Action of cortisol on the proliferation of rainbow trout fibroblasts

The effect of cortisol on the proliferation of the rainbow trout fibroblast cell line, RTG-2, was examined in synchronous and asynchronous cultures. When the transition from G1 to S was synchronized by restoring serum to serum-deprived cultures, the addition of cortisol at the time of serum restoration delayed the entry of cells into S phase. However, if cortisol was added 24 h after serum restoration, at the G1/S transition point, the subsequent peak of DNA synthesis was unaffected. In asynchronous cultures cortisol inhibited [3H]-thymidine and [3H]-uridine but not [3H]-leucine incorporation into acid-insoluble material. If the exogenous nucleoside concentration was raised, [3H]-thymidine but not [3H]-uridine incorporation continued to be inhibited by cortisol. This suggested that cortisol's effect on [3H]-thymidine incorporation reflected a change in entry into S phase and not just on thymidine uptake and metabolism. Cortisol inhibited the proliferation of RTG-2 in asynchronous cultures. At 1000 ng/ml of cortisol a reduction in cell number became apparent before the RTG-2 cultures were confluent, whereas at 100 ng/ml the reduction only became evident in confluent cultures. The synthetic antiglucocorticoid, RU 486, which acts at the level of the corticosteroid receptor, blocked the growth inhibition by cortisol. These results suggest that cortisol regulates rainbow trout fibroblast proliferation via the corticosteroid receptor and that the G1/S transition is one point at which this regulation occurs.     

Effects of cortisol on aggression and locomotor activity in rainbow trout

Noninvasive administration of cortisol through the diet resulted in relatively rapid (<1.5 h) and highly reproducible increases in plasma cortisol in rainbow trout, comparable to changes seen in fish subjected to substantial stress. Juvenile rainbow trout were reared in isolation for 1 week, before their daily food ration was replaced by a meal of cortisol-treated food corresponding to 6 mg cortisol kg(-1). All fish were observed for 30 min, beginning at 1 or 48 h following the introduction of cortisol-treated food. Additional cortisol (75% of the original dose on Day 2, and 50% on Day 3) was administered to the long-term cortisol-treated group. The resulting blood plasma concentrations of cortisol were similar in short- and long-term treated fish, and corresponded to those previously seen in stressed rainbow trout. Controls were fed similar food without cortisol. Half of the fish from each treatment group (controls and short- and long-term cortisol) were subjected to an intruder test (a smaller conspecific introduced into the aquarium), while half of the fish were observed in isolation. In fish challenged by a conspecific intruder, short-term cortisol treatment stimulated locomotor activity, while long-term treatment inhibited locomotion. Aggressive behavior was also inhibited by long-term cortisol treatment, but not by short-term exposure to cortisol. Cortisol treatment had no effect on locomotor activity in undisturbed fish, indicating that the behavioral effects of cortisol were mediated through interaction with other signal systems activated during the simulated territorial intrusion test. This study demonstrates for the first time that cortisol has time- and context-dependent effects on behavior in teleost fish.     

Branchial release of free cortisol and melatonin by rainbow trout

Individual rainbow trout Oncorhynchus mykiss were held in a specially constructed tank that enabled water to be collected separately from the anterior and posterior ends of the fish. Measurement by radioimmunoassay showed that >95% of the cortisol and melatonin released into the water originated from the anterior end (dominated by the gills). High performance liquid chromatography confirmed the identity of both hormones.     

Induction and decay of thermotolerance in rainbow trout fibroblasts

Thermotolerance was studied in the rainbow trout fibroblast cell line RTG-2. RTG-2 cultures that had been incubated at 28 degrees C for 24 h were better able to withstand ultimately lethal temperatures above 28 degrees C than RTG-2 cultures that had been maintained at the routine growth temperature of 22 degrees C. This thermotolerance developed rapidly between 3 and 6 h and was fully developed by 24 h at 28 degrees C. After development for 24 hr at 28 degrees C, thermotolerance showed little change over 72 h at 0 and 5 degrees C but approximately a 40 and 60% reduction at 10 and 22 degrees C, respectively. This is the first demonstration of heat-induced thermal resistance in the cells of a poikilothermic vertebrate.     

