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.

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Pro-opiomelanocortin (POMC) is the precursor of a number of biologically active peptides, including adrenocorticotropic hormone, -melanocyte-stimulating hormone and -endorphin, which are released by the pituitary glands of fish as well as mammals. To quantify the levels of expression of the two POMC mRNAs relative to one another during the response of the common carp to temperature-induced stress, we used reverse transcriptase PCR combined with capillary electrophoresis and laserinduced fluorescence detection. The ratio of POMC-I mRNA to POMC-II mRNA determined in wild-type and four isogenic carp strains was found to be straindependent and influenced by temperature. In strain E20 R8, the ratio had altered in favour of POMC-I from 1:3·2 (POMC-I:POMC-II) in fish adapted to 24 C to 1:1·2 in fish adapted to a decrease of 9 C in ambient temperature. A rapid drop in temperature from 24 to 15 C decreased the POMC mRNA ratio at the expense of POMC-I from 1:1·9 in the control fish (strain E4 R3R8) to 1:4·2 3 h after the temperature drop of 9 C. We conclude that both POMC genes are expressed in the common carp and that their expression ratio is strain-dependent and changes in response to ambient temperature.

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.

Carp -endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the -endorphins. Forms of -endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the -endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of -endorphins by the pituitary gland was studied after stimulation with corticotrophinreleasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl -endorphin(1-33) coeluted with N-acetyl -endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetylendorphin(1-15) (30·8% of total immunoreactivity) and N-acetyl -endorphin(1-10) (3·1%) via two different cleavage pathways. The acetylated carp homologues of mammalian -and -endorphin were also found. N-acetyl -endorphin(1-15) and (1-29) and/or (1-33) were the major products to be released in vitro, and were the only acetylated -endorphins found in blood plasma, although never together. CRF stimulated the release of opioid -endorphin from the pars distalis. This nonacetylated -endorphin represents the full length peptide and is the most abundant form in plasma.