The present study which ascertained lipid profiles of 50 Atlantic salmon families confirmed previous results showing important inter-family variation in the ability to retain n-3 LC-PUFA in the flesh when fish are fed diets with low levels of these fatty acids . Furthermore, even though a high correlation was found between flesh lipid levels and n-3 LC-PUFA contents, families with the same total lipid level varied significantly in n-3 LC-PUFA contents. In the present study we did not examine whether these differences have a genetic basis, as this was established previously , but instead aimed to identify molecular pathways whose transcriptional regulation might underlie the phenotypic differences, independent of lipid content.
Differences in flesh n-3 LC-PUFA content in individuals fed the same diet is likely to arise from either selective incorporation and retention of fatty acids supplied by the diet or from biosynthesis from precursors in tissues such as the liver. In the present study we performed a transcriptomic study to identify molecular mechanisms potentially underlying flesh n-3 LC-PUFA phenotypes. Expression of candidate genes of the LC-PUFA biosynthesis pathway were also quantified as there was good evidence that these genes are transcriptionally regulated and that mRNA levels correlate with enzymatic activity of this pathway [13, 14], and so this appeared a likely mechanism that required specific investigation. Flesh was the target tissue for analysis of the n-3 LC-PUFA retention trait because salmon accumulate lipid reserves in muscle and this is the main product for human consumption, and so its composition will affect the health-promoting properties of salmon. However, hepatic tissue was analyzed for effects on gene expression since the production of both LC-PUFA and the lipoproteins that transport them to the tissues takes place mainly in the liver .
The transcriptomic analysis revealed few effects of the n-3 LC-PUFA factor on metabolism in general and, in particular, a lack of effect on lipid metabolism genes, when the statistical analysis employed multiple testing correction. However, this correction is typically not used when examining effects of diet and genetic background on metabolic genes, which tend to show subtle, but physiologically relevant, changes [9, 11, 16]. Without multiple testing correction we were able to identify pathways of lipid metabolism that might be altered in response to this factor, although a clear mechanism for the observed inter-family differences in n-3 LC-PUFA content was not identified. Potential effects on lipid transport and lipoprotein metabolism were indicated by the presence of two apolipoprotein A4 transcripts (apoa4a and b), a low density lipoprotein (LDL) receptor-related protein (lrp1) and a lipoprotein lipase (lpl) transcript in the microarray analysis, albeit these were not validated by RT-qPCR. In contrast, the RT-qPCR results clearly confirmed that the flesh n-3 LC-PUFA phenotype cannot be explained by transcriptional modulation of genes of LC-PUFA biosynthesis and so other mechanisms must be in operation. One hypothesis might be that phenotypic differences between families originates from the presence of different alleles of fatty acyl desaturases and/or elongases encoding proteins with altered biological activity or specificity, as described for the nematode Caenorhabditis elegans.
Effects of n-3 LC-PUFA flesh contents on hepatic cholesterol biosynthesis
Within the lipid metabolism genes that were differentially expressed in the liver between fish showing higher or lower n-3 LC-PUFA contents in flesh, the category of cholesterol biosynthesis and its regulation was the most apparent, based on the number of probes for interrelated genes present in this list, all with coordinated regulation indicating reduced cholesterol biosynthesis in salmon having higher flesh n-3 LC-PUFA. In addition, and inferred by the magnitude of change (i.e., fold-changes), effects were more pronounced in fish containing lower flesh lipid levels. These results were confirmed by quantifying the expression of three enzymes catalyzing steps in cholesterol biosynthesis (mev, ipi and 7dchr) as well as srebp2, a transcription factor that regulates cholesterol synthesis . Furthermore, the RT-qPCR analysis indicated that this regulation was only associated with lower flesh lipid levels given that in the high lipid group only 7dchr was down-regulated. Therefore, this experiment confirmed previous studies suggesting an association between flesh adiposity and n-3 LC-PUFA in the regulation of cholesterol biosynthesis in Atlantic salmon families, with lean fish showing a higher responsiveness to n-3 LC-PUFA . However, an important novel outcome of the present study was the demonstration that the previous results were not solely a consequence of a higher dietary intake of cholesterol supplied by a FO diet in contrast to a VO diet  but also resulted from higher incorporation and increased tissue levels of n-3 LC-PUFA. The likely explanation for these results is the role of n-3 LC-PUFA as regulators of gene transcription, including some implicated in cholesterol biosynthesis, mediated by srebp2[18–20]. Nonetheless, the mechanism for why this response was only observed when associated with low flesh lipid levels requires clarification. Recent studies showed that lean humans are also more responsive, in terms of plasma lipid and lipoprotein composition, to cholesterol-reducing diets containing lower levels of saturated fatty acids and cholesterol than obese individuals, and several mechanisms have been proposed to explain this . In the present case, the absolute, rather than the relative, level of n-3 LC-PUFA may be the determinant factor affecting gene transcription and, in the high lipid group, absolute levels of these fatty acids might have been sufficiently high to repress cholesterol biosynthesis genes, even at lower relative n-3 LC-PUFA contents (i.e., group HL). This hypothesis is supported by the RT-qPCR analysis comparing the families with regards to lipid level, HL/LL and HH/LH. In the HL/LL comparison, contrasting absolute n-3 LC-PUFA levels of 427 versus 363 mg/100 g flesh, there was down-regulation of both ipi and srebp2 (−3.95 and −2.09, respectively), whereas comparison of the families HH/LH, containing 554 versus 468 mg/100 g flesh, showed no difference in the expression of the genes. Similarly, genes involved in lipoprotein metabolism, which are also regulated by LC-PUFA through different mechanisms , also showed more significant changes when comparing fatter and leaner salmon with lower LC-PUFA levels, indicating that a similar regulatory mechanism might occur. Therefore, the present study is consistent with previous work identifying cholesterol and lipoprotein metabolism as pathways significantly and differentially affected by n-3 LC-PUFA depending on flesh adiposity .
