Exploring transcriptional and post-transcriptional epigenetic regulation of crf and 11ßhsd2 in rainbow trout brain during chronic social stress.

Using dominance hierarchies in juvenile rainbow trout (Oncorhynchus mykiss) as a model of chronic social stress in fish, we explored whether epigenetic transcriptional and post-transcriptional mechanisms are involved in the gene expression of corticotropin-releasing factor (crf) and 11ß-hydroxysteroid dehydrogenase (11ßhsd2), key factors involved in the regulation of the endocrine stress axis response. In juvenile rainbow trout pairs, subordinate individuals display sustained elevation of circulating cortisol concentrations. Cortisol production is controlled by the hypothalamic-pituitary-interrenal (HPI) axis in fish and initiated by CRF release from the preoptic area (POA). Given that crf is modulated during chronic social stress, and that such stress has been implicated in the epigenetic regulation of crf in other taxa, we probed a role for epigenetic regulation of crf transcript abundance in chronically stressed rainbow trout. We also investigated the regulation of the cortisol-metabolising enzyme 11ßhsd2 in the POA, which is upregulated in subordinates. The potential involvement of DNA methylation and microRNAs (miRNAs) in the regulation of crf transcript abundance was investigated during social stress in the POA of fish, as was the potential involvement of miRNAs in 11ßhsd2 regulation. Although transcript abundances of crf were elevated in subordinate fish after 4 days, DNA methylation profiles within putative promoter sequences upstream of the crf gene were not significantly affected by chronic stress. An inverse relationship between crf and its predicted posttranscriptional regulator miR-103a-3p in the POA suggests that miRNAs may be involved in mediating the effects of chronic social stress on key components of the endocrine stress axis.

Negative feedback regulation in the hypothalamic-pituitary-interrenal axis of rainbow trout subjected to chronic social stress.

The formation of dominance hierarchies in pairs of juvenile rainbow trout (Oncorhynchus mykiss) results in subordinate individuals exhibiting chronically elevated plasma cortisol concentrations. Cortisol levels reflect a balance between cortisol production, which is coordinated by the hypothalamic-pituitary-interrenal (HPI) axis in teleost fish, and negative feedback regulation and hormone clearance, which act to lower cortisol levels. However, the mechanisms contributing to the longer-term elevation of cortisol levels during chronic stress are not well established in fishes. The current study aimed to determine how subordinate fish maintain elevated cortisol levels, by testing the prediction that negative feedback and clearance mechanisms are impaired by chronic social stress. Plasma cortisol clearance was unchanged by social stress based on a cortisol challenge trial, hepatic abundance of the cortisol-inactivating enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11ßHSD2), and tissue fate of labelled cortisol. The capacity for negative feedback regulation in terms of transcript and protein abundances of corticosteroid receptors in the preoptic area (POA) and pituitary appeared stable. However, changes in 11ßHSD2 and mineralocorticoid receptor (MR) expression suggest subtle regulatory changes in the pituitary that may alter negative feedback. The chronic cortisol elevation observed during social subordination likely is driven by HPI axis activation and compounded by dysregulated negative feedback.

The relationship between hypoxia exposure and circulating cortisol levels in social and solitary African mole-rats: An initial report.

Hypoxemia from exposure to intermittent and/or acute environmental hypoxia (lower oxygen concentration) is a severe stressor for many animal species. The response to hypoxia of the hypothalamic-pituitary-adrenal axis (HPA-axis), which culminates in the release of glucocorticoids, has been well-studied in hypoxia-intolerant surface-dwelling mammals. Several group-living (social) subterranean species, including most African mole-rats, are hypoxia-tolerant, likely due to regular exposure to intermittent hypoxia in their underground burrows. Conversely, solitary mole-rat species, lack many adaptive mechanisms, making them less hypoxia-tolerant than the social genera. To date, the release of glucocorticoids in response to hypoxia has not been measured in hypoxia-tolerant mammalian species. Consequently, this study exposed three social African mole-rat species and two solitary mole-rat species to normoxia, or acute hypoxia and then measured their respective plasma glucocorticoid (cortisol) concentrations. Social mole-rats had lower plasma cortisol concentrations under normoxia than the solitary genera. Furthermore, individuals of all three of the social mole-rat species exhibited significantly increased plasma cortisol concentrations after hypoxia, similar to those of hypoxia-intolerant surface-dwelling species. By contrast, individuals of the two solitary species had a reduced plasma cortisol response to acute hypoxia, possibly due to increased plasma cortisol under normoxia. If placed in perspective with other closely related surface-dwelling species, the regular exposure of the social African mole-rats to hypoxia may have reduced the basal levels of the components for the adaptive mechanisms associated with hypoxia exposure, including circulating cortisol levels. Similarly, the influence of body mass on plasma cortisol levels cannot be ignored. This study demonstrates that both hypoxia-tolerant rodents and hypoxia-intolerant terrestrial laboratory-bred rodents may possess similar HPA-axis responses from exposure to hypoxia. Further research is required to confirm the results from this pilot study and to further confirm how the cortisol concentrations may influence responses to hypoxia in African mole-rats.

