Osmotic and Ionic Regulation in the Prosobranch Gastropod Mollusc, Viviparus Viviparus Linn

J. Exp. Bio!. (1965), 43, 23-37 23 With 5 text-figwret Printed in Great Britain OSMOTIC AND IONIC REGULATION IN THE PROSOBRANCH GASTROPOD MOLLUSC, VIVIPARUS VIVIPARUS LINN. BY C. LITTLE Department of Zoology, Cambridge*

{Received 23 November 1964)

Investigations of the composition of the blood and tissues of freshwater invertebrates, and the relations that these bear to the composition of the external medium, have in the main been confined to crustaceans and insect larvae, whilst the molluscs have been somewhat neglected. Potts (1954, 1958) and Florkin & Duchiteau (1950a) have studied the lamellibranch Anodonta cygnaea in some detail. Duval & Portier (1927) and Florkin & Duchateau (19506) have given some figures for the pulmonate Limnaea stagnalis. Among the prosobranchs, published work appears to provide nothing more than values for the depression of freezing-point of the blood of Viviparus viviparus (Fredericq, 1904), V. fasciatus (Obuchowicz, 1958), and Theodoxus fluviatiUs(Neu - mann, i960). In the present paper, the composition of the blood and that of the opercular muscle of the freshwater prosobranch Viviparus viviparus Linn, are considered, and the relations of the composition of the blood to that of the external medium are examined.

MATERIAL AND METHODS Material. Viviparus were supplied by Haig's of Newdigate, and were kept in aquaria of approximately 10 1. capacity, containing streamwater and a 2 in. layer of mud collected from a local stream. The water was circulated and aerated by use of an air lift. Under these conditions the snails lived for long periods, some surviving for over a year. In general, animals with shells of 2-5-3-0 cm. in length, weighing 1-2 g. (wet weight without the shell) were used. Removal of blood samples A hole about 5 mm. square was filed in the shell near the umbilicus, care being taken not to puncture the body wall. Blood was withdrawn from the efferent renal sinuses and from the afferent branchial sinus, using a Pyrex pipette of diameter 1 mm. (diameter at tip approximately o-i mm.) into which liquid paraffin had first been drawn. The sample was transferred either to a watch-glass or to a small Pyrex tube and stored under liquid paraffin. The area of shell round the hole was painted with silicone (' Repelcote') and a wax cover was placed over it. When melted at the edges, this adhered firmly, and animals treated in this way opened within an hour of the operation. No anaesthetics were used. • Present address: Institute of Marine Science, University of Miami, Florida. 24 C. LITTLE For comparison, blood samples were taken from the head sinus; no difference was found in composition.

Measurement of the depression of freezing-point (A) Preliminary experiments showed that A of blood tended to increase if samples were kept for several hours. Samples for freezing-point determination were therefore frozen within 5 min. of removal from the animal, and A was determined by the method of Ramsay & Brown (1955). The apparatus was calibrated with solutions of sodium chloride, and A is expressed in terms of mM./l. NaCl. Estimations of concentrations of individual ions. The concentrations of individual ions should strictly be expressed as mg. ions/1., but since it has become conventional to regard mM./l. as synonymous with mg. ions/1., the concentrations of ions will here be expressed as mM./l.; or, when the balance of charges is at issue, as m-equiv./l. Chloride. Samples were kept under liquid paraffin, or for short periods on a waxed slide in a Petri dish containing damp filter paper. Depending upon the quantity of liquid available, one of the two electrometric titration methods of Ramsay, Brown & Croghan (1955) was used. Bicarbonate. Samples were centrifuged under liquid paraffin to remove corpuscles, and 0-05 or o-i ml. of blood was used in Conway's (1962) microdiffusion method. Sodium, potassium, calcium and magnesium. Samples were centrifuged under liquid paraffin and then diluted in de-ionized water. Determinations were made using either an EEL flame photometer working at its maximum sensitivity (for sodium only) or a Unicam flame photometer. No interference was found in the measurement of sodium or calcium, but there was considerable interference when potassium was measured. True potassium concentrations were found by dividing the sample, adding a known quantity of potassium to one-half, and taking readings for both sample and sample- plus-added-potassium. If the 'background reading' (average of readings 5 ra/i to either side of the potassium wavelength) is taken into account, then

