Daily rhythms of physiological processes in fishsynchronization to light and feeding cycles and effects on the molecular clock, epigenetics and welfare

Zusammenfassung

Objectives: 1. Investigate the presence of a daily rhythm in the stress response in European sea bass and how the season of the year modulate the response. 2. Explore the presence of a daily rhythm in the genes involved in the regulation of feeding and digestion in European sea bass. 3. Study the presence of a daily rhythm in epigenetic mechanism and their connection to the clock genes in European sea bass. 4. Understand how different stressor combined (food deprivation and density) can affect the clock genes and genes related to epigenetic mechanisms in European sea bass. 5. Investigate the daily rhythm of the stress response of Zebrafish knockout for glucocorticoid receptor (gr-/-) and the influence of feeding time. Metodology 1. The fish have been kept in natural conditions before the sampling. During the sampling, control fish have been sampled directly while treated fish have been exposed to air for 1 minute and sampled 1h later. The sampling has been conducted in a 24h cycle with 7 sampling point where samples of plasma and liver have been collected and the experiments have been repeated in December and June. 2. The fish have been kept in natural condition and fed at two different moments of the day. Hypothalamus and liver have been collected in a 24h sampling at 7 different sampling point to analyze factors which regulate appetite and digestion. 3. The fish have been kept in natural condition and fed at two different moments of the day. Liver and hypothalamus have been collected in a 24h sampling at 7 different sampling points to check the presence of a daily rhythm in the clock genes or factors involved in epigenetic processes. 4. Fish have been kept in natural condition and divided in 4 groups with different density and feeding conditions. To test the chronic effect of the stress we sampled them in different days during the day or during the night. Liver samples were collected to test the response of the clock gene and epigenetic genes. 5. Mutants and wild type fish have been fed respectively at ML or MD and after one month sampled in a 24-hours cycle to collect brain. Q-PCR analysis have been carried out to analyze the rhythm of clock and epigenetic genes. Conclusions 1. European sea bass is particularly sensitive to stress but the response change not only during the day but also during the year. 2. Feeding time is a strong synchronizer and is important that match the phase of activity of the animal. Feeding European sea bass in a moment that alter their resting phase, has led in most cases to a loss of the rhythm. 3. Clock and epigenetic genes are rhythmically expressed in the brain and liver of European sea bass and feeding time had major effect on liver, promoting the loss of the rhythm of epigenetic genes when fish were fed during the resting phase. 4. Density and low calories restriction can affect differently clock and epigenetic genes in European sea bass, but the methylation processes are the most affected. 5. The glucocorticoid system and feeding time exert a strong effect on the genes of the secondary loop of the clock genes in the brain of Zebrafish. On the contrary, the effect on the epigenetic genes was lower.