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Abstract Background Extensive sequencing efforts have been taking place for the Atlantic cod (Gadus morhua) in recent years, the number of ESTs in the Genbank has reached more than 140.000. Despite its importance in North Atlantic fisheries and potential use in aquaculture, relatively few gene expression examination exists for this species, and systematic evaluations of reference gene stability in quantitative real-time RT-PCR (qRT-PCR) studies are lacking. Results The stability of 10 potential reference genes was examined in six tissues of Atlantic cod obtained from four populations, to determine the most suitable genes to be used in qRT-PCR analyses. Relative transcription levels of genes encoding β-actin (ACTB), elongation factor 1A (EF1A), actin-related protein-2 (ARP-2), glyceraldehyde-3P-dehydrogenase (GAPDH), ubiquitin (Ubi), acidic ribosomal protein (ARP), ribosomal protein S9 (S9), ribosomal protein L4 (RPL4), RPL22 and RPL37 were quantified in gills, brain, liver, head kidney, muscle and middle intestine in six juvenile fish from three wild populations and from farmed Atlantic cod. Reference gene stability was investigated using the geNorm and NormFinder tools. Based on calculations performed with the geNorm, which determines the most stable genes from a set of tested genes in a given cDNA sample, ARP, Ubi, S9 and RPL37 were among the most stable genes in all tissues. When the same calculations were done with NormFinder, the same genes plus RPL4 and EF1A were ranked as the preferable genes. Conclusion Overall, this work suggests that the Ubi and ARP can be useful as reference genes in qRT-PCR examination of gene expression studying wild populations of Atlantic cod.

Abstract: Decades after being banned in many countries, DDT and its metabolites are still considered major environmental hazards. The p,p′‐DDE isomer, the DDT metabolite found in highest concentration in aquaculture feeds, is an endocrine disruptor with demonstrated ability to induce epigenetic effects. This study aimed at examining the impact of p,p′‐DDE on Atlantic salmon. Primary hepatocytes were exposed to four concentrations of p,p′‐DDE (0.1, 1, 10, 100 μ m) for 48 hours, and endpoints included cytotoxicity, global DNA methylation, targeted transcription and metabolomics profiling (100 μ m). p,p′‐DDE was moderately cytotoxic at 100 μ m. No impact was seen on global DNA methylation. Vtg1 and esr1 transcription, markers of endocrine disruption, was most strongly induced at 10 μ m p,p′‐DDE, while ar showed strongest response at 100 μ m. Metabolomics profiling showed that p,p′‐DDE at 100 μ m most strongly affected carbohydrate metabolism, primary bile acid metabolism, leucine, isoleucine and valine metabolism, diacylglycerol and sphingolipid metabolism. Observed changes in lipid levels suggest that p,p′‐DDE interferes with phospholipid membrane biosynthesis. Elevation of bile acid levels in p,p′‐DDE‐exposed hepatocytes indicates upregulation of synthesis of bile acids after cytochrome P450 activation. Pathway analysis showed that the superpathway of methionine degradation was the most significantly affected pathway by p,p′‐DDE exposure, while endocrine system disorder topped the diseases and disorder ranking. In conclusion, this work predicts an endocrine response to p,p′‐DDE exposure, and demonstrates how this legacy pesticide might interfere with mechanisms linked to DNA methylation in Atlantic salmon hepatocytes. [ABSTRACT FROM AUTHOR]

Global warming may alter the bioavailability of contaminants in aquatic environments. In this work, mercury (Hg) toxicity was studied in cells obtained from Atlantic salmon smolt kept at 15 °C (optimal growth temperature) for 3 months or at a stepwise increase to 20 °C (temperature-stress) during 3 months prior to cell harvest to evaluate whether acclimation temperature affects Hg toxicity. To examine possible altered dietary requirements in warmer seas, one group of fish following the stepwise temperature regimes was fed a diet spiked with antioxidants. Atlantic salmon hepatocytes were exposed in vitro to 0, 1.0, or 100 μM Hg for 48 h. Cytotoxicity, determined as electrical impedance changes with the xCELLigence system, and transcriptional responses, determined with RT-qPCR, were assessed as measures of toxicity. The results showed that inorganic Hg at a concentration up to 100 μM is not cytotoxic to Atlantic salmon hepatocytes. Significance and directional responses of the 18 evaluated target genes suggest that both Hg and temperature stress affected the transcription of genes encoding proteins involved in the protection against ROS-generated oxidative stress. Both stressors also affected the transcription of genes linked to lipid metabolism. Spiking the diet with antioxidants resulted in higher concentrations of Se and vitamin C and reduced concentration of Hg in the liver in vivo, but no interactions were seen between the dietary supplementation of antioxidants and Hg toxicity in vitro. In conclusion, no evidence was found suggesting that inorganic Hg is more toxic in cells harvested from temperature-stressed fish. [ABSTRACT FROM AUTHOR]