X+A _ D2-Dm or (D.-DJxA where Dx = reading given by blood sample, D2 = reading given by blood with added potassium, Dm = background reading, A = added potassium, X = potassium in blood (true value). Usually the background reading was so low as to be negligible. This method was also used for magnesium. pH. The sample was drawn up under paraffin into a Pyrex pipette, and transferred to a small glass tube (volume 1 ml.). From there it was sucked into a capillary glass- electrode of 20 fil. capacity. The pH rose after the sample was withdrawn from the animal, so determinations were made within 30 sec. of taking the sample. Estimation of intracellular ion concentrations in muscle. The largest uniform tissue Osmotic and ionic regulation in V. viviparus 25 for analysis is the large opercular muscle. This was dissected out and a slice approximately 1 mm. thick and weighing 50-100 mg. was washed rapidly (10 sec.) in dextrose solution isotonic with the blood, dried between filter papers and weighed. The water content was found by drying for 2 hr. at ioo° C. in an oven and re-weighing. The muscle was digested in a silica tube at 60-800 C, with concentrated nitric acid and a known volume of o-i N silver nitrate to ensure that no chloride escaped as hydrochloric acid vapour. When digestion was complete the solution was evaporated to dryness and re-dissolved in a known volume of distilled water; o-i N hydrochloric acid was added to neutralize the silver nitrate exactly. Sodium, potassium, calcium and chloride in the solution were determined as described above; from these values and from the dilution factor, the concentrations in total muscle could be calculated. In order to find the intracellular ion concentrations the extracellular space must be known. This was calculated by injecting a solution of 20 % inulin (in Ringer solution isotonic with the blood) into the blood system, leaving the animal overnight to equilibrate, and measuring the ratio of inulin concentrations in blood and muscle. Inulin was extracted from the muscle by frequent shaking in a known volume of Ringer solution and was measured by the method of Roe, Epstein & Goldstein (1949).

THE FREEZING-POINT DEPRESSION AND COMPOSITION OF THE BLOOD (1) Snails living in stream water The composition of the blood of six animals taken straight from the aquaria is given in Table 1. All the snails were healthy and had been feeding. The total cations in the blood add up to 46-6 m-equiv./l., whereas the anions add up to only 42-0 m-equiv./t This suggests that either some anions are present which have not been detected, or that some of the cations are bound. Since the total anions add up to 42-0 mM./l., which is actually greater than the depression of freezing-point

Table 1. Composition of the blood of Viviparus from stream water Stream Blood Blood water Measurement (mM./l. ±8.E.) (m-equiv./l.) (m-equiv./L) A as NaCl 4°-5±o-8 (6) 4°-5 5 Sodium 34-o±o-8(6) 34° Potassium I-2±OI (6) 1-2 0-2 Calcium 5 7 ±0-4 (6) 114 6-6 Magnesium < o-s (2) < i-o — Total cations 409 466 103 Chloride 310±04 (6) 31-0 80 Bicarbonate II-O±I-4 (2) II-O — Total anions 42-0 420 80 pH 7-73±002 (10) Brackets denote number of observations. Note. (1) No magnesium was detectable using the flame photometer. The concentration is cer- tainly less than 0-5 mM./l. (2) Determinations of A for very dilute solutions are inaccurate and less accurate than the chloride determinations. The chloride concentration of stream water is therefore a truer measure of its ic content than the A value. 26 C. LITTLE (40-5 mM./l. NaCl), the second alternative seems more probable. Of the catiorJ| calcium is known to form complexes with proteins, and Schoffeniels (1951) has shown that in Anodonta 29 % of the plasma calcium is non-diffusible. In a second paper on Viviparus evidence is given concerning the relative composition of blood and peri- cardial fluid which also suggests that some calcium is bound (Little, 1965). Since all later experiments were carried out on animals that were not feeding, so as to prevent excessive fouling of the water, preliminary experiments were performed to note the effects of starvation. In tap water A of the blood stayed constant for up to at least 15 days, although the concentration of chloride decreased slightly. In 2% sea water both A and the concentration of chloride of the blood remained constant.

