I I

THE PHYSIOLOGY OF CONTRACTILE VACUOLES

II. THE CONTROL OF BODY VOLUME IN MARINE PERITRICHA

BY J. A. KITCHING.

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

(Received July 4, 1935.)

(With Eight Text-figures.)

INTRODUCTION.

IN a previous paper (Kitching, 1934) it was shown that, when a marine PeritrichCiliate was transferred from ordinary to hypotonic sea water, the body volumeincreased, and there was (generally) a great increase in the rate of output of fluidfrom the contractile vacuole. The increase in body volume was ascribed to theosmotic uptake of water through a cell membrane which was relatively impermeableto salts; and it was suggested that the contractile vacuole, in the lower concentrationsof sea water, was acting as a controller of the body volume of the organism. Thisinvestigation has now been continued by an examination of the effects of certaincomparatively inert non-electrolytes and also of various narcotics.

The effects of certain non-electrolytes were investigated in order to see whetherthe results previously obtained were due solely to changes in the osmotic pressureof the medium, and not to any other consequences of a decreased salt concentration.Pantin (1931), using this method of enquiry in his investigation of amoeboid move-ment, found that glycerol was practically inert and harmless, whereas urea andcane-sugar had definite specific effects. The object of the present experiments hasbeen to vary the concentration of sea water in the medium, and at the same time tokeep the osmotic pressure of the medium constant by means of an inert non-electrolyte.

In order to obtain evidence as to the nature of the vacuolar process, an investi-gation has also been made of the effects of various narcotics which are believed toact more or less specifically on particular parts of the cell mechanism. In additionit was hoped that a narcotic might be found which would quickly and reversiblyinhibit vacuolar activity, in order that the effects of stoppage of the vacuole on theorganism itself might be studied, with special reference to the body volume.

MATERIAL AND METHODS.

(1) Species used. Cotkurnia curvula Entz. and Cothurnia sp.? socialis Gruber(see Hamburger and von Buddenbrock, 1911) formed the material for most of theexperiments. Occasional experiments were performed on other species.

12 J. A. KlTCHING

(2) Irrigation. The organisms were irrigated throughout the experiments in theway already described (Kitching, 1934). The room temperature was maintained at14-5-15-5° C, and the water-jackets of the supply units were discarded. Solutionscontaining H,S or alcohols were supplied from pipettes, the lower ends of which hadbeen drawn out to capillary apertures, so that the fluid dripped sufficiently slowlywithout the need for any screw clip or tap. In this way these solutions were pre-vented from coming into contact at any time with rubber or grease.

(3) Solutions. Non-electrolytes (unless it is stated otherwise) were made up to astrength of 1 -04 molar, which is approximately isotonic withPlymouth "outside" sea water. All mixtures of sea waterwith non-electrolyte solutions were corrected where necessarytopW 8-0-8-1 with NaOH; when pure solutions of non-elec-trolytes were used the />H was not corrected.

For each experiment with a narcotic 2 litres of dilute seawater of known concentration at pH 8-i were prepared bymixing sea water and London tap water. To a portion of thisdilute sea water was added sufficient of the narcotic to givethe required concentration. The remainder of the dilute seawater was required for the " control" part of the experiment,before and after the administration of the narcotic (seebelow). Solutions of HCN or HSS in dilute sea water wereprepared by the addition of NaCN or NajS and by neutralisa-tion with HC1 to pH 8 1 . A few drops of the HC1 weregenerally added before the NaCN or NajS, and in no casedid the alkalinity become so great as to cause any precipita-tion. The other narcotics had no effect on the />H of dilutesea water.

(4) Body volume measurements. 1 he body volumes wereestimated in the way already described (Kitching, 1934, Fig , Diag ram illustratingp. 370), except that a correction was made to the formula, the method of estimating theThe correct formula is: v = 2vyA, and not ^A, as pre- £ ^ f r of P i t r i h

viously stated. Consequently the volume measurementsgiven in the previous paper require to be halved; this, however, does not affect theform of the curves given, or the nature of the results.

(5) Surface area measurements. The surface areas of the organisms wereestimated in the following way:

(a) The surface areas of the circular free and attached ends of the organismwere estimated as TTT2 in each case—the diameter, zr, having been measureddirectly.

