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Sodium uptake in the isopod sphaeroma rugicauda leach. During acclimatization to high and low salinities
Comp. Biochem. Physiol., 1970, Vol. 32, pp. 763 to 773. Pergamon Press. Printed in Great Britain
SODIUM UPTAKE IN T H E ISOPOD S P H A E R O M A R U G I C A U D A LEACH. DURING A C C L I M A T I Z A T I O N TO H I G H AND LOW SALINITIES R. R. HARRIS*
Department of Oceanography, The University, Southampton
(Received 19 May 1969) A b s t r a c t - - 1 . Sphaeroma tolerates salinities in the range 2-180 per cent sea
water. 2. Blood is maintained hypo- and hypertonic respectively to media more concentrated and less concentrated than 100 per cent sea water. 3. Active sodium uptake decreases on transferance of animals from 2-100 per cent sea water and is markedly lower than the uptake by steady-state animals with the same blood concentration. 4. On transference from 180-2 per cent sea water uptake of sodium at a blood concentration of 500 mM/l NaCI is higher than that of steady-state animals with this blood concentration. 5. It is concluded that the absolute blood concentration is not the only factor involved in regulating rate of sodium uptake. INTRODUCTION THE MAINTENANCEof the haemolymph hyperosmotic to the surrounding medium by fresh-water and brackish-water Crustacea depends on active uptake of ions across the body surface. Since sodium is the predominant cation in the haemolymph of these forms it is not surprising that the sodium uptake mechanism is of prime importance in osmotic and ionic regulation. That sodium uptake takes place independently of chloride, the major anion, has been shown by a number of investigators (Krogh, 1938, 1939; Shaw, 1960a, b). T h e regulation of haemolymph concentration at a constant level and within defined limits requires a means of regulating the rate of uptake of sodium. T h e influence of external and internal sodium concentrations on the sodium uptake rate has been studied in a variety of species from both fresh water and brackish water. It has been found that the active uptake of sodium by these animals is determined, at low-medium concentrations, by the amount of sodium present in the medium. Furthermore, various crustacean species have sodium uptake systems with different affinities for sodium. T h e value Kin, the medium concentration at which the uptake system is half-saturated, is lower in fresh-water species than in brackish-water forms (Shaw, 1961a; Croghan & Lockwood, 1968). * Present address: Department of Biology, The Royal University of Malta, Msida,
Malta. 763
764
R.R. HARRIS
T h e effect of the sodium concentration of the haemolymph on the rate of sodium transport has also been investigated. Here the picture is not so clear. It has been suggested that lowering the sodium concentration below the "normal" level causes an increase in the rate of sodium uptake (Shaw, 1964 for review; Bryan, 1960). A simple feedback system has been proposed for Austrapotamobius (Shaw, 1959) where a fall in the sodium concentration elicits an increase in the sodium uptake rate. As the lost sodium is replaced, or a new steady-state haemolymph concentration is attained, the active uptake system is suppressed and the normal uptake rate is re-established. This is considered to be the general pattern of the control of sodium balance in aquatic Crustacea. Lockwood (1964) has shown that in the brackish-water amphipod Gammarus duebeni, the sodium concentration of the haemolymph may not be the only factor controlling the sodium active uptake system. A rapid fall in the haemolymph concentration, caused by washing animals acclimatized to high salinities in deionised water, resulted in high rates of sodium uptake. This treatment elicited higher rates of uptake than those measured in control animals with similar haemolymph concentrations. Sphaeroma rugicauda is a brackish-water isopod often found in the field with G. duebeni. A typical habitat is the saking pools and creeks of an estuary. Sudden and often large changes in salinity are encountered by Sphaeroma in these environments. In this paper the effect of a transfer from a high to a low salinity on the sodium uptake system is studied. Active uptake of sodium is determined in experimental conditions where interference by exchange diffusion is minimal, and also where the passive component of the uptake is small. T h e effect on the sodium uptake system of an increase in the medium concentration is also studied. METHODS AND MATERIALS
Sphaeroma rugicauda were collected by hand net from tidal creeks and salting pools at Totton, an area at the head of Southampton Water. They were kept in the laboratory at room temperature (16-20°C). Haemolymph samples were collected in a micropipette inserted intersegmentally in the region of the heart. The animals were discarded after sampling. The sodium concentration of both haemolymph and medium was measured by flame photometry on a Unicam SP 900 flame spectrophotometer. Duplicate analyses were possible on haemolymph samples from single animals. Determinations of a known standard were accurate to S.D. + 2%. This value includes both pipetting and instrument errors. Sodium influx was determined using the radioisotope ~2Na. The activity of this isotope was measured in an Ekco Universal scintillation counter with a well NaI (Th) crystal in conjunction with an Ekco scaler. Individual animals were placed in 2 ml of inactive medium in a test-tube which fitted into the well of the scintillation crystal. RESULTS
The sodium concentration of the haemolymph T h e concentration of sodium in the haemolymph of Sphaeroma acclimatized to a range of m e d i u m concentration is shown in Fig. 1. Sphaeroma were able to
S O D I U M U P T A K E I N T H E ISOPOD S P H A E R O M A R U G I C A U D A
765
tolerate direct transfer into salinities from 2-140 per cent sea water (10-694 mM/l NaC1). The animals survived in higher salinities (up to 185 per cent sea water) only if the medium concentration was increased gradually by increments of 5 per cent. The sodium concentration of the haemolymph is similar to that of the medium in' 100 per cent sea water. Below and above this medium concentration the sodium concentration of the haemolymph is respectively higher and lower than that of the medium. 900
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FIG. 1. The relation between the sodium concentration of the haemolymph and that of the external medium.
