Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon

BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1A(A )and EF1A(B)) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1A(B)>EF1A(A)>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1A(B)>EF1A(A)>S20>β-actin>18S rRNA>GAPDH. CONCLUSION: Overall, this work suggests that the EF1A(A )and EF1A(B )genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon

Brain Distribution of 10 cart Transcripts and Their Response to 4 Days of Fasting in Atlantic Salmon (Salmo salar L.)

Cocaine- and amphetamine-regulated transcript (CART) has been known to be involved in feeding and energy balance in mammals, acting as an anorexigenic neuropeptide in hypothalamus. In Atlantic salmon, little is known about Cart brain localization and its function. In this study, in silico analysis revealed the existence of 10 cart paralogs, here named cart1a, 1b1, 1b2, 2a, 2b1, 2b2, 3a1, 3a2, 3b, and 4. The Atlantic salmon Cart sequences shared from 19 to 50% of identity with the human homolog and between 25 and 90% of sequence identity among paralogs, except for Cart4 which only shared 18–23% of identity. We further explored cart mRNA expressions in 8 brain regions (Olfactory Bulb-OB, Telencephalon-TEL, Midbrain-MB, Cerebellum-CE, Hypothalamus-HYP, Saccus vasculosus-SV, Pituitary-PT, and Brain Stem-BS) of Atlantic salmon smolt under 4 days of fasting and continuous fed conditions. The cart paralogs analyzed were widely distributed among the brain regions and OB, TEL, HYP, MB, and BS seemed to be the major sites of expression. The expression of cart1a and 1b showed quite similar pattern in MB, HYP, and BS. The expression of cart2a had the highest in MB followed by HYP and TEL. The cart3a transcript was widely distributed in rostrocaudal regions of brain except in OB and SV whereas cart3b was predominantly expressed in BS followed by MB. Expression of cart4 was high in HYP followed by TEL. With regards to effect of feeding status the Atlantic salmon cart2b, which is the most abundant among the paralogs, was upregulated after 4 days of fasting in OB, MB, and HYP compared to fed group. This may suggest an unexpected, but possible orexigenic role of cart2b in Atlantic salmon or a fasting induced stress effect. No other significant effect was observed. Collectively, the differential expressions of the cart paralogs in different brain regions suggest that they may have roles in regional integration of appetite signals and are possibly involved in regulating other brain functions in Atlantic salmon. The fact that salmon has 10 cart paralogs, while mammalians only one, opens interesting perspectives for comparative research on evolutionary adaptations of gene function in the control of appetite and energy homeostasis.publishedVersio

Cloning, tissue and ontogenetic expression of the taurine transporter in the flatfish Senegalese sole (Solea senegalensis)

Flatfish species seem to require dietary taurine for normal growth and development. Although dietary taurine supplementation has been recommended for flatfish, little is known about the mechanisms of taurine absorption in the digestive tract of flatfish throughout ontogeny. This study described the cloning and ontogenetic expression of the taurine transporter (TauT) in the flatfish Senegalese sole (Solea senegalensis). Results showed a high similarity between TauT in Senegalese sole and other vertebrates, but a change in TauT amino acid sequences indicates that taurine transport may differ between mammals and fish, reptiles or birds. Moreover, results showed that Senegalese sole metamorphosis is an important developmental trigger to promote taurine transport in larvae, especially in muscle tissues, which may be important for larval growth. Results also indicated that the capacity to uptake dietary taurine in the digestive tract is already established in larvae at the onset of metamorphosis. In Senegalese sole juveniles, TauT expression was highest in brain, heart and eye. These are organs where taurine is usually found in high concentrations and is believed to play important biological roles. In the digestive tract of juveniles, TauT was more expressed in stomach and hindgut, indicating that dietary taurine is quickly absorbed when digestion begins and taurine endogenously used for bile salt conjugation may be recycled at the posterior end of the digestive tract. Therefore, these results suggest an enterohepatic recycling pathway for taurine in Senegalese sole, a process that may be important for maintenance of the taurine body levels in flatfish species

The combined impact of plant-derived dietary ingredients and acute stress on the intestinal arachidonic acid cascade in Atlantic salmon (Salmo salar)

A study was conducted to assess the effect of substituting high levels of dietary fish oil (FO) and fishmeal (FM) for vegetable oil (VO) and plant protein (PP) on the intestinal arachidonic acid (AA) cascade in the carnivorous fish species Atlantic salmon. Four diets were fed to salmon over a period of 12 months, including a control FMFO diet, with varying replacements of plant-derived ingredients: 80 % PP and 35 % VO; 40 % PP and 70 % VO; 80 % PP and 70 %VO. Subsequently, fish were examined pre- (0 h) and post- (1 h) acute stress for blood parameters and intestinal bioactive lipidic mediators of inflammation (prostaglandins). Plasma cortisol responses were greatest in the FMFO group, while 80 % PP and 70 % VO fish exhibited increased plasma chloride concentrations. The n-3:n-6 PUFA ratio in intestinal glycerophospholipids from 70 % VO groups significantly decreased in both proximal and distal regions due to elevated levels of 18 : 2n-6 and the elongation/desaturation products 20 : 2n-6 and 20 : 3n-6. Increases in n-6 PUFA were not concomitant with increased AA, although the AA:EPA ratio did vary significantly. The 40 % PP and 70 % VO diet produced the highest intestinal AA:EPA ratio proximally, which coincided with a trend in elevated levels of PGF2α, PGE2 and 6-keto-PGF1α in response to stress. PGE2 predominated over PGF2α and 6-keto-PGF1α (stable metabolite of PGI2) with comparable concentrations in both intestinal regions. Cyclo-oxygenase-2 (COX-2) mRNA expression was an order of magnitude higher in distal intestine, compared with proximal, and was significantly up-regulated following stress. Furthermore, the 80 % PP and 70 % VO diet significantly amplified proximal COX-2 induction post-stress. Results demonstrate that high replacements with plant-derived dietary ingredients can enhance COX-2 induction and synthesis of pro-inflammatory eicosanoids in the intestine of salmon in response to acute physiological stress

The C4B6⁻ cells change morphology upon stimulation with various mitogens.