Social status-dependent regulation and function of the somatotropic axis in juvenile rainbow trout.

Juvenile rainbow trout (Oncorhynchus mykiss) develop social hierarchies when competing for resources in a constrained environment. Among the physiological consequences of social status are changes in organismal energy metabolism, which generally favour anabolic pathways in dominant fish and catabolic pathways in subordinate fish. The somatotropic axis is an important regulator of metabolism and growth that could be involved in mediating metabolic changes in response to social status in juvenile rainbow trout. Here we used juvenile trout housed either in dyads or individually (sham controls) to determine whether social status changes indices of somatotropic axis function. Although pituitary growth hormone expression (gh1 and gh2) did not differ among groups, circulating growth hormone (GH) increased ∼12-fold in subordinate fish compared to sham and dominant fish. Social status caused consistent differential expression of GH receptor paralogues in liver and muscle, two principal target tissues of GH. Compared to dominant and/or sham fish, ghra paralogue expression (ghra1 and ghra2) was lower, while ghrb1 expression was higher in subordinate fish. Across tissues, ghra paralogue expression was generally positively correlated with expression of insulin growth factors (igf1, igf2), while ghrb1 expression was positively correlated with transcript abundance of hormone sensitive lipase (hsl1). Because igf and hsl expression are subject to context-dependent GH control in rainbow trout, these results suggest that increased circulating GH in conjunction with differential expression of ghr paralogues may translate into prioritization of downstream catabolic lipolytic pathways in subordinate rainbow trout. These findings support a social context-dependent role for GH signalling in mediating metabolic changes in juvenile rainbow trout.

Regulation of cortisol production during chronic social stress in rainbow trout.

Chronic stress resulting from social interactions impacts the endocrine stress response in many vertebrates, including teleost fishes. Juvenile rainbow trout held in pairs form a dominance hierarchy with the subordinate individual exhibiting chronic elevation of plasma cortisol and an attenuated cortisol response to an additional acute stressor. The current study investigated the mechanisms underlying this apparent dichotomy in cortisol production at the level of the head kidney (adrenal homolog). Following four days of chronic social stress, subordinate rainbow trout exhibited elevated plasma cortisol levels that correlated with basal cortisol production by the head kidney in vitro. Subordinate trout had higher transcript abundances of steroidogenic acute regulatory protein and cytochrome p450 side chain cleavage enzyme, which facilitate key steps in steroidogenesis, as well as two paralogs of steroidogenic factor 1. Despite elevation of basal steroidogenesis, acute cortisol production in response to ACTH (in vivo and in vitro) was lower in subordinate trout. Transcript abundances of the ACTH receptor accessory proteins were elevated in subordinate fish, but head kidney cortisol production in response to a cAMP analogue was lower than in dominant fish. Together, the data suggest that the attenuated acute cortisol response of subordinate trout reflects limitations on cortisol production downstream of cAMP signalling in steroidogenic cells of the head kidney, despite the increased basal abundance of key components of the steroidogenic pathway.

Chronic social stress alters protein metabolism in juvenile rainbow trout, Oncorhynchus mykiss.

