J. Exp. Bio!. (1965), 43, 23-37 2 3With 5 text-figwret

Printed in Great Britain

OSMOTIC AND IONIC REGULATION IN THEPROSOBRANCH GASTROPOD MOLLUSC,

VIVIPARUS VIVIPARUS LINN.

BY C. LITTLEDepartment of Zoology, Cambridge*

{Received 23 November 1964)

Investigations of the composition of the blood and tissues of freshwater inverte-brates, 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 molluscshave 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 pulmonateLimnaea stagnalis. Among the prosobranchs, published work appears to provide nothingmore 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 muscleof the freshwater prosobranch Viviparus viviparus Linn, are considered, and the re-lations 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 inaquaria of approximately 10 1. capacity, containing streamwater and a 2 in. layer ofmud collected from a local stream. The water was circulated and aerated by use of anair lift. Under these conditions the snails lived for long periods, some surviving forover a year.

In general, animals with shells of 2-5-3-0 cm. in length, weighing 1-2 g. (wet weightwithout the shell) were used.

Removal of blood samples

A hole about 5 mm. square was filed in the shell near the umbilicus, care beingtaken not to puncture the body wall. Blood was withdrawn from the efferent renalsinuses and from the afferent branchial sinus, using a Pyrex pipette of diameter 1 mm.(diameter at tip approximately o-i mm.)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 beendrawn. The sample was transferred either to a watch-glass or to a small Pyrex tube andstored under liquid paraffin.

The area of shell round the hole was painted with silicone (' Repelcote') and a waxcover was placed over it. When melted at the edges, this adhered firmly, and animalstreated 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 wasfound in composition.

Measurement of the depression of freezing-point (A)

Preliminary experiments showed that A of blood tended to increase if samples werekept for several hours. Samples for freezing-point determination were thereforefrozen within 5 min. of removal from the animal, and A was determined by themethod of Ramsay & Brown (1955). The apparatus was calibrated with solutions ofsodium chloride, and A is expressed in terms of mM./l. NaCl.

Estimations of concentrations of individual ions. The concentrations of individual ionsshould strictly be expressed as mg. ions/1., but since it has become conventional toregard mM./l. as synonymous with mg. ions/1., the concentrations of ions will here beexpressed 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 awaxed slide in a Petri dish containing damp filter paper. Depending upon thequantity 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 liquidparaffin and then diluted in de-ionized water. Determinations were made using eitheran EEL flame photometer working at its maximum sensitivity (for sodium only) or aUnicam flame photometer. No interference was found in the measurement of sodiumor calcium, but there was considerable interference when potassium was measured.True potassium concentrations were found by dividing the sample, adding a knownquantity 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 toeither 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 theanimal, 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 sliceapproximately 1 mm. thick and weighing 50-100 mg. was washed rapidly (10 sec.) indextrose 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 acidand a known volume of o-i N silver nitrate to ensure that no chloride escaped ashydrochloric acid vapour. When digestion was complete the solution was evaporatedto dryness and re-dissolved in a known volume of distilled water; o-i N hydrochloricacid was added to neutralize the silver nitrate exactly.

Sodium, potassium, calcium and chloride in the solution were determined asdescribed above; from these values and from the dilution factor, the concentrationsin total muscle could be calculated.

In order to find the intracellular ion concentrations the extracellular space must beknown. This was calculated by injecting a solution of 20 % inulin (in Ringer solutionisotonic with the blood) into the blood system, leaving the animal overnight toequilibrate, 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 Ringersolution and was measured by the method of Roe, Epstein & Goldstein (1949).

THE FREEZING-POINT DEPRESSION AND COMPOSITIONOF THE BLOOD

(1) Snails living in stream water

The composition of the blood of six animals taken straight from the aquaria is givenin 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 addup to only 42-0 m-equiv./t This suggests that either some anions are present whichhave not been detected, or that some of the cations are bound. Since the total anionsadd 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

Measurement

A as NaClSodiumPotassiumCalciumMagnesiumTotal cationsChlorideBicarbonateTotal anionsp H

Blood(mM./l. ±8.E.)

