364

THE PHYSIOLOGY OF CONTRACTILE VACUOLES

I. OSMOTIC RELATIONS

BY J. A. KITCHING.

(From the Department of Zoology, Birkbeck College, London.)

(Received \th November, 1933.)

(With Eight Text-figures.)

INTRODUCTION.CONSIDERABLE discussion has centred around the contractile vacuoles of Protozoa,and both structure and functions are discussed in Lloyd's review of the subject(1928). Of the various suggestions which have been made as to what is theirfunction, the only two probable ones are (i) that they are organs for the excretionof waste matter (other than water), and (ii) that they control the internal osmoticpressure. These two possibilities are not mutually inconsistent. The contractilevacuole may have more than one function in the same organism, or differentfunctions in different species, but it is very unlikely that the mechanism is notfundamentally the same in all species. There is as yet no convincing positive evidencefor the theory that they are excretory, and this is not surprising in view of theirsmall size; but this possibility need not affect an examination of the second alter-native, which is the purpose of this paper. The osmotic control theory was suggestedby the supposed occurrence of contractile vacuoles in fresh-water Protozoa, butnot in marine or parasitic forms. Actually they do occur in a number of marineCiliates, but this does not invalidate the argument. A marine form might havean internal osmotic pressure either greater than or the same as that of the externalmedium, and so might or might not have a contractile vacuole. A fresh-water form,however, must have its internal osmotic pressure greater than that of the sur-rounding medium, and so would have to have a contractile vacuole, unless it couldmaintain its internal concentration in some other way.

If the contractile vacuole is a controller of the internal osmotic pressure onewould expect it to be affected by large changes in the osmotic pressure of themedium. Various workers have reported a loss of the contractile vacuole fromfresh-water forms which were placed in sea water, and a reappearance of it when theorganisms were replaced in fresh water {e.g. Ziilzer, 1910), but in such cases it canalways be objected that the stoppage of the vacuole was due to a toxic effect of theunnatural environment. Hogue (1923) found that contractile vacuoles appeared ina marine amoeba which was placed in fresh water—a piece of evidence the im-portance of which has not been sufficiently stressed. A more detailed analysis of therelations between the rate of vacuolar output and the osmotic pressure of theexternal medium requires the measurement of the durations and of the ultimate

Physiology of Contractile Vacuoles 365

diameters of the vacuoles. (By "duration" of a vacuole is meant the time intervalbetween the systole of that vacuole and of the one before it; by "ultimate diameter "the diameter just before systole, which is the maximum diameter.) In some of theearlier work on contractile vacuoles the diameters were unfortunately not measured.Adolf (1926), however, measured both diameters and durations for Amoeba proteus,and found that there was no significant decrease in the rate of output when theamoebae were placed in solutions of NaCl and of other salts of concentrations upto MJ20; and that there was no direct relation between the surface area of theorganism and the rate of vacuolar output. He puts this forward as evidence againstthe osmotic control theory. When the amoeba is in equilibrium with its environ-ment, the rate of output of water from the contractile vacuole must equal the rateof intake of water through the body surface. If the external osmotic pressure wereraised above the internal osmotic pressure—as may have been the case in Adolf sexperiments—then the contractile vacuole might be expected to stop. There arefour possible explanations of Adolf s results:

(1) that the surface of the amoeba is freely permeable to the solutes used;(2) that the internal osmotic pressure of the amoeba is high, and that the increase

in external osmotic pressure was too small to have any effect;(3) that the internal osmotic pressure was raised by shrinkage of the body, and

that the contractile vacuole was maintaining it above that of the new externalmedium;

(4) that the contractile vacuole was drawing on the internal water supply of theamoeba without there being any entry of water to replace the water evacuated.

(1) and (2) are both unlikely. As regards (4), Chalkey (1929) has shown that,in solutions of non-electrolytes of approximately the same osmotic pressures aswere used by Adolf, amoebae go on shrinking for about 2 hours. There is no proof,in the absence of volume measurements, that Adolf s amoebae had reached equili-brium with the new outside medium in respect of body volume, and that water waspassing into them from the outside.

In the experiments about to be described the body volume was measured inorder to meet the above objections; and since more significance could be attachedto an increase (if such were to occur) than to a decrease in rate of output, hypotonicas well as hypertonic media were used.

MATERIAL AND METHODS.

The species used were chosen from among the Peritrich Ciliates, which havethe following advantages:

(i) The contractile vacuole contracts very frequently (once in 30-60 sec. at150 C.) in fresh-water species, and is present and contracts fairly frequently (oncein 1-20 min. at 150 C.) in marine species.

