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Volume regulation in Nereis diversicolor—II. the effect of calcium
Camp. B&hem.
Physiol., 1974, Vol. 47A, pp. 1215 to 1220. Perganwn Press. Printed in Great Blitoin
VOLUME REGULATION IN NEREIS DIVERSICOLOR-II. THE EFFECT OF CALCIUM C. R. FLETCHER Department of Pure and Applied Zoology, University of Leeds, Leeds LS2 9JT, England (Received 3 May 1973) Abstract-l. The effect of lack of calcium on the water permeability of Nereis diversicolor has been examined. 2. Calcium lack does not result in a significantly increased water permeability measured either by exchange of labelled water molecules or by osmotic flow. 3. It is concluded that swelling in calcium-free media, which commences after a delay of more than 4 hr, is due to reduction in the rate of urine flow, probably caused by loss of internal calcium. 4. The requirement for internal calcium probably lies in the dependence of ciliary activity on extracellular calcium. INTRODUCTION
been known for many years that calcium is necessary for volume regulation in Nereis diversicolor, and animals transferred from sea water to diluted sea water in which calcium is lacking fail to control their volume, unlike animals transferred to diluted sea water containing normal amount of calcium (Beadle, 1931). This was confirmed by Ellis (1937) w h o also showed that even when the animals’ volumes had stabilized in normal 20% sea water withdrawal of calcium resulted in swelling after a delay of about 12 hr, and such changes were reversible. Neither author showed any change in volume regulation until the worms had been in calcium-free media for several hours, and both authors interpreted their observations in terms of an increase in water permeability in the absence of calcium. This is reasonable since calcium is known to be necessary for cellular adhesion (Gasic & Galanti, 1966) which is necessary for epithelial integrity, and is also known to be necessary to maintain a low permeability in some cells (e.g. McCutcheon Sz Lucke, 1928). However, it has also been shown that the calcium fluxes of N. diverskolor are substantial whilst its ability to regulate its internal calcium in the presence of changes in the calcium to chloride ratio of its environment is minimal, and it has been proposed that the swelling in calcium-free media could result from the cessation of urine production when the internal calcium levels have dropped excessively (Fletcher, 1970). In order to distinguish between these two hypotheses experiments have been conducted to determine the effect of removal of the external calcium on the water permeability of these animals. IT HAS
1215
1216
C. R. FLETCHER MATERIALS
AND
METHODS
Animals were acquired and maintained as already described (Fletcher, 1973), and were acclimatised to 20% sea water for at least 6 days, at 12-14°C before experiments were conducted. This salinity was chosen because it is towards the middle of the animals’ range of tolerance but they are substantially hyperosmotic in this salinity. Sea water was acquired from the Wellcome Marine Laboratory at Robin Hood’s Bay, and calcium-free artificial sea water was prepared according to the data of Hale (1965), omitting the calcium chloride or replacing it with sodium chloride. Chemicals were of Analar grade. Firstly, the weight changes of animals transferred from normal to calcium-free 20% sea water were followed, and the condition of the animals recorded. Details of the weighing technique have already been given (Fletcher, 1974). The diffusional water permeabilities of animals were measured by the influx of tritiated water, using calcium-free loading solutions where appropriate. The osmotic water permeabilities were determined by rates of change of weight of animals, relative to controls, when subjected to a change of external osmotic pressure of -t 150 mOsmo1 approximately, and the solutions were calcium free where appropriate. Details of these methods have been given already (Fletcher, 1973). During experiments worms were kept individuaily in beakers containing 50 ml of media which was changed at least once. Efflux of calcium from the animals could not have raised the calcium concentrations of these solutions by more than 0.02 mM. The chlorosity and osmotic concentrations of each solution used were checked with a Cotlove amperometric chloride titrator and a vapour pressure osmometer. All experiments were conducted at 12 f 0.5%.
The changes of weight of twenty-two animals after transfer to calcium-free media are shown in Fig. 1, together with the data for twenty-two controls remaining in normal medium. Each point is shown as mean + SD. with standard errors denoted by the box. The acclimatization media were 195 and 196 mosmolal, and experimental and control animals were taken at random from these two media, The controls were transferred to 191 mosmolal, and the experimental animals to 207 mosmolal.
