Scott D. Reid

Localization and characterization of phenamil-sensitive Na+influx in isolated rainbow trout gill epithelial cells

SUMMARY Percoll density-gradient separation, combined with peanut lectin agglutinin (PNA) binding and magnetic bead separation, was used to separate dispersed fish gill cells into sub-populations. Functional characterization of each of the sub-populations was performed to determine which displayed acid-activated phenamil- and bafilomycin-sensitive Na+ uptake. Analysis of the mechanism(s) of 22Na+ influx was performed in control and acid-activated (addition of 10 mmoll-1 proprionic acid) cells using a variety of Na+ transport inhibitors (ouabain, phenamil, HOE-694 and bumetanide) and a V-type ATPase inhibitor (bafilomycin). We found that cells migrating to a 1.03-1.05 g ml-1 Percoll interface [pavement cells (PVCs)] possessed the lowest rates of Na+ uptake and that influx was unchanged during either bafilomycin (10 nmoll-1) treatment or internal acidification with addition of proprionic acid (10 mmoll-1). Mitochondria-rich (MR) cells that migrated to the 1.05-1.09 g ml-1 interface of the Percoll gradient demonstrated acidification-activated bafilomycin and phenamil-sensitive Na+ influx. Further separation of the MR fraction into PNA+ and PNA- fractions using magnetic separation demonstrated that only the PNA- cells (α-MR cells) demonstrated phenamil-and bafilomycin-sensitive acid-activated 22Na+ uptake. We confirm the coupling of a V-type H+-ATPase with phenamil-sensitive Na+ uptake activity and conclude that high-density α-MR cells function in branchial Na+ uptake in freshwater fish.

The Metabolic Costs and Physiological Consequences to Juvenile Rainbow Trout of a Simulated Winter Warming Scenario in the Presence or Absence of Sublethal Ammonia

Abstract This experiment examined the metabolic costs and physiological consequences of growth and energetics of juvenile rainbow trout Oncorhynchus mykiss in a warmer, more polluted winter environment. Growth under the warm-winter conditions was approximately three times less than equivalent growth of experimental and control groups previously observed under warm-summer conditions. However, during winter exposure, wet weights and total lengths were roughly 30% higher in the “warmed” fish than in the base temperature group due to a combination of greater appetite and higher energy conversion efficiency. Oxygen consumption and nitrogenous (ammonia + urea) waste excretion rates were 30–40% higher for “warmed” fish but were less than one-third of levels recorded in the summer. A corresponding increase in food intake was associated with elevations in whole-body protein and lipid but not carbohydrate. Addition of 70 μmol ammonia/L elevated nitrogenous waste excretion much like in the previous summer exposure, ...

Effects of chronic environmental acidification and a summer global warming scenario: protein synthesis in juvenile rainbow trout (Oncorhynchus mykiss)

Protein synthesis ( Ks), net accretion ( Kg), and degradation ( Kd) in liver, gills, and white muscle were measured using a flooding dose of [ 3H]phenylalanine in juvenile rainbow trout chronically exposed (90 days) to softwater in the presence or absence of sublethal acidity (H 2SO4, pH 5.2) alone or in combination with a 2°C elevation in the normal temperature profile over the months of June–September 1993 (control temperature range 13–24°C). Chronic sublethal exposure to low pH reduced protein synthesis and degradation in both the gill and liver with little apparent impact on white muscle. As a result, protein was increased in the affected tissues. This suggested that both liver and gill have some capacity to compensate for the effects of acid exposure. The 2°C elevation in the normal temperature profile resulted in a slight increase in protein turnover in both gills and liver. However, during the period of peak water temperature, the 2°C elevation in temperature triggered a dramatic reduction in the protein turnover rates in these tissues. The exact mechanism by which these modifications in protein turnover occurred could not be clearly established. Overall, environmental acidification in combination with a summer global warming scenario would decrease fish growth and survival, most notably during periods of

Extracellular fluid volume measurements in tissues of the rainbow trout (Oncorhynchus mykiss)in vivo and their effects on intracellular pH and ion calculations.

Extracellular fluid volume (ECFV) estimates were determined in various tissues and whole body of resting, chronically cannulated rainbow trout (Oncorhynchus mykiss). Fish were infused with14C-inulin,3H-polyethylene glycol (PEG, M.W. 4000),3H-mannitol, or14C-mannitol, and values of ECFV determined from tissue: plasma water distribution ratios after 6 h or 13 h equilibration. Overall,3H-PEG provided the most conservative and reliable estimates after 13 h equilibration, with ECFV values in the order: brain < white muscle < red muscle < liver < heart < gill tissue.14C-inulin yielded generally similar values to3H-PEG at 13 h, but probably overestimated ECFV in liver.3H-mannitol and14C-mannitol spaces were similar to each other and far greater than3H-PEG or14C-inulin values in most tissues.3H-mannitol values increased significantly between 6 h and 13 h, in contrast to14C-inulin. Mannitol clearly overestimated ECFV in liver and gill, and probably also heart and whole body, but may have provided more realistic estimates in brain due to better penetration of the blood-brain barrier. The Cl−/K+ space technique overestimated ECFV in gills, but was satisfactory in white muscle. Measurements and model calculations evaluated sources of error in intracellular pH (by14C-DMO) and ion determinations. Trapped red cells in the gills have negligible influence. Errors in ECFV are much more influential in a tissue with a high ECFV (gills) than a low ECFV (white muscle).3H-PEG is the marker of choice for intracellular pH determinations. However, even when3H-PEG is used, the potential for absolute errors in intracellular ion concentrations remains high.

