Long-chain Polyunsaturated Fatty Acids Biosynthesis Research Articles

A complete biosynthetic pathway of the long-chain polyunsaturated fatty acids in an amphidromous fish, ayu sweetfish Plecoglossus altivelis (Stomiati; Osmeriformes)

The biosynthetic capability of the long-chain polyunsaturated fatty acids (LC-PUFA) in teleosts are highly diversified due to evolutionary events such as gene loss and subsequent neo- and/or sub-functionalisation of enzymes encoded by existing genes. In the present study, we have comprehensively characterised genes potentially involved in LC-PUFA biosynthesis, namely one front-end desaturase (fads2) and eight fatty acid elongases (elovl1a, elovl1b, elovl4a, elovl4b, elovl5, elovl7, elovl8a and elovl8b) from an amphidromous teleost, Ayu sweetfish, Plecoglossus altivelis. Functional analysis confirmed Fads2 with Δ6, Δ5 and Δ8 desaturase activities towards multiple PUFA substrates and several Elovl enzymes exhibited elongation capacities towards C18–20 or C18–22 PUFA substrates. Consequently, P. altivelis possesses a complete enzymatic capability to synthesise physiologically important LC-PUFA including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) from their C18 precursors. Interestingly, the loss of elovl2 gene in P. altivelis was corroborated by genomic and phylogenetic analyses. However, this constraint would possibly be overcome by the function of alternative Elovl enzymes, such as Elovl1b, which has not hitherto been functionally characterised in teleosts. The present study contributes novel insights into LC-PUFA biosynthesis in the relatively understudied teleost group, Osmeriformes (Stomiati), thereby enhancing our understanding of the complement of LC-PUFA biosynthetic genes within teleosts.

Characterization and Functional Analysis of Fads Reveals Δ5 Desaturation Activity during Long-Chain Polyunsaturated Fatty Acid Biosynthesis in Dwarf Surf Clam Mulinia lateralis.

Fatty acid desaturases (Fads), as key enzymes in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs), catalyze the desaturation between defined carbons of fatty acyl chains and control the degree of unsaturation of fatty acids. In the present study, two Fads genes, designated MulFadsA and MulFadsB, were identified from the genome of the dwarf surf clam Mulinia lateralis (Mollusca, Mactridae), and their spatiotemporal expression was examined. MulFadsA and MulFadsB contained the corresponding conserved functional domains and clustered closely with their respective orthologs from other mollusks. Both genes were expressed in the developmental stages and all tested adult tissues of M. lateralis, with MulFadsA exhibiting significantly higher expression levels in adult tissues than MulFadsB. Subsequently, the effects of dietary microalgae on Fads expressions in the dwarf surf clam were investigated by feeding clams with two types of unialgal diets varying in fatty acid content, i.e., Chlorella pyrenoidosa (Cp) and Platymonas helgolandica (Ph). The results show that the expressions of MulFads were significantly upregulated among adult tissues in the Cp group compared with those in the Ph group. In addition, we observed the desaturation activity of MulFadsA via heterologous expression in yeasts, revealing Δ5 desaturation activity toward PUFA substrates. Taken together, these results provide a novel perspective on M. lateralis LC-PUFA biosynthesis, expanding our understanding of fatty acid synthesis in marine mollusks.

Can nutritional programming in Atlantic salmon (Salmo salar) be optimised with a reduced stimulus period?

New strategies are required to enhance the efficient assimilation and bioconversion of plant-based ingredients in Atlantic salmon (Salmo salar) diets, especially relating to the essential long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). Our study investigated nutritional programming and specifically evaluated the optimal duration of dietary ‘stimulus’ and whether it could be reduced compared to a previous study using a three- week ‘stimulus’. Fish were fed an experimental ‘stimulus’ vegetable-based diet (VS, 5% marine meals [MM]/0% fish oil [FO]) or a standard marine-based control (MS, 82% MM/4% FO) for either one (V1) or two weeks (V2 and M) from first exogenous feeding. All groups were then fed a standard marine based formulation, for an ‘intermediate’ grow-out phase to the end of 16 weeks post-first feeding, prior to a ‘challenge’ phase of six weeks when all fish were fed a vegetable-based diet (VC, 10% MM/0% FO). Compared to M, fish from both V1 and V2 groups were significantly smaller at the end of the ‘stimulus’ phase, but there were no statistical differences in overall growth, proximate or fatty acid compositions at the end of the trial. However, hepatosomatic and viscerosomatic indices were significantly lower in V1 compared to V2 fish and there was an overall trend of improved performance in V1 fish throughout the ‘intermediate’ and ‘challenge’ phases. During the ‘challenge’ phase, M fish displayed a greater net gain of DHA than V1 fish, whilst V2 was a net consumer of all n-3 LC-PUFA over the same period. Compared to M, n-3 LC-PUFA biosynthesis genes in pyloric caeca were downregulated in both experimental groups indicating possible post-transcriptional modification of this pathway in either V1 or V2, considering the differences in DHA retention levels between groups. Taken together, the results suggested that nutritional programming was not initiated by a one- or two- week ‘stimulus’. However, more studies are required to elucidate the mechanism behind enhanced performance of V1 fish.

