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Combinations of stressors occur regularly throughout an animal's life, especially in agriculture and aquaculture settings. If an animal fails to acclimate to these stressors, stress becomes chronic, and a condition of allostatic overload arises with negative results for animal welfare. In the current study, we describe effects of exposing Atlantic salmon parr to an unpredictable chronic stressor (UCS) paradigm for 3 weeks. The paradigm involves exposure of fish to seven unpredictable stressors three times a day. At the end of the trial, experimental and control fish were challenged with yet another novel stressor and sampled before and 1 h after that challenge. Plasma cortisol decreased steadily over time in stressed fish, indicative of exhaustion of the endocrine stress axis. This was confirmed by a lower cortisol response to the novel stressor at the end of the stress period in chronically stressed fish compared with the control group. In the preoptic area (POA) and pituitary gland, chronic stress resulted in decreased gene expression of 11βhsd2, gr1 and gr2 in the POA and increased expression of those genes in the pituitary gland. POA crf expression and pituitary expression of pomcs and mr increased, whereas interrenal gene expression was unaffected. Exposure to the novel stressor had no effect on POA and interrenal gene expression. In the pituitary, crfr1, pomcs, 11βhsd2, grs and mr were down-regulated. In summary, our results provide a novel overview of the dynamic changes that occur at every level of the hypothalamic-pituitary gland-interrenal gland (HPI) axis as a result of chronic stress in Atlantic salmon., (© 2015. Published by The Company of Biologists Ltd.)

A characteristic feature of the behavioural response to intensely acute or chronic stressors is a reduction in appetite. In fish, as in other vertebrates, the corticotropin-releasing factor (CRF) system plays a key role in coordinating the neuroendocrine, autonomic, and behavioural responses to stress. The following review documents the evidence implicating the CRF system as a mediator of the appetite-suppressing effects of stress in fish. Central injections of CRF or the related peptide, urotensin I (UI), or pharmacological treatments or stressors that result in an increase in forebrain CRF and UI gene expression, can elicit dose-dependent reductions in food intake that are at least partially reversed by pre-treatment with a CRF receptor antagonist. In addition, the appetite suppressing effects of various environmental, pathological, physical, and social stressors are associated with elevated levels of forebrain CRF and UI gene expression and with an activation of the hypothalamic-pituitary-interrenal (HPI) stress axis. In contrast, although stressors can also be associated with an increase in caudal neurosecretory system CRF and UI gene expression and an endocrine role for CRF-related peptides has been suggested, the physiological effects of peripheral CRF-related peptides on the gastrointestinal system and in the regulation of appetite have not been investigated. Overall, while CRF and UI appear to participate in the stress-induced changes in feeding behaviour in fish, the role of other know components of the CRF system is not known. Moreover, the extent to which the anorexigenic effects of CRF-related peptides are mediated through the hypothalamic feeding center, the HPI axis and cortisol, or via actions on descending autonomic pathways remains to be investigated.

In response to most stressors, fish will elicit a generalized physiological stress response, which involves the activation of the hypothalamic-pituitary-interrenal axis (HPI). As in other vertebrates, this generalized stress response comprises physiological responses that are common to a wide range of environmental, physical and biological stressors. Recently, several families of heat shock proteins (hsps) have been proposed as indicators of a generalized stress response at the cellular level. Recent findings that hsp levels, in various fish tissues, respond to a wide range of stressors have supported the use of these proteins as indicators of stressed states in fish. However, the cellular stress response can vary, for example, according to tissue, hsp family and type of stressor. This brief overview of these responses in fish asks the question of whether changes in levels and families of hsps can be used as a suitable indicator of stressed states in fish. By casting this question in the context of the well-established generalized physiological stress response in fish, we argue that the use of hsps as indicators of stressed states in fish in general is premature.