4°-5±o-8 (6)34-o±o-8(6)

I-2±OI (6)5 7 ±0-4 (6)

< o-s (2)4 0 9310±04 (6)I I - O ± I - 4 (2)42-0

7-73±002 (10)

Blood(m-equiv./l.)

4°-534°

1-2

1 1 4< i -o

4 6 631-0I I - O

4 2 0

Streamwater

(m-equiv./L)

5

0 - 2

6-6—

1 0 38 0—

8 0

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 itsic 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 shownthat in Anodonta 29 % of the plasma calcium is non-diffusible. In a second paper onViviparus 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 asto prevent excessive fouling of the water, preliminary experiments were performed tonote the effects of starvation. In tap water A of the blood stayed constant for up to atleast 15 days, although the concentration of chloride decreased slightly. In 2% seawater 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-sizedrivers, 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). Toshow the effect of increased salt concentrations of the medium on the concentrationof 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 withstandard deviations are given in Table 2.

Table 2. Depression of freezing-point of the blood of Viviparus living indifferent concentrations of sea water

Medium

Sea water(%)

De-ionized water125

101520

A(mM./l.NaCl)

04-5

10295985

n o

BloodA±8.D.

(no. of observations)(mM./l. NaCl)

40-013-32 (10)42-o±3-24 (14)45-°±2-57 (10)50-911-70 (10)655±5-2i (10)87-7± 2-50(7)

II2'O±4II (io)

Significance of differencebetween A of blood and

of the medium

t

420640563942-87i-54

Probability

< o-ooi< O'OOI

O-OI-O-OOI0-02-001O'2-O-I

The snails appeared quite healthy in all concentrations up to 15% sea water. In20% 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 thatonly in 20% sea water are the animals isotonic with the medium (i.e. no significantdifference 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 limitfor 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 equalonly in 20% sea water.

Osmotic and ionic regulation in V. viviparus 27

The blood chloride presents rather a different picture (Fig. 2) Below an externalconcentration of about 40 mM./l- the concentration of chloride in the blood is greaterthan that in the medium, but above 40 mM./l. it is lower. Wigglesworth (1938)

100

80

•S 60

s

40

20

s

\ SI

2 0 - 4 0 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 dif-ferent solutions of sea water. Each point represents a sample from one individual. Thediagonal is the iso-ionic line.

1201-

:? ioo

80J3

.5

•g 60

40

20

20 40 60 80 100 120Concentration of chloride in external medium (mM./l.)

Fig. 2. The concentration of chloride in the blood of snails after a period of io days indifferent dilutions of sea water. Each point represents a sample from one individual. Thediagonal is the iso-ionic line.

28 C. LITTLE

described the same relationship for mosquito larvae, and Beadle & Shaw (1950) havSfound that in the larvae of Sialis lutaria the non-chloride fraction of the blood isregulated by non-protein nitrogen. In Viviparus the non-chloride fraction is made upby 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 about10 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

MediumBlood

Seawater(%)

1

51 0

IS2 0

K(mM./l.)

—0-2

I I

i-51-9

Ca(mM./l.)

—0-9

i'52-53 2

K (mM./l.)Mean±s.E.

1-3 ±0-3 (6)2-1 ± 0 2 (6)I-2±O2 (6)3i±o-8 (6)26±0-4 (4)

Ca (mM./l.)Mean±s.E.

S-i±o-s(6)3-sio-7 (6)4-0+01 (6)40 ±0-2 (6)8-6±!-i(4)

Brackets denote the number of observations.