Cloning, tissue specificity and regulation of expression of genes of four key enzymes related to long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis by ambient salinity during embryogenesis in the marine teleost Siganus guttatus.

The rabbitfish Siganus canaliculatus was the first marine teleost reported to possess long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic ability, and the related molecular mechanisms have been well clarified. Here, we investigated the LC-PUFA biosynthesis capability of the congeneric rabbitfish S. guttatus. First, cDNAs of genes for four key enzymes related to LC-PUFA biosynthesis, namely Δ6Δ5 fatty acyl desaturase (fads2) (1335 bp; 445 aa), Δ4 fads2 (1335 bp; 445 aa), and elongation of very long chain fatty acid proteins (elovl5) (873 bp; 291 aa) and elovl4 (906 bp; 302 aa) were cloned from the liver of S. guttatus. The Δ6Δ5 fads2, Δ4 fads2 and elovl5 genes showed high expression in brain, liver, spleen, gallbladder, and intestine but relatively low expression in eye, whereas the elovl4 gene showed specific and high expression in eye. During embryogenesis, mRNA expression of Δ4 fads2 and elovl4 was detected from 8 h post-fertilization (hpf) and then maintained a high level to 24 hpf, while mRNA expression of Δ6Δ5 fads2 and elovl5 reached a peak at 14 hpf but then declined. In addition, ambient salinity (32 ppt and 20 ppt) exerted some regulatory influence on the expression of the four genes during embryogenesis. The levels of C18 PUFA precursors and, especially, PUFA and DHA of the embryos, decreased from 17 hpf to 24 hpf. These results suggested that S. guttatus, similar to the congeneric S. canaliculatus, would have capability for LC-PUFA biosynthesis, which is still not activated at the fertilized egg stage.

The role of polyunsaturated fatty acids in the neurobiology of major depressive disorder and suicide risk.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are obtained from diet or derived from essential shorter-chain fatty acids, and are crucial for brain development and functioning. Fundamentally, LC-PUFAs' neurobiological effects derive from their physicochemical characteristics, including length and double bond configuration, which differentiate LC-PUFA species and give rise to functional differences between n(omega)-3 and n-6 LC-PUFAs. LC-PUFA imbalances are implicated in psychiatric disorders, including major depression and suicide risk. Dietary intake and genetic variants in enzymes involved in biosynthesis of LC-PUFAs from shorter chain fatty acids influence LC-PUFA status. Domains impacted by LC-PUFAs include 1) cell signaling, 2) inflammation, and 3) bioenergetics. 1) As major constituents of lipid bilayers, LC-PUFAs are determinants of cell membrane properties of viscosity and order, affecting lipid rafts, which play a role in regulation of membrane-bound proteins involved in cell-cell signaling, including monoaminergic receptors and transporters. 2) The n-3:n-6 LC-PUFA balance profoundly influences inflammation. Generally, metabolic products of n-6 LC-PUFAs (eicosanoids) are pro-inflammatory, while those of n-3 LC-PUFAs (docosanoids) participate in the resolution of inflammation. Additionally, n-3 LC-PUFAs suppress microglial activation and the ensuing proinflammatory cascade. 3) N-3 LC-PUFAs in the inner mitochondrial membrane affect oxidative stress, suppressing production of and scavenging reactive oxygen species (ROS), with neuroprotective benefits. Until now, this wealth of knowledge about LC-PUFA biomechanisms has not been adequately tapped to develop translational studies of LC-PUFA clinical effects in humans. Future studies integrating neurobiological mechanisms with clinical outcomes may suggest ways to identify depressed individuals most likely to respond to n-3 LC-PUFA supplementation, and mechanistic research may generate new treatment strategies.