In the present study the response of white sea-bream (Diplodus sargus), used as experimental model, to different stocking densities was tested to assess whether the induced stress conditions were able to activate a chemical communication. Once a good recovery was evident, six days after capture and transportation, experiments started to evaluate eventual changes in cortisol plasma levels as well as in both plasma and pituitary alpha-melanocyte stimulating hormone (alpha-MSH) levels. These studies demonstrated that a low stocking density (2.5kg/1000L) is not deleterious for this species while a higher one (10kg/1000L) induces the activation of the hypothalamus-pituitary-interrenal axis (HPI). Thus, in these fish were evident both a significant increase of cortisol plasma levels and a significant decrease of alpha-MSH pituitary levels, while no significant changes were detected in alpha-MSH plasma concentrations. The most relevant result reported in this study, for the first time in marine species, is the presence of a chemical communication among stressed fish. It also should be noted that the data obtained from cortisol and alpha-MSH detections strongly indicate a gender specificity of this chemical signal.

Two F1 lines of rainbow trout Oncorhynchus mykiss, divergent for plasma cortisol responsiveness, were generated by individual selection for post-stress cortisol values within the F0 generation. Adult females of the F1 generation were transferred to rearing in social isolation in observation tanks. After 6 days, locomotor activity in high-responding (HR) and low-responding (LR) individuals was quantified as time spent moving during a 20 min observation period. Behavioural observations were repeated the next day with a smaller conspecific intruder present in each observation tank. Differential hypothalamus-pituitary-interrenal axis activity in the two lines was subsequently confirmed by a standardised confinement stress test, which resulted in significantly higher plasma cortisol concentrations in HR than LR fish. HR fish displayed higher levels of locomotor activity than LR fish in the presence of an intruder, but not when in isolation. Aggressive behaviour towards the intruder was not seen, suggesting either a state-dependent lack of territorial aggression, or chronic stress in the experimental fish. A significantly higher incidence of feed intake was seen in LR trout when held in observation tanks (40% versus 0% of the fish took food when in isolation), suggesting that these fish acclimated more successfully to the experimental conditions than HR fish did. These results suggest that selection for stress responsiveness in salmonid fish leads to behavioural alterations, which are of potential importance to the performance of these fish in aquaculture rearing operations.

Dynamics of adrenocorticotropin (ACTH), alpha melanocyte-stimulating hormone (alpha-MSH), N-acetylated-beta-endorphin (N-ac-beta-END), cortisol, and growth hormone (GH) were investigated in gilthead sea bream (Sparus aurata) stressed by handling plus confinement. As indices of the secondary stress response, plasma levels of glucose, lactate, and plasma ions were monitored. Within 1 h, plasma cortisol and ACTH levels increased above the control values but GH levels decreased. Subsequently, at 24 h cortisol and ACTH levels had declined, but were still higher than in controls, whereas GH levels had recovered after 4 h. Regarding the melanotrope peptides, there were no differences in plasma levels of alpha-MSH and N-ac-beta-END, but pituitary stores of these peptides were severely depleted already after 1 h, as were ACTH stores. Pituitary contents of proopiomelanocortin (POMC)-derived hormones did not show significant differences from 72 h onward. Therefore, the results indicate that both handling and confinement affected the corticotropes of the pars distalis and the melanotropes of the neurointermediate lobe but at different magnitudes. The possible involvement of corticotropin-releasing hormone (CRH) in the regulation of pituitary POMC-producing cell types under these conditions was indicated by the in vitro dose-dependent effect of the peptide on release of ACTH, alpha-MSH, and N-ac-beta-END. The corticocotropes appeared more responsive, and approximately 10-fold more sensitive, to CRH compared with the melanotropes. The ACTH-releasing potency of 1 nM CRH was inhibited 75% following pretreatment of the whole pituitary gland with 400 nM of the CRH antagonist alpha-helical CRH(9-41)., (Copyright 2001 Academic Press.)

