Laura Martinez-Rubio

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.

Conservation of lipid metabolic gene transcriptional regulatory networks in fish and mammals.

Lipid content and composition in aquafeeds have changed rapidly as a result of the recent drive to replace ecologically limited marine ingredients, fishmeal and fish oil (FO). Terrestrial plant products are the most economic and sustainable alternative; however, plant meals and oils are devoid of physiologically important cholesterol and long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic (EPA), docosahexaenoic (DHA) and arachidonic (ARA) acids. Although replacement of dietary FO with vegetable oil (VO) has little effect on growth in Atlantic salmon (Salmo salar), several studies have shown major effects on the activity and expression of genes involved in lipid homeostasis. In vertebrates, sterols and LC-PUFA play crucial roles in lipid metabolism by direct interaction with lipid-sensing transcription factors (TFs) and consequent regulation of target genes. The primary aim of the present study was to elucidate the role of key TFs in the transcriptional regulation of lipid metabolism in fish by transfection and overexpression of TFs. The results show that the expression of genes of LC-PUFA biosynthesis (elovl and fads2) and cholesterol metabolism (abca1) are regulated by Lxr and Srebp TFs in salmon, indicating highly conserved regulatory mechanism across vertebrates. In addition, srebp1 and srebp2 mRNA respond to replacement of dietary FO with VO. Thus, Atlantic salmon adjust lipid metabolism in response to dietary lipid composition through the transcriptional regulation of gene expression. It may be possible to further increase efficient and effective use of sustainable alternatives to marine products in aquaculture by considering these important molecular interactions when formulating diets.

Coastal fish farms are settlement sites for juvenile fish.

Two south-west Mediterranean fish farms were monitored over a period of 22 months to test if sea-cage fish farms act as settlement habitats for juvenile fish. Twenty juvenile fish species were found to settle at farms throughout the year. Fish assemblage composition varied markedly over time and was dependent on the spawning period for each species. The most abundant species were Obladamelanura, Atherina sp., Diplodussargus, Boopsboops and Lizaaurata. Up to 3783+/-1730 individuals/cage were found closely associated with the cages. Highest densities were observed during the warmer summer and autumn months. Zooplankton sampling and stomach content analyses of the most abundant species were done to assess prey availability, selectivity and diet overlap among species. Copepods were the main prey item for all juvenile fish species, irrespective of fish size. Ivlevs Index indicated that food was not a limiting factor for juvenile fish at farms. Furthermore, food pellets from the farm affected the food chain by modifying the fatty acid profiles of farm-associated zooplankton and juveniles of L. aurata and O. melanura. These results show that aquaculture can directly influence the body composition of juvenile fish that recruit to sea-cage fish farms.

Functional Feeds Reduce Heart Inflammation and Pathology in Atlantic Salmon (Salmo salar L.) following Experimental Challenge with Atlantic Salmon Reovirus (ASRV)

Heart and Skeletal Muscle Inflammation (HSMI), recently associated with a novel Atlantic salmon reovirus (ASRV), is currently one of the most prevalent inflammatory diseases in commercial Atlantic salmon farms in Norway. Mortality varies from low to 20%, but morbidity can be very high, reducing growth performance and causing considerable financial impact. Clinical symptoms, including myocarditis, myocardial and red skeletal muscle necrosis, correlate with the intensity of the inflammatory response. In the present study, the effects of two functional feeds (FF1 and FF2) were compared to a standard commercial reference feed (ST) in Atlantic salmon subjected to an ASRV challenge. The functional feeds had reduced levels of total lipid and digestible energy, and different levels and proportions of long-chain polyunsaturated fatty acids (LC-PUFA). The objective was to determine whether these feeds could provide effective protection by decreasing the inflammatory response associated with HSMI. Histopathology, viral load, fatty acid composition and gene expression of heart tissue were assessed over a period of 16 weeks post-infection with ASRV. The viral load and histopathology scores in heart tissue in response to ASRV infection were reduced in fish fed both functional feeds, with FF1 showing the greatest effect. Microarray hierarchical cluster analysis showed that the functional feeds greatly affected expression of inflammation/immune related genes over the course of the ASRV infection. Viral load correlated with up-regulation of pro-inflammatory genes at the early-mid stages of infection in fish fed the ST diet. Expression of inflammatory genes 16-weeks after ASRV challenge reflected the difference in efficacy between the functional feeds, with fish fed FF1 showing lower expression. Thus, severity of the lesions in heart tissue correlated with the intensity of the innate immune response and was associated with tissue fatty acid compositions. The present study demonstrated that dietary modulation through clinical nutrition had major influences on the development and severity of the response to ASRV infection in salmon. Thus, HSMI was reduced in fish fed the functional feeds, particularly FF1. The modulation of gene expression between fish fed the different feeds provided further insight into the molecular mechanisms and progression of the inflammatory and immune responses to ASRV infection in salmon.

Effect of functional feeds on fatty acid and eicosanoid metabolism in liver and head kidney of Atlantic salmon (Salmo salar L.) with experimentally induced Heart and Skeletal Muscle Inflammation

Heart and Skeletal Muscle Inflammation (HSMI) is an emerging viral disease caused by a novel Atlantic salmon reovirus (ASRV) affecting farmed fish. Primary symptoms associated with HSMI include myocardial and skeletal muscle necrosis indicating a severe inflammatory process. Recently, we applied the concept of clinical nutrition to moderate the long-term inflammatory process associated with HSMI in salmon subjected to experimental ASRV challenge. The use of functional feeds with lower lipid (hence energy) content reduced the inflammatory response to ASRV infection and the severity of associated heart lesions. The aim of the present study was to elucidate possible mechanisms underpinning the observed effects of the functional feeds, focussing on eicosanoid and fatty acid metabolism in liver and head kidney. Here we show that liver was also a site for histopathological lesions in HSMI showing steatosis reflecting impaired lipid metabolism. This study is also the first to evaluate the expression of a suite of key genes involved in pathways relating diet and membrane phospholipid fatty acid compositions, and the inflammatory response after ASRV infection. The expression of hepatic Δ6 and Δ5 desaturases was higher in fish fed the functional feeds, potentially increasing their capacity for endogenous production and availability of anti-inflammatory EPA. Effects on mobilization of lipids and changes in the LC-PUFA composition of membrane phospholipids, along with significant changes in the expression of the genes related to eicosanoid pathways, showed the important role of the head kidney in inflammatory diseases caused by viral infections. The results from the present study suggest that clinical nutrition through functional feeding could be an effective complementary therapy for emerging salmon viral diseases associated with long-term inflammation.

