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Seasonal variations in osmoregulatory and respiratory responses to seawater exposure of juvenile Atlantic salmon (Salmo salar) maintained in freshwater
Aquaculture, 82 (1989) 219-228 Elsevier Science Publishers B.V., Amsterdam
219
-
Printed
in The Netherlands
Seasonal Variations in Osmoregulatory and Respiratory Responses to Seawater Exposure of Juvenile Atlantic Salmon (Salmo salar) Maintained in Freshwater R.M. STAGG’*, C. TALBOT’,
F.B. EDDY3 and M. WILLIAMS3
‘Heriot- Watt University, Department of Brewing and Biological Sciences, Chambers Street, Edinburgh EHI 1HX (Great Britain) ‘Freshwater Fisheries Laboratory, Pitlochry, Perthshire PH16 5LB (Great Britain) 3University of Dundee, Department of Biological Sciences, Dundee DDl 4H (Great Britain)
ABSTRACT Stagg, R.M., Talbot, C., Eddy, F.B. and Williams, M., 1989. Seasonal variations in osmoregulatory and respiratory responses to seawater exposure of juvenile Atlantic salmon (Salmo salar ) maintained in freshwater. Aquaculture, 82: 219-228. Three- or four-year-old Atlantic salmon (Salmo salar) which first smolted in their second spring (S2 smolts) and had been kept in freshwater since hatching have the external characteristics of parr in December and smolts in April. Such fish were used to investigate the acute effects (up to 48 h) of rapid changeover from freshwater to seawater on respiratory, acid-base and ionoregulatory homeostasis. Over the first 18 h of seawater exposure the plasma sodium, chloride and total osmotic concentrations rose rapidly in a similar manner for both groups of fish. In the smolt-like fish levels then stabilised whereas in Parr-like fish they continued to rise to the end of the experiment. Immediately after exposure to seawater, there was a rapid rise in blood pH and a fall in arterial P, 0, which were transient in the smolt-like and more severe and prolonged in the parrlike fish. These results are discussed in the context of branchial dehydration and cell volume control. It is suggested that an important aspect of smolting is the ability to regulate branchial dehydration since this must precede activation of the branchial ion excretory mechanisms regulating extracellular fluid composition following seawater transfer.
INTRODUCTION
Smolting in Atlantic salmon (Salmo sakzr ) is a complex series of physiological, morphological, biochemical and behavioural changes that occur in the *Present address and address for correspondence: Department of Agriculture and Fisheries for Scotland, Marine Laboratory, P.O. Box 101, Victoria Road, Torry, Aberdeen AB9 8DB (Great Britain).
0044-8486/89/$03.50
0 1989 Elsevier Science Publishers
B.V.
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spring (Folmar and Dickhoff, 1980, 1981; McCormick and Saunders, 1987). These changes result in the stenohaline, freshwater parr becoming euryhaline, and accompany the migration of the smolt from its natal river to the sea. The changes that occur during smolting are reversible and smolts which are prevented from migrating will revert to having Parr-like characteristics (Hoar, 1976). Abrupt transfer of euryhaline salmonids from freshwater to seawater elicits an acute adaptive or crisis phase, characterised by a rapid rise in plasma electrolyte concentrations, and a chronic regulatory phase when plasma ions decline to constant levels (Holmes and Donaldson, 1969; Bath and Eddy, 1979a). However, euryhalinity must also be related to the ability to tolerate the profound initial changes that occur, in the gill epithelium, upon transfer from freshwater to seawater because this tissue is in direct contact with the external medium. Shuttleworth and Freeman (1973) showed that in the eel (Anguillu dieffenbachii) there was a marked reduction in gill intracellular space during the first 6 h after transfer from freshwater to seawater which suggests that the gill was undergoing extensive shrinkage. In rainbow trout (Salmo guirdneri) one of the consequences of such effects for gill function is a transient reduction in oxygen transfer factor during the crisis period following exposure to seawater (Bath and Eddy, 1979b). Acute changes in respiratory parameters may contribute to mortalities following seawater transfer but to date such effects have not been studied in salmon. The objectives of this study were: (1) to compare the acute ionoregulatory ability of large Atlantic salmon (Salmo s&r) in December (Parr-like) and April (smolt-like) following rapid transfer from freshwater to seawater, and (2) to determine the consequences of such transfer for the respiratory and extracellular acid-base status of the animal. Such a study involves the use of cannulated fish in excess of 300 g. For Atlantic salmon smolts this can only be achieved using fish reared in freshwater for a number of years to a suitable size on the assumption that once the first smolting has occurred the seasonal smolting process subsequently occurs annually and is independent of age. MATERIALS AND METHODS
