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 their
function, the only two probable ones are (i) that they are organs for the excretion
of waste matter (other than water), and (ii) that they control the internal osmotic
pressure. These two possibilities are not mutually inconsistent. The contractile
vacuole may have more than one function in the same organism, or different
functions in different species, but it is very unlikely that the mechanism is not
fundamentally the same in all species. There is as yet no convincing positive evidence
for the theory that they are excretory, and this is not surprising in view of their
small size; but this possibility need not affect an examination of the second alternative, which is the purpose of this paper. The osmotic control theory was suggested
by the supposed occurrence of contractile vacuoles in fresh-water Protozoa, but
not in marine or parasitic forms. Actually they do occur in a number of marine
Ciliates, but this does not invalidate the argument. A marine form might have
an internal osmotic pressure either greater than or the same as that of the external
medium, 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 surrounding medium, and so would have to have a contractile vacuole, unless it could
maintain its internal concentration in some other way.
If the contractile vacuole is a controller of the internal osmotic pressure one
would expect it to be affected by large changes in the osmotic pressure of the
medium. Various workers have reported a loss of the contractile vacuole from
fresh-water forms which were placed in sea water, and a reappearance of it when the
organisms were replaced in fresh water {e.g. Ziilzer, 1910), but in such cases it can
always be objected that the stoppage of the vacuole was due to a toxic effect of the
unnatural environment. Hogue (1923) found that contractile vacuoles appeared in
a marine amoeba which was placed in fresh water—a piece of evidence the importance of which has not been sufficiently stressed. A more detailed analysis of the
relations between the rate of vacuolar output and the osmotic pressure of the
external 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 interval
between 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 the
earlier 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 the
amoebae were placed in solutions of NaCl and of other salts of concentrations up
to MJ20; and that there was no direct relation between the surface area of the
organism and the rate of vacuolar output. He puts this forward as evidence against
the osmotic control theory. When the amoeba is in equilibrium with its environment, the rate of output of water from the contractile vacuole must equal the rate
of intake of water through the body surface. If the external osmotic pressure were
raised above the internal osmotic pressure—as may have been the case in Adolf s
experiments—then the contractile vacuole might be expected to stop. There are
four 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 external
medium;
(4) that the contractile vacuole was drawing on the internal water supply of the
amoeba 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 as
were 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 equilibrium with the new outside medium in respect of body volume, and that water was
passing into them from the outside.
In the experiments about to be described the body volume was measured in
order to meet the above objections; and since more significance could be attached
to an increase (if such were to occur) than to a decrease in rate of output, hypotonic
as well as hypertonic media were used.
MATERIAL AND METHODS.
The species used were chosen from among the Peritrich Ciliates, which have
the following advantages:
(i) The contractile vacuole contracts very frequently (once in 30-60 sec. at
150 C.) in fresh-water species, and is present and contracts fairly frequently (once
in 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 conjunction 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 remain 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 of
the 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 from
rot-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 to
wriggle. The adjustment of the cover-slip is critical, and long experiments are
frequently 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, Cothurnia
curvula Entz., Cothurnia ingenita* O.F.M.
All the marine forms were found on Cladophora in the Drake's Island tank of
the Plymouth Aquarium. The Cladophora came originally from intertidal rockpools 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 that
material would arrive from Plymouth in a healthy state so long as the weather was
cool, and would remain healthy in the laboratory in London for a week if it was
kept below 150 C. No material was used which did not appear to be in good
condition.
The Protozoa were kept in a continuous flow of fluid while under observation
by means of the apparatus shown in Fig. 1. Fluid flows from the capillary opening
of the supply unit on to the slide at the edge of the cover-slip, at a rate of a drop in
1-2 sec. It flows under the cover-slip, out through the blotting paper at the other
side, into the funnel, and away. In all experiments in which room temperature
could not be maintained as low as 15-160 C , the temperature of the fluid supplied
was controlled by means of the water-jacket. When the room temperature was not
excessively high, the temperature of the fluid under the cover-slip (as determined
by 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-jacket
was adjusted so as to give a temperature under the cover-slip of 15-16° C. A number
of supply units of this apparatus were fitted up. By changing the supply unit a
1
1
1
1
Kindly named for me by Prof. Mackinnon.
Supplied to me by Mr T. T. Macan, to whom I am grateful.
