Vegetable omega-3 and omega-6 fatty acids differentially modulate the antiviral and antibacterial immune responses of Atlantic salmon.

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

Interacting Effects of Sea Louse (Lepeophtheirus salmonis) Infection and Formalin-Killed Aeromonas salmonicida on Atlantic Salmon Skin Transcriptome.

Lepeophtheirus salmonis (sea lice) and bacterial co-infection threatens wild and farmed Atlantic salmon performance and welfare. In the present study, pre-adult L. salmonis-infected and non-infected salmon were intraperitoneally injected with either formalin-killed Aeromonas salmonicida bacterin (ASAL) or phosphate-buffered saline (PBS). Dorsal skin samples from each injection/infection group (PBS/no lice, PBS/lice, ASAL/no lice, and ASAL/lice) were collected at 24 h post-injection and used for transcriptome profiling using a 44K salmonid microarray platform. Microarray results showed no clear inflammation gene expression signatures and revealed extensive gene repression effects by pre-adult lice (2,189 down and 345 up-regulated probes) in the PBS-injected salmon (PBS/lice vs. PBS/no lice), which involved basic cellular (e.g., RNA and protein metabolism) processes. Lice repressive effects were not observed within the group of ASAL-injected salmon (ASAL/lice vs. ASAL/no lice); on the contrary, the observed skin transcriptome changes -albeit of lesser magnitude (82 up and 1 down-regulated probes)- suggested the activation in key immune and wound healing processes (e.g., neutrophil degranulation, keratinocyte differentiation). The molecular skin response to ASAL was more intense in the lice-infected (ASAL/lice vs. PBS/lice; 272 up and 11 down-regulated probes) than in the non-infected fish (ASAL/no lice vs. PBS/no lice; 27 up-regulated probes). Regardless of lice infection, the skin's response to ASAL was characterized by the putative activation of both antibacterial and wound healing pathways. The transcriptomic changes prompted by ASAL+lice co-stimulation (ASAL/lice vs. PBS/no lice; 1878 up and 3120 down-regulated probes) confirmed partial mitigation of lice repressive effects on fundamental cellular processes and the activation of pathways involved in innate (e.g., neutrophil degranulation) and adaptive immunity (e.g., antibody formation), as well as endothelial cell migration. The qPCR analyses evidenced immune-relevant genes co-stimulated by ASAL and lice in an additive (e.g., mbl2b, bcl6) and synergistic (e.g., hampa, il4r) manner. These results provided insight on the physiological response of the skin of L. salmonis-infected salmon 24 h after ASAL stimulation, which revealed immunostimulatory properties by the bacterin with potential applications in anti-lice treatments for aquaculture. As a simulated co-infection model, the present study also serves as a source of candidate gene biomarkers for sea lice and bacterial co-infection.

Influence of Varying Dietary ω6 to ω3 Fatty Acid Ratios on the Hepatic Transcriptome, and Association with Phenotypic Traits (Growth, Somatic Indices, and Tissue Lipid Composition), in Atlantic Salmon (Salmo salar).