Compositional and metabolic responses of European seabass (Dicentrarchus labrax) to a dietary gradient of long-chain (≥ C20) polyunsaturated fatty acids

Decrease in the inclusion of fish oil (FO) in current aquafeed formulations reduces the availability of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) to marine fish like the European seabass Dicentrarchus labrax. Here we investigated the compositional and physiological responses of European seabass to a dietary gradient of n-3 LC-PUFA. Six isoenergetic and isonitrogenous diets (D1-D6) were fed to triplicate groups of juvenile seabass (23 g), containing blends of FO and vegetable oil (VO) that resulted in a gradient of n-3 LC-PUFA varying from 0.23 to 3.40% of diet as fed. Sampling was carried out after four months feeding and biochemical composition, as well as lipid metabolic gene expression in liver and mid-intestine analysed. The only effect of diet on proximate composition was a decrease in hepatic lipid content as dietary n-3 LC-PUFA increase whereas, in contrast, fatty acid (FA) compositions of both tissues were substantially modified by diet. Concentrations of LC-PUFA biosynthesis intermediates decreased in both tissues as dietary n-3 LC-PUFA increased with some preference detected towards n-6 FA. Two genes, namely fads2 and igf1, were moderately influenced by dietary VO in liver while genes involved in lipogenesis (srebp1, srebp2 and fas) and LC-PUFA biosynthesis (fads2 and elovl5) were markedly down-regulated, in mid-intestine of seabass in response to increasing dietary n-3 LC-PUFA. The expression of genes of β-oxidation (ppara, cpt1a and fabp1) did not vary among treatment. Collectively, our data suggest that the nutritional regulation of key lipid metabolic genes in European seabass was more pronounced in mid-intestine than in liver. Overall, when compared to a previous study of similar design in gilthead seabream (Sparus aurata), the European seabass seems less sensitive to a dietary gradient of vegetable oil.

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.

Effects of Three Microalgal Diets Varying in LC-PUFA Composition on Growth, Fad, and Elovl Expressions, and Fatty Acid Profiles in Juvenile Razor Clam Sinonovacula constricta

The razor clam Sinonovacula constricta is the first marine mollusk demonstrated to possess the complete long-chain polyunsaturated fatty acids (LC-PUFA) biosynthetic pathway. This study explored the impact of different microalgae on growth, Fad and Elovl expressions, and fatty acid (FA) profiles in juvenile S. constricta. Results revealed that juveniles fed with Isochrysis galbana (rich in DHA) or Chaetoceros calcitrans (rich in EPA) consistently exhibited higher growth than those fed Chlorella sp. (rich in LA and ALA), underscoring the importance of dietary LC-PUFA in S. constricta’s development. Expression of most Fad and Elovl in C. calcitrans and I. galbana-fed juveniles were initially up-regulated, then down-regulated, suggesting LC-PUFA demand for faster growth. Although Chlorella sp.-fed juveniles exhibited decreased mRNA levels for most genes, levels were notably higher lately compared to those fed C. calcitrans or I. galbana, hinting at potential LC-PUFA biosynthesis induction. FA profiles in S. constricta generally mirrored those in ingested microalgae, implying direct FA accumulation from diets. Some microalgal FA were absent in farmed S. constricta, while others emerged, indicating S. constricta’s ability to selectively accumulate and synthesize FA. This study enhances the understanding of dietary FA metabolism in S. constricta, valuable for selecting appropriate microalgae in its farming practices.

Comparative analysis of nutritional and transcriptional regulation of hacd1 in large yellow croaker (Larimichthys crocea) and rainbow trout (Oncorhynchus mykiss).

