Heat resistance and contractile vacuolar activity of paramecium caudatum acclimated to different temperatures

Camp. Biochem. Physiol. Vol. 77A, No. 4, pp. 641-645, 1984 Printed in Great Britain

8

HEAT RESISTANCE AND CONTRACTILE VACUOLAR OF PARAMECIUM CAUDATUM ACCLIMATED DIFFERENT TEMPERATURES

0300-9629/84 $3.00 + 0.00 1984 Pergamon Press Ltd

ACTIVITY TO

HIROKO TSUKUDA and YOSHIO TAKEUCHI Department of Biology, Faculty of Science, Osaka City University, Sugimoto-3, Sumiyoshi-ku, Osaka, Japan

(Recei~d 28 April 1983) Abstract-l. Paramecium acclimated to 25°C was more heat resistant than lO”C-acclimated Paramecium. 2. Change in heat resistance after transfer to a new acclimation temperature was completed in 6-9 days. 3. The vacuolar frequency-temperature curve of ZYC-acclimated Paramecium shifted toward higher

temperature. 4. Thus Paramecium

is possessed

of the ability of capacity

adaptation

as well as resistance

adaptation

to

temperature.

INTRODUCTION

Temperature adaptation has been divided by the response of animals into two categories (Precht et al., 1955): one is the adaptation to the lethal effect of temperature, “resistance adaptation” and the other is the adaptation in various rate functions at sublethal or physiological temperature, “capacity adaptation”. Phenomena of temperature adaptation in Paramecia have been summarized by Wichterman (1953) and Poljansky and Sukhanova (1967). It has been found that Paramecia have the ability to acclimate gradually to a high temperature (Jacobs, 1919), to cold (Efimoff and Efimoff, 1924) and to subzero temperatures (Wolfson, 1935). Mendelssohn (1902) noted that optimum temperature rose in Paramecia kept at a higher temperature for some hours. Jollos (1913, 1914, 1921) found that the thermal death point could be raised l-3°C in long-continued experiments for acclimatization. Poljansky (1957, 1959) showed that the thermoresistance examined in clone material of Paramecium caudatum was under the direct influence of the environmental temperature. However, these are studies principally on “resistance adaptation” and phenomena comparable to “capacity adaptation” in Paramecia have been not found. Investigation of the capacity adaptation in various physiological functions is important to elucidate the mechanism of physiological regulation against change in environmental temperature. This paper is the report for the time course of changing heat resistance and for “capacity adaptation” in pulsation rate of the contractile vacuole in Paramecium caudatum acclimated to two different temperatures. MATERIALS

AND

METHODS

The Paramecia used in the present study were three clones (A, B and C) originated from a strain of the Paramecium caudatum which has been cultured for several years under constant conditions in our laboratory. Each clone was divided into two lines one ofwhich was cultured at lO”C(cold 641

acclimation) and the other at 25°C (warm acclimation). This acclimation culture was continued above three months before determination of heat resistance and vacuolar activity. The heat resistance was determined by the survival ratio after exposure to a given high temperature for 5-40 min. Small test tubes containing 0.5 ml of suspension of the Paramecia were immersed into a warm bath controlled at a constant temperature and taken out successively from the bath after 5, 10, 20, 30 and 40 min for count of the survival ratio by the following procedure. The suspension of Paramecia was dropped on a glass slide with viscous 6% methylcellulose solution and survival or death was microscopically observed. The survival ratio per 250-300 Paramecia was calculated from the random observation of several fields. The activity of the contractile vacuole was microscopically observed under control of temperature using a glass vessel connected to a water circulation system. A known volume of the suspension of Paramecia kept at a test temperature was dropped into the glass vessel and 0.1% NiSO, solution of l/l0 volume of the suspension was added. The time taken for two contractions of the anterior contractile vacuole was recorded. This was initiated by measurement at the acclimation temperature to avoid the shocking effect of an abrupt temperature change. Before the succeeding measurement, the temperature of the system was raised or lowered by 3°C at a rate of 0.5’C per min and so measurement was continued from the acclimation temperature to the highest or to the lowest temperature. The effect of the medium concentration on the vacuolar frequency at various temperatures was determined using media of different concentrations prepared by addition of sucrose. RESULTS