The importance of dietary omega-6 to omega-3 (ω6:ω3) fatty acid (FA) ratios for human health has been extensively examined. However, its impact on fish physiology, and the underlying molecular mechanisms, are less well understood. This study investigated the influence of plant-based diets (12-week exposure) with varying ω6:ω3 (0.4-2.7) on the hepatic transcriptome of Atlantic salmon. Using 44 K microarray analysis, genes involved in immune and inflammatory response (lect2a, itgb5, helz2a, p43), lipid metabolism (helz2a), cell proliferation (htra1b), control of muscle and neuronal development (mef2d) and translation (eif2a, eif4b1, p43) were identified; these were differentially expressed between the two extreme ω6:ω3 dietary treatments (high ω6 vs. high ω3) at week 12. Eight out of 10 microarray-identified transcripts showed an agreement in the direction of expression fold-change between the microarray and qPCR studies. The PPARα activation-related transcript helz2a was confirmed by qPCR to be down-regulated by high ω6 diet compared with high ω3 diet. The transcript expression of two helz2 paralogues was positively correlated with ω3, and negatively with ω6 FA in both liver and muscle, thus indicating their potential as biomarkers of tissue ω6:ω3 variation. Mef2d expression in liver was suppressed in the high ω6 compared to the balanced diet (ω6:ω3 of 2.7 and 0.9, respectively) fed fish, and showed negative correlations with ω6:ω3 in both tissues. The hepatic expression of two lect2 paralogues was negatively correlated with viscerosomatic index, while htra1b correlated negatively with salmon weight gain and condition factor. Finally, p43 and eif2a were positively correlated with liver Σω3, while these transcripts and eif4b2 showed negative correlations with 18:2ω6 in the liver. This suggested that some aspects of protein synthesis were influenced by dietary ω6:ω3. In summary, this nutrigenomic study identified hepatic transcripts responsive to dietary variation in ω6:ω3, and relationships of transcript expression with tissue (liver, muscle) lipid composition and other phenotypic traits.

Influence of Dietary Long-Chain Polyunsaturated Fatty Acids and ω6 to ω3 Ratios on Head Kidney Lipid Composition and Expression of Fatty Acid and Eicosanoid Metabolism Genes in Atlantic Salmon (Salmo salar).

The interaction of dietary eicosapentaenoic acid and docosahexaenoic acid (EPA+DHA) levels with omega-6 to omega-3 ratios (ω6:ω3), and their impact on head kidney lipid metabolism in farmed fish, are not fully elucidated. We investigated the influence of five plant-based diets (12-week exposure) with varying EPA+DHA levels (0.3, 1.0, or 1.4%) and ω6:ω3 (high ω6, high ω3, or balanced) on tissue lipid composition, and transcript expression of genes involved in fatty acid and eicosanoid metabolism in Atlantic salmon head kidney. Tissue fatty acid composition was reflective of the diet with respect to C18 PUFA and MUFA levels (% of total FA), and ω6:ω3 (0.5-1.5). Fish fed 0.3% EPA+DHA with high ω6 (0.3% EPA+DHA↑ω6) had the highest increase in proportions (1.7-2.3-fold) and in concentrations (1.4-1.8-fold) of arachidonic acid (ARA). EPA showed the greatest decrease in proportion and in concentration (by ~½) in the 0.3% EPA+DHA↑ω6 fed fish compared to the other treatments. However, no differences were observed in EPA proportions among salmon fed the high ω3 (0.3 and 1.0% EPA+DHA) and balanced (1.4% EPA+DHA) diets, and DHA proportions were similar among all treatments. Further, the transcript expression of elovl5a was lowest in the 0.3% EPA+DHA↑ω6 fed fish, and correlated positively with 20:3ω3, 20:4ω3 and EPA:ARA in the head kidney. This indicates that high dietary 18:3ω3 promoted the synthesis of ω3 LC-PUFA. Dietary EPA+DHA levels had a positive impact on elovl5a, fadsd5 and srebp1 expression, and these transcripts positively correlated with tissue ΣMUFA. This supported the hypothesis that LC-PUFA synthesis is positively influenced by tissue MUFA levels in Atlantic salmon. The expression of pparaa was higher in the 0.3% EPA+DHA↑ω6 compared to the 0.3% EPA+DHA↑ω3 fed fish. Finally, significant correlations between head kidney fatty acid composition and the expression of eicosanoid synthesis-related transcripts (i.e., 5loxa, 5loxb, cox1, cox2, ptges2, ptges3, and pgds) illustrated the constitutive relationships among fatty acids and eicosanoid metabolism in salmon.

Interaction between ω6 and ω3 fatty acids of different chain lengths regulates Atlantic salmon hepatic gene expression and muscle fatty acid profiles.

