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.     

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.     

Cortisol receptor expression differs in the brains of rainbow trout selected for divergent cortisol responses

In rainbow trout (Oncorhynchus mykiss), selection for divergent post-stress plasma cortisol levels has yielded low (LR)- and high (HR) responsive lines, differing in behavioural and physiological aspects of stress coping. For instance, LR fish display prolonged retention of a fear response and of previously learnt routines, compared to HR fish. This study aims at investigating putative central nervous system mechanisms controlling behaviour and memory retention. The stress hormone cortisol is known to affect several aspects of cognition, including memory retention. Cortisol acts through glucocorticoid receptors 1 and 2 (GR1 and 2) and a mineralcorticoid receptor (MR), all of which are abundantly expressed in the salmonid brain. We hypothesized that different expressions of MR and GRs in LR and HR trout brains could be involved in the observed differences in cognition. We quantified the mRNA expression of GR1, GR2 and MR in different brain regions of stressed and non-stressed LR and HR trout. The expression of MR was higher in LR than in HR fish in all brain parts investigated. This could be associated with reduced anxiety and enhanced memory retention in LR fish. MR and GR1 expression was also subject to negative regulation by stress in a site-specific manner.     

Extinction of a conditioned response in rainbow trout selected for high or low responsiveness to stress

Two lines of rainbow trout (Oncorhynchus mykiss) that exhibit divergent endocrine responsiveness to stressors also display disparate behavioral traits. To investigate whether the high-responding (HR) and low-responding (LR) fish also differ in cognitive function, the rate of extinction of a conditioned response was compared between the two lines. Groups of HR and LR fish were exposed to a paired conditioned stimulus (CS; water off) and unconditioned stimulus (US; confinement stressor). After exposure to 18 CS-US pairings, at least 70% of individuals of both lines acquired a conditioned response (CR) manifested as an elevation of blood cortisol levels on presentation of the CS only. Post-conditioning, the fish were tested by presentation of the CS at weekly intervals, for 4 weeks, with no further reinforcement, and the extinction of the CR in the two lines was compared. The decline in mean plasma cortisol levels after exposure to the CS over successive tests suggested that the CR was retained for a shorter period among the HR (<14 days) than LR fish (<21 days). The frequency of individuals within each line whose plasma cortisol levels indicated a stress response when exposed to the CS was significantly greater among the LR than HR fish at 14 and 21 days with no HR fish falling into this category at 21 days. At 28 days post-conditioning, there were no HR fish and only three LR fish were categorized as "stressed". These results suggest that there are differences in cognitive function between the two lines. Possible mechanisms underlying these differences are discussed.     

ACTH does not mediate divergent stress responsiveness in rainbow trout.

Two lines of rainbow trout selected for high (HR) and low (LR) responsiveness to a standardised confinement stressor displayed a sustained divergence in plasma cortisol levels during a 3-h period of confinement (max.: HR: 167+/-13 ng ml(-1); LR: 103+/-8 ng ml(-1); P<0.001). However, no significant difference in plasma ACTH levels was evident (max: HR: 153+/-9 pg ml(-1); LR: 142+/-7 pg ml(-1)). Dexamethasone (DEX) was administered to HR and LR fish to block endogenous adrenocorticotropin (ACTH) release. Administration of a weight-adjusted dose of ACTH to the DEX-blocked fish elevated plasma cortisol levels to a significantly greater extent in HR (233+/-24 ng ml(-1)) than LR (122+/-14 ng ml(-1)) fish (P<0.001). Plasma cortisol levels in DEX-blocked HR and LR fish after sham injection were low but also significantly different (HR: 6.7+/-1 ng ml(-1); LR: 2.2+/-0.2 ng ml(-1); P<0.001). These results indicate that modulation of cortisol responsiveness to stressors in HR and LR fish resides, at least in part, downstream of the hypothalamic-pituitary axis.     

Physiological and genetic correlates of boldness: characterising the mechanisms of behavioural variation in rainbow trout, Oncorhynchus mykiss

Bold, risk-taking animals have previously been putatively linked with a proactive stress coping style whereas it is suggested shyer, risk-averse animals exhibit a reactive coping style. The aim of this study was to investigate whether differences in the expression of bold-type behaviour were evident within and between two lines of rainbow trout, Oncorhynchus mykiss, selectively bred for a low (LR) or high (HR) endocrine response to stress, and to link boldness and stress responsiveness with the expression of related candidate genes. Boldness was determined in individual fish over two trials by measuring the latency to approach a novel object. Differences in plasma cortisol concentrations and the expression of eight novel candidate genes previously identified as being linked with divergent behaviours or stress were determined. Bold and shy individuals, approaching the object within 180 s or not approaching within 300 s respectively, were evident within each line, and this was linked with activity levels in the HR line. Post-stress plasma cortisol concentrations were significantly greater in the HR line compared with the LR line, and six of the eight tested genes were upregulated in the brains of LR fish compared with HR fish. However, no direct relationship between boldness and either stress responsiveness or gene expression was found, although clear differences in stress physiology and, for the first time, gene expression could be identified between the lines. This lack of correlation between physiological and molecular responses and behavioural variation within both lines highlights the complexity of the behavioural-physiological complex.     

