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Plasma Prolactin, Cortisol, and Thyroid Responses of the Brown Trout (Salmo trutta) Exposed to Lethal and Sublethal Aluminium in Acidic Soft Waters
General and Comparative Endocrinology 102, 377 – 385 (1996) Article No. 0081
Plasma Prolactin, Cortisol, and Thyroid Responses of the Brown Trout (Salmo trutta) Exposed to Lethal and Sublethal Aluminium in Acidic Soft Waters C. P. Waring,1 J. A. Brown, J. E. Collins, and P. Prunet* Department of Biological Sciences, Hatherly Laboratories, University of Exeter, Exeter, Devon EX4 4PS, United Kingdom; and *Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France Accepted February 6, 1996
Brown trout, with indwelling dorsal aortic cannulae, were exposed to various concentrations of aluminium (Al; 50 mg liter01, 100% mortality over 48 hr; 25 mg liter01, 50% mortality over 120 hr; 12.5 mg liter01, 0% mortality over 120 hr) in acidic (pH 5.0) soft water. The plasma concentrations of prolactin (PRL), cortisol, thyroxine (T4), and triiodothyronine (T3) were monitored. Plasma PRL concentrations were transiently depressed (to less than 20% of resting concentrations) after 12 hr in trout in the two highest water Al concentrations, but were unchanged in the trout exposed to 12.5 mg liter01 Al. Plasma cortisol concentrations were elevated in response to all water Al levels and remained elevated in trout in the lethal conditions. The sublethally exposed trout showed a recovery in plasma cortisol concentrations by 120 hr. Plasma T4 concentrations were significantly elevated in trout exposed to both the lethal and the sublethal Al concentrations (from mean resting concentrations of 1–2 ng ml01 to peaks of 8.9 and 9.0 ng ml01 in the 50 and 12.5 mg liter01 Al groups, respectively), although a recovery in plasma concentrations was evident in the sublethally exposed trout from 72 hr onwards. Plasma T3 concentrations were relatively stable in the trout exposed to the two highest doses of Al, whereas the trout under the lowest, sublethal, Al condi1 Present address: MAFF, Fisheries Laboratory, Pakefield Road, Lowestoft, Suffolk, NR33 0HT, UK.
tions exhibited a sustained (12–72 hr) elevation in plasma T3 concentrations (from a mean resting concentration of 0.9 ng ml01 to a peak of 4.2 ng ml01 at 48 hr). No clear relationship was apparent between the plasma PRL concentrations and the previously reported ionoregulatory status of the trout. q 1996 Academic Press, Inc.
Freshwater fish species acutely exposed to aluminium (Al) in acidic soft waters show ionoregulatory, cardiovascular, and respiratory disruptions (Wood et al., 1988; Playle and Wood, 1989; Playle et al., 1989). The respiratory and/or ionoregulatory disturbances resulting from exposure to Al have been suggested to depend upon water pH (Playle et al., 1989) and the Al species present (Pole´o, 1995; Brown and Waring, 1995). During prolonged exposure to acidic water with or without the presence of Al, some fish species or individuals show at least a partial recovery of hydromineral balance and respiratory function (Wood et al., 1988; McDonald et al., 1991) and hormones are likely to play a significant role in this recovery. In this respect, many studies have shown that plasma cortisol concentrations are elevated after exposure to acidic waters and acidic soft waters containing Al (Goss and Wood, 1988; Whitehead and Brown, 1989; Brown et al., 1990), although, at least in rainbow trout, this response is not always apparent when water Al concentrations are low
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(Balm and Pottinger, 1993). Plasma catecholamines have been reported to be elevated in acidic waters containing Al (Witters et al., 1991; Brown and Whitehead, 1995), while again some studies did not show mobilisation of plasma catecholamines in salmonids exposed to acidic conditions without Al (Audet and Wood, 1993). In a recent study of brown trout exposed to lethal Al concentrations (50 mg liter01; 100% mortality), intermediate Al concentrations (25 mg liter01; 50% mortality), and sublethal Al concentrations (12.5 mg liter01; 0% mortality) in pH 5.0 soft water for up to 5 days, fish exposed to the sublethal Al concentration showed transiently reduced plasma Na/ and Cl0 concentrations, whereas the fish exposed to the two highest water Al concentrations had stable plasma monovalent ion concentrations (Waring and Brown, 1995). The marked differences in mortality and the physiological responses to different water Al concentrations prompted the measurement of prolactin (PRL), cortisol, thyroxine (T4 ), and triiodothyronine (T3 ) concentrations in the plasma samples from these trout to assess whether these hormones might be implicated in their recovery and/or adaptation. PRL has a well-known ionoregulatory role in freshwater salmonids (reviewed by Bern and Madsen, 1992) and has been suggested to play a role in counteracting H/-induced ionic disturbances in tilapia, Oreochromis mossambicus (Flik et al., 1989). Studies on the tilapia have suggested that PRL is released in fish exposed to low-pH conditions alongside declining plasma monovalent ions (Wendelaar Bonga et al., 1988; Flik et al., 1989). Salmonids exposed to low-pH water with or without Al present show structural alterations in pituitary PRL cells, indicating inhibition rather than stimulation of PRL release (Notter et al., 1976; Fryer et al., 1988). More recently, rainbow trout showed stable plasma PRL concentrations when sampled 14 days after exposure to pH 4.0 soft water (Balm et al., 1995). Plasma cortisol concentrations increase in salmonids exposed to Al in acidic waters (Goss and Wood, 1988; Whitehead and Brown, 1989; Brown et al., 1990) and may play an important role in the amelioration of ionoregulatory disturbances due to its stimulation of branchial chloride cell proliferation (Laurent and Perry, 1990) and branchial Na//K/ and Ca2/ – ATPase activities (Flik and Perry, 1989; Madsen, 1990). Plasma thyroid hormones have been measured in relatively few species of fish under low-pH conditions or
Al-containing acidic waters. Increased plasma T4 but stable plasma T3 concentrations have been consistently reported to occur in brown trout exposed to low-pH water or to Al in acidic water (Edwards et al., 1987; Brown et al., 1989; Whitehead and Brown, 1989), whereas the thyroidal responses of the rainbow trout in these waters have been inconsistent (Brown et al., 1984, 1986, 1990). The thyroidal system, particularly T3 , has been shown to be important for rainbow trout and brown trout adapting to salinity changes (Leloup and Lebel, 1993) and may have an important ionoregulatory role in these species.