The influx of sodium at various haemolymph concentrations It has been found (Harris, 1967) that in 10 mM/l NaCI the sodium active transport system in S. rugicauda is approaching full saturation. Exchange diffusion has been shown to be neglible at this medium concentration and the passive influx of sodium ions has been calculated to be only 9 per cent of the total influx. That active transport of sodium does occur is indicated by the electrochemical gradient present across the gills of Sphaeroma. Uptake of 2~Na from a 10 mM/l NaC1 loading medium after correction for the small exchange diffusion and passive influx components will therefore reflect active transport of sodium. However, the sodium concentration of the haemolymph of an animal placed in the loading medium will be initially that corresponding to the steady-state concentration attained in the acclimatization medium. In the loading medium loss of sodium will occur as the sodium concentration of the haemolymph falls to a new steadystate value. It is probable that a large backflux of ~2Na will take place and therefore influx of ~Na into the animal will only represent active uptake for an initial period
766
R.R. HARRIS
in the loading medium. A loading period of 30 min was selected for all experiments after considering, in preliminary experiments, the rate of efflux of 22Na,from animals which had been fully loaded with the isotope, into inactive 10 mM/1NaC1. As a result of falling concentration of the haemolymph it is possible that the active transport system may be stimulated, for this reason also a short loading period was used. The active uptake of sodium by Sphaeromapreviously acclimatized to media ranging from 4-95 per cent sea water was investigated. Animals of similar size (12-15 mg) were placed into the loading medium of 10 mM]l NaC1 containing Z2Na for a period of 30 min. After this time they were removed, rinsed quickly in inactive medium and counted. Since the same loading medium was used for all animals, differences in the amount of 22Na taken up, after correction for the exchange diffusion and passive influx components, can be assumed to be mainly owing to differences in active uptake of sodium occurring in the acclimatization media prior to loading. At the end of each experiment, the animals were placed in a large volume of sea water and allowed to exchange all their Z2Na over a period of 10 days. They were then acclimatized to another medium concentration and the previous experiment repeated. Thus the amount of 22Na taken up during the loading period was measured in groups of animals acclimatized to high and low salinities. The results of these experiments are shown in Fig. 2.
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FIG. 2. T h e uptake of 2~Na from a 10 m M / 1 NaC1 solution by Sphaeroma rugicauda acclimatized to a range of salinities. The solid circles and vertical lines indicate the mean and the standard error.
Considerable variation in the amount of 22Na taken up during the loading period by individual animals is observed. Comparing the amounts of tracer
S O D I U M U P T A K E I N T H E ISOPOD S P H A E R O M A R U G I C A U D A
767
taken up by animals acclimatized to high and low salinities, 3-58 times as much is taken up by animals from 4~o sea water than by those from 95~/o sea water.
Sodium influx under conditions of a falling haemolymph sodium concentration Active uptake of sodium in Sphaeroma after transfer from a high to a low salinity was investigated using the same loading medium as before. A group of 50 Sphaeroma was acclimatized to 180~/o sea water. These animals were rinsed quickly in deionized water, to remove traces of the acclimatization medium, and placed in 2 1 of 2% sea water. Haemolymph samples for sodium estimation were removed from three individuals taken directly from the acclimatization medium. The haemolymph samples were kept in watch-glasses coated with Repelcote, under liquid paraffin, for analysis at the end of the experiment. Three animals were also placed in the 10 raM/1 loading medium and allowed to take up ~2Na for a period of 30 min. These animals were then counted. At frequent intervals after transfer into the 2% sea water medium, similar groups of 3 animals were removed for simultaneous haemolymph sampling and the determination of 22Na uptake. Figure 3(a) shows the fall in the sodium concentration of the haemolymph of animals, acclimatized to 180~o and transferred to 2~o sea water, plotted against time. Initially there is a rapid fall in sodium concentration. After about 3 hr a decrease in the rate of fall in concentration is evident. After 10 hr the sodium concentration is close to that measured in animals acclimatized to 2~o sea water. The mean sodium concentrations at various times after transfer are used in Fig. 3(a) where they are plotted against the number of counts, corrected for exchange diffusion and passive influx, taken up by animals removed simultaneously and placed in the 10 mM/l, NaC1 loading medium for 30 rain. The amount of 22Na taken up by Sphaeroma during the loading period increases as the sodium concentration of the haemolymph falls. A rate of active uptake close to that measured previously in animals acclimatized to 4°/0 sea water can be elicited in animals with a haemolymph sodium concentration of 500 mM/1 Na. In animals acclimatized to 95% sea water, whose haemolymph concentration is about this value, the amount of 2~Na taken up in an identical loading period is far less than that taken up by the experimental animals. A maximal rate of sodium uptake seems to be reached. This is close to that rate recorded in animals acclimatized to 4 ~/o sea water.
Sodium influx after transfer from a dilute to a concentrated medium Animals were acclimatized to 2% sea water and, after rinsing in deionized water, placed in 2 1 of 100% sea water. Haemolymph samples were taken and the sampled animals discarded. Groups of 3 animals were allowed to take up 22Na from the loading medium for a 30 min period as before. A second group of animals was acclimatized to 40% sea water and similarly transferred to 2% sea water. Figure 4(a) shows the increase of the sodium concentration of the haemolymph of Sphaeroma after transfer from 2%-100% sea water.
