We investigated short-term effects (up to 24 h) of air exposure and confinement, and long-term effects (up to 11 days) of confinement, to elucidate signalling pathways in the stress response of gilthead sea bream Sparus aurata L. Plasma glucose and lactate were taken as indicators of sympathetic activation, and -melanocyte stimulating hormone ( -MSH), adrenocorticotrophic hormone (ACTH) and cortisol as indicators of activation of the brainpituitary-interrenal (BPI) axis. Air exposure for 3 min resulted, within 30 min, in an increase in plasma concentrations of cortisol, -MSH, glucose, lactate, osmolality and plasma Na, Cl and Mg. Plasma ACTH and -endorphin and plasma K, Ca and P did not change. We conclude that air exposure mainly activates the brainsympathetic-chromaffin cell (BSC) axis. In fish confined at a density of 70 kg/m 3 (compared with 4 kg/m 3 in controls), cortisol, ACTH and -MSH increased within 1 h, indicating activation of the BPI axis. Plasma glucose, Na, Cl and Mg increased with an 8 h delay compared with the response to air exposure. No changes in plasma lactate, osmolality, K, Ca and P were observed. Long-term confinement induced a biphasic cortisol response with peaks at 1 h and at 2 and 3 days. A gradual increase in plasma -endorphin concentrations peaked at 7 days; the concentration of -MSH increased rapidly within 1 h and then declined to control values 4 days after the onset of confinement. No changes in ACTH were detected. Our data provide evidence that a stressor-specific activation of the BSC and BPI axes may occur in Sparus aurata.

MSH is a pituitary hormone derived by post-translational processing from POMC and involved in stress and background adaptation. N-terminal acetylation of MSH to monoacetyl -MSH or diacetyl -MSH increases the bioactivity of the peptide. The aim of this study was to characterize -MSH acetylation in the sea bream (Sparus aurata L.) pituitary gland in response to the stressors air exposure and confinement, as well as in fish adapted for 15 days to a white, gray or black background. Pituitary homogenates were purified by reversed-phase HPLC (RP-HPLC). The -MSH content of fractions was measured by RIA. Immunoreactive RP-HPLC fractions were further analyzed by electrospray mass spectrometry and the peptide sequence determined as SYSMEHFRWGKPV-NH 2 . In the pituitary gland of sea bream, des-, mono-and diacetyl -MSH were identified. Then plasma -MSH levels were measured in sea bream adapted to different backgrounds. Surprisingly, we found the highest plasma -MSH levels in white-adapted as compared with black-adapted sea bream with intermediate values for gray-adapted fish. This observation is in contrast with results that have been obtained in eel, trout or terrestrial vertebrates. Next, des-, mono-and diacetyl -MSH forms were measured in homogenates of the pituitary gland and in plasma of sea bream exposed to air, to confinement, or to different backgrounds. Monoacetyl -MSH was the predominant form in all control and experimental groups. The lowest content of monoacetyl -MSH relative to des-and diacetyl -MSH was found in white-adapted fish. Levels of des-and diacetyl -MSH forms were similar under all conditions. We observed that monoacetyl -MSH is the most abundant isoform in the pituitary gland after background adaptation, confinement and air exposure, in sea bream. These data indicate that the physiologically most potent isoform of -MSH may vary from species to species.