MATERIALS AND METHODS Fish Husbandry and Water Quality Manipulations Animal husbandry, water quality manipulations, and water quality measurements were as described previously (Waring and Brown, 1995). Briefly, sexually immature brown trout of both sexes (200 – 500 g) were obtained from Roadwater Fisheries, Somerset, and allowed to recover from transport for at least 7 days in dechlorinated Exeter tap water (0.54 mM Na/, 0.36 mM Ca2/, 0.07 mM K/, 0.07 mM Si0, 0.004 mM F0, 6 mg liter01 Al, pH 7.1, 10 – 127) prior to study. Fish were fed every second day to satiation with a floating pelleted diet. The trout were then transferred to a 400-liter tank and acclimated to artificial soft water (ASW; 0.02 mM Ca2/; 0.03 mM Na/, 0.01 mM K/, 0.04 mM Cl0, 0.001 0 mM F0, 0.002 mM NO30, 0.001 mM PO20 4 , 0.001 mM Si , 20 0.03 mM SO4 , pH 7.0, 10 – 127) for at least 10 days. The fish were held under a 12L:12D photoperiod and were fed to satiation every 3rd day until cannulation. Trout were prepared by cannulation of the dorsal aorta and allowed to recover for 48 hr in one of three individual 6-liter darkened perspex aquaria, connected in series, with flow-through ASW (18 liters hr01). Water quality was manipulated where necessary with stock solutions of H2SO4 and Al(SO4 )3 to give five experimental groups of fish, i.e., pH 7.0 (n Å 6), pH 5.0 in the absence of Al (n Å 5), and pH 5.0 containing 50 mg liter01 Al (n Å 5), 25 mg liter01 Al (n Å 6), and 12.5 mg liter01 Al (n Å 8). Waring and Brown (1995) give full details of the method for water quality manipulation
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and for blood sampling collection. All fish were sampled at 0 (at pH 7.0, premanipulation of water pH or Al addition), 6, 12, 24, 48, 72, 96, and 120 hr postaddition of acid and Al. Plasma samples were stored at 0607 prior to analysis.
Plasma Hormone Analysis Plasma T4 and plasma T3 concentrations were measured by RIA (Edwards et al., 1987; Brown et al., 1989), except that the double antibody separation procedure used a second antibody covalently coupled to cellulose (Sac-Cel; ImmunoDiagnostic Systems, Boldon, UK). Plasma PRL concentrations were determined by RIA (Prunet et al., 1985) as has been previously used for plasma PRL measurements in brown trout (Tanguy et al., 1994). Plasma cortisol concentrations were determined by RIA of ethanol-extracted samples (Edwards et al., 1987; Brown et al., 1989). Anti-cortisol, anti-T4 , and anti-T3 sera were obtained from the Scottish Antibody Production Unit (SAPU; Law Hospital, Carluke, Strathclyde, Scotland). Cross-reactivities (mole for mole) of anti-T4 serum with T3 , anti-T3 serum with T4 , and anticortisol serum with other major corticosteroids were õ2.5, õ0.25, and õ0.5%, respectively (SAPU measurements). Our own measurements show that the anti-T3 serum and the anti-T4 serum cross-react with reverse T3 by 0.88 and 0.98%, respectively (mole for mole).
Statistical Analysis The data were log-transformed when necessary to achieve normality and homogeneity of variance. The data were analysed using an unbalanced one-way ANOVAR with repeated measures (PROC GLM; SAS Institute) excluding data for late time points where õ50% of fish survived at the two higher Al concentrations. When the F value indicated significance, a priori com-
FIG. 1. Plasma PRL concentrations in individual brown trout exposed to water of pH 7.0, pH 5.0, pH 5.0 plus 12.5 mg liter01 Al, pH 5.0 plus 25 mg liter01 Al, and pH 5.0 plus 50 mg liter01 Al. Fish which died during the exposure period are represented by open symbols, surviving fish are represented by closed symbols. 50 and 25 mg liter01 Al groups P õ 0.05 at 12 hr compared to 0 hr; all other time points in all groups NS.
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parisons of values at time 0 hr with values at each later sampling point were made using the CONTRAST option.
RESULTS No fish died in water of pH 5.0 or in water of pH 5.0 containing 12.5 mg liter01 Al. In water of pH 5.0 containing 25 mg liter01 Al, 50% of fish died over the 120hr period and all fish died in water of pH 5.0 containing 50 mg liter01 Al. The figures showing individual physiological parameters identify the fish which died due to exposure to Al (open symbols) from those surviving (closed symbols). Cannula failures prevented continued sample collection from some of the fish (two fish in the pH 5.0 group, one fish in pH 5.0 plus 25 mg liter01 Al, and four fish in pH 5.0 plus 12.5 mg liter01 Al). Plasma PRL concentrations exhibited wide variations at time 0 hr both within and between experimental groups (Fig. 1). Irrespective of the initial concentration, exposure to 25 and 50 mg liter01 Al in ASW of pH 5.0 caused a significant depression in plasma PRL concentrations at the 12-hr sampling point (Fig. 1). At 25 mg liter01 Al, plasma PRL concentrations were restored to values not significantly different from resting concentrations within the next 12 hr. Although plasma PRL concentrations were lower at 12 hr in trout exposed to 12.5 mg liter01 Al compared to the 0-hr concentrations (reduced to a mean of 38% of resting concentrations), this was not statistically significant. Exposure to water of pH 7.0 and 5.0 had no effect on plasma PRL concentrations. Repeated blood sampling of trout exposed to pH 7.0 or pH 5.0 ASW had no effect on plasma T4 concentrations (Fig. 2). Plasma T4 concentrations were, however, significantly greater in fish exposed to water of pH 5.0 con-
FIG. 2. Plasma T4 concentrations in individual brown trout exposed to water of pH 7.0, pH 5.0, pH 5.0 plus 12.5 mg liter01 Al, pH 5.0 plus 25 mg liter01 Al, and pH 5.0 plus 50 mg liter01 Al. Fish which died during the exposure period are represented by open symbols, surviving fish are represented by closed symbols. 50 mg liter01 Al group P õ 0.05 at 6 hr, P õ 0.01 at 12 hr compared to 0 hr; 12.5 mg liter01 Al group P õ 0.01 at 6, 24, 72, and 96 hr, P õ 0.05 at 12 and 48 hr compared to 0 hr; all other time points in all groups NS.
