Suzanne Currie

Influence of bioenergetic stress on heat shock protein gene expression in nucleated red blood cells of fish

The physiological and biochemical signals that induce stress protein (HSP) synthesis remain conjectural. In this study, we used the nucleated red blood cells from rainbow trout, Oncorhynchus mykiss, to address the interaction between energy status and HSP gene expression. Heat shock (25°C) did not significantly affect ATP levels but resulted in an increase in HSP70 mRNA. Hypoxia alone did not induce HSPtranscription in these cells despite a significant depression in ATP. Inhibition of oxidative phosphorylation with azide, in the absence of thermal stress, decreased ATP by 56% and increased lactate production by 62% but did not induce HSP gene transcription. Inhibition of oxidative phosphorylation and glycolysis with azide and iodoacetic acid respectively, decreased ATP by 79% and prevented lactate production, but did not induce either HSP70 or HSP30 gene transcription in these cells. This study demonstrates that a reduction in the cellular energy status will not induce stress protein gene transcription in rainbow trout red blood cells and may, in fact, limit induction during extreme metabolic inhibition.

Transcriptome responses to heat stress in the nucleated red blood cells of the rainbow trout (Oncorhynchus mykiss)

The retention of a nucleus in the mature state of fish red blood cells (RBCs) and the ability to easily collect and manipulate blood in nonterminal experiments make blood an ideal tissue on which to study the cellular stress response in fish. Through the use of the cGRASP 16K salmonid microarray, we investigated differences in RBC global gene transcription in fish held under control conditions (11 degrees C) and exposed to heat stress (1 h at 25 degrees C followed by recovery at 11 degrees C). Repeated blood sampling (via a dorsal aorta cannula) enables us to examine the individual stress response over time. Samples were taken preheat stress (representing individual control) and at 4 and 24 h postheat stress (representing early and late transcriptional regulation). Approximately 3,000 microarray features had signal above threshold when hybridized with RBC RNA-derived targets, and cannulation did not have a detectable effect on RBC mRNA expression at the investigated time points. Genes involved in the stress response, immune response, and apoptosis were among those showing the highest dysregulation during both early and late transcriptional regulation. Additionally, genes related to the differentiation and development of blood cells were transcriptionally upregulated at the 24 h time point. This study provides a broader understanding of the mechanisms underpinning the stress response in fish and the discovery of novel genes that are regulated in a stress specific manner. Moreover, salmonid transcripts that are consistently dysregulated in blood in response to heat stress are potential candidates of nonlethal biomarkers of exposure to this particular stressor.

Modulation of steroidogenesis and estrogen signalling in the estuarine killifish (Fundulus heteroclitus) exposed to ethinylestradiol.

Previous studies have shown that mummichog (Fundulus heteroclitus; a lunar, asynchronous-spawning killifish of the western Atlantic) exposed to 17alpha-ethynylestradiol (EE2) exhibit decreased plasma reproductive steroid levels, decreased gonadal steroid production, increased plasma vitellogenin, decreased fecundity and impaired fertilization. The objective of this study was to determine the potential mechanisms by which EE2 depresses gonadal steroidogenesis and influences estrogen signalling in the mummichog. Adult recrudesced fish were exposed to the potent synthetic estrogen, ethinylestradiol (EE2; 0-270ng/L) for 14 days. Following exposure, gonadal tissue was removed and incubated for 24h with stimulators of steroidogenesis, including forskolin; 25-OH cholesterol; or pregnenolone. Testosterone production was decreased in basal, forskolin-stimulated and pregnenolone-stimulated EE2-exposed males, indicating effects on the steroidogenic pathway both at and downstream of cholesterol mobilization to P450 side-chain cleavage (P450scc) and/or P450scc conversion of cholesterol to pregnenolone. Hepatic transcript levels of estrogen receptor alpha (ERalpha) and vitellogenin were increased in EE2-treated males compared to control recrudescing males and females confirming an estrogenic response. Hepatic heat shock protein 90 (Hsp90), a chaperoning molecule involved in estrogen signalling, was not affected by EE2 exposure at either the transcript or protein level. However, higher levels of Hsp90 observed in the membrane fractions of female fish raise interesting questions regarding the influence of gender on Hsp90s role in estrogen signalling. These results demonstrate that EE2 can alter steroid production at specific sites within the steroidogenic pathway and can stimulate hepatic estrogen signalling, providing important information regarding the molecular mechanisms underlying the endocrine response of the mummichog to exogenous estrogen.

