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.     

Development of lamellar epithelial hyperplasia in gills of pantothenic acid-deficient rainbow trout, Salmo gairdneri Richardson

Two semisynthetic diets differing in the levels of pantothenic acid (PA) supplementation (0 or 40 mg kg⁻¹ diet) were fed to rainbow trout, Salmo gairdneri Richardson, initially weighing 0.7 g fish⁻¹. Each diet was fed to two tanks of fish and, at the start of the feeding trial, each tank contained 200 animals. The experiment was conducted for 28 days during which time, every 2 days, gill tissue was sampled and food consumption was determined. Lamellar hyperplasia was first detected in the gills of deficient fish on the 12th day sample, while reduced feed intake was first manifest at 16 days. Hyperplasia first appeared in the distal regions of the gill filaments, but the lesion rapidly progressed in a proximal direction and 35 of 40 fish examined on days 22–28 exhibited hyperplasia on more than 75% of the filament surface. Gill lamellar hyperplasia is a sensitive indicator of PA deficiency in the rainbow trout. Moreover, the lesion is specific to PA deficiency since its developmental pattern differs histologically from the lamellar hyperplasia of the non-nutritional gill diseases.     

Comparative ionic flux and gill mucous cell histochemistry: effects of salinity and disease status in Atlantic salmon (Salmo salar L.)

Two experiments were conducted to assess the physiological effects of freshwater exposure and amoebic gill disease (AGD) in marine Atlantic salmon (Salmo salar L.). The first experiment monitored marine salmon during a 3 h freshwater exposure, the standard treatment for AGD in Tasmania. The second experiment described the gill mucous cell histochemistry for freshwater adapted and seawater acclimated fish (AGD affected and unaffected) for possible correlations to ionoregulation. When exposed to freshwater, marine Atlantic salmon experienced a minor ionoregulatory dysfunction represented by a net efflux of Cl(-) ions at 3 h. AGD affected fish experienced the net efflux of Cl(-) ions 1 h sooner, and had a significantly greater net efflux of total ammonia. Changes to gill mucous cell populations corresponded to differing salinity and the presence of AGD. In AGD affected fish, these populations significantly differed between lesion and non-lesion associated areas of the gill filament. Our results have shown changes in the ionoregulatory capacity of Atlantic salmon due to freshwater exposure and AGD. Gill mucous cell histochemistry indicates the potential importance of the mucous layer in ionoregulation and disease. In comparison to previous studies on rainbow trout, these results suggest that Atlantic salmon have a greater short-term ionoregulatory capacity.     

Osmoregulatory action of 17beta-estradiol in the gilthead sea bream Sparus auratus

The osmoregulatory action of 17beta-estradiol (E2) was examined in the euryhaline teleost Sparus auratas. In a first set of experiments, fish were injected once with vegetable oil containing E2 (1, 2 and 5 microg/g body weight), transferred 12h after injection from sea water (SW, 38 ppt salinity) to hypersaline water (HSW, 55 ppt) or to brackish water (BW, 5 ppt salinity) and sampled 12h later (i.e. 24 h post-injection). In a second experiment, fish were injected intraperitoneally with coconut oil alone or containing E2 (10 microg/g body weight) and sampled after 5 days. In the same experiment, after 5 days of treatment, fish of each group were transferred to HSW, BW and SW and sampled 4 days later (9 days post-implant). Gill Na+,K+ -ATPase activity, plasma E2 levels, plasma osmolality, and plasma levels of ions (sodium and calcium), glucose, lactate, protein, triglyceride, and hepatosomatic index were examined. Transfer from SW to HSW produced no significant effects on any parameters assessed. E2 treatment did not affect any parameter. Transfer from SW to BW resulted in a significant decrease in plasma osmolality and plasma sodium but did not affect gill Na+,K+ -ATPase activity. A single dose of E2 attenuated the decrease in these parameters after transfer from SW to BW, but was without effect on gill Na+,K+ -ATPase activity. An implant of E2 (10 microg/g body weight) for 5 days significantly increased plasma calcium, hepatosomatic index, plasma metabolic parameters, and gill Na+,K+ -ATPase activity. In coconut oil-implanted (sham) fish, transfer from SW to HSW or BW during 4 days significantly elevated gill Na+,K+ -ATPase. Gill Na+,K+ -ATPase activity remained unaltered after transfer of E2-treated fish to HSW or BW. However, in E2-treated fish transferred from SW to SW (9 days in SW after E2-implant), gill Na+,K+ -ATPase activity decreased with respect to HSW- or BW-transferred fish. Shams transferred to HSW showed increased levels of lactate, protein, and trygliceride in plasma, while those transferred to BW only displayed increased trygliceride levels. E2-treated fish transferred to HSW showed higher protein levels without any change in other plasmatic parameters, while those transferred to BW displayed elevated plasma glucose levels but decreased osmolality and protein levels. These results substantiate a chronic stimulatory action of E2 on gill Na+,K+ -ATPase activity in the euryhaline teleost Sparus auratas.     

