Hierarchical clustering analysis of gene differentially expressed between FW and SW ionocytes.

<p>Analysis was performed based on a log₂-transformed ratio value of 138 genes differentially expressed. Row and columns represent genes and samples respectively. Expression levels of log₂-transformed ratio are represented by a color tag: red and green for high and low levels of expression respectively.</p

<i>In situ</i> hybridization of 3 genes (purple labelling) in fish gill from freshwater (A, C, E) or seawater (B, D, F) acclimated rainbow trout associated with immunocytochemistry of ionocyte with Na/K-ATPase antibody (brown labelling).

<p>AB: SLC26A6, CD: NBC, EF: CIC2.</p

Annotated genes exhibiting differential expression between FW and SW ionocytes.

<p>Genes in italic were upregulated in SW and others genes were upregulated in FW.</p><p>Annotated genes exhibiting differential expression between FW and SW ionocytes.</p

Plasma ion concentrations after sea water transfer.

<p>Mean plasma sodium (A), chloride (B), and calcium (C) from time 0 to 21 days post sea water transfer. Open and filled circles represent respectively freshwater and seawater transferred trout. Values represent means ± s.e.m. of six fishes. Differences between freshwater and seawater at each time point were assessed with non-parametric Mann-Whitney U-test after non-parametric analysis of variance (Kruskal-Wallis test). * and **, significantly different from the corresponding values in freshwater at P<0.05 and P<0.01.</p

Primer pairs for real time quantitative RT-PCR.

<p>Primer pairs for real time quantitative RT-PCR.</p

Table_2_Acute Stress and an Electrolyte- Imbalanced Diet, but Not Chronic Hypoxia, Increase Oxidative Stress and Hamper Innate Immune Status in a Rainbow Trout (Oncorhynchus mykiss) Isogenic Line.DOCX

<p>In aquaculture, fish may be exposed to sub-optimal rearing conditions, which generate a stress response if full adaptation is not displayed. However, our current knowledge of several coexisting factors that may give rise to a stress response is limited, in particular when both chronic and acute stressors are involved. This study investigated changes in metabolic parameters, oxidative stress and innate immune markers in a rainbow trout (Oncorhynchus mykiss) isogenic line exposed to a combination of dietary (electrolyte-imbalanced diet, DEB 700 mEq Kg^-1) and environmental (hypoxia, 4.5 mg O₂ L^-1) challenges and their respective controls (electrolyte-balanced diet, DEB 200 mEq Kg^-1 and normoxia, 7.9 or mg O₂ L^-1) for 49 days. At the end of this period, fish were sampled or subjected to an acute stressor (2 min of handling/confinement) and then sampled. Feeding trout an electrolyte-imbalanced diet produced a reduction in blood pH, as well as increases in cortisol levels, hepato-somatic index (HSI) and total energy content in the liver. The ratio between the lactate dehydrogenase (LDH) and isocitrate dehydrogenase (IDH) activities decreased in the liver of trout fed the DEB 700 diet, but increased in the heart, suggesting a different modulation of metabolic capacity by the dietary challenge. Several markers of oxidative stress in the liver of trout, mainly related to the glutathione antioxidant system, were altered when fed the electrolyte-imbalanced diet. The dietary challenge was also associated with a decrease in the alternative complement pathway activity (ACH₅₀) in plasma, suggesting an impaired innate immune status in that group. Trout subjected to the acute stressor displayed reduced blood pH values, higher plasma cortisol levels as well as increased levels of metabolic markers associated with oxidative stress in the liver. An interaction between diet and acute stressor was detected for oxidative stress markers in the liver of trout, showing that the chronic electrolyte-imbalance impairs the response of rainbow trout to handling/confinement. However, trout reared under chronic hypoxia only displayed changes in parameters related to energy use in both liver and heart. Taken together, these results suggest that trout displays an adaptative response to chronic hypoxia. Conversely, the dietary challenge profoundly affected fish homeostasis, resulting in an impaired physiological response leading to stress, which then placed constraints on a subsequent acute challenge.</p