Chronic social stress impairs thermal tolerance in the rainbow trout (Oncorhynchus mykiss).

SUMMARY When faced with limited resources, juvenile salmonid fish form dominance hierarchies that result in social stress for socially subordinate individuals. Social stress, in turn, can have consequences for the ability of the fish to respond to additional stressors such as pathogens or exposure to pollutants. In the present study, the possibility that social stress affects the ability of rainbow trout (Oncorhynchus mykiss) to tolerate acute increases in water temperature was investigated. To this end, we first evaluated physiological and cellular stress responses following a 1 h heat shock in juvenile fish in dominance hierarchies. We measured stress hormone (cortisol and catecholamines) concentrations and blood, brain and liver tissue levels of three heat shock proteins (HSPs), the stress inducible HSP70, the constitutive HSC70 and HSP90, in dominant and subordinate trout. No effects of social status on the hormonal response to the heat stress were detected, but the cellular heat shock response in the brain and liver of dominant and subordinate individuals was inhibited. We then assessed thermal tolerance in dominant and subordinate fish through critical thermal maximum temperature (CTmax) trials and measured HSPs following the heat shock. Subordinate fish were less thermally tolerant than their dominant counterparts. We conclude that social stress impacts the ability of fish to respond, on a cellular scale and in a tissue-specific manner, to increases in water temperature, with likely consequences for overall fitness.

Hormonal modulation of the heat shock response: insights from fish with divergent cortisol stress responses

Acute temperature stress in animals results in increases in heat shock proteins (HSPs) and stress hormones. There is evidence that stress hormones influence the magnitude of the heat shock response; however, their role is equivocal. To determine whether and how stress hormones may affect the heat shock response, we capitalized on two lines of rainbow trout specifically bred for their high (HR) and low (LR) cortisol response to stress. We predicted that LR fish, with a low cortisol but high catecholamine response to stress, would induce higher levels of HSPs after acute heat stress than HR trout. We found that HR fish have significantly higher increases in both catecholamines and cortisol compared with LR fish, and LR fish had no appreciable stress hormone response to heat shock. This unexpected finding prevented further interpretation of the hormonal modulation of the heat shock response but provided insight into stress-coping styles and environmental stress. HR fish also had a significantly greater and faster heat shock response and less oxidative protein damage than LR fish. Despite these clear differences in the physiological and cellular responses to heat shock, there were no differences in the thermal tolerance of HR and LR fish. Our results support the hypothesis that responsiveness to environmental change underpins the physiological differences in stress-coping styles. Here, we demonstrate that the heat shock response is a distinguishing feature of the HR and LR lines and suggest that it may have been coselected with the hormonal responses to stress.

Development-dependent differences in intracellular localization of stress proteins (hsps) in rainbow trout, Oncorhynchus mykiss, following heat shock.

Using antibodies specific for salmonid fish, we have determined the intracellular localization of hsp70, hsc70 and hsp90 before and after an acute heat shock in juvenile and mature rainbow trout. We found that both hsp70 and hsp90 were primarily located outside the nucleus in both the liver and the heart of juvenile and mature fish and heat shock resulted in an increase in these proteins in all cellular fractions examined. In mature fish, liver hsp70 was predominantly found in the membranes and organelles after heat shock, while in juvenile fish, hsp70 was mostly cytoplasmic. Hsc70 was found in all cellular compartments examined both before and after heat shock in the livers and hearts of juvenile and mature fish. Heat shock resulted in a significant induction of hsp90 in the liver tissue of both juvenile and mature fish; however, in juvenile fish, this increase was seen in the membranes and organelles whereas in mature fish, hsp90 increased in the nucleus and cytoplasm. Hsp90 was only induced in the hearts of mature fish with heat shock, where it increased in both the nucleus and the cytoplasm. These results indicate that the cells of juvenile and mature fish respond differently to acute temperature stress. While the nucleus appears to be an important target for hsp protection following heat shock, the presence of hsps in all subcellular fractions examined suggests multifunctional roles for these proteins in the cellular response to temperature stress in fish.