(b) Let X, A, B, Y (see Fig. 1) be points on the periphery of the large-scaledrawing already prepared for body volume measurement (Kitching, 1934, p. 370),such that XY represents the central axis of the organism and that XA and YB areradii of the circular free and attached ends of the organism respectively. Thedistance of the centre of gravity (y) of the irregular line AB from the central

ga r e a o f a P e r i t r i c h

The Physiology of Contractile Vacuoles 13

axis ZFwas obtained by means of a geometrical construction (Henrici and Turner,1903, p. 61).

(c) The surface area of the body, excluding the circular ends, was estimated fromA = 2-nyL, where A = the area in question, and L = the length of the irregular line AB.

(d) The total surface area of the body was obtained by the addition of these twocomponents. The circular ends of the organism amounted to only a very smallproportion of the whole.

RESULTS.

Series i. Non-electrolytes.

In some experiments (Fig. 2) organisms were subjected to (1) 100 per cent, seawater, (2) 25 per cent, sea water, (3) 25 per cent, sea water in which had beendissolved sufficient non-electrolyte to make the total osmotic pressure equal to that

a 2OJ0OO

I 15,000

o 10,000 -IXI3

1u

13U

§ 15,000 •

6

ic

J3

•g

a

I-au

5 30 60 90T i m e in minutes

150 180

ig. 2. T h e effect of urea, glycerol, and cane-sugar on the body volume and rate of vacuolaroutput of Cothurnia socialii. Broken line indicates change of medium.

14 J. A. KlTCHING

of ioo per cent, sea water. Transference of the organism to (2) resulted immediatelyin an increase in body volume and in rate of output; transference to (3) was im-mediately followed by a return of the body volume approximately to its original(100 per cent, sea water) value or less, and by a decrease in the rate of vacuolaroutput to slightly below its original value. In the experiments with glycerol therewas after some hours in (3) a slight increase in body volume. In all cases while in(3) the organisms at first underwent a series of violent retractions of the body (asis usual for Peritricha after stimulation), but later they expanded and their cilia beatnormally. However, in some experiments, particularly when urea was used,this normal behaviour was after a time discontinued, and the body was againretracted.

Table I.

Composition of medium

S.W.S.W. 1: Glyc. 9S.W.S.W.Glyc.S.W.S.W.S.W. 1: Urea 4S.W.s.w.S.W. 1: Urea 9S.W.S.W

UreaS.W.

Mean rate ofoutput in

cubic micraper sec.

0-940 3 1

0-941-24o-oo2 7 0

0-560-160-26

o-75c-15o-6o0-56

decreasing toO'CO

0-93

No. ofvacuolar

cyclesmeasured

656

14—2 0

162917

337

3

—

Mean bodyvolume in

cubic micra

7,6007,9007,700

25,30025,00027,800

6,5007,700

7,8008,2008,300

12,000

13.00012,500

Duration oftreatment in

min.

356428

352 0

127

62357182

2 47955

33

615O

N.B. S.W. = 100 per cent, sea water; Glyc. = isotonic glycerol; Urea = iaotonic urea.

In other experiments the organisms were subjected to (1) sea water, (2) a mixtureof sea water with an isotonic solution of glycerol or urea, (3) sea water. Someexamples of the results are given in Table I. There were no significant changes inthe body volume during these experiments, but in mixtures containing urea, and toa less extent in mixtures containing glycerol, there was a decrease in the rate ovacuolar output. The organisms behaved more normally in mixtures containingglycerol, and after a short period of repeated retractions of the body they expandedand their cilia beat as usual. In mixtures containing urea the organisms did notremain expanded for long, but soon went into a state of permanent retraction. Inpure isotonic solutions of glycerol or urea they remained permanently retracted,and all activity of the contractile vacuole was stopped. In all cases there wascomplete recovery after the organisms had been replaced in sea water.

The Physiology of Contractile Vacuoles 15

Series 2. Narcotics.

Organisms were acclimatised for 1 hour in 20 per cent, sea water under experi-mental conditions, and then observed in (1) 20 per cent, sea water, for about half anhour, (2) 20 per cent, sea water + known amount of narcotic, for about 1 hour,(3) 20 per cent, sea water for about half an hour.

Control experiments. Organisms irrigated with 20 per cent, sea water for2\ hours maintained a constant rate of output which was unaffected by changes ofthe supply unit.

