The effect of micronutrient supplementation on growth and hepatic metabolism in diploid and triploid Atlantic salmon (Salmo salar) parr fed a low marine ingredient diet.

The effects of low marine ingredient diets supplemented with graded levels (L1, L2, L3) of a micronutrient package (NP) on growth and metabolic responses were studied in diploid and triploid salmon parr. Diploids fed L2 showed significantly improved growth and reduced liver, hepatic steatosis, and viscerosomatic indices, while fish fed L3 showed suppressed growth rate 14 weeks post feeding. In contrast, dietary NP level had no effect on triploid performance. Whole body mineral composition, with exception of copper, did not differ between diet or ploidy. Whole fish total AAs and N-metabolites showed no variation by diet or ploidy. Free circulating AAs and white muscle N-metabolites were higher in triploids than diploids, while branch-chained amino acids were higher in diploids than triploids. Diploids had higher whole body α-tocopherol and hepatic vitamins K1 and K2 than triploids. Increased tissue B-vitamins for niacin and whole-body folate with dietary NP supplementation were observed in diploids but not triploids, while whole body riboflavin was higher in diploids than triploids. Hepatic transcriptome profiles showed that diploids fed diet L2 was more similar to that observed in triploids fed diet L3. In particular, sterol biosynthesis pathways were down-regulated, whereas cytochrome P450 metabolism was up-regulated. One­carbon metabolism was also affected by increasing levels of supplementation in both ploidies. Collectively, results suggested that, for optimised growth and liver function, micronutrient levels be supplemented above current National Research Council (2011) recommendations for Atlantic salmon when fed low marine ingredient diets. The study also suggested differences in nutritional requirements between ploidy.

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.

A comparative analysis of the response of the hepatic transcriptome to dietary docosahexaenoic acid in Atlantic salmon (Salmo salar) post-smolts.

BACKGROUND: The present study aimed to explore the impact of dietary docosahexaenoic acid (DHA) on aspects of the metabolism of Atlantic salmon (Salmo salar). The effects of diets containing increasing levels of DHA (1 g kg(-1), 3 g kg(-1), 6 g kg(-1), 10 g kg(-1) and 13 g kg(-1)) on the liver transcriptome of post-smolt salmon was examined to elucidate patterns of gene expression and responses of specific metabolic pathways. Total RNA was isolated from the liver of individual fish and analyzed using a custom gene expression 44K feature Atlantic salmon oligo-microarray. RESULTS: The expression of up to 911 unique annotated genes was significantly affected by dietary DHA inclusion relative to a low DHA reference diet. Analysis of a total of 797 unique genes were found with a significant linear correlation between expression level and dietary DHA. Gene-Set Enrichment Analysis (GSEA) identified a range of pathways that were significantly affected by dietary DHA content. CONCLUSIONS: Pathways that showed a significant response to dietary DHA level included those for long-chain polyunsaturated fatty acid biosynthesis, fatty acid elongation, steroid biosynthesis, glycan biosynthesis, protein export and protein processing in the endoplasmic reticulum. These findings suggest that in addition to clear roles in influencing lipid metabolic pathways, DHA might also have key functional roles in other pathways distinct from lipid metabolism.

Replacement of dietary soy- with air classified faba bean protein concentrate alters the hepatic transcriptome in Atlantic salmon (Salmo salar) parr.

The production of carnivorous fish such as Atlantic salmon (Salmo salar) is dependent on the availability of high quality proteins for feed formulations. For a number of nutritional, strategic and economic reasons, the use of plant proteins has steadily increased over the years, however a major limitation is associated with the presence of anti-nutritional factors and the nutritional profile of the protein concentrate. Investigating novel raw materials involves understanding the physiological consequences associated with the dietary inclusion of protein concentrates. The primary aim of the present study was to assess the metabolic response of salmon to increasing inclusion of air-classified faba bean protein concentrate (BPC) in feeds as a replacement for soy protein concentrate (SPC). Specifically, we tested treatments with identical contents of fishmeal (222.4gkg(-1)) and progressively higher inclusion of BPC (0gkg(-1), 111.8gkg(-1), 223.6gkg(-1), 335.4gkg(-1), 447.2gkg(-1)) substituting SPC. This study demonstrated a dose-dependent metabolic response to a plant ingredient and was the first to compare the nutrigenomic transcriptional responses after substitution of terrestrial feed ingredients such as BPC and SPC without withdrawal of marine ingredients. It was found that after eight weeks a major physiological response in liver was only evident above 335.4gkg(-1) BPC and included decreased expression of metabolic pathways, and increased expression of genes regulating transcription and translation processes and the innate immune response. Furthermore, we showed that the nutritional stress caused by BPC resembled, at least at hepatic transcriptional level, that caused by soybean meal (included as a positive control in our experimental design). The outcomes of the present study suggested that Atlantic salmon parr might efficiently utilize moderate substitution of dietary SPC with BPC, with the optimum inclusion level being around 120gkg(-1)in the type of feeds tested here.

