The Physiology of Contractile Vacuoles: V. The Effects of Short-Term Variations Of Temperature on a Fresh-Water Peritrich Ciliate

It has long been known that increase of temperature, within reasonable limits, causes an increase in the frequency of pulsation of the contractile vacuoles of various Protozoa. (For references see the review by Kitching, 1938 b, and the recent paper by Smyth, 1942.) Unfortunately, earlier workers did not follow the changes in maximal diameter of the contractile vacuole which may result from changes in temperature, so that no information has hitherto been available as to the relation between temperature and rate of vacuolar output.

In many Protozoa, and especially in fresh-water forms, there is a considerable exchange of water between the body and the outside. The contractile vacuole bales out water continually, and there must be a corresponding uptake into the body from the external medium. For the purpose of the present investigation it is necessary to consider more closely the various components of this exchange, since the effects of temperature on vacuolar output can only be understood in relation to the water balance as a whole.

Uptake of water from the external medium takes place in part by means of food vacuoles, but these account only for a minor portion of the output in those freshwater Protozoa in which this question has been investigated (Kitching, 1938b, p. 413, and 1939). The greater part of the uptake must take place either through the general body surface or through some restricted part of this. There is as yet no satisfactory evidence by means of which a choice can be made between these two alternatives. Eisenberg (1925), Frisch (1937), and Mast (1947) have concluded that uptake is restricted to the ‘oesophageal sac’ at the base of the pharynx. The evidence rests on the observations that neutral red staining first appears in this neighbourhood (Frisch) and that food particles become concentrated in the oesophageal sac shortly before a food vacuole is pinched off (Mast); it does not seem sufficient to justify the claim that water is exclusively or even predominantly taken up through the oesophageal membrane. Accordingly the other hypothesis, being the simpler, will be utilized here for purposes of discussion; the conclusions drawn in this paper are not affected by this choice. In any case the uptake of water is presumably due to osmosis; it is difficult otherwise to explain the susceptibility of vacuolar output to changes in the osmotic concentration of the external medium (Kitching, 1938a).

Loss of water from the body to the external medium takes place through the contractile vacuole; this probably is the chief means in most fresh-water Protozoa. There is also some loss of water by extrusion of food vacuoles at the close of their digestive cycles. In Vorticella, according to Mast & Bowen (1944), the amount of water lost in spent food vacuoles does not fall far short of that which was taken up in these vacuoles when they were first formed; and the same may be true of Paramecium (Mast, 1947). In these cases the rate of output of the contractile vacuoles must be approximately equal to the rate of osmotic uptake of water into the body from the external medium. It is difficult to observe the elimination of spent food vacuoles in the smaller peritrich ciliates, but I am very doubtful whether the amount of water lost in this way is as great as that taken up in food vacuoles. However, the rate of uptake of water in food vacuoles is in any case fairly low (see p. 406), so that here also the rate of output of the contractile vacuole cannot differ greatly from the rate of osmotic uptake.

Maintenance of the normal body volume demands that the rates of uptake and of loss of water remain equal, though not necessarily constant; any change in one must be met by a compensatory change in the other. The present investigation, therefore, deals with the effects of temperature on the rate of vacuolar output and on the body volume of certain peritrich ciliates. From these the effect of temperature on the rate of uptake of water from the external medium can be inferred. This rate cannot easily be measured directly, but additional evidence relating to it is also presented in the following paper.

Peritrich ciliates have been used throughout this series of investigations both because of their regular body shape and because it is much easier to work with a sessile organism. Carchesium aselli (previously referred to incorrectly as Zoothamnium sp. by Kitching, 1938a) was used for the experiments described in this paper. It is commonly found on the hairs on the legs of the fresh-water isopod Asellus aquaticus. The Asellus were collected from a pond near Bristol, and were kept for 2 weeks or longer in a large aquarium tank containing pond water at pH 7·7–8·0 and 12–15°C. and well supplied with organic detritus.

In a number of preliminary experiments a leg of Asellus was pulled off and mounted in a hanging drop of pond water under a cover-glass sealed with vaseline over a small glass cell. The cell and cover-glass were surrounded by well-stirred water at controlled temperature in a water-bath mounted on the microscope stage. The objective was immersed, and for low temperatures dry air was blown through the microscope to prevent condensation on the lenses. This method gave good control of the temperature down to freezing-point, but was found unsatisfactory for general use because in the majority of cases the rate of output of the contractile vacuole fell off in control experiments at constant temperature (15° C.). It was therefore abandoned except for experiments at the lowest temperatures.