Cortisol stimulates the zinc signaling pathway and expression of metallothioneins and ZnT1 in rainbow trout gill epithelial cells

Intracellular zinc signaling is important in the control of a number of cellular processes. Hormonal factors that regulate cellular zinc influx and initiate zinc signals are poorly understood. The present study investigates the possibility for cross talk between the glucocorticoid and zinc signaling pathways in cultured rainbow trout gill epithelial cells. The rainbow trout metallothionein A (MTA) gene possesses a putative glucocorticoid response element and multiple metal response elements 1042 base pairs upstream of the start codon, whereas metallothionein B (MTB) and zinc transporter-1 (ZnT1) have multiple metal response elements but no glucocorticoid response elements in this region. Cortisol increased MTA, MTB, and ZnT1 gene expression, and this stimulation was enhanced if cells were treated with cortisol together with zinc. Cells treated with zinc showed increased zinc accumulation, transepithelial zinc influx (apical to basolateral), and intracellular labile zinc concentrations. These responses were also significantly enhanced in cells pretreated with cortisol and zinc. The cortisol-mediated effects were blocked by the glucocorticoid receptor (GR) antagonist RU-486, indicating mediation via a GR. In reporter gene assays, zinc stimulated MTA promoter activity, whereas cortisol did not. Furthermore, cortisol significantly reduced zinc-stimulated MTA promoter activity in cells expressing exogenous rainbow trout GR. These results demonstrate that cortisol enhances cellular zinc uptake, which in turn stimulates expression of MTA, MTB, and ZnT1 genes.     

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.     

The effect of 3,5,3'-triiodo-L-thyronine on gill Na+/K+-ATPase of rainbow trout Salmo gairdneri, in fresh water

Gill and liver microsomal Na+/K+-adenosine triphosphatase (ATPase) activities and plasma levels of 3,5,3'-triiodo-L-thyronine (T3) were measured in rainbow trout (100-300 g) immersed in a freshwater solution of T3 for 6 or 7 days at 11 degrees C. Ambient T3 (1.25 or 2 micrograms T3/100 ml H2O) elevated plasma T3 within a physiologic range; an ambient concentration of 10 micrograms T3/100 ml produced supranormal plasma T3 levels. All T3 treatments depressed gill ATPase to a similar degree. Liver ATPase was lower than gill ATPase and was elevated by a physiologic T3 treatment.     

Heat shock protein (Hsp70) induced by a mild heat shock slightly moderates plasma osmolarity increases upon salinity transfer in rainbow trout (Oncorhynchus mykiss)

We have investigated whether mild heat shock, and resulting Hsp70 expression, can confer cross-protection against the stress associated with transfer from freshwater (FW) to seawater (SW) in juvenile rainbow trout (Oncorhynchus mykiss). In experimental Series I, juvenile trout reared in FW were transferred from 13.5 degrees C to 25.5 degrees C in FW, held for 2 h, returned to 13.5 degrees C for 12 h, and then transferred to 32 ppt SW at 13.5 degrees C. Branchial Hsp70 increased approximately 10-fold in the heat-shocked fish relative to the control by the end of recovery and remained high 2, 8, and 24 h post-salinity transfer. However, no clear differences could be detected in blood parameters (blood hemoglobin, hematocrit, MCHC, plasma Na(+) and plasma osmolarity) or muscle water content between heat-shocked and sham-shocked fish in SW at any sampling interval (0, 2, 8, 24, 48, 120, 240 and 360 h post-SW transfer). In experimental Series II, trout acclimated to 8 degrees C were heat-shocked at 22 degrees C for 2 h, allowed to recover 18 h, and exposed to a more severe salinity transfer (either 36 or 45 ppt) than in Series I. Branchial Hsp70 levels increased approximately 6-fold in heat-shocked fish, but had declined to baseline after 120 h in SW. Plasma osmolarity and chloride increased in both groups upon transfer to 36 ppt; however, the increase was significantly less in heat-shocked fish when compared to the increase observed in sham-shocked fish at 24 h. No significant differences could be detected in branchial Na(+)/K(+)-ATPase activity or Na(+)/K(+)-ATPase alpha1a and alpha1b mRNA expression between the two groups. Our data indicate that a mild temperature shock has only modest effects on the ability of rainbow trout to resist osmotic stress during FW to SW transfer.     