This form wa3 always solitary, but otherwise resembled Cothurnia (Pyxicola) socialis.
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 sometimes
but not always.
Physiology of Contractile Vacuoles
367
rapid change of the external medium of the organism can be brought about. Such
a change of units can be effected in a few seconds. The constant flow of fluid
ensures 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 fluid
to organism
A unit of the
irrigation 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 London
or Plymouth tap water at />H 7-9-8-2. The pH was taken with indicators, and was
corrected where necessary with NaOH.
The Protozoa were in no way compressed during the experiments. The material
on 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 under
observation. The contractile vacuoles of all species were, just before systole,
perfectly circular in outline from whatever direction they were viewed. They are
therefore held to have been spherical at that time, although they were frequently
of irregular shape during the earlier part of diastole. They emptied themselves
completely to the exterior, and there were no signs of any "canals" such as occur
in Parameeium. The time of systole was estimated generally within a second. The
ultimate diameter was measured with a Watson screw micrometer eyepiece. For
this 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 zero
or 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 by
dividing the ultimate volume of that vacuole by its duration. The mean rate of
output for a longer period of time was obtained by dividing the total output by
that time; this eliminates practically all error in the measurement of time, but the
error involved in measuring the vacuolar diameter remains. The rate of output is
extraordinarily constant under constant conditions, although there is occasionally
a 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 mean
r
v n(n — 1)
rate of output and rate of output for any one vacuolar duration, and n = number
of these rates of output observed. It was generally for fresh-water species 5-10 per
cent., and for marine species 5-15 per cent, of the mean rate of output. The number
of readings was in some cases too small for the standard error of the mean to have
much statistical value. For measurements of the body volume only two, three, or
four readings could be obtained under a given set of conditions. The maximum
deviation from the mean was generally between 5 and 10 per cent, of the total body
volume.
Each organism was kept in a constant flow of fluid (fresh water for fresh-water
forms, sea water for marine forms) under control experimental conditions for an
hour before observations began. Measurements for estimation of the body volume
were taken at intervals throughout the experiment. While these measurements
were being taken, or sometimes for purposes of rest for the observer, there were
intervals during which observations of the contractile vacuole ceased. In the tables
of results (p. 369) the actual time is given during which the organisms were kept
under any given set of conditions, inclusive of such intervals, but exclusive of the
hour for acclimatisation at the beginning of the experiments. The organisms used
(with the exception of VorticeUa marina) are practically perfectly symmetrical about
an axis of rotation. An accurately reproduced side view therefore contains all the
data necessary for an estimation of the body volume. Each of these estimations
was 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 offresh 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 performed, and gave similar results.
Concentration
of sea
water in
medium
Mean rate of
output in cubic
microns per second
Mean
ultimate
diameter
in microns
, 0
Number of
vacuolar
cycles
measured and
included
( = «)
Volume of
organism in
cubic microns
Duration
of treatment in
min.
RJuibdottyla brevipes (fresh water)
0
4
0
0
| ->
0
17-2
io-i
16-9
10-2
±0-9
+ o-6
+ i-o
±0-7
0-28 + O'O2
lo-o
+O'6
10-3
8-7
13
17
10-5
20
«-s
5'5
8-7
11
13
15
44
—
25
25
184
56
3°
Cutlmrma curvula (marine)
100
40
0-96 + 0-13
18-3 +0-4
100
I-I4±O-14
100
o-6i ±0-05
22-6 ±0-55
0-90 + 0-15
25
100
100
100
1-10 + 0-17
2O-2
±O"5
0-63 + o-o(>
7-0
9-6
7-1
24
59
12
IO-I
6-7
5'6
9-6
5'4
7
10
3°
6
20,700
33,4OO
20,000
18,400
42,600
17,600
201
44
32
12
—
48
85
59
—
16
15
13
56
9
44
34
Cothurnia ingenita (marine)
100
25
100
9 6 3 + 1-34
!S6-i ±4*5
4-10 + 1-92
•9-3
18-2
167
2
13
2
3°
Votfturnia imuitti (marine)
5O
o-8i + o-i i
15-7 ± 1-2
100
°'35 ±o-oi
100
54
8-6
5'5
8
56
24
4i
61
3
—
Vorticella marina (marine)
100
55
100
55
100
5O
100
20
3-11 ±0-44
lyh ±2-5
2'48 + O'OQ
11'9 +0-97
1'06 +0-09
14-3 ±0-57
I-IO + O-I8
O'O
2O'2
19-6
Variable
18-8
131
15-9
149
3
7
2
21
53
—
4
3
6
2
+O-O
104
46
55
17
Blisters under
pellicle
65
Zoot/iammum niveum (marine)
100
5°
100
100
12}
IOO
9-0
90-1
7-0
10-4
167-3
io-o
+ 1-3
± 2-5
± 0-5
+ o'5
± I2'2
± 1-5
i5-4
194
16-2
18-6
24-8
192
8
17
—
9
241,500
487,500
13
8
15
33
53
50
36
60
J. A. KlTCHING
37°
Table I I . Results of further experiments in which marine species were subjected to
dilute sea water; examples showing a marked falling off in rate of output while the
organisms were still in the hypotonic medium.