3-hydroxyacyl-CoA dehydratases 1 (Hacd1) is a critical enzyme in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. The difference in expression of hacd1 might account for the stronger capacity of LC-PUFA biosynthesis in freshwater fish than in marine fish, but little is known about fish hacd1. Therefore, this study compared the responses of large yellow croaker and rainbow trout hacd1 to different oil sources or fatty acids, and also examined transcriptional regulation of this gene. In this study, hacd1 was highly expressed in the liver of large yellow croaker and rainbow trout, which is the main organ for LC-PUFA biosynthesis. Therefore, we cloned the hacd1 coding sequence, with a phylogenetic analysis showing that this gene is evolutionarily conserved. Its localization to the endoplasmic reticulum (ER), likely also indicates a conserved structure and function. The expression of hacd1 in the liver was significantly decreased after the substitution of soybean oil (SO) for fish oil but was not significantly affected after palm oil (PO) substitution. Linoleic acid (LA) incubation significantly promoted hacd1 expression in primary hepatocytes of large yellow croaker and eicosapentaenoic acid (EPA) incubation significantly promoted hacd1 expression in primary hepatocytes of rainbow trout. Transcription factors STAT4, C/EBPα, C/EBPβ, HNF1, HSF3 and FOXP3 were identified in both large yellow croaker and rainbow trout. HNF1 had a stronger activation effect in rainbow trout than in large yellow croaker. FOXP3 inhibited hacd1 promoter activity in large yellow croaker but had no effect in rainbow trout. Therefore, the differences between HNF1 and FOXP3 affected the expression of hacd1 in the liver thus being responsible for the high capacity of LC-PUFA biosynthesis in rainbow trout.

Determination of dietary essential fatty acids in a deep-sea fish, the splendid alfonsino Beryx splendens: functional characterization of enzymes involved in long-chain polyunsaturated fatty acid biosynthesis

The splendid alfonsino Beryx splendens is a commercially important deep-sea fish in East Asian countries. Because the wild stock of this species has been declining, there is an urgent need to develop aquaculture systems. In the present study, we investigated the long-chain polyunsaturated fatty acid (LC-PUFA) requirements of B. splendens, which are known as essential dietary components in many carnivorous marine fish species. The fatty acid profiles of the muscles, liver, and stomach contents of B. splendens suggested that it acquires substantial levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from its natural diet. The functional characterization of a fatty acid desaturase (Fads2) and three elongases (Elovl5, Elovl4a, and Elovl4b) from B. splendens confirmed their enzymatic capabilities in LC-PUFA biosynthesis. Fads2 showed Δ6 and Δ8 bifunctional desaturase activities. Elovl5 showed preferential elongase activities toward C18 and C20 PUFA substrates, whereas Elovl4a and Elovl4b showed activities toward various C18-22 substrates. Given that Fads2 showed no Δ5 desaturase activity and no other fads-like sequence was found in the B. splendens genome, EPA and arachidonic acid cannot be synthesized from C18 precursors; hence, they can be categorized as dietary essential fatty acids in B. splendens. EPA can be converted into DHA in B. splendens via the so-called Sprecher pathway. However, given that fads2 is only expressed in the brain, it is unlikely that the capacity of B. splendens to biosynthesize DHA from EPA can fulfill its physiological requirements. These results will be useful to researchers developing B. splendens aquaculture methods.

Biosynthesis of LC-PUFA in Ruditapes philippinarum: Cloning and tissue distribution of Fad and Elovl, and effects of microalgae diets varied in LC-PUFA composition on their expressions and fatty acids profile of this bivalve

To reveal the biosynthetic pathway of long-chain polyunsaturated fatty acids (LC-PUFA) in Ruditapes philippinarum, herein, two fatty acid desaturases (Fads, including one Δ5 Fad and one Δ6 Fad-like) and three elongases of very long-chain fatty acids (Elovls, including one Elovl2/5 and two Elovl4-like) genes were firstly cloned from this bivalve and their tissue distributions were examined. Results showed that the newly cloned Fads and Elovls contained the corresponding conserved functional domains and clustered closely with their orthologs, respectively. Meanwhile, they were expressed significantly higher in the digestive glands and intestine. Subsequently, to further understand the LC-PUFA biosynthesis in R. philippinarum, the effects of dietary LC-PUFA on Fad and Elovl expressions and the fatty acid (FA) profile in this bivalve were investigated by feeding with three microalgae varied in LC-PUFA compositions [including Chlorella sp. (rich in 18:2n-6 and 18:3n-3), Chaetoceros calcitrans (rich in eicosapentaenoic acid, EPA), and Isochrysis galbana (rich in docosahexaenoic acid, DHA]. Results showed that, throughout the experiment, the expressions of Fad and Elovl were significantly up-regulated in the visceral mass (digestive glands and intestine) of R. philippinarum fed with Chlorella sp., while no significant changes or slightly decreases were observed in those fed with I. galbana. Furthermore, in those fed with C. calcitrans, the expressions of Fad were not significantly changed, whereas the expressions of Elovl were firstly up-regulated but then restored to its initial level at the end of experiment. These results suggested that R. philippinarum could modulate Fad and Elovl expressions to adapt to the dietary LC-PUFA composition. The FA analysis showed that a significantly higher amount of DHA and EPA was found in the R. philippinarum fed with I. galbana and C. calcitrans, respectively, which reflected well of the dietary FA. However, the R. philippinarum fed with Chlorella sp. exhibited a significant decrease of 18:2n-6 and 18:3n-3 but with a significant increase of their products such as 20:3n-6 and 22:5n-3, indicating that R. philippinarum had a certain capacity for LC-PUFA biosynthesis. Collectively, this study provided valuable insights into the biosynthesis of LC-PUFA in R. philippinarum.

