Tom G. Pottinger

Stress responses and disease resistance in salmonid fish: Effects of chronic elevation of plasma cortisol

Basal levels of plasma cortisol in unstressed salmonid fish are normally in the range 0–5 ng ml−1. An acute stress such as handling or 1 h confinement caused a temporary elevation of the plasma cortisol levels of both brown trout,Salmo trutta L., and rainbow trout,Salmo gairdneri Richardson, in the range 40–200 ng ml−1 with a return to basal levels within 24–48 h. The extent of the cortisol elevation in response to an acute stress was dependent upon both the species and strain of trout. Chronic stresses, such as prolonged confinement or crowding, resulted in an elevation of plasma cortisol levels to approximately 10 ng ml−1. Under these circumstances, blood cortisol levels remained elevated for periods of up to 4 weeks before acclimation finally occurred.It is shown, by means of intraperitoneal implantation of cortisol, that chronic elevation of plasma cortisol levels in the brown trout results in a dose-dependent increase in mortality due to common bacterial and fungal diseases. This effect is apparent at plasma cortisol levels as low as 10 ng ml−1, levels below those often reported as being representative of ‘unstressed’ fish. These findings are discussed in relation to the known immunosuppressive effects of corticosteroids in teleost fish.

Evolutionary background for stress-coping styles: relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates.

Reactions to stress vary between individuals, and physiological and behavioral responses tend to be associated in distinct suites of correlated traits, often termed stress-coping styles. In mammals, individuals exhibiting divergent stress-coping styles also appear to exhibit intrinsic differences in cognitive processing. A connection between physiology, behavior, and cognition was also recently demonstrated in strains of rainbow trout (Oncorhynchus mykiss) selected for consistently high or low cortisol responses to stress. The low-responsive (LR) strain display longer retention of a conditioned response, and tend to show proactive behaviors such as enhanced aggression, social dominance, and rapid resumption of feed intake after stress. Differences in brain monoamine neurochemistry have also been reported in these lines. In comparative studies, experiments with the lizard Anolis carolinensis reveal connections between monoaminergic activity in limbic structures, proactive behavior in novel environments, and the establishment of social status via agonistic behavior. Together these observations suggest that within-species diversity of physiological, behavioral and cognitive correlates of stress responsiveness is maintained by natural selection throughout the vertebrate sub-phylum.

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

Abstract In rainbow trout the magnitude of the cortisol response to stress shows both consistency over time and a moderate to high degree of heritability, and high responding (HR) and low responding (LR) lines of rainbow trout have been generated by individual selection for consistently high or low post-stress cortisol values. Using 2nd and 3rd generation fish, we tested the hypothesis that differential stress responsiveness is associated with behavioral alterations in the HR-LR trout model. LR fish showed a tendency to become socially dominant, a rapid recovery of food intake after transfer to a novel environment, and a reduced locomotor response in a territorial intrusion test. Furthermore, stress induced elevation of brain stem and optic tectum concentrations of the monoamine neurotransmitters serotonin, dopamine, and norepinephrine and their metabolites suggests that both synthesis and metabolism of these transmitters were elevated after stress to a larger degree in HR than in LR trout. A divergent pattern was seen in the hypothalamus, where LR fish displayed elevated levels 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 both behavior and central signaling systems. The apparent parallel to genetically determined stress coping styles in mammals, and the existence of similar trait associations in unselected populations of rainbow trout, suggests an evolutionarily conserved correlation between multiple traits. Continuing studies on the HR and LR trout lines are aimed at providing the physiological and genetic basis for new marker-assisted selection strategies in the rapidly developing finfish aquaculture industry, as well as increased knowledge of the function and evolution of central neuroendocrine signaling systems.

The effects of acute and chronic stress on the levels of reproductive hormones in the plasma of mature male brown trout, Salmo trutta L

Chronic confinement for 1 month caused a significant elevation of plasma cortisol but suppressed the levels of plasma testosterone and 11-ketotestosterone in sexually mature male brown trout. An acute handling stress for 1 hr elevated blood cortisol and ACTH levels and also suppressed circulating androgens. This androgen suppression in response to acute stress was accompanied by an elevation of plasma gonadotropin levels. These findings are discussed in relation to stress-induced suppression of reproductive function in mammals and the possible biological consequences of such a suppression in fish are outlined.

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.

The deleterious effects of cortisol implantation on reproductive function in two species of trout, Salmo trutta L. and Salmo gairdneri Richardson