Accordingly I reverted to the method used in previous papers of this series (Kitching, 1934, fig. 1), by which the organism is irrigated throughout the experiment with a solution at controlled temperature. The solution used for irrigation was allowed to drip at the edge of the cover-slip from a glass ‘supply tube’, and was led away at the other side of the cover-slip by means of filter-paper to a funnel. The supply tube passed through a water jacket the temperature of which could be altered quickly, and the actual temperature under the cover-glass was measured by means of a thermocouple junction placed within 2 mm. of the organism. Pond water at pH 7·7–8·0 was used. In each experiment the organism was irrigated under experimental conditions at 15 ±0·5° C. for 30 min. before observations were begun, and this preliminary period is not included in the durations of treatment given in Table 1.

The maximal diameter of the contractile vacuole (attained just before systole) was measured with a screw micrometer eyepiece. The time of systole was recorded to the nearest second. From this information it is possible to estimate the rate of output for each cycle of the vacuole observed, and this has been done for purposes of illustration in Fig. 2. However, since (except in certain specified cases) there was no evidence to suggest other than random fluctuations in ultimate diameter (as measured) and in the interval between systoles, the mean rate of output for each temperature was calculated from the mean ultimate diameter and the mean interval. The range of variation found in typical experiments is illustrated in Fig. 2; the significance of the readings is so apparent as to render further statistical treatment superfluous.

In some experiments observations were also made of the approximate size of the food vacuoles and of their time of uptake. The exact size of food vacuoles is difficult to determine as these are not spherical when first taken up, and no significant changes in size are claimed in this paper. The significance of the changes observed in the interval between the times of uptake of successive food vacuoles can be judged from the means and standard errors given in Table 2.

In view of the importance of knowing how much food vacuoles contribute to the general water balance, a more precise estimation of the upper limit of this contribution was attempted in a few cases at 15° C. Food vacuoles originate in a spindle-shaped depression, known as the oesophageal sac, at the base of the pharynx. This sac is periodically pinched off as a food vacuole and is reformed. The volume of the oesophageal sac, at its maximum, was estimated from the length and breadth on the assumption that the shape was that of a sphere capped by two tangential cones. Figures obtained in this way give an upper limit for the uptake of water by means of food vacuoles.

Observations were also made on the activity of the adoral cilia which encircle the disk at the free end of the organism. In some experiments these stopped beating, and in some they underwent periodic intermissions.

An indication of possible changes in the body volume of the organisms under experiment was obtained from measurements of the length and breadth. The length as measured in this work usually did not include the disk, as the constriction or neck immediately below this provided a more clearly defined line from which to measure. The breadth was, of course, measured at its greatest. The range of variation in estimates of the length and breadth under constant environmental conditions did not normally exceed 1 μ in either measurement, so long as the organism remained extended in the plane of focus.

In a few experiments organisms were subjected to a series of temperatures, starting with 15° C. In most cases, however, for reasons explained later, the organism was observed (a) at 15 ± 0·5° C., (b) at a selected experimental temperature ±0·5°C., (c) at 15±0·5°C.

During irrigation at 15° C. the vacuolar output was maintained at an approximately steady level. This was shown in the initial period of all experiments, and throughout control experiments lasting up to 2 hr. The limited scatter recorded during the initial period of the experiment illustrated in Fig. 2 is typical. Food vacuoles were also taken up at regular intervals, the cilia beat actively, and the size of the body remained constant.

A few experiments were carried out in which the organisms were subjected to various temperatures in succession. Results are given in Fig. 1. It will be seen that the rate of vacuolar output rises steeply with increase of temperature. However, towards the higher temperatures, the results are somewhat irregular, and for this reason the experimental procedure was modified in the hope that a well-defined curve relating temperature and rate of output might be obtained. Accordingly in all the experiments described below the organisms were observed at 15° C., and then at a selected experimental temperature, and finally at 15° C. again.

Decrease of temperature, in experiments carried out as just indicated, was accompanied by reduction in the rate of vacuolar output. Although the maximal vacuolar diameter was greater at low temperatures, and very markedly so in the region of o° C., this was more than counteracted by the much longer interval between successive systoles (Table 1). In the region of o° C. the rate of output was reduced, in experiments carried out with the hanging-drop method, to just under 20% of its value at 15° C. However, the contractile vacuole continued to operate even when ice crystals formed in the hanging drop (after the addition of freezing mixture to the water-bath), and was only stopped when the organism was destroyed by the complete freezing of the external medium. Except in cases of complete freezing, restoration of the temperature to 15° C. led to a rapid recovery of the rate of output to a level of the same order as that maintained initially at that temperature.