Nutrigenomic profiling of transcriptional processes affected in liver and distal intestine in response to a soybean meal-induced nutritional stress in Atlantic salmon (Salmo salar).

The aim of the present study was to generate an experimental model to characterize the nutrigenomic profile of a plant-derived nutritional stress. Atlantic salmon (Salmo salar) was used as the model species. The nutritional stress was induced by inclusion of dietary defatted soybean meal (SBM), as this ingredient had been previously demonstrated to induce enteropathy in the distal intestine and reduce growth in salmon. Triplicate groups of Atlantic salmon were fed concentrations of 0, 100, 200 and 300 g kg(-1) SBM for 12 weeks and reduced growth performance was used as the indicator of nutritional stress. The transcriptome was analyzed in two tissues, liver and distal intestine, with the hypothesis being that the liver transcriptome would be characterized by gene expression responses related to overall growth and health performance, whereas intestinal gene expression would be dominated by specific responses to SBM. A set of 133 genes was differentially expressed in liver including 44 genes in common with the intestinal response. The liver-specific response included up-regulation of genes involved in protein digestion, energy metabolism and immune functions, whereas genes in other metabolic pathways were generally anabolic and down-regulated. These responses may be more related to general nutritional stress than to SBM per se. The transcriptomic profile in the distal intestine was consistent with the enteritis response as described previously. This study provides a comprehensive report on the profiles of liver and distal intestine transcriptomes, specifically highlighting the role of the liver in fish undergoing SBM-induced nutritional stress.

Otx2 expression and implications for olfactory imprinting in the anemonefish, Amphiprion percula.

The otx2 gene encodes a transcription factor (OTX2) essential in the formation of the brain and sensory systems. Specifically, OTX2-positive cells are associated with axons in the olfactory system of mice and otx2 is upregulated in odour-exposed zebrafish, indicating a possible role in olfactory imprinting. In this study, otx2 was used as a candidate gene to investigate the molecular mechanisms of olfactory imprinting to settlement cues in the coral reef anemonefish, Amphiprion percula. The A. percula otx2 (Ap-otx2) gene was elucidated, validated, and its expression tested in settlement-stage A. percula by exposing them to behaviourally relevant olfactory settlement cues in the first 24 hours post-hatching, or daily throughout the larval phase. In-situ hybridisation revealed expression of Ap-otx2 throughout the olfactory epithelium with increased transcript staining in odour-exposed settlement-stage larval fish compared to no-odour controls, in all scenarios. This suggests that Ap-otx2 may be involved in olfactory imprinting to behaviourally relevant settlement odours in A. percula.

Abundance of myostatin gene transcripts and their correlation with muscle hypertrophy during the development of barramundi, Lates calcarifer.

Myostatin (MSTN) is a pivotal protein that regulates vertebrate muscle growth and development. Teleost fish possess two MSTN paralogs (MSTN-1 and MSTN-2) whose respective physiological functions are still largely unclear. To clarify the role of each of these paralogs the transcript abundance of Mstn-1 and Mstn-2 was quantified during embryonic and larval development of the teleosts, barramundi, Lates calcarifer. Histological analyses of developing muscle fibers were also obtained to correlate Mstn paralog expression with muscle hypertrophy as larvae undergo metamorphosis. Mstn-1 and Mstn-2 transcripts were detected as early as immediately postfertilization of eggs, with the level of expression observed to increase during embryonic development and reach a peak near hatching. Mstn-2 expression was thousands-fold higher than that observed for Mstn-1. Close to metamorphosis, the expression of Mstn-1 was non-significantly, although positively associated, with muscle hypertrophy (r=0.384, p=0.064), while that of Mstn-2 showed a highly significant negative correlation (r=-0.691, p<0.0001) suggesting that this paralog may be responsible for muscle hypertrophy that occurs post-metamorphosis. Altogether, findings from this study support the hypothesis that Mstn paralogs are differentially regulated during various phases of fish development and that they may have evolved different functions in fish, particularly that related to muscle hypertrophy.

Growing backwards: an inverted role for the shrimp ortholog of vertebrate myostatin and GDF11.