Atlantic salmon, Sulmo s&r which had been maintained in freshwater at the Freshwater Fisheries Laboratory since hatching were used. The fish had first smolted in their second spring (S2 smolts) and were subsequently retained in freshwater for an additional 2 years. On the basis of external appearance the fish were considered to be smolt-like in April (silvered, low condition factor) but reverted to Parr-like in December (prominent parr marks, high condition factor). They weighed (mean + se., n in parentheses) 441+ 19 (15) g and 557 rt 42 (9) g, respectively. Fish were anaesthetised with 20-40 mg 1-l benzocaine and cannulated, for
SEASONAL RESPONSES TO SEAWATER OF S. SALAR MAINTAINED IN FRESHWATER
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blood sampling, via the dorsal aorta with a short length of polyethylene tubing (i.d. 0.58 mm, o.d. 0.96 mm) containing heparinised saline (Smith and Bell, 1964). Fish were then placed in individual tanks containing freshwater and allowed to recover for at least 16 h. The tanks could be supplied with either running freshwater (4.2 2 0.3’ C) or recirculating seawater (31 ppt, 6.7 + 0.5 ’ C ). A salinity of 29 ppt was achieved within 10 min of seawater being introduced into the tanks. 700~1 of blood was taken and replaced with an equal volume of saline with the fish in freshwater. Similar samples were subsequently taken at 0.25,0.5,1, 2, 6, 18, 24 and 48 h after exposure to seawater. Arterial oxygen tension (P, 0,) and pH were measured immediately in whole blood using a Radiometer BMS Mk II blood gas analyser with a Radiometer PHM73 meter. Aliquots of whole blood were taken for determination of haematocrit (%packed cell volume), total haemoglobin (cyanomethaemoglobin method, Sigma) and the remainder centrifuged and the plasma frozen ( - 20’ C ) for subsequent analysis. Mean corpuscular haemoglobin was calculated for each sample from the haematocrit and haemoglobin concentration. The samples were analysed for lactate (Boehringer diagnostic assay), sodium and potassium by flame photometry (Eel loo), magnesium and calcium by atomic absorption spectrophotometry (Pye Unicam SP9), chloride by electrochemical titration (Radiometer CMTlO) and plasma osmotic pressure by freezing point depression osmometry (Camlab). Data were analysed for each experimental group by a l-way ANOVA and the significance of individual points assessed by calculating the least significant difference at the 5% level of probability. RESULTS In April the fish remained quiescent throughout the transfer period whereas in December the fish were much more agitated during and immediately after the changeover to seawater. The mortalities during the experiments were much greater in December compared to April (50% and 11% respectively over the 48 h period). Osmotic and ionic regulation In both spring and autumn fish there was a progressive increase in the plasma sodium, chloride, and osmotic concentrations following abrupt salinity change (Fig. 1). In smolt-like fish this was characterised by an initial rapid rate of increase which slowed after 2-6 h and reached a plateau after 18 h. The parrlike fish had almost identical responses up to 18 h after exposure to seawater but thereafter concentrations continued to rise for the duration of the experimental period to levels which might be expected to be lethal to fish (e.g. an osmotic pressure of 450 mOsmo1 l- ’ after 48 h in seawater).
R.M. STAGG ET AL.
Osmotic. pressure
7
Fig. 1. Plasma sodium, chloride and osmotic concentrations in Atlantic salmon maintained in freshwater following abrupt exposure to seawater in April and December. Mean values k se. (n = 49 ) . Control, 0; smolt, m; Parr, 0. * Significantly different from equivalent control at P < 0.05.
Fig. 2. Plasma magnesium, potassium and calcium in Atlantic salmon maintained in freshwater following abrupt exposure to seawater in April and December. Legend as in Fig. 1.
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In both groups of fish plasma, magnesium rose rapidly over the first hour after transfer from a mean value of 0.78 mmol 1-l in freshwater (Fig. 2). In smolt-like fish the magnesium concentration subsequently stabilised at 1.5 mmol l-l by 6 h whereas in Parr-like fish it continued to increase albeit at a lower rate reaching 2.8 mmol 1-l by 48 h after seawater transfer. Concentrations of potassium and calcium in the plasma did not change significantly following change-over from freshwater to seawater in either smolt-like or parrlike fish (Fig. 2). Plasma ion and osmotic concentrations of control fish were unaffected by the sampling regime (Figs. 1 and 2). Respiratory parameters
Changeover from freshwater to seawater was associated with a reduction in arterial P, O2 and an increase in both plasma lactate and arterial pH (Fig. 3 ) .