40-

30-

j»h

| 30-

3 20-

1 , 0 -0

1 o-U^ 30-

•s

10-

0-

20% on wilrr

20% »a w>ltr

2 0 % m witrr

^. 20% sra witrr + ryanMlf 10 s moUr (

—_ _ —

20% KM water + cyanide

10 * molar

'*• • • •• . •.

20 %iea water

20% i n water + cyanide 10 ' molar

*. 20%sca watCT

• •" • • •

30 60

Time in minutes90 120

Fin. 3. The effect of cyanide on the rate of vacuolar output of Cothurnia socialis.Broken line indicates change of medium.

Cyanide. In concentrations as low as io~5 molar, cyanide caused an immediatereduction in the rate of vacuolar output and in the ultimate diameter of the con-tractile vacuole. In higher concentrations cyanide either completely stopped thecontractile vacuole or caused a very great diminution in the rate of output. In somecases there was still an exceedingly small though steady output in M/200 cyanide.Return of the organism to 20 per cent, sea water without cyanide led to immediaterecovery of the contractile vacuole, but the rate of output then generally decreasedsomewhat to a value lower than that found at the beginning of the experiment (in(1)). (See Fig. 3.)

i 6 J. A. KlTCHING

In concentrations of not lower than 10 * molar, sulphide at firstSulphide.caused fluctuations in the rate of vacuolar output, and soon a very considerabledecrease. Recovery was less definite than in the case of cyanide (see Fig. 4), andthe results obtained were variable.

Alcohols. Ethyl alcohol Af/10 and butyl alcohol M/100 had little effect on thecontractile vacuole; ethyl alcohol iM and butyl alcohol M/10 caused a gradualdecrease in the rate of output; ethyl alcohol 10M and butyl alcohol iM rapidly ledto irreversible stoppage of the vacuole and death of the organism (see Figs. 5 and 6).

« •

30-•aa 20-

O" 10-

1 ouIS3 i<H.9

uo

I 10-

20% >ea water

20%iea water

20%iea water

20% tea water + wlphide 1 0 * molar 20% K < water

20% te* water | 20% tea water- -ttalpUde lO-'rooUr

20% ra water+ Mlphi<le 10 "molar

-t

20% tea water

* 120

Time in minutes

Fig. 4. The effect of sulphide on the rate of vacuolar output of Cothumia tocialis.Broken line indicates change of medium.

Ethyl carbamate (urethane). Urethane M/io had only a slight or moderate in-hibitory effect on the rate of vacuolar output, which was rapidly reversible; buturethane iM rapidly led to irreversible stoppage of the vacuole and death of theorganism.

Series 3. Cyanide and body volume.

In each experiment the organism was acclimatised to experimental conditionsfor 1 hour in 100 per cent, sea water, and then observed in (1) 100 per cent, seawater; (2) dilute sea water of known concentration; (3) dilute sea water of sameconcentration + M/200 or M/500 cyanide; (4) same as (2); (5) same as (1). Measure-

The Physiology of Contractile Vacuoles 17

ments were made of the body volume of the organism and of the rate of output ofthe contractile vacuole. The results of experiments performed in this way aresummarised below:

(1) In 100 per cent.'sea water the body volume and rate of vacuolar outputremained constant.

(2) In dilute sea water the body volume and rate of vacuolar output eachincreased rapidly to a new steady value.

20-

15-

T3 10-

J

e o-o

tput

in

cubi

c IT

icuo

lar

ou

£ o-a l5"3,

10-

5-

0 J

20%va»ater

20% sea water

20% sea water

—

- "

;

20% sea water +• ethyl alcohol M/lO

. ' — ' .

20 % sea water + ethyl alcohol 1M

_

20% H « water

20% M * water —

bo%!B.W't ihyj 20% tea water

\

1

120 150n 30 60 90

Time in minutesFig. 5. The effect of ethyl alcohol on the rate of vacuolar output of Cothumia tocialis.

Broken line indicates change of medium.