The concentrations of calcium and potassium in the blood have also been measuredin various dilutions of sea water (Table 3). There appears to be no tendency for theseto increase in proportion to the external concentrations as the concentration of seawater increases. Experiments on potassium tolerance showed that animals will nottolerate 5 mM./l. KC1 and that they are only active in 1 mM./l. KC1 when this ispresent in 5 % sea water. In contrast, two animals placed in 70 mM./l. CaCljj survivedand 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 eventswhen an animal is placed in de-ionized water. Salt is initially lost until a certain con-centration in the medium is reached. At this concentration the rate of salt uptakebalances the rate of salt loss, and the animal comes into a steady state. If the animal isthen transferred to a new volume of de-ionized water it again loses salt and comes intoa steady state at a lower external concentration. This process can be repeated untileventually the external steady-state concentration reaches a value which cannot bereduced by transferring the animal to another volume of de-ionized water. Shawcalls this the minimum equilibrium concentration. Although it is strictly a steadystate or dynamic equilibrium, the term minimum equilibrium concentration will beretained to avoid confusion, and similarly other steady states will be termedequilibria.

In order to examine the relations between blood and external medium in Viviparusin the lower ranges of concentration, snails were placed separately in polythenebeakers fitted with polythene aerator tubes and covers and filled with 100 ml. ofde-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 onlyreached a constant level after 4-7 days.

To determine the minimum equilibrium concentration animals were allowed tocome to equilibrium in 100 ml. of de-ionized water as described above; the water waschanged and the animals again came to equilibrium. This procedure was repeateduntil the equilibrium concentrations showed no further decrease. Minimum equi-librium concentrations for sodium and calcium in six animals are given in Table 4.

Table 4. Minimum equilibrium concentrations of sodium and calcium

Sodium(mM./l.)

000900030-00500100-0040-006

Mean o-oo6

Calcium(mM./l.)

O-2O0-07O-2OO-20O-2O0 3 2

0-20

100-p

S- 80O

==• 60 -

£1

O20 -

-

-

• •

m go • o # o o*Oo CD • »

Oo O •o

1 1 1

•ItO o "

iOQ

• O

J-/

100

80

- 60O

2

- 20 S

0 001 001 0-1 10 10-0 100Total salt concentration of external medium, calculated as mM./L NaCl

Fig. 3. Equilibrium concentrations for Viviparui placed in various dilutions of sea waterand in de-ionized water. Dots represent depression of freezing-point; open circles repre-sent sodium concentrations. The total salt concentration of the external medium is calculatedfrom the sum of measured calcium and sodium. The curve is the iso-ionic line.

Minimum equilibrium concentrations of sodium are somewhat lower than thosefound by Shaw (1959) for Astacus (av. 0-04 mM./l). This difference is probablyrelated to the much lower sodium content of Viviparus blood as compared to crusta-cean blood. In contrast, the minimum equilibrium concentrations of calcium arerelatively high. The figures obtained by allowing snails to come to equilibrium inde-ionized water can be used to provide an extended graph showing the relationbetween internal and external concentrations. Since these latter equilibrium con-centrations 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 calculatedfrom 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 bloodto a constant low value, and a decrease of the concentration of sodium in the blood to aconstant low value followed by a sudden fall below concentrations in the medium ofo-i mM./l. NaCl. This suggests that A of the blood can be maintained even thoughsodium is lost, and this possibility has been further investigated.

Blood composition in washed-out animals. The ' washing-out' of snails was effectedby 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 com-position 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 normalvalue. Sodium is reduced to about one-fifth of the normal concentration, whilecalcium increases slightly, thus balancing some of the sodium loss. Chloride is de-creased greatly, while the concentration of bicarbonate shows an enormous increase.This compensates for the loss of chloride, and indeed in two cases the concentrationof bicarbonate is greater than A of the blood. The explanation of this 'overshoot' isnot known. The source of additional calcium and possibly of bicarbonate is discussedlater.

Table 5. Composition of the blood of snails washed out in de-ionized water

A(mM./l.)

2O-0

I7'51 5 °23-016-015-0

Na(mM./l.)

6 210-57-5———

K(mM./l.)

i-o0 32 8———

Ca(mM./l.)

13-35 0

5-5———

Cl(mM./l.)

4-S9-54 0

5°3-o2 5

HCO,(mM./l.)

2 6 516s2 0 0

• —

—

—

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 livingin stream water and in 1-20 % sea water

MediumSea

water(%)

1

S1 0

IS2 0

Stream water

Extracellularspace as % of

total muscle water%±8.E.