I_1
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’
J
r
7.6
L
Lactate
II! 0 2
6
12
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Fig. 3. Arterial blood oxygen tension (P. O,), pH and plasma lactate in Atlantic salmon maintained in freshwater following abrupt exposure to seawater in April and December. Legend as in Fig. 1.
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For smolt-like fish these changes were small and transient. P, 0, fell to a minimum of 16.8 kPa 15-30 min after transfer and then progressively returned to control values (18.4 kPa). Lactate rose from 1.03 mmol 1-l to a maximum of 2.00 mmol 1-l within 1 h of exposure but then rapidly ( ~6 h) returned to control levels. Similarly arterial pH rose rapidly over the first hour and then returned to control values within 6 h. In Parr-like fish the changes were comparatively larger and lasted for the duration of the experiment. P, O2 fell very rapidly down to 13.3 kPa within 15 min of seawater exposure and then recovered slightly but remained less than 15.3 kPa for the duration of the sampling period. Plasma lactate rose steadily for the first 12 h and then stabilised between 2.5-3 mmol 1-l. Whole bloodpH rose rapidly within 15 min of changeover to seawater and remained elevated by approximately 0.1 of a pH unit for the duration of the experiment. Haematological parameters
In both experimental and control groups haematocrit and haemoglobin fell throughout the experiment to values 50-60% of the initial levels (Fig. 4) presumably due to the sampling regime and the quantity of blood removed. There were no significant differences between control and experimental fish (Fig. 4). Mean corpuscular haemoglobin did not change significantly from freshwater values in either controls or smolts throughout the experiment or in parr up to 24 h after transfer to seawater. In Parr, 48 h after immersion in seawater there
Fig. 4. Blood haematocrit (Hct), haemoglobin (Hb) and mean corpuscular haemoglobin concentration (MCHC) in Atlantic salmon maintained in freshwater following abrupt exposure to seawater in April and December. Legend as in Fig. 1.
SEASONAL RESPONSES TO SEAWATER OF S. SALAR MAINTAINED IN FRESHWATER
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was a significant increase in corpuscular haemoglobin. This is presumably a reflection of a breakdown in cell volume regulation and erythrocyte shrinkage as a consequence of the elevated plasma osmotic pressure. DISCUSSION
Although the steady state mechanisms of osmotic and ionic regulation in fish have been extensively studied (see reviews by Evans, 1980; Foskett et al., 1983) there are comparatively few studies which report the kinetics of changes following abrupt freshwater to seawater transfer. In this study, the first blood sample was taken 15 min after changeover to seawater which is approximately 5 min’after the water in the experimental system attained a salinity of 30 ppt. Transfer of fish from freshwater to seawater results in a complete reversal of the osmotic and ionic gradients and it is unlikely that the branchial mechanisms of ion excretion, characteristic of seawater-adapted fish, are fully developed during this initial phase. For example, chloride efflux in Atlantic salmon smolts takes about 18 h to reach seawater intensities (Potts et al., 1970) and in fish in general it often takes several days in seawater for many of the parameters associated with long term survival, such as water permeability (Ogasawara and Hirano, 1984) and branchial Na+/K+-ATPase activity (Boeuf et al., 1978) to fully develop. Other regulatory mechanisms involved in maintenance of extracellular fluid.homeostasis may be operating during this initial phase including increased drinking (Bath and Eddy, 1979a), a change in body/muscle ions and a redistribution of body water (Holmes and Donaldson, 1969). The sharp rise in plasma magnesium seen in this study (Fig. 2) may be a reflection of the onset of the drinking response or a delayed renal excretion in the face of an influx of magnesium across the body surface. The response of plasma ions and osmotic concentrations in the Parr-like fish were almost identical to those in the smolt-like fish during the first 18 h in seawater and this suggests comparable physiological mechanisms are operating during this acute initial phase. Subsequent regulation seen in the smoltlike fish is absent in the Parr-like fish and plasma osmotic and ionic concentrations continue to rise. There was a small, transient fall in P, 0, following transfer of smolt-like fish to seawater (Fig. 3) and an increase in circulating lactate. Since these changes were of such short duration one could assume that respiratory homeostasis was restored relatively quickly. In contrast the Parr-like fish showed a severe and sustained fall in P, O2 within 15 min of exposure to seawater and a parallel rise in plasma lactate. Bath and Eddy (1979b) showed that, in rainbow trout, oxygen transfer factor fell by approximately 30% for up to 5 h after transfer of the fish to seawater. Such effects, per se, may be caused by an active redistribution of branchial blood flow to reduce epithelial water and ion permeability with a concommitant reduction in respiratory efficiency or, more likely,