(3) In dilute sea water + cyanide the rate of output immediately and rapidlydecreased, until the contractile vacuole either disappeared entirely or maintainedan exceedingly low but steady output. Except in only slightly hypotonic sea waterthe body volume began to increase as soon as the rate of output had decreased, andsoon attained a new steady value. In cases when the body had almost completelyfilled the lorica while the organism was in dilute sea water without cyanide, so thatthere was little room for further swelling except at the unattached end, the pelliclewas frequently raised up on a blister.

i8 J. A. KlTCHING

(4) On a return of the organism to dilute sea water without cyanide the con-tractile vacuole became active again almost immediately; and from the time of thefirst systole the body volume began to decrease until it attained a new steady value,which was rather below the previous value in dilute sea water before treatment withcyanide. The diameter of the contractile vacuole just before the first systole duringrecovery from cyanide was often exceptionally large, and sometimes it was possibleto observe unmistakably the rapid shrinkage of the body which coincided with thissystole. This shrinkage of the body sometimes resulted in a wrinkling of the pellicle.

15-

10-

5-

&»

.9 15- •

u3

20% tea water w«lrr+- bul)l alcohol M/100 20? s-i »«t<-r

waitr+ bulvl ak-ohol M/10 201

•* , 2 0 % sc« wal t r

30 60 90Time in minutes

120 150

Fig. 6. The effect of butyl alcohol on the rate of vacuolar output of Cotfturma socialis.Broken line indicates change of medium.

(5) In 100 per cent, sea water the rate of output returned to a value not greaterthan the original one at the beginning of the experiment; and the body volumedecreased to a value lower than the original. A typical experiment is illustrated inFig. 7.

In experiments in which the dilute sea water was very dilute (30-10 per cent.),the changes in the body volume which resulted from cyanide treatment could beobserved directly in relation to the lorica of the organism. Generally while in dilutesea water before treatment with cyanide the organism did not completely fill its lorica,during cyanide treatment it filled its lorica and extended to a considerable distanceoutside, and after cyanide treatment it occupied only a small part of its lorica.

2 0 J. A. KlTCHING

The relation of body volume to concentration of sea water, with and withoutthe presence of cyanide, is shown in Fig. 8. Each pair of points (for the same con-centration of sea water with and without cyanide) is derived from a single experi-ment. The more dilute was the sea water the greater was the difference between thebody volumes of the organism in the absence and in the presence of cyanide. In75 and in ioo per cent, sea water there were no detectable differences, but it mustbe remembered that the method of body volume estimation is not a sensitive one.

100 80 60 40 20 0%

Concentration of tea water in medium

Fig. 8. T h e relation of body volume with concentration of medium in the absence and in the presenceof cyanide, for Cothurma curvula. ® without cyanide; <•> with cyanide.

DISCUSSION.

The osmotic action of non-electrolytes.

The body of a Peritrich Ciliate swells up when the organism is placed in hypo-tonic sea water (Kitching, 1934). This may be due either to osmotic uptake of waterthrough a cell membrane which is relatively impermeable to salts, or (much lessprobably) to the imbibition of water by the cell proteins, owing to the Donnaneffect. In this latter case the cell membrane need not be impermeable to salts.Since the addition of urea, glycerol, or cane-sugar to the outside medium causes adecrease in body volume, it must be assumed that the cell membrane is relativelyimpermeable to these substances; and since the power of a known decrease in theexternal salt concentration to produce an increase in the body volume is exactlycounteracted by the addition to the external medium of an osmotically equivalentamount of non-electrolyte, it is difficult to avoid the conclusion that the cell mem-brane is relatively impermeable to salts. It therefore appears that the external

The Physiology of Contractile Vacuoles 21

osmotic pressure is the paramount factor in the control of the body volume and ofthe activity of the contractile vacuole.

If the body surface is impermeable to salts, the salt concentration of the vacuolarfluid must be very low, for (as already stated, Kitching, 1934, p. 377) no more saltscan be leaving the organism in the vacuolar fluid than are entering through the bodysurface. The vacuolar surface must be, like the body surface, relatively imperme-able to salts. Morita and Chambers (1929) have already shown by micro-injectionexperiments that in the case of Amoeba dubia the vacuolar surface resembles the bodysurface by being impermeable to HC1. There may, however, be excretory matterpresent in the vacuolar fluid, and it might even be suggested that water is drawninto the contractile vacuole by the osmotic action of catabolic products. Forcatabolic products to be present in sufficient concentration to make the vacuolarfluid isotonic just before systole with the general cytoplasm, it would be necessaryfor catabolism to proceed at a rate which could not for long be maintained. Forinstance, if urea were the substances in question, an individual Cotkumia curvulaplaced in 25 per cent, sea water would require to produce its own weight1 of ureain not more than 14 hours; and this would involve the breakdown of its own weightof amino acid in less than 5^ hours. Actually only a small proportion of the bodysubstance could really be available for such a purpose, and since marine Peritrichacan maintain vacuolar activity at a high rate for many hours without taking in anyfood vacuoles, the production of sufficient urea would clearly be impossible. Andsimilar considerations apply to other possible breakdown products. It musttherefore be concluded that water is actively secreted by the vacuolar walls.