Effects of a summer temperature regime representative of a global warming scenario on growth and protein synthesis in hardwater- and softwater-acclimated juvenile rainbow trout (Oncorhynchus mykiss)

Abstract 1. 1. Growth, appetite, gross conversion efficiency and protein turnover rates of liver, gills and white muscle were measured in juvenile rainbow trout chronically exposed (90 days) to soft and hardwater at two temperatures (ambient, ambient temp. +2°C). The temperature regime followed that of inshore Lake Ontario over the months of June–September 1993 as temperature rose from ∼13 to 24°C. 2. 2. Over the initial 60 days of exposure, the addition of 2°C to the ambient temperature increased growth, appetite, gross conversion efficiency and protein turnover by an average of 16%. However, further exposure during the period of peak ambient temperatures, led to an average 20% reduction in growth, appetite, gross conversion efficiency and protein turnover. 3. 3. Increased rates of gill protein turnover and arguably lower rates of growth indicate that the cost of living for a trout acclimated and maintained in synthetic softwater is higher than that of hardwater fish. In addition, lower appetite in softwater fish suggest that life in softwater is itself a mild form of environmental stress.

Effects of a Restricted Ration on the Growth and Energetics of Juvenile Rainbow Trout Exposed to a Summer of Simulated Warming and Sublethal Ammonia

Abstract Laboratory tests were conducted to determine the potential effects of a warmer and more polluted environment on the growth and energetics of juvenile rainbow trout Oncorhynchus mykiss fed a fixed restricted ration (1% wet body weight/d) during summer. The fish were exposed either to the naturally fluctuating ambient thermal regime (base, representative of inshore Lake Ontario) or to the ambient regime + 2°C (base + 2°C), both in the presence or absence of 70 μmol total ammonia(T amm)/L (0.013 mg NH3-N/L at 15°C, pH = 7.6). The 90-d exposures lasted from June to September 1994 and were designed to mimic an earlier study in which juvenile rainbow trout were fed to satiation. Relative to the earlier study, the restricted ration markedly increased (4–9-fold) the metabolic costs of nitrogen retention, that is, oxygen consumption per unit protein growth. Rainbow trout from the present study exhibited O2 consumption, and specific growth rates that were 50–75% and 13–20%, respectively, of the O2 and grow...

Physiological impact of acute molybdenum exposure in juvenile kokanee salmon (Oncorhynchus nerka)

A series of experiments were conducted to determine the physiological impact of acute sublethal molybdenum exposure to juvenile kokanee salmon (Oncorhynchus nerka Kennerlyi). Molybdenum was found to be relatively non-toxic to kokanee as the 96 h LC(50) was greater than 2,000 mg Mo l(-1). Exposure to either 25 or 250 mg Mo x l(-1) for 7 days was found to stimulate a significant 1.6- to 1.7-fold increase in ventilation which was later characterized to be dose-dependent between 5 and 250 mg Mo l(-1). Acute sublethal molybdenum exposure was found to have little or no impact on kokanee oxygen consumption at rest or immediately following a bout of forced activity or on physiological indicators of stress such as plasma lactate, sodium and cortisol. Despite these findings, prior exposure to 25 or 250 mg Mo l(-1) resulted in post-exercise loss of equilibrium and exercise-induced delayed mortality that were not observed in controls. Molybdenum accumulation in gill and liver of kokanee was also characterized. The findings of this study suggest that despite the non-toxic nature of molybdenum, acute sublethal exposure to this metal has physiological consequences to those fish exposed even for only a brief period. Further studies are needed to more fully elucidate the metabolism and mode of action of this metal in fish.

Long-term exposure to small temperature increase and sublethal ammonia in hardwater acclimated rainbow trout: does acclimation occur?