RNAseq reveals modulation of genes involved in fatty acid biosynthesis in chicken liver according to genetic background, sex, and diet.

Increases in chicken production are mainly due to specialised breeds. However, local breeds are of increasing importance, known for ability to adapt to the environment and unique products. Conventional poultry products contain lower levels of n-3 fatty acids (FAs) compared to those obtained from local breeds, therefore the aim of this study was to evaluate the modulation of expression of genes involved in long-chain polyunsaturated FA (PUFA) biosynthesis pathways according to genetic background, diet conditions, and sex. Animals from two local breeds and a commercial line were fed different diets: control and experimental diet (10% linseed supplementation). For each breed and diet group, both sexes were reared. The RNA was extracted from 36 liver samples and sequenced by RNAseq method. Bioinformatic analysis was carried out to find differentially expressed genes from comparisons between experimental groups. Results showed low impact of diet on differentially expressed genes related to FA biosynthesis, but linseed diet increased percentage of n-3 FAs of liver. Sex and genetic background determined the differential expression of genes related to long-chain PUFA biosynthesis. Specifically, females of local breeds shared 23 up-regulated genes when compared to their respective commercial line groups. Some of the shared genes had a role in de novo triglyceride biosynthesis (MTTPL and GPAM), and in de novo FA biosynthesis (ACACA and SCD) was detected. In conclusion, local breeds are able to better adapt to a diet rich in PUFA, by triggering certain transcriptomic shifts in the liver that allow birds to process the high PUFA content provided by diet.

GPR120-ERK1-Srebp1c signaling pathway regulates long-chain polyunsaturated fatty acids biosynthesis in marine teleost Siganus canaliculatus.

The rabbitfish Siganus canaliculatus is the first marine teleost reported to possess long-chain polyunsaturated fatty acids (LC-PUFA) biosynthetic ability; its regulatory mechanisms have been investigated at the transcriptional and posttranscriptional levels, but little is known about its regulation at the cellular signaling level. The present study investigated the regulatory role of the G-protein-coupled receptor 120 (GPR120) signaling pathway in LC-PUFA biosynthesis in rabbitfish. S. canaliculatus hepatocyte line (SCHL) cells treated with GRP120 agonists (TUG891 and GW9508) showed significantly lower docosahexaenoic acid (DHA) content and mRNA levels of the key genes involved in LC-PUFA biosynthesis, encoding Δ6/Δ5 Fads2, Elovl5, and transcriptional factor Srebp1c. Transcriptome analysis of the treated SCHL cells showed significantly lower mRNA levels of genes encoding extracellular signal-regulated kinase 1 (ERK1), AMP-activated protein kinase (AMPKα2), target of rapamycin (TORC2) and Srebp1c, suggesting that these proteins are potentially involved in the GRP120 signaling pathway. Moreover, treatment of SCHL cells with signaling chemicals of ERK1, AMPKα2, TORC2, and Srebp1c confirmed the involvement of the ERK1-Srebp1c signaling pathway in the regulation of LC-PUFA biosynthesis. The mRNA levels of Srebp1c, Δ6/Δ5 fads2 and elovl5 were significantly lower in cells treated with PUFAs (linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, DHA) but higher in those treated with ERK1 inhibitors (U0126 and CI-1040). CI-1040-treated cells showed significantly higher DHA content, but the other treatment groups (except PD98059) showed significantly lower DHA content. These results indicate that the GPR120-ERK1-Srebp1c signaling pathway regulates rabbitfish LC-PUFA biosynthesis, representing a novel regulatory mechanism in vertebrates.