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taining 50 mg liter01 Al with a 5- to 10-fold increase prior to death. High plasma T4 concentrations occurred in the two fish surviving at 24 hr in 50 mg liter01 Al. Plasma T4 concentrations were unchanged by exposure to 25 mg liter01 Al in pH 5.0 ASW. Exposure to 12.5 mg liter01 Al in pH 5.0 ASW caused a significant increase in plasma T4 concentrations. There was a partial recovery in the T4 concentrations evident at 120 hr in the trout in this group. Plasma T3 concentrations were constant in all experimental groups except for fish exposed to 12.5 mg liter01 Al at pH 5.0 in which there was a sustained elevation in plasma T3 concentrations (Fig. 3). In trout exposed to 50 mg liter01 Al plasma cortisol concentrations were elevated at the 6-hr sampling point, and in the two fish that survived to the 24-hr timepoint, plasma cortisol levels were high (Fig. 4). Similarly, plasma cortisol concentrations were significantly increased from 6 hr onwards in the trout exposed to 25 mg liter01 Al and remained high in the two fish which survived up to 120 hr. Exposure to 12.5 mg liter01 Al at pH 5.0 induced the most dramatic, but more transient, elevation in plasma cortisol concentrations, with a recovery evident from 24 hr onwards.
DISCUSSION The initial PRL concentrations measured in the plasma of brown trout in the present study were variable and in some fish rather high compared to reported values in other salmonids (Pottinger et al., 1992), including recent measurements in the brown trout (Tanguy et al., 1994). Irrespective of these variations, the data clearly show that Al exposure depressed plasma PRL concentrations at the 12-hr sampling point in fish exposed to the two highest Al concentrations. However, PRL concentrations were stable in trout exposed to the sublethal Al concentration of 12.5 mg liter01. The declining circulating PRL concentrations of brown trout in
FIG. 3. Plasma T3 concentrations in individual brown trout exposed to water of pH 7.0, pH 5.0, pH 5.0 plus 12.5 mg liter01 Al, pH 5.0 plus
25 mg liter01 Al, and pH 5.0 plus 50 mg liter01 Al. Fish which died during the exposure period are represented by open symbols, surviving fish are represented by closed symbols. 12.5 mg liter01 Al group P õ 0.05 at 12, 24, 48, and 72 hr compared to 0 hr; all other time points in all groups NS.
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these early stages of exposure to 50 and 25 mg liter01 Al in acidic soft waters supports previous suggestions of decreased pituitary PRL cell activity in acid or acid plus Al-exposed brook trout, Salvelinus fontinalis (Notter et al., 1976; Fryer et al., 1988). After the initial decline in plasma PRL shown by fish exposed to 25 mg liter01 Al, there was a wide variability in the pattern of change in plasma PRL concentrations with little apparent correlation between the plasma concentrations and the ultimate fate of the fish. Salmonids would thus appear to differ from tilapia which shows an activation of the pituitary PRL cells in response to acidic waters and Al stress (Wendelaar Bonga et al., 1984a,b, 1988). In longer-term acid-exposed tilapia, the activation of pituitary PRL cells was associated with a reduced diffusional Na/ loss and a reestablishment of a positive Na/ balance (Flik et al., 1989). There was no correlation between the changes in plasma PRL concentrations and the plasma Na/ and Cl0 concentrations in the present study. Plasma PRL concentrations declined at the two highest Al concentrations whereas plasma Na/ and Cl0 concentrations were stable (Waring and Brown, 1995). Trout exposed to a sublethal concentration of Al showed stable plasma PRL concentrations but plasma Na/ and Cl0 concentrations declined and subsequently recovered (Waring and Brown, 1995). The decline in plasma PRL concentrations demonstrated by brown trout was only apparent in arguably a more stressful environment where mortalities were common. Thus the plasma PRL responses may reflect a rather more general stress-induced effect in salmonids. This idea is supported by the decline in plasma PRL concentrations in physically stressed rainbow trout with or without declining water quality (Pottinger et al., 1992). Plasma cortisol concentrations have been reported to increase significantly in fish exposed to acidic waters containing Al (Goss and Wood, 1988; Wood et al., 1988; Whitehead and Brown, 1989; Brown et al., 1990; Witters et al., 1991). However, none of these studies examined
FIG. 4. Plasma cortisol concentrations in individual brown trout exposed to water of pH 7.0, pH 5.0, pH 5.0 plus 12.5 mg liter01 Al,
pH 5.0 plus 25 mg liter01 Al, and pH 5.0 plus 50 mg liter01 Al. Fish which died during the exposure period are represented by open symbols, surviving fish are represented by closed symbols. 50 mg liter01 Al group P õ 0.05 at 6 hr, P õ 0.001 at 12 hr compared to 0 hr; 25 mg liter01 Al group P õ 0.05 at 6, 12, and 24 hr compared to 0 hr; 12.5 mg liter01 Al group P õ 0.05 at 6, 12, and 24 hr compared to 0 hr; all other time points in all groups NS.
