| 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.
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