Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss)

Eurasian perch (Perca fluviatilis) is a promising aquaculture candidate, but the growth performance of this non-domesticated species may be negatively affected by its stress responsiveness to intensive culture conditions. To evaluate this potential problem, juvenile Eurasian perch were exposed to a standardized handling stressor twice a week for an 8-week period. A similar study was conducted on domesticated rainbow trout (Oncorhynchus mykiss) for comparison of intra- and inter-specific differences. The stressed fish of both species showed lower body growth than the non-stressed control fish, however, the final mean body mass was 35.4% lower in the stressed Eurasian perch than in the non-stressed controls, compared to 22.8% difference between the two groups in rainbow trout. The stress responsiveness was examined by comparing the post-stress cortisol and glucose levels in repeatedly stressed fish and fish exposed to the stressor only once. The cortisol stress response in both species strongly indicated a habituation to the repeated stressor. Thus, repeatedly stressed Eurasian perch reached maximum cortisol levels of 130 ng/mL after 0.5 h compared to 200 ng/mL in the fish stressed once, while considerably smaller differences in cortisol levels were shown between the repeatedly and single stressed rainbow trout. Rainbow trout also showed lower post-stress glucose levels in the repeatedly stressed fish compared to the single stressed fish. In contrast, the glucose levels in both groups of Eurasian perch increased abruptly after stress treatment and remained elevated at approximately 19 mM for 6 h; levels were three times as high as the peak levels 3 h post-stress in rainbow trout. Together, the habituation of the stress response shown in both species did not eliminate the growth difference found in the repeatedly stressed fish versus the control fish. Further, the lower growth performance of Eurasian perch compared to rainbow trout could partly be due to the increased energy consumption in the more stress responsive Eurasian perch.     

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.     

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.     

Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.     

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.     

A cDNA microarray assessment of gene expression in the liver of rainbow trout (Oncorhynchus mykiss) in response to a handling and confinement stressor

A purpose-designed microarray platform (Stressgenes, Phase 1) was utilised to investigate the changes in gene expression within the liver of rainbow trout during exposure to a prolonged period of confinement. Tissue and blood samples were collected from trout at intervals up to 648 h after transfer to a standardised confinement stressor, together with matched samples from undisturbed control fish. Plasma ACTH, cortisol, glucose and lactate were analysed to confirm that the neuroendocrine response to confinement was consistent with previous findings and to provide a phenotypic context to assist interpretation of gene expression data. Liver samples for suppression subtractive hybridisation (SSH) library construction were selected from within the experimental groups comprising “early” stress (2–48 h) and “late” stress (96–504 h). In order to reduce redundancy within the four SSH libraries and yield a higher number of unique clones an additional subtraction was carried out. After printing of the arrays a series of 55 hybridisations were executed to cover 6 time points. At 2 h, 6 h, 24 h, 168 h and 504 h 5 individual confined fish and 5 individual control fish were used with control fish only at 0 h. A preliminary list of 314 clones considered differentially regulated over the complete time course was generated by a combination of data analysis approaches and the most significant gene expression changes were found to occur during the 24 h to 168 h time period with a general approach to control levels by 504 h. Few changes in expression were apparent over the first 6 h. The list of genes whose expression was significantly altered comprised predominantly genes belonging to the biological process category (response to stimulus) and one cellular component category (extracellular region) and were dominated by so-called acute phase proteins. Analysis of the gene expression profile in liver tissue during confinement revealed a number of significant clusters. The major patterns comprised genes that were up-regulated at 24 h and beyond, the primary examples being haptoglobin, β-fibrinogen and EST10729. Two representative genes from each of the six k-means clusters were validated by qPCR. Correlations between microarray and qPCR expression patterns were significant for most of the genes tested. qPCR analysis revealed that haptoglobin expression was up-regulated approximately 8-fold at 24 h and over 13-fold by 168 h.     