Effect of Gyrodactylus derjavini infections on cortisol production in rainbow trout fry

Cortisol production in fry of rainbow trout (367-456 mg body weight) infected with the ectoparasitic monogenean Gyrodactylus derjavini (mean intensities 4.7 and 4.9 parasites per fish) was studied at two temperature levels, 4-6 degrees C and 11-12 degrees C, respectively. Due to difficulties in obtaining an adequate amount of plasma from the small sized fish, the corticosteroid concentration was measured in the body fluid recovered (as supernatant) after sonication and centrifugation of whole fry. Infected fry at 11-12 degrees C showed an elevated level of cortisol compared to uninfected fry. However, the cortisol concentration was lower than in fish exposed to handling stress. At 4 degrees C, the cortisol level in infected fish compared to uninfected was insignificantly increased. The findings are discussed in relation to the role of monogeneans as inducers of secondary infections.     

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.     

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 aluminium and cortisol on in vitro calcium deposition on otoliths in rainbow trout

Aluminium (1–100 μM) reduced in vitro calcium deposition on otoliths concentration-dependently in rainbow trout, but cortisol (1 and 10 μg ml⁻¹) had no effect.     

Evaluation of oral administration of cortisol and metyrapone: the effects on serum cortisol in rainbow trout (Salmo gairdneri)

The effect of manipulating dietary levels of cortisol and metyrapone on peripheral concentrations of serum cortisol was examined in rainbow trout, Salmo gairdneri. Fish were fed 0, 10, 20, 30, 40 or 50 mg/kg body weight/day of metyrapone or cortisol for 7 days. All levels of cortisol tested increased peripheral levels of serum cortisol. Feeding a level of 30-40 mg/kg body weight/day of metyrapone depressed serum cortisol. We describe, herein, a noninvasive technique for suppression or stimulation of serum cortisol in rainbow trout.     

Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts

Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.     

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.     

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.     

Temperature ranges over which rainbow trout fibroblasts survive and synthesize heat-shock proteins

Cultures of the rainbow trout fibroblast cell line RTG-2 withstood temperatures from 0 degrees C to 28 degrees C. At 0 degrees C and 28 degrees C, no proliferation occurred, but cells persisted for at least 7 days. If the cultures were placed back at 22 degrees C, proliferation returned to normal in those that had been kept at 0 degrees C but was reduced in cultures that had been kept at 28 degrees C. Above 28 degrees C, cultures survived for only short periods. If RTG-2 cells that were grown routinely at 22 degrees C were shifted to 26, 28, and 30 degrees C, heat shock proteins (hsps) of 100, 87, 70, 68, 60, 39, 27, and 19 kilodaltons were synthesized. Synthesis was most pronounced at 28 degrees C, and at this temperature hsp synthesis was maximal by 2 hr and had returned to control levels by 36 hr. Individual hsps were synthesized maximally at slightly different times and temperatures, but under all conditions hsps 87 and 70 were most abundant. If cultures were shifted to 24 degrees C or 32 degrees C, hsp synthesis was not observed. Neither the placement of cultures at 5 degrees C nor the shift of cultures that had been maintained at 0 degrees C or 5 degrees C back to 22 degrees C induced the synthesis of hsps. However, cultures incubated at 5 degrees C for 24 hr did synthesize hsps at 26 degrees C, 28 degrees C, and 30 degrees C.     

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.     