768
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FIG. 3a.
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FIG. 3b. T h e relation b e t w e e n h a e m o l y m p h s o d i u m concentration and the uptake o f 2aNa from a 10 m M / 1 N a C I solution after transfer from 1 8 0 % - 2 % sea water.
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increase in h a e m o l y m p h s o d i u m concentration of Sphaeroma to 2% sea water and transferred to 1 0 0 % sea water. T h e m e a n and the standard deviation are indicated.
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FIo. 4b. T h e relation b e t w e e n the s o d i u m and the uptake o f 22Na from a 10 m M / 1 NaC1 a h i g h salinity. O p e n circles refer to animals refer to animals acclimatized
o 500
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concentration o f the h a e m o l y m p h solution after transfer from a l o w to from 2% sea water. Closed circles to 4 0 % sea water.
770
R.R. HARRIS
The concentration rises rapidly initially, slowing as the steady-state level is approached. This takes about 10 hr after transfer from 2% sea water. In Fig. 4(b) the counts, corrected as before, taken up by animals during the course of the experiment are plotted against the prevailing sodium concentration of the haemolymph. The latter is taken as the mean of the sodium determinations made. Active uptake of sodium, of animals acclimatized to 2~o sea water and subsequently placed in 100% sea water, decreases rapidly. The amount of 22Na taken up during the loading period in this case is much less than that taken up by animals which are maintained in 2% sea water and not transferred to the higher salinity. This reduction in the rate of sodium uptake occurs after a rise in haemolymph sodium concentration of only about 50 mM/l Na, i.e. within 1 hr after transfer. The rate of uptake is maintained at this low level as the concentration of the haemolymph rises and also at steady-state conditions. Animals acclimatized to 10% sea water with a haemolymph concentration of about the same level, 250 mM/1 Na, showed a high rate of sodium uptake. It seems that transfer into a more concentrated medium affects the rate of sodium uptake rapidly. Animals treated in this way show much lower rates of sodium uptake than animals in steady-state conditions even though the sodium concentrations of their haemolymph are comparable. In animals acclimatized to 40% and transferred to 100% sea water a similar result was obtained, although the initial haemolymph concentration was above that of animals acclimatized to 2% sea water and consequently the number of counts taken up less. DISCUSSION A considerable amount of evidence has been collected in recent years concerning the regulation of the ion transport mechanisms responsible for the maintenance of salt balance in Crustacea. The sodium transport system has been shown to be saturable and rate limited (Shaw, 1960a, 1961a, b; Shaw & Sutcliffe, 1961; Sutcliffe, 1967; Croghan & Lockwood, 1968). Provided the external sodium concentration is high enough to saturate the sodium active uptake system, it was thought that the rate of uptake was related to the concentration of that ion in the haemolymph. Thus Bryan, (1960) found in Astacusfluviatilis that sodium influx is negligible when the haemolymph concentration is above 300 mM/l Na. As the haemolymph concentration fall below the "normal" level of 200 mM/1 Na, there is an initially slow but eventually fast increase in the rate of sodium influx. Similarly, Shaw (1961b) proposed that in Carcinus, a fall in the sodium concentration of the haemolymph below about 400 mM/1 Na of only 10 to 20 mM/1 Na leads to full activation of the sodium uptake mechanism. Both authors therefore suggested that the uptake system was related to haemolymph concentration and that increases in the rate of active transport occur as a result of a fall in haemolymph concentration below a defined level. Similar suggestions have been put forward by Shaw (1964) for Astacus and Lockwood (1960) for Asellus aquaticus. In the case of S. rugicauda however other factors beside that of absolute
SODIUM UPTAKE IN THE ISOPOD S P H A E R O M A R U G 1 C A U D A
771
haemolymph concentration appear to affect the rate of sodium uptake. High rates of sodium uptake can be elicited in animals with haemolymph concentrations which in steady-state conditions have been shown to be associated with low rates of sodium uptake. This has been found to occur when Sphaeroma is transferred from a concentrated to a dilute medium concentration. Lockwood (1964) has found similar results in G. duebeni. Treatment of acclimatized animals to a period in deionized water resulted in influxes of sodium several times greater than untreated control Gammarus. One factor that the system controlling active transport may be responding to is the relative changes in a number of the ions in the haemolymph. Shaw (1960b) has shown in Astacus pallipes that during salt depletion the animals may lose more sodium than chloride. Restoration of the normal sodium chloride concentration of the haemolymph involves, firstly, independent uptake of sodium or chloride to regain the initial ion ratio. This requires synchronization of the two uptake mechanisms. Secondly, when the new steady-state haemolymph concentration is reached, both uptake systems are suppressed by a primary feedback controller which responds to absolute haemolymph concentration. In Sphaeroma preliminary experiments have indicated that after transfer into a dilute medium there is no significantly faster fall in the haemolymph concentration of either sodium or chloride. One would expect that differential loss rates of these ions over a period of 10 hr would be detectable in terms of haemolymph concentration. Transferring Sphaeroma from a dilute to a concentrated medium results in a rapid suppression of sodium uptake. In Sphaeroma it seems unlikely that the sodium uptake system is responding to relative changes in sodium and chloride ion concentrations in the haemolymph. The rapid response of the uptake system in the latter situation, i.e. transfer from a low to a high salinity, adds weight to this suggestion. Animals acclimatized to high salinities and transferred to low ones will lose sodium rapidly. If the haemolymph is diluted at a rate faster than the intracellular concentration can be adjusted, water is likely to pass from the haemolymph into the cells. Harris (1969) has suggested that the characteristic curve of falling haemolymph concentration in Sphaeroma transferred from a high to a low salinity is a result of a water shift of this kind. Under these conditions the haemolymph volume is reduced by removal of water which passes into the cells. Lockwood (personal communication) has found that removal of a quantity of haemolymph from G. duebenicauses an increase in the rate of active uptake of sodium. Thus in Sphaeroma one factor that might affect a system controlling the sodium uptake mechanism is a change in the haemolymph volume. SUMMARY 1. The sodium concentration of the haemolymph of S. rugicauda acclimatized to salinities ranging from 2-185~o sea water was measured. In concentrations below and above 100% sea water the sodium concentration of the haemolymph is respectively higher and lower than that of the medium.
