.

Long chain polyunsaturated fatty acid synthesis in a marine vertebrate: ontogenetic and nutritional regulation of a fatty acyl desaturase with Δ4 activity.

Solea senegalensis is an unusual marine teleost as it has very low dietary requirement for long-chain 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 characterized fatty acyl desaturase and elongase cDNAs corresponding to Δ4fad (with some Δ5 activity for the n-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 higher or lower dietary n-3 LC-PUFA was assessed during larval and post-larval stages. There was a marked pattern of regulation during early ontogenesis, with both transcripts showing peak expression coinciding with the start of exogenous feeding. Although elovl5 transcripts were present in fertilized eggs, Δ4fad only appeared at hatching. However, eggs have high proportions of DHA (~20%) and high DHA/EPA ratio (~11) to meet the high demands for early embryonic development. The fatty acid profile of larvae after the start of exogenous feeding closely reflected dietary composition. Nonetheless, Δ4fad 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.

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.

A rearing system for the culture of ornamental decapod crustacean larvae

Abstract The design and operation of a small research scale and a mass commercial scale rearing system for the culture of marine ornamental decapod crustacean larvae are described in the present paper. Preliminary data on the culture of the Mediterranean cleaner shrimp ( Lysmata seticaudata ), peppermint shrimp ( Lysmata wurdemanni ), blue-white partner shrimp ( Periclimenes sagittifer ), sponge crab ( Cryptodromiopsis antillensis ) and green emerald crab ( Mithraculus sculptus ) are also presented. The use of these “plantonkreisel” based systems allowed the complete larval development of the above-mentioned species, inducing minimal mechanical stress while keeping an excellent water quality. Higher survival rates (up to 70% and 60% for L. seticaudata and L. wurdemanni , respectively) to the post-larval stage and a shorter larval stage duration (27 and 22 days for L. seticaudata and L. wurdemanni , respectively) were achieved, in comparison to conventional rearing systems. This culture technology may play a key role in the realisation of a commercial culture of these highly priced crustacean species and therefore the reduction of wild specimen collection.

Lipid enrichment for Senegalese sole (Solea senegalensis) larvae: effect on larval growth, survival and fatty acid profile

Results from three larval Senegalese sole (Solea senegalensis) feeding trials using non-enriched Artemia and Artemia enriched with Super HUFA®, Arasco®, sunflower oil and microalgae are presented and the effects on larval survival, growth and fatty acid (FA) composition are reported. The FA profile of Senegalese sole eggs was analysed to gather information about the nutritional requirements of the early larval stages and a quite high DHA/EPA ratio (4.3) was found. However, there was no evidence of a high dietary demand for DHA or EPA, given that no relationship was found between dietary HUFA concentration and larval growth and survival. When larvae were fed non-enriched Artemia a significantly better growth and comparable survival were obtained than with Artemia enriched with Super HUFA® (containing the highest HUFA level and DHA/EPA ratio). The FA profiles of the larvae generally reflected those of their diets. DHA was an exception, as it was present in high proportions, even in larvae fed DHA-deficient prey. Total FAME concentration decreased during larval development, with SFA, MUFA and PUFA being equally consumed; HUFA appeared to be less used, with its relative concentration being either kept constant (particularly EPA and ARA) or increased (DHA). A specific requirement for ARA in the first larval stages could not be confirmed but it was always present in considerable amounts, even in larvae fed an ARA poor diet.

Transcriptomic analyses of intestinal gene expression of juvenile Atlantic cod (Gadus morhua) fed diets with Camelina oil as replacement for fish oil.