Effects of cortisol implants on the PKX myxosporean causing proliferative kidney disease in rainbow trout, Salmo gairdneri

The myxosporean (PKX) that causes proliferative kidney disease in salmonid fishes is found primarily in the kidney interstitium of clinically affected fish, where it invokes a severe inflammatory response. Incomplete spores are observed in the lumen of kidney tubules in recovering fish. PKX-infected rainbow trout, Salmo gairdneri, were immunosuppressed with cortisol implants to determine if a reduction in renal interstitial inflammation would enhance the development of the parasite. Immunosuppression of these fish was indicated by high plasma cortisol levels, chronic mortality due to opportunistic pathogens, and depressed leucocrits when compared to infected fish not receiving cortisol implants. Cortisol-treated PKX-infected fish had 20 times the density of interstitial PKX compared with nontreated fish, but showed less interstitial inflammation. All of the former group examined at 4 wk postimplantation exhibited intraluminal sporogonic forms of PKX, whereas no sporogonic forms were detected in the nontreated fish. Suppression of the inflammatory response clearly enhanced the parasite's ability to reach the sporogonic stage, but did not induce complete development of the spore. Possibly the kidney tubules of rainbow trout do not provide the proper physiological environment for complete sporulation of PKX and a nonsalmonid fish may be the primary host.     

Poststress levels of lysozyme and cortisol in adult rainbow trout: heritabilities and genetic correlations

Lysozyme response in stressed rainbow trout was compared with measurements of poststress cortisol activity. Estimates of heritability of lysozyme and cortisol were both moderate-to-high and both traits displayed positive genetic correlations in pair-wise comparisons of stress exposures. Genetic correlations between lysozyme and cortisol in stressed rainbow trout tended to be negative, although insignificant. Neither lysozyme, nor cortisol exhibited significant correlations with serum haemolytic activity. It is concluded that the data do not confirm earlier suggestions that lysozyme should be superior to cortisol in consistency of stress response in rainbow trout.     

The effect of exercise on plasma cortisol and blood sugar levels in the rainbow trout, Salmo gairdnerii Richardson

Preliminary experiments were performed to determine the diurnal variation in cortisol, using trout which had been cannulated three days previously. These results indicated that cortisol levels were reasonably stable between 10.00 and 18.00 hours, thus permitting experimentation during this period without diurnal fluctuations masking the cortisol response. Uncannulated fish were exercised in a flume for 2 h at 1, 2.6 and 5 bl s^-1 and plasma samples taken from groups of five animals at 15, 30, 60 and 120 min after the start of exercise and at 1½, 12 and 24 h after the exercise ceased. The cortisol levels in all cases were elevated after 15 min, but the magnitude of the elevation increased with swimming speed. At 1 bl s^-1 the cortisol levels increased from 76.4 (± 20.4) to 129.2 (± 20.4) ng ml^-1 [mean (± s.d. )]. At 2.6 bl s^-1 the increase was from 72.4 (± 17.1) to 254.4 (± 34.4) ng ml^-1 and at 5 bl s^-1 the increase was from 69.5 (± 27.5) to 326.4 (± 39.0) ng ml^-1. The cortisol levels were stable over the exercise period and all groups recovered to baseline levels after 24 h, though the sample taken 12 h after the termination of exercise was elevated due to regular nocturnal increases in cortisol levels. There were no dramatic changes in blood sugar levels during and after exercise at 1 and 3.2 bl s^-1.     

In vitro effects of pure microcystin-LR on the lymphocyte proliferation in rainbow trout (Oncorhynchus mykiss)

The aim of this study was to determine the in vitro influence of microcystin-LR on the viability and mitogenic response of rainbow trout (Oncorhynchus mykiss) lymphocytes since few data are available in the literature on the influence of cyanotoxins on fish immunocompetent cells. Lymphocytes were isolated from blood and haematopoietic organs (pronephros and spleen) and cultivated in RPMI 1640 medium with different concentrations of the toxin (1, 5, 10, 20, 40 mg ml-1 of cell suspension). Dose-dependent effects of microcystin-LR on the lymphocyte viability were shown. The lymphocyte proliferation was inhibited after application of microcystin at a concentration of 40 mg ml-1 but significantly increased at a concentration of 1mg ml-1 in comparison to the control group. The results suggest the modulatory effects of microcystin-LR dependent on the applied concentration.