772
R . R . HARRIS
2. Using a m e t h o d which precluded interference by exchange diffusion, the active uptake of sodium by Sphaeroma, acclimatized to salinities f r o m 4 - 9 5 % sea water, was investigated. Animals f r o m 4 % s e a w a t e r t o o k u p a b o u t t h r e e t i m e s m o r e ~ N a during the loading period than animals f r o m 95% sea water. 3. Active uptake of sodium was measured in Sphaeroma during transfer f r o m a high to low salinity and vice versa. 4. Animals acclimatized to 180% and transferred to 2 % sea water took up more 22Na during the loading period than animals in steady-state conditions with comparable h a e m o l y m p h sodium concentrations. 5. Animals acclimatized to 2 % sea water and transferred to 100% sea water showed a rapid decrease in the active uptake of sodium. 6. T h e sodium uptake in Sphaeroma appears to respond to a factor other than the change in h a e m o l y m p h concentration f r o m a " n o r m a l " value. H i g h rates of sodium uptake, normally associated with steady-state conditions in low salinities and the corresponding h a e m o l y m p h concentrations, can be elicited at high haemol y m p h concentrations in animals transferred f r o m high to low salinity. Acknowledgements--I wish to thank Dr. A. P. M. Lockwood, under whose supervision this work was carried out, for his advice and criticism. I am indebted to the Science Research Council for a maintenance grant.
REFERENCES BRYAN G. W. (1960) Sodium regulation in the crayfish Astacus fluviatilis--I. Experiments with NaCl-loaded animals. J. exp. Biol. 37, 113-128. CROGHANP. C. ~ LOCKWOODA. P. M. (1968) Ionic regulation of the Baltic and freshwater races of the isopod Mesidotea (Saduria) entomon L. J. exp. Biol. 48, 141-158. HARRIS R. R. (1967) Ph.D. Thesis, The University of Southampton. HARRIS R. R. (1969) Free amino acid and haemolymph concentration changes in Sphaeroma rugicauda (Isopoda) during adaptation to a dilute salinity, ft. exp. Biol. 50, 319-326. KROGH A. (1938) The active absorption of ions in some fresh water animals. Z. vergI. Physiol. 25, 335-350. KROCH A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press. LOCKWOODA. P. M. (1960) Some effects of temperature and concentration of the medium on the ionic regulation of the isopod Asellus aquaticus L. J. exp. Biol. 37, 614-630. LOCKWOODA. P. M. (1964) Activation of the sodium uptake system at high blood concentration in the amphipod Gammarus duebeni. J. exp. Biol. 41, 447-458. SHAW J. (1959) The absorption of sodium ions by the crayfish, Astacus pallipes Lereboullet--I. The effect of external and internal sodium concentration. J. exp. Biol. 36, 126--144. SHAW J. (1960a) The absorption of sodium ions by the crayfish, Astacus pallipes Lereboullet--II. The effect of the external anion. J. exp. Biol. 37, 543-547. SHAWJ. (1960b) The absorption of chloride ions by the crayfish, Astacus pallipes Lereboullet. J. exp. Biol. 37, 557-572. SHAW J. (1961a) Sodium balance in Eriocheir sinensis (M. Edw.). The adaptation of the Crustacea to fresh water. J. exp. Biol. 38, 153-162. SHAW J. (1961b) Studies on ionic regulation in Carcinus maenas L . - - I . Sodium balance. J. exp. Biol. 38, 135-152.
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SHAW J. (1964) T h e control of salt balance in the Crustacea. Syrup. Soc. exp. Biol. 18, 237-254. SHAW J. & SUTCLIFFE D. W. (1961) Studies on sodium balance in Gammarus duebeni Lilljeborg and G. pulex pulex L. J. exp. Biol. 38, 1-15. SUTCLIFFED. W. (1967) Sodium regulation in the fresh water amphipod, Gammarus pulex L. ft. exp. Biol. 46, 499-518.
