Divergence of endocrine and metabolic responses to stress in two rainbow trout lines selected for differing cortisol responsiveness to stress.

Rainbow trout (Oncorhynchus mykiss) of two lines selected for low (LR) and high (HR) cortisol stress-responsiveness were subjected to confinement for a period of 336 h. Endocrine (plasma cortisol, hepatic cortisol binding) and metabolic (plasma glucose, lactate, amino acids; hepatic glycogen and alanine aminotransferase levels) indices of stress were measured at intervals in confined and unconfined fish of both lines. During confinement plasma cortisol concentration reached maximum values earlier in HR fish (2h) than in LR fish (6h) returning to control values within 336 h in both lines. Paradoxically, although both HR and LR lines displayed a characteristic metabolic stress response, these changes were more pronounced in LR fish. Plasma glucose and lactate levels increased during confinement in both lines but to a significantly greater extent in LR fish. Confinement significantly elevated plasma amino acids to a greater extent in LR fish than in HR fish. Liver glycogen concentration was depleted most rapidly in LR fish but was significantly higher in confined fish of both lines than controls at the end of the experiment. No significant changes were observed in hepatic alanine aminotransferase activity during confinement. Confined fish of both lines displayed a decrease in hepatic cortisol receptor abundance within 24h and this was more sustained in HR fish. The more pronounced disturbance of a broad range of indicators of stress in confined LR fish, compared to HR fish, throws doubt on the magnitude of the cortisol response being the primary driver of these differences.

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.

Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness.

This paper investigates whether two lines of rainbow trout displaying genetically determined variation in stress responsiveness and behavior also show differences in brain monoaminergic activity. In several brain regions, strains of rainbow trout selected for consistently high or low post-stress cortisol levels displayed differences in tissue concentrations of monoamines and/or monoamine metabolites, or in metabolite/monoamine ratios. High-responsive trout reacted to stress by an increase in the concentrations of both serotonin (brain stem), dopamine (brain stem), and norepinephrine (optic tectum, telencephalon), whereas low-responsive fish did not. Brain stem and optic tectum concentrations of monoamine metabolites were also elevated after stress in high responders, but not in low-responsive fish. The simultaneous increase in the concentration of monoamines and their metabolites suggests that both synthesis and metabolism of these transmitters were elevated after stress in high-responsive trout. A divergent pattern was seen in the hypothalamus, where low-responsive fish displayed elevated levels of 5-hydroxyindoleacetic acid (a serotonin metabolite) and 3-methoxy-4-hydroxyphenylglycol (a norepinephrine metabolite). In the telencephalon, both populations had elevated concentrations of these metabolites after stress. These results clearly suggest that selection for stress responsiveness in rainbow trout is also associated with changes in the function of brain monoaminergic systems. The possible functional significance of these observations is discussed with respect to the physiological and behavioral profile of these strains of fish. Literature is reviewed showing that several factors affecting brain monoaminergic activity might be altered by selection for stress responsiveness, or alternatively be under direct influence of circulating glucocorticoids.

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.

High blood cortisol levels and low cortisol receptor affinity: is the chub, Leuciscus cephalus, a cortisol-resistant teleost?

In contrast to the relatively minor intra- and interspecies differences in blood cortisol levels reported for salmonid species, there is a more pronounced distinction between cortisol levels among the Salmonidae and the Cyprinidae, with both basal and stress-induced cortisol levels markedly higher in the latter. This study shows that in the chub, Leuciscus cephalus, a widely distributed European cyprinid, mean blood cortisol levels during stress (1500 ng mL(-1)) exceeded those reported for most other species of fish and, even in unstressed chub, cortisol levels (50-100 ng mL(-1)) were within the range known to cause immunosuppression, growth retardation, and reproductive dysfunction in salmonid fish. The chub appears to be atypical only with respect to plasma cortisol levels; the levels of plasma glucose and plasma lactate in unstressed and stressed chub are similar to those reported for other species. Plasma levels of 11-ketotestosterone in males and 17beta-estradiol in females are lower than those reported for salmonids but similar to those reported for other cyprinid species and display clear stress-induced reduction. Comparative analysis of the binding characteristics of the trout and chub gill cortisol receptor revealed that the total number of binding sites in gill tissue for each species was similar (B(max); approximately 50-100 fmol mg(-1) protein). However, the affinity of the binding site for cortisol displayed an eightfold difference between the species (rainbow trout: K(d) approximately 6 nM; chub: K(d) approximately 50 nM). Therefore, the potentially adverse effects of high circulating levels of cortisol found both at rest and under conditions of stress in chub may be offset by the lower affinity of the cortisol receptor, rather than the abundance of target-tissue receptor sites. This strategy is similar to that reported for some glucocorticoid-resistant rodents and New World primates.

Modification of the plasma cortisol response to stress in rainbow trout by selective breeding.