Atlantic salmon smolts (approx. 20-months old) were fed experimental diets with different combinations of omega-6:omega-3 fatty acids (FAs) (high-ω6, high-ω3, or balanced) and eicosapentaenoic acid plus docosahexaenoic acid (EPA + DHA) levels (0.3, 1.0 or 1.4%) for 12 weeks. Muscle FA (% total FA) reflected dietary C18-polyunsaturated FA; however, muscle EPA per cent and content (mg g-1) were not different in salmon fed high-ω3 or balanced diets. Muscle DHA per cent was similar among treatments, while DHA content increased in fish fed 1.4% EPA + DHA, compared with those fed 0.3-1.0% EPA + DHA combined with high-ω6 FA. Muscle 20:3ω6 (DGLA) content was highest in those fed high-ω6 with 0.3% EPA + DHA. Quantitative polymerase chain reaction analyses on liver RNA showed that the monounsaturated FA synthesis-related gene, scdb, was upregulated in fish fed 1.0% EPA + DHA with high-ω6 compared to those fed 0.3% EPA + DHA. In high-ω3-fed salmon, liver elovl2 transcript levels were higher with 0.3% EPA + DHA than with 1.0% EPA + DHA. In high-ω6-fed fish, elovl2 did not vary with EPA + DHA levels, but it was positively correlated with muscle ARA, 22:4ω3 and DGLA. These results suggest dietary 18:3ω3 elongation contributed to maintaining muscle EPA + DHA levels despite a two- to threefold change in dietary proportions, while 18:2ω6 with 0.3% EPA + DHA increased muscle DGLA more than arachidonic acid (ARA). Positive correlations between hepatic elovl2 and fabp10a with muscle ω6:ω3 and EPA + DHA + ARA, respectively, were confirmed by reanalysing data from a previous salmon trial with lower variations in dietary EPA + DHA and ω6:ω3 ratios. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

Cardiac mitochondrial function, nitric oxide sensitivity and lipid composition following hypoxia acclimation in sablefish.

In fishes, the effect of O2 limitation on cardiac mitochondrial function remains largely unexplored. The sablefish (Anoplopoma fimbria) encounters considerable variations in environmental oxygen availability, and is an interesting model for studying the effects of hypoxia on fish cardiorespiratory function. We investigated how in vivo hypoxia acclimation (6 months at 40% then 3 weeks at 20% air saturation) and in vitro anoxia-reoxygenation affected sablefish cardiac mitochondrial respiration and reactive oxygen species (ROS) release rates using high-resolution fluorespirometry. Further, we investigated how hypoxia acclimation affected the sensitivity of mitochondrial respiration to nitric oxide (NO), and compared mitochondrial lipid and fatty acid (FA) composition between groups. Hypoxia acclimation did not alter mitochondrial coupled or uncoupled respiration, or respiratory control ratio, ROS release rates, P50 or superoxide dismutase activity. However, it increased citrate synthase activity (by ∼20%), increased the sensitivity of mitochondrial respiration to NO inhibition (i.e., the NO IC50 was 25% lower), and enhanced the recovery of respiration (by 21%) and reduced ROS release rates (by 25-30%) post-anoxia. In addition, hypoxia acclimation altered mitochondrial FA composition [increasing arachidonic acid (20:4ω6) and eicosapentaenoic acid (20:5ω3) proportions by 11 and 14%, respectively], and SIMPER analysis revealed that the phospholipid:sterol ratio was the largest contributor (24%) to the dissimilarity between treatments. Overall, these results suggest that hypoxia acclimation may protect sablefish cardiac bioenergetic function during or after periods of O2 limitation, and that this may be related to alterations in mitochondrial sensitivity to NO and to adaptive changes in membrane composition (fluidity).

Effect of plant-based diets with varying ratios of ω6 to ω3 fatty acids on growth performance, tissue composition, fatty acid biosynthesis and lipid-related gene expression in Atlantic salmon (Salmo salar).