Key Word Index--Crustacea; sodium; active uptake; osmoregulation.
z6
SODIUM UPTAKE IN T H E ISOPOD S P H A E R O M A R U G I C A U D A LEACH. DURING A C C L I M A T I Z A T I O N TO H I G H AND LOW SALINITIES R. R. HARRIS*
Department of Oceanography, The University, Southampton
(Received 19 May 1969) A b s t r a c t - - 1 . Sphaeroma tolerates salinities in the range 2-180 per cent sea
water. 2. Blood is maintained hypo- and hypertonic respectively to media more concentrated and less concentrated than 100 per cent sea water. 3. Active sodium uptake decreases on transferance of animals from 2-100 per cent sea water and is markedly lower than the uptake by steady-state animals with the same blood concentration. 4. On transference from 180-2 per cent sea water uptake of sodium at a blood concentration of 500 mM/l NaCI is higher than that of steady-state animals with this blood concentration. 5. It is concluded that the absolute blood concentration is not the only factor involved in regulating rate of sodium uptake. INTRODUCTION THE MAINTENANCEof the haemolymph hyperosmotic to the surrounding medium by fresh-water and brackish-water Crustacea depends on active uptake of ions across the body surface. Since sodium is the predominant cation in the haemolymph of these forms it is not surprising that the sodium uptake mechanism is of prime importance in osmotic and ionic regulation. That sodium uptake takes place independently of chloride, the major anion, has been shown by a number of investigators (Krogh, 1938, 1939; Shaw, 1960a, b). T h e regulation of haemolymph concentration at a constant level and within defined limits requires a means of regulating the rate of uptake of sodium. T h e influence of external and internal sodium concentrations on the sodium uptake rate has been studied in a variety of species from both fresh water and brackish water. It has been found that the active uptake of sodium by these animals is determined, at low-medium concentrations, by the amount of sodium present in the medium. Furthermore, various crustacean species have sodium uptake systems with different affinities for sodium. T h e value Kin, the medium concentration at which the uptake system is half-saturated, is lower in fresh-water species than in brackish-water forms (Shaw, 1961a; Croghan & Lockwood, 1968). * Present address: Department of Biology, The Royal University of Malta, Msida,
Malta. 763
764
R.R. HARRIS
T h e effect of the sodium concentration of the haemolymph on the rate of sodium transport has also been investigated. Here the picture is not so clear. It has been suggested that lowering the sodium concentration below the "normal" level causes an increase in the rate of sodium uptake (Shaw, 1964 for review; Bryan, 1960). A simple feedback system has been proposed for Austrapotamobius (Shaw, 1959) where a fall in the sodium concentration elicits an increase in the sodium uptake rate. As the lost sodium is replaced, or a new steady-state haemolymph concentration is attained, the active uptake system is suppressed and the normal uptake rate is re-established. This is considered to be the general pattern of the control of sodium balance in aquatic Crustacea. Lockwood (1964) has shown that in the brackish-water amphipod Gammarus duebeni, the sodium concentration of the haemolymph may not be the only factor controlling the sodium active uptake system. A rapid fall in the haemolymph concentration, caused by washing animals acclimatized to high salinities in deionised water, resulted in high rates of sodium uptake. This treatment elicited higher rates of uptake than those measured in control animals with similar haemolymph concentrations. Sphaeroma rugicauda is a brackish-water isopod often found in the field with G. duebeni. A typical habitat is the saking pools and creeks of an estuary. Sudden and often large changes in salinity are encountered by Sphaeroma in these environments. In this paper the effect of a transfer from a high to a low salinity on the sodium uptake system is studied. Active uptake of sodium is determined in experimental conditions where interference by exchange diffusion is minimal, and also where the passive component of the uptake is small. T h e effect on the sodium uptake system of an increase in the medium concentration is also studied. METHODS AND MATERIALS
Sphaeroma rugicauda were collected by hand net from tidal creeks and salting pools at Totton, an area at the head of Southampton Water. They were kept in the laboratory at room temperature (16-20°C). Haemolymph samples were collected in a micropipette inserted intersegmentally in the region of the heart. The animals were discarded after sampling. The sodium concentration of both haemolymph and medium was measured by flame photometry on a Unicam SP 900 flame spectrophotometer. Duplicate analyses were possible on haemolymph samples from single animals. Determinations of a known standard were accurate to S.D. + 2%. This value includes both pipetting and instrument errors. Sodium influx was determined using the radioisotope ~2Na. The activity of this isotope was measured in an Ekco Universal scintillation counter with a well NaI (Th) crystal in conjunction with an Ekco scaler. Individual animals were placed in 2 ml of inactive medium in a test-tube which fitted into the well of the scintillation crystal. RESULTS
The sodium concentration of the haemolymph T h e concentration of sodium in the haemolymph of Sphaeroma acclimatized to a range of m e d i u m concentration is shown in Fig. 1. Sphaeroma were able to
S O D I U M U P T A K E I N T H E ISOPOD S P H A E R O M A R U G I C A U D A
765
tolerate direct transfer into salinities from 2-140 per cent sea water (10-694 mM/l NaC1). The animals survived in higher salinities (up to 185 per cent sea water) only if the medium concentration was increased gradually by increments of 5 per cent. The sodium concentration of the haemolymph is similar to that of the medium in' 100 per cent sea water. Below and above this medium concentration the sodium concentration of the haemolymph is respectively higher and lower than that of the medium. 900
800
--
700
E
600
_
_~o8
go
500
E 400 -
~
3OO
80§!;
osFJ-
o
g 2oo E
0
I00
200
300
i
400
I
500
!
600
S o d i u m conc. of m e d i u m ,
I
700
[
800
L
900
I
I000
mMIL
FIG. 1. The relation between the sodium concentration of the haemolymph and that of the external medium.