It is interesting that the cell membrane of marine Peritricha is relativelyimpermeable to urea. The same is true of the Elasmobranch gill membraneand of marine Amoebae (see Pantin, 1931). The surface of the Arbacia egg is onlyslightly permeable to urea (Stewart, 1931). The great permeability of the erythro-cyte and of the kidney glomerulus to urea is perhaps exceptional; the surfaces ofthese tissues are inside the body, and it is not surprising that they should differ fromexternal surfaces.

Glycerol, as regards its effects on marine Peritricha, appears to be more inertthan urea. Even the depressing effect on the contractile vacuole may really be duenot to the glycerol but to the reduction in salt concentration. The slight increasein body volume at the end of some of the experiments with glycerol (see Fig. 2)must be ascribed to a slow penetration of glycerol either through the body surfaceor possibly via food vacuoles, of which a large number were formed.

Body volume control.

Transference of the organism from dilute sea water to the same dilute sea water+ cyanide led to an increase in body volume, and a return of the organism to dilutesea water without cyanide led to a decrease of the body volume. The addition of

1 I.e. the body weight when in i o o p e r cent, lea water, and assuming a specific gravity of I - I ,which is probably a little too high.

22 J. A. KlTCHING

NaCN to dilute sea water, and neutralisation with HC1, will lead to: (a) the forma-tion of HCN and CN', (b) the formation of NaCl, and (c) a slight increase in theosmotic pressure of the solution. The change in the proportion of Na* and Cl' tothe other ions present will be quite insignificant. The slight increase in osmoticpressure would produce a decrease, if anything, and not an increase, in the bodyvolume; but this would be too small to be detected. It is therefore concluded thatthe increase in body volume is due to the presence of cyanide.

The increase in body volume which results from treatment with cyanide mightbe due (a) to some effect of cyanide on the physical state of the protoplasm, or(b) to the inactivation of some volume-controlling mechanism. A similar volumeincrease was observed in a few cases in which organisms were treated with iMethyl or I / IO butyl alcohol (experiments of series 2), and there was also the corre-sponding volume decrease on recovery. It is therefore concluded that the volumechanges in question were due to the inactivation and recovery of a volume-control-ling mechanism. Since in the case both of cyanide and of the alcohols these volumechanges occurred when the concentration of narcotic was such as to depress veryconsiderably the activity of the contractile vacuole, it seems not unlikely that thecontractile vacuole is the volume-controlling mechanism.

If the contractile vacuole is really controlling the body volume by ejecting waterwhich is almost or completely free from salts, then the rate of output during re-covery from cyanide must be sufficient to account for the rate of decrease in bodyvolume. According to the theory here supported, at any instant during recoveryfrom cyanide the rate of output will exceed the rate of body volume decrease by therate of entry of water through the body surface. Only at the first instant of re-covery, when (it may be assumed) the internal osmotic pressure of the organism isequal to the osmotic pressure of the external medium, will the rate of entry of waterbe zero. At the end of recovery the rate of body-volume decrease is zero, and thewhole of the rate of output is equal to the rate of entry of water through the bodysurface. If the time during which the organism is recovering from cyanide is dividedinto a series of sufficiently short periods (in this case of 5 min. each), it is possibleto estimate approximately for each period the rate of output of the contractilevacuole which will account for the observed body-volume decrease. This estimationinvolves certain assumptions:

(a) That the permeability of the body surface to water is constant. This assump-tion is not founded on any evidence, and is not likely to be strictly correct.