/-.-.I ,
5 5
,,,?I -
&
25 5 t ::
I.10-
L
+
e
bO5-
/~ t p%q...~+________t_____________~_ o-95
0
1
4
I
8 Time,
I
12
i
I6
I
20
I
24'
hr
FIG. 1. Weight as a fraction of original weight of animals acclimatized to 20% sea water and transferred to calcium-free 20% sea water (full line) or normal 2076 sea water (broken line). S.D. shown by vertical lines, SE. by the central block.
VOLUME
REGULATION
1217
IN NEREZS DZVERSZCOLORII
The initial slight fall in weight is probably due to the effects of handling and to 2°C drop in temperature. These factors cannot account for the difference between control and experimental animals in the first 2 hr (P < 0.005 at 1 hr, P < 0.02 at 2 hr), nor is the slight difference in osmotic pressures of the control and calcium-free solutions sufficient to account for it, so these differences may represent an early effect of lack of calcium on the animals. At later times the animals in calcium-free media show a marked swelling, being significantly swollen after 8 hr (P
Diffusional permeability This was measured as a rate constant K for twelve animals. The first group was acclimatized measured from such a medium. The other groups from 0 to 24 hr, and K was determined from such
water exchange, using groups of ten to to normal 20% sea water and the influx had been in calcium-free media for times a solution.
TABLE~-RATEOFDIFFUSIONALWATERINFLUXASAFUNCTIONOFRESIDENCETIMEINCALCIUMFREE MEDIA
Control
K
I
0 hr
2 hr
6 hr
24 hr
3.47 k 0.32 4.13 + 0.25 3.57 + 0.48 4.08 f 044 4.34 f 0.50 S.E. = 0.16 (10) SE. = 0.13 (12) SE. = 0.10 (12) S.E. = 0.14 (12) S.E. = 0.09 (12) 30.0 SE. = 1.1
28.2 S.E. = 0.9
27.9 S.E = 0.7
24.8 S.E. = 1.0
28.8 S.E. = 0.8
K is the rate constant, in hr-l, shown with S.D., S.E. and No. of observations in parentheses. I is the gross influx in kg water/kg ash-free dry weight of animal per hr, and the S.E.‘s for I include uncertainty in K and the degree of swelling. The controls were measured in normal calcium media. The results are given in Table 1, and show no increase in K in calcium free conditions, but rather a decline. However, the rate constant is affected by the water content of the animals, which is increasing as the animals swell. This may be corrected for by expressing the gross water influx in terms of the ash-free dry weight of the animals, which may be assumed to be constant. This has been done assuming that these animals had a water content initially like those in the preceding paper and swelled like those shown in Fig. 1. The resulting gross influxes, 1, are also given in Table 1. The data show no evidence for an increase in permeability to labelled water molecules either immediately after calcium removal or when swelling commences. Indeed there is some suggestion of a decrease in permeability at 2 hr and 6 hr after transfer, though between sample variance is not significantly larger than within sample variance (P> 0.1).