Male and female rainbow trout were segregated into high- and low-responding individuals (HR, LR) on the basis of their plasma cortisol response to a 3-h period of confinement imposed at monthly intervals for 5 months. Consistent divergence was obtained in the responsiveness of the two groups, although the difference between LR and HR groups was greater in female fish (56 c.f. 116 ng ml(-1)) than in males (45 c.f. 69 ng ml(-1)). Progeny groups (full-sib families) were obtained from the pairing of HR males and females and LR males and females. A third progeny group (US) was obtained by random pairing of parents which were not selected as HR or LR. Poststress plasma cortisol levels in the progeny were first tested at 6 months after hatch and were significantly correlated with the response of the corresponding parental groups, HR > US > LR (178, 126, 81 ng ml(-1), respectively). The difference in responsiveness between LR and HR groups was demonstrated in all four subsequent tests over a 12-month period. There were no significant differences in baseline plasma cortisol levels in LR and HR groups prior to confinement. During a 4-h period of confinement, the differences in plasma cortisol levels between LR and HR fish were sustained throughout, indicating that the trait upon which the fish were selected was related to absolute maximum levels of circulating cortisol and not to the rate of change of cortisol levels during exposure to a stressor. A moderately high heritability (h(2)) for confinement-induced plasma cortisol of 0.41 was obtained by a parent-progeny regression. Manipulation of stress responsiveness in fish by selective breeding offers scope for optimizing performance under intensive rearing conditions but also provides a useful research tool for investigating the operation of the endocrine stress response.

Testosterone, 11-ketotestosterone, and estradiol-17 beta modify baseline and stress-induced interrenal and corticotropic activity in trout.

Estradiol-17 beta (E), 11-ketotestosterone (KT), and testosterone (T) were administered to immature rainbow and brown trout by implantation of steroid-containing cocoa butter pellets. This procedure elevated the levels of these hormones in the blood of the treated fish and had significant effects on plasma ACTH and cortisol levels in both unstressed and stressed rainbow trout and in stressed brown trout. E treatment significantly elevated resting levels of ACTH and cortisol and KT significantly suppressed resting ACTH levels in rainbow trout, although no effect of KT was noted on baseline cortisol levels. One hour of confinement stress increased ACTH levels in rainbow trout, but less so in T- and KT-implanted fish than in sham-implanted fish. A similar pattern was observed in stress-induced plasma cortisol levels where T and KT treatment of rainbow trout resulted in a more than 50% attenuation of plasma cortisol levels while E implantation significantly increased stress-induced plasma cortisol levels. In brown trout subjected to confinement stress for 96 hr, within 1 hr of the onset of confinement the stress-induced increase in plasma ACTH and plasma cortisol was significantly lower in T- and KT-implanted fish than in sham-implanted controls. However, these differences were not sustained at subsequent sample points during the 96-hr period of continuous confinement. Nonetheless, overall mean ACTH levels for the entire confinement period were significantly enhanced in E-implanted brown trout and significantly reduced in KT-implanted fish. Overall mean cortisol levels were significantly lower in T- and KT-implanted fish. The enhancement of stress responsiveness observed in E-treated immature fish was not observed during confinement stress in untreated mature female trout, with naturally high plasma E levels. However, untreated mature male trout displayed a significantly reduced cortisol response to confinement. It is suggested that gonadal steroids are involved in the regulation of both baseline and stress-induced activity of the pituitary-interrenal axis in salmonid fish.

An automatic respirometer for determining oxygen uptake in crayfish (Austropotamobius pallipes (Lereboullet)) over periods of 3-4 days.

1. An automatic respirometer has been developed for continuous measurements over 3-4 days on 1-15 g crayfish. The sensor is a modified Mackereth oxygen electrode. Respiration is recorded on a millivolt potentiometric pen recorder during closed periods when the O2 concentration in the medium falls to a predetermined level. A solenoid-operated valve is then opened via a relay circuit energized by a reed switch mounted on the recorder. Medium flows through the respirometer until the O2 concentration is raised back to another predetermined level. Artificial media containing little or no nutrient salts are used to restrict the growth of microbes. 2. Respiration was determined chiefly on crayfish (Austropotamobius pallipes) with body wet weights of 7-0-12-5 g at 10-0 degrees C. In unrestrained but relatively quiescent animals, standard metabolism is described by the regression equation, ln O2 uptake = 3-3037 + 1-002 ln body wt. In restless crayfish active metabolism is described by the equation, ln O2 uptake = 4-4412 + 0-861 ln body wt.

Respiration in relation to ion uptake in the crayfish Austropotamobius pallipes (Lereboullet).

1. O2 uptake was determined for periods of 23-46 h in salt-depleted crayfish held in deionized water (DW) or Na-free media at 10 degrees C. These media were replaced by artificial lakewater media (ALW) containing 0-2-0-6 mM Na and O2 uptake was again determined for periods of 24-66 h. 2. During net ion uptake in ALW the metabolic rate was either elevated or depressed. Standard metabolism in ALW altered by amounts equivalent to 0-1 - 15-5% (mean 6-4 (15) +/- 4-4% S. D.) of the metabolic rate measured during salt-depletion. On three occasions the metabolic rate was elevated by 22-0 - 66-7%, but some of this increase may have been due to locomotor activity. 3. The calculated values for thermodynamic work involved in ion transport were 0-056 - 0-268 J/10 g. h at 10 degrees C, or 1-5 - 7-2% of the mean standard metabolic rate. Most of the observed changes in metabolic rate lie within the limits of experimental error (ca. +/- 7%). Hence the energetic cost of ion transport is too small for direct measurement in intact crayfish.