The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons

Heat shock (25° C) of 10° C-acclimated rainbow trout Oncorhynchus mykiss led to increases in heat shock protein 70 (hsp70) mRNA in blood, brain, heart, liver, red and white muscle, with levels in blood being amongst the highest. Hsp30 mRNA also increased with heat shock in all tissues with the exception of blood. When rainbow trout blood was heat shocked in vitro , both hsp70 and hsp30 mRNA increased significantly. In addition, these in vitro experiments demonstrated that blood from fish acclimated to 17° C water had a lower hsp70 mRNA heat shock induction temperature than did 5° C acclimated fish (20 v. 25° C). The hsp30 mRNA induction temperature (25° C), however, was unaffected by thermal acclimation. While increases in hsp70 mRNA levels in blood may serve as an early indicator of temperature stress in fish, tissue type, thermal history and the particular family of hsp must be considered when evaluating stress by these molecular means.     

A physiological approach to quantifying thermal habitat quality for Redband Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss gairdneri)</Emphasis> in the south Fork John Day River,Oregon

We examined tissue-specific levels of heat shock protein 70 (hsp70) and whole body lipid levels in juvenile redband trout (Oncorhynchus mykiss gairdneri) from the South Fork of the John Day River (SFJD), Oregon, with the goal of determining if these measures could be used as physiological indicators of thermal habitat quality for juvenile redband trout. Our objectives were to determine the hsp70 induction temperature in liver, fin, and white muscle tissue and characterize the relation between whole body lipids and hsp70 for fish in the SFJD. We found significant increases in hsp70 levels between 19 and 22°C in fin, liver, and white muscle tissue. Maximum hsp70 levels in liver, fin, and white muscle tissue occurred when mean weekly maximum temperatures (MWMT) exceeded 20–22°C. In general, the estimated hsp70 induction temperature for fin and white muscle tissue was higher than liver tissue. Whole body lipid levels began to decrease when MWMT exceeded 20.4°C. There was a significant interaction between temperature and hsp70 in fin and white muscle tissue, but not liver tissue. Collectively, these results suggest that increased hsp70 levels in juvenile redband trout are symptomatic of thermal stress, and that energy storage capacity decreases with this stress. The possible decrease in growth potential and fitness for thermally stressed individuals emphasizes the physiological justification for thermal management criteria in salmon-bearing streams.     

Physiological causes and consequences of social status in salmonid fish

Social interactions in small groups of juvenile rainbow trout(Oncorhynchus mykiss) lead to the formation of dominance hierarchies.Dominant fish hold better positions in the environment, gaina larger share of the available food and exhibit aggressiontowards fish lower in the hierarchy. By contrast, subordinatefish exhibit behavioural inhibition, including reduced activityand feeding. The behavioural characteristics associated withsocial status are likely the result of changes in brain monoaminesresulting from social interactions. Whereas substantial physiologicalbenefits, including higher growth rates and condition factor,are experienced by dominant trout, low social status appearsto be a chronic stress, as indicated by sustained elevationof circulating cortisol concentrations in subordinate fish.High cortisol levels, in turn, may be responsible for many ofthe deleterious physiological consequences of low social status,including lower growth rates and condition factor, immunosuppressionand increased mortality. Circulating cortisol levels may alsobe a factor in determining the outcome of social interactionsin pairs of rainbow trout, and hence in determining social status.Rainbow trout treated with cortisol were significantly morelikely to become subordinate in paired encounters with smalleruntreated conspecifics.     

Heat shock protein (Hsp70) induced by a mild heat shock slightly moderates plasma osmolarity increases upon salinity transfer in rainbow trout (Oncorhynchus mykiss)