Susceptibility of two strains of rainbow trout (one with suspected resistance to whirling disease) to Myxobolus cerebralis infection

The susceptibility of 2 strains of rainbow trout Oncorhynchus mykiss, 1 from North America (TL) and 1 from Germany (GR), to Myxobolus cerebralis (the cause of salmonid whirling disease) was assessed following exposure to the infectious stages (triactinomyxons). Two laboratory experiments were conducted with age-matched rainbow trout of each strain. At the beginning of the study, the 2 trout strains were aged ca. 570 degree-days in Expt 1, and ca. 999 degree-days in Expt 2. In both experiments, replicate groups of each trout strain were exposed to 10, 100, 1000 or 10000 triactinomyxons (TAMs) fish(-1) for 2 h. The fish were then held in aquaria receiving 15 degrees C well-water. Severity of infection was evaluated 5 mo after exposure by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts. Clinical signs of whirling disease were evident only in the younger fish exposed in Expt 1: These occurred first among TL rainbow trout at the highest dose at 6 to 7 wk post exposure and then 2 wk later in fish at the 1000 TAMs dose. Black tail was also observed among GR rainbow trout at the 10000 TAMs dose only, but in fewer fish. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among GR rainbow trout were less at all doses compared to TL rainbow trout. Risk of infection analyses showed that TL rainbow trout were more prone to infection at the lower doses than GR trout. Mean spore counts were consistently (10- to 100-fold) less in GR than TL trout at doses of 1000 TAMs or lower. Microscopic lesions increased with increasing dose in both strains of rainbow trout. The mechanisms underlying the greater resistance of the GR strain to M. cerebralis infections are unknown, but are under investigation as part of a long-term project to determine the basis for resistance and susceptibility of salmonid fishes to whirling disease.     

The effect of pH and/or calcium-enriched freshwater on gill Ca2+-ATPase activity and osmotic water inflow in rainbow trout (Salmo gairdneri)

Rainbow trout (Salmo gairdneri) were exposed to pH 5.0-5.1, 6.6 and/or calcium-enriched freshwater for 14 days. Hematocrit, gill Ca2+-ATPase enzyme activities, gill osmotic water inflow, plasma calcium and osmolarity were measured. No significant changes in plasma calcium ion levels were found. The typical increase in hematocrit usually associated with exposure of fish to acidified water was not found in the present study and is discussed. Plasma osmolarity decreased in fish exposed to calcium-enriched freshwater (60 mg Ca2+ X 1(-1) ) in comparison to fish exposed to control freshwater conditions (2 mg Ca2+ X 1(1) ), irrespective of the pH level. Gill Ca2+-ATPase enzyme activities were measured for both low affinity (3 mM Ca2+) and high affinity (100 microM) activity. Exposure of rainbow trout to low pH (pH 5.0-5.1) did not affect the specific activity of Ca2+-ATPase enzyme. However, low affinity Ca2+-ATPase activity in fish exposed to calcium-enriched freshwater did show a significant reduction. The increase in gill osmotic water permeability in fish exposed to calcium-enriched freshwater is interpreted as a result of the increase in osmolarity of the ambient media.     

