RESUMO
BACKGROUND: One important physiological response to environmental stress in animals is change in gene expression. To obtain reliable data from gene expression studies using RT-qPCR it is important to evaluate a set of possible reference genes as normalizers for expression. The expression of these candidate genes should be analyzed in the relevant tissues during normal and stressed situations. To find suitable reference genes it was crucial that the genes were stably expressed also during a situation of physiological stress. For poikilotermic animals like cod, changes in temperature are normal, but if the changes are faster than physiological compensation, the animals respond with typical stress responses. It has previously been shown that Atlantic cod show stress responses when elevation of water temperature is faster than 1 degree/day, for this reason we chose hyperthermia as stress agent for this experiment. FINDINGS: We here describe the expression of eight candidate reference genes from Atlantic cod (Gadus morhua l.) and their stability during thermal stress (temperature elevation of one degree C/day for 5 days). The genes investigated were: Eukaryotic elongation factor 1 alpha, ef1a; 18s ribosomal RNA; 18s, Ubiquitin conjugate protein; ubiq, cytoskeletal beta-actin; actb, major histcompatibility complex I; MHC-I light chain, beta-2 -microglobulin; b2m, cytoskeletal alpha-tubulin; tba1c, acidic ribosomal phosphoprotein; rplp1, glucose-6-phosphate dehydrogenase; g6pd. Their expression were analyzed in 6 tissues (liver, head kidney, intestine, spleen, heart and gills) from cods exposed to elevated temperature and compared to a control group. Although there were variations between tissues with respect to reference gene stability, four transcripts were more consistent than the others: ubiq, ef1a, 18s and rplp1. We therefore used these to analyze the expression of stress related genes (heat shock proteins) induced during hyperthermia. We found that both transcripts were significantly upregulated in several tissues in fish exposed to increased temperature. CONCLUSION: This is the first study comparing reference genes for RT-qPCR analyses of expression during hyperthermia in Atlantic cod. ef1a, 18s, rplp1 and ubiq transcripts were found to be well suited as reference genes during these experimental conditions.
RESUMO
Acoustic stimuli within the sonic range are effective triggers of C-type escape behaviours in fish. We have previously shown that fish have an acute sensitivity to infrasound also, with acceleration thresholds in the range of 10(-5) m s(-2). In addition, infrasound at high intensities around 10(-2) m s(-2) elicits strong and sustained avoidance responses in several fish species. In the present study, the possible triggering of C-escapes by infrasonic single-cycle vibrations was examined in juvenile roach Rutilus rutilus. The fish were accelerated in a controlled and quantifiable manner using a swing system. The applied stimuli simulated essential components of the accelerations that a small fish would encounter in the hydrodynamic flow field produced by a predatory fish. Typical C- and S-type escape responses were induced by accelerations within the infrasonic range with a threshold of 0.023 m s(-2) for an initial acceleration at 6.7 Hz. Response trajectories were on average in the same direction as the initial acceleration. Unexpectedly, startle behaviours mainly occurred in the trailing half of the test chamber, in which the fish were subjected to linear acceleration in combination with compression, i.e. the expected stimuli produced by an approaching predator. Very few responses were observed in the leading half of the test chamber, where the fish were subjected to acceleration and rarefaction, i.e. the stimuli expected from a suction type of predator. We conclude that particle acceleration is essential for the directionality of the startle response to infrasound, and that the response is triggered by the synergistic effects of acceleration and compression.