(2) Increasing salt content of the external medium Viviparus viviparus is found in England ' as far north as Yorks. in slow fair-sized rivers, canals and large draining ditches' (Boycott, 1936). In the Baltic, however, Viviparus is found in up to T>%O salinity (approx. 8-5% sea water) (Ankel, 1936). To show the effect of increased salt concentrations of the medium on the concentration of the blood, animals were placed in de-ionized water, and in 1, 2, 5, 10, 15 and 20% sea water. There is considerable variation between individuals and the means with standard deviations are given in Table 2.

Table 2. Depression of freezing-point of the blood of Viviparus living in different concentrations of sea water Medium Significance of difference Blood between A of blood and A A±8.D. of the medium Sea water (mM./l. (no. of observations) (%) NaCl) (mM./l. NaCl) t Probability De-ionized water 0 40-013-32 (10) 1 4-5 42-o±3-24 (14) 2 10 45-°±2-57 (10) 4206 < o-ooi 5 29 50-911-70 (10) 4056 < O'OOI 10 59 655±5-2i (10) 394 O-OI-O-OOI 15 85 87-7± 2-50(7) 2-87 0-02-001 20 no II2'O±4II (io) i-54 O'2-O-I

The snails appeared quite healthy in all concentrations up to 15% sea water. In 20% sea water many remained closed for long periods, and at the end of 10 days (the time allowed for equilibrium) none were crawling. The results are of the usual type for freshwater invertebrates, with the snails main- taining their blood hypertonic to the medium in the more dilute solutions but be- coming isotonic at higher concentrations. A 't' test (results in Table 2) shows that only in 20% sea water are the animals isotonic with the medium (i.e. no significant difference between the concentration of the blood and that of the medium at a 5 % level of probability). This concentration appears also to be the upper tolerance limit for the species, as snails placed in 25 % sea water died after a few days. Data for the concentration of sodium in the blood lead to similar conclusions (Fig. 1), with the concentration of sodium in the blood and in the medium being equal only in 20% sea water. Osmotic and ionic regulation in V. viviparus 27 The blood chloride presents rather a different picture (Fig. 2) Below an external concentration of about 40 mM./l- the concentration of chloride in the blood is greater than that in the medium, but above 40 mM./l. it is lower. Wigglesworth (1938)

100

•S 60 s 40 \ SI

20 s

20-40 60 80 100 Concentration of sodium in external medium (mM./L) Fig. 1. The concentration of sodium in the blood of snails after a period of io days in different solutions of sea water. Each point represents a sample from one individual. The diagonal is the iso-ionic line.

1201-

:? ioo

80 J3 .5

•g 60

20 40 60 80 100 120 Concentration of chloride in external medium (mM./l.) Fig. 2. The concentration of chloride in the blood of snails after a period of io days in different dilutions of sea water. Each point represents a sample from one individual. The diagonal is the iso-ionic line. 28 C. LITTLE described the same relationship for mosquito larvae, and Beadle & Shaw (1950) havS found that in the larvae of Sialis lutaria the non-chloride fraction of the blood is regulated by non-protein nitrogen. In Viviparus the non-chloride fraction is made up by bicarbonate, and as shown below this can compensate for a decrease of chloride. At levels of blood chloride of 50 miw./l. or higher the non-chloride fraction is about 10 mM./l. The average of nine bicarbonate determinations was also 10 mM./l.

Table 3. Concentrations of calcium and potassium in the blood, at different concentrations of sea water Medium Blood Sea water K Ca K (mM./l.) Ca (mM./l.) (%) (mM./l.) (mM./l.) Mean±s.E. Mean±s.E. 1 — — 1-3 ±0-3 (6) S-i±o-s(6) 5 0-2 0-9 2-1 ±02 (6) 3-sio-7 (6) 10 II i'5 I-2±O2 (6) 4-0+01 (6) IS i-5 2-5 3i±o-8 (6) 40 ±0-2 (6) 20 1-9 32 26±0-4 (4) 8-6±!-i(4) Brackets denote the number of observations.

