Essential fatty acid metabolism and requirements of the cleaner fish, ballan wrasse Labrus bergylta: Defining pathways of long-chain polyunsaturated fatty acid biosynthesis

Ballan wrasse (Labrus bergylta) is an effective counter-measure against sea lice used by Atlantic salmon farmers, proving to be more effective and economical than drugs or chemical treatments alone. There are currently efforts underway to establish a robust culture system for this species, however, essential fatty acid dietary requirements are not known for ballan wrasse. In the present study, we isolated and functionally characterised ballan wrasse fatty acid desaturase (Fads) and elongation of very long-chain fatty acids (Elovl) protein to elucidate their long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic capability. Sequence and phylogenetic analysis demonstrated that the cloned genes were fads2 and elovl5 orthologues of other teleost species. Functional characterisations of fads2 and elovl5 were performed using the yeast (Saccharomyces cerevisiae) heterologous expression system. The Fads2 showed Δ6 desaturase activity towards 18:3n–3, 18:2n–6 and 24:5n–3, and Δ8 desaturase activity towards 20:3n–6 and 20:2n–6. The Elovl5 showed elongase activities towards various C18 and C20 fatty acids. Therefore, 20:4n–3 and 20:3n–6 can be synthesised from 18:3n–3 and 18:2n–6, respectively in ballan wrasse via two possible pathways, the Δ6 (Δ6 desaturation – elongation) and Δ8 (elongation – Δ8 desaturation) pathways. However, due to the absence of Δ5 desaturase activity and no other Fads2 in their genome, 20:5n–3 (eicosapentaenoic acid, EPA) and 20:4n–6 (arachidonic acid, ARA) cannot be synthesised from C18 PUFA precursors and they could consequently be regarded as dietary essential fatty acids for ballan wrasse. Since no Δ4 desaturase activity was detected in ballan wrasse Fads2, 22:6n–3 (docosahexaenoic acid, DHA) can only be synthesised from EPA via the Sprecher pathway comprising two sequential elongation steps to produce 24:5n–3 followed by Δ6 desaturation and chain shortening. Although ballan wrasse Elovl5 had no elongase activity towards C22, other elongases such as Elovl4 exist in the ballan wrasse genome that may be able to produce 24:5n–3. Therefore, as ballan wrasse Fads2 can desaturate 24:5n–3 to produce 24:6n-­3, it can be assumed that ballan wrasse can synthesise DHA from EPA

Biosynthesis of Polyunsaturated Fatty Acids in Sea Urchins: Molecular and Functional Characterisation of Three Fatty Acyl Desaturases from Paracentrotus lividus (Lamark 1816)

Sea urchins are broadly recognised as a delicacy and their quality as food for humans is highly influenced by their diet. Lipids in general and the long-chain polyunsaturated fatty acids (LC-PUFA) in particular, are essential nutrients that determine not only the nutritional value of sea urchins but also guarantee normal growth and reproduction in captivity. The contribution of endogenous production (biosynthesis) of LC-PUFA in sea urchins remained unknown. Using Paracentrotus lividus as our model species, we aimed to characterise both molecularly and functionally the repertoire of fatty acyl desaturases (Fads), key enzymes in the biosynthesis of LC-PUFA, in sea urchins. Three Fads, namely FadsA, FadsC1 and FadsC2, were characterised. The phylogenetic analyses suggested that the repertoire of Fads within the Echinodermata phylum varies among classes. On one hand, orthologues of the P. lividus FadsA were found in other echinoderm classes including starfishes, brittle stars and sea cucumbers, thus suggesting that this desaturase is virtually present in all echi- noderms. Contrarily, the FadsC appears to be sea urchin-specific desaturase. Finally, a fur- ther desaturase termed as FadsB exists in starfishes, brittle stars and sea cucumbers, but appears to be missing in sea urchins. The functional characterisation of the P. lividus Fads confirmed that the FadsA was a Δ5 desaturase with activity towards saturated and polyun- saturated fatty acids (FA). Moreover, our experiments confirmed that FadsA plays a role in the biosynthesis of non-methylene interrupted FA, a group of compounds typically found in marine invertebrates. On the other hand, both FadsC desaturases from P. lividus showed Δ8 activity. The present results demonstrate that P. lividus possesses desaturases that account for all the desaturation reactions required to biosynthesis the physiological essential eicosapentaenoic and arachidonic acids through the so-called “Δ8 pathway”

Two alternative pathways for docosahexaenoic acid (DHA, 22:6n-3) biosynthesis are widespread among teleost fish

