Characterization and comparison of fatty acyl Delta 6 desaturase cDNAs from freshwater and marine teleost fish species

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterise and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead seabream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b5 domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Δ6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, seabream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Δ5 desaturase activity, but none of the products showed Δ4 desaturase activity. The cloning and characterisation of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur

The autophagy response during adipogenesis of primary cultured rainbow trout (Oncorhynchus mykiss) adipocytes

Adipogenesis is a tightly regulated process, and the involvement of autophagy has been recently proposed in mammalian models. In rainbow trout, two well-defined phases describe the development of primary cultured adipocyte cells: proliferation and differentiation. Nevertheless, information on the transcriptional profile at the onset of differentiation and the potential role of autophagy in this process is scarce. In the present study, the cells showed an early and transient induction of several adipogenic transcription factors genes' expression (i.e., cebpa and cebpb) along with the morphological changes (round shape filled with small lipid droplets) typical of the onset of adipogenesis. Then, the expression of various lipid metabolism-related genes involving the synthesis (fas), uptake (fatp1 and cd36), accumulation (plin2) and mobilization (hsl) of lipids, characteristic of the mature adipocyte, increased. In parallel, several autophagy markers (i.e., atg4b, gabarapl1 and lc3b) mirrored the expression of those adipogenic-related genes, suggesting a role of autophagy during in vitro fish adipogenesis. In this regard, the incubation of preadipocytes with lysosomal inhibitors (Bafilomycin A1 or Chloroquine), described to prevent autophagy flux, delayed the process of adipogenesis (i.e., cell remodelling), thus suggesting a possible relationship between autophagy and adipocyte differentiation in trout. Moreover, the disruption of the autophagic flux altered the expression of some key adipogenic genes such as cebpa and pparg. Overall, this study contributes to improve our knowledge on the regulation of rainbow trout adipocyte differentiation, and highlights for the first time in fish the involvement of autophagy on adipogenesis, suggesting a close-fitting connection between both processes

What determines growth potential and juvenile quality of farmed fish species?

Enhanced production of high quality and healthy fry is a key target for a successful and competitive expansion of the aquaculture industry. Although large quantities of fish larvae are produced, survival rates are often low or highly variable and growth potential is in most cases not fully exploited, indicating significant gaps in our knowledge concerning optimal nutritional and culture conditions. Understanding the mechanisms that control early development and muscle growth are critical for the identification of time windows in development that introduce growth variation, and improve the viability and quality of juveniles. This literature review of the current state of knowledge aims to provide a framework for a better understanding of fish skeletal muscle ontogeny, and its impact on larval and juvenile quality as broadly defined. It focuses on fundamental biological knowledge relevant to larval phenotype and quality and, in particular, on the factors affecting the development of skeletal muscle. It also discusses the available methodologies to assess growth and larvae/juvenile quality, identifies gaps in knowledge and suggests future research directions. The focus is primarily on the major farmed non-salmonid fish species in Europe that include gilthead sea bream, European sea bass, turbot, Atlantic cod, Senegalese sole and Atlantic halibut

Environmental and dietary influences on highly unsaturated fatty acid biosynthesis and expression of fatty acyl desaturase and elongase genes in liver of Atlantic salmon (Salmo salar)

Highly unsaturated fatty acid (HUFA) synthesis in Atlantic salmon (Salmo salar) was known to be influenced by both nutritional and environmental factors. Here we aimed to test the hypothesis that both these effectors involved similar molecular mechanisms. Thus, HUFA biosynthetic activity and the expression of fatty acyl desaturase and elongase genes were determined at various points during an entire two year production cycle in salmon fed diets containing either 100% fish oil or diets in which a high proportion (75% and 100%) of fish oil was replaced by C18 polyunsaturated fatty acid-rich vegetable oil. The results showed that HUFA biosynthesis in Atlantic salmon varied during the growth cycle with peak activity around seawater transfer and subsequent low activities in seawater. Consistent with this, gene expression of Δ6 desaturase, the rate-limiting step in the HUFA biosynthetic pathway, was highest around the point of seawater transfer and lowest during the seawater phase. In addition, the expression of both Δ6 and Δ5 desaturase genes was generally higher in fish fed the vegetable oil-substituted diets compared to fish fed fish oil, particularly in the seawater phase. Again, generally consistent with this, the activity of the HUFA biosynthetic pathway was invariably higher in fish fed diets in which fish oil was substituted by vegetable oil compared to fish fed only fish oil. In conclusion, these studies showed that both nutritional and environmental modulation of HUFA biosynthesis in Atlantic salmon involved regulation of fatty acid desaturase gene expression