Figure 1 shows the heat resistance of three clones of caudatum in survival ratio at three high temperatures. The Paramecia acclimated to 25°C survived after 40 min exposure to 36.5 and 38°C in all clones, while 10”C-acclimated Paramecia were subjected to lethal effect after 5 min at 36.5”C. The effect of 41°C was lethal on either of lO’Cand 25”C-acclimated Paramecia, but median survival time of the former was obviously shorter than that of the latter in any clone. The Paramecia acclimated to 25°C Paramecium

642

HIROKO TSUKUDA and

‘;jy-T~f~ 0

20 Duration

20

400 of

400

heating

Fig. 1. Survival curves of three C (right), of the Paramecium (solid circles) and to 2YC measured at 41°C (top), 38°C Each point is the mean

20

40

(min)

clones, A (left), B (middle) and caudatum acclimated to 10°C (empty circles). These were (middle) and 36.5’C (bottom). value for 10 Paramecia.

distinctly heat resistant in comparison with lO”C-acclimated Paramecia. Figure 2 shows the time course of change in heat resistance after transfer from one to another temperature of 10 and 25°C. Conditions of heat treatment were at 41°C for 10 min, so as to bring on 100% survival in 25”C-acclimated Paramecia and zero in lo”Cacclimated ones. When the Paramecia were transferred from 10 to 25°C the heat resistance comparable to 25”C-acclimated Paramecia was gained after about seven days and in the opposite case the heat resistance declined to the level of 10’Cacclimated Paramecia within 10 days in any clones. Figure 3 shows activity of the anterior contractile vacuole at various temperatures in mean frequency per are

Y~SHKI TAKEUCHI

min for 10 Paramecia. The vacuo!ar frequency generally increased with rising temperature up to a high temperature above which the frequency decreased. The frequency of 25”C-acclimated Paramecia was higher above about 17’C in clone A, 14.5’C in clone B and 15°C in clone C than that of IOC-acclimated Paramecia and below these temperatures this relation was reversed. These facts indicate that environmental temperature affects temperature dependence of the vacuolar frequency and the temperature-frequency curve of the warm acclimated Paramecia shifts toward higher temperature. The vacuolar frequency in clone C was generally lowest among the three clones, especially at higher temperatures. To confirm the differences between clones or between temperature acclimation lines, statistical analysis of variance was tried for values measured at 9,15,24 and 30°C. These results are given in Table 1.The difference between clones was not significant at any of the four temperatures. The difference between lO”C- and 25’C-acclimated lines was significant at 9, 24 and 30-C but not significant at 15°C. The interaction between clones and acclimation lines was non-significant at any temperature. The effect of the medium concentration on the vacuolar frequency was determined at 15, 20, 25 and 30°C. Results are given in Fig. 4 and Table 2. Each point in Fig. 4 is the mean vacuolar frequency for 10 Paramecia at different sucrose concentrations equivalent to (r690 sea-water. The vacuolar frequency seemed to decrease linearly with increasing concentration, so regression lines calculated were added in Fig. 4. Table 2 shows the test of linearity and the ditference between temperature acclimation lines in the regression lines. In many cases, especially in all at 20 and

0

OOTime

(

days)

Fig. 2. Time course of change in the heat resistance of the Paramecium caudatum transferred from IO to 25°C (empty circles) and in reverse (solid circles). Top : clone A, middle : clone B, bottom : clone C.

10 Temperature

20

30

40

( “C)

Fig. 3. Temperature dependence of the anterior contractile vacuole of the Paramecium cuudutum acclimated to 10 C (solid circles) and to 25’C (empty circles) of three clones, A (top), B (middle) and C (bottom).