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the responses of fish to more than one water Al concentration. In fish exposed to acidic conditions with no Al present a dose-dependent increase in plasma concentrations with increasing acidity has occasionally been observed (Brown et al., 1984; Scherer et al., 1986). In the brown trout, plasma cortisol concentrations did not increase to a greater degree with increasing water Al concentrations. Indeed, sublethally exposed fish tended to show a higher plasma cortisol response, with 5 of 8 fish having a plasma concentration ú100 ng ml01 in the first 24 hr. In trout in the higher water Al concentrations only 1 of the 11 fish had a ú100 ng ml01 cortisol concentration during the same period, when the mortalities usually occurred, despite similar resting concentrations. However, Brown and Whitehead (1995) measured much greater plasma cortisol concentrations (400 – 2000 ng ml01) in Al-exposed brown trout in pH 5.0 soft water, where ú60% mortality occurred, which suggests that the plasma cortisol peak level-to-survival relationship in this salmonid is not straightforward. Elevated plasma T4 concentrations over a 5-day period in brown trout exposed to 50 and 12.5 mg liter01 Al in the present experiments are consistent with previous studies (of 2- to 7-day exposures) on the brown trout in acidic soft water and acidic soft water containing Al (Edwards et al., 1987; Brown et al., 1989; Whitehead and Brown, 1989). These observations appear to contrast markedly with the apparent lack of a consistent thyroid response in another salmonid, the rainbow trout. Rainbow trout exposed to pH 4.7 or pH 5.2 water for 21 days exhibited no changes in plasma T4 or T3 concentrations, whereas fish exposed to pH 4.2 water had elevated plasma T4 concentrations (Table 1 in Brown et al., 1984). In a more detailed investigation of the temporal changes in plasma thyroid hormone concentrations in rainbow trout exposed to pH 4.7 water, plasma T4 and T3 concentrations were initially stable (Brown et al., 1984). However, the same study showed that plasma T3 concentrations significantly decreased relative to controls (pH 7.7) at 8 – 21 days and a decreased plasma T4 concentration relative to controls was evident at 21 days. In a further study, 21 days of exposure of rainbow trout to pH 4.8 water had no significant effect on plasma thyroid hormones (Brown et al., 1986) nor did a 7-day exposure of rainbow trout to pH 4.7 water containing 540 mg liter01 Al affect plasma T4 or T3 concentrations (Brown et al., 1990). However, plasma T4 concentrations
in rainbow trout can respond to certain types of stress. Acute physical disturbance, i.e., injection and handling stress, significantly elevates plasma T4 concentrations in the first few hours after the stress (Brown et al., 1978; Himick and Eales, 1990). Interestingly, a similar acute physical stress has been reported not to affect plasma T4 concentrations in the brown trout (Pickering et al., 1982). Plasma T3 concentrations were either not reported (Pickering et al., 1982) or were not affected by acute physical stress (Brown et al., 1978; Himick and Eales, 1990). In the previous experiments on brown trout in fairly harsh environmental conditions [low pH (pH 4.0) alone or pH 5.0 containing ú50 mg liter01 Al], despite the elevated circulating T4 concentrations, the plasma concentrations of T3 were unchanged (Edwards et al., 1987; Brown et al., 1989; Whitehead and Brown, 1989). In the present studies, plasma T3 concentrations were again stable in acidic waters containing lethal Al concentrations. However, during exposure to a sublethal concentration of Al (12.5 mg liter01) brown trout exhibited a sustained increase in plasma T3 concentrations for 12 – 72 hr. There is no explanation for this differential increase in plasma T3 concentrations, but it does show that the physiologically more active plasma thyroid hormone (at least in rainbow trout; Bres and Eales, 1985) can increase in response to stress in the brown trout. In summary, the increased plasma T3 concentrations in Al-exposed brown trout at timepoints associated with a transient decline in their plasma Na/ and Cl0 concentrations, with subsequent recovery (Waring and Brown, 1995), may suggest that T3 may play a role in this recovery. No clear role for PRL in this recovery was evident. One possibility is that T3 may act synergistically with other ionoregulatory hormones such as cortisol and growth hormone. For example, Leloup and Lebel (1993) suggest that the seawater-adapting actions of growth hormone in brown and rainbow trout require T3 . Alternatively, the endocrine changes in Al-stressed brown trout may be a nonspecific effect of stress on the endocrine system.
ACKNOWLEDGMENTS We thank the Royal Society and the Fisheries Society of the British Isles for their assistance in the travel between our laboratories. Thanks
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are also due to Phil and Jan Shears for their help with fish husbandry. This work was funded by the Natural Environment Research Council.
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Aluminium Effects on Brown Trout Endocrine Status (Oncorhynchus mykiss) in fresh water. Gen. Comp. Endocrinol. 88, 454 – 460. Prunet, P., Boeuf, G., and Houdebine, L. M. (1985). Plasma and pituitary prolactin levels in rainbow trout during adaptation to different salinities. J. Exp. Zool. 235, 187 – 196. Scherer, E., Harrison, S. E., and Brown, S. B. (1986). Locomotor activity and blood plasma parameters of acid-exposed lake whitefish, Coregonus clupeaformis. Can. J. Fish. Aquat. Sci. 43, 1556 – 1561. Tanguy, J. M., Ombredane, D., Baglinie`re, J. L., and Prunet, P. (1994). Aspects of parr – smolt transformation in anadromous and resident forms of brown trout (Salmo trutta) in comparison with Atlantic salmon (Salmo salar). Aquaculture 121, 51 – 63. Waring, C. P., and Brown, J. A. (1995). Ionoregulatory and respiratory responses of brown trout, Salmo trutta, to lethal and sublethal aluminium in acidic soft waters. Fish Physiol. Biochem. 14, 81 – 91. Wendelaar Bonga, S. E., Van Der Meij, J. C. A., and Flik, G. (1984a). Prolactin and acid stress in the teleost Oreochromis (formerly Sarotherodon) mossambicus. Gen. Comp. Endocrinol. 55, 323 – 332.