23±i-523±i-626± I-I28±i-833±2-323±i-6

No. ofobservations

(7)(5)(6)(4)(3)

(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 forion content of whole muscle the intracellular ion concentrations have been calculated(Table 7). In animals from stream water intracellular sodium and potassium make upa 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 adifference of 18-2 mM./l. unaccounted for. It is possible that part of this is made up bycalcium ions, but calcium in fact has an average concentration of 96 mM./l. of totalmuscle water. It is shown later that much of this calcium is in deposits outside themuscle 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 thesewere probably bound while the rest made up the depression of freezing-point not due

Table 7. Concentrations of ions in the1-20% sea water, and

muscles of snails living in stream water and inin those of two washed-out snails

MediumSea

water0//o

Stream water

1

5

1 0

I S

2 0

Washed-outWashed-out

Whole muscle(mM./kg. muscle

Na

1 3 6± 0 6

1 3 6± 0 8

1 8 3±0-5

29-1±2-74 1 3

±i-766-8

± 3 3

3-75-5

K

1 4 6±2-51 8 9

±1-2

15-3± 0 9

19-2

±i-72 6 s

± 1 9178

±1-25"34-8

water±s.E.)

Ca

96±55

63± 1 2

IOI

±69167

± 2 2

198± 9 5

174±76

8 3246

Cl

IO'O

±o-814-0

±i-41 4 2

+ 072 3 7

± 1'4

36-5±i-7

533±1-2

2-54-8

Blood(mM./l. ±s

Na

3 4 °±i-o

343±1-239-8

±o-75 1 9

±i-576-1

±i-5

9 2 5±2-4

7'5n - 5

K

1-2

±0-3

i"3

±o-62-1

± 0 2

1-2

±o-i3 1

±o-82 6

±0-42 80 4

.E.)

Cl

3 1 0

± 0 5

30-7± 0 8

35-7± I - I

51-2± 1 8

72-3±1-3

90-0

± 0 9

3-712-5

Muscle—intracellularwater

(mM./kg. intracellularwater ±s.E.

Na

7-6± 0 2

7'4±2-S

n - 8± 0 8

2 I - I

±3-327-7

±2-S

54-1±5-3

2 6

3-7

K

1 5 1±2-42 4 2

± 1 6

1 9 3±1-22 5 5

±2-33SO

±2-525-2

±2-36 06-i

)

Cl

3 9± 0 6

9 0± 1 8

8 2

±i-o1 4 1

± 1 6

22-8

±i"5

354±i-7

2 1

1-3

obs

5

6

6

6

6

4

1

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 outsidethe 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. theintracellular concentration of chloride stays at a very low value. Above this the con-centration 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 betweenthe chloride concentration in the blood and that in the muscle fibres, and he showedthat the redistribution of chloride between blood and tissues at varying concentrationsof the blood is brought about by normal diffusion processes. This may be true forViviparus in concentrations above 25 mM./l. but below this the cells must activelyretain chloride.

Sodium. The blood:muscle ratio is very similar to that found for chloride. Theintracellular concentration of sodium is constant below a concentration in the bloodof about 30 mM./l. and above this it increases proportionately to the concentration inthe blood (Fig. 5).

Potassium. The concentration of potassium in the blood does not appear to rise whenthe 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 theblood was never found to be greater than 7 mM./l. and it was assumed that highinternal concentrations of potassium were lethal. Consequently it is not possible toevaluate the relationship between blood potassium and intracellular potassium.

= 40r

•S &•8 3

TV

30

20

10

-

-

tt

•

•

•

• •

••

1

•

1

•

•

•

20 40 60 80

Concentration of chloride in blood (mM./l.)

100

Fig. 4. The relation of chloride concentration in the blood to chloride concentration inmuscle cells. Each point represents a sample from one individual.

1

B P.S 3

11

80

60

40

g£

sco

O20 40 60 80

Concentration of sodium in blood (mM./l.)

100

Fig. 5. T h e relation of sodium concentration in the blood to sodium concentration in musclecells. Each point represents a sample from one individual.

