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Yusuke Kumai, Nicholas J Bernier and Steve F Perry

The contribution of the renin–angiotensin system (RAS) to Na+ uptake was investigated in larval zebrafish (Danio rerio). At 4 days post fertilization (dpf), the level of whole-body angiotensin-II (ANG-II) was significantly increased after 1- or 3-h exposure to acidic (pH=4.0) or ion-poor water (20-fold dilution of Ottawa tapwater), suggesting rapid activation of the RAS. Long-term (24 h) treatment of 3 dpf larvae with ANG-I or ANG-II significantly increased Na+ uptake which was accompanied by an increase in mRNA expression of the Na+-Cl cotransporter (zslc12a10.2). Induction of Na+ uptake by exposure to ANG-I was blocked by simultaneously treating larvae with lisinopril (an angiotensin-converting enzyme inhibitor). Acute (2 h) exposure to acidic water or ion-poor water led to significant increase in Na+ uptake which was partially blocked by the ANG-II receptor antagonist, telmisartan. Consistent with these data, translational knockdown of renin prevented the stimulation of Na+ uptake following exposure to acidic or ion-poor water. The lack of any effects of pharmacological inhibition (using RU486), or knockdown of glucocorticoid receptors on the stimulation of Na+ uptake during acute exposure to acidic or ion-poor environments, indicates that the acute effects of RAS occur independently of cortisol signaling. The results of this study demonstrate that the RAS is involved in Na+ homeostasis in larval zebrafish.

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Nicholas J Bernier, Sarah L Alderman and Erin N Bristow

Corticotropin-releasing factor (CRF)- and urotensin I (UI)-expressing cells of the preoptic area (POA) and caudal neurosecretory system (CNSS) are considered key contributors to the regulation of the stress response in fish; however, the expression pattern of these neurons to environmental and social challenges have not been compared in a single study. Therefore, we characterized in rainbow trout (Oncorhynchus mykiss) the central distribution of CRF and UI expression and quantified the POA and CNSS mRNA levels of both transcripts in response to hyperammonemia, hypoxia, isolation, or subordination. The tissue distribution demonstrated that the POA and the CNSS are dominant sites of CRF and UI expression. Comparison of the plasma cortisol levels in response to the diverse treatments showed that subordination was the most severe stressor followed by hyperammonemia, isolation, and hypoxia. In the POA, with the exception of subordination that had no effect on UI expression, all stressors resulted in increase in CRF and UI mRNA levels. In the CNSS, while hyperammonemia was associated with increase in CRF and UI mRNA levels, and hypoxia induced an increase in CRF expression, isolation caused a decrease in the expression of both transcripts, and subordination had no effect. Independent of the stressor, we found strong positive correlations between CRF and UI expression in the POA and the CNSS, and no correlation in the expression of either gene between regions. Overall, the results demonstrate that the contribution of POA and CNSS CRF and UI neurons to the stress response in rainbow trout is stressor-, time-, and region-specific.

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Barry N Madison, Patrick T K Woo and Nicholas J Bernier

Despite clear physiological duress, rainbow trout (Oncorhynchus mykiss) infected with the pathogenic haemoflagellate Cryptobia salmositica do not appear to mount a cortisol stress response. Therefore, we hypothesized that the infection suppresses the stress response by inhibiting the key effectors of the hypothalamic–pituitary–interrenal (HPI) axis. To test this, we characterized the basal activity of the HPI axis and the cortisol response to air exposure in saline- and parasite-injected fish. All fish were sampled at 4 and 6 weeks post-injection (wpi). While both the treatment groups had resting plasma cortisol levels, the parasite-infected fish had lower levels of plasma ACTH than the control fish. Relative to the control fish, the infected fish had higher mRNA levels of brain pre-optic area corticotrophin-releasing factor (CRF) and pituitary CRF receptor type 1, no change in pituitary POMC-A1, -A2 and -B gene expression, higher and lower head kidney melanocortin 2 receptor mRNA levels at 4 and 6 wpi respectively and reduced gene expression of key proteins regulating interrenal steroidogenesis: StAR, cytochrome P450scc and 11β-hydroxylase. The parasite-infected fish also had a reduced plasma cortisol response to a 60-s air exposure stressor. Superfusion of the head kidney tissues of the parasite-infected fish led to significantly lower ACTH-stimulated cortisol release rates than that observed in the control fish. These novel findings show that infection of rainbow trout with C. salmositica results in complex changes in the transcriptional activity of both central and peripheral regulators of the HPI axis and in a reduction in the interrenal capacity to synthesize cortisol.