The influx of sodium at various haemolymph concentrations It has been found (Harris, 1967) that in 10 mM/l NaCI the sodium active transport system in S. rugicauda is approaching full saturation. Exchange diffusion has been shown to be neglible at this medium concentration and the passive influx of sodium ions has been calculated to be only 9 per cent of the total influx. That active transport of sodium does occur is indicated by the electrochemical gradient present across the gills of Sphaeroma. Uptake of 2~Na from a 10 mM/l NaC1 loading medium after correction for the small exchange diffusion and passive influx components will therefore reflect active transport of sodium. However, the sodium concentration of the haemolymph of an animal placed in the loading medium will be initially that corresponding to the steady-state concentration attained in the acclimatization medium. In the loading medium loss of sodium will occur as the sodium concentration of the haemolymph falls to a new steadystate value. It is probable that a large backflux of ~2Na will take place and therefore influx of ~Na into the animal will only represent active uptake for an initial period
766
R.R. HARRIS
in the loading medium. A loading period of 30 min was selected for all experiments after considering, in preliminary experiments, the rate of efflux of 22Na,from animals which had been fully loaded with the isotope, into inactive 10 mM/1NaC1. As a result of falling concentration of the haemolymph it is possible that the active transport system may be stimulated, for this reason also a short loading period was used. The active uptake of sodium by Sphaeromapreviously acclimatized to media ranging from 4-95 per cent sea water was investigated. Animals of similar size (12-15 mg) were placed into the loading medium of 10 mM]l NaC1 containing Z2Na for a period of 30 min. After this time they were removed, rinsed quickly in inactive medium and counted. Since the same loading medium was used for all animals, differences in the amount of 22Na taken up, after correction for the exchange diffusion and passive influx components, can be assumed to be mainly owing to differences in active uptake of sodium occurring in the acclimatization media prior to loading. At the end of each experiment, the animals were placed in a large volume of sea water and allowed to exchange all their Z2Na over a period of 10 days. They were then acclimatized to another medium concentration and the previous experiment repeated. Thus the amount of 22Na taken up during the loading period was measured in groups of animals acclimatized to high and low salinities. The results of these experiments are shown in Fig. 2.
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0
00
0 0
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Acclimatization medium,
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80
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90
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% sea water
FIG. 2. T h e uptake of 2~Na from a 10 m M / 1 NaC1 solution by Sphaeroma rugicauda acclimatized to a range of salinities. The solid circles and vertical lines indicate the mean and the standard error.
Considerable variation in the amount of 22Na taken up during the loading period by individual animals is observed. Comparing the amounts of tracer
S O D I U M U P T A K E I N T H E ISOPOD S P H A E R O M A R U G I C A U D A
767
taken up by animals acclimatized to high and low salinities, 3-58 times as much is taken up by animals from 4~o sea water than by those from 95~/o sea water.
Sodium influx under conditions of a falling haemolymph sodium concentration Active uptake of sodium in Sphaeroma after transfer from a high to a low salinity was investigated using the same loading medium as before. A group of 50 Sphaeroma was acclimatized to 180~/o sea water. These animals were rinsed quickly in deionized water, to remove traces of the acclimatization medium, and placed in 2 1 of 2% sea water. Haemolymph samples for sodium estimation were removed from three individuals taken directly from the acclimatization medium. The haemolymph samples were kept in watch-glasses coated with Repelcote, under liquid paraffin, for analysis at the end of the experiment. Three animals were also placed in the 10 raM/1 loading medium and allowed to take up ~2Na for a period of 30 min. These animals were then counted. At frequent intervals after transfer into the 2% sea water medium, similar groups of 3 animals were removed for simultaneous haemolymph sampling and the determination of 22Na uptake. Figure 3(a) shows the fall in the sodium concentration of the haemolymph of animals, acclimatized to 180~o and transferred to 2~o sea water, plotted against time. Initially there is a rapid fall in sodium concentration. After about 3 hr a decrease in the rate of fall in concentration is evident. After 10 hr the sodium concentration is close to that measured in animals acclimatized to 2~o sea water. The mean sodium concentrations at various times after transfer are used in Fig. 3(a) where they are plotted against the number of counts, corrected for exchange diffusion and passive influx, taken up by animals removed simultaneously and placed in the 10 mM/l, NaC1 loading medium for 30 rain. The amount of 22Na taken up by Sphaeroma during the loading period increases as the sodium concentration of the haemolymph falls. A rate of active uptake close to that measured previously in animals acclimatized to 4°/0 sea water can be elicited in animals with a haemolymph sodium concentration of 500 mM/1 Na. In animals acclimatized to 95% sea water, whose haemolymph concentration is about this value, the amount of 2~Na taken up in an identical loading period is far less than that taken up by the experimental animals. A maximal rate of sodium uptake seems to be reached. This is close to that rate recorded in animals acclimatized to 4 ~/o sea water.
Sodium influx after transfer from a dilute to a concentrated medium Animals were acclimatized to 2% sea water and, after rinsing in deionized water, placed in 2 1 of 100% sea water. Haemolymph samples were taken and the sampled animals discarded. Groups of 3 animals were allowed to take up 22Na from the loading medium for a 30 min period as before. A second group of animals was acclimatized to 40% sea water and similarly transferred to 2% sea water. Figure 4(a) shows the increase of the sodium concentration of the haemolymph of Sphaeroma after transfer from 2%-100% sea water.