Calcium. Concentrations of calcium in the muscle are exceedingly large, as notedabove, and the readings show an enormous scatter. Concentrations of calcium appearto be similar in the muscles of all animals from 1 to 20% sea water, but they arereduced in washed-out animals. Since most of the calcium must be osmoticallyinactive 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 coulddiffuse 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 olderindividuals the cells 'en sont litte"ralement bourr6es'. Numanoi (1939) examined thedistribution of calcium in the tissues of a freshwater bivalve, Cristaria plicata, anddecided that the gills acted as a calcium reservoir, but found very little calcium in theshell muscles. To investigate the site of calcium in the opercular muscle of Viviparusmuscle 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 cellmembranes. These concretions consist of an outer red-staining layer and a centralnon-staining part. As staining proceeds, the central core diminishes in size, while thered-staining layer expands. Since the central core appears crystalline, these obser-vations have been interpreted to mean that the original body is calcium in crystallineform (this will not stain) and that this reacts with the stain and dissolves, producinga red coloration. It is these crystalline calcium deposits and not the calcium inside themuscle cells which determines the concentrations of calcium in the muscle givenin Table 7.

DISCUSSION

The blood of Viviparus viviparus has a depression of freezing-point intermediatebetween 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, insnails from stream water, 1-20% sea water, and in washed-out snails

Medium

Stream wateri *

S#

1 0 *

I S *2O»

Washed-out

NaJNa,±S.D.

4-58±O-754-85±O-OI3-4810-583-3012-332-831O-76I-78iO-2830 10-14

K./K.is.D.

I3'8l4-2i16-711-719-8012-9423-7i3-o6I4-I13-6810-611-598-7l6-58

cuci,is.D.

84612073-3iii-524-6212-154-2412-163-3010-342-5810-3057 13-92

No. ofobs.

5466642

Significance ofdifference between

(K./K,,)

t

623461523395559O428743450390

and (Clo/Cl,)

Probability

< 001

< o-oi002-001

< o-oi< o-oi

005-002

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 toprevent 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 Aafter 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 isfurther emphasized by the high value of the minimum equilibrium concentration forcalcium. It is interesting to consider the calcium content of waters in which Viviparusnormally lives, i.e. the ecological tolerance range for calcium. Boycott (1936) classedV. 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 con-centrations, and it is possible that when other ecological conditions are favourablethe 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 intra-cellular: extracellular potassium and chloride (Table 8) shows that these differ signi-ficantly. In general, the concentrations of potassium and chloride ions in and outsidecells are thought to obey a Donnan equilibrium, in which case

[Cl0][CL]'

and the low intracellular concentration of sodium is produced by the active transportof sodium out of the cells. However, Robertson (1957) found that of many inverte-brates, including Mytilus edulis, the only one in which ionic ratios approximated to a

Table 9. Values of b for muscle cells

permeability to potassium0 =

permeability

Medium

Stream wateri*

5*

2 O #

Washed-out

• Percentage se

to sodium

b

0 0 30 1 8o-p60 1 3O-I2O I 2

O-IO

a water.

Donnan equilibrium was Carcinus mamas. He postulated an active uptake of potas-sium by the cells to account for the difference. Potts (1958) found that of the musclesof Pecten, Mytilus and Anodonta, only the fast adductor of Pecten showed ratios forpotassium and chloride close to a Donnan equilibrium. He suggested the possibilityof a third phase in the muscle, other than blood or sarcoplasm, inaccessible to inulinbut containing large quantities of sodium and chloride. However, he was unable tofind large amounts of connective tissue in Mytilus byssus retractor; and he calculatedthat if this third phase were in the sarcolemma, the latter would have to contain thevery large quantity of 100 mM./kg. muscle. An examination of the opercular musclein Viviparus after fixation in Zenker and staining in Mallory's triple stain showed thatthere 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 whichthere 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 potas-sium 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% seawater, and for two washed-out animals (Table 9). In general, values of b are similar inall media, but two points remain unexplained. First, although there is a significantdifference between K)/Ko and CIQ/CIJ in animals from stream water, b in this case isvery low. Secondly, in washed-out animals (admittedly only two individuals) there isno significant difference between Kj/K0 and C1O/C1,, while b is still relatively high. Thetheory 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 cationsas represented by the sum of K and Na

Medium(% s.w.)