(ii) The contractile vacuole is always in the same place.(iii) The organisms are sessile—a fact which enables them to be used in con-

junction with the apparatus about to be described.

366 J. A. KlTCHING

(iv) The organisms are nearly perfectly symmetrical about an axis of rotation;this enables estimations of their volume and surface area to be made.

(v) The ultimate vacuolar diameter and the rate of output of these forms re-main constant, within the limits of experimental error, under constant conditions.

The species used were:Fresh-water forms (see Kent, 1880-1):

Rhabdostyla brevipes1 (C. and L.). This species was sessile on the larvae2 ofthe mosquito Aedes geniculatus, and was obtained from a rot-hole in a beech tree.Individuals of Rhabdostyla brevipes lived healthily in the laboratory, in water fromrot-holes, until the skins of the larvae on which they were growing were cast off.They are, however, difficult material to use, as the mosquito larvae are liable towriggle. The adjustment of the cover-slip is critical, and long experiments arefrequently ended prematurely by the movement of the larva.

Marine forms (see Hamburger and Von Buddenbrock, 1911):Vorticella marina Greef, Zoothamnium niveum Ehrbg., Zoothamnium marinum

Mereschk., Cothurnia innata O.F.M., Cothurnia3 sp. ? socialis Gruber, Cothurniacurvula Entz., Cothurnia ingenita* O.F.M.

All the marine forms were found on Cladophora in the Drake's Island tank ofthe Plymouth Aquarium. The Cladophora came originally from intertidal rock-pools in Plymouth Sound. Most of the work on marine forms was done in Plymouth,but a few experiments were done in London in winter time. It was found thatmaterial would arrive from Plymouth in a healthy state so long as the weather wascool, and would remain healthy in the laboratory in London for a week if it waskept below 150 C. No material was used which did not appear to be in goodcondition.

The Protozoa were kept in a continuous flow of fluid while under observationby means of the apparatus shown in Fig. 1. Fluid flows from the capillary openingof the supply unit on to the slide at the edge of the cover-slip, at a rate of a drop in1-2 sec. It flows under the cover-slip, out through the blotting paper at the otherside, into the funnel, and away. In all experiments in which room temperaturecould not be maintained as low as 15-160 C , the temperature of the fluid suppliedwas controlled by means of the water-jacket. When the room temperature was notexcessively high, the temperature of the fluid under the cover-slip (as determinedby a thermocouple) was found to be nearly the same as that of the water-jacket.Accordingly in the majority of experiments a thermocouple junction was not used,since it adds considerably to the difficulties of manipulation, and the water-jacketwas adjusted so as to give a temperature under the cover-slip of 15-16° C. A numberof supply units of this apparatus were fitted up. By changing the supply unit a

1 Kindly named for me by Prof. Mackinnon.1 Supplied to me by Mr T. T. Macan, to whom I am grateful.1 This form wa3 always solitary, but otherwise resembled Cothurnia (Pyxicola) socialis.1 The present writer has followed Hamburger and Von Buddenbrock in including under this

name both Vagimcola crystallina Ehrbg. and Ttiuricola S.K. An operculum was present sometimesbut not always.

Physiology of Contractile Vacuoles 367rapid change of the external medium of the organism can be brought about. Sucha change of units can be effected in a few seconds. The constant flow of fluidensures that there can be no lack of oxygen nor accumulation of carbon dioxide.

The fluids used for fresh-water organisms were mixtures of sea water and London

Supply of fluidto organism

A unit of theirrigation apparatus

(.o\rrgla*s

Itlotting |MI|HT

plale

"Microscope stage

^ " ^ Funnel

Fig. i. Diagram of irrigation apparatus.

tap water atpH 7-9-8-0, and for the marine forms mixtures of sea water and Londonor Plymouth tap water at />H 7-9-8-2. The pH was taken with indicators, and wascorrected where necessary with NaOH.

The Protozoa were in no way compressed during the experiments. The materialon which they were growing supported the cover-slip, and particles or free-swim-