The concentrations of calcium and potassium in the blood have also been measured in various dilutions of sea water (Table 3). There appears to be no tendency for these to increase in proportion to the external concentrations as the concentration of sea water increases. Experiments on potassium tolerance showed that animals will not tolerate 5 mM./l. KC1 and that they are only active in 1 mM./l. KC1 when this is present in 5 % sea water. In contrast, two animals placed in 70 mM./l. CaCljj survived and indeed crawled about and appeared quite normal. Their blood A was 100 mM./l. NaCl, with 11 mM./l. sodium and 78 mM./l. calcium.

(3) Decreasing salt content of the external medium and washing out Shaw (1959), working with Astacus pallipes, has described the sequence of events when an animal is placed in de-ionized water. Salt is initially lost until a certain concentration in the medium is reached. At this concentration the rate of salt uptake balances the rate of salt loss, and the animal comes into a steady state. If the animal is then transferred to a new volume of de-ionized water it again loses salt and comes into a steady state at a lower external concentration. This process can be repeated until eventually the external steady-state concentration reaches a value which cannot be reduced by transferring the animal to another volume of de-ionized water. Shaw calls this the minimum equilibrium concentration. Although it is strictly a steady state or dynamic equilibrium, the term minimum equilibrium concentration will be retained to avoid confusion, and similarly other steady states will be termed equilibria. In order to examine the relations between blood and external medium in Viviparus in the lower ranges of concentration, snails were placed separately in polythene beakers fitted with polythene aerator tubes and covers and filled with 100 ml. of de-ionized water. The concentrations of sodium and calcium in the medium were Osmotic and ionic regulation in V. viviparus 29 measured at intervals. Sodium came into equilibrium in 2 days, but calcium only reached a constant level after 4-7 days. To determine the minimum equilibrium concentration animals were allowed to come to equilibrium in 100 ml. of de-ionized water as described above; the water was changed and the animals again came to equilibrium. This procedure was repeated until the equilibrium concentrations showed no further decrease. Minimum equilibrium concentrations for sodium and calcium in six animals are given in Table 4.

Table 4. Minimum equilibrium concentrations of sodium and calcium

Sodium Calcium (mM./l.) (mM./l.) 0009 O-2O 0003 0-07 0-005 O-2O 0010 O-20 0-004 O-2O 0-006 032 Mean o-oo6 0-20

100-p 100 i S- 80 - OQ 80 O • O ==• 60 -- - 60 O J- 2 •• £1 #o •IO ot " O m go •o o* S 20 - Oo CD •» / - 20 Oo O • o 1 1 1 0 001 001 0-1 10 10-0 100 Total salt concentration of external medium, calculated as mM./L NaCl Fig. 3. Equilibrium concentrations for Viviparui placed in various dilutions of sea water and in de-ionized water. Dots represent depression of freezing-point; open circles represent sodium concentrations. The total salt concentration of the external medium is calculated from the sum of measured calcium and sodium. The curve is the iso-ionic line.

Minimum equilibrium concentrations of sodium are somewhat lower than those found by Shaw (1959) for Astacus (av. 0-04 mM./l). This difference is probably related to the much lower sodium content of Viviparus blood as compared to crusta- cean blood. In contrast, the minimum equilibrium concentrations of calcium are relatively high. The figures obtained by allowing snails to come to equilibrium in de-ionized water can be used to provide an extended graph showing the relation between internal and external concentrations. Since these latter equilibrium concentrations are very low the results are best plotted on a logarithmic scale (Fig. 3). The total salt concentration of the medium at very low concentrations was calculated from the sum of measured calcium and sodium concentrations (no potassium was 30 C. LITTLE detected in these equilibrium solutions). The graph 9hows a decrease of A of the blood to a constant low value, and a decrease of the concentration of sodium in the blood to a constant low value followed by a sudden fall below concentrations in the medium of o-i mM./l. NaCl. This suggests that A of the blood can be maintained even though sodium is lost, and this possibility has been further investigated. Blood composition in washed-out animals. The ' washing-out' of snails was effected by placing individuals in 500 ml. of de-ionized water and changing this once very day. Animals treated in this way survived for up to 30 days. Some figures for the composition of the blood after about 20 days in de-ionized water are given in Table 5, from which it can be seen that A of the blood can be decreased to about half its normal value. Sodium is reduced to about one-fifth of the normal concentration, while calcium increases slightly, thus balancing some of the sodium loss. Chloride is decreased greatly, while the concentration of bicarbonate shows an enormous increase. This compensates for the loss of chloride, and indeed in two cases the concentration of bicarbonate is greater than A of the blood. The explanation of this 'overshoot' is not known. The source of additional calcium and possibly of bicarbonate is discussed later. Table 5. Composition of the blood of snails washed out in de-ionized water A Na K Ca Cl HCO, (mM./l.) (mM./l.) (mM./l.) (mM./l.) (mM./l.) (mM./l.)

