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Scales tell a story on the stress history of fish.

Aerts J, Metz JR, Ampe B, Decostere A, Flik G, De Saeger S - PLoS ONE (2015)

Bottom Line: An increase in scale cortisol content was found in STRESS and CORT but not in CTR and DEX fish.Plasma analyses confirmed that (i) CTR, DEX and CORT treatments were effective, (ii) plasma cortisol of STRESS fish showed no signs of chronic HPI-axis activation, and (iii) plasma cortisol is a poor predictor for chronic stress.We conclude that the cortisol content in ontogenetic and regenerated scales is an innovative biomarker for chronic stress offering ample applications in science and industry.

View Article: PubMed Central - PubMed

Affiliation: Bio-analysis Research Group, Animal Sciences Unit, Institute for Agriculture and Fisheries Research, Melle, Belgium; Laboratory of Food Analysis, Department of Bio-analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.

ABSTRACT
Fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal (HPI-) axis to release cortisol into the blood. Scientifically validated biomarkers to capture systemic cortisol exposure over longer periods of time are of utmost importance to assess chronic stress in governmental, wildlife, aquaculture and scientific settings. Here we demonstrate that cortisol in scales of common carp (Cyprinus carpio L.) is the long-sought biomarker for chronic stress. Undisturbed (CTR) and daily stressed (STRESS) carp were compared. Dexamethasone (DEX) or cortisol (CORT) fed fish served as negative and positive controls, respectively. Scale cortisol was quantified with a validated ultra-performance liquid chromatography tandem mass spectrometry method. An increase in scale cortisol content was found in STRESS and CORT but not in CTR and DEX fish. Scale cortisol content reflects its accumulation in a stressor and time dependent manner and validates the scale cortisol content as biomarker for chronic stress. Plasma analyses confirmed that (i) CTR, DEX and CORT treatments were effective, (ii) plasma cortisol of STRESS fish showed no signs of chronic HPI-axis activation, and (iii) plasma cortisol is a poor predictor for chronic stress. The expression of HPI key genes crf, pomc, and star were up-regulated in STRESS fish in the absence of a plasma cortisol response, as was the target gene of cortisol encoding subunit α1 of the Na+/K+-ATPase in gills. When lost, scales of fish regenerate fast. Regenerated scales corroborate our findings, offering (i) unsurpassed time resolution for cortisol incorporation and as such for stressful events, and (ii) the possibility to investigate stress in a well defined and controlled environment and time frame creating novel opportunities for bone physiological research. We conclude that the cortisol content in ontogenetic and regenerated scales is an innovative biomarker for chronic stress offering ample applications in science and industry.

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Plasma analyses of cortisol (nM) (n = 47, F(3,39) = 12.80, p<0.0001), glucose (mM) (n = 47, F(3,39) = 13.12, p<0.0001), lactate (mM) (n = 47, F(3,39) = 16.01, p<0.0001), total calcium (mM) (n = 47, F(3,39) = 1.59, p = 0.2068), and osmolality (mOsmol kg-1) (n = 47, F(3,39) = 0.69, p = 0.5655) at day 42 of treatment.Fifty percent of the observations occurs between the lower and upper edges of the box (the first and third quartiles) and the whiskers extend to the most extreme observation which is no more than 1.5 times the interquartile range from the box; open circles represent values outside the range mentioned. For statistics, see S2 Table.
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pone.0123411.g001: Plasma analyses of cortisol (nM) (n = 47, F(3,39) = 12.80, p<0.0001), glucose (mM) (n = 47, F(3,39) = 13.12, p<0.0001), lactate (mM) (n = 47, F(3,39) = 16.01, p<0.0001), total calcium (mM) (n = 47, F(3,39) = 1.59, p = 0.2068), and osmolality (mOsmol kg-1) (n = 47, F(3,39) = 0.69, p = 0.5655) at day 42 of treatment.Fifty percent of the observations occurs between the lower and upper edges of the box (the first and third quartiles) and the whiskers extend to the most extreme observation which is no more than 1.5 times the interquartile range from the box; open circles represent values outside the range mentioned. For statistics, see S2 Table.