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Barry N Madison, Sara Tavakoli, Sarah Kramer and Nicholas J Bernier

To gain a better understanding of the mechanisms by which cortisol suppresses growth during chronic stress in fish, we characterized the effects of chronic cortisol on food intake, mass gain, the expression of appetite-regulating factors, and the activity of the GH/IGF axis. Fish given osmotic pumps that maintained plasma cortisol levels at ∼70 or 116 ng/ml for 34 days were sampled 14, 28 and 42 days post-implantation. Relative to shams, the cortisol treatments reduced food intake by 40–60% and elicited marked increases in liver leptin (lep-a1) and brain preoptic area (POA) corticotropin-releasing factor (crf) mRNA levels. The cortisol treatments also elicited 40–80% reductions in mass gain associated with increases in pituitary gh, liver gh receptor (ghr), liver igfI and igf binding protein (igfbp)-1 and -2 mRNA levels, reduced plasma GH and no change in plasma IGF1. During recovery, while plasma GH and pituitary gh, liver ghr and igfI gene expression did not differ between treatments, the high cortisol-treated fish had lower plasma IGF1 and elevated liver igfbp1 mRNA levels. Finally, the cortisol-treated fish had higher plasma glucose levels, reduced liver glycogen and lipid reserves, and muscle lipid content. Thus, our findings suggest that the growth-suppressing effects of chronic cortisol in rainbow trout result from reduced food intake mediated at least in part by increases in liver lep-a1 and POA crf mRNA, from sustained increases in hepatic igfbp1 expression that reduce the growth-promoting actions of the GH/IGF axis, and from a mobilization of energy reserves.

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Lauren E MacDonald, Sarah L Alderman, Sarah Kramer, Patrick T K Woo and Nicholas J Bernier

Leptin is a potent anorexigen, but little is known about the physiological conditions under which this cytokine regulates food intake in fish. In this study, we characterized the relationships between food intake, O2-carrying capacity, liver leptin-A1 (lep-a1) gene expression, and plasma leptin-A1 in rainbow trout infected with a pathogenic hemoflagellate, Cryptobia salmositica. As lep gene expression is hypoxia-sensitive and Cryptobia-infected fish are anemic, we hypothesized that Cryptobia-induced anorexia is mediated by leptin. A 14-week time course experiment revealed that Cryptobia-infected fish experience a transient 75% reduction in food intake, a sharp initial drop in hematocrit and hemoglobin levels followed by a partial recovery, a transient 17-fold increase in lep-a1 gene expression, and a sustained increase in plasma leptin-A1 levels. In the hypothalamus, peak anorexia was associated with decreases in mRNA levels of neuropeptide Y (npy) and cocaine- and amphetamine-regulated transcript (cart), and increases in agouti-related protein (agrp) and pro-opiomelanocortin A2 (pomc). In contrast, in non-infected fish pair-fed to infected animals, lep-a1 gene expression and plasma levels did not differ from those of non-infected satiated fish. Pair-fed fish were also characterized by increases in hypothalamic npy and agrp, no changes in pomc-a2, and a reduction in cart mRNA expression. Finally, peak infection was characterized by a significant positive correlation between O2-carrying capacity and food intake. These findings show that hypoxemia, and not feed restriction, stimulates leptin-A1 secretion in Cryptobia-infected rainbow trout and suggest that leptin contributes to anorexia by inhibiting hypothalamic npy and stimulating pomc-a2.

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Marnix Gorissen, Nicholas J Bernier, Sander B Nabuurs, Gert Flik and Mark O Huising

We describe duplicate leptin genes in zebrafish (Danio rerio) that share merely 24% amino acid identity with each other and only 18% with human leptin. We were also able to retrieve a second leptin gene in medaka (Oryzias latipes). The presence of duplicate leptin genes in these two distantly related teleosts suggests that duplicate leptin genes are a common feature of teleostean fishes. Despite low primary sequence conservation, we are confident in assigning orthology between mammalian and zebrafish leptins for several reasons. First, both zebrafish leptins share their characteristic gene structure and display key features of conserved synteny with mammalian leptin genes. Secondly, the cysteine residues that make up leptin's single disulphide bridge are equally spaced in mammalian and zebrafish leptins and are unique among all members of the class-I helical cytokine family. Thirdly, the zebrafish leptins cluster with other fish leptins and mammalian leptins in phylogenetic analysis, supported by high bootstrap values. Within the leptin cluster, leptin-b forms a separate clade with the leptin-b orthologue from medaka. Finally, our prediction of the tertiary structures shows that both leptins conform to the typical four α-helix bundle structure of the class-I α-helical cytokines. The zebrafish leptins are differentially expressed; the liver shows high leptin-a expression (in concordance with what we observed for carp leptins), while leptin-b is expressed at much lower levels, which are downregulated further upon fasting. The finding of duplicate leptin genes in teleosts adds to our understanding of the evolution of leptin physiology in the early vertebrate lineage.