Concentration
of sea
water in
medium l"o
I
Ultimate
diameter in
microns
Rate of output in
cubic microns
per second
Duration
Body
volume in
of treatment
cubic microns
in min.
Cothurnia curvula
ICO
75
IOO
0-13
OI9
1OO
7i
IOO
First io min : 35-47
20-32
Later:
°75
(rather variable]
IOO
5
IOO
44
76
134
First io min.: o-9S
O'2O
Later:
o-54
First io min : 70-78
24-32
Later:
0-48
101
I2-2-I3-2
9-2-11-3
Variable
100
14-6-15-6
11-2—12-3
Variable
16,400
23,200
19,000
12,400
5°
134
63
37
7«
54
Cothurnia sp. ? socialis
100
25
IOO
25
IOO
25
1 09
First 10 min.: Abt. 48
16-20
Later:
0-83
0-58
First 10 min.: 35-41
I^ater:
9-8-11-8
1-17
162
First 10 min.: 34-4O
10-16
Later:
2-5
104
145
11-9—129
Variable
8-2
11-9-12-9
10-5-12-9
109—126
7-2
II-2-I2-O
8-S-IO-O
5-1-8-5
51
1052
717
48
1614
946
139
816
446
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 was
constructed, with a linear magnification of about x 3000.
(3) The figure so obtained is divided into two halves—mirror images of each
other—by the central axis. The distance (y) of the centre of gravity of one of these
halves from the central axis was found by means of a geometrical construction and
calculations (see Henrici and Turner, 1903). The measurement of areas which this
method 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 sufficiently
small to be neglected entirely. The transparent sheath which surrounds the contractile 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 for
observation should lie accurately in the same optical plane, since otherwise the
length measurements are inaccurate.
EXPERIMENTAL RESULTS.
(1) Fresh-water forms.
The contractile vacuole of Rhabdostyla brevipes was found to have a duration
of 30-60 sec, and an ultimate diameter of 7-11 microns, at 150 C. The average rate
of output under these conditions was 11-6 cubic microns per second. Individuals
were subjected successively to (1) tap water, (2) a known mixture of tap water and
sea water, (3) tap water. Fig. 2 illustrates a typical experiment. Transference of the
organisms to (2) led to an immediate decrease in the rate of output and in the ultimate
diameter until these reached a new steady value. Also in many cases a decrease in
body volume occurred which was very noticeable, although the body volume could
not be measured accurately for this species owing to the fact that an individual
does not remain for long in the same optical plane. In Fig. 5 is shown the relation
of concentration of sea water in the medium with mean rate of output. In calculating
the latter no readings were included which were taken immediately after a change
of medium and before the rate of output had settled down to a steady value. In
spite of individual variations it is clear that there was a falling off of rate of output
with increasing concentration of sea water, until in about 12 per cent, sea water
the rate of output was zero. The ultimate diameter was also decreased. Both mean
rate of output and mean ultimate diameter returned in most cases to their original
values when the organism was replaced in tap water.