The Autophagic Flux Inhibitor Bafilomycine A1 Affects the Expression of Intermediary Metabolism-Related Genes in Trout Hepatocytes

Autophagy is an evolutionarily conserved process of cellular self-eating which emerged these last years as a major adaptive metabolic response to various stresses such as fasting, hypoxia, or environmental pollutants. However, surprisingly very few data is currently available on its role in fish species which are directly exposed to frequent environmental perturbations. Here, we report that the treatment of fasted trout hepatocytes with the autophagy inhibitor Bafilomycine A1 lowered the mRNA levels of many of the gluconeogenesis-related genes and increased those of genes involved in intracellular lipid stores. Concurrently, intracellular free amino acid levels dropped and the expression of the main genes involved in the endoplasmic reticulum (ER) stress exhibited a sharp increase in autophagy inhibited cells. Together these results highlight the strong complexity of the crosstalk between ER, autophagy and metabolism and support the importance of considering this function in future studies on metabolic adaptation of fish to environmental stresses

Expression of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis genes during zebrafish Danio rerio early embryogenesis

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential in important physiological processes, many of which are particularly vital during embryonic development. This study investigated the expression of genes encoding enzymes involved in LC-PUFA biosynthesis, namely fatty acyl desaturase (Fad) and Elovl5- and Elovl2-like elongases, during early embryonic development of zebrafish. Firstly, zebrafish elovl2 cDNA was isolated and functionally characterised in yeast, showing high specificity towards C20 and C22 PUFAs, compared to C18 substrates. Secondly, spatial-temporal expression for elovl2 and the previously cloned fad and elovl5 were studied during zebrafish early embryonic development. Temporal expression shows that all three genes are expressed from the beginning of embryogenesis (zygote), suggesting maternal mRNA transfer to the embryo. However, a complete activation of the biosynthetic pathway seems to be delayed until 12 hpf, when noticeable increases of fad and elovl2 transcripts were observed, in parallel with high docosahexaenoic acid levels in the embryo. Spatial expression was studied by whole-mount in situ hybridization in 24 hpf embryos, showing that fad and elovl2 are highly expressed in the head area where neuronal tissues are developing. Interestingly, elovl5 shows specific expression in the pronephric ducts, suggesting an as yet unknown role in fatty acid metabolism during zebrafish early embryonic development. The yolk syncytial layer also expressed all three genes, suggesting an important role in remodelling of yolk fatty acids during zebrafish early embryogenesis. Tissue distribution in zebrafish adults demonstrates that the target genes are expressed in all tissues analysed, with liver, intestine and brain showing the highest expression

Physiological roles of fatty acyl desaturases and elongases in marine fish: Characterisation of cDNAs of fatty acyl delta6 desaturase and elovl5 elongase of cobia (Rachycentron canadum)

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)

Fish larval nutrition and feed formulation: knowledge gaps and bottlenecks for advances in larval rearing

Despite considerable progress in recent years, many questions regarding fish larval nutrition remain largely unanswered, and several research avenues remain open. A holistic understanding of the supply line of nutrients is important for developing diets for use in larval culture and for the adaptation of rearing conditions that meet the larval requirements for the optimal presentation of food organisms and/or microdiets. The aim of the present review is to revise the state of the art and to pinpoint the gaps in knowledge regarding larval nutritional requirements, the nutritional value of live feeds and challenges and opportunities in the development of formulated larval diets.Norwegian Ministry of Fisheries; Research Council of Norway [CODE-199482, GutFeeling-190019]; Spanish Ministry of Science and Innovation MICINN + FEDER/ERDF [AGL2007-64450-C02-01, CSD2007-0002]; project HYDRAA [PTDC/MAR/71685/2006]; Fundacao para a Ciencia e a Tecnologia (FCT), Portugal; FEDER; EC [LIFECYCLE- 222719]; EU RTD [FA0801]info:eu-repo/semantics/publishedVersio