When confined in pairs, juvenile rainbow trout (Oncorhynchus mykiss) form dominance hierarchies in which subordinate fish exhibit characteristic physiological changes including reduced growth rates and chronically elevated plasma cortisol concentrations. We hypothesized that alterations in protein metabolism contribute to the reduced growth rate of socially stressed trout, and predicted that subordinate trout would exhibit reduced rates of protein synthesis coupled with increases in protein degradation. Protein metabolism was assessed in dominant and subordinate fish after 4 days of social interaction, and in fish that were separated after 4 days of interaction for a 4 days recovery period, to determine whether effects on protein metabolism recovered when social stress was alleviated. Protein metabolism was assessed in liver and white muscle by measuring the fractional rate of protein synthesis and markers of protein degradation. In the white muscle of subordinate fish, protein synthesis was inhibited and activities of the ubiquitin-proteasome pathway (UPP) and the autophagy lysosomal system (ALS) were elevated. By contrast, the liver of subordinate fish exhibited increased rates of protein synthesis and activation of the ALS. When allowed to recover from chronic social stress for 4 days, differences in protein metabolism observed in white muscle of subordinate fish during the interaction period disappeared. In liver, protein synthesis returned to baseline levels during recovery from social stress, but markers of protein degradation did not. Collectively, these data support the hypothesis that inhibition of muscle protein synthesis coupled with increases in muscle protein breakdown contribute to the reduced growth rates of subordinate rainbow trout.

Loss of hypoxia-inducible factor 1α affects hypoxia tolerance in larval and adult zebrafish (Danio rerio).

The coordination of the hypoxic response is attributed, in part, to hypoxia-inducible factor 1α (Hif-1α), a regulator of hypoxia-induced transcription. After the teleost-specific genome duplication, most teleost fishes lost the duplicate copy of Hif-1α, except species in the cyprinid lineage that retained both paralogues of Hif-1α (Hif1aa and Hif1ab). Little is known about the contribution of Hif-1α, and specifically of each paralogue, to hypoxia tolerance. Here, we examined hypoxia tolerance in wild-type (Hif1aa+/+ab+/+) and Hif-1α knockout lines (Hif1aa-/-; Hif1ab-/-; Hif1aa-/-ab-/-) of zebrafish (Danio rerio). Critical O2 tension (Pcrit; the partial pressure of oxygen (PO2) at which O2 consumption can no longer be maintained) and time to loss of equilibrium (LOE), two indices of hypoxia tolerance, were assessed in larvae and adults. Knockout of both paralogues significantly increased Pcrit (decreased hypoxia tolerance) in larval fish. Prior exposure of larvae to hypoxia decreased Pcrit in wild-type fish, an effect mediated by the Hif1aa paralogue. In adults, individuals with a knockout of either paralogue exhibited significantly decreased time to LOE but no difference in Pcrit. Together, these results demonstrate that in zebrafish, tolerance to hypoxia and improved hypoxia tolerance after pre-exposure to hypoxia (pre-conditioning) are mediated, at least in part, by Hif-1α.

Cortisol modulates metabolism and energy mobilization in wild-caught pumpkinseed (Lepomis gibbosus).

Acute elevation of cortisol via activation of the hypothalamic-pituitary-interrenal (HPI) axis aids the fish in dealing with a stressor. However, chronic elevation of cortisol has detrimental effects and has been studied extensively in lab settings. However, data pertaining to wild teleosts are lacking. Here, we characterized the metabolic consequences of prolonged cortisol elevation (96 h) in wild-caught pumpkinseed (Lepomis gibbosus). Pumpkinseed were implanted with cocoa butter alone (sham) or containing cortisol (25 mg kg-1 body weight), and at 24, 48, 72, and 96 h, tissue samples were collected, whole-body ammonia excretion was determined, and whole-organism metabolism was assessed using intermittent flow respirometry. Cortisol-treated pumpkinseed exhibited the highest plasma cortisol concentration at 24 h post-implantation, with levels decreasing over the subsequent time points although remaining higher than in sham-treated fish. Cortisol-treated fish exhibited higher standard and maximal metabolic rates than sham-treated fish, but the effect of cortisol treatment on aerobic scope was negligible. Indices of energy synthesis/mobilization, including blood glucose concentrations, hepatosomatic index, hepatic glycogen concentrations, and ammonia excretion rates, were higher in cortisol-treated fish compared with controls. Our work suggests that although aerobic scope was not diminished by prolonged elevation of cortisol levels, higher metabolic expenditures may be of detriment to the animal's performance in the longer term.

Epigenetics in teleost fish: From molecular mechanisms to physiological phenotypes.