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passive dehydration of the gills as a result of the reversed osmotic gradient. Bert (1871) first expressed the notion that the gills of freshwater fish undergo shrinkage leading to an arrest of the branchial circulation when placed in seawater and it has since been shown that gills of fish transferred from freshwater to seawater are considerably dehydrated for several days (Shuttleworth and Freeman, 1973; Leray et al., 1981). Using chloride space as a marker Shuttleworth and Freeman (1973) showed a pronounced reduction in intracellular volume of gill tissue which reached a minimum 6 h after transfer of AnguiZZu dieffenbachii from freshwater to seawater. These authors attributed such changes to an effect of the external medium rather than any changes in the extracellular fluid osmotic pressure since changes in the latter occurred much more slowly. Since dehydration may result in shrinkage of the gills and a reduction in lamellar surface area (Bath and Eddy, 1979b ), it is clear that euryhalinity will not only be related to the ability to stabilise plasma ion and osmotic concentrations but will also be related to the ability to regulate the initial dehydration of the gills. Presumably the latter is a prerequisite of the former with regard to the role of branchial ion excretion in overall ionic homeostasis. If changes in dorsal aortic P, 0, are an index of the disruption of branchial function due to dehydration, then this also provides an additional commentary on the distinction between “pa&’ and “smolts”. The small changes which occur in the smoltlike fish are rapidly reversed whereas the more severe and sustained responses in the Parr-like fish must reflect an inability to reverse branchial dehydration and control cell volume. Milne and Randall (1976) and Bath and Eddy (1979b) were unable to detect significant changes in the acid-base status of rainbow trout transferred to seawater. This is in contrast to the work reported by Leray et al. (1981) who recorded a sharp rise in pH followed by a large decrease until 16 h after seawater transfer. In this study whole blood pH was elevated within 15 min of exposure to seawater which was again transient in smolt-like and sustained in Parr-like fish. This occurred despite an increase in metabolic production of acid which is suggested by the profile of lactate production in the blood (Fig. 3 ) . The mechanisms of acid-base regulation in fish are principally through the branchial Na+/H+ or NH: and Cl-/HCO, exchanges (Heisler, 1984). However, the effect of transfer to seawater on metabolic or respiratory acid/base production or the effects of changes in branchial integrity on the capacity to excrete acid or base equivalents by the gill is largely unknown and is obviously an area with scope for further work. In conclusion, with respect to ionic regulation in salmon, the initial period following rapid changeover of salmon from freshwater to seawater is identical in the Parr-like and smolt-like fish but subsequent regulation occurs only in smolts. Respiratory function is much more affected in the Parr-like fish compared to the smolt-like fish and this may reflect an inability of these fish to
SEASONAL RESPONSES TO SEAWATER OF S. SALAR MAINTAINED IN FRESHWATER
control branchial dehydration the gills.
with concomitant
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effects on gas exchange by
ACKNOWLEDGEMENTS
The authors would like to thank M.S. Miles, D.S. Keay and J. Muir for their assistance during the course of this study and for maintaining the experimental facilities used.