Temperature adaptation in

643

Paramecium

Table 1. Analysis of variance of the vacuolar frequency in three clones of Parcrmecium caudarum acclimated to 10 and 25°C. C: between clones; T: between acclimation temperatures; C x T: clone-acclimation temperature interaction; E: within subclones Test temp. (‘Cl

9

15

24

30

Source of variation

Degrees of freedom

Sum of squares

C T CXT E

2 1 2 54

0.665 141.681 1.241 0.858

C T CXT E

2

54

9.847 0.140 2.500 263.032

C T CxT E

2 t 2 54

104.094 271.363 53.576 573.150

c T CxT E

2

316.756 3796.922 47.341 1867.703

I 2

I 2 54

25°C the frequencyyconcentration relation was regarded as linear. The regression coefficient(b), or slope of regression line, increased with rising temperature in any clone. There was no significant difference in the slopes of the regression lines between lO”C- and 25”Cacclimated Paramecia in any case except for clone C at 30°C. Mean frequency (j), or level of regression line, increased too with rising temperature, although the difference between temperature acclimation lines was r

r

L-L-L-

024602460246 Sucrose concentration

(Ye

sea

wateres.1

Fig. 4. Regression lines of the vacuolar frequency on the sucrose concentration at 30, 25, 20 and 15°C from top to bottom in the Paramecium caudatum acclimated to 10°C (solid circles and solid lines) and to 25°C (empty circles and broken lines). Left : cloneA, middle : clone B, right : clone C.

Variance ratio(F)

Levels of significance

0.388 165.065 0.723

0.05 < P P < 0.001 0.05 < P

1.011 0.029 0.257

4.904 25.567 2.524 4.579 109.779 0.684

0.05 < P 0.05 < P 0.05 < P 0.01 < P < 0.05 P
significant at 25 and 30°C but not at 15and 20°C. This is reasonable from the fact that the vacuolar frequency of the cold acclimated Paramecia was higher below about 15°C and lower above it than that of the warm acclimated ones.

DISCUSSION

Heat resistance of the Paramecium caudatum of three clones changed in the parallel direction with acclimation temperature (Fig. 1) as found in other previous investigations (Wichterman, 1953 ; Poljansky and Sukhanova, 1967). Jollos (1913, 1914, 1921) found the following fact in one clone of Paramecium aurelia subjected to 31°C for some years, that the heat tolerance increase during long time culture at the high temperature lasted in some cases up to six months after removal from the high temperature, but the tolerance finally disappeared. According to Poljansky (1957) change in thermoresistance of Paramecium caudatum proceeded very quickly to the maximum, sometimes within several hours after transfer to a warm environment. In the present experiment, the rate of change in the heat resistance was not as high as Poljansky described, but the heat resistance gained did not last much longer than Puramecium aurelia cited above. Both gain and loss of the heat resistance reached the maximum in about a week after transfer from one to another temperature (Fig. 2). It has been found in a teleost, guppy, that gain of the heat resistance was generally faster than loss ofit (Doudoroff, 1942), while the rate of the gain and loss was dependent on the temperature of the second acclimation as well as on the width of the temperature range between the first and second acclimation (Tsukuda, 1960). In Paramecium also the rate of change in the heat resistance could vary with ambient temperature. Since Paramecium divided once every two days at 10°C and three times a day at 25°C under the conditions in our laboratory. the rate of change in the heat resistance may be independent of the fission rate.

644

HIROK~ Table 2. Linearity

A B C A B C

25

A B C

30

A B C

*Temperature

YOSHIO TAKEUCHI

Difference between acclimation temperature (Levels of sig.) h J

Test of linearity (Levels of sig.)

77-8

20

and

of regression lines of vacuolar frequency of medium concentration and test of differences between acclimation temperature (AT) in Paramecium caudatum

(“C)