While the field of epigenetics is increasingly recognized to contribute to the emergence of phenotypes in mammalian research models across different developmental and generational timescales, the comparative biology of epigenetics in the large and physiologically diverse vertebrate infraclass of teleost fish remains comparatively understudied. The cypriniform zebrafish and the salmoniform rainbow trout and Atlantic salmon represent two especially important teleost orders, because they offer the unique possibility to comparatively investigate the role of epigenetic regulation in 3R and 4R duplicated genomes. In addition to their sequenced genomes, these teleost species are well-characterized model species for development and physiology, and therefore allow for an investigation of the role of epigenetic modifications in the emergence of physiological phenotypes during an organism's lifespan and in subsequent generations. This review aims firstly to describe the evolution of the repertoire of genes involved in key molecular epigenetic pathways including histone modifications, DNA methylation and microRNAs in zebrafish, rainbow trout, and Atlantic salmon, and secondly, to discuss recent advances in research highlighting a role for molecular epigenetics in shaping physiological phenotypes in these and other teleost models. Finally, by discussing themes and current limitations of the emerging field of teleost epigenetics from both theoretical and technical points of view, we will highlight future research needs and discuss how epigenetics will not only help address basic research questions in comparative teleost physiology, but also inform translational research including aquaculture, aquatic toxicology, and human disease.

Cortisol elevation post-hatch affects behavioural performance in zebrafish larvae.

Maternal cortisol is essential for cortisol stress axis development and de novo production of this steroid commences only after hatch in zebrafish (Danio rerio). However, very little is known about the effect of elevated cortisol levels, during the critical period of stress axis activation, on larval performance. We tested the hypothesis that elevated cortisol levels post-hatch affect behavioural performance and this is mediated by glucocorticoid receptor (GR) activation in zebrafish larvae. The behavioural response included measuring larval activity in response to alternating light and dark cycles, as well as thigmotaxis. Zebrafish larvae at 3days post-fertilization were exposed to waterborne cortisol for 24h to mimic a steroid response to an early-life stressor exposure. Also, larvae were exposed to waterborne RU-486 (a GR antagonist) either in the presence or absence of cortisol to confirm GR activation. Co-treatment with RU-486 completely abolished the upregulation of cortisol-induced 11ß-hydroxysteroid dehydrogenase type 2 transcript abundance, confirming GR signalling. Cortisol-exposed larvae displayed increased locomotor activity irrespective of light condition, but showed no changes in thigmotaxis. This cortisol-mediated behavioural response was not affected by co-treatment with RU-486. Cortisol exposure also did not modify the transcript abundances of GR and mineralocorticoid receptor (MR) in zebrafish larvae. Altogether, cortisol stress axis activation post-hatch increases locomotor activity in zebrafish larvae. Our results suggest that GR signalling may not be involved in this behavioural response, leading to the proposal that cortisol action via MR signalling may influence locomotor activity in zebrafish larvae.

Maternal cortisol stimulates neurogenesis and affects larval behaviour in zebrafish.

Excess glucocorticoid transferred from stressed mother to the embryo affects developing vertebrate offspring, but the underlying programming events are unclear. In this study, we tested the hypothesis that increased zygotic glucocorticoid deposition, mimicking a maternal stress scenario, modifies early brain development and larval behaviour in zebrafish (Danio rerio). Cortisol was microinjected into the yolk at one cell-stage, to mimic maternal transfer, and the larvae [96 hours post-fertilization (hpf)] displayed increased activity in light and a reduction in thigmotaxis, a behavioural model for anxiety, suggesting an increased propensity for boldness. This cortisol-mediated behavioural phenotype corresponded with an increase in primary neurogenesis, as measured by incorporation of EdU at 24 hpf, in a region-specific manner in the preoptic region and the pallium, the teleostean homolog of the hippocampus. Also, cortisol increased the expression of the proneural gene neurod4, a marker of neurogenesis, in a region- and development-specific manner in the embryos. Altogether, excess zygotic cortisol, mimicking maternal stress, affects early brain development and behavioural phenotype in larval zebrafish. We propose a key role for cortisol in altering brain development leading to enhanced boldness, which may be beneficial in preparing the offspring to a stressful environment and enhancing fitness.

Maternal stress-associated cortisol stimulation may protect embryos from cortisol excess in zebrafish.