REFERENCES Bath, R.N. and Eddy, F.B., 1979a. Salt and water balance in rainbow trout (Salmo gairdneri) rapidly transferred from freshwater to seawater. J. Exp. Biol., 83: 193-202. Bath, R.N. and Eddy, F.B., 1979b. Ionic and respiratory regulation in rainbow trout during rapid transfer to seawater. J. Comp. Physiol., 134: 351-357. Bert, M.P, 18’71. Sur les phenomenes et les causes de la mart des animaux d’eau deuce que l’on plonge dans l’eau mer. CR. Acad. Sci. Paris, 73: 382-385. Boeuf, G., Lasserre, P. and Harache, Y., 1978. Osmotic adaptation of Oncorhynchus kisutch Walbaum. II. Plasma osmotic and ionic variations, and gill Na +, K+-ATPase activity of yearling coho salmon transferred to seawater. Aquaculture, 15: 35-52. Evans, D.H., 1980. Kinetic studies of ion transport by fish gill epithelium. Am. J. Physiol., 238: R225-R230. Folmar, L.C. and Dickhoff, W.W., 1980. The Parr-smolt transformation (smoltification) and seawater adaptation in salmonids. Aquaculture, 21: l-37. Folmar, L.C. andDickhoff, W.W., 1981. Evaluation of some physiological parameters as predictive indices of smoltification. Aquaculture, 23: 309-324. Foskett, J.K., Bern, H.A., Machen, T.E. and Conner, M., 1983. Chloride cells and the hormonal control of teleost osmoregulation. J. Exp. Biol., 105: 255-281. Heisler, N., 1984. Acid-base regulation in fishes. In: W.S. Hoar and D.J. Randall (Editors), Fish Physiology, Vol. XA. Academic Press, Orlando, FL, pp. 239-283. Hoar, W.S., 1976. Smolt transformation evolution, behaviour and physiology. J. Fish. Res. Board Can., 33: 1233-1252. Holmes, W.N. and Donaldson, E.M., 1969. The body compartments and the distribution of electrolytes. In: W.S. Hoar and D.J. Randall (Editors), Fish Physiology, Vol. I. Academic Press, Orlando, FL, pp. l-89. Leray, C., Colin, D.A. and Florentz, A., 1981. Time course of osmotic adaptation and gill energetics of rainbow trout (Salmo gairdneri R.) following abrupt changes in external salinity. J. Comp. Physiol., 144: 175-181. McCormick, S.D. and Saunders, R.L., 1987. Preparatory physiological adaptations for marine life of salmonids: osmoregulation, growth and metabolism. Am. Fish. Sot. Symp., 1: 211-229. Milne, R.S. and Randall, D.J., 1976. Regulation of arterialpH during freshwater to seawater transfer in the rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol., 53A: 157-160. Ogasawara, T. and Hirano, T., 1984. Changes in osmotic water permeability of the eel gills during seawater and freshwater adaptation. J. Comp. Physiol., 154: 3-11. Potts, W.T.W., Foster, M.A. and Stather, J.W., 1970. Salt and water balance in salmon smolts. J. Exp. Biol., 52: 553-564. Shuttleworth, T.J. and Freeman, R.F.H., 1973. The role of the gills in seawater adaptation in
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dieffenbachii. I. Osmotic and ionic composition of the blood and gill tissue. J. Comp. Physiol., 86: 293-313. Smith, L.S. and Bell, G.R., 1964. A technique for prolonged blood sampling in free-swimming salmon. J. Fish. Res. Board Can., 21: 711-717.
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Seasonal Variations in Osmoregulatory and Respiratory Responses to Seawater Exposure of Juvenile Atlantic Salmon (Salmo salar) Maintained in Freshwater R.M. STAGG’*, C. TALBOT’,
F.B. EDDY3 and M. WILLIAMS3
‘Heriot- Watt University, Department of Brewing and Biological Sciences, Chambers Street, Edinburgh EHI 1HX (Great Britain) ‘Freshwater Fisheries Laboratory, Pitlochry, Perthshire PH16 5LB (Great Britain) 3University of Dundee, Department of Biological Sciences, Dundee DDl 4H (Great Britain)
ABSTRACT Stagg, R.M., Talbot, C., Eddy, F.B. and Williams, M., 1989. Seasonal variations in osmoregulatory and respiratory responses to seawater exposure of juvenile Atlantic salmon (Salmo salar ) maintained in freshwater. Aquaculture, 82: 219-228. Three- or four-year-old Atlantic salmon (Salmo salar) which first smolted in their second spring (S2 smolts) and had been kept in freshwater since hatching have the external characteristics of parr in December and smolts in April. Such fish were used to investigate the acute effects (up to 48 h) of rapid changeover from freshwater to seawater on respiratory, acid-base and ionoregulatory homeostasis. Over the first 18 h of seawater exposure the plasma sodium, chloride and total osmotic concentrations rose rapidly in a similar manner for both groups of fish. In the smolt-like fish levels then stabilised whereas in Parr-like fish they continued to rise to the end of the experiment. Immediately after exposure to seawater, there was a rapid rise in blood pH and a fall in arterial P, 0, which were transient in the smolt-like and more severe and prolonged in the parrlike fish. These results are discussed in the context of branchial dehydration and cell volume control. It is suggested that an important aspect of smolting is the ability to regulate branchial dehydration since this must precede activation of the branchial ion excretory mechanisms regulating extracellular fluid composition following seawater transfer.
INTRODUCTION
Smolting in Atlantic salmon (Salmo sakzr ) is a complex series of physiological, morphological, biochemical and behavioural changes that occur in the *Present address and address for correspondence: Department of Agriculture and Fisheries for Scotland, Marine Laboratory, P.O. Box 101, Victoria Road, Torry, Aberdeen AB9 8DB (Great Britain).
0044-8486/89/$03.50
0 1989 Elsevier Science Publishers
B.V.