Increase of temperature (above 15° C.) up to about 34° C. was accompanied by an increase of vacuolar output (Fig. 2). There was always an increase in vacuolar frequency, and near the upper end of the temperature range there was also a considerable increase in ultimate diameter of the contractile vacuole, so that the rate of output was increased by several-fold. In an experiment in which a Carchesium was subjected to 34° C. the considerable immediate increase in rate of output was only maintained for a few minutes; then the contractile vacuole slowed down, the body swelled, and death ensued. Except after such extreme treatment the rate of vacuolar output decreased rapidly on restoration of the temperature to 15° C. The final level was usually slightly below that of the first period at 15° C. In all these experiments the change from one temperature to another took several minutes. The changes in rate of vacuolar output appeared to accompany closely the changes in temperature, but it would not have been possible to detect a slight lag.

The relation of mean rate of vacuolar output to temperature, as indicated by these experiments, is shown in Fig. 3. Here each plotted point represents one experiment, the rate of vacuolar output at the test temperature (that is during the second of the three periods) being expressed as a percentage of that at 15° C. (first period). In this way the results of all the experiments have been scaled to the same level (100%) for the initial rate of output at 15° C. This procedure is justified by the fact that the points lie close to a smooth curve and that where points represent independent results for the same temperatures they lie very close together.

The curve plotted in Fig. 3 indicates a Q₁₀ for rate of vacuolar output of about 2·5–3·2. If the fit of these data to the form demanded by the Arrhenius equation is tested by a plot of the logarithm of the rate of vacuolar output against the reciprocal of the absolute temperature, the points are found to be scattered not unreasonably about a straight line over the temperature range 0–30° C-; the value for μ is about 17,000.

Decrease in temperature was accompanied by a decrease in frequency of uptake of food vacuoles (Table 2). At, or slightly below, o° C. no food vacuoles were taken up, and the cilia stopped. On return to a temperature of 15° C. the frequency of uptake of food vacuoles was restored to a level of the same order as that in the initial period at that temperature, and, after exposure to o° C., the cilia resumed their activity. In one case, after o° C., recovery of food-vacuolar and ciliary activity was delayed for some minutes; in the other cases no such delay was observed.

Increase of temperature up to 24° C. was accompanied by an increase in frequency of uptake of food vacuoles. On a return of the organism to a temperature of 15° C. the frequency of uptake of food vacuoles was reduced to a value in most cases somewhat lower than the original for that temperature. No food vacuoles were taken up at temperatures above about 30° C., and the disk cilia stopped beating. In such cases on return of the organism to a temperature of 15°C. recovery of food-vacuolar and ciliary activity was delayed for some time (Fig. 2).

The relation between frequency of uptake of food vacuoles and temperature is shown in Fig. 4. Frequency’ is expressed as 10³ × the reciprocal of the mean interval between times of uptake of successive food vacuoles. Since no significant differences could be found in the sizes of food vacuoles taken up at different temperatures, this curve approximately represents the relation of rate of uptake of water by food vacuoles to temperature.

Measurements were made of the volume of the oesophageal sac, which periodically becomes cut off as a food vacuole, in the case of four individuals at 15° C. From these values and from the frequency of uptake in each of these cases it was calculated that the rate of uptake of fluid in food vacuoles was 10–14% of the rate of output by the contractile vacuole.

No significant changes were observed in the length or breadth of the body during any of the experiments described above (except for the swelling noted at excessively high temperature). A few additional experiments of longer duration were carried out, in which the temperature control was slightly less rigid. Results are summarized in Table 3. In no case was there any suggestion of a change in body volume, although sometimes there was a slight tendency towards shortening and widening of the body at higher temperatures.

The chief components of the water exchange of a peritrich ciliate are osmotic uptake and the output of the contractile vacuole (p. 406). The part played by food vacuoles, which is in any case of minor importance, becomes reduced to zero at unnaturally high temperatures (30° C. and over; see Fig. 4). Accordingly, the effects of temperature will be considered in relation to the balance between osmotic uptake and vacuolar output.

No changes could be detected in the body volume of Carchesium aselli exposed to various temperatures near the limits of the biological range for periods of 2 hr. (and in one case hr.) even though the rate of output of the contractile vacuole was greatly altered. Clearly the maintenance of a constant body volume would require a simultaneous compensatory change in the rate of uptake of water from the external medium. The rate of osmotic entry of water into the body is presumably determined by the difference of osmotic pressure between the inside and the outside and by the permeability of the body surface to water. An appropriate alteration in one or both of these would produce the necessary change in rate of uptake of water.

However, before we can conclude that such an alteration has taken place, it is necessary to consider both the rate and the magnitude of the change in body volume which would otherwise have been expected; it must be shown that this change in body volume could have been detected during the experiments. Various factors might tend to retard and to reduce such a change—for instance, the presence of osmotically inactive non-aqueous material within the body of the organism or of non-penetrating solutes in the external medium.

