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Identifier 000406616
Title Cortisol dynamics in European sea bass, Dicentrarchus labrax L. : factors inducing variability and its molecular and endocrine regulation
Alternative Title Η δυναμική της κορτιζόλης στο Λαβράκι, Dicentrarchus Labrax : παράγοντες που προκαλούν μεταβλητότητα και η ρύθμιση της σε μοριακό κα ενδοκρινικό επίπεδο
Author Σαμαράς, Αθανάσιος
Thesis advisor Παυλίδης, Μιχάλης
Reviewer Κεντούρη, Μαρουδιώ
Flik, Gert
Abstract The aim of the present study was to get a better insight into environmental, husbandry, and genetic factors that affect cortisol regulation and variability, as well as control the cortisol stress response in a Mediterranean marine teleost, the European sea bass, Dicentrarchus labrax. In order to examine how the environment can modify cortisol regulation, the effects of water temperature on cortisol dynamics so before stress as after exposure to acute stress were studied. Three water temperatures were examined, i.e. 15, 20, and 25°C, which reflect the range of temperature fluctuation in Mediterranean aquaculture. Fish were acclimated at the respective temperature for 2 weeks before sampling. Blood and water samples were collected prior and at 0.5, 1, 2, 4, and 8 hours post-stress for cortisol analysis. Results showed that water temperature affected the resting and post-stress levels of cortisol. Specifically, higher resting concentrations were observed at 20°C and 25°C than at 15°C. In terms of response, cortisol in all temperatures examined responded with higher post-stress levels. However, the rapidity of the response was greater at the higher temperatures, in both terms of time to peak and to recover, while at the lowest temperature the response was delayed and prolonged. Additionally, the overall outcome of the response, as indicated by the AUC, was greater at 15°C. The release of cortisol in the water was also affected by temperature, being slower and of lower intensity at the low temperature examined. In conclusion, differences in the pattern of response between temperatures for E. sea bass could have resulted from temperature-derived differences in cortisol synthesis and/or clearance rates, but also due to differences in the rate of cortisol release in the water. A repeated predictable stress protocol of various intensities was developed to investigate how husbandry stress of increasing intensity can exert different allostatic loads on fish and how this could affect cortisol dynamics and peripheral regulation in tissues like liver and head kidney. Fish were left undisturbed (controls) or exposed to three levels of repeated predictable stress for three weeks and then subjected to an additional acute stress test. The stress protocol used a combination of common husbandry practices, such as chasing with a net, confinement and air-exposure, and was categorized as low, medium and high according to its intensity. Specifically, low stress consisted of subjecting fish to confinement at 50 % of the tank for 30 min every 2nd day; medium stress consisted of both confinement (conducted as previously described) and chasing for 5 min with a net every 2nd day; high stress consisted of confining fish at 25% of the tank for 30 min, and chasing for 5 min every 2nd day, coupled with a 1-min air-exposure stressor once per week. Two days after the end of the application of this protocol, 10 fish per tank were immediately sampled (T0), while the remaining 10 fish were acutely stressed by chasing them for 5 min and exposing them to air for 1 min, and sampled 1 hour later. Results showed that body weight gain was significantly reduced as stress load increased, leading eventually to body weight loss in the high stress group. Feeding was also reduced in all stress groups compared to controls. In terms of cortisol, fish exposed to high stress exhibited high basal cortisol levels and an inability to further respond to acute stress. At the molecular level, upregulation of the expression of adrenocorticotropic hormone (ACTH) receptor, mc2r, and of cortisol biosynthesis enzyme 11β-hydroxylase was observed in the head kidney of all stress load groups compared to controls. Additionally, in the high load group the dysregulation of the balance between the expression of glucocorticoid, gr, and mineralocorticoid, mr. receptors, as well as the lower hepatic gene expression of 11β-hsd2, an enzyme that inactivates cortisol, possible indicate the basis behind the high cortisol levels seen in this group. Genetic background along with the environment is well known to affect the phenotype of individuals. For that reason, it was aimed to assess how the genetic background can influence the variability of cortisol response at both family and individual level and subsequently explore the hepatic transcriptome profile of fish showing consistently low (LR) or high (HR) cortisol responsiveness. The progeny (full sibs) of six families was used, and sampled for plasma cortisol after an acute stress challenge once per month, for four consecutive months. Results suggested that cortisol response was affected by the genetic background, as seen by the familybased differences, and that individual responsiveness was a repeatable trait. Subsequently, LR and HR fish were identified, and showed low or high resting, free and post-stress cortisol concentrations, respectively. These differences could not be explained by differences in the plasma ACTH concentrations. Finally, the liver transcription profiles of LR and HR fish showed some important differences, indicating differential hepatic regulation between these divergent phenotypes. These transcription differences were related to various metabolic and immunological processes, with 169 transcripts being transcribed exclusively in LR and 161 in HR fish. Mechanisms regulating different cortisol responsiveness between LR and HR individuals have been poorly studied. In this context, it was aimed to study these mechanisms at the level of the head kidney in LR and HR fish of E. sea bass. To do so, initially resting plasma cortisol and ACTH concentrations were estimated in LR and HR approximately 1.5 years after their characterization as such. The head kidneys of these individuals were superfused through an in vitro superfusion system, and stimulated with the same dose of ACTH to assess their cortisol biosynthetic capacity. Moreover, the expression of important for cortisol regulation genes was estimated in the head kidneys. Results showed that LR and HR fish differed in the resting cortisol levels, although no differences existed in the circulating levels of ACTH. Additionally, the biosynthetic capacity of HR was higher than that of LR fish when in vitro stimulated with the same concentration of ACTH. At the molecular level, differences in resting cortisol between LR and HR fish could be attributed to a higher expression of the ACTH receptor, mc2r, and the 2.3-fold higher expression of 11β-hydroxylase, an enzyme involved in cortisol biosynthesis in the HR fish. Finally, a significant downregulation of 11β-hsd2, an enzyme involved in cortisol inactivation was observed in HR when compared to LR fish, indicating for the first time that post-production regulation of cortisol in the head kidney can also explain the differences observed between these divergent phenotypes.
Language English
Subject Adrenocorticotropic hormone
Allostatic load
Chronic stress
Genetic background
Water Temperatute
Αδρενοκορτικοτρόπος ορμόνη
Αλλοστατικό φορτίο
Γενετικό υπόβαθρο
Θερμοκρασία νερού
Χρόνιο στρές
Issue date 2017-03-09
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Biology--Doctoral theses
  Type of Work--Doctoral theses
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