Temporal changes in stress and tissue-specific metabolic responses to municipal wastewater effluent exposure in rainbow trout

Sub-chronic exposure to municipal wastewater effluent (MWWE) in situ was recently shown to impact the acute response to a secondary stressor in rainbow trout (Oncorhynchus mykiss). However, little is known about whether MWWE exposure in itself is stressful to the animal. To address this, we carried out a laboratory study to examine the organismal and cellular stress responses and tissue-specific metabolic capacity in trout exposed to MWWE. Juvenile rainbow trout were exposed to 0, 20 and 90% MWWE (from a tertiary wastewater treatment plant), that was replenished every 2d, for 14 d. Fish were sampled 2, 8 or 14 d post-exposure. Plasma cortisol, glucose and lactate levels were measured as indicators of organismal stress response, while inducible heat shock protein 70 (hsp70), constitutive heat shock protein 70 (hsc70) and hsp90 expression in the liver were used as markers of cellular stress response. Impact of MWWE on cortisol signaling was ascertained by determining glucocorticoid receptor protein (GR) expression in the liver, brain and, heart, and metabolic capacity was evaluated by measuring liver glycogen content and tissue-specific activities of key enzymes in intermediary metabolism. Plasma glucose and lactate levels were unaffected by exposure to MWWEs, whereas cortisol showed a transient increase in the 20% group at 8d. Liver hsc70 and hsp90, but not hsp70 expression, were higher in the 90% MWWE group after 8d. There was a temporal change in GR expression in the liver and heart, but not brain of trout exposed to MWWE. Liver glycogen content and activities liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and alanine aminotransferase (AlaAT) were significantly affected by MWWE exposure. The glycolytic enzymes pyruvate kinase (PK) and hexokinase (HK) activities were significantly higher temporally by MWWE exposure in the gill and heart, but not in the liver and brain. Overall, a 14 d exposure to MWWE evokes a cellular stress response and perturbs the cortisol stress response in rainbow trout. The tissue-specific temporal changes in the metabolic capacity suggest enhanced energy demand in fish exposed to MWWE, which may eventually lead to reduced fitness.     

Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Krøyer) influences the physiological response to an acute stressor

The influence of infection with the juvenile stages of the sea louse, Lepeophtheirus salmonis (Kr?yer) on the response of rainbow trout Oncorhynchus mykiss (Walbaum) to a net confinement protocol was investigated. The experiment consisted of two groups of seawater-adapted rainbow trout, one which was exposed to a total of 4000 nauplii/copepodid stages of L. salmonis 30, 25 and 14 days prior to confinement. Confinement elicited a greater stress response in the lice-exposed fish, than in the controls, as seen by higher plasma cortisol and glucose levels. A reduced spleen somatic index in exposed fish following 6 h confinement coincided with increased erythrocyte and lymphocyte numbers in the blood. Circulating lymphocyte numbers were significantly reduced in both groups 24 h post-confinement, when a lower alternative complement activity was recorded in control fish. Prior to confinement, lice-exposed fish had an elevated serum lysozyme activity and reduced oxygen radical production by blood leukocytes. Following confinement, lysozyme activity was gradually reduced in lice-exposed trout. During confinement, oxygen radical production decreased in control fish and increased in infested fish. Overall, transient exposure to juvenile lice altered the response to a second stressor, which has implications for management procedures of L. salmonis exposed fish.     

Contrasting seasonal modulation of the stress response in male and female rainbow trout

Between June and September the magnitude of the plasma cortisol response of maturing male and female rainbow trout to confinement was indistinguishable. A progressive increase in confinement-induced cortisol levels in both sexes occurred during May to September, associated with the seasonal rise in water temperature. Between September and January a reduction of >50% in the magnitude of the cortisol response to confinement in male fish (but no decline in females) coincided with declining water temperature and significant increases in plasma 11-ketotestosterone and elevated plasma testosterone levels. Plasma oestradiol-17β levels were significantly greater in females than males throughout the study period and this difference was maximal between September and January. However, plasma testosterone was also elevated in females during this period and levels overall were higher than those in male fish. Previous studies have shown oestradiol-17β and testosterone to have diametrically opposed effects on stress responsiveness in trout, with the former enhancing, and the latter suppressing, the cortisol response to a stressor. The relative roles of androgens, estrogen and water temperature in modulating the stress responsiveness of rainbow trout are discussed.     

CRF-related peptides contribute to stress response and regulation of appetite in hypoxic rainbow trout