1218
C.R.FLETCHER
Osmotic water permeability This was calculated from the rates of change of weight of animals subjected to an increase or decrease of salinity of 15% sea water, as already detailed. Eighty experimental animals and forty controls were used to determine the permeability of animals acclimatized to normal 20% sea water, and groups of about half these sizes to determine permeabilities after 0,s and 18 hr in calcium-free media, using calcium-free experimental solutions. The water content required for calculations was derived from the water content of animals acclimatized to normal 20% sea water and the increase in weight; the internal osmotic pressure from the value for animafs acclimatized to 20% sea water diluted by the fractional increase in weight (water). Neither of these parameters is of critical importance. Since the permeabilities calculated are relative to the weight of the animal which is variable the results have been corrected to the weight that the animal had before transfer to calcium-free media. A few results have been discarded where the animal was obviously damaged and leakage of coelomic fluid was observed. TABLE
%--OSMOTIC
WATER PERMEABILITY,
PO,, AS
A FUNCTION
OF RESIDENCE TIME IN CALCIUM-
FREE MEDIA
Controls
PO,
0.139 + 0.008 (76)
0 hr 0*160f0*012 (39)
5 hr
18 hr
0~152~0*011 (37)
0.154 _+0.012 (35)
PoB in kg HzO/kg of animal in normal calcium medium per hr per 1 osmolal concentration difference. P,, of controls was measured in normal calcium media; P,, of the others were measured in calcium-free media. The results are given as mean rt S.E. with the number of experimental animals in parentheses. The results are given in Table 2, and it can be seen that there is an indication of a slight increase in permeability on transfer to calcium-free solution, but this is small, and not statistically significant (P>O*l). During the subsequent 18 hr there is no significant change in permeability. DISCUSSION
The swelling in calcium-free conditions does not commence until the animals have been in calcium-free conditions for more than 4 hr. Ellis’s (1937) data show no measurable swelling until 12 hr after calcium removal. However, he also showed marked differences between the time course of volume regulation in different populations, and this may be another aspect of regional variation. The water permeabilities, both diffusional and osmotic, are in good agreement with those already reported (Fletcher, 1973), and any increase in permeability on transfer to calcium-free sea water is small, and could not possibly account for the rate of swelling observed in these conditions. The lack of change in both osmotic and diffusional permeabilities shows that if the discrepancy found between the two is caused by porosity of the body wall the dimensions of the pores are not dependent on external calcium; equally if the discrepancy is caused by unstirred layers these are unaffected by lack of calcium. The body wall does not show the increased
VOLUME
REGULATION
IN NEREIS
DIVERSICOLOR-II
1219
hydration and breakdown of intercellular cement seen in other epithelia (Robertson, 1941; Mannery, 1966). This may be a result of properties of the cuticle which has been found to be resistant to hydrolysis by trypsin and papain (unpublished observations). It may also be the cuticle which limits water movements. The delayed swelling is not caused by an increase in water permeability. Two other hypotheses are possible ; either the internal osmotic pressure is raised in calcium-free conditions resulting in a raised water influx or the normal rate of urine production is reduced. The first hypothesis is contrary to Beadle’s (1937) observations that the swelling in calcium-free conditions is accompanied by a reduction in internal osmotic pressure to nearly iso-osmotic, and also refuted by Ellis’s (1937) observations of a continuing but small net loss of chloride during this time. Thus the last hypothesis must be correct. It is not improbable since it has already been deduced that the main cause of urine flow is ciliary beating, and calcium is known to be required for ciliary beating (Gray, 1924), and at least in some instances the response of the cell to stimuli is mediated by the entry of calcium and regenerative depolarisation, the responses being depressed by reduced levels of extracellular calcium (Eckert, 1972; Murakami & Eckert, 1972). It is not known how the activity of nephridial cilia is controlled, though a future paper will show that good control exists. For the swelling to be caused by cessation of nephridial activity in response to lack of internal calcium requires that the internal calcium levels be reduced in calcium-free media. It