Abnormal embryo cortisol level causes developmental defects and poor survival in zebrafish (Danio rerio). However, no study has demonstrated that maternal stress leads to higher embryo cortisol content in zebrafish. We tested the hypothesis that maternal stress-associated elevation in cortisol levels increases embryo cortisol content in this asynchronous breeder. Zebrafish mothers were fed cortisol-spiked food for 5 days, to mimic maternal stress, followed by daily breeding for 10 days to monitor temporal embryo cortisol content. Cortisol treatment increased mean embryo yield, but the daily fecundity was variable among the groups. Embryo cortisol content was variable in both groups over a 10-day period. A transient elevation in cortisol levels was observed in the embryos from cortisol-fed mothers only on day 3, but not on subsequent days. We tested whether excess cortisol stimulates 11ßHSD2 expression in ovarian follicles as a means to regulate embryo cortisol deposition. Cortisol treatment in vitro increased 11ß HSD2 levels sevenfold, and this expression was regulated by actinomycin D and cycloheximide suggesting tight regulation of cortisol levels in the ovarian follicles. We hypothesize that cortisol-induced upregulation of 11ßHSD2 activity in the ovarian follicles is a mechanism restricting excess cortisol incorporation into the eggs during maternal stress.

The antidepressant venlafaxine disrupts brain monoamine levels and neuroendocrine responses to stress in rainbow trout.

Venlafaxine, a serotonin-norepinephrine reuptake inhibitor, is a widely prescribed antidepressant drug routinely detected in the aquatic environment. However, little is known about its impact on the physiology of nontarget organisms. We tested the hypothesis that venlafaxine perturbs brain monoamine levels and disrupts molecular responses essential for stress coping and feeding activity in fish. Rainbow trout (Oncorhynchus mykiss) were exposed to waterborne venlafaxine (0.2 and 1.0 µg/L) for 7 days. This treatment elevated norepinephrine, serotonin, and dopamine levels in the brain in a region-specific manner. Venlafaxine also increased the transcript levels of genes involved in stress and appetite regulation, including corticotropin releasing factor, pro-opiomelanocortin B, and glucose transporter type 2 in distinct brain regions of trout. The drug treatment reduced the total feed consumed per day, but did not affect the feeding behavior of the dominant and subordinate fish. However, the subordinate fish from the venlafaxine-exposed group had significantly higher plasma cortisol levels compared to the subordinate fish in the control group. Collectively, our results demonstrate that venlafaxine, at environmentally realistic levels, is a neuroendocrine disruptor, impacting the stress and feeding responses in rainbow trout. We propose the midbrain region as a key target for venlafaxine impact and the mode of action involves abnormal monoamine content in trout.

Environmental levels of the antidepressant venlafaxine impact the metabolic capacity of rainbow trout.

The antidepressant venlafaxine is detected at parts per billion levels in tertiary-treated municipal wastewater effluent. However, the impact of this serotonin-norepinephrine reuptake inhibitor (SNRI) on non-target aquatic animals is poorly understood. We tested the hypothesis that environmentally relevant levels of venlafaxine disrupt the highly conserved cortisol and glucose response to stress in rainbow trout (Oncorhynchus mykiss). Juvenile trout were exposed to venlafaxine (0, 0.2 and 1.0 µg/L) in a static system with daily renewal for seven days. The fish were then subjected to an acute handling disturbance and sampled either prior to (0 h) or 1, 4 and 24h after stressor exposure. Venlafaxine exposure did not affect the handling disturbance-mediated transient elevation in plasma cortisol levels or target tissue glucocorticoid receptor expression. The drug exposure disrupted the interrenal steroidogenic capacity, including altered handling stressor-mediated changes in mRNA abundances of steroidogenic acute regulatory protein and cytochrome P450 side chain cleavage. The handling stressor-induced transient elevations in plasma glucose levels were significantly reduced in the venlafaxine-exposed fish. This was not accompanied by changes in liver glycogen content, glucose transporter 2 mRNA abundance or the glycolytic capacity, whereas the capacity for gluconeogenesis and amino acid catabolism were enhanced. Venlafaxine also brought about changes in the gill of trout, including enhanced lactate dehydrogenase activity and Na(+)-K(+) ATPase protein expression, while the Na(+)-K(+) ATPase enzyme activity was reduced. Collectively, our results demonstrate that venlafaxine at levels detected in the aquatic environment impacts tissue metabolic capacities and may compromise the adaptive responses to an acute stressor in rainbow trout.