Little is known about how variation in omega-6 to omega-3 (ω6:ω3) fatty acid (FA) ratios affects lipid metabolism and eicosanoid synthesis in salmon, and the potential underlying molecular mechanisms. The current study examined the impact of five plant-based diets (12-week exposure) with varying ω6:ω3 (0.3-2.7) on the growth, tissue lipid composition (muscle and liver), and hepatic transcript expression of lipid metabolism and eicosanoid synthesis-related genes in Atlantic salmon. Growth performance and organ indices were not affected by dietary ω6:ω3. The liver and muscle FA composition was highly reflective of the diet (ω6:ω3 of 0.2-0.8 and 0.3-1.9, respectively) and suggested elongation and desaturation of the ω3 and ω6 precursors 18:3ω3 and 18:2ω6. Furthermore, proportions of ω6 and ω3 PUFA in both tissues showed significant positive correlations with dietary inclusion (% of diet) of soy and linseed oils, respectively. Compound-specific stable isotope analysis (CSIA) further demonstrated that liver long-chain polyunsaturated fatty acid (LC-PUFA) synthesis (specifically 20:5ω3 and 20:4ω6) was largely driven by dietary 18:3ω3 and 18:2ω6, even when 20:5ω3 and 22:6ω3 were supplied at levels above minimum requirements. In addition, significant positive and negative correlations were identified between the transcript expression of LC-PUFA synthesis-related genes and liver ω6 and ω3 LC-PUFA, respectively, further supporting FA biosynthesis. Liver ω3 LC-PUFA also correlated negatively with the eicosanoid synthesis-related transcripts pgds and cox1. This is the first study to use CSIA, hepatic transcriptome, and tissue lipid composition analyses concurrently to demonstrate the impact of plant-based diets with varying ω6:ω3 on farmed Atlantic salmon.

Transcriptome profiling reveals that feeding wild zooplankton to larval Atlantic cod (Gadus morhua) influences suites of genes involved in oxidation-reduction, mitosis, and selenium homeostasis.

BACKGROUND: Larval nutrition and growth are key issues for wild and cultured cod. While it was shown previously that larval cod fed wild zooplankton grow faster than those fed only rotifers, the mechanisms involved in this enhanced growth are not completely understood. We used microarrays to identify larval cod transcripts that respond to feeding with small amounts of wild zooplankton (5-10 % of live prey items). The larval transcriptome was compared between 3 treatment groups [fed rotifers (RA), rotifers with protein hydrolysate (RA-PH), or rotifers with zooplankton (RA-Zoo)] at 9-10 mm length [26-30 days post-hatch (dph)] to identify a robust suite of zooplankton-responsive genes (i.e. differentially expressed between RA-Zoo and both other groups). RESULTS: The microarray experiment identified 147 significantly up-regulated and 156 significantly down-regulated features in RA-Zoo compared with both RA and RA-PH. Gene ontology terms overrepresented in the RA-Zoo responsive gene set included "response to selenium ion" and several related to cell division and oxidation-reduction. Ten selenoprotein-encoding genes, and 2 genes involved in thyroid hormone generation, were up-regulated in RA-Zoo compared with both other groups. Hierarchical clustering of RA-Zoo responsive genes involved in oxidation-reduction and selenium homeostasis demonstrated that only the zooplankton treatment had a considerable and consistent impact on the expression of these genes. Fourteen microarray-identified genes were selected for QPCR involving 9-13 mm larvae, and 13 of these were validated as differentially expressed between RA-Zoo and both other groups at ~9 mm. In contrast, in age-matched (34-35 dph; ~11 mm RA and RA-PH, ~13 mm RA-Zoo) and size-matched (~13 mm) older larvae, only 2 and 3 genes, respectively, showed the same direction of RA-Zoo-responsive change as in ~9 mm larvae. CONCLUSIONS: The modulation of genes involved in selenium binding, redox homeostasis, and thyroid hormone generation in ~9 mm RA-Zoo larvae in this study may be in response to the relatively high levels of selenium, iodine, and LC-PUFA (potentially causing oxidative stress) in zooplankton. Nonetheless, only a subset of zooplankton-responsive genes in ~9 mm larvae remained so in older larvae, suggesting that the observed transcriptome changes are largely involved in initiating the period of growth enhancement.