368 J. A. KlTCHING

ming Ciliates often passed in between the cover-slip or slide and the organism underobservation. The contractile vacuoles of all species were, just before systole,perfectly circular in outline from whatever direction they were viewed. They aretherefore held to have been spherical at that time, although they were frequentlyof irregular shape during the earlier part of diastole. They emptied themselvescompletely to the exterior, and there were no signs of any "canals" such as occurin Parameeium. The time of systole was estimated generally within a second. Theultimate diameter was measured with a Watson screw micrometer eyepiece. Forthis purpose the lines of the micrometer were kept continually set on the vacuole,and the scale was read when systole occurred. The scale was then returned to zeroor thereabouts until the vacuole next appeared. A \ in. objective was used. The

ultimate volume of the contractile vacuole was calculated as - --- -- '-,0

and the average rate of output for the duration of each vacuole was obtained bydividing the ultimate volume of that vacuole by its duration. The mean rate ofoutput for a longer period of time was obtained by dividing the total output bythat time; this eliminates practically all error in the measurement of time, but theerror involved in measuring the vacuolar diameter remains. The rate of output isextraordinarily constant under constant conditions, although there is occasionallya sharp deviation for a single cycle of the vacuole. The standard error of the mean

rate of output was estimated as » / . ., where d — difference between meanr v n(n — 1)rate of output and rate of output for any one vacuolar duration, and n = numberof these rates of output observed. It was generally for fresh-water species 5-10 percent., and for marine species 5-15 per cent, of the mean rate of output. The numberof readings was in some cases too small for the standard error of the mean to havemuch statistical value. For measurements of the body volume only two, three, orfour readings could be obtained under a given set of conditions. The maximumdeviation from the mean was generally between 5 and 10 per cent, of the total bodyvolume.

Each organism was kept in a constant flow of fluid (fresh water for fresh-waterforms, sea water for marine forms) under control experimental conditions for anhour before observations began. Measurements for estimation of the body volumewere taken at intervals throughout the experiment. While these measurementswere being taken, or sometimes for purposes of rest for the observer, there wereintervals during which observations of the contractile vacuole ceased. In the tablesof results (p. 369) the actual time is given during which the organisms were keptunder any given set of conditions, inclusive of such intervals, but exclusive of thehour for acclimatisation at the beginning of the experiments. The organisms used(with the exception of VorticeUa marina) are practically perfectly symmetrical aboutan axis of rotation. An accurately reproduced side view therefore contains all thedata necessary for an estimation of the body volume. Each of these estimationswas obtained as follows:

(1) During the experiment a small drawing of the side view of the organism was

Table I. Results of experiments in which fresh-water and marine Peritricha were

subjected to known mixtures of fresh water and sea water. The mean rates of output

are given ± the standard error of the mean. For further explanation see p. 368.Only a few typical experiments are included in this table; many others were per-

formed, and gave similar results.

Concen-trationof sea

water inmedium

, 0

0

40

0| ->

0

100

40100

100

25100

100

100

10025

100

1005O

100

100

55100

55100

5O100

20

100

5°100

10012}

IOO

Mean rate ofoutput in cubic

microns per second

Meanultimatediameter

in microns

Number ofvacuolar

cyclesmeasured and

included( = «)

RJuibdottyla brevipes (fresh water)

17-2 ±0-9io-i + o-616-9 + i-o

10-2 ±0-70-28 + O'O2

lo-o +O'6

0-96 + 0-1318-3 +0-4

I - I 4 ± O - 1 4

o-6i ±0-0522-6 ±0-55

0-90 + 0-15

1-10 + 0-172O-2 ±O"5

0-63 + o-o(>

963 + 1-34!S6-i ±4*5

4-10 + 1-92

o-8i + o-i i15-7 ± 1-2°'35 ±o-oi

3-11 ±0-44lyh ±2-52'48 + O'OQ

11'9 +0-971'06 +0-09

14-3 ±0-57I- IO + O-I8O'O +O-O

10-38-7

10-5

«-s5'58-7

131720

11

13

3°

Cutlmrma curvula (marine)

7-09-67-1

5 9I O - I

6-7

5'69-65'4

72410

123°

6

13569

Cothurnia ingenita (marine)

•9-318-2167

2

132

Votfturnia imuitti (marine)

5 48-65'5

824

3

Vorticella marina (marine)

2O'219-6

Variable18-8

13115-9149

3724362

Zoot/iammum niveum (marine)

9-0 + 1-390-1 ± 2-5

7-0 ± 0-5

10-4 + o'5167-3 ± I 2 ' 2

io-o ± 1-5

i5-419416-2

18-624-8192

817

913

8

Volume oforganism in

cubic microns

—

20,70033,4OO20,00018,40042,60017,600

—

—

—

—

Blisters underpellicle

—

241,500487,500

Durationof treat-ment in

min.

154425

2518456

4434

201

443212

488559

16

153°

564i61

21

5310446

551765

153353503660

37° J. A. KlTCHING

Table II . Results of further experiments in which marine species were subjected todilute sea water; examples showing a marked falling off in rate of output while theorganisms were still in the hypotonic medium.

Concen-trationof sea

water inmedium l"o

Rate of output incubic microns

per second

Ultimatediameter in

microns

I

Bodyvolume in

cubic microns

Durationof treatment

in min.