15

TSUKUDA

IO 25 10 25 IO 25

P
2.14 2.62 2.12 2.05 2.22 2.59

-0.362 -0.353 -0.380 PO.381 -0.362 -0.388

IO 25 10 25 10 25

0.05 0.05 0.05 0.05 0.05 0.05

< < < < < <

P P P P P P

3.93 3.88 3.73 3.78 3.12 3.55

-0.437 -0.491 -0.501 -0.561 -0.459 -0.527

IO 25 10 2s IO 25

0.05 0.05 0.05 0.05 0.05 0.05

< < < < < <

P P P P P P

4.57 5.20 4.37 5.09 4.79 5.70

-0.585 - 0.605 -0.636 PO.625 PO.565 PO.608

6.1 1 7.01 6.80 7.15 5.91 6.47

-0.784 -0.724 -0.844 -0.839 -0.791 -0.593

IO 25 IO 25 IO 25

0.01 < P < 0.05 0.05 < P P < 0.001 0.05 < P 0.001 < P < 0.01 0.05 i P

0.05 < P

0.05 < P

0.05 < P

0.05 < P

0.05 < P

0.05 < P

0.05 < P

0.05 < P

0.05 < P

0.05 < P

0.05 < P

0.05 < P

P < 0.001

0.05 < P

P < 0.001

0.05 < P

P < 0.001

0.05 < P

P < 0.001

0.05 < P

0.01 < P < 0.05

0.05 < P

P < 0.001

0.001 < P < 0.01

of test.

It has been shown that there were some differences in pulsation rate between the anterior and posterior contractile vacuole (Child, 19 14 ; Unger, 1926 ; Frisch, 1937) and that the anterior vacuole pulsated more rapidly than the posterior one in Paramecium caudatum (Unger, 1926). In the present experiment the pulsation of only the anterior vacuole was observed. The vacuolar frequency increases with rising temperature in some protozoa (Cole, 1925; Gaw, 1936; Kitching, 1954). The vacuolar frequency of the Paramecium caudatum in the present experiment also increased with rising temperature up to a temperature above which the frequency decreased. This conforms to general rate-temperature relation in responses of poikilotherms. The rate-temperature curves of the warm acclimated Paramecia shifted toward higher temperature as compared with the curves of the cold acclimated Paramecia (Fig. 3) as found in many rate functions of thermally acclimated poikilotherms (Tsukuda and Ohsawa, 1959; Tsukuda, 1961; Takamatsu and Tsukuda, 1977). This suggests the adaptive effect of environmental temperatures. However, it seems different from the capacity adaptation in various multicellular poikilotherms at the point that the rate-temperature curves of the warm and cold acclimated Paramecia intersected at relatively lower temperatures. Herfs (1922) showed that the vacuolar frequency of Paramecium caudatum linearly decreased with increasing concentration of sodium chloride from 0 to 0.75% at about 20°C. In the present experiment also, the vacuolar frequency-concentration relation was linear, especially at 20 and 25°C (Table 2). This indicates that the water volume discharged from contractile vacuoles is equal to water inflow. Discharged volume is the

product of vacuolar frequency and volume, while inflow is proportional to permeability and surface area of a cell and concentration gradient between intracellular and extracellular fluids. If the vacuolar volume, surface area and intracellular concentration are constant, a linear relation between the vacuolar frequency and the extracellular concentration suggests that the permeability is constant. Between the cold and the warm acclimated Paramecia there was no significant difference in slopes of regression lines (regression coefficient) in many cases, while there was a significant difference in levels of regression lines (mean frequency) (Table 2). These facts suggest that permeability of the Paramecia is not variable, but intracellular osmotic concentration varies in relation to environmental temperature. Recently, effects of temperature on the behavioral responses have been found by Tawada and Oosawa (1972), Nakaoka and Oosawa (1977) and Kawakubo and Tsuchiya (1981). These are interesting problems for further study on temperature adaptation in Paramecia. Acknowledgements-We are grateful to Prof. W. Ohsawa for helpful suggestion in statistical analyses. We wish also to thank Prof. T. Daikoku for initiating culture method of Paramecia.

REFERENCES

Child C. M. (1914) The axial gradient Biol.

Bull.

mas.

biol.

Lab.,

Woods

in Ciliate infusoria. Hole

26, 36.

Cole W. H. (19251 Pulsation of the contractile vacuole of Paramecium as affected by temperature. J. gen. Physiol. 7, 581-587.

