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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.

Thyroid hormones are involved in many developmental and physiological processes, including osmoregulation. The regulation of the thyroid system by environmental salinity in the euryhaline gilthead seabream (Sparus aurata) is still poorly characterized. To this end seabreams were exposed to four different environmental salinities (5, 15, 40 and 55 ppt) for 14 days, and plasma free thyroid hormones (fT3, ff4), outer ring deiodination and Na+/K+ -ATPase activities in gills and kidney, as well as other osmoregulatory and metabolic parameters were measured. Low salinity conditions (5 ppt) elicited a significant increase in fT3 (29%) and ff4 (184%) plasma concentrations compared to control animals (acclimated to 40 ppt, natural salinity conditions in the Bay of Cadiz, Spain), while the amount of pituitary thyroid stimulating hormone subunit 13 (tshb) transcript abundance remained unchanged. In addition, plasma fT4 levels were positively correlated to renal and branchial deiodinase type 2 (dio2) mRNA expression. Gill and kidney T4-outer ring deiodination activities correlated positively with dio2 mRNA expression and the highest values were observed in fish acclimated to low salinities (5 and 15 ppt). The high salinity (55 ppt) exposure caused a significant increase in tshb expression (65%), but deiodinase gene expression (diol and dio2) and activity did not change and were similar to controls (40 ppt). In conclusion, acclimation to different salinities led to changes in the peripheral regulation of thyroid hormone metabolism in seabream. Therefore, thyroid hormones are involved in the regulation of ion transport and osmoregulatory physiology in this species. The conclusions derived from this study may also allow aquaculturists to modulate thyroid metabolism in seabream by adjusting culture salinity. (C) 2016 Elsevier Inc. All rights reserved. ; Socrates/Erasmus Grant from the European Union ; University of Cadiz [UCA 2009-074-FPI] ; Ministerio de Education y Ciencia, Spain [AGL2007-61211/ACU] ; FEDER, Spain ...