Wendelaar Bonga, S. E., Van Der Meij, J. C. A., Van Der Krabben, W. A. W. A., and Flik, G. (1984b). The effects of water acidification on prolactin cells and pars intermedia PAS-positive cells in the teleost fish Oreochromis (formerly Sarotherodon) mossambicus and Carassius auratus. Cell Tissue Res. 238, 601 – 609. Wendelaar Bonga, S. E., Balm, P., and Flik, G. (1988). Control of prolactin secretion in the teleost Oreochromis mossambicus: Effects of water acidification. Gen. Comp. Endocrinol. 72, 1 – 12. Whitehead, C., and Brown, J. A. (1989). Endocrine responses of brown trout, Salmo trutta L., to acid, aluminium and lime dosing in a Welsh hill stream. J. Fish Biol. 35, 59 – 71. Witters, H. E., Van Puymbroeck, S., and Vanderborght, O. L. J. (1991). Adrenergic response to physiological disturbances in rainbow trout, Oncorhynchus mykiss, exposed to aluminum at acid pH. Can. J. Fish. Aquat. Sci. 48, 414 – 420. Wood, C. M., Simons, B. P., Mount, D. R., and Bergman, H. L. (1988). Physiological evidence of acclimation to acid/aluminum stress in adult brook trout (Salvelinus fontinalis). 2. Blood parameters by cannulation. Can. J. Fish. Aquat. Sci. 45, 1597 – 1605.
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Plasma Prolactin, Cortisol, and Thyroid Responses of the Brown Trout (Salmo trutta) Exposed to Lethal and Sublethal Aluminium in Acidic Soft Waters C. P. Waring,1 J. A. Brown, J. E. Collins, and P. Prunet* Department of Biological Sciences, Hatherly Laboratories, University of Exeter, Exeter, Devon EX4 4PS, United Kingdom; and *Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France Accepted February 6, 1996
Brown trout, with indwelling dorsal aortic cannulae, were exposed to various concentrations of aluminium (Al; 50 mg liter01, 100% mortality over 48 hr; 25 mg liter01, 50% mortality over 120 hr; 12.5 mg liter01, 0% mortality over 120 hr) in acidic (pH 5.0) soft water. The plasma concentrations of prolactin (PRL), cortisol, thyroxine (T4), and triiodothyronine (T3) were monitored. Plasma PRL concentrations were transiently depressed (to less than 20% of resting concentrations) after 12 hr in trout in the two highest water Al concentrations, but were unchanged in the trout exposed to 12.5 mg liter01 Al. Plasma cortisol concentrations were elevated in response to all water Al levels and remained elevated in trout in the lethal conditions. The sublethally exposed trout showed a recovery in plasma cortisol concentrations by 120 hr. Plasma T4 concentrations were significantly elevated in trout exposed to both the lethal and the sublethal Al concentrations (from mean resting concentrations of 1–2 ng ml01 to peaks of 8.9 and 9.0 ng ml01 in the 50 and 12.5 mg liter01 Al groups, respectively), although a recovery in plasma concentrations was evident in the sublethally exposed trout from 72 hr onwards. Plasma T3 concentrations were relatively stable in the trout exposed to the two highest doses of Al, whereas the trout under the lowest, sublethal, Al condi1 Present address: MAFF, Fisheries Laboratory, Pakefield Road, Lowestoft, Suffolk, NR33 0HT, UK.
tions exhibited a sustained (12–72 hr) elevation in plasma T3 concentrations (from a mean resting concentration of 0.9 ng ml01 to a peak of 4.2 ng ml01 at 48 hr). No clear relationship was apparent between the plasma PRL concentrations and the previously reported ionoregulatory status of the trout. q 1996 Academic Press, Inc.
Freshwater fish species acutely exposed to aluminium (Al) in acidic soft waters show ionoregulatory, cardiovascular, and respiratory disruptions (Wood et al., 1988; Playle and Wood, 1989; Playle et al., 1989). The respiratory and/or ionoregulatory disturbances resulting from exposure to Al have been suggested to depend upon water pH (Playle et al., 1989) and the Al species present (Pole´o, 1995; Brown and Waring, 1995). During prolonged exposure to acidic water with or without the presence of Al, some fish species or individuals show at least a partial recovery of hydromineral balance and respiratory function (Wood et al., 1988; McDonald et al., 1991) and hormones are likely to play a significant role in this recovery. In this respect, many studies have shown that plasma cortisol concentrations are elevated after exposure to acidic waters and acidic soft waters containing Al (Goss and Wood, 1988; Whitehead and Brown, 1989; Brown et al., 1990), although, at least in rainbow trout, this response is not always apparent when water Al concentrations are low
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(Balm and Pottinger, 1993). Plasma catecholamines have been reported to be elevated in acidic waters containing Al (Witters et al., 1991; Brown and Whitehead, 1995), while again some studies did not show mobilisation of plasma catecholamines in salmonids exposed to acidic conditions without Al (Audet and Wood, 1993). In a recent study of brown trout exposed to lethal Al concentrations (50 mg liter01; 100% mortality), intermediate Al concentrations (25 mg liter01; 50% mortality), and sublethal Al concentrations (12.5 mg liter01; 0% mortality) in pH 5.0 soft water for up to 5 days, fish exposed to the sublethal Al concentration showed transiently reduced plasma Na/ and Cl0 concentrations, whereas the fish exposed to the two highest water Al concentrations had stable plasma monovalent ion concentrations (Waring and Brown, 1995). The marked differences in mortality and the physiological responses to different water Al concentrations prompted the measurement of prolactin (PRL), cortisol, thyroxine (T4 ), and triiodothyronine (T3 ) concentrations in the plasma samples from these trout to assess whether these hormones might be implicated in their recovery and/or adaptation. PRL has a well-known ionoregulatory role in