Evaluation of cottonseed oil as a substitute for dietary fish oil of juvenile black seabream (Acanthopagrus schlegelii): Based on growth, lipid metabolism, antioxidant capacity and pi3k/akt pathway

An eight weeks feeding trial was conducted to evaluate the potential of cottonseed oil (CSO) as a substitute for fish oil (FO) on juvenile black seabream (Acanthopagrus schlegelii) (2.29 ± 0.01 g). Five diets were formulated with pure FO (FO) or partial CSO replacement of FO: 20% CSO (CSO20), 40% CSO (CSO40), 60% CSO (CSO60) and 80% CSO (CSO80), respectively. Results showed that fish fed CSO60 diet significantly increased growth performance compared to control group. Hepatic expression of genes related to long-chain polyunsaturated fatty acid biosynthesis were significantly up-regulated by dietary CSO60 compared to FO group. Hepatic paraffin section and serum biochemical indices showed that fish fed dietary CSO80 could increase vacuolar fat drops and cause liver damage. The expression levels of peroxisome proliferators-activated receptor alpha and its downstream lipolysis related genes were markedly up-regulated with the increasing of replacement levels of CSO, opposite results were recorded in sterol regulatory element-binding protein-1c and lipogenesis related genes. The malondialdehyde content in liver was decreased with the increasing replacement levels of CSO, whereas the antioxidant parameters were significantly higher in CSO60 group compared to FO group. Phosphatidylinositol-3 kinase and protein kinase B expression levels were significantly up-regulated by dietary CSO80, triggering inflammation by activating nuclear factor kappa b. Dietary CSO60 dramatically promote target of rapamycin expression level. With the increasing replacement levels of FO by CSO, the expression level of ribosomal protein S6 kinase 1 was significantly increased, whereas opposite result was found for the eukaryotic translation initiation factor 4E binding protein 2. Collectively, based on weight gain, double broken line model analysis showed that a maximum growth occurred at 59.13% replacement of dietary FO by CSO. These findings demonstrate that dietary CSO60 can improve growth and antioxidant capacity. However, dietary CSO80 can result in lipid deposition, liver injury and inflammation, hence, suppress growth performance.

Effects of low salinities on growth, fatty acid composition, and transcriptome in Florida pompano (Trachinotus carolinus) at early developmental stages

The Florida pompano, Trachinotus carolinus, is a prime marine finfish candidate for commercial production in the United States (U.S.). Low salinity conditions (i.e., < 30 parts per thousand, ppt) can affect the early development of T. carolinus by increasing osmoregulatory stress. To address the knowledge gap regarding the impact of low salinity on Florida pompano health, we conducted an experiment to determine the optimal salinity for the culture of fingerlings. Larvae were reared in three salinities (10, 20, and 30 ppt) at local producers' farms and growth, fatty acid profiles, and transcriptome responses were quantified every three days after hatching (3, 6, 9, 12, 15, 18, and 24 days post hatch) and results were compared across salinities. Larvae reared at 10 ppt grew at a normal rate and the developmental stage had a greater impact than salinity on fatty acid composition and larval gene expression. Although there were few differentially expressed genes between salinities (n = 17), there was an upregulation of gene sets associated with ribosomes and oxidative phosphorylation and a downregulation of glycerophospholipid and carbon metabolism at lower salinities in comparison to higher salinities. In the early days of development, the fatty acids linoleic acid (LA), alpha linoleic acid (LNA), and 18:3 n-6 were proven to be important as potential energy fuels and precursors for long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis, which was supported by the whole transcriptomics analysis. The gene expression of fatty acid synthesis genes coincided with changes in the fatty acid levels suggesting the ability of Florida pompano larvae to biosynthesize LC-PUFAs from LA and LNA precursors. Overall, this study suggests that it is possible to raise Florida pompano larvae for 24 DPH under low salinity conditions as low as 10 ppt. However, it is important to run this study for a longer period to investigate the long-term effects of low salinity on larval health and development. Importantly, this research serves as a model study for future on-farm collaborations and helps build a bridge between scientists and farmers in aquaculture.