Plasma levels of ACTH, alpha-MSH, and N-ac-beta-END, and in vitro interrenal ACTH sensitivity were investigated in rainbow trout (Oncorhynchus mykiss) stressed by confinement and in unstressed fish treated with exogenous cortisol. Within 3 hr after the onset of confinement, plasma cortisol and ACTH levels were significantly elevated above control values, while plasma alpha-MSH, but not N-ac-beta-END, levels were significantly decreased compared with those of unstressed fish. At 3 hr, sensitivity of the interrenal tissue to ACTH stimulation in vitro was reduced in stressed fish compared to that of unstressed controls. This hyposensitivity cannot be due to the intervention of alpha-MSH or N-ac-beta-END, because after 48 hr of confinement levels of both POMC-derived peptides were significantly lower than in controls, whereas interrenal tissue of stressed fish still responded significantly less to an ACTH challenge than tissue from control fish. Plasma cortisol and ACTH levels in confined fish at this time point were similar to those at 3 hr. Within 96 hr of the onset of confinement, plasma ACTH levels in stressed fish had returned to baseline levels. Plasma cortisol levels in stressed fish at 96 hr had also declined significantly, but were still higher than those in controls. The circulating cortisol level cannot be the regulatory factor responsible for the ACTH hyposensitivity observed after 3 and 48 hr of stress, because treatment of unstressed fish with exogenous cortisol (which resulted in elevated plasma cortisol and lower plasma ACTH and alpha-MSH levels compared to those of controls) did not induce a reduction in interrenal sensitivity to ACTH. It is suggested, instead, that these data support the contention that not only the initiation of the interrenal stress response, but also the habituation of the response, are regulated at the level of the hypothalamus via circulating ACTH levels.

A significant reduction in stress-induced plasma cortisol levels is apparent in mature male rainbow trout compared to immature fish of both sexes and of the same age and strain. Mean plasma cortisol levels in groups of immature fish subjected to a standard 1-hr confinement stress were consistently higher (range 93.9 +/- 4.9-114.8 +/- 4.1 ng ml-1) than mean levels in mature males exposed to the same procedure (range 47.0 +/- 4.3-71.7 +/- 5.7 ng ml-1), throughout the 4-month period around spawning (P < 0.001). Body weight was not found to be a significant determinant of poststress cortisol level. The dissimilarity in plasma cortisol levels between mature and immature fish following confinement does not stem from differences in the dynamics of the response; during a 24-hr period of confinement the rate of elevation of plasma cortisol levels was similar for both mature male and immature fish, but mature male fish attained a significantly lower peak cortisol level (51.6 +/- 5.2 ng ml-1) than immature fish (89.5 +/- 7.7 ng ml-1), a disparity which was maintained throughout the period of stress (P < 0.05-P < 0.001). The reduced responsiveness of the interrenal tissue of mature male fish during stress appears to be modulated by the hypothalamus/pituitary. Plasma ACTH levels in mature male trout (44 +/- 9 pg ml-1) are significantly lower than those in immature fish (71 +/- 9 pg ml-1, P < 0.01) within 30 min of the onset of confinement and remain so during a 3-hr period of confinement. These data suggest that the cortisol/ACTH feedback equilibrium has been modified in mature fish to a lower "set point." Furthermore, although stress caused a significant decline of plasma alpha-MSH levels in both immature fish and mature males, N-acetyl-beta-endorphin levels were reduced only in mature male fish during confinement stress.

This study examines ACTH-like immunoreactivity in the pituitary pars distalis and pars intermedia of the freshwater teleost Oreochromis mossambicus (tilapia). Two products (tACTHA and tACTHB) were present in both lobes. These two products also accounted for the majority of the ACTH i.r. when in vitro pars distalis incubation medium was analyzed by HPLC. In a homologous bioassay the two tilapia ACTH-like molecules and human ACTH1-39 possessed similar corticotropic potency. The peptides were quantified using a newly validated radioimmunoassay, which was also used to measure ACTH in plasma of unstressed and stressed fish. Short-term (< 12 min) stress rapidly increased plasma cortisol, reaching levels of around 300 ng/ml in 5 min. Surprisingly, this initial elevation was not accompanied by a rise in plasma ACTH levels. A more prolonged (3 hr) confinement in pairs resulted in high plasma cortisol and ACTH levels in one fish of every pair. The second fish had control ACTH levels and only marginally elevated cortisol levels. Therefore, in this species social interactions seem to influence the reaction of the pituitary-interrenal axis to stress. The short-term cortisol response to disturbance could be abolished completely by pretreating fish in vivo with cortisol for 48 hr. This treatment did not alter circulating ACTH levels. It is concluded that tilapia did not rely on circulating ACTH for a rapid elevation of plasma cortisol levels. Both neuronal mechanisms and cortisol feedback may regulate the pituitary-interrenal axis at the level of the interrenal.