has already been shown (Fletcher, 1970) that the calcium fluxes are large, and the gross efflux in 20% sea water is such that about half of the total calcium would be lost in 10 hr in calcium-free conditions, if the body wall potential were maintained. If it fell under these conditions the loss would be even more rapid, and these observations were made on animals from a different location which may have been more like those of Ellis (1937). The loss of activity and muscle tone by the time swelling commences suggests that the internal calcium levels have fallen sufficiently to interfere with nervous and muscular functioning It is thus concluded that the swelling results from reduction of nephridial flow caused by loss of internal calcium, The rate of urine production in normal 20% sea water has been deduced to be 27.5 g H,O/kg of animal per hr (Fletcher, 1973), which is substantially greater than the rate of swelling. If we assume that the total quantity of internal solutes remains unaltered the osmotic concentration difference between the animal and its environment is falling. We may deduce that after 18 hr in Ca2+-free media the rate of urine flow calculated as the difference between the osmotic influx and the rate of swelling is about a quarter of the normal rate, and the true value must be even lower as the total internal solutes are falling (Ellis, 1937). Thus urine production is very markedly depressed by this time. REFERENCES L. C. (1931) The effect of salinity changes on the water content and respiration of marine invertebrates. J. exp. Biol. 8, 211-227. BEADLE L. C. (1937) Adaptation to changes of salinity in the polychaetes-I. Control of body volume and body fluid concentration in Nereis diversicolor. J. exp. Biol. 14, 56-70
BEADLE
1220
C. R. FLETCHER
ECKERTR. (1972) Bioelectric control of ciliary activity. Science, N. Y. 176, 473-481. ELLISW. G. (1937) The water and electrolyte exchanges of Nereis diversicolor (Mfiller J. exp. Biol. 14, 340-350. FLETCHERC. R. (1970) The regulation of calcium and magnesium in the brackish water polychaete Nereis diversicolor O.F.M. J. exp. Biol. 53, 425-443. FLETCHERC. R. (1974) Volume regulation in Nereis diversicolor-I. The steady state. Camp. Biochem. Physiol. 47A, 1199-1214. GASIC G. J. & GALANTIN. L. (1966) Proteins and disulphide groups in the aggregation of dissociated cells of sea sponges. Science, N.Y. 151, 203-205. GRAY J. (1924). The mechanism of ciliary movement-IV. The relation of ciliary activity to oxygen consumption. PYOC.R. Sot. B 96, 95-114. HALE L. J. (1965). Biological Laboratory Data, 2nd edn. Methuen, London. MCCUTCHEONM. & LWCKEB. (1928). The effect of certain electrolytes and non-electrolytes on permeability of living cells to water. J. gen. PhysioE. 12, 129-138. MANNERYJ. F. (1966). Effect of Ca on cell membranes. Fedn Proc. Fedn Am Sots exp. Biol. 25, 1804-1810. MURAKAMIA. & ECK~RTR. (1972) Cilia: activation coupled to a mechanical stimulation by calcium influx. Science, N. Y. 175, 13751377. ROBERTSON J. D. (1941) The function and the metabolism of calcium in the invertebrata. Biol, Rev. 16, 106-133. Key Word Index-Nereis diversicolor; Polychaete; estuarine; euryhaline; brackish water; calcium ions; water permeability; effect of calcium on epithelia.
Physiol., 1974, Vol. 47A, pp. 1215 to 1220. Perganwn Press. Printed in Great Blitoin
VOLUME REGULATION IN NEREIS DIVERSICOLOR-II. THE EFFECT OF CALCIUM C. R. FLETCHER Department of Pure and Applied Zoology, University of Leeds, Leeds LS2 9JT, England (Received 3 May 1973) Abstract-l. The effect of lack of calcium on the water permeability of Nereis diversicolor has been examined. 2. Calcium lack does not result in a significantly increased water permeability measured either by exchange of labelled water molecules or by osmotic flow. 3. It is concluded that swelling in calcium-free media, which commences after a delay of more than 4 hr, is due to reduction in the rate of urine flow, probably caused by loss of internal calcium. 4. The requirement for internal calcium probably lies in the dependence of ciliary activity on extracellular calcium. INTRODUCTION