freshwater salmonids (reviewed by Bern and Madsen, 1992) and has been suggested to play a role in counteracting H/-induced ionic disturbances in tilapia, Oreochromis mossambicus (Flik et al., 1989). Studies on the tilapia have suggested that PRL is released in fish exposed to low-pH conditions alongside declining plasma monovalent ions (Wendelaar Bonga et al., 1988; Flik et al., 1989). Salmonids exposed to low-pH water with or without Al present show structural alterations in pituitary PRL cells, indicating inhibition rather than stimulation of PRL release (Notter et al., 1976; Fryer et al., 1988). More recently, rainbow trout showed stable plasma PRL concentrations when sampled 14 days after exposure to pH 4.0 soft water (Balm et al., 1995). Plasma cortisol concentrations increase in salmonids exposed to Al in acidic waters (Goss and Wood, 1988; Whitehead and Brown, 1989; Brown et al., 1990) and may play an important role in the amelioration of ionoregulatory disturbances due to its stimulation of branchial chloride cell proliferation (Laurent and Perry, 1990) and branchial Na//K/ and Ca2/ – ATPase activities (Flik and Perry, 1989; Madsen, 1990). Plasma thyroid hormones have been measured in relatively few species of fish under low-pH conditions or
Al-containing acidic waters. Increased plasma T4 but stable plasma T3 concentrations have been consistently reported to occur in brown trout exposed to low-pH water or to Al in acidic water (Edwards et al., 1987; Brown et al., 1989; Whitehead and Brown, 1989), whereas the thyroidal responses of the rainbow trout in these waters have been inconsistent (Brown et al., 1984, 1986, 1990). The thyroidal system, particularly T3 , has been shown to be important for rainbow trout and brown trout adapting to salinity changes (Leloup and Lebel, 1993) and may have an important ionoregulatory role in these species.
MATERIALS AND METHODS Fish Husbandry and Water Quality Manipulations Animal husbandry, water quality manipulations, and water quality measurements were as described previously (Waring and Brown, 1995). Briefly, sexually immature brown trout of both sexes (200 – 500 g) were obtained from Roadwater Fisheries, Somerset, and allowed to recover from transport for at least 7 days in dechlorinated Exeter tap water (0.54 mM Na/, 0.36 mM Ca2/, 0.07 mM K/, 0.07 mM Si0, 0.004 mM F0, 6 mg liter01 Al, pH 7.1, 10 – 127) prior to study. Fish were fed every second day to satiation with a floating pelleted diet. The trout were then transferred to a 400-liter tank and acclimated to artificial soft water (ASW; 0.02 mM Ca2/; 0.03 mM Na/, 0.01 mM K/, 0.04 mM Cl0, 0.001 0 mM F0, 0.002 mM NO30, 0.001 mM PO20 4 , 0.001 mM Si , 20 0.03 mM SO4 , pH 7.0, 10 – 127) for at least 10 days. The fish were held under a 12L:12D photoperiod and were fed to satiation every 3rd day until cannulation. Trout were prepared by cannulation of the dorsal aorta and allowed to recover for 48 hr in one of three individual 6-liter darkened perspex aquaria, connected in series, with flow-through ASW (18 liters hr01). Water quality was manipulated where necessary with stock solutions of H2SO4 and Al(SO4 )3 to give five experimental groups of fish, i.e., pH 7.0 (n Å 6), pH 5.0 in the absence of Al (n Å 5), and pH 5.0 containing 50 mg liter01 Al (n Å 5), 25 mg liter01 Al (n Å 6), and 12.5 mg liter01 Al (n Å 8). Waring and Brown (1995) give full details of the method for water quality manipulation
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and for blood sampling collection. All fish were sampled at 0 (at pH 7.0, premanipulation of water pH or Al addition), 6, 12, 24, 48, 72, 96, and 120 hr postaddition of acid and Al. Plasma samples were stored at 0607 prior to analysis.
Plasma Hormone Analysis Plasma T4 and plasma T3 concentrations were measured by RIA (Edwards et al., 1987; Brown et al., 1989), except that the double antibody separation procedure used a second antibody covalently coupled to cellulose (Sac-Cel; ImmunoDiagnostic Systems, Boldon, UK). Plasma PRL concentrations were determined by RIA (Prunet et al., 1985) as has been previously used for plasma PRL measurements in brown trout (Tanguy et al., 1994). Plasma cortisol concentrations were determined by RIA of ethanol-extracted samples (Edwards et al., 1987; Brown et al., 1989). Anti-cortisol, anti-T4 , and anti-T3 sera were obtained from the Scottish Antibody Production Unit (SAPU; Law Hospital, Carluke, Strathclyde, Scotland). Cross-reactivities (mole for mole) of anti-T4 serum with T3 , anti-T3 serum with T4 , and anticortisol serum with other major corticosteroids were õ2.5, õ0.25, and õ0.5%, respectively (SAPU measurements). Our own measurements show that the anti-T3 serum and the anti-T4 serum cross-react with reverse T3 by 0.88 and 0.98%, respectively (mole for mole).
Statistical Analysis The data were log-transformed when necessary to achieve normality and homogeneity of variance. The data were analysed using an unbalanced one-way ANOVAR with repeated measures (PROC GLM; SAS Institute) excluding data for late time points where õ50% of fish survived at the two higher Al concentrations. When the F value indicated significance, a priori com-
FIG. 1. Plasma PRL concentrations in individual brown trout exposed to water of pH 7.0, pH 5.0, pH 5.0 plus 12.5 mg liter01 Al, pH 5.0 plus 25 mg liter01 Al, and pH 5.0 plus 50 mg liter01 Al. Fish which died during the exposure period are represented by open symbols, surviving fish are represented by closed symbols. 50 and 25 mg liter01 Al groups P õ 0.05 at 12 hr compared to 0 hr; all other time points in all groups NS.
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parisons of values at time 0 hr with values at each later sampling point were made using the CONTRAST option.