Divergence in behavioural responses to stress in two strains of rainbow trout (Oncorhynchus mykiss) with contrasting stress responsiveness

The aim of this study was to establish whether two lines of rainbow trout divergent for their plasma cortisol response to a standardized stressor would show consistent differences in their behavioural response to a range of challenging situations. Our results show that the high- and low-responding (HR and LR) lines of rainbow trout did not differ in the aggression shown towards an intruder or in their response to the introduction of a novel object to their home environment. However, there was a difference in behaviour between the two selection lines when they were exposed to two unfamiliar environments. These results suggest that the behaviour of the HR and LR fish differs when they are challenged in unfamiliar environments, while their behaviour does not differ when they are challenged in their home environment. These observations are in agreement with studies on mammals that show that individuals with reactive coping styles perform similarly to proactive animals when they are challenged in a familiar environment, while they show different behaviour when they are challenged in unfamiliar environments. Thus, these results provide further evidence that the HR and LR selection lines of rainbow trout exemplify the two different coping styles described in mammals.     

Behavioral and Neuroendocrine Correlates of Selection for Stress Responsiveness in Rainbow Trout--a Review

In rainbow trout the magnitude of the cortisol response to stressshows both consistency over time and a moderate to high degreeof heritability, and high responding (HR) and low responding(LR) lines of rainbow trout have been generated by individualselection for consistently high or low post-stress cortisolvalues. Using 2nd and 3rd generation fish, we tested the hypothesisthat differential stress responsiveness is associated with behavioralalterations in the HR-LR trout model. LR fish showed a tendencyto become socially dominant, a rapid recovery of food intakeafter transfer to a novel environment, and a reduced locomotorresponse in a territorial intrusion test. Furthermore, stressinduced elevation of brain stem and optic tectum concentrationsof the monoamine neurotransmitters serotonin, dopamine, andnorepinephrine and their metabolites suggests that both synthesisand metabolism of these transmitters were elevated after stressto a larger degree in HR than in LR trout. A divergent patternwas seen in the hypothalamus, where LR fish displayed elevatedlevels of 5-hydroxyindoleacetic acid (a serotonin metabolite)and 3-methoxy-4-hydroxyphenylglycol (a norepinephrine metabolite).Thus, selection for a single trait, cortisol responsiveness,in rainbow trout is associated with concurrent changes in bothbehavior and central signaling systems. The apparent parallelto genetically determined stress coping styles in mammals, andthe existence of similar trait associations in unselected populationsof rainbow trout, suggests an evolutionarily conserved correlationbetween multiple traits. Continuing studies on the HR and LRtrout lines are aimed at providing the physiological and geneticbasis for new marker-assisted selection strategies in the rapidlydeveloping finfish aquaculture industry, as well as increasedknowledge of the function and evolution of central neuroendocrinesignaling systems.     

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.     

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.     

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.     

Effects of maternal stress coping style on offspring characteristics in rainbow trout (Oncorhynchus mykiss)

Maternal size, age, and allostatic load influence offspring size, development, and survival. Some of these effects have been attributed to the release of glucocorticoids, and individual variation in these stress hormones is related to a number of traits. Correlated traits are often clustered and used to define the proactive and reactive stress coping styles. Although stress coping styles have been identified in a number of animal groups, little is known about the coupling between stress coping style and offspring characteristics. In the present study, plasma cortisol levels in ovulated mothers and cortisol levels in non-fertilized eggs from two rainbow trout (Oncorhynchus mykiss) strains selected for high (HR) and low (LR) post-stress plasma cortisol levels were compared. Offspring characteristics such as egg size, larval growth, and energy reserves also were compared between the two strains. Maternal plasma and egg cortisol levels were correlated, but no difference between the HR and LR strains was detected in either parameter. LR females produced larger eggs, and larvae with larger yolk sacs compared to HR females, however no differences in larval body size (excluding the yolk) was detected between strains. Considering that the HR and LR strains have a number of correlated behavioral and physiological traits that resemble the reactive and proactive stress coping styles, respectively, the results suggest that proactive mothers invest more energy into their offspring, producing larvae with larger energy reserves. It is possible that larger energy reserves in proactive larvae support the energy requirement for establishing and defending territory in salmonid fish. Furthermore, in the present study we found a positive relationship between mother plasma cortisol and egg cortisol; however neither mother plasma cortisol nor egg cortisol differed between strains. These results indicate that cortisol endowment from the mother to the offspring plays a minor role in the transfer of the behavioral and physiological traits which separates these strains.     

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.     