Implantation of a cortisol-releasing pellet (60 mg kg-1 fish) into the peritoneal cavity of brown trout, Salmo trutta L. (sexually maturing males and females), and rainbow trout, Salmo gairdneri Richardson (maturing males and immature fish of both sexes), significantly elevated their plasma cortisol level. At 18 days postimplantation, cortisol-implanted sexually maturing male brown trout had smaller gonads, a lower plasma testosterone level, and less gonadotropin in their pituitary gland than control fish. Plasma levels of 11-ketotestosterone and gonadotropin were not significantly affected. Cortisol-implanted sexually maturing female brown trout had smaller gonads, reduced plasma levels of 17 beta-oestradiol, testosterone, and vitellogenin, and a lower pituitary gland gonadotropin content than control fish. The plasma gonadotropin level was unaffected. At 36 days post-implantation, cortisol treatment of maturing male rainbow trout significantly suppressed plasma gonadotropin levels. Plasma levels of testosterone, 11-ketotestosterone, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, pituitary gonadotropin content, and gonad size were not significantly affected. In sexually immature female rainbow trout, cortisol administration suppressed the level of vitellogenin in the plasma, compared to control-implanted fish. The 17 beta-oestradiol level was not affected. Cortisol implantation did not affect the plasma testosterone level in sexually immature male trout. These results suggest that prolonged elevation of plasma cortisol, to levels well within physiological range, can affect a wide range of reproductive parameters in both brown and rainbow trout. Further, some effects are manifest in immature as well as in mature fish. These findings are discussed in relation to the effects of cortisol treatment on the state of health of the treated fish.

Preliminary evidence that chronic confinement stress reduces the quality of gametes produced by brown and rainbow trout

This study presents preliminary evidence that periods of chronic confinement stress experienced during the final stages of reproductive development not only disrupt the reproductive endocrinology of trout, but also result in reduced egg size in rainbow trout (Oncorhynchus mykiss) and, most importantly, significantly lower survival rates for progeny from both stressed brown (Salmo trutta) and rainbow trout compared to progeny from unstressed controls. Groups of male and female brown and rainbow trout were subjected to one or two episodes of confinement stress in the months immediately prior to spawning. Plasma levels of cortisol, testosterone, 17β-estradiol and vitellogenin were determined following the 2-week period of confinement. Plasma levels of cortisol were significantly elevated in all groups of stressed fish, whereas plasma levels of testosterone were significantly reduced in some, but not all, groups of trout. Plasma levels of 17β-estradiol were unaffected by confinement stress, whereas the plasma vitellogenin levels of the stressed female rainbow trout were significantly reduced. Female rainbow trout subjected to two episodes of confinement stress, experienced 1 and 3 months prior to spawning, produced eggs which were significantly smaller than eggs from control fish. Eggs from ovulated brown and rainbow trout females were fertilised with milt from males subjected to the corresponding treatment regime. Subsequent development of the fertilised eggs was then monitored through the stages of eying, hatch and swim-up. The significantly reduced survival rates observed in the progeny from the stressed crosses compared to the controls indicates that stress reduces the quality of gametes produced by trout.

Effects of acute and chronic stress on the levels of circulating growth hormone in the rainbow trout, Oncorhynchus mykiss.

The acute stress of handling followed by confinement for a period of 1 or 24 hr caused a typical stress response in rainbow trout (elevation of plasma ACTH and cortisol) and a significant reduction in the concentration of circulating growth hormone. The chronic stress of low oxygen levels in both crowded and uncrowded tanks of fish caused a significant elevation of circulating GH levels, an effect which was abolished by the provision of additional aeration to the rearing tanks. This chronic elevation of GH levels was closely correlated with an elevation of plasma cortisol in the same fish. These findings are discussed in relation to stress-induced growth suppression and to the links between the hypothalamic-pituitary-interrenal axis and somatotrope activity.

The effect of starvation on growth and plasma growth hormone concentrations of rainbow trout, Oncorhynchus mykiss

Two experiments, one using 0+ the other 1+ rainbow trout, were conducted to investigate the effect of prolonged starvation on plasma growth hormone levels. The results from both experiments were essentially the same. As expected, starvation resulted in cessation of growth and in a lower coefficient of condition, whereas fed fish continued to grow and remained in good condition. Starvation had relatively little effect on the plasma cortisol level; in one experiment levels were elevated temporarily in starved fish, although by the end of the experiment there was no longer any difference between starved and fed fish, and in the other experiment plasma cortisol levels remained very low throughout the course of the experiment in both starved and fed fish. In contrast, in both experiments starvation had a pronounced effect on the plasma growth hormone level, which rose steadily during both experiments, such that it was six times higher after 1 month of starvation in 0+ fish, and five times higher after 6 weeks of starvation in 1+ fish. Thus, paradoxically, fed fish had very low plasma growth hormone levels and grew rapidly, whereas starved fish had elevated plasma growth hormone levels but did not grow. In both experiments a strong negative correlation was observed between the plasma growth hormone level and the coefficient of condition of the fish. The results are discussed with regard to the well-established metabolic changes that occur during starvation, and it is suggested that a major role of growth hormone during starvation is to aid in the mobilisation of fatty acids and glycerol from adipose stores.

Seasonal and diel changes in plasma cortisol levels of the brown trout, Salmo trutta L

A marked diel variation in plasma cortisol concentration was demonstrated in the brown trout, Salmo trutta L. For most of the year this variation took the form of an elevation of cortisol levels during the hours of darkness. There was some evidence of a phase shift from a peak at 2400 hr during the spring and early summer to a peak at 0400 hr during the late summer and early autumn. During the winter months a nocturnal elevation of plasma cortisol was not evident. A shorter, episodic elevation of plasma cortisol was associated with feeding in the brown trout. These findings are discussed in relation to the effects of stress and intensive cultivation on teleost fish.