2O-0 62 i-o 13-3 4-S 265 I7'5 10-5 03 50 9-5 16s 15° 7-5 28 5-5 40 200 23-0 — — — 5° •— 16-0 — — — 3-o — 15-0 — — — 25 — Mean±s.E. i7-8±i-2 8i±o-3 i'4±o-7 7'9±2'3 4-8±o-9 2i-o±o-9

Table 6. Extracellular (inulin) spaces in the muscles of snails living in stream water and in 1-20 % sea water Medium Extracellular Sea space as % of water total muscle water No. of (%) %±8.E. observations 1 23±i-5 (7) S 23±i-6 (5) 10 26± I-I (6) IS 28±i-8 (4) 20 33±2-3 (3) Stream water 23±i-6 (S)

THE IONIC COMPOSITION OF THE OPERCULAR MUSCLE The opercular muscles of animals from stream water, of animals living in 1-20% sea water, and of two animals washed out in de-ionized water, have been analysed. Extracellular (inulin) spaces are shown in Table 6, and using these and the figures for ion content of whole muscle the intracellular ion concentrations have been calculated (Table 7). In animals from stream water intracellular sodium and potassium make up a total of 22-7 mM./l. The total cations in the blood add up to 40-9 mM./l. If the blood Osmotic and ionic regulation in V. viviparus 31 and the intracellular fluid have the same depression of freezing-point, this leaves a difference of 18-2 mM./l. unaccounted for. It is possible that part of this is made up by calcium ions, but calcium in fact has an average concentration of 96 mM./l. of total muscle water. It is shown later that much of this calcium is in deposits outside the muscle fibres. In the ventral adductor muscle oiAnodonta Potts (1958) found 11 mM./l. of free amino acids and 19*8 mM./l. phosphate, and concluded that some of both these were probably bound while the rest made up the depression of freezing-point not due

Table 7. Concentrations of ions in the muscles of snails living in stream water and in 1-20% sea water, and in those of two washed-out snails Muscle—intracellular water Medium Whole muscle Blood (mM./kg. intracellular Sea (mM./kg. muscle water±s.E.) (mM./l. ±s.E.) water ±s.E.) water 0/ Na K Ca Cl Na K Cl Na K Cl obs /o Stream water 136 146 96 IO'O 34° 1-2 310 7-6 151 39 5 ±06 ±2-5 ±55 ±o-8 ±i-o ±0-3 ±05 ±02 ±2-4 ±06 1 136 189 63 14-0 343 i"3 30-7 7'4 242 90 6 ±08 ±1-2 ±12 ±i-4 ±1-2 ±o-6 ±08 ±2-S ±16 ±18 5 183 15-3 IOI 142 39-8 2-1 35-7 n-8 193 82 6 ±0-5 ±09 ±69 + 07 ±o-7 ±02 ±I-I ±08 ±1-2 ±i-o 10 29-1 19-2 167 237 519 1-2 51-2 2I-I 255 141 6 ±2-7 ±i-7 ±22 ± 1'4 ±i-5 ±o-i ±18 ±3-3 ±2-3 ±16 IS 413 26s 198 36-5 76-1 31 72-3 27-7 3SO 22-8 6 ±i-7 ±19 ±95 ±i-7 ±i-5 ±o-8 ±1-3 ±2-S ±2-5 ±i"5 20 66-8 178 174 533 925 26 90-0 54-1 25-2 354 4 ±33 ±1-2 ±76 ±1-2 ±2-4 ±0-4 ±09 ±5-3 ±2-3 ±i-7 Washed-out 3-7 5"3 83 2-5 7'5 28 3-7 26 60 21 1 Washed-out 5-5 4-8 246 4-8 n-5 04 12-5 3-7 6-i 1-3 1 to sodium, potassium and chloride. Similar conditions probably obtain in Viviparus, but amino acids and phosphates have not been estimated. The distributions of sodium, potassium, calcium and chloride inside and outside the muscle cells, at a variety of blood concentrations, are discussed individually. Chloride. Up to a concentration of chloride in the blood of about 25 mM./l. the intracellular concentration of chloride stays at a very low value. Above this the concentration in the muscle cells increases in proportion to the concentration in the blood (Fig. 4). Shaw (1955), working on Carcinus maenas, found a constant relation between the chloride concentration in the blood and that in the muscle fibres, and he showed that the redistribution of chloride between blood and tissues at varying concentrations of the blood is brought about by normal diffusion processes. This may be true for Viviparus in concentrations above 25 mM./l. but below this the cells must actively retain chloride. Sodium. The blood:muscle ratio is very similar to that found for chloride. The intracellular concentration of sodium is constant below a concentration in the blood of about 30 mM./l. and above this it increases proportionately to the concentration in the blood (Fig. 5). Potassium. The concentration of potassium in the blood does not appear to rise when the concentration of the medium is increased over the range 1-20% sea water. In 32 C. LITTLE experiments carried out on potassium tolerance the concentration of potassium in the blood was never found to be greater than 7 mM./l. and it was assumed that high internal concentrations of potassium were lethal. Consequently it is not possible to evaluate the relationship between blood potassium and intracellular potassium.