I

51 0

152 0

BloodA

(mM./l.)

4 2496487

1 1 2

Intra-cellular

totalcations

(Na + K,mM./kg.

cellwater)

3 1 63 1 14 6 66 2 77 9 3

A ofblood— totalcations

(mM./kg.)

9 41 7 917-42 4 332-7

Waterin

totalmuscle

(%)

87-186-68768448 1 6

Extra-cellularspacea s %

of totalmusclewater(%)

23232628

33

Waterintra-

cellular(%)

6 7 166-76 4 86 0 8

54-7

Calculateddifference

(due toshrinkageof fibres)(mM./kg.)

9-49-49 7

1 0 4I I - S

One final point may be noted concerning the fact that the intracellular concentra-tions of sodium and potassium combined do not equal A of the blood. Assumingisotonicity between blood and cells calcium may make up a part of this difference, butsince much of the calcium in the muscle is osmotically inactive other radicals probablymake a large contribution to the total cations. Magnesium may be present, althoughnone is detectable in the blood. Amino acids probably account for a major part of thedifference.

If the difference between total cations in the cells and the total expected on thebasis of A values for the blood is largely made up of organic radicals, the shrinkage offibres in higher sea water concentrations will produce a concentration effect, i.e. thedifference between total cations and A of the blood will rise. Calculated and observedvalues are compared in Table 10. The observed rise is very much greater than thecalculated one. It has been demonstrated in many invertebrates, including Anodontaand Mytilus (Potts, 1958,) Carcinus (Shaw, 1958), Nereis and Leander (Florkin, 1962)that the concentrations of amino acids in muscle cells rise when individuals areadapted to higher concentrations of sea water, and that this effect is reversed in dilutesea 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 Aof the blood and total cations has increased, i.e. regulation has been achieved entirelyby the fraction which makes up this difference. At 10% sea water and in higher con-centrations salt enters the cells. The animal is never found in concentrations of seawater greater than 8-5% sea water (Ankel, 1936), and it may be that this increase inintracellular sodium, although not harmful over short periods, is a limiting factor inlong-term distribution.

This paper forms part of a dissertation for the degree of Ph.D. at CambridgeUniversity. 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 IndustrialResearch for financial support.

SUMMARY

1. The inorganic composition of the blood of Viviparus has been examined. Themean 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. ThepHis773 .

2. When the concentration of the external medium is increased, A of the bloodincreases 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 cancome 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 itsnormal value. Chloride is reduced to about 5 mM./l. and is to some extent replaced bybicarbonate.

5. The ionic composition of the opercular muscle has been analysed. Much cal-cium is held in solid concretions. The ratios of internal: external potassium andchloride 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 cellsincreases when A of the blood is increased.

REFERENCES

ANKEL, W. E. (1936). Prosobranchia. Tierwelt N.-u. Ostsee, 39, (96), I.BEADLE, L. C. & SHAW, J. (1950). The retention of salt and the regulation of the non-protein fraction of

the blood of the aquatic larva Sialis lutaria. J. Exp. Biol. 37, 96-109.BOYCOTT, A. E. (1936). The habitats of fresh-water mollusca in Britain. J. Amm. Ecol. 5, 116-86.CONWAY, E. S. (1962). Microdiffution Analysis and Volumetric Error. London: Crosby and Lockwood.CUENOT, L. (1900). L'excretion chez les mollusques. Arch. Biol., Paris, 16, 49-96.DUVAL, M. & PORTIHR, P. (1927). Sur la teneur en gaz carbonique total du sang des invert^bres d'eau

douce et des invertebr^s marins. CJI. Acad. Sci., Paris, 184, 1594-6.FLORKIN, M. (1962). La regulation isosmotique intracellulaire chez les invert6br£s marins euryhalins.