For aquaculture of marine species to continue to expand, dietary fish oil (FO) must be replaced with more sustainable vegetable oil (VO) alternatives. Most VO are rich in n-6 polyunsaturated fatty acids (PUFA) and few are rich in n-3 PUFA but Camelina oil (CO) is unique in that, besides high 18:3n-3 and n-3/n-6 PUFA ratio, it also contains substantial long-chain monoenes, commonly found in FO. Cod (initial mass ~1.4 g) were fed for 12 weeks diets in which FO was replaced with CO. Growth performance, feed efficiency and biometric indices were not affected but lipid levels in liver and intestine tended to increase and those of flesh, decrease, with increasing dietary CO although only significantly for intestine. Reflecting diet, tissue n-3 long-chain PUFA levels decreased whereas 18:3n-3 and 18:2n-6 increased with inclusion of dietary CO. Dietary replacement of FO by CO did not induce major metabolic changes in intestine, but affected genes with potential to alter cellular proliferation and death as well as change structural properties of intestinal muscle. Although the biological effects of these changes are unclear, given the important role of intestine in nutrient absorption and health, further attention should be given to this organ in future.

Dietary protein:lipid ratio and lipid nature affects fatty acid absorption and metabolism in a teleost larva

Studies with teleost larvae have reported poor performance associated with quantitative lipid imbalances in the diet. The present study examined the effect of dietary protein:neutral lipid ratio on fatty acid (FA) absorption efficiency and metabolism in larval Senegalese sole. In addition, the effect of lipid class (triolein (TRI) and l-3-phosphatidylcholine-1,2-di-oleoyl (PC)), carbon number and degree of saturation of the labelled NEFA, stearic acid (SA), oleic acid (OA) and DHA) was tested. FA absorption was determined by tube feeding [1-14C]-labelled lipids and NEFA after a single meal of either non-enriched Artemia (NEA) or Artemia enriched on a soyabean oil emulsion (EA), or after feeding these diets over an extended period of time (18 d). The tested dietary protein:lipid ratios had no short-term influence but long-term feeding of a diet higher in neutral lipid (EA) increased lipid accumulation within the gut epithelium and resulted in lower FA absorption (higher label evacuation and lower retention of dietary FA), which may partially explain the trend for lower growth observed with this diet. The lipids and NEFA, showed different digestive and metabolic properties, independent of feeding regime. FA absorption increased with unsaturation, being lowest for SA, followed by OA, and highest for DHA. In addition, sole larvae had a lower capacity to digest and absorb FA esterified to TRI, compared with PC, with the order of decreasing absorption being NEFA>PC>>TRI. Moreover, larvae appeared to discriminate between the source of OA, as this FA in the free form or esterified to PC was catabolised less than TRI.

Dietary TAG source and level affect performance and lipase expression in larval sea bass (Dicentrarchus labrax)

The influence of dietary TAG source (fish oil, triolein, and coconut oil) and level (7.5 and 15% of the diet) on growth, lipase activity, and mRNA level was studied in sea bass larvae, from mouth opening until day 24 and from day 37 to 52. Fish oil and triolein induced better growth in both experiments, this being significant at a higher dietary level. Coconut oil significantly decreased growth at the higher level, possibly as the result of an excessive supply of medium-chain TAG. Growth was not related to lipase specific activity, suggesting a production in excess to dietary needs. Body lipid content was positively related to dietary lipid level and was affected by lipid quality. In addition, larval FA composition generally reflected that of the diet. The source of dietary lipid, but not the quantity, was shown to affect lipase activity significantly. Coconut oil diets induced the highest lipase activity, whereas the effect of fish oil was age dependent-it was similar to coconut oil at day 24 but induced the lowest lipase activity in 52-d-old larvae. The differential lipase response was probably caused by differences in the FA composition of the diet, related to the specificity of lipase toward FA differing in chain length and degree of saturation. No significant differences were found in lipase/glyceraldehyde-3-phosphate dehydrogenase mRNA, which suggests the existence of a posttranscriptional regulation mechanism.