Hypoxia stress suppresses appetite in a variety of fish species, but the mechanisms mediating this response are not known. Therefore, given their anorexigenic and hypophysiotropic properties, we investigated the contribution of forebrain corticotropin-releasing factor (CRF) and urotensin I (UI) to the regulation of food intake and the hypothalamic-pituitary-interrenal (HPI) stress axis in hypoxic rainbow trout. Exposure to 50 and 35% O(2) saturation for 24 h decreased food intake by 28 and 48%, respectively. The 35% O(2) treatment also increased forebrain CRF and UI mRNA levels, plasma cortisol, and lactate. Exposure for 72 h to the same conditions resulted in similar reductions in food intake, increases in plasma cortisol proportional to the hypoxia severity, and increases in forebrain CRF and UI mRNA levels in the 50% O(2) treatment. Relative to saline-infused fish, chronic intracranial infusion of the CRF receptor antagonist alpha-helical CRF((9-41)) reduced the appetite-suppressing effects of 24-h exposure to 35% O(2) and blocked the hypoxia-induced increase in plasma cortisol. Finally, forebrain microdissection revealed that 50 and 35% O(2) exposure for 24 h specifically increases preoptic area CRF and UI mRNA levels in proportion to the severity of the hypoxic challenge and either has no effect or elicits small decreases in other forebrain regions. These results show that CRF-related peptides play a physiological role in regulating the HPI axis and in mediating at least a portion of the reduction in food intake under hypoxic conditions in rainbow trout and demonstrate that the response of forebrain CRF and UI neurons to this stressor is region specific.     

Stress responsiveness affects dominant-subordinate relationships in rainbow trout.

The magnitude by which plasma cortisol levels increase following exposure to a stressor is a heritable trait in rainbow trout. The relative growth in coculture of F1 lines selected for high responsiveness (HR) and low responsiveness (LR) to a confinement stressor suggested that behavioral characteristics related to food acquisition, aggression, or competitive ability might differ between the two lines. This hypothesis was tested using the F2 generation of the selected lines. The F2 lines clearly exhibited the characteristics of the F1 parents, displaying significantly divergent plasma cortisol responses to a 1-h confinement stressor and a high heritability for the trait. Behavioral differences between the lines were assessed by observing the outcome of staged fights for dominance in size-matched pairs of HR and LR fish. The identification of dominant and subordinate fish within each pair on the basis of their behavior was supported by the levels of blood cortisol in the fish attributed to each group (dominant < subordinate). Fish from the LR line were identified as dominant in significantly more trials than were HR individuals. The results suggest that behavioral attributes that affect the outcome of rank-order fights are closely linked to the magnitude of the plasma cortisol response to stress in rainbow trout. Whether the link is causal or circumstantial is not yet evident.     

Effects of copper on the acute cortisol response and associated physiology in rainbow trout

The aim of this study was to determine the effects of chronic waterborne copper (Cu) exposure on the acute stress-induced cortisol response and associated physiological consequences in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 30 μg Cu/L in moderately hard water (120 mg/L as CaCO(3)) for 40 days, following which time the acute cortisol response was examined with a series of stressors. At 40 days, a 65% increase in Cu was observed in the gill, but no accumulation was observed in the liver, brain or head kidney. Stressors such as air exposure or confinement did not elicit an increase in circulating cortisol levels for Cu-exposed fish, in contrast to controls. However, this inhibitory effect on the acute cortisol response appeared to have few implications on the ability of Cu-exposed fish to maintain ion and carbohydrate homeostasis. For example, plasma Na(+), Ca(2+) and glucose levels as well as hepatic glycogen levels were the same post-stress in control and Cu-exposed fish. Trout were also challenged with exposure to 50% seawater for 48 h, where Cu-exposed trout maintained plasma Na(+), glucose and hepatic glycogen levels. However, Cu-exposed fish experienced decreased plasma K(+) levels throughout the Cu exposure and stress tests. In conclusion, chronic Cu exposure resulted in the abolition of an acute cortisol response post-stress. There was no Cu accumulation in the hypothalamus-pituitary-interrenal axis (HPI axis) suggesting this was not a direct toxic effect of Cu on the cortisol regulatory pathway. However, the lack of an acute cortisol response in Cu-exposed fish did not impair the ability of the fish to maintain ion and carbohydrate homeostasis. This effect on cortisol may be a strategy to reduce costs during the chronic stress of Cu exposure, and not endocrine disruption as a result of toxic injury.     

Gene expression in the liver of rainbow trout, Oncorhynchus mykiss, during the stress response

To better appreciate the mechanisms underlying the physiology of the stress response, an oligonucleotide microarray and real-time RT-PCR (QRT-PCR) were used to study gene expression in the livers of rainbow trout (Oncorhynchus mykiss). For increased confidence in the discovery of candidate genes responding to stress, we conducted two separate experiments using fish from different year classes. In both experiments, fish exposed to a 3 h stressor were compared to control (unstressed) fish. In the second experiment some additional fish were exposed to only 0.5 h of stress and others were sampled 21 h after experiencing a 3 h stressor. This 21 h post-stress treatment was a means to study gene expression during recovery from stress. The genes we report as differentially expressed are those that responded similarly in both experiments, suggesting that they are robust indicators of stress. Those genes are a major histocompatibility complex class 1 molecule (MHC1), JunB, glucose 6-phosphatase (G6Pase), and nuclear protein 1 (Nupr1). Interestingly, Nupr1 gene expression was still elevated 21 h after stress, which indicates that recovery was incomplete at that time.     

Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness

The aim for this study was to examine whether the F4 generation of two strains of rainbow trout divergent in their plasma cortisol response to confinement stress (HR: high responder or LR: low responder) would also differ in stress-induced effects on forebrain concentrations of mRNA for corticotropin-releasing factor (CRF), arginine vasotocin (AVT), CRF receptor type 1 (CRF-R1), CRF receptor type 2 (CRF-R2) and AVT receptor (AVT-R). In addition, plasma cortisol concentrations, brainstem levels of monoamines and monoamine metabolites, and behaviour during confinement were monitored. The results confirm that HR and LR trout differ in their cortisol response to confinement and show that fish of these strains also differ in their behavioural response to confinement. The HR trout displayed significantly higher locomotor activity while in confinement than LR trout. Moreover, following 180 min of confinement HR fish showed significantly higher forebrain concentrations of CRF mRNA than LR fish. Also, when subjected to 30 min of confinement HR fish showed significantly lower CRF-R2 mRNA concentrations than LR fish, whereas there were no differences in CRF-R1, AVT or AVT-R mRNA expression between LR and HR fish either at 30 or 180 min of confinement. Differences in the expression of CRF and CRF-R2 mRNA may be related to the divergence in stress coping displayed by these rainbow trout strains.     

Quantitative trait loci for magnitude of the plasma cortisol response to confinement in rainbow trout

Better understanding of the mechanisms underlying interindividual variation in stress responses and their links with production traits is a key issue for sustainable animal breeding. In this study, we searched for quantitative trait loci (QTL) controlling the magnitude of the plasma cortisol stress response and compared them to body size traits in five F2 full‐sib families issued from two rainbow trout lines divergently selected for high or low post‐confinement plasma cortisol level. Approximately 1000 F2 individuals were individually tagged and exposed to two successive acute confinement challenges (1 month interval). Post‐stress plasma cortisol concentrations were determined for each fish. A medium density genome scan was carried out (268 markers, overall marker spacing less than 10 cM). QTL detection was performed using qtlmap software, based on an interval mapping method ( http://www.inra.fr/qtlmap ). Overall, QTL of medium individual effects on cortisol responsiveness (<10% of phenotypic variance) were detected on 18 chromosomes, strongly supporting the hypothesis that control of the trait is polygenic. Although a core array of QTL controlled cortisol concentrations at both challenges, several QTL seemed challenge specific, suggesting that responses to the first and to a subsequent exposure to the confinement stressor are distinct traits sharing only part of their genetic control. Chromosomal location of the steroidogenic acute regulatory protein (STAR) makes it a good potential candidate gene for one of the QTL. Finally, comparison of body size traits QTL (weight, length and body conformation) with cortisol‐associated QTL did not support evidence for negative genetic relationships between the two types of traits.     

Stress from air emersion fails to alter chloride cell numbers in the gills of rainbow trout

Air emersion stress caused an elevation in blood plasma cortisol of rainbow trout Oncorhynchus mykiss but this acute stressor did not elicit chloride cell proliferation in the gill tissue within 2 weeks following the disturbance.     

Differences in the sensitivity of brown trout, Salmo trutta L., and rainbow trout, Salmo gairdneri Richardson, to physiological doses of cortisol

Interspecific differences in the stress response of fish may be due, in part, to differences in the sensitivity of target tissues to cortisol. The relative response of brown and rainbow trout to a standardized dose of cortisol was assessed by monitoring condition (K factor), the number of circulating lymphocytes and mortality due to disease, following cortisol treatment. Cortisol implantation resulted in a significant decline in K factor and number of circulating lymphocytes in immature brown trout, but not in immature rainbow trout, despite plasma cortisol levels being similar in both cases. Cortisol implantation in mature brown and rainbow trout significantly increased the mortality rate due to bacterial and fungal infection compared with control fish. Furthermore, the mortality rate due to disease was significantly greater in brown trout than rainbow trout, despite both groups receiving similar doses of steroid.