220
R.M. STAGG ET AL.
spring (Folmar and Dickhoff, 1980, 1981; McCormick and Saunders, 1987). These changes result in the stenohaline, freshwater parr becoming euryhaline, and accompany the migration of the smolt from its natal river to the sea. The changes that occur during smolting are reversible and smolts which are prevented from migrating will revert to having Parr-like characteristics (Hoar, 1976). Abrupt transfer of euryhaline salmonids from freshwater to seawater elicits an acute adaptive or crisis phase, characterised by a rapid rise in plasma electrolyte concentrations, and a chronic regulatory phase when plasma ions decline to constant levels (Holmes and Donaldson, 1969; Bath and Eddy, 1979a). However, euryhalinity must also be related to the ability to tolerate the profound initial changes that occur, in the gill epithelium, upon transfer from freshwater to seawater because this tissue is in direct contact with the external medium. Shuttleworth and Freeman (1973) showed that in the eel (Anguillu dieffenbachii) there was a marked reduction in gill intracellular space during the first 6 h after transfer from freshwater to seawater which suggests that the gill was undergoing extensive shrinkage. In rainbow trout (Salmo guirdneri) one of the consequences of such effects for gill function is a transient reduction in oxygen transfer factor during the crisis period following exposure to seawater (Bath and Eddy, 1979b). Acute changes in respiratory parameters may contribute to mortalities following seawater transfer but to date such effects have not been studied in salmon. The objectives of this study were: (1) to compare the acute ionoregulatory ability of large Atlantic salmon (Salmo s&r) in December (Parr-like) and April (smolt-like) following rapid transfer from freshwater to seawater, and (2) to determine the consequences of such transfer for the respiratory and extracellular acid-base status of the animal. Such a study involves the use of cannulated fish in excess of 300 g. For Atlantic salmon smolts this can only be achieved using fish reared in freshwater for a number of years to a suitable size on the assumption that once the first smolting has occurred the seasonal smolting process subsequently occurs annually and is independent of age. MATERIALS AND METHODS
Atlantic salmon, Sulmo s&r which had been maintained in freshwater at the Freshwater Fisheries Laboratory since hatching were used. The fish had first smolted in their second spring (S2 smolts) and were subsequently retained in freshwater for an additional 2 years. On the basis of external appearance the fish were considered to be smolt-like in April (silvered, low condition factor) but reverted to Parr-like in December (prominent parr marks, high condition factor). They weighed (mean + se., n in parentheses) 441+ 19 (15) g and 557 rt 42 (9) g, respectively. Fish were anaesthetised with 20-40 mg 1-l benzocaine and cannulated, for
SEASONAL RESPONSES TO SEAWATER OF S. SALAR MAINTAINED IN FRESHWATER
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blood sampling, via the dorsal aorta with a short length of polyethylene tubing (i.d. 0.58 mm, o.d. 0.96 mm) containing heparinised saline (Smith and Bell, 1964). Fish were then placed in individual tanks containing freshwater and allowed to recover for at least 16 h. The tanks could be supplied with either running freshwater (4.2 2 0.3’ C) or recirculating seawater (31 ppt, 6.7 + 0.5 ’ C ). A salinity of 29 ppt was achieved within 10 min of seawater being introduced into the tanks. 700~1 of blood was taken and replaced with an equal volume of saline with the fish in freshwater. Similar samples were subsequently taken at 0.25,0.5,1, 2, 6, 18, 24 and 48 h after exposure to seawater. Arterial oxygen tension (P, 0,) and pH were measured immediately in whole blood using a Radiometer BMS Mk II blood gas analyser with a Radiometer PHM73 meter. Aliquots of whole blood were taken for determination of haematocrit (%packed cell volume), total haemoglobin (cyanomethaemoglobin method, Sigma) and the remainder centrifuged and the plasma frozen ( - 20’ C ) for subsequent analysis. Mean corpuscular haemoglobin was calculated for each sample from the haematocrit and haemoglobin concentration. The samples were analysed for lactate (Boehringer diagnostic assay), sodium and potassium by flame photometry (Eel loo), magnesium and calcium by atomic absorption spectrophotometry (Pye Unicam SP9), chloride by electrochemical titration (Radiometer CMTlO) and plasma osmotic pressure by freezing point depression osmometry (Camlab). Data were analysed for each experimental group by a l-way ANOVA and the significance of individual points assessed by calculating the least significant difference at the 5% level of probability. RESULTS In April the fish remained quiescent throughout the transfer period whereas in December the fish were much more agitated during and immediately after the changeover to seawater. The mortalities during the experiments were much greater in December compared to April (50% and 11% respectively over the 48 h period). Osmotic and ionic regulation In both spring and autumn fish there was a progressive increase in the plasma sodium, chloride, and osmotic concentrations following abrupt salinity change (Fig. 1). In smolt-like fish this was characterised by an initial rapid rate of increase which slowed after 2-6 h and reached a plateau after 18 h. The parrlike fish had almost identical responses up to 18 h after exposure to seawater but thereafter concentrations continued to rise for the duration of the experimental period to levels which might be expected to be lethal to fish (e.g. an osmotic pressure of 450 mOsmo1 l- ’ after 48 h in seawater).
