BEHAVIORAL BIOLOGY, 14,499-504 (1975), Abstract No. 5128
BRIEF REPORT Thermotaxis in a Slime Mold, Physarum polycephalum I
W I ~ G - W A I TSO and TAG E. MANSOUR 2
Department of Pharmacology, Stanford University Medical School, Stanford, California 94305 Physarum polycephalum is thermotactic toward 29 ± I°C avoiding both higher and lower temperatures. 29°C appears to be a-combined optima for growth and locomotion. It is likely that thermotaxis is a more efficient way of avoiding unfavorable temperature than transforming into spherules.
The plasmodium of Physarum polycephalum, an acellular slime mold, migrates by a net transfer of the protoplasmic mass through shuttle streaming (Seifriz, 1943; Kamiya, 1950). Its migration can be directed by an external stimulus such as a chemical gradient (in chemotaxis: Coman, 1940; Carlile, 1970) or an electric field (in galvanotaxis: Anderson, 1951). Carlile found that several carbohydrates were attractants and that their chemotactic properties paralleled their ability to support growth (Carlile, 1970). Anderson pointed out that in galvanotaxis the effect of the electric current was that of inhibition of migration toward the anode rather than stimulation toward the cathode (Anderson, 1951). We report here the thermotactic (directed movement in response to a temperature gradient) behaviour of the organism. The plasmodium of Physarum polycephalum Mac was grown by spreading a suspension o f microplasmodia onto sterile 13-mm Millipore filters laid on the surface of agar supplemented with semidefined growth medium with hemin and citrate (Daniel and Baldwin~ 1964). The filters, when covered with freshly grown plasmodium at log phase, were lifted and excess nutrients were washed off by floating the filters on distilled water and drying them on blotting paper repeatedly for three times. Each specimen on a 13-ram filter was counted as an individual in the thermotaxis experiment. Washed filters of plasmodium were placed on the wet surface of the filter support in the observation dish atop the bisecting line (Fig. 1), thus producing a temperature gradient across the organism. 1This investigation was supported by U.S. Public Health Service MH 23464. We gratefully acknowledge the receipt of the original spherule samples of Physarurn polycephalum from Professor Harold Rusch of the University of Wisconsin, Madison. 2To whom correspondence should be addressed. 499 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
500
TSO AND MANSOUR
\
Fig. 1. Schematic drawing of the appartus for thermotaxis study. It consists of a plastic Petri dish cemented on top of another one having a midiine partition. The lower sealed dish has inlets for circulating water into each of the two separated chambers which serve as water jackets for the observation dish above. Water at controlled temperatures was circulated into each of these chambers, thus generating a temperature gradient of various steepness across the midline when different temperatures were chosen. At 22°C room temperature, operating at temperature pairs between 15 and 40°C (range within which protoplasm streams) with water driven by peristaltic pumps at 150 ml/min, the temperature of the exit water measured by a thermometer is less than I°C different from the incoming water. In determining optimal temperature, a narrow temperature pair was chosen which raised the accuracy to within +_ I°C. In the observation dish, a wet Millipore filter was laid on top of another sheet of wet Whatman filter to provide moisture and support for the plasmodium. Individuals tested responded to the temperature gradient by moving away from 19°C and toward 29°C (Fig. 2a-d). This migration was due neither to growth nor to a following-up event of a previous physiological programmed migration pattern such as the directional growth of the advancing edge of the plasmodium. The thermotactic behavior was studied in the absence of growth medium and completed in 2 hr, about one-sixth of the doubling time (12 hr) of the organism in nutrient medium. The plasmodium, when grown on solid support, does grow by expansion and filters containing plasmodium lifted from an advancing edge may contain a predetermined migration pattern. But we found that rotating the filter of plasmodium in any direction has no effect on the thermotactic response. Filters supporting plasmodium having no directional bias have been obtained by lifting covered filters and placing new filters in the clearing zone to allow the plasmodium to migrate in from all directions. The thermotactic response of these organisms were equal to those which were directionally biased by growth.
B
C
D
E
Fig. 2. Migration of four individual plasmodia in a temperature gradient, a,b,c,d are photographs at 0,45,60, and 90 min after a gradient of 10°C is applied. The left side is 19°C and the right side is 29°C. In e, the migration of the organism between two extreme temperatures at the end of 2 hr (22°C on the left and 37°C on the right; 29°C is the preferred temperature, see text and Fig. 3).