I C O

75

I O O

1 O O

7iI O O

I O O

5

I O O

First io min.Later:

First io minLater:

(rather

First io minLater:

0 - 1 3

: o-9SO'2OO I 9

: 35-4720-32

°75variable]

o-54: 70-78

24-320-48

Cothurnia curvula

101I2-2-I3-29-2-11-3Variable

10014-6-15-611-2—12-3Variable

16,40023,20019,00012,400

44

76

134

5°134

63

37

7«

54

10025

IOO25

IOO25

First 10 min.Later:

First 10 min.I^ater:

First 10 min.Later:

1 09: Abt. 48

16-200-83

0-58: 35-419-8-11-8

1-171 6 2

: 34-4O10-16

2 - 5

Cothurnia sp. ? socialis

104145

1 1 - 9 — 1 2 9Variable

8-211-9-12-910-5-12-9109—126

7-2I I - 2 - I 2 - O8-S-IO-O5-1-8-5

51

1052

71748

1614946

139816446

made, and measurements of a number of its salient dimensions (e.g. total length,breadth at various levels, depth of rim) were taken.

(2) From the above, after the experiment was over, a large-scale drawing wasconstructed, with a linear magnification of about x 3000.

(3) The figure so obtained is divided into two halves—mirror images of eachother—by the central axis. The distance (y) of the centre of gravity of one of thesehalves from the central axis was found by means of a geometrical construction andcalculations (see Henrici and Turner, 1903). The measurement of areas which thismethod entails was done with a planimeter.

(4) The body volume (v) was calculated from

where A = area represented by one-half of the scale drawing described in (2).The ciliary disc, in respect of which the Protozoa used are asymmetrical, and the

Physiology of Contractile Vacuoles 371

contractile fibre present in the stalk of some species, were considered sufficientlysmall to be neglected entirely. The transparent sheath which surrounds the con-tractile fibre and which forms the bulk of the stalk is a secretion and is dead material.No allowance was made for the gullet. It is important that individuals chosen forobservation should lie accurately in the same optical plane, since otherwise thelength measurements are inaccurate.

EXPERIMENTAL RESULTS.(1) Fresh-water forms.

The contractile vacuole of Rhabdostyla brevipes was found to have a durationof 30-60 sec, and an ultimate diameter of 7-11 microns, at 150 C. The average rateof output under these conditions was 11-6 cubic microns per second. Individualswere subjected successively to (1) tap water, (2) a known mixture of tap water andsea water, (3) tap water. Fig. 2 illustrates a typical experiment. Transference of theorganisms to (2) led to an immediate decrease in the rate of output and in the ultimatediameter until these reached a new steady value. Also in many cases a decrease inbody volume occurred which was very noticeable, although the body volume couldnot be measured accurately for this species owing to the fact that an individualdoes not remain for long in the same optical plane. In Fig. 5 is shown the relationof concentration of sea water in the medium with mean rate of output. In calculatingthe latter no readings were included which were taken immediately after a changeof medium and before the rate of output had settled down to a steady value. Inspite of individual variations it is clear that there was a falling off of rate of outputwith increasing concentration of sea water, until in about 12 per cent, sea waterthe rate of output was zero. The ultimate diameter was also decreased. Both meanrate of output and mean ultimate diameter returned in most cases to their originalvalues when the organism was replaced in tap water.

In the higher concentrations (10-15 Pe r cent, sea water) the cilia stopped andthe organisms were contorted by shrinkage, but in all cases after the organisms hadbeen returned to fresh water they appeared perfectly healthy and normal.

(2) Marine forms.

The contractile vacuole of marine forms generally had a duration of about 10-15min. (though for Cothurnia curvula it was about 3-5 min.), and an ultimate diameterof 10-20 microns. The rates of output ranged from about 0-5 (for Cothurnia curvula)to 10 cubic microns per second (for Cothurnia ingenita and Zoothamnium niveum).

In the series of experiments described below (see also Tables I and II, pp.369,370), various marine Peritricha were subjected successively to (1) 100 per cent, seawater, (2) hypotonic sea water of known dilution, (3) 100 per cent, sea water. Ingeneral it may be stated that dilution of the sea water led to an increase in bodyvolume and in rate of output. A typical experiment is illustrated graphically inFig. 3. This series of experiments may be summarised briefly as follows:

(1) In 100 per cent, sea water the body volume, rate of output, and ultimatediameter remained constant.