= T40V r • • •

30 - • •S & • • •8 3 20 - • • • t t 10 • •

1 1 20 40 60 80 100 Concentration of chloride in blood (mM./l.) Fig. 4. The relation of chloride concentration in the blood to chloride concentration in muscle cells. Each point represents a sample from one individual.

80 1

B P 60 .S 3

11 40

g£

s c o O 20 40 60 80 100 Concentration of sodium in blood (mM./l.) Fig. 5. The relation of sodium concentration in the blood to sodium concentration in muscle cells. Each point represents a sample from one individual.

Calcium. Concentrations of calcium in the muscle are exceedingly large, as noted above, and the readings show an enormous scatter. Concentrations of calcium appear to be similar in the muscles of all animals from 1 to 20% sea water, but they are reduced in washed-out animals. Since most of the calcium must be osmotically inactive it was thought possible that it might be a form of calcium store, to be used, as it were, in time of need, as in the case of washed-out animals. The calcium could diffuse into the blood, as calcium is lost from the blood to the external de-ionized Osmotic and ionic regulation in V. viviparus 33 Pvater. Concentrations of calcium do tend to rise in the blood of washed-out animals (Table 5). Cue"not (1900) says that the connective tissue cells of Paludina (= Viviparus) contain yellowish granules of purines, sometimes melanin, and calcium. In older individuals the cells 'en sont litte"ralement bourr6es'. Numanoi (1939) examined the distribution of calcium in the tissues of a freshwater bivalve, Cristaria plicata, and decided that the gills acted as a calcium reservoir, but found very little calcium in the shell muscles. To investigate the site of calcium in the opercular muscle of Viviparus muscle slices were fixed in neutral formalin and stained with Erichochrome Black T (calcium deposits stain bright red, tissues stain blue; method given by Pearse, 1961). Scattered throughout the muscle are concretions, apparently enclosed by thin cell membranes. These concretions consist of an outer red-staining layer and a central non-staining part. As staining proceeds, the central core diminishes in size, while the red-staining layer expands. Since the central core appears crystalline, these observations have been interpreted to mean that the original body is calcium in crystalline form (this will not stain) and that this reacts with the stain and dissolves, producing a red coloration. It is these crystalline calcium deposits and not the calcium inside the muscle cells which determines the concentrations of calcium in the muscle given in Table 7.