<p>Left panels, inverted microscope pictures. Middle panels, fluorescence microscopy pictures of cells stained with anti-TO antiserum, captured with 40× objective. Right panels, fluorescence microscopy pictures of cells stained with anti-TO antiserum, captured with 63× objective. (A) Cells without mitogen, 24 hrs incubation. (B) Cells stimulated with Con A and PMA for 18 hrs. (C) Cells stimulated with LPS for 24 hrs.</p

The C4B6⁻ cells have potent phagocytic activity.

<p>(A) A representative histogram showing the phagocytic activity (left panel) of the C4B6⁻ cells. Increased peak fluorescence indicates an increased number of ingested beads. The colors of the markers reflect the colors in the dot plot (middle panel). The dot plot shows the distribution of cells with various number of cells; cells without beads are shown as black dots, cells with one bead are red, cells with two beads are green, cells with three beads are blue and cells with more than three beads is shown as purple. Right panel, Diff Quick stained cells with various numbers of ingested beads. (B) TEM analyses of C4B6⁻ cells with beads. Left panel, a representative cell containing several 1 µm beads (magnification = 20 000×, scale bar = 1 µm). Middle panels, cells containing beads that are 2 µm (magnification = 15 000×, scale bar = 1 µm). Right panel is an enlargement of the delimited area of the middle, right panel showing beads that are just about to be ingested (magnification = 30 000×, scale bar = 0.5 µm). (C) SEM of C4B6⁻ cells ingesting beads that are 1 µm (two left panels) and 2 µm (two right panels). Notice the cell membrane that is about to enclose around the beads. A typical thrombocyte, containing one 2 µm bead, is shown in the right panel for comparison. In panels from left to right: Magnification (scale bar); 10 000× (1 µm), 30 000× (0.3 µm), 10 000× (1 µm) and 5000× (1 µm). (D). Different stages of bead capturing. Notice the veil (arrow). In panels from left to right: Magnification (scale bar); 8000× (1 µm), 5000× (1 µm) and 6500× (1 µm).</p

C4B6⁻ cells are small, round cells which are abundant among the PBL.

<p>(A) Double immunostaining with anti-TO (green) and MAb C4B6 (red) revealed small, round cells that were C4B6⁻ (white arrows). (B) Flow cytometry histograms of PBL prior and after MACS separation. The bound fraction (C4B6⁺ cells) contained both polymorphonuclear (PML) and mononuclear cells (L = leukocytes, M = monocytes) while the cells in the unbound fraction (C4B6⁻ cells) were small, mononuclear cells with low level of granularity. Cells with large, round nuclei were present in most C4B6⁻ cells (labeled with *), although cells with other nuclei forms were observed. C4B6⁺ cells are shown as red dots in the dot plots (middle panels). The markers define the C4B6⁺ cells. Diff Quick stained cells are shown in the right panels. Representative single cells are enlarged. The diff quick pictures are captured with 63× objective. Scale bar = 5 µm.</p

Oligonucleotids used as primers in qRT-PCR.

<p>Oligonucleotids used as primers in qRT-PCR.</p

The C4B6⁻ cells are negative for lymphocyte-, neutrophil and monocyte/macrophage markers, but express CD83 and MHC class II.

<p>(A) Flow cytometry analyses of the unbound fraction after MACS show the C4B6⁻ cells' reactivity with MAb C4B6 (anti-leukocytes), C7G7 and G2H3 (B-cells), E3D9 (neutrophils) and the polyclonal anti-human CD3 antiserum (T- and IgM⁺ cells). Representative histograms are shown. Grey filled curves are negative controls. Positive cells are shown as red dots. The markers represent positive cells. The antibodies reactivity against PBL prior to MACS is shown in the lowest panels. (B) A silver stained SDS- polyacrylamide gel, left panel, and an immunoblot using polyclonal IgM serum developed with ECL, right panel. Lane 1, molecular mass markers; lane 2, PBL; lane 3, unbound fraction after MACS (C4B6⁻ cells); lane 4, bound fraction after MACS (C4B6⁺ cells); lane 5, salmon IgM. The 66 KDa band (*) in lane 3 and 4 is most likely BSA present in the MACS buffer. (C) qRT-PCR analysis of the C4B6⁻ cells. Gene expression of the following genes; IgM (B-cell marker), CD3, CD8 and TCRα (T-cell markers), MCSF-R (monocyte/macrophage marker), CD86 (involved in antigen presentation), CD83 (DC marker), MHC class II (APC), GATA-1 and G6F (thrombocyte/erythrocyte markers) is presented as mean normalized expression (MNE) using EF1α as reference gene (n = 4). The average of triplicates from four fish with standard error is shown. Note different scale in the inserted histogram.</p