Physiology of Contractile Vacuoles 373(2) In hypotonic sea water the body volume increased rapidly and immediately,

and in most cases remained constant at a new high level. In a few experimentsthere was a falling off in the body volume after the initial increase (Fig. 4). Inmany experiments in which very dilute sea water (approximately 5 per cent, orless) was used, the organisms swelled up until they were globular; and then cleardrops of fluid raised the pellicle up in blisters, which often swelled and becamenearly spherical. In a few cases the protoplasm flowed out into the blisters. Therate of output increased rapidly and immediately, and then either remained constant

Ikxly voluin

©

I III) |M 1 m i l .

v \ t walcr

60 120Time in minutes

300 360

Fig. 4. The effect of dilute sea water on the body volume and rate of output of Cothitrnia curvula;a case in which there was a falling off in body volume while the organism was still in the hypotonicmedium. N.B. For 12.I per cent, sea water the average rates of output for groups of three vacuolardurations have been plotted.

at a new high level, or decreased at first but subsequently became constant at alevel which was still considerably higher than the original (ioo per cent, sea water)level. In one case the organism (Cothurnia sp. ? sodalis) was still maintaining asteady rate of output 1600 min. after it had been transferred to 25 per cent, seawater. In the case of some individuals which had been transferred to 75 per cent,sea water (or stronger) the rate of output rose temporarily and then fell approxi-mately to its original level. When blisters had been formed, in very dilute seawater, the contractile vacuole generally slowed down and stopped. The ultimate

374 J. A. KlTCHING

diameter was liable to sharp variations after a change of medium, but subsequentlybecame constant. In moderately hypotonic (50-100 per cent.) sea water it wasgenerally less, and in more dilute sea water generally more, than what it had beenoriginally (in 100 per cent, sea water). In very dilute sea water the contractilevacuole sometimes failed to empty itself completely.

o-o . . ,4 8 12 16%

Concentration of sea water in medium

Fig. 5. The relation of rate of output with concentration of medium for Rlmbdoslyla hrcvipcs.

2-5

2-0

E 1-5

i-n100 80 60 40 20

Concentration of sea water in medium

0%

Fig. 6. T h e relation of body volume with concentration of medium for Znnthamnium marinum andCothuntia curvula. ® Zoothamnium marinum; <J> Cothurnia curvula.

(3) In 100 per cent, sea water the body volume returned immediately andrapidly to its original value, or less. In those cases in which the hypotonic seawater was very dilute the pellicle wrinkled when the organism was replaced in100 per cent, sea water, and the wrinkles remained for some time (e.g. 15-30 min.).It is therefore probable that volume measurements obtained under these conditionsare too high. The rate of output returned approximately to its original value

Physiology of Contractile Vacuoles 375

immediately and rapidly. It was generally more variable than formerly. Theultimate diameter became variable, but returned approximately to its original value.

The relation of concentration of sea water with body volume and rate of outputare shown in Figs. 6 and 7 respectively. Readings taken over a period of 5-10 min.after the change of medium were discarded, so that no readings were included

100 HO 60 40 20

Concentration of sea water in medium

0%

Fig. 7. The relation of rate of output with concentration of medium for Zoothammum marinum andCothumia curvula.

Curve A: ® Zoothammum marinum; <$> Cothumia curvula. Experimental treatment as describedon p. 371.

Curves B and C: /2\ and • two single individuals of Cothumia curvula. W.S.W. = Wemburystream water; P.T.W. = Plymouth tap water. Experimental treatment as described on this page.

which were taken before a steady level was reached. The maximum increase inrate of output was x 70, in 12J per cent, sea water. In greater dilutions the rate ofoutput fell off.

In another series of experiments on marine Peritricha the organisms (Cothumiacurvula and Cothumia ingenita) were subjected by successive steps to more andmore dilute mixtures of sea water and Plymouth tap water. The relation betweenrate of output and concentration of sea water (Fig. 7, curves B and C) is similar

! B B - x i i v 25

376 J. A. KlTCHING

to that found in the experiments described above. In one experiment (Fig. 8) theorganism was taken in steps down to 1 per cent, sea water, and then back to 100per cent, sea water by the same steps in the reverse order. The body volume andrate of output were much lower on the return journey than they had been on thesame steps on the outward journey. In two experiments the organism was subjectedto Wembury stream water (/>H not corrected) and then to Plymouth tap water(/>H not corrected). In Wembury stream water a fairly steady rate of output wasmaintained for a long period (40 hours in one experiment, 15 hours in the other),

6 0 r

100 60 60 40 20

Concentrat ion of sea water in med ium

Fig. 8. T h e relations of body volume and rate of output with concentration of medium for a singleindividual of Cothurnia curvula, which was transferred by successive steps to more and more dilutesea water, and then back by the same steps in the reverse order to 100 per cent, sea water.

but in Plymouth tap water the pellicle was raised up in blisters and the contractilevacuole stopped.