Mentions: Results for five blood parameters commonly used as stress indicators confirm that DEX fish may be considered as negative, and CORT fish as positive controls, respectively (Fig 1). Plasma cortisol, the most commonly used parameter for stress, had elevated in the CORT group (CORT vs. CTR: P<0.0001), confirming the absorption of cortisol from feed into the blood; in STRESS fish cortisol levels did not differ from negative controls indicating the unsuitability of the latter for chronic stress assessment. The secondary stress parameter plasma glucose, reflecting predicted glucocorticoid actions (gluconeogenesis), had increased in DEX (DEX vs. CTR: P<0.0001), while CORT (CORT vs. CTR: P = 0.2004) and STRESS (STRESS vs. CTR: P = 0.0531) did not differ significantly, in line with the more potent action of DEX. Plasma lactate, an indicator of anaerobic metabolism and fatigue, had increased in DEX (DEX vs. CTR: P<0.0001) and CORT (CORT vs. CTR: P = 0.0004), but not significantly in STRESS (STRESS vs. CTR: P = 0.4224) in line with the downstream effects of DEX and CORT. Fish regulate calcium levels tightly (ionic, physiologically important calcium more strictly than total calcium) as calcium is pivotal to vision, muscle contraction, signal transduction, membrane permeability, etc. Even minor disruptions induce stress and disturbance of calcium balance. Plasma osmolality discloses disturbances in water and ionic balance, characteristic aspects of stress in fish which occur due to the intimate relationship between the body fluids in the gills and the aquatic environment. Plasma total calcium and osmolality were not affected indicating that the treatments did not exceed the resilience of the fish and did not evoke distress [13, 18–19]. We conclude that (i) CTR, DEX and CORT treatments elicited the intended glucocorticoid actions; (ii) in plasma of STRESS fish no signs of activation of HPI-axis were observed; (iii) plasma parameters are poor predictors for chronic stress as they reflect no more than a snap-shot of the stress response at a given moment.


Scales tell a story on the stress history of fish.

Aerts J, Metz JR, Ampe B, Decostere A, Flik G, De Saeger S - PLoS ONE (2015)

Plasma analyses of cortisol (nM) (n = 47, F(3,39) = 12.80, p<0.0001), glucose (mM) (n = 47, F(3,39) = 13.12, p<0.0001), lactate (mM) (n = 47, F(3,39) = 16.01, p<0.0001), total calcium (mM) (n = 47, F(3,39) = 1.59, p = 0.2068), and osmolality (mOsmol kg-1) (n = 47, F(3,39) = 0.69, p = 0.5655) at day 42 of treatment.Fifty percent of the observations occurs between the lower and upper edges of the box (the first and third quartiles) and the whiskers extend to the most extreme observation which is no more than 1.5 times the interquartile range from the box; open circles represent values outside the range mentioned. For statistics, see S2 Table.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414496&req=5