In the higher concentrations (10-15 P e r cent, sea water) the cilia stopped and
the organisms were contorted by shrinkage, but in all cases after the organisms had
been 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 10-15
min. (though for Cothurnia curvula it was about 3-5 min.), and an ultimate diameter
of 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, sea
water, (2) hypotonic sea water of known dilution, (3) 100 per cent, sea water. In
general it may be stated that dilution of the sea water led to an increase in body
volume and in rate of output. A typical experiment is illustrated graphically in
Fig. 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 ultimate
diameter 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 experiments
there was a falling off in the body volume after the initial increase (Fig. 4). In
many experiments in which very dilute sea water (approximately 5 per cent, or
less) was used, the organisms swelled up until they were globular; and then clear
drops of fluid raised the pellicle up in blisters, which often swelled and became
nearly spherical. In a few cases the protoplasm flowed out into the blisters. The
rate of output increased rapidly and immediately, and then either remained constant
Ikxly voluin
©
I III)
|M
1 mil.
v \ t walcr
300
360
120
Time in minutes
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 hypotonic
medium. N.B. For 12.I per cent, sea water the average rates of output for groups of three vacuolar
durations have been plotted.
60
at a new high level, or decreased at first but subsequently became constant at a
level 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 a
steady rate of output 1600 min. after it had been transferred to 25 per cent, sea
water. 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 approximately to its original level. When blisters had been formed, in very dilute sea
water, the contractile vacuole generally slowed down and stopped. The ultimate
J. A. KlTCHING
374
diameter was liable to sharp variations after a change of medium, but subsequently
became constant. In moderately hypotonic (50-100 per cent.) sea water it was
generally less, and in more dilute sea water generally more, than what it had been
originally (in 100 per cent, sea water). In very dilute sea water the contractile
vacuole 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-n
100
80
60
40
20
Concentration of sea water in m e d i u m
0%
Fig. 6. T h e relation of body volume with concentration of medium for Znnthamnium
Cothuntia curvula. ® Zoothamnium marinum; <J> Cothurnia curvula.
marinum and
(3) In 100 per cent, sea water the body volume returned immediately and
rapidly to its original value, or less. In those cases in which the hypotonic sea
water was very dilute the pellicle wrinkled when the organism was replaced in
100 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 conditions
are 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. The
ultimate diameter became variable, but returned approximately to its original value.
The relation of concentration of sea water with body volume and rate of output
are 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
0%
Concentration of sea water in medium
Fig. 7. T h e relation of rate of output with concentration of medium for Zoothammum marinum and
Cothumia curvula.
Curve A: ® Zoothammum marinum; <$> Cothumia curvula. Experimental treatment as described
on p. 371.
Curves B and C: /2\ and • two single individuals of Cothumia curvula. W.S.W. = Wembury
stream 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 in
rate of output was x 70, in 12J per cent, sea water. In greater dilutions the rate of
output fell off.
In another series of experiments on marine Peritricha the organisms (Cothumia
curvula and Cothumia ingenita) were subjected by successive steps to more and
more dilute mixtures of sea water and Plymouth tap water. The relation between
rate of output and concentration of sea water (Fig. 7, curves B and C) is similar
!BB-xiiv
25
376
J. A. KlTCHING
to that found in the experiments described above. In one experiment (Fig. 8) the
organism was taken in steps down to 1 per cent, sea water, and then back to 100
per cent, sea water by the same steps in the reverse order. The body volume and
rate of output were much lower on the return journey than they had been on the
same steps on the outward journey. In two experiments the organism was subjected
to 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 was
maintained for a long period (40 hours in one experiment, 15 hours in the other),
60r
100
60
60
40
20
Concentration of sea water in m e d i u m
Fig. 8. T h e relations of body volume and rate of output with concentration of m e d i u m for a single
individual of Cothurnia curvula, which was transferred by successive steps to more and more dilute
sea 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 contractile
vacuole stopped.
In both these series of experiments on marine Peritricha the cilia stopped
beating when the organism was placed in the more dilute mixtures (e.g. sea water
25 per cent, or less), although there was considerable individual variation in this
respect. Also sometimes they started beating again in the diluted sea water, but
at other times they remained stopped until some time after the organism had been
replaced in 100 per cent, sea water. Except in some cases in which blisters had been
formed, the organisms appeared to be perfectly healthy at the end of the experiments, and the cilia beat again normally. On several occasions individuals divided
soon 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 total
number of systoles observed was over 4000.
DISCUSSION.