In both these series of experiments on marine Peritricha the cilia stoppedbeating when the organism was placed in the more dilute mixtures (e.g. sea water25 per cent, or less), although there was considerable individual variation in thisrespect. Also sometimes they started beating again in the diluted sea water, butat other times they remained stopped until some time after the organism had beenreplaced in 100 per cent, sea water. Except in some cases in which blisters had beenformed, the organisms appeared to be perfectly healthy at the end of the experi-ments, and the cilia beat again normally. On several occasions individuals dividedsoon after experiments had been performed on them.

Physiology of Contractile Vacuoles 377

In all these experiments, both on fresh-water and on marine forms, the totalnumber of systoles observed was over 4000.

DISCUSSION.The increase in the body volume of marine Peritricha, consequent on treat-

ment with hypotonic sea water, may be explained in two ways:(1) Osmotic swelling due to a cell membrane which is relatively impermeable

to salts, and yet freely permeable to water, or(2) Ionisation of the cell proteins due to the reduction in salt concentration.If (1) is true, the cell membrane must be relatively impermeable to salts. If (2)

is true, the cell membrane need only be impermeable to proteins, and may befreely permeable to salts. Evidence is as yet inconclusive as to which is the rightexplanation; (2) may possibly play a small part, even if (1) accounts for most of theswelling. There is strong evidence that in many different kinds of animal cells thecell boundary is semi-permeable with regard to salts and water (Luck£ andMcCutcheon, 1932). Some preliminary experiments with marine Peritricha onthe effect of ammonia, an alkaline substance likely to penetrate the cell and there toinfluence the ionisation of the cell proteins in the same way as would a reductionin the salt concentration, have indicated that there is no change in volume whilethe organism is alive. Again, when an individual of Cotkurnia curvula was treatedwith a mixture of a glycerol solution and sea water such that the salt concentrationwas reduced to one-sixth while the osmotic pressure remained unaltered, therewas no change in body volume or in rate of output. Thus there is evidence, thoughas yet incomplete, for believing that the changes in volume observed were due tothe fact that the cell surface of these Protozoa is semi-permeable with regard tosalts. If this is so, information can be deduced concerning the osmotic pressure ofthe vacuolar fluid.

The osmotic pressure of the vacuolar fluid has never been measured, but it canbe inferred that it is probably near that of pure water, at least for fresh-water species,unless excretory matter is present. For no more salts can leave the organism thanenter it, unless its salt content is to be depleted (which could not go on indefinitely);and no appreciable amount of salts can enter a fresh-water organism from freshwater except occasionally in the food. On the other hand, the internal osmoticpressure of fresh-water Protozoa, though low, must be greater than that of thesurrounding fresh water, so that the contractile vacuole must be separating waterfrom the internal solutes of the organism. And in marine forms, if the cell mem-brane is relatively impermeable to salts, the same argument can be applied, namely,that since no more salts can leave the organism than enter it, the contractile vacuolemust be separating fluid of very low osmotic pressure from an internal solution ofosmotic pressure not less than that of sea water. Assuming the semi-permeabilityof the cell membrane with regard to salts, the contractile vacuole must in both casesbe doing work, and it must therefore be regarded as an active mechanism involvingthe expenditure of energy. Its operation will raise the internal osmotic pressure of theorganism until a steady state is reached, which will depend partly on the rate of inflow

25-3

378 J. A. KITCHING

of water, and hence on the permeability of the cell membrane to water. The magni-tude of the difference between the internal and the external osmotic pressures willdepend on the rate of vacuolar output and on the permeability of the cell membraneto water, and may be insignificant if the" latter is great as compared with theformer.

The secretory theory of diastole, as outlined above, is entirely contradictory toany suggestion that the contractile vacuole grows larger by osmotic uptake of waterfrom the surrounding protoplasm. For osmotic uptake there would have to beinside the vacuole a quantity of solute such that at the greatest volume of thevacuole the osmotic concentration of the vacuolar fluid was not less than that of thesurrounding protoplasm. At the beginning of diastole, when the volume of thevacuole is much less, the concentration of the vacuolar fluid would have to becorrespondingly greater. In marine Peritricha, whose internal osmotic pressuremust be not less than that of sea water, the initial concentration of the vacuolarfluid would have to be extremely great, and it seems unlikely that such a concen-tration is actually produced. And for Amoeba proteus, a fresh-water form, Adolfhas shown that the relation between vacuolar volume and time is linear during theperiod of a single diastole. Such a relation is inconsistent with simple osmoticuptake. In view of these objections to the theory of osmotic uptake of water by thevacuole, the validity of the secretory theory is assumed in the discussion whichfollows.

