Acute phase gene expression in rainbow trout (Oncorhynchus mykiss) after exposure to a confinement stressor: A comparison of pooled and individual data.

This study set out to investigate whether differential expression of genes for acute phase proteins in rainbow trout (Oncorhynchus mykiss) could be induced by confinement stress, a non-invasive method of activating the neuroendocrine stress response. In addition, a second objective was to assess the variation in gene expression between individual fish within the population of stressed fish in an attempt to identify APP genes having uniform and consistent changes in expression during stress. The liver was chosen for this investigation as it is the primary site of acute phase protein synthesis. Relative expression of the eight genes including transferrin, fibrinogen-like protein 2 (flp2), alpha-1-anti-proteinase-like protein (alpha1-antiprot), leukocyte cell-derived chemotaxin 2 (LECT2), pentraxin, serum amyloid A (SAA), haptoglobin (Hp), and differentially regulated trout protein 1 (DRTP1) was analysed by quantitative real-time PCR (qPCR) over 5 experimental time points spanning the course of a week. The results showed that the expression of three genes, SAA, haptoglobin and DRTP1, were most altered as a result of exposure to confinement stress. A correlation was identified between the expression of haptoglobin and DRTP1. Gene expression analyses in individual fish found that the transcript levels of haptoglobin and DRTP1 genes varied much less between individuals than was the case for SAA. The increase of haptoglobin and DRTP1 gene expression and its uniformity in response to stress make these genes potential biomarkers for stress in trout.

Characterisation of the differentially regulated trout protein 1 (DRTP1) gene in rainbow trout (Oncorhynchus mykiss).

Increased levels of differentially regulated trout protein 1 (DRTP1) mRNA transcripts have been reported in fish after activation of the acute phase response. While the function of the DRTP1 protein still remains to be elucidated, this study focused on the genomic organisation of the gene, the quantification of the DRTP1 transcript in various tissues, and the isolation and analysis of the 5' regulatory region of the DRTP1 gene in rainbow trout (Oncorhynchus mykiss). Analysis of the DRTP1 genomic and cDNA sequences showed the gene to consist of four exons separated by three introns. Tissue localisation of the DRTP1 gene was performed by Northern analysis and validated by quantitative real-time PCR (qPCR). Six tissues (liver, intestine, spleen, brain, pituitary, and hypothalamus) were analysed. The tissues with the most abundant transcripts were the liver and the pituitary, with lesser amounts detected in the intestine, hypothalamus, brain and spleen. Genome walking allowed the isolation of a 934 bp sequence of the 5' regulatory region of the gene which was cloned, sequenced and in which potential transcription factor binding sites were identified. Promoter fragments of decreasing size were generated and transiently transfected into the human hepatoma cell line (HepG2). Inducibility of the promoter was determined by stimulation of the HepG2 cells containing the constructs with dexamethasone, polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNFalpha). One construct, containing two potential C-EBP/beta sites and two NF-kappaB sites, exhibited the highest promoter induction (6.34 fold +/- SEM 0.5) when stimulated with human TNFalpha. A slightly shorter fragment containing one C-EBP/beta site and one NF-kappaB site did not show any significant inducibility when treated with TNFalpha. The loss of the C-EBP/beta and NF-kappaB in the shorter construct suggests that these sites, either individually or in combination, are critical for the induction of the DRTP1 promoter by TNFalpha.