Elucidation of Functional Genes Associated with Long Chain-Polyunsaturated Fatty Acids (LC-PUFAs) Metabolism in Oleaginous Diatom Phaeodactylum tricornutum

Phaeodactylum tricornutum is a marine diatom rich in omega-3 fatty acids, a nutraceutical-relevant product. Long chain-polyunsaturated fatty acids (LC-PUFAs) are crucial dietary components for human development and growth. With the availability of genome information and genetic engineering tools, the productivities of OMEGAs have improved, but the functional and organizational relationship of such protein-encoding genes associated with LC-PUFAs biosynthesis is still not clear. Henceforth, our study highlights the conservation pattern, functionality and interaction of LC-PUFAs protein-encoding genes via in silico analysis. The transcriptome and quantitative PCR analysis demonstrates downregulation of ACS4, ELO6b, PTD5a, and MYB106 genes associated with LC-PUFAs synthesis and upregulation of ECoAH and ACAT1 genes associated with β-oxidation in nitrogen-depleted conditions in P. tricornutum. Phylogenomics studies of LC-PUFAs protein-encoding genes show a highly conserved evolutionary pattern in various microalgal lineages. Further, for elucidating the interaction of LC-PUFA metabolic genes, subcellular networks were predicted and pathway enrichment analysis was performed, providing new insights on the crosstalk between LC-PUFA protein-encoding genes (ELO6, PTD5, ACS, and ACL1), regulatory elements (LEC2, MYB, WIN) and transporters (ABCD1). In conclusion, such extensive functional enrichment analysis will undoubtedly aid in the development of genetically engineered algal strains with enhanced production of biomolecules i.e., LC-PUFAs.

Long-chain polyunsaturated fatty acid biosynthesis in a land-crab with advanced terrestrial adaptations: Molecular cloning and functional characterization of two fatty acyl elongases.

Depending on the presence and activities of the front-end fatty acyl desaturases and elongation of very long-chain fatty acid (Elovl) enzymes, animals have different capacities for long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis. Successful land colonisation in brachyuran crabs requires a shift towards terrestrial food chain with limited LC-PUFA availability. We cloned and functionally characterised two elovl genes from the purple land crab Gecarcoidea lalandii. The two Elovl contained all the necessary motifs of a typical polyunsaturated fatty acids (PUFA) Elovl and phylogenetically clustered in the Elovl1 and Elovl6 clades, respectively. The G. lalandii Elovl1 elongated saturated fatty acids, with low activities towards C20 and C22 PUFA substrates. Moreover, the G. lalandii Elovl6 was particularly active in the elongation of C18 PUFA, although it also recognised monounsaturated fatty acids as substrates for elongation. Collectively, the herein characterised G. lalandii elovl paralogues fulfil all the elongation steps involved in the LC-PUFA biosynthetic pathways. Tissue distribution of the G. lalandii elovl genes, along with the FA composition analyses, suggest the hepatopancreas and gill as key metabolic sites for fatty acid elongation. However, current data suggest that G. lalandii is unable to rely solely on biosynthesis to fulfil LC-PUFA requirements, since front-end desaturase appears to be absent in this species and other decapods.

Can Artemia franciscana produce essential fatty acids? Unveiling the capacity of brine shrimp to biosynthesise long-chain polyunsaturated fatty acids

Artemia nauplii are widely used as live preys for feeding early life-cycle stages of marine finfish and shrimp. However, a major drawback associated to using Artemia is their deficient nutritional value for marine larvae that is primarily linked to suboptimal levels of long-chain polyunsaturated fatty acids (LC-PUFA), essential nutrients that guarantee normal growth and development of animals. While common marine hatchery procedures involve enrichment processes to address such nutritional deficiencies, the specific drivers accounting for the naturally occurring variability in LC-PUFA of Artemia are largely unknown. Biosynthesis, along diet, is one of the main factors determining the LC-PUFA profiles in animals and it depends upon the repertoire and function of fatty acyl elongases and desaturases existing in a particular species. The aim of this study was the molecular and functional characterisation of all elongase and desaturase enzymes involved in the LC-PUFA biosynthesis from Artemia franciscana, arguably the most commonly used Artemia species. Seven out of eight elongases (termed “Elo1-8”) found in A. franciscana had distinctive features of LC-PUFA biosynthesising elongases. Consistently, functional assays showed these elongases were able to elongate multiple substrates and thus enabling A. franciscana to perform all elongation reactions of the LC-PUFA biosynthetic pathways. While all seven functionally characterised elongases from A. franciscana showed activity towards multiple substrates including C18 to C22 polyunsaturated fatty acids, particularly high activity was detected for the Elovl8 orthologue (termed herein as “Elo7”). Additionally, A. franciscana was found to have three genes of the so-called “first” desaturases (“Des1-3”), and lack other key LC-PUFA biosynthesising desaturases such as methyl-end and front-end desaturases. Two desaturases have the expected ∆9 desaturase activity, whereas a third one showed ∆12 activity. Neither methyl-end nor front-end desaturases were found in A. franciscana. In conclusion, this study demonstrated that A. franciscana has high elongation capacity but its overall LC-PUFA biosynthesis can be regarded as limited due to the lack of an adequate complement of fatty acyl desaturases in its genome.