The increase in the body volume of marine Peritricha, consequent on treatment 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 be
freely permeable to salts. Evidence is as yet inconclusive as to which is the right
explanation; (2) may possibly play a small part, even if (1) accounts for most of the
swelling. There is strong evidence that in many different kinds of animal cells the
cell boundary is semi-permeable with regard to salts and water (Luck£ and
McCutcheon, 1932). Some preliminary experiments with marine Peritricha on
the effect of ammonia, an alkaline substance likely to penetrate the cell and there to
influence the ionisation of the cell proteins in the same way as would a reduction
in the salt concentration, have indicated that there is no change in volume while
the organism is alive. Again, when an individual of Cotkurnia curvula was treated
with a mixture of a glycerol solution and sea water such that the salt concentration
was reduced to one-sixth while the osmotic pressure remained unaltered, there
was no change in body volume or in rate of output. Thus there is evidence, though
as yet incomplete, for believing that the changes in volume observed were due to
the fact that the cell surface of these Protozoa is semi-permeable with regard to
salts. If this is so, information can be deduced concerning the osmotic pressure of
the vacuolar fluid.
The osmotic pressure of the vacuolar fluid has never been measured, but it can
be 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 than
enter 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 fresh
water except occasionally in the food. On the other hand, the internal osmotic
pressure of fresh-water Protozoa, though low, must be greater than that of the
surrounding fresh water, so that the contractile vacuole must be separating water
from the internal solutes of the organism. And in marine forms, if the cell membrane 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 vacuole
must be separating fluid of very low osmotic pressure from an internal solution of
osmotic pressure not less than that of sea water. Assuming the semi-permeability
of the cell membrane with regard to salts, the contractile vacuole must in both cases
be doing work, and it must therefore be regarded as an active mechanism involving
the expenditure of energy. Its operation will raise the internal osmotic pressure of the
organism 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 magnitude of the difference between the internal and the external osmotic pressures will
depend on the rate of vacuolar output and on the permeability of the cell membrane
to water, and may be insignificant if the" latter is great as compared with the
former.
The secretory theory of diastole, as outlined above, is entirely contradictory to
any suggestion that the contractile vacuole grows larger by osmotic uptake of water
from the surrounding protoplasm. For osmotic uptake there would have to be
inside the vacuole a quantity of solute such that at the greatest volume of the
vacuole the osmotic concentration of the vacuolar fluid was not less than that of the
surrounding protoplasm. At the beginning of diastole, when the volume of the
vacuole is much less, the concentration of the vacuolar fluid would have to be
correspondingly greater. In marine Peritricha, whose internal osmotic pressure
must be not less than that of sea water, the initial concentration of the vacuolar
fluid would have to be extremely great, and it seems unlikely that such a concentration is actually produced. And for Amoeba proteus, a fresh-water form, Adolf
has shown that the relation between vacuolar volume and time is linear during the
period of a single diastole. Such a relation is inconsistent with simple osmotic
uptake. In view of these objections to the theory of osmotic uptake of water by the
vacuole, the validity of the secretory theory is assumed in the discussion which
follows.
The factors which are likely to affect the secretory activity of the vacuole are of
two types: (a) those dependent on the concentration of the sea water outside at the
time in question, and (b) those governed by the state of activity of the organism
or 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 was
considerable individual variation in the rate of output among members of the same
species, and that this could not be attributed to size. Specimens which had been
sent from Plymouth to London in cold weather had a high rate of output, while
those which had been sent up in hot weather had an extraordinarily low rate of
output, and the vacuolar duration was as high as half an hour. Those that had been
kept for any length of time in the laboratory in London also had a low rate of
output. Hogue (1923) observed that old cultures of amoeba developed a low rate of
output, and this has been confirmed by the present writer. It is therefore probable
that the rate of output is considerably influenced by the state of the organism.
Small differences in condition might account for the differences found by Adolf
in the rates of output of amoebae.