DISCUSSION The blood of Viviparus viviparus has a depression of freezing-point intermediate between that of Anodonta (25 mM./l. NaCl, Potts, 1954) and that of Limnaea stagnalis (66 mM./l. NaCl, Picken, 1937). The value of 40-5 mM./NaCl given in the present

Table 8. Ratios of concentrations of Na, K and Cl inside and outside muscle cells, in snails from stream water, 1-20% sea water, and in washed-out snails Significance of difference between (K./K,,) and (Clo/Cl,) NaJNa, K./K. cuci, No. of Medium ±S.D. is.D. is.D. obs. t Probability Stream water 4-58±O-75 I3'8l4-2i 8461207 5 6234 < 001 i* 4-85±O-OI 16-711-71 3-3iii-52 4 6152 < o-oi S# 3-4810-58 9-8012-94 4-6212-15 6 3395 002-001 10* 3-3012-33 23-7i3-o6 4-2412-16 6 559O < o-oi IS* 2-831O-76 I4-I13-68 3-3010-34 6 4287 < o-oi 2O» I-78iO-28 10-611-59 2-5810-30 4 4345 005-002 Washed-out 30 10-14 8-7l6-58 57 13-92 2 0390 0-8-0-7 • Percentage sea water.

paper may be compared with a range of A = o-i7-o-2i° C. found by Fredericq (1904) for liquid extracted from various tissues. This is equivalent to 50-60 mM./l. NaCl, but the results must be regarded with suspicion because no precautions were taken to prevent the change of A after samples were obtained. Potts (quoted in Potts & Parry, 1964) gives the concentration as 94 m-osmole/kg. water, or approximately 50 mM./l. NaCl, but again no precautions are mentioned concerning possible increases of A after sampling. The blood is characterized by a high bicarbonate content and a 3 Exp. BioL 43. 1 34 C. LITTLE relatively high calcium content. These have been related to the presence of a cal- careous shell by Potts & Parry (1964). The importance of calcium in the relationship of Viviparus to its environment is further emphasized by the high value of the minimum equilibrium concentration for calcium. It is interesting to consider the calcium content of waters in which Viviparus normally lives, i.e. the ecological tolerance range for calcium. Boycott (1936) classed V. viviparus as a hard-water species, requiring at least 20 mg. calcium/1. (0-5 mM./l.), but he also recorded it from one location where there was only 11-13 mg./l (0-275- 0-325 mM./l.). These concentrations are very close to the minimum equilibrium concentrations, and it is possible that when other ecological conditions are favourable the minimum equilibrium concentration for calcium is truly a limiting factor. First impressions of the ionic composition of the opercular muscle are also domi- nated by the large deposits of calcium; but besides this, analysis of the ratios of intracellular: extracellular potassium and chloride (Table 8) shows that these differ signi- ficantly. In general, the concentrations of potassium and chloride ions in and outside cells are thought to obey a Donnan equilibrium, in which case

[Cl0] [CL]' and the low intracellular concentration of sodium is produced by the active transport of sodium out of the cells. However, Robertson (1957) found that of many invertebrates, including Mytilus edulis, the only one in which ionic ratios approximated to a

Table 9. Values of b for muscle cells permeability to potassium 0 = permeability to sodium Medium b Stream water 003 i* 018 5* o-p6 013 O-I2 2O# OI2 Washed-out O-IO • Percentage sea water.

Donnan equilibrium was Carcinus mamas. He postulated an active uptake of potassium by the cells to account for the difference. Potts (1958) found that of the muscles of Pecten, Mytilus and Anodonta, only the fast adductor of Pecten showed ratios for potassium and chloride close to a Donnan equilibrium. He suggested the possibility of a third phase in the muscle, other than blood or sarcoplasm, inaccessible to inulin but containing large quantities of sodium and chloride. However, he was unable to find large amounts of connective tissue in Mytilus byssus retractor; and he calculated that if this third phase were in the sarcolemma, the latter would have to contain the very large quantity of 100 mM./kg. muscle. An examination of the opercular muscle in Viviparus after fixation in Zenker and staining in Mallory's triple stain showed that there was no appreciable amount of connective tissue. Osmotic and ionic regulation in V. viviparus 35 Potts (1958) applied an equation, derived by Hodgkin (1958), for a system in which there is a small but not negligible permeability to sodium compared with potassium, and where a neutral pump maintains the cell in a steady state by absorbing one potassium ion for each sodium ion extruded. In such a system, the ionic ratios are given by

[KJ + flNaJ _ [Clt] [KJ + fttNa,] [Cl0]' where b is the permeability to sodium relative to the permeability to potassium. Values of b have been calculated for Viviparus living in stream water, in 1-20% sea water, and for two washed-out animals (Table 9). In general, values of b are similar in all media, but two points remain unexplained. First, although there is a significant difference between K)/Ko and CIQ/CIJ in animals from stream water, b in this case is very low. Secondly, in washed-out animals (admittedly only two individuals) there is no significant difference between Kj/K0 and C1O/C1,, while b is still relatively high. The theory of a neutral pump does not provide an explanation of all the results.