Salinity acclimation affects the somatotropic axis in rainbow trout

In this study, we set out to examine the role of the somatotropic axis in the ion-regulation process in rainbow trout. Specifically, our objective was to examine whether plasma insulin-like growth factor-binding proteins (IGFBPs) are modulated by gradual salinity exposure. To this end, freshwater (FW)-adapted rainbow trout were subjected to gradual salinity increases, up to 66% seawater, over a period of 5 days. During this acclimation process, minimal elevations in plasma Ca2+ and Cl- were seen in the salinity-acclimated groups compared with FW controls. There were no changes in plasma Na+ levels, and only a minor transient change in plasma cortisol levels was seen with salinity exposure. The salinity challenged animals responded with elevations in plasma growth hormone (GH) and IGF-I levels and gill Na+-K+-ATPase activity. We identified IGFBPs of 21, 32, 42, and 50 kDa in size in the plasma of these animals, and they were consistently higher with salinity. Despite the overall increase in IGFBPs with salinity, transient changes in individual BPs over the 5-day period were noted in the FW and salinity-exposed fish. Specifically, the transient changes in plasma levels of the 21-, 42-, and 50-kDa IGFBPs were different between the FW and salinity groups, while the 32-kDa IGFBP showed a similar trend (increases with sampling time) in both groups. Considered together, the elevated plasma IGFBPs suggest a key role for these binding proteins in the regulation of IGF-I during salinity acclimation in salmonids.     

Environment-phenotype interactions: Influences of brackish-water rearing on lake trout (<Emphasis Type="Italic">Salvelinus namaycush</Emphasis>) physiology

Fertilization and development in salmonids occurs almost exclusively within freshwater environments (< 1 ppt). A less common life history strategy in this group of fishes is the brackish-water resident life history, where entire life cycles occur in brackish water (> 1 ppt). In the present study, we tested the hypothesis that differences in rearing environment (fresh or brackish water) results in significant differences in the ability of lake trout to ionoregulate when faced with a salinity challenge later in life. To test this, genetically similar lake trout were fertilized and raised at either 0 or 5 ppt saltwater. At approximately 240 days post hatch, lake trout from both rearing environments were acutely transferred to 20 ppt salt water or their respective rearing environments as a control. Individuals were sampled at time 0, 1, 7, and 14 days post transfer. Fish raised in 5 ppt transferred to 20 ppt saltwater had significantly higher gill Na⁺ K⁺-ATPase activity, gill Na⁺ K⁺-ATPase α1b expression, and lower plasma osmolality when compared to freshwater reared lake trout transferred to 20 ppt across various time points. Additionally, the 5 ppt control treatment had greater overall aerobic scope than 0 ppt control fish and those transferred from 0 ppt to 20 ppt. These data imply that populations exhibiting a brackish-water resident life history, as has been observed in Arctic Canada, may have an advantage over freshwater reared conspecifics when foraging in marine influenced environments and colonizing new locations in coastal regions.     

A comparison of cadmium-induced metallothionein gene expression and Me2+ distribution in the tissues of cadmiumsensitive (rainbow trout; Salmo gairdneri) and tolerant (stone loach; Noemacheilus barbatulus) species of freshwater fish

1. The accumulation of cadmium in the liver, kidney and gills of rainbow trout and stone loach was measured during exposure of the fish to the metal at 3 smg/l in their aquarium water. The pattern of accumulation of the toxic metal in the individual organs was different between the two species.2. The tissue concentrations of metallothionein-specific mRNA and metallothionein protein were also determined in these organs from the same fish. In rainbow trout, the induction of metallothionein gene expression resulted in a gradual increase in metallothionein concentration in gill over the course of the experiment whereas increases in metallothionein in the liver and kidney were detected only at the later time points of analysis (beyond 19 weeks). By contrast, in the same tissues from stone loach, relatively minor changes were quantified in specific mRNA and metallothionein concentrations.3. Throughout the experimental period, tissue concentrations of zinc and copper were determined in the liver, kidney and gills of the rainbow trout and stone loach. Subtle decreases were observed in the zinc concentration of gills in rainbow trout and substantial increases were observed in the hepatic copper concentrations in both species at the later time points of analysis.4. The ability of cadmium to induce metallothionein gene expression and its subsequent ability to compete for the sequestration sites on the newly-synthesized protein is discussed with regard to the relative levels of cadmium, zinc and copper in the organs studied and differing regimes of cadmium administration.