Effects of total lipid level on lipid metabolism
Lipid level significantly affected expression of lipid metabolism genes, although effects were still relatively small (8% of all genes assigned to a biological function category). A noteworthy result was the down-regulation of elovl2 (confirmed by RT-qPCR) in salmon presenting higher flesh lipid, independent of LC-PUFA content. Elovl2 has substrate specificity towards LC-PUFA and is highly responsive to dietary n-3 LC-PUFA levels in salmon . However, the expression of this gene is often co-ordinately regulated with other genes of LC-PUFA biosynthesis, such as Δ5fad and Δ6fad, which was not the case here. Hence, the biological significance of this result is not clear and may indicate other roles of elovl2 in lipid metabolism. For instance, an association between overexpression of elovl2 and enhanced triacylglycerol synthesis and lipid droplet accumulation, as well as induction of PPARγ target genes, was shown in mouse preadipocyte cell lines . In addition, elovl2 was up-regulated in the liver transcriptome of rats with nephrotic syndrome, a condition characterized by hyperlipidemia . Elovl2 was only recently characterized in salmon , and this is the first indication of an association between its expression and lipid accumulation in a non-mammalian vertebrate, with results suggesting that increased lipid level in salmon flesh repressed elovl2 expression independent of n-3 LC-PUFA level although this requires further investigation. Another gene down-regulated at higher lipid levels was a mitochondrial acyl carrier protein, involved in acyl transfer steps, including roles in fatty acid synthesis and functioning of the electron transport chain , which could conceptually be responding to similar regulatory mechanisms affecting elovl2. In contrast, stearoyl-CoA desaturase, responsible for the synthesis of monounsaturated fatty acids from saturated precursors, was up-regulated in salmon with higher flesh lipid levels. This gene was positively correlated with fat accumulation in bovine skeletal muscle , consistent with up-regulation in salmon families with increased fat stores.
Possible association between flesh n-3 LC-PUFA contents and immune response
The predominance of immune response genes responding to total lipid level and, particularly, n-3 LC-PUFA contents in salmon flesh was unexpected. This was a true over-representation as GO enrichment analysis enabled identification of several GO terms related to regulation of immune and inflammatory responses in relation to the total lipid factor. However, as mentioned above, the transcriptomic comparison, although balanced for total lipid, was not balanced for viral disease resistance (specifically IPN in this case) and, as a consequence, higher contrast between families was imposed on the high lipid group (families HL and HH) due to the fortuitous selection of family HH presenting a much higher viral resistance EBV. Nonetheless, if family HH biased the results of the two-way ANOVA we would expect a preponderance of immune-related genes to occur only when comparing these two families, presenting higher and lower flesh n-3 LC-PUFA contents at the higher lipid level. In order to assess this, t-tests were performed comparing separately the higher versus lower n-3 LC-PUFA families at each total lipid level, i.e., LH/LL and HH/HL. A Venn diagram contrasting the two t-test significant lists was then performed and when analyzing the genes that were similarly affected by n-3 LC-PUFA contents at both higher and lower total lipid level, a similar preponderance (33%) of immune response genes was observed (Additional file 4). Finally, examination of the fold changes of immune-related genes, indicating magnitude of effects, between families with higher and lower contents of n-3 LC-PUFA at either higher or lower total lipid levels (Tables 2 and 5), showed no clear evidence of the effect being more marked for the high lipid comparison, which is what would be expected if results were caused simply by inclusion of family HH in the transcriptomic analysis.