pone.0123411.g001: Plasma analyses of cortisol (nM) (n = 47, F(3,39) = 12.80, p<0.0001), glucose (mM) (n = 47, F(3,39) = 13.12, p<0.0001), lactate (mM) (n = 47, F(3,39) = 16.01, p<0.0001), total calcium (mM) (n = 47, F(3,39) = 1.59, p = 0.2068), and osmolality (mOsmol kg-1) (n = 47, F(3,39) = 0.69, p = 0.5655) at day 42 of treatment.Fifty percent of the observations occurs between the lower and upper edges of the box (the first and third quartiles) and the whiskers extend to the most extreme observation which is no more than 1.5 times the interquartile range from the box; open circles represent values outside the range mentioned. For statistics, see S2 Table.
Mentions: Results for five blood parameters commonly used as stress indicators confirm that DEX fish may be considered as negative, and CORT fish as positive controls, respectively (Fig 1). Plasma cortisol, the most commonly used parameter for stress, had elevated in the CORT group (CORT vs. CTR: P<0.0001), confirming the absorption of cortisol from feed into the blood; in STRESS fish cortisol levels did not differ from negative controls indicating the unsuitability of the latter for chronic stress assessment. The secondary stress parameter plasma glucose, reflecting predicted glucocorticoid actions (gluconeogenesis), had increased in DEX (DEX vs. CTR: P<0.0001), while CORT (CORT vs. CTR: P = 0.2004) and STRESS (STRESS vs. CTR: P = 0.0531) did not differ significantly, in line with the more potent action of DEX. Plasma lactate, an indicator of anaerobic metabolism and fatigue, had increased in DEX (DEX vs. CTR: P<0.0001) and CORT (CORT vs. CTR: P = 0.0004), but not significantly in STRESS (STRESS vs. CTR: P = 0.4224) in line with the downstream effects of DEX and CORT. Fish regulate calcium levels tightly (ionic, physiologically important calcium more strictly than total calcium) as calcium is pivotal to vision, muscle contraction, signal transduction, membrane permeability, etc. Even minor disruptions induce stress and disturbance of calcium balance. Plasma osmolality discloses disturbances in water and ionic balance, characteristic aspects of stress in fish which occur due to the intimate relationship between the body fluids in the gills and the aquatic environment. Plasma total calcium and osmolality were not affected indicating that the treatments did not exceed the resilience of the fish and did not evoke distress [13, 18–19]. We conclude that (i) CTR, DEX and CORT treatments elicited the intended glucocorticoid actions; (ii) in plasma of STRESS fish no signs of activation of HPI-axis were observed; (iii) plasma parameters are poor predictors for chronic stress as they reflect no more than a snap-shot of the stress response at a given moment.

Bottom Line: An increase in scale cortisol content was found in STRESS and CORT but not in CTR and DEX fish.Plasma analyses confirmed that (i) CTR, DEX and CORT treatments were effective, (ii) plasma cortisol of STRESS fish showed no signs of chronic HPI-axis activation, and (iii) plasma cortisol is a poor predictor for chronic stress.We conclude that the cortisol content in ontogenetic and regenerated scales is an innovative biomarker for chronic stress offering ample applications in science and industry.

View Article: PubMed Central - PubMed

Affiliation: Bio-analysis Research Group, Animal Sciences Unit, Institute for Agriculture and Fisheries Research, Melle, Belgium; Laboratory of Food Analysis, Department of Bio-analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.

ABSTRACT
Fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal (HPI-) axis to release cortisol into the blood. Scientifically validated biomarkers to capture systemic cortisol exposure over longer periods of time are of utmost importance to assess chronic stress in governmental, wildlife, aquaculture and scientific settings. Here we demonstrate that cortisol in scales of common carp (Cyprinus carpio L.) is the long-sought biomarker for chronic stress. Undisturbed (CTR) and daily stressed (STRESS) carp were compared. Dexamethasone (DEX) or cortisol (CORT) fed fish served as negative and positive controls, respectively. Scale cortisol was quantified with a validated ultra-performance liquid chromatography tandem mass spectrometry method. An increase in scale cortisol content was found in STRESS and CORT but not in CTR and DEX fish. Scale cortisol content reflects its accumulation in a stressor and time dependent manner and validates the scale cortisol content as biomarker for chronic stress. Plasma analyses confirmed that (i) CTR, DEX and CORT treatments were effective, (ii) plasma cortisol of STRESS fish showed no signs of chronic HPI-axis activation, and (iii) plasma cortisol is a poor predictor for chronic stress. The expression of HPI key genes crf, pomc, and star were up-regulated in STRESS fish in the absence of a plasma cortisol response, as was the target gene of cortisol encoding subunit α1 of the Na+/K+-ATPase in gills. When lost, scales of fish regenerate fast. Regenerated scales corroborate our findings, offering (i) unsurpassed time resolution for cortisol incorporation and as such for stressful events, and (ii) the possibility to investigate stress in a well defined and controlled environment and time frame creating novel opportunities for bone physiological research. We conclude that the cortisol content in ontogenetic and regenerated scales is an innovative biomarker for chronic stress offering ample applications in science and industry.

Show MeSH
Related in: MedlinePlus