Intraspecific Variation in Thermal Tolerance and Acclimation Capacity in Brook Trout (Salvelinus fontinalis): Physiological Implications for Climate Change*

Cold-water fishes are becoming increasingly vulnerable as changing thermal conditions threaten their future sustainability. Thermal stress and habitat loss from increasing water temperatures are expected to impact population viability, particularly for inland populations with limited adaptive resources. Although the long-term persistence of cold-adapted species will depend on their ability to cope with and adapt to changing thermal conditions, very little is known about the scope and variation of thermal tolerance within and among conspecific populations and evolutionary lineages. We studied the upper thermal tolerance and capacity for acclimation in three captive populations of brook trout (Salvelinus fontinalis) from different ancestral thermal environments. Populations differed in their upper thermal tolerance and capacity for acclimation, consistent with their ancestry: the northernmost strain (Lake Nipigon) had the lowest thermal tolerance, while the strain with the most southern ancestry (Hill’s Lake) had the highest thermal tolerance. Standard metabolic rate increased following acclimation to warm temperatures, but the response to acclimation varied among strains, suggesting that climatic warming may have differential effects across populations. Swimming performance varied among strains and among acclimation temperatures, but strains responded in a similar way to temperature acclimation. To explore potential physiological mechanisms underlying intraspecific differences in thermal tolerance, we quantified inducible and constitutive heat shock proteins (HSP70 and HSC70, respectively). HSPs were associated with variation in thermal tolerance among strains and acclimation temperatures; HSP70 in cardiac and white muscle tissues exhibited similar patterns, whereas expression in hepatic tissue varied among acclimation temperatures but not strains. Taken together, these results suggest that populations of brook trout will vary in their ability to cope with a changing climate.

β‐Adrenergic Stimulation Enhances the Heat‐Shock Response in Fish

We have taken advantage of the unique properties of nucleated rainbow trout (Oncorhynchus mykiss) red blood cells (rbcs) to demonstrate that β‐adrenergic stimulation with the agonist, isoproterenol, significantly enhanced the heat‐induced induction of heat‐shock proteins (Hsps) in trout rbcs without affecting hsp expression on its own. Furthermore, this β‐adrenergic potentiation of hsp expression occurred only at physiologically relevant concentrations of adrenergic stimulation. In further experiments, we found that adrenaline increased 100‐fold and noradrenaline increased 50‐fold in trout after a 1‐h heat shock at 25°C, ∼12°C above acclimation temperature. This is the first time the adrenergic heat‐shock response has been described for a temperate fish species. We conclude that β‐adrenergic stimulation enhances hsp expression in trout rbcs following heat stress, indicating physiological regulation of the cellular stress response in fish.

High survivorship after catch-and-release fishing suggests physiological resilience in the endothermic shortfin mako shark (Isurus oxyrinchus)

We used satellite tags and blood based analyses to examine the impacts of catch and release angling on the shortfin mako shark. We report high survivorship seemingly unaffected by fight time or physiological stress and recommend that circle hooks should be used to reduce physical damage to these sharks.

The curious case of the chemical composition of hagfish tissues—50 years on☆

Modern hagfishes are considered to be the most primitive of the living craniates and along with their close jawless agnathan relative, the lamprey, take us back an astonishing 500 million years to the base of the vertebrate evolutionary tree. The unique osmoconforming strategy of the hagfish, whereby the osmotic constituents of the blood plasma bear more of a resemblance to marine invertebrates than vertebrates, has been classically depicted in comparative physiology textbooks for many years. Fifty years ago in this journal, Bellamy and Chester Jones [Bellamy and Chester Jones, 1961. CBP 3, 173-183] published a paper on the chemical composition of the tissues of the Atlantic hagfish, Myxine glutinosa. This publication was one of a flurry of papers published in the 50s, 60s and early 70s focused on describing the ionic and osmotic components of this bizarre fish. Here we take a retrospective look at the research that has taken place on these intriguing animals prior to and following the Bellamy and Chester Jones manuscript, focusing on tissue chemical compositions, the possible role of amino acids, and our current view on ion regulation, metabolism and hypoxia tolerance.