been known for many years that calcium is necessary for volume regulation in Nereis diversicolor, and animals transferred from sea water to diluted sea water in which calcium is lacking fail to control their volume, unlike animals transferred to diluted sea water containing normal amount of calcium (Beadle, 1931). This was confirmed by Ellis (1937) w h o also showed that even when the animals’ volumes had stabilized in normal 20% sea water withdrawal of calcium resulted in swelling after a delay of about 12 hr, and such changes were reversible. Neither author showed any change in volume regulation until the worms had been in calcium-free media for several hours, and both authors interpreted their observations in terms of an increase in water permeability in the absence of calcium. This is reasonable since calcium is known to be necessary for cellular adhesion (Gasic & Galanti, 1966) which is necessary for epithelial integrity, and is also known to be necessary to maintain a low permeability in some cells (e.g. McCutcheon Sz Lucke, 1928). However, it has also been shown that the calcium fluxes of N. diverskolor are substantial whilst its ability to regulate its internal calcium in the presence of changes in the calcium to chloride ratio of its environment is minimal, and it has been proposed that the swelling in calcium-free media could result from the cessation of urine production when the internal calcium levels have dropped excessively (Fletcher, 1970). In order to distinguish between these two hypotheses experiments have been conducted to determine the effect of removal of the external calcium on the water permeability of these animals. IT HAS
1215
1216
C. R. FLETCHER MATERIALS
AND
METHODS
Animals were acquired and maintained as already described (Fletcher, 1973), and were acclimatised to 20% sea water for at least 6 days, at 12-14°C before experiments were conducted. This salinity was chosen because it is towards the middle of the animals’ range of tolerance but they are substantially hyperosmotic in this salinity. Sea water was acquired from the Wellcome Marine Laboratory at Robin Hood’s Bay, and calcium-free artificial sea water was prepared according to the data of Hale (1965), omitting the calcium chloride or replacing it with sodium chloride. Chemicals were of Analar grade. Firstly, the weight changes of animals transferred from normal to calcium-free 20% sea water were followed, and the condition of the animals recorded. Details of the weighing technique have already been given (Fletcher, 1974). The diffusional water permeabilities of animals were measured by the influx of tritiated water, using calcium-free loading solutions where appropriate. The osmotic water permeabilities were determined by rates of change of weight of animals, relative to controls, when subjected to a change of external osmotic pressure of -t 150 mOsmo1 approximately, and the solutions were calcium free where appropriate. Details of these methods have been given already (Fletcher, 1973). During experiments worms were kept individuaily in beakers containing 50 ml of media which was changed at least once. Efflux of calcium from the animals could not have raised the calcium concentrations of these solutions by more than 0.02 mM. The chlorosity and osmotic concentrations of each solution used were checked with a Cotlove amperometric chloride titrator and a vapour pressure osmometer. All experiments were conducted at 12 f 0.5%.
The changes of weight of twenty-two animals after transfer to calcium-free media are shown in Fig. 1, together with the data for twenty-two controls remaining in normal medium. Each point is shown as mean + SD. with standard errors denoted by the box. The acclimatization media were 195 and 196 mosmolal, and experimental and control animals were taken at random from these two media, The controls were transferred to 191 mosmolal, and the experimental animals to 207 mosmolal.
/-.-.I ,
5 5
,,,?I -
&
25 5 t ::
I.10-
L
+
e
bO5-
/~ t p%q...~+________t_____________~_ o-95
0
1
4
I
8 Time,
I
12
i
I6
I
20
I
24'
hr
FIG. 1. Weight as a fraction of original weight of animals acclimatized to 20% sea water and transferred to calcium-free 20% sea water (full line) or normal 2076 sea water (broken line). S.D. shown by vertical lines, SE. by the central block.
VOLUME
REGULATION
1217
IN NEREZS DZVERSZCOLORII
The initial slight fall in weight is probably due to the effects of handling and to 2°C drop in temperature. These factors cannot account for the difference between control and experimental animals in the first 2 hr (P < 0.005 at 1 hr, P < 0.02 at 2 hr), nor is the slight difference in osmotic pressures of the control and calcium-free solutions sufficient to account for it, so these differences may represent an early effect of lack of calcium on the animals. At later times the animals in calcium-free media show a marked swelling, being significantly swollen after 8 hr (P
Diffusional permeability This was measured as a rate constant K for twelve animals. The first group was acclimatized measured from such a medium. The other groups from 0 to 24 hr, and K was determined from such
water exchange, using groups of ten to to normal 20% sea water and the influx had been in calcium-free media for times a solution.