R.M. STAGG ET AL.
Osmotic. pressure
7
Fig. 1. Plasma sodium, chloride and osmotic concentrations in Atlantic salmon maintained in freshwater following abrupt exposure to seawater in April and December. Mean values k se. (n = 49 ) . Control, 0; smolt, m; Parr, 0. * Significantly different from equivalent control at P < 0.05.
Fig. 2. Plasma magnesium, potassium and calcium in Atlantic salmon maintained in freshwater following abrupt exposure to seawater in April and December. Legend as in Fig. 1.
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In both groups of fish plasma, magnesium rose rapidly over the first hour after transfer from a mean value of 0.78 mmol 1-l in freshwater (Fig. 2). In smolt-like fish the magnesium concentration subsequently stabilised at 1.5 mmol l-l by 6 h whereas in Parr-like fish it continued to increase albeit at a lower rate reaching 2.8 mmol 1-l by 48 h after seawater transfer. Concentrations of potassium and calcium in the plasma did not change significantly following change-over from freshwater to seawater in either smolt-like or parrlike fish (Fig. 2). Plasma ion and osmotic concentrations of control fish were unaffected by the sampling regime (Figs. 1 and 2). Respiratory parameters
Changeover from freshwater to seawater was associated with a reduction in arterial P, O2 and an increase in both plasma lactate and arterial pH (Fig. 3 ) .
I_1
1
1
’
’
J
r
7.6
L
Lactate
II! 0 2
6
12
I
I
I
18
2L
48 nours
Fig. 3. Arterial blood oxygen tension (P. O,), pH and plasma lactate in Atlantic salmon maintained in freshwater following abrupt exposure to seawater in April and December. Legend as in Fig. 1.
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For smolt-like fish these changes were small and transient. P, 0, fell to a minimum of 16.8 kPa 15-30 min after transfer and then progressively returned to control values (18.4 kPa). Lactate rose from 1.03 mmol 1-l to a maximum of 2.00 mmol 1-l within 1 h of exposure but then rapidly ( ~6 h) returned to control levels. Similarly arterial pH rose rapidly over the first hour and then returned to control values within 6 h. In Parr-like fish the changes were comparatively larger and lasted for the duration of the experiment. P, O2 fell very rapidly down to 13.3 kPa within 15 min of seawater exposure and then recovered slightly but remained less than 15.3 kPa for the duration of the sampling period. Plasma lactate rose steadily for the first 12 h and then stabilised between 2.5-3 mmol 1-l. Whole bloodpH rose rapidly within 15 min of changeover to seawater and remained elevated by approximately 0.1 of a pH unit for the duration of the experiment. Haematological parameters
In both experimental and control groups haematocrit and haemoglobin fell throughout the experiment to values 50-60% of the initial levels (Fig. 4) presumably due to the sampling regime and the quantity of blood removed. There were no significant differences between control and experimental fish (Fig. 4). Mean corpuscular haemoglobin did not change significantly from freshwater values in either controls or smolts throughout the experiment or in parr up to 24 h after transfer to seawater. In Parr, 48 h after immersion in seawater there
Fig. 4. Blood haematocrit (Hct), haemoglobin (Hb) and mean corpuscular haemoglobin concentration (MCHC) in Atlantic salmon maintained in freshwater following abrupt exposure to seawater in April and December. Legend as in Fig. 1.