Gonad development and fatty acid composition of Patella depressa Pennant (Gastropoda : Prosobranchia) populations with different patterns of spatial distribution, in exposed and sheltered sites

The present study examines the effect of shore exposure on the feeding performance (assessed by fatty acid analyses of the whole body) and gonad condition (stage of development and gonad somatic index, GSI) of Patella depressa populations. Male and female limpets were collected at exposed and sheltered sites, during winter and summer. The population at the exposed site was at a more advanced stage of gonad development, with a higher dispersion of gonad stages, both in winter and summer. Additionally, limpets from the exposed site, particularly the males, presented a higher GSI than the corresponding stage in the sheltered site. The quantitatively most important fatty acids were the saturated fatty acids (SFA) 16:0, 14:0, and 18:0, the monounsaturated fatty acids (MUFA) 18:1(n-7), 18:1(n-9), 16:1(n-7) and 20:1(n-9) and the polyunsaturated fatty acids (PUFA) 20:5(n-3) and 20:4(n-6). Females had a significantly higher fatty acid methyl esters (FAME) content (in summer and winter) and higher amounts of SFA and MUFA (in summer), which points to a higher degree of storage of neutral lipids in this sex. Male and female limpets at the exposed site had a significantly higher FAME, SFA, MUFA, PUFA and highly unsaturated fatty acids (HUFA) content than the corresponding sex in the sheltered site in summer. In addition, an inversion in the eicosapentaenoic acid (EPA)/arachidonic acid (ARA) and (n-3)/(n-6) ratios was observed in the sheltered site, as a result of the significantly higher levels of ARA and (n-6) fatty acids and lower amounts of EPA and (n-3) fatty acids found in the sheltered limpets. A high variability among patches in the fatty acid composition in the exposed site was found in winter, possibly related to the aggregation of limpets at this time. The differences found between limpets from the exposed and sheltered sites suggest qualitative and quantitative differences in their diets. Additionally, the results show that the spatial aggregation strategy adopted by limpets in sites of great wave and wind exposure does not affect their feeding and reproductive success, at least in the site examined here. In fact, more developed gonads, a higher GSI and an elevated FAME content was found in the exposed population. Possible factors are suggested and discussed to explain these observations.

Biochemical changes during the embryonic development of Norway lobster, Nephrops norvegicus

The objectives of the present study were to investigate the total and free amino acid profiles and lipid dynamics (lipid classes and fatty acids) during embryogenesis of Nephrops norvegicus, in order to understand the early larval protein and lipid requirements. There was a significant increase in total essential (EAA) and nonessential amino acid (NEAA) contents during embryonic development (P<0.05). The major EAA were arginine, histidine and leucine, while the most important NEAA were glutamic acid, aspartic acid, glycine and proline. The higher percent increase occurred in respect to NEAA (19.9%), mainly due to the significant increase of glycine (51.8%) and alanine (35.1%). The free amino acid (FAA) content, especially the free nonessential amino acids (FNEAA), also increased significantly, being the quantitatively most important glycine, proline, taurine and glutamic acid. The free essential amino acids (FEAA) also revealed significant variations and the most important were arginine, lysine and leucine. A higher percent increase was attained with FNEAA (66.8%) in comparison to FEAA (49.6%), mainly due to the significant increase of homocystine (87.7%), valine (83.5%), glutamine (82.5%) and glutamic acid (76.1%). The absorption of dissolved organic compounds from seawater can explain the increase in free and total amino acid (FAA and TAA) contents, because a significant increase in the water content during development was observed (P<0.05). A substantial decrease in all neutral lipid classes (P<0.05) was observed during embryonic development, namely diacylglycerols (DAG) (97.9% of utilization), triacylglycerols (TAG) (93.2%), sterol esters (StE) (91.3%) and monoacylglycerols (MAG) (90.4%). The quantitatively most important fatty acids were the saturates (SFA) 14:0, 16:0 and 18:0, the monounsaturates (MUFA) 16:1n-7, 18:1n-9 and 18:1n-7, and the polyunsaturates (PUFA) 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3. The unsaturated fatty acids (UFA) are used up at a higher rate (54.5% of utilization) than SFA (42.8%); within the UFA, MUFA are more consumed than PUFA (59.3% and 52.7%, respectively). In terms of the utilization of individual fatty acids, there was a preferential consumption of 20:4n-3, 22:6n-3, 18:1n-9, 22:5n-3 and 16:1n-7. It is evident that N. norvegicus depends primarily on lipid reserves of the egg during early ontogeny. On the contrary, this species tends to conserve EAA and increase NEAA and FNEAA contents during embryonic development.