768
R . R . HARRIS
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(,9
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I
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4
5
I
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7
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8
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decrease in h a e m o l y m p h s o d i u m concentration of Sphaeroma to 180% sea water and transferred to 2% sea water. T h e solid circles and vertical lines indicate the m e a n and the standard deviation.
FIG. 3a.
The
rugicauda acclimatized
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800
700
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500
400
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S o d i u m conc. of h o e m o l y m p h ,
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FIG. 3b. T h e relation b e t w e e n h a e m o l y m p h s o d i u m concentration and the uptake o f 2aNa from a 10 m M / 1 N a C I solution after transfer from 1 8 0 % - 2 % sea water.
SODIUM
500
UPTAKE
IN THE
ISOPOD
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769
S P H A E R O M A RUGICAUDA
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F I c . 4a.
The
increase in h a e m o l y m p h s o d i u m concentration of Sphaeroma to 2% sea water and transferred to 1 0 0 % sea water. T h e m e a n and the standard deviation are indicated.
rugicauda acclimatized
16--
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Sodium conc.of the haemolymph,
FIo. 4b. T h e relation b e t w e e n the s o d i u m and the uptake o f 22Na from a 10 m M / 1 NaC1 a h i g h salinity. O p e n circles refer to animals refer to animals acclimatized
o 500
mM/t
concentration o f the h a e m o l y m p h solution after transfer from a l o w to from 2% sea water. Closed circles to 4 0 % sea water.
770
R.R. HARRIS
The concentration rises rapidly initially, slowing as the steady-state level is approached. This takes about 10 hr after transfer from 2% sea water. In Fig. 4(b) the counts, corrected as before, taken up by animals during the course of the experiment are plotted against the prevailing sodium concentration of the haemolymph. The latter is taken as the mean of the sodium determinations made. Active uptake of sodium, of animals acclimatized to 2~o sea water and subsequently placed in 100% sea water, decreases rapidly. The amount of 22Na taken up during the loading period in this case is much less than that taken up by animals which are maintained in 2% sea water and not transferred to the higher salinity. This reduction in the rate of sodium uptake occurs after a rise in haemolymph sodium concentration of only about 50 mM/l Na, i.e. within 1 hr after transfer. The rate of uptake is maintained at this low level as the concentration of the haemolymph rises and also at steady-state conditions. Animals acclimatized to 10% sea water with a haemolymph concentration of about the same level, 250 mM/1 Na, showed a high rate of sodium uptake. It seems that transfer into a more concentrated medium affects the rate of sodium uptake rapidly. Animals treated in this way show much lower rates of sodium uptake than animals in steady-state conditions even though the sodium concentrations of their haemolymph are comparable. In animals acclimatized to 40% and transferred to 100% sea water a similar result was obtained, although the initial haemolymph concentration was above that of animals acclimatized to 2% sea water and consequently the number of counts taken up less. DISCUSSION A considerable amount of evidence has been collected in recent years concerning the regulation of the ion transport mechanisms responsible for the maintenance of salt balance in Crustacea. The sodium transport system has been shown to be saturable and rate limited (Shaw, 1960a, 1961a, b; Shaw & Sutcliffe, 1961; Sutcliffe, 1967; Croghan & Lockwood, 1968). Provided the external sodium concentration is high enough to saturate the sodium active uptake system, it was thought that the rate of uptake was related to the concentration of that ion in the haemolymph. Thus Bryan, (1960) found in Astacusfluviatilis that sodium influx is negligible when the haemolymph concentration is above 300 mM/l Na. As the haemolymph concentration fall below the "normal" level of 200 mM/1 Na, there is an initially slow but eventually fast increase in the rate of sodium influx. Similarly, Shaw (1961b) proposed that in Carcinus, a fall in the sodium concentration of the haemolymph below about 400 mM/1 Na of only 10 to 20 mM/1 Na leads to full activation of the sodium uptake mechanism. Both authors therefore suggested that the uptake system was related to haemolymph concentration and that increases in the rate of active transport occur as a result of a fall in haemolymph concentration below a defined level. Similar suggestions have been put forward by Shaw (1964) for Astacus and Lockwood (1960) for Asellus aquaticus. In the case of S. rugicauda however other factors beside that of absolute
SODIUM UPTAKE IN THE ISOPOD S P H A E R O M A R U G 1 C A U D A
771
haemolymph concentration appear to affect the rate of sodium uptake. High rates of sodium uptake can be elicited in animals with haemolymph concentrations which in steady-state conditions have been shown to be associated with low rates of sodium uptake. This has been found to occur when Sphaeroma is transferred from a concentrated to a dilute medium concentration. Lockwood (1964) has found similar results in G. duebeni. Treatment of acclimatized animals to a period in deionized water resulted in influxes of sodium several times greater than untreated control Gammarus. One factor that the system controlling active transport may be responding to is the relative changes in a number of the ions in the haemolymph. Shaw (1960b) has shown in Astacus pallipes that during salt depletion the animals may lose more sodium than chloride. Restoration of the normal sodium chloride concentration of the haemolymph involves, firstly, independent uptake of sodium or chloride to regain the initial ion ratio. This requires synchronization of the two uptake mechanisms. Secondly, when the new steady-state haemolymph concentration is reached, both uptake systems are suppressed by a primary feedback controller which responds to absolute haemolymph concentration. In Sphaeroma preliminary experiments have indicated that after transfer into a dilute medium there is no significantly faster fall in the haemolymph concentration of either sodium or chloride. One would expect that differential loss rates of these ions over a period of 10 hr would be detectable in terms of haemolymph concentration. Transferring Sphaeroma from a dilute to a concentrated medium results in a rapid suppression of sodium uptake. In Sphaeroma it seems unlikely that the sodium uptake system is responding to relative changes in sodium and chloride ion concentrations in the haemolymph. The rapid response of the uptake system in the latter situation, i.e. transfer from a low to a high salinity, adds weight to this suggestion. Animals acclimatized to high salinities and transferred to low ones will lose sodium rapidly. If the haemolymph is diluted at a rate faster than the intracellular concentration can be adjusted, water is likely to pass from the haemolymph into the cells. Harris (1969) has suggested that the characteristic curve of falling haemolymph concentration in Sphaeroma transferred from a high to a low salinity is a result of a water shift of this kind. Under these conditions the haemolymph volume is reduced by removal of water which passes into the cells. Lockwood (personal communication) has found that removal of a quantity of haemolymph from G. duebenicauses an increase in the rate of active uptake of sodium. Thus in Sphaeroma one factor that might affect a system controlling the sodium uptake mechanism is a change in the haemolymph volume. SUMMARY 1. The sodium concentration of the haemolymph of S. rugicauda acclimatized to salinities ranging from 2-185~o sea water was measured. In concentrations below and above 100% sea water the sodium concentration of the haemolymph is respectively higher and lower than that of the medium.
