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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.)

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.

An exposure to ambient temperature of 25 degrees C had no perceptible effect on interrenal function but further increase of temperature to 35 degrees C caused nuclear hypertrophy with increase of nuclear diameter, RNA concentration, acid phosphatase and alkaline phosphatase activities, accompanied by quantitative depletions of cholesterol (free, esterified and total) and ascorbic acid levels in the interrenal gland of the soft-shelled turtle Lissemys p. punctata. Similar manifestations of stimulation, except in the nucleus, were marked after exposure to 38 degrees C, but the degree of response in respect of esterified and free cholesterol levels was higher at 38 degrees C than at 35 degrees C. Moreover, withdrawal of 38 degrees C temperature and subsequently maintaining at 25 degrees C for 15 days showed reverse manifestations to those of 35 degrees C/38 degrees C, leading to a tendency towards normalcy. It is suggested that high a ambient temperature of 35 degrees C significantly stimulates interrenal function of Lissemys turtles, but further increase of 38 degrees C does not cause further overall stimulation, and withdrawal of higher temperature (38 degrees C) shows a tendency towards normalcy. It is also suggested that (a) high ambient temperature causes thermal stress, (b) it is reversible and (c) it acts on interrenal activity presumably via CRF-ACTH-axis in turtles.

The zebrafish ftz-f1 gene, ff1b, is activated in two cell clusters lateral to the midline in the trunk during late embryogenesis. These cell clusters coalesce to form a discrete organ at around 30 hpf, which then begins to acquire a steroidogenic identity as evidenced by the expression of the steroidogenic enzyme genes, cyp11a and 3beta-hsd. The migration of the cell clusters to the midline is impaired in zebrafish midline signaling mutants. Knockdown of Ff1b activity by antisense ff1b morpholino oligonucleotide (ff1bMO) leads to phenotypes that are consistent with impaired osmoregulation. Injection of ff1bMO was also shown to downregulate the expression of cyp11a and 3beta-hsd. Histological comparison of wild-type and ff1b morphants at various embryonic and juvenile stages revealed the absence of interrenal tissue development in ff1b morphants. The morphological defects of ff1b morphants could be mimicked by treatment with aminoglutethimide, an inhibitor of de novo steroid synthesis. Based on these data, we propose that ff1b is required for the development of the steroidogenic tissue of the interrenal organ.

Steroids are synthesized mainly from the adrenal cortex. Adrenal deficiencies are often associated with problems related to its development, which is not fully understood. To better understand adrenocortical development, we studied zebrafish because of the ease of embryo manipulation. The adrenocortical equivalent in zebrafish is called the interrenal, because it is embedded in the kidney. We find that interrenal development parallels that of the embryonic kidney (pronephros). Primordial interrenal cells first appear as bilateral intermediate mesoderm expressing ff1b in a region ventral to the third somite. These cells then migrate toward the axial midline and fuse together. The pronephric primordia are wt1-expressing cells located next to the interrenal. They also migrate to the axial midline and fuse to become glomeruli at later developmental stages. Our gene knockdown experiments indicate that wt1 is required for its initial restricted expression in pronephric primordia, pronephric cell migration and fusion. wt1 also appears to be involved in interrenal development and ff1b expression. Similarly, ff1b is required for interrenal differentiation and activation of the differentiated gene, cyp11a1. Our results show that the zebrafish interrenal and pronephros are situated close together and go through parallel developmental processes but are governed by different signaling events.

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.