Abstract |
The aims of this dissertation were to study the ontogeny of the endocrine stress
response, to characterize the molecular programming of the Hypothalamus-Pituitary-
Interrenal (HPI) axis, and to determine the impact of long term chronic mild stress
applied early in life on the performance of fish at subsequent stages of development,
in a Mediterranean marine teleost, the European sea bass (Dicentrarchus labrax).
Sea bass embryos, pre-larvae and larvae at specific points of development were
exposed to acute stressors and the temporal patterns of cortisol and α-MSH whole
body concentrations and the expression of genes involved in corticosteroid
biosynthesis, degradation and both cortisol and α-MSH signaling were determined.
Expression of genes involved into the corticoid response regulation (gr1, gr2, mr, crf)
combined with histological data indicated that, although a cortisol stress response is
evident for the first time around first feeding, a pattern becomes progressively
established in larvae at flexion until the formation of all fins. Moreover, mRNA
transcript levels of 11β-hydroxylase and 11β-hsd2 which are involved in cortisol
synthesis and deactivation/metabolism, respectively, showed a strong correlation with
the whole body cortisol concentrations. An α-MSH stress response, an additional to
cortisol pathway regulating stress in teleosts, is evident for the first time in the early
development of European sea bass at the stage of mouth opening showing a specific
pattern characterized by elevated levels that becomes established around the
formation of all fins. mRNA transcript levels of pomc and mc2r were altered after the
acute stress application in a consistent elevated pattern especially as development
proceeds, at the stages of flexion and after the formation of all fins, showing at the
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same time a similar pattern with the whole body α-MSH concentrations. The acute
stress application had no effect on the expression of mc1r but in the case of mc4r
resulted in an increased transcription even as early as at the stage of mouth opening.
In fish, stress research is focused on the effects of acute or chronic severe noxius
stimuli of physical, chemical and husbandry nature applied in juveniles or adult
individuals, and there is no information on the effects of early exposure to long term
chronic mild stressors on the development and performance of fish at subsequent
phases of the life-cycle, but also no valid chronic low intensity stress protocol exists
for fish at early development. To this end an unpredictable chronic low intensity stress
(UCLIS) protocol was developed and evaluated for the first time in early development
of E. sea bass. UCLIS protocol was based on the unpredictability, variety, frequency
and moderate intensity of the applied stressors, providing a relatively realistic model
of everyday aquaculture husbandry practices. The UCLIS application lasted for 14
consecutive days, starting at three different phases of early ontogeny (first feeding,
flexion and development of all fins). Evaluation of the UCLIS protocol was
performed through the determination of water-born cortisol concentrations of the
larvae rearing tanks at regular intervals, recording of mortality and measurements of
growth performance. In addition, its effects on subsequent developmental phases were
evaluated by measurement of growth characteristics and by the determination of
plasma cortisol in juvenile fish, prior and 1h after the application of an acute stressor.
Our data show that European sea bass larvae are sensitive to mild husbandry stimuli
with consequences even at subsequent phases of the life-cycle, with the stages of first
feeding and all fins being the most critical, pointing out the necessity to reconsider
common rearing practices. In particular, UCLIS application resulted in higher water
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cortisol release rates in all groups where the stress was applied compared to the
controls, proving to be a reliable non-invasive indicator of stress even during early
ontogeny. Performance of fish in terms of growth rate was also affected by
application of the stress protocol, as larvae that had been exposed to UCLIS at the
beginning of first feeding and the formation of all fins displayed worst performance
than fish exposed to UCLIS at flexion and controls. Early life stress did not affect
plasma cortisol levels of juveniles exposed to additional acute stressors. However,
fish were very sensitive to common handling practices and in addition, significant
higher plasma cortisol concentrations were found in juveniles exposed to UCLIS at
the stages of first feeding and the formation of all fins, compared to the other two
groups, in accordance with the differences observed in growth rates.
Concluding, the data obtained from this study provide a better insight into the onset
and regulation of the stress response in early development of E. sea bass and show for
the first time that early life stress, in the form of common husbandry practices, has an
impact both on larvae performance and also on later stages of the life-cycle in this
species, as life history affected the growth performance and the stress response in
juvenile fish.
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