772
R . R . HARRIS
2. Using a m e t h o d which precluded interference by exchange diffusion, the active uptake of sodium by Sphaeroma, acclimatized to salinities f r o m 4 - 9 5 % sea water, was investigated. Animals f r o m 4 % s e a w a t e r t o o k u p a b o u t t h r e e t i m e s m o r e ~ N a during the loading period than animals f r o m 95% sea water. 3. Active uptake of sodium was measured in Sphaeroma during transfer f r o m a high to low salinity and vice versa. 4. Animals acclimatized to 180% and transferred to 2 % sea water took up more 22Na during the loading period than animals in steady-state conditions with comparable h a e m o l y m p h sodium concentrations. 5. Animals acclimatized to 2 % sea water and transferred to 100% sea water showed a rapid decrease in the active uptake of sodium. 6. T h e sodium uptake in Sphaeroma appears to respond to a factor other than the change in h a e m o l y m p h concentration f r o m a " n o r m a l " value. H i g h rates of sodium uptake, normally associated with steady-state conditions in low salinities and the corresponding h a e m o l y m p h concentrations, can be elicited at high haemol y m p h concentrations in animals transferred f r o m high to low salinity. Acknowledgements--I wish to thank Dr. A. P. M. Lockwood, under whose supervision this work was carried out, for his advice and criticism. I am indebted to the Science Research Council for a maintenance grant.
REFERENCES BRYAN G. W. (1960) Sodium regulation in the crayfish Astacus fluviatilis--I. Experiments with NaCl-loaded animals. J. exp. Biol. 37, 113-128. CROGHANP. C. ~ LOCKWOODA. P. M. (1968) Ionic regulation of the Baltic and freshwater races of the isopod Mesidotea (Saduria) entomon L. J. exp. Biol. 48, 141-158. HARRIS R. R. (1967) Ph.D. Thesis, The University of Southampton. HARRIS R. R. (1969) Free amino acid and haemolymph concentration changes in Sphaeroma rugicauda (Isopoda) during adaptation to a dilute salinity, ft. exp. Biol. 50, 319-326. KROGH A. (1938) The active absorption of ions in some fresh water animals. Z. vergI. Physiol. 25, 335-350. KROCH A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press. LOCKWOODA. P. M. (1960) Some effects of temperature and concentration of the medium on the ionic regulation of the isopod Asellus aquaticus L. J. exp. Biol. 37, 614-630. LOCKWOODA. P. M. (1964) Activation of the sodium uptake system at high blood concentration in the amphipod Gammarus duebeni. J. exp. Biol. 41, 447-458. SHAW J. (1959) The absorption of sodium ions by the crayfish, Astacus pallipes Lereboullet--I. The effect of external and internal sodium concentration. J. exp. Biol. 36, 126--144. SHAW J. (1960a) The absorption of sodium ions by the crayfish, Astacus pallipes Lereboullet--II. The effect of the external anion. J. exp. Biol. 37, 543-547. SHAWJ. (1960b) The absorption of chloride ions by the crayfish, Astacus pallipes Lereboullet. J. exp. Biol. 37, 557-572. SHAW J. (1961a) Sodium balance in Eriocheir sinensis (M. Edw.). The adaptation of the Crustacea to fresh water. J. exp. Biol. 38, 153-162. SHAW J. (1961b) Studies on ionic regulation in Carcinus maenas L . - - I . Sodium balance. J. exp. Biol. 38, 135-152.
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773
SHAW J. (1964) T h e control of salt balance in the Crustacea. Syrup. Soc. exp. Biol. 18, 237-254. SHAW J. & SUTCLIFFE D. W. (1961) Studies on sodium balance in Gammarus duebeni Lilljeborg and G. pulex pulex L. J. exp. Biol. 38, 1-15. SUTCLIFFED. W. (1967) Sodium regulation in the fresh water amphipod, Gammarus pulex L. ft. exp. Biol. 46, 499-518.
Key Word Index--Crustacea; sodium; active uptake; osmoregulation.
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