Docosahexaenoic acid (DHA) plays important physiological roles in vertebrates. Studies in rats and rainbow trout confirmed that DHA biosynthesis proceeds through the so-called “Sprecher pathway”, a biosynthetic process requiring a Δ6 desaturation of 24:5n-3 to 24:6n-3. Alternatively, some teleosts possess fatty acyl desaturases 2 (Fads2) that enable them to biosynthesis DHA through a more direct route termed the “Δ4 pathway”. In order to elucidate the prevalence of both pathways among teleosts, we investigated the Δ6 ability towards C24 substrates of Fads2 from fish with different evolutionary and ecological backgrounds. Subsequently, we retrieved public databases to identify Fads2 containing the YXXN domain responsible for the Δ4 desaturase function, and consequently enabling these species to operate the Δ4 pathway. We demonstrated that, with the exception of Δ4 desaturases, fish Fads2 have the ability to operate as Δ6 desaturases towards C24 PUFA enabling them to synthesise DHA through the Sprecher pathway. Nevertheless, the Δ4 pathway represents an alternative route in some teleosts and we identified the presence of putative Δ4 Fads2 in a further 11 species and confirmed the function as Δ4 desaturases of Fads2 from medaka and Nile tilapia. Our results demonstrated that two alternative pathways for DHA biosynthesis exist in teleosts

Functional diversification of teleost Fads2 fatty acyl desaturases occurs independently of the trophic level

The long-chain (≥C20) polyunsaturated fatty acid biosynthesis capacity of fish varies among species, with trophic level hypothesised as a major factor. The biosynthesis capacity is largely dependent upon the presence of functionally diversified fatty acyl desaturase 2 (Fads2) enzymes, since many teleosts have lost the gene encoding a Δ5 desaturase (Fads1). The present study aimed to characterise Fads2 from four teleosts occupying different trophic levels, namely Sarpa salpa, Chelon labrosus, Pegusa lascaris and Atherina presbyter, which were selected based on available data on functions of Fads2 from closely related species. Therefore, we had insight into the variability of Fads2 within the same phylogenetic group. Our results showed that Fads2 from S. salpa and C. labrosus were both Δ6 desaturases with further Δ8 activity while P. lascaris and A. presbyter Fads2 showed Δ4 activity. Fads2 activities of herbivorous S. salpa are consistent with those reported for carnivorous Sparidae species. The results suggested that trophic level might not directly drive diversification of teleost Fads2 as initially hypothesised, and other factors such as the species’ phylogeny appeared to be more influential. In agreement, Fads2 activities from P. lascaris and A. presbyter were similar to their corresponding phylogenetic counterparts Solea senegalensis and Chirostoma estor

Retention of fatty acyl desaturase 1 (fads1) in Elopomorpha and Cyclostomata provides novel insights into the evolution of long-chain polyunsaturated fatty acid biosynthesis in vertebrates

Background Provision of long-chain polyunsaturated fatty acids (LC-PUFA) in vertebrates occurs through the diet or via endogenous production from C18 precursors through consecutive elongations and desaturations. It has been postulated that the abundance of LC-PUFA in the marine environment has remarkably modulated the gene complement and function of Fads in marine teleosts. In vertebrates two fatty acyl desaturases, namely Fads1 and Fads2, encode ∆5 and ∆6 desaturases, respectively. To fully clarify the evolutionary history of LC-PUFA biosynthesis in vertebrates, we investigated the gene repertoire and function of Fads from species placed at key evolutionary nodes. Results We demonstrate that functional Fads1Δ5 and Fads2∆6 arose from a tandem gene duplication in the ancestor of vertebrates, since they are present in the Arctic lamprey. Additionally, we show that a similar condition was retained in ray-finned fish such as the Senegal bichir and spotted gar, with the identification of fads1 genes in these lineages. Functional characterisation of the isolated desaturases reveals the first case of a Fads1 enzyme with ∆5 desaturase activity in the Teleostei lineage, the Elopomorpha. In contrast, in Osteoglossomorpha genomes, while no fads1 was identified, two separate fads2 duplicates with ∆6 and ∆5 desaturase activities respectively were uncovered. Conclusions We conclude that, while the essential genetic components involved LC-PUFA biosynthesis evolved in the vertebrate ancestor, the full completion of the LC-PUFA biosynthesis pathway arose uniquely in gnathostomes

Examination of gammarid transcriptomes reveals a widespread occurrence of key metabolic genes from epibiont bdelloid rotifers in freshwater species

Previous data revealed the unexpected presence of genes encoding for long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic enzymes in transcriptomes from freshwater gammarids but not in marine species, even though closely related species were compared. This study aimed to clarify the origin and occurrence of selected LC-PUFA biosynthesis gene markers across all published gammarid transcriptomes. Through systematic searches, we confirmed the widespread occurrence of sequences from seven elongases and desaturases involved in LC-PUFA biosynthesis, in transcriptomes from freshwater gammarids but not marine species, and clarified that such occurrence is independent from the gammarid species and geographical origin. The phylogenetic analysis established that the retrieved elongase and desaturase sequences were closely related to bdelloid rotifers, confirming that multiple transcriptomes from freshwater gammarids contain contaminating rotifers’ genetic material. Using the Adineta steineri genome, we investigated the genomic location and exon–intron organization of the elongase and desaturase genes, establishing they are all genome-anchored and, importantly, identifying instances of horizontal gene transfer. Finally, we provide compelling evidence demonstrating Bdelloidea desaturases and elongases enable these organisms to perform all the reactions for de novo biosynthesis of PUFA and, from them, LC-PUFA, an advantageous trait when considering the low abundance of these essential nutrients in freshwater environments