Myostatin (MSTN) and growth differentiation factor-11 (GDF11) are closely related proteins involved in muscle cell growth and differentiation as well as neurogenesis of vertebrates. Both MSTN and GDF11 negatively regulate their functions. Invertebrates possess a single ortholog of the MSTN/GDF11 family. In order to understand the role of MSTN/GDF11 in crustaceans, the gene ortholog was identified and characterized in the penaeid shrimp Penaeus monodon. The overall protein sequence and specific functional sites were highly conserved with other members of the MSTN/GDF11 family. Gene transcripts of pmMstn/Gdf11, assessed by real-time PCR, were detected in a variety of tissue types and were actively regulated in muscle across the moult cycle. To assess phenotypic function in shrimp, pmMstn/Gdf11 gene expression was downregulated by tail-muscle injection of sequence-specific double-stranded RNA. Shrimp with reduced levels of pmMstn/Gdf11 transcripts displayed a dramatic slowing in growth rate compared with control groups. Findings from this study place the MSTN/GDF11 gene at the centre of growth regulation in shrimp, but suggest that, compared with higher vertebrates, this gene has an opposite role in invertebrates such as shrimp, where levels of gene expression may positively regulate growth.

Normalizing RT-qPCR data: are we getting the right answers? An appraisal of normalization approaches and internal reference genes from a case study in the finfish Lates calcarifer.

Commonly used normalization approaches for quantitative reverse transcription polymerase chain reaction analyses include (a) input nucleic acids standardization (ΔC (q) method), (b) normalizing target gene transcript abundance against a single internal reference gene (ΔΔC (q) method), and (c) geometric averaging of multiple reference gene abundance using the geNorm software. We compared these three approaches to examine expression of a negative muscle growth regulator gene, myostatin-I (mstn-I), in the finfish Lates calcarifer, following 4 weeks of nutritional fasting. Interestingly, these three different approaches led to widely divergent data interpretations. When ΔC (q) and subsequently ΔΔC (q) with α-tub as the reference gene were applied to mstn-I expression data, an ∼threefold upregulation of this gene was observed in fasted compared to fed fish. In contrast, mstn-I appeared unchanged when data was normalized against the geometric average of the two apparently most "stable" reference genes (elongation factor-1 α (ef1-α) and rpl8) selected from a panel comprising seven commonly utilized candidate reference genes (18S, cat-D, ef1-α, rpl8, gapdh, ubq, and α-tub). The geNorm software erroneously suggested that ef1-α, rpl8, and ubq were the most "stable" reference genes, whereas ΔC (q) analysis revealed these genes simply to exhibit similar patterns of regulation in response to fasting. The ΔC (q) approach showed that α-tub was the least variable in its expression level between fasted and fed fish after 4 weeks. The present study also highlights the importance of validating internal references for each time point under investigation when applying ΔΔC (q) and suggests that the more cost-effective ΔC (q) normalization approach, if carefully applied, may in fact produce the most biologically valid results.

Molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer) myostatin gene.

BACKGROUND: Myostatin (MSTN) is a member of the transforming growth factor-beta superfamily that negatively regulates growth of skeletal muscle tissue. The gene encoding for the MSTN peptide is a consolidate candidate for the enhancement of productivity in terrestrial livestock. This gene potentially represents an important target for growth improvement of cultured finfish. RESULTS: Here we report molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer) MSTN-1 gene. The barramundi MSTN-1 was encoded by three exons 379, 371 and 381 bp in length and translated into a 376-amino acid peptide. Intron 1 and 2 were 412 and 819 bp in length and presented typical GT...AG splicing sites. The upstream region contained cis-regulatory elements such as TATA-box and E-boxes. A first assessment of sequence variability suggested that higher mutation rates are found in the 5' flanking region with several SNP's present in this species. A putative micro RNA target site has also been observed in the 3'UTR (untranslated region) and is highly conserved across teleost fish. The deduced amino acid sequence was conserved across vertebrates and exhibited characteristic conserved putative functional residues including a cleavage motif of proteolysis (RXXR), nine cysteines and two glycosilation sites. A qualitative analysis of the barramundi MSTN-1 expression pattern revealed that, in adult fish, transcripts are differentially expressed in various tissues other than skeletal muscles including gill, heart, kidney, intestine, liver, spleen, eye, gonad and brain. CONCLUSION: Our findings provide valuable insights such as sequence variation and genomic information which will aid the further investigation of the barramundi MSTN-1 gene in association with growth. The finding for the first time in finfish MSTN of a miRNA target site in the 3'UTR provides an opportunity for the identification of regulatory mutations on the expression of this gene.