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was a significant increase in corpuscular haemoglobin. This is presumably a reflection of a breakdown in cell volume regulation and erythrocyte shrinkage as a consequence of the elevated plasma osmotic pressure. DISCUSSION
Although the steady state mechanisms of osmotic and ionic regulation in fish have been extensively studied (see reviews by Evans, 1980; Foskett et al., 1983) there are comparatively few studies which report the kinetics of changes following abrupt freshwater to seawater transfer. In this study, the first blood sample was taken 15 min after changeover to seawater which is approximately 5 min’after the water in the experimental system attained a salinity of 30 ppt. Transfer of fish from freshwater to seawater results in a complete reversal of the osmotic and ionic gradients and it is unlikely that the branchial mechanisms of ion excretion, characteristic of seawater-adapted fish, are fully developed during this initial phase. For example, chloride efflux in Atlantic salmon smolts takes about 18 h to reach seawater intensities (Potts et al., 1970) and in fish in general it often takes several days in seawater for many of the parameters associated with long term survival, such as water permeability (Ogasawara and Hirano, 1984) and branchial Na+/K+-ATPase activity (Boeuf et al., 1978) to fully develop. Other regulatory mechanisms involved in maintenance of extracellular fluid.homeostasis may be operating during this initial phase including increased drinking (Bath and Eddy, 1979a), a change in body/muscle ions and a redistribution of body water (Holmes and Donaldson, 1969). The sharp rise in plasma magnesium seen in this study (Fig. 2) may be a reflection of the onset of the drinking response or a delayed renal excretion in the face of an influx of magnesium across the body surface. The response of plasma ions and osmotic concentrations in the Parr-like fish were almost identical to those in the smolt-like fish during the first 18 h in seawater and this suggests comparable physiological mechanisms are operating during this acute initial phase. Subsequent regulation seen in the smoltlike fish is absent in the Parr-like fish and plasma osmotic and ionic concentrations continue to rise. There was a small, transient fall in P, 0, following transfer of smolt-like fish to seawater (Fig. 3) and an increase in circulating lactate. Since these changes were of such short duration one could assume that respiratory homeostasis was restored relatively quickly. In contrast the Parr-like fish showed a severe and sustained fall in P, O2 within 15 min of exposure to seawater and a parallel rise in plasma lactate. Bath and Eddy (1979b) showed that, in rainbow trout, oxygen transfer factor fell by approximately 30% for up to 5 h after transfer of the fish to seawater. Such effects, per se, may be caused by an active redistribution of branchial blood flow to reduce epithelial water and ion permeability with a concommitant reduction in respiratory efficiency or, more likely,
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passive dehydration of the gills as a result of the reversed osmotic gradient. Bert (1871) first expressed the notion that the gills of freshwater fish undergo shrinkage leading to an arrest of the branchial circulation when placed in seawater and it has since been shown that gills of fish transferred from freshwater to seawater are considerably dehydrated for several days (Shuttleworth and Freeman, 1973; Leray et al., 1981). Using chloride space as a marker Shuttleworth and Freeman (1973) showed a pronounced reduction in intracellular volume of gill tissue which reached a minimum 6 h after transfer of AnguiZZu dieffenbachii from freshwater to seawater. These authors attributed such changes to an effect of the external medium rather than any changes in the extracellular fluid osmotic pressure since changes in the latter occurred much more slowly. Since dehydration may result in shrinkage of the gills and a reduction in lamellar surface area (Bath and Eddy, 1979b ), it is clear that euryhalinity will not only be related to the ability to stabilise plasma ion and osmotic concentrations but will also be related to the ability to regulate the initial dehydration of the gills. Presumably the latter is a prerequisite of the former with regard to the role of branchial ion excretion in overall ionic homeostasis. If changes in dorsal aortic P, 0, are an index of the disruption of branchial function due to dehydration, then this also provides an additional commentary on the distinction between “pa&’ and “smolts”. The small changes which occur in the smoltlike fish are rapidly reversed whereas the more severe and sustained responses in the Parr-like fish must reflect an inability to reverse branchial dehydration and control cell volume. Milne and Randall (1976) and Bath and Eddy (1979b) were unable to detect significant changes in the acid-base status of rainbow trout transferred to seawater. This is in contrast to the work reported by Leray et al. (1981) who recorded a sharp rise in pH followed by a large decrease until 16 h after seawater transfer. In this study whole blood pH was elevated within 15 min of exposure to seawater which was again transient in smolt-like and sustained in Parr-like fish. This occurred despite an increase in metabolic production of acid which is suggested by the profile of lactate production in the blood (Fig. 3 ) . The mechanisms of acid-base regulation in fish are principally through the branchial Na+/H+ or NH: and Cl-/HCO, exchanges (Heisler, 1984). However, the effect of transfer to seawater on metabolic or respiratory acid/base production or the effects of changes in branchial integrity on the capacity to excrete acid or base equivalents by the gill is largely unknown and is obviously an area with scope for further work. In conclusion, with respect to ionic regulation in salmon, the initial period following rapid changeover of salmon from freshwater to seawater is identical in the Parr-like and smolt-like fish but subsequent regulation occurs only in smolts. Respiratory function is much more affected in the Parr-like fish compared to the smolt-like fish and this may reflect an inability of these fish to
SEASONAL RESPONSES TO SEAWATER OF S. SALAR MAINTAINED IN FRESHWATER
control branchial dehydration the gills.
with concomitant
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effects on gas exchange by
ACKNOWLEDGEMENTS
The authors would like to thank M.S. Miles, D.S. Keay and J. Muir for their assistance during the course of this study and for maintaining the experimental facilities used.
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