(b) That the osmotic pressure of the organism x the body volume = a constant;or, in other words, that the organism is a perfect osmotic system containing a dilutesolution only. Evidence already given (p. 20) favours the view that the bodysurface acts as a good semipermeable membrane. The amount of osmotically inactivesubstance inside the organisms is unknown.

(c) That the contractile vacuole is ejecting pure water. This has already beendiscussed (p. 21).

(d) That in the presence of cyanide the organism attained osmotic equilibriumwith its environment.

The Physiology of Contractile Vacuoles 23

On these assumptions the rate of output required to account for a given rate ofaody-volume decrease can be calculated as follows:

At any instant R0 = Ri + Rv (I)

and Rt = kS(P-p) (II),

where RQ = theoretical rate of vacuolar output, R( = rate of entry of water throughthe body surface, Rv = rzte of body volume decrease, P= internal osmotic pressure,p = external osmotic pressure, S=surface area of body, and k is a constant repre-senting permeability.

At the end of recovery from cyanide, after the body volume has becomeconstant, Rv = o and R* = R =kS (P — ti\ (IID

R, for any particular instant during recovery can be calculated by substituting inequation (III) the appropriate values. For this purpose {P—p) was calculated fromthe body volume at the time in question relative to the body volume before recoveryfrom cyanide (assumptions (b) and (d)). S was found by the method described onp. 12. The calculated and actual values for the total output in a series of shortperiods during recovery in two experiments are shown in Table II. The resultsagree within the rather wide limits of error. No great accuracy is claimed for thesenecessarily rather crude calculations; they are only presented in order to show thatthe figures obtained are not inconsistent with the theory of vacuolar activity whichis supported in this paper.

Table II.

Min. from be-ginning ofrecovery

o-59-14

20I-25I.29-34

S min. at con-stant body vol.

0-55-i°

10-1515-20

5 min. at con-stant body vol

Calculatedvolume of

water enteringthrough body

surface inperiod, in

cubic micra

Experiment C 17,1000420042004400

—

Experiment C 10,600

150021002400

—

Observed body-volume de-crease forperiod, in

cubic micra

I2i % sea watei80001300

8 0 05 0 0

0

12I % sea water400030002000

8000

Expected out-put for period,in cubic micra

Observed output

-, Cothurma socialis.900055°o50004900

—

, Cothurma socialis.4600450O41003200

—

x>r period, incubic micra

I3.3°°6,8006,6oo6,4006,ooo

5,2008,7003,9OO4,2002,400

The possibility of the exercise of volume control by the contractile vacuole offresh-water amoebae can be tested roughly from data already available. Adolph(1926, Table IV) measured the rate of vacuolar output and the surface area of thebody of a number of individuals of Amoeba proteus. Mast and Fowler (1935), bymeasuring the rate of body shrinkage in hypertonic media, and on the justifiable

24 J. A. KlTCHING

assumption that the internal osmotic pressure of the organism was negligible incomparison with the external pressures which they used, estimated the averagepermeability of the body surface to water as 0-026 cubic micra per square micronper atmosphere per minute. Under normal conditions the rate of output of watermust equal the rate of entry of water through the body surface. The entry of waterat the same rate as that at which Adolph found the contractile vacuole to eject itwould require a pressure of 0-9-3-6 atmospheres, or a difference of osmotic pressureacross the membrane equivalent to 0-02-0-08 molar KC1 (average 0-055 molar).According, therefore, to these figures the contractile vacuole is capable of control-ling the internal osmotic pressure to a significant extent. Gelfan (1928) has measuredthe conductivity of the protoplasm of various Protozoa by means of micro-electrodes,and has obtained values in terms of molarity of KC1 of 001 for Amoeba proteus and0-03-0-06 for various fresh-water Ciliates. The figures are liable to a viscositycorrection of uncertain magnitude, and are therefore possibly rather too low.Agreement is, however, sufficiently close to the value calculated from the figures ofAdolph and of Mast and Fowler.