A
o
© ,-]
~]
502
TSO AND MANSOUR
There is a lag (approx 30 rain) in the behavioral response to the stimulus which is probably due to one or both of the following reasons: (a) injury and/or (b)shock (anaesthetic) reaction (Seifriz and Epstein, 1941) when the specimen was transferred to the observation dish. The possibility that the organism requires a latent period to express the response when started from resting condition seems unlikely since a small intact plasmodium atop a larger filter support exhibits no lag in thermotaxis. When the temperature gradient is reversed, the organism reverses by flowing out from the once posterior edge without making a turn around. This further supports the idea that thermotaxis is independent of previous migration pattern. The manner in which the reversion is made is identical to that observed in a chemotactic response (Coman, 1940). Any part of the organism can be the migrating front. Unlike galvanotaxis (Anderson, 1951), the thermotactic movement is not due to a unilateral inhibition but to an actual directed movement in response to the temperature gradient. This is demonstrated in Fig. 2d, by the gradual flowing out of the plasmodium from the 19°C (cold side) and is not simply a cessation of movement on that side. Not only did it migrate away from the cold side, but also from the hot side (Fig. 3, Expts 1, 3, 6, 10). When both temperatures are at extremes, the individuals tend to move laterally and prefer to remain in the intermediate temperature zone (Fig. 2e) suggesting that there is an optimal temperature in which the organism prefers to stay. When the temperature difference is small, the individuals ignore the gradient, resulting in having 50% of the individuals tested go either direction (Fig. 3, Expts 5, 7, 9). By choosing different temperature pairs, the sensitivity of the organism detected by this method is 3°C in the 15-35°C temperature range (Fig. 3, Expts 4-9). The preferred temperature was determined to be 29 + I°C (Fig. 3, Expts 8, 10-12). Will the magnitude of the cold stimulus be equal to that of the hot stimulus? We chose temperatures on either side of 29°C and found 8 degrees in the hot side balanced 4 degrees in the cold side, thus confining the organism to lateral migration. (Fig. 2e) Light inhibits growth and promotes sporulation in Physarum polycephalum (Gray, 1938). However, in the dark or in fluroescent light, the thermotactic behavior is unaffected by light (Fig. 3, Expt 2). Starved as well as nourished organisms exhibit equal thermotaxis (Fig. 3, Expt 2), indicating that the detection mechanism of the thermal stimulus does not link to the metabolic state. Temperature adaptation and thermoregulation are known to occur in higher organisms (Wessels, 1968). However, P. polycephalum grown at 15, 20, 22, 25, and 30°C showed the same temperature preference. Apparently, this behavior is independent of the growth history of the organism. We have not looked into the possibility of temperature regulation in P. polycephalum.
THERMOTAXIS TERMINAL TEMPERATURE (°C)
EXPT.
1 2•
503
4/4
0/4
0/4
,el
-"- 4/4
0/4
0/4
4/4
4/4
2/4 ''=r-'4=~ 2/4 10
4/4 •
11
2/4~2/4
12
0/4
2/4"=z"--'-'-I~ 2/4
Fig. 3. Direction of plasmodium movement studied at different temperature pairs and different conditions. The two ends of each bar indicate the pair of temperature chosen. The ratio indicates the fraction of individuals migrating in the designated direction scored at the end of 2-hr testing period. (-~-~)Means approaching random and undirected migration. (, ) Means migration as directed by the arrow. ( ~ff ) Means away from two extreme temperatures and migrate laterally as in Fig. 2e. *Identical results were observed from four luminated and four starved samples.
Reports from previous investigators have indicated that the growth rate and protoplasmic streaming rate of the plasmodium are influenced by temperature (Daniel and Baldwin, 1964; Kamiya, 1953; Lomagin, Berstam, and Zheleznjak, 1972). Kamiya observed that the motility of the organism as monitored by rate of streaming is faster at higher temperature, studied up to 25°C (Kamiya, 1953). Recently, Lomagin et al. (1972) reported on the adverse effect of high temperature to P. polycephalum (Lomagin, Bernstam, and Zheleznjak, 1972). The plasmodium stopped protoplasmic streaming and formed sphemles above 38°C. We have confirmed the results by allowing plasmodial inocula to migrate at different temperatures and measured the rate of the area covered by the spreading plasmodium. We found the motility reached a maximum at 30°C and then decreased rapidly until it stopped at 35°C. Daniel and Baldwin (1964) compared the doubling time of the organism at 21.5, 24.0, and 27.5 °, and found that it is shortest at 27.5°C. We have extended this study over a temperature range of 15-37°C and found that it grows fastest in the temperature range 25-300C. Probably a temperature of 29+ I°C is the combined optimum for various physiological processes. Encystment and sporulation are mechanisms for the organism to survive
504
TSO AND MANSOUR
adverse conditions. A p p a r e n t l y t h e r m o t a x i s is a m o r e sensitive and i m m e d i a t e process enabling P. polycephalum to avoid unfavorable t e m p e r a t u r e s .