The factors which are likely to affect the secretory activity of the vacuole are oftwo types: (a) those dependent on the concentration of the sea water outside at thetime in question, and (b) those governed by the state of activity of the organismor of the vacuolar mechanism itself, e.g. general health and condition of organism,possibly food reserves, age, etc. It was observed that in marine Peritricha there wasconsiderable individual variation in the rate of output among members of the samespecies, and that this could not be attributed to size. Specimens which had beensent from Plymouth to London in cold weather had a high rate of output, whilethose which had been sent up in hot weather had an extraordinarily low rate ofoutput, and the vacuolar duration was as high as half an hour. Those that had beenkept for any length of time in the laboratory in London also had a low rate ofoutput. Hogue (1923) observed that old cultures of amoeba developed a low rate ofoutput, and this has been confirmed by the present writer. It is therefore probablethat the rate of output is considerably influenced by the state of the organism.Small differences in condition might account for the differences found by Adolfin the rates of output of amoebae.

The observed increases in the body volume of Peritricha fall short of thosewhich would take place if the cell membrane were perfectly semi-permeable, and ifthe cell contents were no more than a dilute solution of salts. Such a falling shortcould be ascribed to salt loss, to the presence of osmotically inactive substanceswithin the cell, or to volume control by the contractile vacuole. It is very unlikelythat the pellicle, which is extremely delicate, could exert any significant pressure.Cole (1932) has shown that the inward pressure of the cell membrane of the egg of

Physiology of Contractile Vacuoles 379Arbacia is very small, and such pressure may therefore safely be ignored. To whatextent the other factors are operative cannot be discussed until the results of furtherexperiments are available, but it seems probable, from the nature of the curverelating body volume with concentration of external medium (Fig. 6), that in verydilute media salts escape. This would explain the return to a body volume smallerthan the original (p. 374), and also the falling off in body volume while the organismwas still in the hypotonic medium (Fig. 4). Whether the falling off in rate of outputwhich was often observed while the organism was still in the hypotonic medium(Table II) is to be ascribed to a loss of salts, or to the dying away of a stimulus setup by the change of medium, or to fatigue of the vacuolar mechanism, is uncertain.

It is of interest to know whether the increase in vacuolar output which followstransference of the organism to a hypotonic medium involves an increase in theamount of work done. As a rough approximation the osmotic pressure of sea watermay be taken as proportional to the concentration, and the internal osmotic pressureof the organism in 100 per cent, sea water as equal to that of 100 per cent, sea water.The internal osmotic pressure of the organism when it is in dilute sea water cannotbe less than that of the external medium. By assuming that it is the same we shallfind a minimum value for the work done. Assuming that the vacuolar fluid is purewater in all cases, w_

where W=work done, P= internal osmotic pressure of organism, V= volume offluid eliminated by the contractile vacuole.

From the curves given we find:

Concentration of sea water inwhich organism is placed ("„)

Minimum value of relative amountof work done per unit time

IOO

100

75

75

50

250

. 25

1075

20

960

10

600

5

IOO

The contractile vacuole has therefore all the potentialities required not only fora maintainer but also for a regulator of the internal osmotic pressure. Whether it iseffective will depend on the precise adjustment of the mechanism.

There are two possible functions which might be served by osmotic control:(a) Maintenance of the internal osmotic pressure at a level higher than the externalosmotic pressure, even though the internal osmotic pressure is influenced by smallchanges in the external osmotic pressure; (b) Regulation of the internal osmoticpressure so as to keep it constant irrespective of small changes in the externalosmotic pressure. For marine forms the curve relating rate of output with con-centration of external medium is flat between 100 and 75 per cent, sea water.No significant change in output, such as would be required for "regulation," occursbetween these values. Whether any "maintenance" occurs is unknown, but it isunlikely that the internal osmotic pressure of marine forms is much above that ofthe surrounding sea water. Although the contractile vacuole of marine speciesmight be regarded as a relic of an ancestral fresh-water habitat, it is possible that in

380 J . A . KlTCHING

maintenance it performs a useful function. Unless the cell membrane is entirelyimpermeable to salts, these will enter, although perhaps very slowly; a Donnanequilibrium will thereby be set up owing to the presence of indiffusible proteinsinside the cell, so that the internal osmotic pressure will be raised slightly abovethat of the outside sea water. It is possible that the contractile vacuole might berequired to relieve the resulting tension on the pellicle. Against this, as against anyother suggestion of a function for contractile vacuoles in marine Protozoa, may bebrought the objection that many marine Protozoa successfully do without them.