The Peritricha are found in fresh water, brackish water, and sea water; and insome cases the same species has a wide range of tolerance as regards the salinity ofthe water. In 100-75 Pe r cent, sea water the rate of vacuolar output was very low,and stoppage of the vacuole by the action of cyanide caused no appreciable changein body volume. In sea water more dilute than 75 per cent., the contractile vacuolehad a considerably higher output, and stoppage of the vacuole was followed by amarked increase in the body volume. The more dilute was the sea water, the greaterwas the rate of vacuolar output in the absence of cyanide, and the greater was theincrease in body volume on the addition of cyanide. Thus body-volume control andrate of vacuolar output can be correlated. The experiments described in this papersuggest that the Contractile vacuole is of little importance to a marine Peritrich insea water, but that in brackish or fresh water the contractile vacuole may controlthe body volume. The rates of output of the contractile vacuoles of fresh-waterPeritricha are comparable with the rates of output of marine Peritricha in dilute seawater. If the latter can maintain a considerable osmotic difference in dilute seawater, it is probable that the former can do the same in fresh water, although furtherexperiments are required.

Direct values for the permeability of the body surface to water, after the com-pletion of recovery from cyanide, were obtained from equation (III) (p. 23) withthe methods, for estimating the internal osmotic pressure (p. 23) and surface areaof the body (p. 12) which have already been described. Estimations of the perme-ability of the body surface to water which were obtained in this way are given inTable III.

Limiting values may also be set to the permeability of the body surface to waterby a different method.

(a) Minimal permeability. From the evidence of experiments with cyanide itappears that in 100 per cent, sea water a marine Peritrich may be regarded aspractically in osmotic equilibrium with its environment. On treatment with dilute

The Physiology of Contractile Vacuoles

Table III.

Exp. No.

C iC 6C 4

C 1 7

C 10

Concentrationof sea water %

1 03 05 0

f i a *

12 j

Permeability ofbody surface towater, by first

method, in cubicmicra per sq.

micron per atmo-sphere per min.

Cothurma curvuk0-0710-0540-095

Cothurma tocialis00550-049o-ioo

Maxima] perme-ability of body

surface to water,in same units

1.

0-2450-1060-071

—

0145o-349

Minimal perme-ability of body

surface to water,in same units

0-0380-0410-016

0020—

0-031

sea water the organism swells, but not so much as would be expected from thetheoretical relation: internal osmotic pressure x body volume = a constant. Thisrelation is only applicable to perfect osmotic systems, and does not apply if saltsare lost. If we calculate the internal osmotic pressure of the organism when indilute sea water on the assumption that the system is osmotically perfect, we shallobtain a maximal value. Since salts are almost certainly lost when the organism issubjected to hypotonic sea water (Kitching, 1934, p. 379), the value so obtainedmay be regarded as too high. From the maximal value of the internal osmoticpressure is obtained a minimal value for the permeability of the body surface to water.

(b) Maximal permeability. The organism may be regarded as in osmotic equili-brium with its environment when in dilute sea water + cyanide (stage (3), p. 17). Ifwe use this postulate to calculate the internal osmotic pressure of the organism whenin dilute sea water before cyanide treatment, disregarding the possibility of salt lossduring the swelling which follows from cyanide treatment, we shall obtain aminimal value. From a minimal value of the internal osmotic pressure is obtaineda maximal value of the permeability of the body surface to water.

The results obtained for these limiting values, together with those obtained bythe first method, are given in Table III. In view of the changing conditions ofthe experiment, as regards both the concentration of the sea water and presenceor absence of cyanide, it is unlikely that the permeability of the body surface towater is constant. The results obtained are of the same order, namely 0-05—0-10cubic micra per square micron per minute per atmosphere (of difference of pressurebetween the two sides of the membrane). This value is slightly less than that obtainedby Lucke\ Hartline, and McCutcheon (1931) for the unfertilised egg of Arbacia(o-i), and slightly greater than the estimate of Mast and Fowler (1935) for Amoebaproteus (0-026). The results for the Arbacia egg and for Amoeba were obtained byexperiments on the rate of volume change under known osmotic pressures, whilethose for marine Peritricha given in this paper were obtained under conditions ofconstant body volume by an entirely different method. The agreement is thereforeall the more significant.