RESULTS No fish died in water of pH 5.0 or in water of pH 5.0 containing 12.5 mg liter01 Al. In water of pH 5.0 containing 25 mg liter01 Al, 50% of fish died over the 120hr period and all fish died in water of pH 5.0 containing 50 mg liter01 Al. The figures showing individual physiological parameters identify the fish which died due to exposure to Al (open symbols) from those surviving (closed symbols). Cannula failures prevented continued sample collection from some of the fish (two fish in the pH 5.0 group, one fish in pH 5.0 plus 25 mg liter01 Al, and four fish in pH 5.0 plus 12.5 mg liter01 Al). Plasma PRL concentrations exhibited wide variations at time 0 hr both within and between experimental groups (Fig. 1). Irrespective of the initial concentration, exposure to 25 and 50 mg liter01 Al in ASW of pH 5.0 caused a significant depression in plasma PRL concentrations at the 12-hr sampling point (Fig. 1). At 25 mg liter01 Al, plasma PRL concentrations were restored to values not significantly different from resting concentrations within the next 12 hr. Although plasma PRL concentrations were lower at 12 hr in trout exposed to 12.5 mg liter01 Al compared to the 0-hr concentrations (reduced to a mean of 38% of resting concentrations), this was not statistically significant. Exposure to water of pH 7.0 and 5.0 had no effect on plasma PRL concentrations. Repeated blood sampling of trout exposed to pH 7.0 or pH 5.0 ASW had no effect on plasma T4 concentrations (Fig. 2). Plasma T4 concentrations were, however, significantly greater in fish exposed to water of pH 5.0 con-
FIG. 2. Plasma T4 concentrations in individual brown trout exposed to water of pH 7.0, pH 5.0, pH 5.0 plus 12.5 mg liter01 Al, pH 5.0 plus 25 mg liter01 Al, and pH 5.0 plus 50 mg liter01 Al. Fish which died during the exposure period are represented by open symbols, surviving fish are represented by closed symbols. 50 mg liter01 Al group P õ 0.05 at 6 hr, P õ 0.01 at 12 hr compared to 0 hr; 12.5 mg liter01 Al group P õ 0.01 at 6, 24, 72, and 96 hr, P õ 0.05 at 12 and 48 hr compared to 0 hr; all other time points in all groups NS.
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taining 50 mg liter01 Al with a 5- to 10-fold increase prior to death. High plasma T4 concentrations occurred in the two fish surviving at 24 hr in 50 mg liter01 Al. Plasma T4 concentrations were unchanged by exposure to 25 mg liter01 Al in pH 5.0 ASW. Exposure to 12.5 mg liter01 Al in pH 5.0 ASW caused a significant increase in plasma T4 concentrations. There was a partial recovery in the T4 concentrations evident at 120 hr in the trout in this group. Plasma T3 concentrations were constant in all experimental groups except for fish exposed to 12.5 mg liter01 Al at pH 5.0 in which there was a sustained elevation in plasma T3 concentrations (Fig. 3). In trout exposed to 50 mg liter01 Al plasma cortisol concentrations were elevated at the 6-hr sampling point, and in the two fish that survived to the 24-hr timepoint, plasma cortisol levels were high (Fig. 4). Similarly, plasma cortisol concentrations were significantly increased from 6 hr onwards in the trout exposed to 25 mg liter01 Al and remained high in the two fish which survived up to 120 hr. Exposure to 12.5 mg liter01 Al at pH 5.0 induced the most dramatic, but more transient, elevation in plasma cortisol concentrations, with a recovery evident from 24 hr onwards.
DISCUSSION The initial PRL concentrations measured in the plasma of brown trout in the present study were variable and in some fish rather high compared to reported values in other salmonids (Pottinger et al., 1992), including recent measurements in the brown trout (Tanguy et al., 1994). Irrespective of these variations, the data clearly show that Al exposure depressed plasma PRL concentrations at the 12-hr sampling point in fish exposed to the two highest Al concentrations. However, PRL concentrations were stable in trout exposed to the sublethal Al concentration of 12.5 mg liter01. The declining circulating PRL concentrations of brown trout in
FIG. 3. Plasma T3 concentrations in individual brown trout exposed to water of pH 7.0, pH 5.0, pH 5.0 plus 12.5 mg liter01 Al, pH 5.0 plus
25 mg liter01 Al, and pH 5.0 plus 50 mg liter01 Al. Fish which died during the exposure period are represented by open symbols, surviving fish are represented by closed symbols. 12.5 mg liter01 Al group P õ 0.05 at 12, 24, 48, and 72 hr compared to 0 hr; all other time points in all groups NS.