Effects of functional feeds on the lipid composition, transcriptomic responses and pathology in heart of Atlantic salmon (Salmo salar L.) before and after experimental challenge with Piscine Myocarditis Virus (PMCV)

BackgroundCardiomyopathy syndrome (CMS) is a severe cardiac disease of Atlantic salmon (Salmo salar) recently associated with a double-stranded RNA virus, Piscine Myocarditis Virus (PMCV). The disease has been diagnosed in 75-85 farms in Norway each year over the last decade resulting in annual economic losses estimated at up to €9 million. Recently, we demonstrated that functional feeds led to a milder inflammatory response and reduced severity of heart lesions in salmon experimentally infected with Atlantic salmon reovirus, the causal agent of heart and skeletal muscle inflammation (HSMI). In the present study we employed a similar strategy to investigate the effects of functional feeds, with reduced lipid content and increased eicosapentaenoic acid levels, in controlling CMS in salmon after experimental infection with PMCV.ResultsHepatic steatosis associated with CMS was significantly reduced over the time course of the infection in fish fed the functional feeds. Significant differences in immune and inflammatory responses and pathology in heart tissue were found in fish fed the different dietary treatments over the course of the infection. Specifically, fish fed the functional feeds showed a milder and delayed inflammatory response and, consequently, less severity of heart lesions at earlier and later stages after infection with PMCV. Decreasing levels of phosphatidylinositol in cell membranes combined with the increased expression of genes related with T-cell signalling pathways revealed new interactions between dietary lipid composition and the immune response in fish during viral infection. Dietary histidine supplementation did not significantly affect immune responses or levels of heart lesions.ConclusionsCombined with the previous findings on HSMI, the results of the present study highlight the potential role of clinical nutrition in controlling inflammatory diseases in Atlantic salmon. In particular, dietary lipid content and fatty acid composition may have important immune-modulatory effects in Atlantic salmon that could be potentially beneficial in fish balancing the immune and tissue responses to viral infections.

Influence of Development and Dietary Phospholipid Content and Composition on Intestinal Transcriptome of Atlantic Salmon (Salmo salar)

The inclusion of intact phospholipids in the diet is essential during larval development and can improve culture performance of many fish species. The effects of supplementation of dietary phospholipid from marine (krill) or plant (soy lecithin) sources were investigated in Atlantic salmon, Salmo salar. First feeding fry were fed diets containing either krill oil or soybean lecithin supplying phospholipid at 2.6%, 3.2%, 3.6% and 4.2% of diet. Fish were sampled at ~ 2.5 g (~1,990°day post fertilization, dpf) and ~10 g (2,850°dpf). By comparison of the intestinal transcriptome in specifically chosen contrasts, it was determined that by 2,850°dpf fish possessed a profile that resembled that of mature and differentiated intestinal cell types with a number of changes specific to glycerophospholipid metabolism. It was previously shown that intact phospholipids and particularly phosphatidylcholine are essential during larval development and that this requirement is associated with the inability of enterocytes in young fry to endogenously synthesize sufficient phospholipid for the efficient export of dietary lipid. In the immature phase (~1,990°dpf), the dietary phospholipid content as well as its class composition impacted on several biochemical and morphological parameters including growth, but these differences were not associated with differences in intestinal transcriptomes. The results of this study have made an important contribution to our understanding of the mechanisms associated with lipid transport and phospholipid biosynthesis in early life stages of fish.

Molecular mechanism of dietary phospholipid requirement of Atlantic salmon, Salmo salar, fry.

The phospholipid (PL) requirement in fish is revealed by enhanced performance when larvae are provided PL-enriched diets. To elucidate the molecular mechanism underlying PL requirement in Atlantic salmon, Salmo salar, were fed a minimal PL diet and tissue samples from major lipid metabolic sites were dissected from fry and parr. In silico analysis and cloning techniques demonstrated that salmon possess a full set of enzymes for the endogenous production of PL. The gene expression data indicated that major PL biosynthetic genes of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylinositol (PtdIns) display lower expression in intestine during the early developmental stage (fry). This is consistent with the hypothesis that the intestine of salmon is immature at the early developmental stage with limited capacity for endogenous PL biosynthesis. The results also indicate that intact PtdCho, PtdEtn and PtdIns are required in the diet at this stage. PtdCho and sphingomyelin constitute the predominant PL in chylomicrons, involved in the transport of dietary lipids from the intestine to the rest of the body. As sphingomyelin can be produced from PtdCho in intestine of fry, our findings suggest that supplementation of dietary PtdCho alone during early developmental stages of Atlantic salmon would be sufficient to promote chylomicron formation. This would support efficient transport of dietary lipids, including PL precursors, from the intestine to the liver where biosynthesis of PtdEtn, PtdSer, and PtdIns is not compromised as in intestine facilitating efficient utilisation of dietary energy and the endogenous production of membrane PL for the rapidly growing and developing animal.