Inclusion of duckweed (Lemna minor) in the diet improves flesh omega-3 long-chain polyunsaturated fatty acid profiles but not the growth of farmed Nile tilapia (Oreochromis niloticus)

A 12-week experiment was conducted to determine the effect of dietary duckweed (Lemna minor) on growth performance, body composition and fatty acid profiles of juvenile Nile tilapia (Oreochromis niloticus). Five isonitrogenous (30 % crude protein) diets were prepared with the inclusion of dry ground L. minor at 0 %, 5 %, 10 %, 15 % and 20 % of diet dry weight. Each diet was randomly allocated to triplicate tanks stocked with juvenile tilapia that had an initial mean weight of 2.00 ± 0.01 g. The juvenile tilapia were reared in a flow-through system with each diet fed twice a day at a total ratio of 5 % body weight per day. While weight gain and specific growth rate (SGR) were generally higher, and food conversion ratio (FCR) generally lower in fish fed the control diet than fish fed L. minor, there were no significant differences in these performance parameters between tilapia fed L. minor at 15 % inclusion and fish fed the control diet. Survival ranged from 80 % to 96 % and was significantly higher in fish fed the control diet. Dietary L. minor significantly reduced whole-body total lipid contents and increased moisture contents. The dietary inclusion of L. minor significantly increased the proportions of omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) in muscle, with eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids increasing by over 10-fold from 0.5 % each of total fatty acids, respectively, in fish fed the control diet to 5 % each in fish fed 20 % L. minor. Since the inclusion of L. minor increased the contents of 18:3n-3, but not EPA and DHA in the diets, this indicates that the increased proportions of EPA and DHA in the muscle of tilapia fed L. minor was due to endogenous biosynthesis of LC-PUFA with the conversion of dietary 18:3n-3 to EPA and DHA. Therefore, the present study indicated that dietary inclusion of 15–20 % L. minor in feeds for Nile tilapia can significantly improve the omega-3 fatty acid profile of muscle for human consumers, with only minor effects on growth and feed utilization. These findings demonstrated the potential for the utilization of L. minor as an ingredient to increase omega-3 LC-PUFA, EPA and DHA, contents of farmed tilapia while reducing the use of fish oil and fish meal in tilapia feeds.

Insulin can up-regulate LC-PUFA biosynthesis with the involvement of Srebp-1c and stimulatory protein 1 (Sp1) in marine teleost Siganus canaliculatus

The rabbitfish Siganus canaliculatus is the first marine teleost found to have the biosynthetic ability of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors catalyzed by fatty acyl desaturases (Δ6/Δ5 Fads, Δ4 Fads) and elongases of very long chain fatty acids (Elovls). Previously, we predicted the existence of insulin (INS) response elements (IREs) including nuclear factor Y (NF-Y) and sterol regulatory element (SRE) in the core promoter region of rabbitfish Δ6/Δ5 fads and Δ4 fads. To clarify the potential regulatory effect and mechanism of INS in LC-PUFA biosynthesis, INS responding region was identified at −456 bp to + 51 bp of Δ6/Δ5 fads core promoter, but not in Δ4 fads promoter. Moreover, a unique stimulatory protein 1 (Sp1) element was predicted in the INS responding region of Δ6/Δ5 fads. Subsequently, SRE, NF-Y and Sp1 elements were proved as IREs in Δ6/Δ5 fads promoter. The up-regulation of INS on gene expression of Srebp-1c, Sp1, Δ6/Δ5 fads and elovl5 as well as the LC-PUFA biosynthesis was further demonstrated in S. canaliculatus hepatocyte line (SCHL) cells, but no influence was detected on Δ4 fads. Besides, inhibitors of transcription factors Srebp-1c (Fatostatin, PF-429242) and Sp1 (Mithramycin) could inhibit the gene expression of Srebp-1c, Δ6/Δ5 fads and elovl5, and abolish the up-regulation of INS on these genes’ expression and LC-PUFA biosynthesis. These results indicated that INS could up-regulate LC-PUFA biosynthesis with the involvement of Srebp-1c and Sp1 in rabbitfish S. canaliculatus, which is the first report in teleost.