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 effect of short term confinement stress on binding characteristics of sex steroid binding protein (SBP) in female black bream (Acanthopagrus butcheri) and rainbow trout (Oncorhynchus mykiss)

The effect of short term confinement stress on sex steroid binding protein (SBP) binding characteristics was examined in female black bream (Acanthopagrus butcheri), and rainbow trout (Oncorhynchus mykiss). Black bream were sampled immediately after capture from the wild and again after 1, 6 or 24 h confinement. Rainbow trout were sampled before and after 5 h confinement. Confinement of black bream for 6 h after capture significantly reduced the binding capacity of SBP. Binding affinity also tended to be lower after confinement. There were no differences in binding affinity or capacity of black bream SBP after 1 or 24 h confinement, or rainbow trout SBP after 5 h confinement. Plasma from rainbow trout at 3 and 6 h after treatment with cortisol was compared to plasma from saline-injected controls. No significant differences in binding characteristics were detected, but there was a trend of decreased binding capacity in cortisol-injected fish compared to controls at 6 h post-injection. Relative binding studies indicated that plasma cortisol at concentrations 100x or more greater than plasma estradiol (E(2)) may displace E(2) from SBP in black bream, and act to reduce circulating levels of E(2) through increased clearance of free steroid. Physiological levels of cortisol did not displace E(2) from SBP in trout. The observed changes in SBP and the competition of physiological concentrations of cortisol for SBP binding sites may generate a component of the stress-induced falls in plasma levels of E(2) reported across a range of species.     

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.     

The effect of elevated blood cortisol levels on the extinction of a conditioned stress response in rainbow trout

Following previously published observations that a conditioned response (CR) was lost more quickly by rainbow trout (Oncorhynchus mykiss) exhibiting a high responsiveness to stressors than by low responding individuals this study was designed to investigate the effects of exogenous cortisol on the retention of a CR in unselected rainbow trout. Fish held in isolation were conditioned over a 10-day period by pairing an innocuous signal (conditioned stimulus, CS: a water jet played on the surface of the tank water) with a mild stressor (unconditioned stimulus, US: 30 min of confinement). This resulted in a brief elevation of plasma cortisol levels (the CR) when the fish was exposed to the CS only. The effect of exogenous cortisol on the retention of the CR was evaluated by comparing the performance of fish that received cortisol-containing slow-release intraperitoneal implants, with fish receiving vehicle-only implants. Retention of the CR was assessed at intervals up to 35 days after conditioning ceased. The CR was considered to be evident when 30 min following presentation of the CS, mean plasma cortisol levels were significantly higher in conditioned than untrained fish. On day 1 both cortisol-implanted and vehicle-implanted conditioned fish exhibited a CR. However, from day 5 onwards the CR was observed only in the vehicle-implanted and conditioned group. This finding indicates that administration of cortisol accelerated the extinction of the CR in the cortisol-implanted fish, suggesting that elevated plasma cortisol levels can impair memory processes in rainbow trout.     

Relationship between stress, feeding and plasma ghrelin levels in rainbow trout, Oncorhynchus mykiss

Sexually immature rainbow trout were acclimated to small-volume (1 m³) holding tanks and then exposed to short-term stress to examine the relationship between feeding, stress, plasma ghrelin levels and other plasma stress parameters. Plasma ghrelin levels showed an increase 24 h after a single feed, plasma lactate and glucose levels decreased over the same period and plasma cortisol levels were low and constant. One hour of confinement stress resulted in elevations of plasma cortisol, glucose and lactate and depression of plasma ghrelin levels. In a separate experiment, 2 h of confinement stress also depressed feeding immediately after stress, concomitant with increases in plasma cortisol, lactate and glucose; however, in this case there was no change in plasma ghrelin concentrations. A repeat of the 2-h confinement experiment using fish that had not been acclimated to small-volume holding tanks produced a more marked elevation in plasma cortisol and a stronger suppression of feeding post-stress but in this case also, there was no change in plasma ghrelin levels. The results of this study confirm that feeding in rainbow trout is suppressed by confinement stress although the effect is transitory in this domesticated stock. Similar to that in other fishes, plasma ghrelin levels appear to be modulated by feeding status and may be influenced by stress, suggesting an orexigenic role for ghrelin in rainbow trout.     

Does socially induced stress in rainbow trout cause chloride cell proliferation?

At the end of a 2-week confinement period, subordinate rainbow trout Oncorhynchus mykiss had significantly higher circulating concentrations of plasma cortisol than did the dominant fish with which they were paired. Physiological effects linked to elevated plasma cortisol concentrations in subordinate fish included loss of weight and a lowering of condition factor. However, there were no significant differences in gill epithelium chloride cell numbers or blood plasma ion concentrations between dominant and subordinate fish. It is concluded that elevated plasma cortisol concentrations elicited by the social stressors of the present study did not cause proliferation of chloride cells.