Temperature

adaptation

Doudoroff P. (1942) The resistance and acclimatization of marine fishes to temperature changes. I. Experiments with Girella nigricans (Ayres). Biol. Bull. mas. biol. Lab., Woods Hole 83,2 19-244. Efimoff A. and Efimoff W. (1924) tiber Ausfrieren und Uberkaltung der Protozoen. Arch. Protistenk. 49,433446. Frisch J. A. (1937) The rate of pulsation and the function of the contractile vacuole in Paramecium multimicronuclealum. Arch. Profistenk. 90, 123-161. Quoted in Wichterman R. (1953). Gaw H. Z. (1936) Physiology of the contractile vacuole in Ciliates. I. Effects of osmotic uressure. II. Effects of hydrogen ion concentration. 111. l%ect of temperature. IV. Effect of heavy water. Arch. Proristenk. 81, 185-224. Quoted in Wichterman R. (1953). Her% A. (1922) Die pulsierendk Va&ole der Protozoen ein Schutzorgan gegen Aussiissung. Studien uber Anpassung der Organismen and das Leben im Siisswasser. Arch. Protistenk. 44, 227-260. Jacobs M. H. (1919) Acclimatization as a factor affecting the ;;;$ifrrnal death points of organisms. J. Exp. Zo61.27, Jollos V. (1913) Experimentelle Untersuchungen as Infusorien. Biol Zbl. 33, 222-236. Jollos V. (1914) Variabilitat und Vererbung bei Mikroorganismen. Z. Indukt. Abstamm.-u. Verebl Lehre. 12, 1435. Quoted in Wichterman R. (1953). Jollos V. (1921) Experimantelle Protistenstunden: I. Untersuchungen fiber Variabilitat und Vererbung bei Infusorien. Arch. Protistenk. 43, l-222. Kawakubo T. and Tsuchiya Y. (1981) Diffusion coefficient of Paramecium as a function of temperature. J. Protozool. 28. 342-344. Kitching J. A. (1952) The physiology of contractile vacuoles. IX. Effects of sudden changes in temperature on the contractile vacuole of a sucto;an ; with a discussion of the mechanism of contraction. J. exp. Biol. 31, 68-75. Mendelssohn M. (1902) Recherches sur la thermotaxie des organismes unicellulaires. J. Physiol. Path. gin. 4, 393409. Quoted in Wichtennan R. (19531. Nakaoka Y. gnd Oosawa F. (1977) Temperiture-sensitive behavior of Paramecium caudatum. J. Protozool. 24, 575580.

in Paramecium

645

Poljansky G. I. (1957) Temperature adaptation in infusoria. 1. Relation of heat-resistance of Paramecium caudatum to the temperature conditions of its existence. J. Zool. USSR 36, 163&1646. Quoted in Poljansky G. and Sukhanova K. M. (1967). Poljansky G. 1. (1959) Temperature adaptation in infusoria. II. The changes of Paramecium caudatum thermo- and coldstability during cultivation at low temperatures. Cytology, USSR 1, 714727. Quoted in Polianskv _ _ G. and Sukhanova K. M. (1967). Poljansky G. 1. and Sukhanova K. M. (1967) Some peculiarities in temperature adaptations of protozoa as compared to multicellular poikilotherms. In The Cell and Environmental Temperature (Edited by Troshin A. S.), pp. 200-208. Pergamon Press, Oxford. Precht H., Christophersen J. and Hensel H. (1955) Temperatur und Leben, p. 206. Springer, Berlin. Takamatsu K. and Tsukuda H. (1977) Effects of acclimation temperature on the temperature dependence of gliding rate in the planarian, Dugesia japonica. Annome. zool. japon. 50, 63-69. Tawada K. and Oosawa F. (1972) Responses of Paramecium to temperature change. J. Protozool. 19, 53-57. Tsukuda H. (1960) Temperature adaptation of fishes. IV. Change in the heat and cold tolerances of the guppy in the process of temperature acclimatization. J. Inst. Polytech. Osaka City Univ. D 11,43-55. Tsukuda H. (1961) Temperature acclimatization on different organization levels in fishes. J. Biol. Osaka City Univ. 12, 1545. Tsukuda H. and Ohsawa W. (1959) Temperature dependence and acclimatization of the rate of heart beat of a red snail, Physa sp. in relation to size. J. Inst. Poltech. Osaka City Univ. D 10, 105-l 13. Unger W. B. (1926) The relationship of rhythms to nutrition and excretion in Paramecium. J. exp. Zoo/. 43, 373-412. Wichterman R. (1953) The Biology of Paramecium, pp. 127-128, 191, 203-205, 321. Blakiston, New York. Wolfson C. (1935) Observations on Paramecium during exposure to sub-zero temperatures. Ecology 16, 63& 639.