TABLE~-RATEOFDIFFUSIONALWATERINFLUXASAFUNCTIONOFRESIDENCETIMEINCALCIUMFREE MEDIA
Control
K
I
0 hr
2 hr
6 hr
24 hr
3.47 k 0.32 4.13 + 0.25 3.57 + 0.48 4.08 f 044 4.34 f 0.50 S.E. = 0.16 (10) SE. = 0.13 (12) SE. = 0.10 (12) S.E. = 0.14 (12) S.E. = 0.09 (12) 30.0 SE. = 1.1
28.2 S.E. = 0.9
27.9 S.E = 0.7
24.8 S.E. = 1.0
28.8 S.E. = 0.8
K is the rate constant, in hr-l, shown with S.D., S.E. and No. of observations in parentheses. I is the gross influx in kg water/kg ash-free dry weight of animal per hr, and the S.E.‘s for I include uncertainty in K and the degree of swelling. The controls were measured in normal calcium media. The results are given in Table 1, and show no increase in K in calcium free conditions, but rather a decline. However, the rate constant is affected by the water content of the animals, which is increasing as the animals swell. This may be corrected for by expressing the gross water influx in terms of the ash-free dry weight of the animals, which may be assumed to be constant. This has been done assuming that these animals had a water content initially like those in the preceding paper and swelled like those shown in Fig. 1. The resulting gross influxes, 1, are also given in Table 1. The data show no evidence for an increase in permeability to labelled water molecules either immediately after calcium removal or when swelling commences. Indeed there is some suggestion of a decrease in permeability at 2 hr and 6 hr after transfer, though between sample variance is not significantly larger than within sample variance (P> 0.1).
1218
C.R.FLETCHER
Osmotic water permeability This was calculated from the rates of change of weight of animals subjected to an increase or decrease of salinity of 15% sea water, as already detailed. Eighty experimental animals and forty controls were used to determine the permeability of animals acclimatized to normal 20% sea water, and groups of about half these sizes to determine permeabilities after 0,s and 18 hr in calcium-free media, using calcium-free experimental solutions. The water content required for calculations was derived from the water content of animals acclimatized to normal 20% sea water and the increase in weight; the internal osmotic pressure from the value for animafs acclimatized to 20% sea water diluted by the fractional increase in weight (water). Neither of these parameters is of critical importance. Since the permeabilities calculated are relative to the weight of the animal which is variable the results have been corrected to the weight that the animal had before transfer to calcium-free media. A few results have been discarded where the animal was obviously damaged and leakage of coelomic fluid was observed. TABLE
%--OSMOTIC
WATER PERMEABILITY,
PO,, AS
A FUNCTION
OF RESIDENCE TIME IN CALCIUM-
FREE MEDIA
Controls
PO,
0.139 + 0.008 (76)
0 hr 0*160f0*012 (39)
5 hr
18 hr
0~152~0*011 (37)
0.154 _+0.012 (35)
PoB in kg HzO/kg of animal in normal calcium medium per hr per 1 osmolal concentration difference. P,, of controls was measured in normal calcium media; P,, of the others were measured in calcium-free media. The results are given as mean rt S.E. with the number of experimental animals in parentheses. The results are given in Table 2, and it can be seen that there is an indication of a slight increase in permeability on transfer to calcium-free solution, but this is small, and not statistically significant (P>O*l). During the subsequent 18 hr there is no significant change in permeability. DISCUSSION
The swelling in calcium-free conditions does not commence until the animals have been in calcium-free conditions for more than 4 hr. Ellis’s (1937) data show no measurable swelling until 12 hr after calcium removal. However, he also showed marked differences between the time course of volume regulation in different populations, and this may be another aspect of regional variation. The water permeabilities, both diffusional and osmotic, are in good agreement with those already reported (Fletcher, 1973), and any increase in permeability on transfer to calcium-free sea water is small, and could not possibly account for the rate of swelling observed in these conditions. The lack of change in both osmotic and diffusional permeabilities shows that if the discrepancy found between the two is caused by porosity of the body wall the dimensions of the pores are not dependent on external calcium; equally if the discrepancy is caused by unstirred layers these are unaffected by lack of calcium. The body wall does not show the increased
VOLUME
REGULATION
IN NEREIS
DIVERSICOLOR-II
1219