Table 10. The difference between A of the blood and the total intracelhilar cations as represented by the sum of K and Na Intra- Extra- cellular cellular total space Calculated cations A of Water as% difference (Na + K, blood in of total Water (due to Blood mM./kg. — total total muscle intra- shrinkage Medium A cell cations muscle water cellular of fibres) (% s.w.) (mM./l.) water) (mM./kg.) (%) (%) (%) (mM./kg.) I 42 316 94 87-1 23 671 9-4 5 49 311 179 86-6 23 66-7 9-4 10 64 466 17-4 876 26 648 97 15 87 627 243 844 28 608 104 20 112 793 32-7 816 33 54-7 II-S

One final point may be noted concerning the fact that the intracellular concentrations of sodium and potassium combined do not equal A of the blood. Assuming isotonicity between blood and cells calcium may make up a part of this difference, but since much of the calcium in the muscle is osmotically inactive other radicals probably make a large contribution to the total cations. Magnesium may be present, although none is detectable in the blood. Amino acids probably account for a major part of the difference. If the difference between total cations in the cells and the total expected on the basis of A values for the blood is largely made up of organic radicals, the shrinkage of fibres in higher sea water concentrations will produce a concentration effect, i.e. the difference between total cations and A of the blood will rise. Calculated and observed values are compared in Table 10. The observed rise is very much greater than the calculated one. It has been demonstrated in many invertebrates, including Anodonta and Mytilus (Potts, 1958,) Carcinus (Shaw, 1958), Nereis and Leander (Florkin, 1962) that the concentrations of amino acids in muscle cells rise when individuals are adapted to higher concentrations of sea water, and that this effect is reversed in dilute sea water. In Viviparus this regulation has been followed in a graded series of 3-2 36 C. LITTLE concentrations of sea water. It is interesting to note that the total intracellular sodium- plus-potassium is the same in 5 % as in 1 % sea water, while the difference between A of the blood and total cations has increased, i.e. regulation has been achieved entirely by the fraction which makes up this difference. At 10% sea water and in higher concentrations salt enters the cells. The animal is never found in concentrations of sea water greater than 8-5% sea water (Ankel, 1936), and it may be that this increase in intracellular sodium, although not harmful over short periods, is a limiting factor in long-term distribution.

This paper forms part of a dissertation for the degree of Ph.D. at Cambridge University. It is a great pleasure to thank Dr J. A. Ramsay, for his advice and con- structive criticism. I am indebted to the Department of Scientific and Industrial Research for financial support. SUMMARY 1. The inorganic composition of the blood of Viviparus has been examined. The mean A is 40-9 mM./l. NaCl, and the blood contains 34 mM./l. sodium, i-2mM./l. potassium, 5-7 mM./l. calcium, 31 mM./l. chloride, and 11 mM./l. bicarbonate. The pHis773. 2. When the concentration of the external medium is increased, A of the blood increases and in 20% sea water the blood is isosmotic with the external medium. Chloride is maintained in lower concentration in the blood than in the external medium. 3. The minimum concentrations of the external medium at which Viviparus can come to equilibrium are 0-006 mM./l. sodium and 0-20 mM./l. calcium. 4. After washing-out in de-ionized water A of the blood can be reduced to half its normal value. Chloride is reduced to about 5 mM./l. and is to some extent replaced by bicarbonate. 5. The ionic composition of the opercular muscle has been analysed. Much calcium is held in solid concretions. The ratios of internal: external potassium and chloride do not appear to obey a Donnan equilibrium. This matter is discussed. 6. The possibility is discussed that the concentration of amino acids in the cells increases when A of the blood is increased.

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