For the integration of Northrop’s equation a relation is necessary between internal osmotic pressure and cell volume. The simplest integrated form, derived on the assumption that the product of internal osmotic pressure and cell volume is constant (Lucké & McCutcheon, 1932, p. 103, equation 3a), has been adjusted for the present purpose (see under Fig. 5) to allow for non-aqueous materials within the body; other more complicated corrections are inappropriate to the present circumstances. The change in the osmotic pressure of the dilute internal solution with temperature will be small over the range under consideration, and has therefore been neglected.

Sample theoretical shrinkage curves, constructed on the assumption of a change in rate of vacuolar output without change in the permeability of the body surface or in the solute content of the body, are given in Fig. 5. They apply to a C. aselli of average size. Since it is not at present possible to say how much non-aqueous material there is within the body, curves have been calculated for a range of possible quantities. However, it should be pointed out that, when C. aselli is transferred to a relatively strong (e.g. molar) solution of sucrose, the body shrinks greatly and becomes strongly wrinkled. The amount of such material must therefore be relatively small, and in any case is not so great as to mask osmotic shrinkage when the difference of concentration is substantial. In other respects the values used for substitution in the equation, and quoted below Fig. 5, are extremes such as would give the slowest changes of body volume.

From the curves given in Fig. 5 it is clear that, on the assumptions on which the treatment described above is based, an increase in rate of vacuolar output to 300–400% would produce a very marked shrinkage well within the durations of the experiments. For instance, with the vacuolar output at 400%, and even with a nonaqueous volume of 25%, C. aselli would be expected to shrink by 50% of its total volume within about 15 min. Even with a greater quantity of non-aqueous materials equilibrium would be approached relatively quickly. Since no change in the body volume was observed it must be concluded that some condition, assumed to be constant in the foregoing treatment, must in fact change with temperature.

The increase in the rate of osmotic uptake, which accompanies an increase of temperature, might be brought about (without change of body volume) either by an increase in the number of solute molecules within the body or by an increase in the ‘permeability’ to water of the membrane through which uptake occurs. The former effect might result from an increased accumulation of metabolites or from an increased ionization of cell proteins. On the other hand, in favour of an increased permeability, it is known (Lucké & McCutcheon, 1932) that the osmotic swelling of Arbacia eggs proceeds more rapidly at higher temperatures, the calculated ‘permeability’ to water having a Q₁₀ of from 2·5 to 3·1 ; this is of the same order as that found for the vacuolar output of Carchesium aselli. Evidence will be presented in the next paper of this series that the effect of temperature is on the permeability of the membrane, and that the resulting change in osmotic stress in turn provokes a change in the rate of vacuolar output.

The increase in rate of output of the contractile vacuole with increase of temperature may be regarded, by those who hold that contractile vacuoles eliminate nitrogenous waste, as a necessary physiological response to an increase of metabolic rate. There is little evidence by which this view may be tested. Attempts by Weatherby (1929) to determine the approximate concentration of urea in the vacuolar fluid of Spirostomum appear to indicate that most of the urea formed in the organism must be eliminated in another way. On the other hand, the body surface of marine ‘limax’ amoebae (Pantin, 1931) and of marine peritrich ciliares (Kitching, 1936) is relatively impermeable to urea in the external medium. Much more information is needed before any conclusions can be reached. Nor must it be assumed that ‘the contractile vacuole’ performs the same functions in the same degrees throughout the sub-kingdom of Protozoa. It may be added, in view of the great turnover in water brought about by vacuolar activity, that it might be expected that the contractile vacuole would exercise some power of retention or resorption of solutes; and, as a speculation, that this active process is likely to be selective.

The effects of temperature on the ciliary activity and on the uptake of food vacuoles of Carchesium aselli are strikingly similar. Both are inhibited at temperatures above 30° C., and on return to a more normal temperature there is a simultaneous delayed recovery. Both also are reversibly inhibited at or about o° C. On the other hand, the contractile vacuole operates, for a limited time at any rate, at temperatures slightly above 30° C. and at 0° C. All this suggests that there is either some fundamental similarity in mechanism or some co-ordination between the post-oesophageal fibrils, which are believed to be responsible for the uptake of food vacuoles (Bozler, 1924; Mast & Bowen, 1944), and the cilia of the disk. It has been shown (Kitching, 1938b) that the initial movements of the food vacuoles of peritrich ciliates can only be accounted for by the action of a contractile process taking place in the surrounding protoplasm—a process which is quite probably the function of the oesophageal fibrils. Their activity is therefore perhaps closely akin to that of cilia, so that they might in any case respond in the same way as the cilia to extremes of temperature. Apart from this, the response of the contractile vacuole to high temperatures may differ from that of other organelles because of the special effect of increased osmotic stress, which is likely to elicit the greatest possible output until the mechanism breaks down.

It is a pleasure to thank Prof. J. E. Harris for much valuable advice given during the course of this work. Specimens of the peritrich ciEate Carchesium aselli were kindly examined and determined for me by Mr A. G. Willis.