We have investigated whether mild heat shock, and resulting Hsp70 expression, can confer cross-protection against the stress associated with transfer from freshwater (FW) to seawater (SW) in juvenile rainbow trout (Oncorhynchus mykiss). In experimental Series I, juvenile trout reared in FW were transferred from 13.5 degrees C to 25.5 degrees C in FW, held for 2 h, returned to 13.5 degrees C for 12 h, and then transferred to 32 ppt SW at 13.5 degrees C. Branchial Hsp70 increased approximately 10-fold in the heat-shocked fish relative to the control by the end of recovery and remained high 2, 8, and 24 h post-salinity transfer. However, no clear differences could be detected in blood parameters (blood hemoglobin, hematocrit, MCHC, plasma Na(+) and plasma osmolarity) or muscle water content between heat-shocked and sham-shocked fish in SW at any sampling interval (0, 2, 8, 24, 48, 120, 240 and 360 h post-SW transfer). In experimental Series II, trout acclimated to 8 degrees C were heat-shocked at 22 degrees C for 2 h, allowed to recover 18 h, and exposed to a more severe salinity transfer (either 36 or 45 ppt) than in Series I. Branchial Hsp70 levels increased approximately 6-fold in heat-shocked fish, but had declined to baseline after 120 h in SW. Plasma osmolarity and chloride increased in both groups upon transfer to 36 ppt; however, the increase was significantly less in heat-shocked fish when compared to the increase observed in sham-shocked fish at 24 h. No significant differences could be detected in branchial Na(+)/K(+)-ATPase activity or Na(+)/K(+)-ATPase alpha1a and alpha1b mRNA expression between the two groups. Our data indicate that a mild temperature shock has only modest effects on the ability of rainbow trout to resist osmotic stress during FW to SW transfer.     

Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress

Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 degrees C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.     

Beta-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 beta-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 beta-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 degrees C, approximately 12 degrees 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 beta-adrenergic stimulation enhances hsp expression in trout rbcs following heat stress, indicating physiological regulation of the cellular stress response in fish.     

Plasma cortisol concentrations before and after social stress in rainbow trout and brown trout

Two related experiments examined the relationship between plasma cortisol concentrations and the development of social hierarchies in fish. In the first, rainbow trout, Oncorhynchus mykiss, and brown trout, Salmo trutta, were observed for dominance interactions when confined within single-species pairs for 4, 48, or 168 h. Subordinate members of a pair exhibited significantly higher cortisol concentrations than dominant and single fish, but the pattern of cortisol elevation differed between the two species, being quicker to rise and increasing to a higher level in rainbow trout. Cortisol concentrations were correlated with behavioural measurements; the more subordinate the behaviour exhibited by a fish, the higher its cortisol concentration. Social stress was a chronic stressor, and no acclimation to social status occurred during the week. In the second experiment, measurements of plasma cortisol were made before pairing of rainbow trout and then after 48 h of confinement in pairs. Subordinate fish demonstrated significantly higher concentrations of plasma cortisol both before and after social stress. It therefore appears that in addition to cortisol being elevated during periods of social stress, an association may exist between initial cortisol levels and the likelihood of a fish becoming subordinate.     

Linking physiological and cellular responses to thermal stress: β-adrenergic blockade reduces the heat shock response in fish

When faced with stress, animals use physiological and cellular strategies to preserve homeostasis. We were interested in how these high-level stress responses are integrated at the level of the whole animal. Here, we investigated the capacity of the physiological stress response, and specifically the β-adrenergic response, to affect the induction of the cellular heat shock proteins, HSPs, following a thermal stress in vivo. We predicted that blocking β-adrenergic stimulation during an acute heat stress in the whole animal would result in reduced levels of HSPs in red blood cells (RBCs) of rainbow trout compared to animals where adrenergic signaling remained intact. We first determined that a 1 h heat shock at 25 °C in trout acclimated to 13 °C resulted in RBC adrenergic stimulation as determined by a significant increase in cell swelling, a hallmark of the β-adrenergic response. A whole animal injection with the β₂-adrenergic antagonist, ICI-118,551, successfully reduced this heat-induced RBC swelling. The acute heat shock caused a significant induction of HSP70 in RBCs of 13 °C-acclimated trout as well as a significant increase in plasma catecholamines. When heat-shocked fish were treated with ICI-118,551, we observed a significant attenuation of the HSP70 response. We conclude that circulating catecholamines influence the cellular heat shock response in rainbow trout RBCs, demonstrating physiological/hormonal control of the cellular stress response.     

Heat shock protein 70 purification and characterization from Cyprinion macrastomus macrastomus and Garra rufa obtusa

Organisms resist stress factors by common mechanisms at cellular and physiological levels. Heat shock proteins (Hsps) play an essential role as a protective mechanism against stressors, and Hsp70 has a central role in the heat shock response. Although several investigations have been made on the cellular functions of Hsps, very little is known about extreme temperature effects on organisms like fish. To address this point, we have studied two fish species, Cyprinion macrostomus macrostomus and Garra rufa obtuse, whose habitat is the Kangal hot spring in Turkey. The hot spring has multiple stress factors; extremely hot temperature, food deprivation, infectious medium and low levels of oxygen. Like other Hsp70s, two Cyprinidae species Hsp70 showed similar ATPase and substrate protein folding activities in vitro. These proteins also showed relatively higher thermal stability. Biochemical characterization of two Hsp70, suggested a Hsp mechanism capable of protecting the cell against stressors even under adverse conditions and in the presence of multiple stress factors.     