The observed increases in the body volume of Peritricha fall short of those
which would take place if the cell membrane were perfectly semi-permeable, and if
the cell contents were no more than a dilute solution of salts. Such a falling short
could be ascribed to salt loss, to the presence of osmotically inactive substances
within the cell, or to volume control by the contractile vacuole. It is very unlikely
that 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
379
Arbacia is very small, and such pressure may therefore safely be ignored. To what
extent the other factors are operative cannot be discussed until the results of further
experiments are available, but it seems probable, from the nature of the curve
relating body volume with concentration of external medium (Fig. 6), that in very
dilute media salts escape. This would explain the return to a body volume smaller
than the original (p. 374), and also the falling off in body volume while the organism
was still in the hypotonic medium (Fig. 4). Whether the falling off in rate of output
which 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 set
up 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 follows
transference of the organism to a hypotonic medium involves an increase in the
amount of work done. As a rough approximation the osmotic pressure of sea water
may be taken as proportional to the concentration, and the internal osmotic pressure
of 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 cannot
be less than that of the external medium. By assuming that it is the same we shall
find a minimum value for the work done. Assuming that the vacuolar fluid is pure
water in all cases,
w_
where W=work done, P= internal osmotic pressure of organism, V= volume of
fluid eliminated by the contractile vacuole.
From the curves given we find:
Concentration of sea water in
which organism is placed ("„)
IOO
75
50
Minimum value of relative amount
of work done per unit time
100
75
250
.
25
20
10
5
1075
960
600
IOO
The contractile vacuole has therefore all the potentialities required not only for
a maintainer but also for a regulator of the internal osmotic pressure. Whether it is
effective 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 external
osmotic pressure, even though the internal osmotic pressure is influenced by small
changes in the external osmotic pressure; (b) Regulation of the internal osmotic
pressure so as to keep it constant irrespective of small changes in the external
osmotic pressure. For marine forms the curve relating rate of output with concentration 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," occurs
between these values. Whether any "maintenance" occurs is unknown, but it is
unlikely that the internal osmotic pressure of marine forms is much above that of
the surrounding sea water. Although the contractile vacuole of marine species
might 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 entirely
impermeable to salts, these will enter, although perhaps very slowly; a Donnan
equilibrium will thereby be set up owing to the presence of indiffusible proteins
inside the cell, so that the internal osmotic pressure will be raised slightly above
that of the outside sea water. It is possible that the contractile vacuole might be
required to relieve the resulting tension on the pellicle. Against this, as against any
other suggestion of a function for contractile vacuoles in marine Protozoa, may be
brought 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 very
dilute sea water, being liable to excessive swelling, but in many cases individuals
of Cothurnia spp. were but little inconvenienced, and in some individuals of
Cothurnia ingenita and Cothurnia curvula the cilia continued to beat, although rather
sporadically, in Wembury stream water. At other times, however, no individuals
of Cothurnia spp. would successfully endure even 10 per cent, sea water. All
individuals of the same batch behaved alike in this respect. It seems probable that
successful adaptation was effected by loss of salt. The rate of vacuolar output falls
off in very low concentrations of sea water, and therefore it is unlikely that in such
concentrations regulation takes place, although the contractile vacuole may have
been preventing excessive swelling by maintenance. The experiments described
above 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 (including
algae as zoospores or as adults) which are devoid of rigid cell walls, but not in those
forms or stages in which rigid cell walls are present. It might be questioned whether
such cellulose walls could withstand the great pressure which would be developed
by a small difference in concentration between the inside and the outside. The
very small size of such cells would make it more possible, but a knowledge of the
strength 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 organism
was placed in a mixture of tap water and sea water. The rate of output returned
to its original value immediately the organism was replaced in tap water. The
contractile vacuole was stopped when the organism was treated with a mixture
containing more than 12 per cent, of sea water.
2. Transference of various species of marine Peritricha from 100 per cent, sea
water to mixtures of sea water and tap water led to an immediate increase of the
body volume to a new and generally steady value. Return of the organism to
100 per cent, sea water led to an immediate decrease of the body volume to its
original 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 mixtures
the rate of output was immediately increased, and then generally fell off slightly
to 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. Return
of the organism to 100 per cent, sea water led to an immediate return of the rate
of output to approximately its original value.
4. When individuals of some marine species were placed in very dilute concentrations of sea water, the pellicle was frequently raised up in blisters by the
formation of drops of fluid underneath it, and the contractile vacuole stopped.
5. Evidence is brought forward to suggest that in the lower concentrations of
sea water marine forms lost salts.
6. The contractile vacuole probably acts as an osmotic controller in fresh-water
Protozoa. 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 for
much encouragement, and to Prof. Sugden and Dr R. G. Cooke for advice on the
physical and mathematical aspects of the work. I was granted the use of the London
University 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 Ausschluss 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.