Adaptation of Peritricha of marine origin to fresh water is probably possible.Zoothamnium spp. and Vorticella marina were completely incapacitated in verydilute sea water, being liable to excessive swelling, but in many cases individualsof Cothurnia spp. were but little inconvenienced, and in some individuals ofCothurnia ingenita and Cothurnia curvula the cilia continued to beat, although rathersporadically, in Wembury stream water. At other times, however, no individualsof Cothurnia spp. would successfully endure even 10 per cent, sea water. Allindividuals of the same batch behaved alike in this respect. It seems probable thatsuccessful adaptation was effected by loss of salt. The rate of vacuolar output fallsoff in very low concentrations of sea water, and therefore it is unlikely that in suchconcentrations regulation takes place, although the contractile vacuole may havebeen preventing excessive swelling by maintenance. The experiments describedabove support Hartog's (1899) suggestion, advocated by Lloyd (1928) in his review,that the contractile vacuole prevents the organism from swelling excessively.Lloyd points out that contractile vacuoles occur in fresh-water organisms (includingalgae as zoospores or as adults) which are devoid of rigid cell walls, but not in thoseforms or stages in which rigid cell walls are present. It might be questioned whethersuch cellulose walls could withstand the great pressure which would be developedby a small difference in concentration between the inside and the outside. Thevery small size of such cells would make it more possible, but a knowledge of thestrength of the cell walls is required to settle this problem.

SUMMARY.1. The rate of output of fluid from the contractile vacuole of a fresh-water

Peritrich Ciliate was decreased to a new steady value immediately the organismwas placed in a mixture of tap water and sea water. The rate of output returnedto its original value immediately the organism was replaced in tap water. Thecontractile vacuole was stopped when the organism was treated with a mixturecontaining more than 12 per cent, of sea water.

2. Transference of various species of marine Peritricha from 100 per cent, seawater to mixtures of sea water and tap water led to an immediate increase of thebody volume to a new and generally steady value. Return of the organism to100 per cent, sea water led to an immediate decrease of the body volume to itsoriginal value or less.

3. Marine Peritricha showed little change in rate of output when treated with

Physiology of Contractile Vacuoles 381

concentrations of sea water between 100 and 75 per cent. In more dilute mixturesthe rate of output was immediately increased, and then generally fell off slightlyto a new steady value which was still considerably above the original (100 per cent,sea water) value. The maximum sustained increase was approximately x 80. Returnof the organism to 100 per cent, sea water led to an immediate return of the rateof output to approximately its original value.

4. When individuals of some marine species were placed in very dilute con-centrations of sea water, the pellicle was frequently raised up in blisters by theformation of drops of fluid underneath it, and the contractile vacuole stopped.

5. Evidence is brought forward to suggest that in the lower concentrations ofsea water marine forms lost salts.

6. The contractile vacuole probably acts as an osmotic controller in fresh-waterProtozoa. Its function in those marine Protozoa in which it occurs remains obscure.

ACKNOWLEDGMENTS.I am grateful to Dr C. F. A. Pantin for suggesting this work to me, and to

Dr J. Gray for much helpful advice and criticism. I am also indebted to Prof.H. G. Jackson and Dr E. J. Allen for laboratory facilities, to Dr E. A. Spaul formuch encouragement, and to Prof. Sugden and Dr R. G. Cooke for advice on thephysical and mathematical aspects of the work. I was granted the use of the LondonUniversity table while working at the Plymouth laboratory.

REFERENCES.

ADOLF, E. F. (1926). Journ. Exp. Zool. 44, 355.CHALKEY, H. W. (1929). Phytiol. Zool. 2, 535.COLE, K. (1932). Journ. Cellular and Comp. Pkysiol. 1, 1.HAMBURGER, C. and VON BUDDENBROCK, W. (1911). Norditchet Plankton, 13. "Ciliata mit Aus-

schluss der Tintinnoidea." Kiel and Leipzig.HARTOC, M. M. (1899). Brit. Assoc. Adv. Sci. 58th Report, London.HENRICI, O. and TURNER, G. C. (1903). Vectors and Rotors, p. 74. London.HOGUE, M. J. (1923). Journ. Elisha Mitchel Sci. Soc. 39, 49.KENT, W. S. (1880—I). A Manual of the Infusoria. London.LLOYD, F. E. (1928). Proc. Camb. Phil. Soc. 3, 329.LUCKE, B. and MCCUTCHEON, M. (1932). Physiol. Reviews, 12, 68.ZOLZER, M. (1910). Arch.f. Enttoickelungsmech. der Organismen, 29, 632.