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these early stages of exposure to 50 and 25 mg liter01 Al in acidic soft waters supports previous suggestions of decreased pituitary PRL cell activity in acid or acid plus Al-exposed brook trout, Salvelinus fontinalis (Notter et al., 1976; Fryer et al., 1988). After the initial decline in plasma PRL shown by fish exposed to 25 mg liter01 Al, there was a wide variability in the pattern of change in plasma PRL concentrations with little apparent correlation between the plasma concentrations and the ultimate fate of the fish. Salmonids would thus appear to differ from tilapia which shows an activation of the pituitary PRL cells in response to acidic waters and Al stress (Wendelaar Bonga et al., 1984a,b, 1988). In longer-term acid-exposed tilapia, the activation of pituitary PRL cells was associated with a reduced diffusional Na/ loss and a reestablishment of a positive Na/ balance (Flik et al., 1989). There was no correlation between the changes in plasma PRL concentrations and the plasma Na/ and Cl0 concentrations in the present study. Plasma PRL concentrations declined at the two highest Al concentrations whereas plasma Na/ and Cl0 concentrations were stable (Waring and Brown, 1995). Trout exposed to a sublethal concentration of Al showed stable plasma PRL concentrations but plasma Na/ and Cl0 concentrations declined and subsequently recovered (Waring and Brown, 1995). The decline in plasma PRL concentrations demonstrated by brown trout was only apparent in arguably a more stressful environment where mortalities were common. Thus the plasma PRL responses may reflect a rather more general stress-induced effect in salmonids. This idea is supported by the decline in plasma PRL concentrations in physically stressed rainbow trout with or without declining water quality (Pottinger et al., 1992). Plasma cortisol concentrations have been reported to increase significantly in fish exposed to acidic waters containing Al (Goss and Wood, 1988; Wood et al., 1988; Whitehead and Brown, 1989; Brown et al., 1990; Witters et al., 1991). However, none of these studies examined
FIG. 4. Plasma cortisol concentrations in individual brown trout exposed to water of pH 7.0, pH 5.0, pH 5.0 plus 12.5 mg liter01 Al,
pH 5.0 plus 25 mg liter01 Al, and pH 5.0 plus 50 mg liter01 Al. Fish which died during the exposure period are represented by open symbols, surviving fish are represented by closed symbols. 50 mg liter01 Al group P õ 0.05 at 6 hr, P õ 0.001 at 12 hr compared to 0 hr; 25 mg liter01 Al group P õ 0.05 at 6, 12, and 24 hr compared to 0 hr; 12.5 mg liter01 Al group P õ 0.05 at 6, 12, and 24 hr compared to 0 hr; all other time points in all groups NS.
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the responses of fish to more than one water Al concentration. In fish exposed to acidic conditions with no Al present a dose-dependent increase in plasma concentrations with increasing acidity has occasionally been observed (Brown et al., 1984; Scherer et al., 1986). In the brown trout, plasma cortisol concentrations did not increase to a greater degree with increasing water Al concentrations. Indeed, sublethally exposed fish tended to show a higher plasma cortisol response, with 5 of 8 fish having a plasma concentration ú100 ng ml01 in the first 24 hr. In trout in the higher water Al concentrations only 1 of the 11 fish had a ú100 ng ml01 cortisol concentration during the same period, when the mortalities usually occurred, despite similar resting concentrations. However, Brown and Whitehead (1995) measured much greater plasma cortisol concentrations (400 – 2000 ng ml01) in Al-exposed brown trout in pH 5.0 soft water, where ú60% mortality occurred, which suggests that the plasma cortisol peak level-to-survival relationship in this salmonid is not straightforward. Elevated plasma T4 concentrations over a 5-day period in brown trout exposed to 50 and 12.5 mg liter01 Al in the present experiments are consistent with previous studies (of 2- to 7-day exposures) on the brown trout in acidic soft water and acidic soft water containing Al (Edwards et al., 1987; Brown et al., 1989; Whitehead and Brown, 1989). These observations appear to contrast markedly with the apparent lack of a consistent thyroid response in another salmonid, the rainbow trout. Rainbow trout exposed to pH 4.7 or pH 5.2 water for 21 days exhibited no changes in plasma T4 or T3 concentrations, whereas fish exposed to pH 4.2 water had elevated plasma T4 concentrations (Table 1 in Brown et al., 1984). In a more detailed investigation of the temporal changes in plasma thyroid hormone concentrations in rainbow trout exposed to pH 4.7 water, plasma T4 and T3 concentrations were initially stable (Brown et al., 1984). However, the same study showed that plasma T3 concentrations significantly decreased relative to controls (pH 7.7) at 8 – 21 days and a decreased plasma T4 concentration relative to controls was evident at 21 days. In a further study, 21 days of exposure of rainbow trout to pH 4.8 water had no significant effect on plasma thyroid hormones (Brown et al., 1986) nor did a 7-day exposure of rainbow trout to pH 4.7 water containing 540 mg liter01 Al affect plasma T4 or T3 concentrations (Brown et al., 1990). However, plasma T4 concentrations
in rainbow trout can respond to certain types of stress. Acute physical disturbance, i.e., injection and handling stress, significantly elevates plasma T4 concentrations in the first few hours after the stress (Brown et al., 1978; Himick and Eales, 1990). Interestingly, a similar acute physical stress has been reported not to affect plasma T4 concentrations in the brown trout (Pickering et al., 1982). Plasma T3 concentrations were either not reported (Pickering et al., 1982) or were not affected by acute physical stress (Brown et al., 1978; Himick and Eales, 1990). In the previous experiments on brown trout in fairly harsh environmental conditions [low pH (pH 4.0) alone or pH 5.0 containing ú50 mg liter01 Al], despite the elevated circulating T4 concentrations, the plasma concentrations of T3 were unchanged (Edwards et al., 1987; Brown et al., 1989; Whitehead and Brown, 1989). In the present studies, plasma T3 concentrations were again stable in acidic waters containing lethal Al concentrations. However, during exposure to a sublethal concentration of Al (12.5 mg liter01) brown trout exhibited a sustained increase in plasma T3 concentrations for 12 – 72 hr. There is no explanation for this differential increase in plasma T3 concentrations, but it does show that the physiologically more active plasma thyroid hormone (at least in rainbow trout; Bres and Eales, 1985) can increase in response to stress in the brown trout. In summary, the increased plasma T3 concentrations in Al-exposed brown trout at timepoints associated with a transient decline in their plasma Na/ and Cl0 concentrations, with subsequent recovery (Waring and Brown, 1995), may suggest that T3 may play a role in this recovery. No clear role for PRL in this recovery was evident. One possibility is that T3 may act synergistically with other ionoregulatory hormones such as cortisol and growth hormone. For example, Leloup and Lebel (1993) suggest that the seawater-adapting actions of growth hormone in brown and rainbow trout require T3 . Alternatively, the endocrine changes in Al-stressed brown trout may be a nonspecific effect of stress on the endocrine system.
ACKNOWLEDGMENTS We thank the Royal Society and the Fisheries Society of the British Isles for their assistance in the travel between our laboratories. Thanks
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are also due to Phil and Jan Shears for their help with fish husbandry. This work was funded by the Natural Environment Research Council.
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