Bull. Acad. Belg. Cl. Sci. 48, 687-9.FLORKIN, M. & DUCHATEAU, G. (1950 a). Concentrations cellulaire et plasmatique du potassium, du

calcium et du magnesium chez Anodonta cygnaea. C.R. Soc. Biol., Paris, 144, 1131—2.FLORKIN, M. & DUCHATEAU, G. (19506). Concentrations cellulaire et plasmatique du potassium, du

calcium et du magnesium chez une s^rie d'animaux dulcicoles. CJi. Soc. Biol., Paris, 114, 1132-3.FREDERICQ, L. (1004). Note sur le concentration moleculaire des tissus solides de quelques animaux

d'eau douce. Arch. int. Physiol. 2, 127-30.

Osmotic and ionic regulation in V. viviparus 37HODGKIN, A. L. (1958). Ionic movements and electrical activity in giant nerve fibres. Proc.Roy. Soc.B,

U&, i-37-LITTLE, C. (1965). The formation of urine by the prosobranch gastropod mollusc, Viviparus viviparus

Linn. J. Exp. Biol. 43, 39-54.NEUMANN, D. (i960). Osmotische Resistenz und Osmoregulation der Flussdeckelschnecke Theodoxus

fluviatUii. Biol. Zbl. 79, 585-605.NUMANOI, H. (1939). Distribution of calcium in the soft parts of the freshwater bivalve Cristaria

pUcala. Jap. J. Zool. 8, 353-6-OBUCHOWICZ, L. (1958). The influence of osmotic pressure of medium on oxygen consumption in

Viviparui fasciatus. Bull. Soc. Amis. Set. Pozndn, B, 14, 367—70.PEAHSE, A. G. E. (1961). Histoehermitry, Theoretical and Applied. London: Churchill.PICKEN, L. E. R. (1937). The mechanism of urine formation in invertebrates. 2. The excretory

mechanism in certain Mollusca J. Exp. Biol. 14, 20-34.POTTS, W. T. W., (1954). The inorganic composition of the blood of Mytilus cdulis and Anodonta

cygnea. J. Exp. Biol. 31, 376-85.POTTS, W. T. W. (1958). The inorganic and amino acid composition of some lamellibranch muscles.

J. Exp. Biol. 35, 749-64.POTTS, W. T. W. & PARRY, G. (1964). Osmotic and Ionic Regulation in Animals. Oxford: Pergamon

Press.RAMSAY, J. A. & BROWN, R. H. J. (1955). Simplified apparatus and procedure for freezing-point

determinations upon small volumes of fluid. J. Sci. Instrum. 33, 372—5.RAMSAY, J. A., BROWN, R. H. J. & CROGHAN, P. C. (1955). Electrometric titration of chloride in small

volumes. J. Exp. Biol. 3a, 822-9.ROBERTSON, J. D. (1957). Osmotic and ionic regulation in aquatic invertebrates. In Recent Advances in

Invertebrate Physiology, ed. Scheer, University of Oregon.ROE, J. H., EPSTEIN, J. H. & GOLDSTEIN, N. P. (1949). A photometric method for determining inulin

in plasma and urine. J. Biol. Chem. 178, 839—45.SCHOFFENIEL8, E. (1951). Distribution du calcium diffusible et non-diffusible dans le plasma sanguin

de l'Anodonte. Arch. Int. Physiol. 59, 49-52.SHAW, J. (1955). Ionic regulation in the muscle fibres of Cardnus maenas. 2. The effect of reduced

blood concentration. J. Exp. Biol. 33, 664-80.SHAW, J. (1958). Further studies on ionic regulation in the muscle fibres of Cardnus maenas. J. Exp.

Biol. 35, 902-19.SHAW, J. (1959). The absorption of sodium ions by Astacus pallipes. 1. The effect of external and

internal sodium concentrations. J. Exp. Biol. 36, 126-44.WIGGLESWORTH, V. B. (1938). The regulation of osmotic pressure and chloride concentration in the

haemolymph of mosquito larvae. J. Exp. Biol. 15, 235-47.