26 J. A. KlTCHING

Cyanide and sulphide in very low concentrations are known to reduce veryconsiderably the respiration of many animal tissues, through their specific effect onoxidase systems, while alcohols and urethanes are only effective in much higherconcentrations (Dixon and Elliott, 1929; Keilin, 1925 and 1930). The action of thecontractile vacuole is inhibited by cyanide and sulphide in similarly small concen-trations, while the other narcotics require to be present in much greater concentra-tions to have any effect. It therefore seems possible that vacuolar activity derivesits energy directly from a respiratory oxidative mechanism, although it is alsopossible that cyanide acts independently on some other enzyme system which isnecessary for the vacuolar process. Experiments on the effect of lack of oxygen bothon marine and on fresh-water Protozoa are required. In whatever way cyanideacts on vacuolar secretion, its effects are not delayed as in the case of muscular andother forms of activity for which there has been postulated an anaerobic preliminaryprocess with an oxidative recovery. The selective process of the kidney tubules, bywhich certain substances are withdrawn from the glomerular filtrate and otherssecreted into it, is rapidly inhibited by cyanide (Starling and Verney, 1925). Itis interesting that the contractile vacuole behaves like the kidney in this respect, inview of the postulated separation of water from salts in the former also.

SUMMARY.1. There was no change in the body volume of marine Peritricha subjected to

reductions in the salt concentration of the medium, so long as the osmotic pressureof the medium was kept constant by the addition of urea, glycerol, or cane-sugar.In mixtures of isotonic non-electrolytes with sea water the rate of vacuolar outputwas decreased—more so in the case of urea than of glycerol. It is concluded that thecell membrane is relatively impermeable to urea, glycerol, and cane-sugar, and alsoto neutral salts.

2. Excretory substances could not be produced in sufficient quantity to attractwater into the contractile vacuole by osmosis at the rate observed. The process ofdiastole therefore involves "secretion" of water by the vacuolar walls.

3. Cyanide and sulphide in very low concentrations rapidly caused a greatreduction in the rate of output of the contractile vacuole of marine Peritricha. Inthe case of cyanide this effect was rapidly reversible. Alcohols and urethane onlydecreased the rate of vacuolar output when present in much higher concentrations.It is suggested that possibly vacuolar activity depends directly on an oxidativeprocess.

4. When marine Peritricha were transferred from dilute sea water to dilute seawater of the same concentration + cyanide M/200 or M/500 (the />H being carefullycontrolled), the contractile vacuole was completely or almost completely stopped,and the body increased in volume. When the organism was transferred back todilute sea water of the same concentration without cyanide, the contractile vacuolebecame active again and the body decreased in volume until a new steady valuewas attained which was rather below the value in dilute sea water before cyanidetreatment.

The Physiology of Contractile Vacuoles 27

5. The increase in body volume consequent on treatment with cyanide wasgreater the more dilute was the sea water. For sea water of concentrations of100-75 Pe r cent, no swelling was detectable when the organism was treated withcyanide.

6. The rate of output of the contractile vacuole is sufficiently great to accountfor the decrease in body volume during recovery from cyanide.

7. The permeability of the body surface to water is estimated as o-05-o-io cubicmicra per square micron per atmosphere per minute.

ACKNOWLEDGMENTS.

I wish to thank Dr J. Gray and Dr C. F. A. Pantin for their advice and criticism.I am also grateful to Prof. H. G. Jackson for his encouragement and interest in thework, and both to him and Mr A. Graham for their criticism of the manuscript. Afew of the experiments were done at the Plymouth Marine Laboratory, where I wasgranted the use of the London University table. I am indebted to Mr A. J. Smithfor his care in obtaining a plentiful supply of Protozoa.

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Nordisches Plankton, 13. Kiel and Leipzig.HENRICI, O. and TURNER, G. C. (1903). Vectors and Rotors, p. 61. London.KEILIN, D. (1925). Proc. roy. Soc. B, 98, 312.

(1930). Proc. roy. Soc. B, 106, 418.KrrcHiNG, J. A. (1934). J- exp. Biol. 11, 364.LUCKE, B., HARTLINE, H. K. and MCCUTCHEON, M. (1931). J. gen. Pkysiol. 14, 405.MAST, S. O. and FOWLER, C. (1935). J. comp. and cell. Physiol. 6, 151.MORITA, Y. and CHAMBERS, R. (1929). Biol. Bull. Wood's Hole, 56, 64.PANTIN, C. F. A. (1931). J. exp. Biol. 8, 365.STARLING, E. H. and VERNEY, E. B. (1925). Proc. roy. Soc. B, 97, 321.STEWART, D. R. (1931). Biol. Bull. Wood's Hole, 60, 152.