hydration and breakdown of intercellular cement seen in other epithelia (Robertson, 1941; Mannery, 1966). This may be a result of properties of the cuticle which has been found to be resistant to hydrolysis by trypsin and papain (unpublished observations). It may also be the cuticle which limits water movements. The delayed swelling is not caused by an increase in water permeability. Two other hypotheses are possible ; either the internal osmotic pressure is raised in calcium-free conditions resulting in a raised water influx or the normal rate of urine production is reduced. The first hypothesis is contrary to Beadle’s (1937) observations that the swelling in calcium-free conditions is accompanied by a reduction in internal osmotic pressure to nearly iso-osmotic, and also refuted by Ellis’s (1937) observations of a continuing but small net loss of chloride during this time. Thus the last hypothesis must be correct. It is not improbable since it has already been deduced that the main cause of urine flow is ciliary beating, and calcium is known to be required for ciliary beating (Gray, 1924), and at least in some instances the response of the cell to stimuli is mediated by the entry of calcium and regenerative depolarisation, the responses being depressed by reduced levels of extracellular calcium (Eckert, 1972; Murakami & Eckert, 1972). It is not known how the activity of nephridial cilia is controlled, though a future paper will show that good control exists. For the swelling to be caused by cessation of nephridial activity in response to lack of internal calcium requires that the internal calcium levels be reduced in calcium-free media. It has already been shown (Fletcher, 1970) that the calcium fluxes are large, and the gross efflux in 20% sea water is such that about half of the total calcium would be lost in 10 hr in calcium-free conditions, if the body wall potential were maintained. If it fell under these conditions the loss would be even more rapid, and these observations were made on animals from a different location which may have been more like those of Ellis (1937). The loss of activity and muscle tone by the time swelling commences suggests that the internal calcium levels have fallen sufficiently to interfere with nervous and muscular functioning It is thus concluded that the swelling results from reduction of nephridial flow caused by loss of internal calcium, The rate of urine production in normal 20% sea water has been deduced to be 27.5 g H,O/kg of animal per hr (Fletcher, 1973), which is substantially greater than the rate of swelling. If we assume that the total quantity of internal solutes remains unaltered the osmotic concentration difference between the animal and its environment is falling. We may deduce that after 18 hr in Ca2+-free media the rate of urine flow calculated as the difference between the osmotic influx and the rate of swelling is about a quarter of the normal rate, and the true value must be even lower as the total internal solutes are falling (Ellis, 1937). Thus urine production is very markedly depressed by this time. REFERENCES L. C. (1931) The effect of salinity changes on the water content and respiration of marine invertebrates. J. exp. Biol. 8, 211-227. BEADLE L. C. (1937) Adaptation to changes of salinity in the polychaetes-I. Control of body volume and body fluid concentration in Nereis diversicolor. J. exp. Biol. 14, 56-70
BEADLE
1220
C. R. FLETCHER
ECKERTR. (1972) Bioelectric control of ciliary activity. Science, N. Y. 176, 473-481. ELLISW. G. (1937) The water and electrolyte exchanges of Nereis diversicolor (Mfiller J. exp. Biol. 14, 340-350. FLETCHERC. R. (1970) The regulation of calcium and magnesium in the brackish water polychaete Nereis diversicolor O.F.M. J. exp. Biol. 53, 425-443. FLETCHERC. R. (1974) Volume regulation in Nereis diversicolor-I. The steady state. Camp. Biochem. Physiol. 47A, 1199-1214. GASIC G. J. & GALANTIN. L. (1966) Proteins and disulphide groups in the aggregation of dissociated cells of sea sponges. Science, N.Y. 151, 203-205. GRAY J. (1924). The mechanism of ciliary movement-IV. The relation of ciliary activity to oxygen consumption. PYOC.R. Sot. B 96, 95-114. HALE L. J. (1965). Biological Laboratory Data, 2nd edn. Methuen, London. MCCUTCHEONM. & LWCKEB. (1928). The effect of certain electrolytes and non-electrolytes on permeability of living cells to water. J. gen. PhysioE. 12, 129-138. MANNERYJ. F. (1966). Effect of Ca on cell membranes. Fedn Proc. Fedn Am Sots exp. Biol. 25, 1804-1810. MURAKAMIA. & ECK~RTR. (1972) Cilia: activation coupled to a mechanical stimulation by calcium influx. Science, N. Y. 175, 13751377. ROBERTSON J. D. (1941) The function and the metabolism of calcium in the invertebrata. Biol, Rev. 16, 106-133. Key Word Index-Nereis diversicolor; Polychaete; estuarine; euryhaline; brackish water; calcium ions; water permeability; effect of calcium on epithelia.