Abstract Juvenile rainbow trout ( Oncorhynchus mykiss ; initially 2–5 g) were exposed for 90 days to either ambient water temperature (natural thermal regime) or to +2 °C superimposed above the ambient water temperature (simulated global warming scenario), in the presence or absence of a nominal 70 μM total ammonia (1290 μg l −1 ionized (NH 4 + ), 10 μg l −1 unionized (NH 3 ) ammonia). The exposures were conducted in moderately hard de-chlorinated water from Lake Ontario ([ Ca 2+ ] = 0.96 ± 0.02 mM, [ Na + ] = 0.55 ± 0.01 mM, [ Cl − ] = 0.737 ± 0.004 mM) on three occasions: over summer (temperature range, 13.0–21.0 °C; pH = 7.57 ± 0.26) and winter (temperature range, 3.5–7.0 °C; pH = 7.46 ± 0.02) without food limitation (satiation feeding), and during summer (temperature range, 13.0–18.5 °C; pH = 7.38 ± 0.09) with food limitation (1% daily, or restricted ration). Lethal temperature, lethal ammonia (1.8 mM total ammonia; approximately 31 700 μg l −1 NH 1 + , 900 μg l −1 NH 3 ), and lethal temperature plus ammonia challenges were conducted after each 90-day exposure to determine whether or not chronic pre-exposure conferred any increased tolerance to elevated temperature or ammonia. In addition, acute sublethal ammonia challenges (1.0 mM total ammonia; approximately 17 800 μg l −1 NH 4 + , 200 μg l −1 NH 3 ), together with unidirectional Na + flux measurements, were conducted after the two summer exposures to gain further insight into the effects of prior sublethal ammonia exposure on Na + regulation, as influenced by ration. The juvenile trout on unlimited ration and exposed to a warming scenario of +2 °C exhibited a slight, but significant elevation in lethal temperatures in both summer and winter, but the effect was not observed in fish fed a restricted ration. A challenge to lethal temperature and ammonia in combination reduced the lethal temperature anywhere from 3–7 °C for fish from all treatments; pre-exposure to ammonia offered some protective effect. However, prior ammonia exposure did not prolong survival times (LT 50 s) during lethal ammonia challenge, and there was no evidence of acclimation to elevated external ammonia with respect to Na + balance. These results suggest that juvenile trout are likely to adapt to a small temperature increase, such as could be associated with a global warming scenario, but their potential for doing so may be restricted by sublethal ammonia and by nutritional status.

The effects of acute waterborne exposure to sublethal concentrations of molybdenum on the stress response in rainbow trout, Oncorhynchus mykiss.

To determine if molybdenum (Mo) is a chemical stressor, fingerling and juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne sodium molybdate (0, 2, 20, or 1,000 mg l-1 of Mo) and components of the physiological (plasma cortisol, blood glucose, and hematocrit) and cellular (heat shock protein [hsp] 72, hsp73, and hsp90 in the liver, gills, heart, and erythrocytes and metallothionein [MT] in the liver and gills) stress responses were measured prior to initiation of exposure and at 8, 24, and 96 h. During the acute exposure, plasma cortisol, blood glucose, and hematocrit levels remained unchanged in all treatments. Heat shock protein 72 was not induced as a result of exposure and there were no detectable changes in total hsp70 (72 and 73), hsp90, and MT levels in any of the tissues relative to controls. Both fingerling and juvenile fish responded with similar lack of apparent sensitivity to Mo exposure. These experiments demonstrate that exposure to waterborne Mo of up to 1,000 mg l-1 did not activate a physiological or cellular stress response in fish. Information from this study suggests that Mo water quality guidelines for the protection of aquatic life are highly protective of freshwater fish, namely rainbow trout.

Quantification of presumptive Na(+)/H (+) antiporters of the erythrocytes of trout and eel.

The presumptive Na+/H+ exchange sites of trout and eel erythrocytes were quantified using amiloride-displaceable 5-(N-methyl-N-[3H]isobutyl)-amiloride (3H-MIA) equilibrium binding to further evaluate the mechanisms of i) hypoxia-mediated modifications in the trout erythrocyte β-adrenergic signal transduction system and ii) the marked differences in the catecholamine responsiveness of this system between the trout and eel. MIA was a more potent inhibitor of both trout apparent erythrocyte proton extrusion (IC50 = 20.1 ± 1.1 μmol l−1, N = 6) activity (as evaluated by measuring plasma pH changes after addition of catecholamine in vitro) and specific 3H-MIA binding (IC50 = 257 ± 8.2 nmol l−1, N = 3) than amiloride, which possessed a proton extrusion IC50 of 26.1 ± 1.6 μmol l−1 (N = 6) and a binding IC50 of 891 ± 113 nmol l−1 (N = 3). The specific Na+ channel blocker phenamil was without effect on adrenergic proton extrusion activity or specific 3H-MIA binding. Trout erythrocytes suspended in Na+-free saline and maintained under normoxic conditions possessed 37,675 ± 6,678 (N = 6) amiloride-displaceable 3H-MIA binding sites per cell (Bmax, presumptive Na+/H+ antiporters) with an apparent dissociation constant (KD) of 244 ± 29 nmol l−1 (N = 6). Acute hypoxia (PO2 = 1.2 kPa; 30 min) did not affect the KD, yet resulted in a 65% increase in the number of presumptive Na+/H+ antiporters. Normoxic eel erythrocytes, similarly suspended in Na+-free saline, possessed only 17,133 ± 3,716 presumptive Na+/H+ antiporters (N = 6), 45% of that of trout erythrocytes, with a similar KD (246 ± 41 nmol l−1, N = 6). These findings suggest that inter- and intra-specific differences in the responsiveness of the teleost erythrocyte β-adrenergic signal transduction system can be explained, in part, by differences in the numbers of Na+/H+ exchange sites.