Hence, there is evidence to suggest that there may be some correlation between flesh n-3 LC-PUFA contents and immune response in the families analysed. An anti-inflammatory role of n-3 LC-PUFA is well established in mammals and fish [27–29]. Immune cells are typically rich in arachidonic acid (ARA), the precursor for eicosanoids with a pro-inflammatory action, whereas EPA and DHA give rise to eicosanoids that are less biologically active, as well as to resolvins and protectins presenting anti-inflammatory properties . Higher incorporation of n-3 LC-PUFA in biological membranes of immune cells can modulate immune responses in several ways [reviewed in [15, 30–33]. They alter the production of inflammatory eicosanoid mediators of which they are precursors, directly affect the organization and properties of the immune cell membranes with effects on signalling pathways, phagocytic capacity and antigen presenting capability, and activate transcription of various genes involved in inflammatory responses. Therefore, families with higher tissue levels of n-3 LC-PUFA may show differential expression of immune response and inflammation-related genes, as well as of genes involved in signalling and regulation of transcription (as observed in the present study). Furthermore, although liver is chiefly a metabolic organ, it has other physiological functions including removal of pathogens and antigens from the blood and modulation of immune responses, as well as the production of inflammatory mediators [34, 35].
Related to the above, microarray analysis revealed the presence of several genes that intervene in eicosanoid synthesis and metabolism including phospholipase A2 (pla2), arachidonate 5-lipoxygenase (alox5), thromboxane-A synthase (thas), prostaglandin I2 synthase (ptgis) and 15-hydroxyprostaglandin dehydrogenase . However, RT-qPCR only confirmed up-regulation of hepatic alox5 in families presenting higher flesh n-3 LC-PUFA and, given that alox5 acts on LC-PUFA of both n-3 and n-6 series and that ARA levels generally accompanied the n-3 LC-PUFA phenotype (Table 1), it cannot be ascertained whether this transcript was responding to higher levels of membrane ARA or EPA and hence if it would result in increased pro-inflammatory 4-series, or less potent 5-series, leukotrienes .
The immune response genes whose expression was correlated with 'n-3 LC-PUFA' are mainly involved in the modulation of inflammatory processes and innate immune response to pathogens, which are particularly important in fish species and that can be easily compromised in aquaculture conditions . We could speculate that the changes in expression may give enhanced protection from inflammation or pathological conditions in fish with higher n-3 LC-PUFA in their tissues. Up-regulation associated with high flesh n-3 LC-PUFA was noted in expression of NACHT domain containing protein, tripartite motif-containing protein 25 (trim25), c-c motif chemokine 13 precursor (ccl13), leukocyte cell-derived chemotaxin 2 precursor (lect2), tissue factor pathway inhibitor a, pentraxin and cathepsin K. In contrast, down-regulation in the high n-3 LC-PUFA families was observed for MHC class I (mostly in the high total lipid group), and for myelin and lymphocyte protein (mal). NACHT domain containing proteins are pathogen-sensing molecules (recognizing intracellular pathogen-associated molecular patterns – PAMPs) implicated in early host defence, inflammation and innate immune signalling pathways in mammals , by activating transcription of MHC class II and the apoptotic pathway. The trim25 protein is involved in antiviral innate immune responses through activation of signalling pathways leading to production of interferons and in teleost cells TRIM genes are induced in response to viral infections [40, 41]. The ccl13 (also known as monocyte chemotactic protein 4) and lect2 proteins are both involved in inflammation, having roles in attracting monocytes and T lymphocytes in tissues exposed to exogenous pathogens, and have neutrophil chemotactic function [42, 43]. Expression of lect2 was increased in fish liver and spleen after bacterial infections . Tissue factor pathway inhibitor inhibits the initial reactions of the blood coagulation cascade and modulates cell proliferation, and may protect vascular tissue in inflammatory conditions in mammals . Cathepsin K mediates immune responses in cells, having a critical role in signalling events proximal to the Toll-like receptor 9 (TLR9) that has a fundamental role in pathogen recognition (recognizing PAMPs) and activation of mammalian innate immunity . Finally, pentraxins are pattern recognition proteins of the innate immune system that play a role in the acute phase response, activating complement pathways to clear pathogens in both mammals and fish [46, 47]. In this case, up-regulation of pentraxin in salmon with higher n-3 LC-PUFA in their flesh was only observed with high lipid levels. Similarly, down-regulation of the MHC class I transcript was observed only in the high lipid group. In mammalian studies, high LC-PUFA contents (EPA, DHA and ARA) reduced cell surface expression of MHC I, decreasing antigen presentation and altering T-cell signalling [34, 35]. Therefore, the high IPN resistance genotype observed in family HH in later genetic evaluations of the families could potentially involve effects on both the complement pathway and T-cell mediated immunity, and involve co- or post-translational modification of proteins by N-linked glycosylation through up-regulation of dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 4 (Table 2; ). Given the high economic impact of IPN in salmonid culture, identification of genes potentially involved in the progression of the disease using transcriptomic approaches is already in progress . Finally, down-regulation of mal, associated with T-cell differentiation and signal transduction , was observed at higher n-3 LC-PUFA levels.
As mentioned above, several immune response-related genes were also affected by the total lipid factor with results validated by RT-qPCR. However, we cannot exclude the possibility that this results from the strong correlation between total lipid levels and absolute LC-PUFA contents, which makes it difficult to dissociate both factors.