The Effects of Acute Waterborne Exposure to Sublethal Concentrations of Molybdenum on the Stress Response in Rainbow Trout,Oncorhynchus mykiss

To determine if molybdenum (Mo) is a chemical stressor, fingerling and juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne sodium molybdate (0, 2, 20, or 1,000 mg l^-1 of Mo) and components of the physiological (plasma cortisol, blood glucose, and hematocrit) and cellular (heat shock protein [hsp] 72, hsp73, and hsp90 in the liver, gills, heart, and erythrocytes and metallothionein [MT] in the liver and gills) stress responses were measured prior to initiation of exposure and at 8, 24, and 96 h. During the acute exposure, plasma cortisol, blood glucose, and hematocrit levels remained unchanged in all treatments. Heat shock protein 72 was not induced as a result of exposure and there were no detectable changes in total hsp70 (72 and 73), hsp90, and MT levels in any of the tissues relative to controls. Both fingerling and juvenile fish responded with similar lack of apparent sensitivity to Mo exposure. These experiments demonstrate that exposure to waterborne Mo of up to 1,000 mg l^-1 did not activate a physiological or cellular stress response in fish. Information from this study suggests that Mo water quality guidelines for the protection of aquatic life are highly protective of freshwater fish, namely rainbow trout.     

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.     

Behavioral plasticity in rainbow trout (Oncorhynchus mykiss) with divergent coping styles: when doves become hawks

Consistent and heritable individual differences in reaction to challenges, often referred to as stress coping styles, have been extensively documented in vertebrates. In fish, selection for divergent post-stress plasma cortisol levels in rainbow trout (Oncorhynchus mykiss) has yielded a low (LR) and a high responsive (HR) strain. A suite of behavioural traits is associated with this physiological difference, with LR (proactive) fish feeding more rapidly after transfer to a new environment and being socially dominant over HR (reactive) fish. Following transport from the UK to Norway, a switch in behavioural profile occurred in trout from the 3rd generation; HR fish regained feeding sooner than LR fish in a novel environment and became dominant in size-matched HR–LR pairs. One year after transport, HR fish still fed sooner, but no difference in social dominance was found. Among offspring of transported fish, no differences in feeding were observed, but as in pre-transported 3rd generation fish, HR fish lost fights for social dominance against size-matched LR opponents. Transported fish and their offspring retained their distinctive physiological profile throughout the study; HR fish showed consistently higher post-stress cortisol levels at all sampling points. Altered risk-taking and social dominance immediately after transport may be explained by the fact that HR fish lost more body mass during transport than did LR fish. These data demonstrate that some behavioural components of stress coping styles can be modified by experience, whereas behavioural plasticity is limited by genetic effects determining social position early in life story.     

The physiology of rainbow trout in social hierarchies: two ways of looking at the same data

Salmonids form dominance hierarchies in environments, where space or food are limiting. Our first objective was to investigate the physiology of individual rainbow trout in 4-fish hierarchies. Our second was to compare conclusions drawn from grouping physiological data on the basis of social rank with those based on relating individual physiology to individual aggressive behavior. To create a social hierarchy, groups of 4 juvenile trout were fed (1 % ration) using a darkened feeding container, twice daily (morning and evening). Each morning feeding was videotaped to record aggressive behavior, thereby facilitating the assignment of a social status rank to each fish. On days 5 and 10–11, physiological parameters were measured in fish fasted for 24 h. Social hierarchies formed in all tested groups. One fish would become dominant, whereas the three subordinate individuals would each assume a stable social rank. When classified according to this social rank, the three subordinate individuals all displayed similar physiology, different from the physiology of the dominant fish. The latter included higher ammonia excretion rate, greater protein utilization in aerobic metabolism, greater feeding, higher specific growth rate, greater increase in condition factor, and lower routine oxygen consumption rate. However, when individual aggression was taken into account, a continuous gradient was observed between aggression and physiology for most parameters, regardless of social status. These relationships could be improved by normalizing the aggression score to the overall level of aggression in each hierarchy. We argue that individual behavior should be considered instead of just social rank when studying the physiology of trout in social hierarchies.     

Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.     

Thermal acclimation and stress in the American lobster, Homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones and polyubiquitin

Using homologous molecular probes, we examined the influence of equivalent temperature shifts on the in vivo expression of genes coding for a constitutive heat shock protein (Hsc70), heat shock proteins (Hsps) (Hsp70 and Hsp90), and polyubiquitin, after acclimation in the American lobster, Homarus americanus. We acclimated sibling, intermolt, juvenile male lobsters to thermal regimes experienced during overwintering conditions (0.4 +/- 0.3 degrees C), and to ambient Pacific Ocean temperatures (13.6 +/- 1.2 degrees C), for 4-5 weeks. Both groups were subjected to an acute thermal stress of 13.0 degrees C, a temperature shift previously found to elicit a robust heat shock response in ambient-acclimated lobsters. Animals were examined after several durations of acute heat shock (0.25-2 hours) and after several recovery periods (2-48 hours) at the previous acclimation temperature, following a 2-hour heat shock. Significant inductions in Hsp70, Hsp90, and polyubiquitin messenger RNA (mRNA) levels were found for the ambient-acclimated group. Alternatively, for the cold-acclimated group, an acute thermal stress over an equivalent interval resulted in no induction in mRNA levels for any of the genes examined. For the ambient-acclimated group, measurements of polyubiquitin mRNA levels showed that hepatopancreas, a digestive tissue, incurred greater irreversible protein damage relative to the abdominal muscle, a tissue possessing superior stability over the thermal intervals tested.     

Heat-shock proteins are induced in unstressed leaves of Nicotiana attenuata (Solanaceae) when distant leaves are stressed

In Nicotiana attenuata, systemic induction of heat-shock proteins (Hsps) was detected in response to the treatment of single leaves by either heat shock, mechanical damage, or exogenous application of methyl jasmonate (MJ). All treatments increased the abundance of members of the 70-kD Hsp (Hsp70) family and induced synthesis of one or more of the small Hsps (sHsp) (16-23 kD) in both treated and untreated leaves. These results provide the first evidence that Hsps can be systemically induced in plants and suggest that systemic induction of Hsps may be important in pre-adapting leaves to stress.     

Quantitative evidence that both Hsc70 and Hsp70 contribute to thermal adaptation in hybrids of the livebearing fishes Poeciliopsis

The 70-kilodalton heat shock protein family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. While the role of the constitutively expressed stress proteins in thermotolerance is largely unknown, de novo expression stress proteins in response to elevated temperatures has been associated with increased thermotolerance in many cell lines, developing embryos and adult organisms. Distinct, hemiclonal hybrids between the livebearing fish species Poeciliopsis monacha and P. lucida varied in their abilities to survive temperature stress, with survival being greatest when rates of temperature increase to 40°C were slowest and when P. monacha genomes were combined with a sympatric P. lucida genome. Quantification of Hsp70 under heat shock conditions and Hsc70 under normal physiological conditions indicated that variation in survival among hemiclones was best explained by the combined effects of these two proteins. Similar complex interactions between maternal and paternal genomes and rate of temperature increase were found to underline patterns of survival, Hsp70 accumulation and Hsc70 abundance. These data suggest that the relationship between Hsps and thermotolerance is more intricate than previously thought and that Hsps contribute to thermal adaptation in these fishes through genetic interactions specific to particular environments.     

Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27

The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration). esophagus; esophagitis; gastroesophageal reflux disease; microvasculature; phosphatidylinositol 3-kinase/Akt; VCAM-1     

Social dominance and interrenal cell activity in rainbow trout,Salmo gairdneri (Pisces,Salmonidae)

Synopsis The relationship between standing in a dominance hierarchy and physiological stress was studied in rainbow trout. Individual fish were assigned relative dominance ranks, based on behavioral observations in a large, simulated stream tank. These ranks were compared to histometric measures of interrenal cell activity. Fish, isolated individually in the stream tank had significantly lower levels of interrenal activity than fish from the crowded holding tank. Groups of fish in the stream tank formed stable, linear dominance hierarchies. Interrenal activity correlated inversely with dominance rank, with the exception that top ranking fish had higher activity than expected. Possible cause and effect relationships are discussed.