Long-chain polyunsaturated fatty acid synthesis in fish: Comparative analysis of Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.) Delta 6 fatty acyl desaturase gene promoters

Fish vary in ability to biosynthesise n-3 long-chain polyunsaturated fatty acids (LC-PUFA), with marine fish such as cod being inefficient in comparison to freshwater and salmonid fish. We investigated differences in the gene promoters of Δ6 fatty acyl desaturase (Δ6 FAD), a critical enzyme in LC-PUFA biosynthesis, in cod and salmon. Progressive deletions and targeted mutations of the promoters were tested for activity in a transfected fish cell line under low or high LC-PUFA treatment, and regions sufficient to direct transcription were identified. Comparison of these regions with sequences of corresponding regions of Δ6 FAD genes from mammals, amphibians and fish indicated a remarkable conservation of binding sites for SREBPs and NF-Y. In addition to these sites, a site was identified in salmon with similarity to that recognised by Sp1 transcription factor, and which was required for full expression of the salmon Δ6 FAD gene. The cod promoter was less active and lacked the Sp1 site. Eicosapentaenoic acid suppressed LC-PUFA synthesis in AS cells and also suppressed activity of the salmon Δ6 FAD promoter although this activity was likely mediated through sites other than Sp1, possibly similar to those recognised by NF-Y and SREBP transcription factors

Highly unsaturated fatty acid synthesis in marine fish: Cloning, functional characterization, and nutritional regulation of fatty acyl delta6 desaturase of Atlantic cod (Gadus morhua L.)

Fish contain high levels of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. Biosynthesis of HUFA requires enzyme-mediated desaturation of fatty acids. Here we report cloning and functional characterisation of a ∆6 fatty acyl desaturase of Atlantic cod (Gadus morhua), and describe its tissue expression and nutritional regulation. PCR primers were designed based on the sequences of conserved motifs in available fish desaturases and used to isolate a cDNA fragment from liver of cod. The full-length cDNA was obtained by Rapid Amplification of cDNA Ends (RACE). The cDNA for the putative fatty acyl desaturase was shown to comprise 1980bp which included a 5’-UTR of 261bp and a 3’-UTR of 375bp. Sequencing revealed that the cDNA included an ORF of 1344 bp that specified a protein of 447 amino acids. The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the haem-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. The cDNA displayed Δ6 desaturase activity in a heterologous yeast expression system. Quantitative real time PCR assay of gene expression in cod showed that the ∆6 desaturase gene, was highly expressed in brain, relatively highly expressed in liver, kidney, intestine, red muscle and gill, and expressed at much lower levels in white muscle, spleen and heart. In contrast, the abundance of a cod fatty acyl elongase transcript was high in brain and gill, with intermediate levels in kidney, spleen, intestine and heart, and relatively low expression in liver. The expression of the Δ6 desaturase gene and the PUFA elongase gene may be under a degree of nutritional regulation, with levels being marginally increased in livers and intestine of fish fed a vegetable oil blend by comparison with levels in fish fed fish oil. However, this was not reflected in increased Δ6 desaturase activity in hepatocytes or enterocytes, which showed very little highly unsaturated fatty acid biosynthesis activity irrespective of diet. The study described has demonstrated that Atlantic cod express a fatty acid desaturase gene with functional Δ6 activity in a yeast expression system. This is consistent with an established hypothesis that the poor ability of marine fish to synthesise HUFA is not due to lack of a Δ6 desaturase, but rather to deficiencies in other parts of the biosynthetic pathway. However, further studies are required to determine why the Δ6 desaturase appears to be barely functional in cod under the conditions tested