Lipid metabolism in Tinca tinca and its n-3 LC-PUFA biosynthesis capacity

Carps, barbels and other cyprinids are the major contributors to freshwater aquaculture at global scale. Nevertheless, freshwater fish aquaculture needs to diversify their production in order to offer consumers new species. Tench (Tinca tinca) is a freshwater species with great interest for the diversification of continental aquaculture. However, up to date, no commercial formulated diet exists for this species in order to optimize their nutritional requirements and the quality of its final product. Using multiple methodological approaches, the aim of this study was to evaluate the long chain polyunsaturated fatty acid (LC-PUFA) metabolism of T. tinca. Firstly, the molecular cloning and functional characterisation by heterologous expression in yeast of a desaturase (Fads2) and two elongases (Elovl2 and Elovl5) involved in LC-PUFA biosynthesis, and the analysis of gene expression among tissues were performed. Secondly, in order to confirm the LC-PUFA biosynthesis capacity of isolated hepatocytes and enterocytes, cells were incubated with [1-14C] labelled linoleic acid (18:2n-6, LA), linolenic acid (18:3n-3, ALA) and eicosapentaenoic acid (20:5n-3, EPA). In yeast, Fads2 showed a Δ6/Δ5 bifunctional activity. Elovl2 was more active over C20 and C22 substrates, whereas Elovl5 was over C18 and C20. Liver displayed the highest expression for the three target genes (fads2, elovl2 and elovl5). Incubated cells also showed Fads2 bifunctional activity as well as elongation products in concordance with yeast heterologous expression results. Importantly, our results demonstrated that tench is able to biosynthesise docosahexaenoic acid (DHA) from 18:3n-3 in both hepatocytes and enterocytes, a capacity that seems to explain in part the surprisingly high levels of DHA found in the fish flesh compared to its dietary supply. Tench is a promising freshwater species with a potential capacity to endogenously increase its flesh DHA contents, reducing the impact that the usage of fish oils from forage fisheries may have on the aquaculture industry

Unraveling the potential of marine invertebrates as a source of omega-3 long-chain polyunsaturated fatty acids for aquaculture

Trabajo presentado en Aquaculture Europe, celebrado en Rimini (Italia), del 27 al 30 de septiembre de 2022

Polyunsaturated fatty acid metabolism in three fish species with different trophic level

Reducing the dependency of fishfeed for marine ingredients and species diversification are both considered crucial factors for the sustainable development of aquaculture. The substitution of fish oil (FO) by vegetable oils (VO) in aquafeeds is an economically feasible solution. However, such substitution may compromise the fish flesh content of essential n-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA) and, therefore, its nutritional value for human consumption. Likewise, there is a wide range of strategies to select new target species for sector diversification, among which, the capacity to biosynthesize n-3 LC-PUFA from their C18 precursors abundant in VO might be considered as a fair preliminary strategy. Therefore, the aim of the present study was to analyze the metabolic fate of [1-14C] labeled 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3 in isolated hepatocytes and enterocytes from wild individuals of three fish species with different trophic level: the marine herbivorous salema (Sarpa salpa), the strict carnivorous sand sole (Pegusa lascaris) and the omnivorous thicklip grey mullet (Chelon labrosus). These species were selected for their phylogenetic proximity to consolidated farmed species such as gilthead seabream (Sparus aurata), senegalese sole (Solea senegalensis), and golden grey mullet (Liza aurata), respectively. The study also assessed the molecular cloning, functional characterization and tissue distribution of the fatty acyl elongase (Elovl) gene, elovl5, involved in the biosynthetic metabolism of n-3 LC-PUFA. The three species were able to biosynthesize docosahexaenoic acid (22:6n-3). S. salpa seems to have similar biosynthetic capacity than S. aurata, with a fatty acyl desaturase 2 (Fads2), with 6, 8 and 5 activities. P. lascaris showed a wider Fads2 activity repertory than S. senegalensis, including 4 and residual 6/5 activities. In C. labrosus, both 8 and 5 activities but not the 6 described for L. aurata were detected in the incubated cells. Elongation from C18 and C20 precursors to C20 and C22 products occurred in hepatocytes and enterocytes as well as in the functional characterization of Elovl5 by heterologous expression in yeast. Elovl5 showed a species specific expression pattern, with the highest rates observed in the liver, gut and brain in S. salpa and P. lascaris, and in the brain for C. labrosus. In summary, the LC-PUFA biosynthesis capacity from S. salpa, P. lascaris and C. labrosus greatly resembled that of their phylogenetic closer species. The three studied species could be further explored as candidates for the aquaculture diversification from their potential ability to biosynthesize LC-PUFA

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

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.Marine ecosystems are responsible for virtually all production of omega-3 (ω3) long- chain polyunsaturated fatty acids (PUFA), essential nutrients for vertebrates. Current consensus is that marine microbes account for this production, given they have key enzymes including methyl-end (or “ωx”) desaturases. ωx desaturases have also been described in a small number of 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 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 represents a fundamental revision in our understanding of ω3 long- chain PUFA production in global food webs, as numerous widespread and abundant invertebrates have the endogenous capacity to make significant contributions beyond that coming from marine microbes.Publisher PDFPeer reviewe