REFERENCES Anderson, J. D. (1951). Galvanotaxis of slime mold. J. Gen. Physiol. 35, 1-16. Carlile, M. J. (1970). Nutrition and chemotaxis in the Myxomycete Physarum polycephalum: the effect of carbohydrates on the plasmodium. J. Gen. Microbiol. 63, 221-226. Coman, D. R. (1940). Additional observations on positive and negative chemotaxis. Arch. Pathol. 29, 220-228. Daniel, J. W. and Baldwin, W. W. (1964). Methods of culture for plasmodial Myxomyceres. In D. M. Prescott (Ed.), "Methods in Cell Physiology," Vol. I, pp. 9-41. New York: Academic Press, Inc. Gray, W. D. (1938). The effect of light on the fruiting of Myxomycetes. Amer. J. Bot. 25, 511-522. Kamiya, N. (1950). The protoplasmic Flow in the Myxomycete plasmodium as revealed by a volumetric analysis. Protoplasma 39, 344-356. Kamiya, N. (1953). The motive force responsible for protoplasmic streaming in the Myxomycete plasmodium. Ann. Repts. Sci. Works Osaka Univ. 1,53-83. Lomagin, A. G., Bernstam, V. A. and Zheleznjak, L. I. (1972). The influence of nutrition on the thermoresistance of Physarum polycephalum. Tsitologia (USSR) 14, 463-471. Seifriz, W. (1943). Protoplasmic streaming. Bot. Rev. 9, 49-123. Seifriz, W. and N. Epstein. (1941). Shock anesthesia in Myxomycetes. Biodynamica 67, 191-197. Wessels, N. K. (Ed.) (1968). "Vertebrate Adaptations," Chap. 50 San Francisco: Freeman & Co.
BRIEF REPORT Thermotaxis in a Slime Mold, Physarum polycephalum I
W I ~ G - W A I TSO and TAG E. MANSOUR 2
Department of Pharmacology, Stanford University Medical School, Stanford, California 94305 Physarum polycephalum is thermotactic toward 29 ± I°C avoiding both higher and lower temperatures. 29°C appears to be a-combined optima for growth and locomotion. It is likely that thermotaxis is a more efficient way of avoiding unfavorable temperature than transforming into spherules.
The plasmodium of Physarum polycephalum, an acellular slime mold, migrates by a net transfer of the protoplasmic mass through shuttle streaming (Seifriz, 1943; Kamiya, 1950). Its migration can be directed by an external stimulus such as a chemical gradient (in chemotaxis: Coman, 1940; Carlile, 1970) or an electric field (in galvanotaxis: Anderson, 1951). Carlile found that several carbohydrates were attractants and that their chemotactic properties paralleled their ability to support growth (Carlile, 1970). Anderson pointed out that in galvanotaxis the effect of the electric current was that of inhibition of migration toward the anode rather than stimulation toward the cathode (Anderson, 1951). We report here the thermotactic (directed movement in response to a temperature gradient) behaviour of the organism. The plasmodium of Physarum polycephalum Mac was grown by spreading a suspension o f microplasmodia onto sterile 13-mm Millipore filters laid on the surface of agar supplemented with semidefined growth medium with hemin and citrate (Daniel and Baldwin~ 1964). The filters, when covered with freshly grown plasmodium at log phase, were lifted and excess nutrients were washed off by floating the filters on distilled water and drying them on blotting paper repeatedly for three times. Each specimen on a 13-ram filter was counted as an individual in the thermotaxis experiment. Washed filters of plasmodium were placed on the wet surface of the filter support in the observation dish atop the bisecting line (Fig. 1), thus producing a temperature gradient across the organism. 1This investigation was supported by U.S. Public Health Service MH 23464. We gratefully acknowledge the receipt of the original spherule samples of Physarurn polycephalum from Professor Harold Rusch of the University of Wisconsin, Madison. 2To whom correspondence should be addressed. 499 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
500
TSO AND MANSOUR
\
Fig. 1. Schematic drawing of the appartus for thermotaxis study. It consists of a plastic Petri dish cemented on top of another one having a midiine partition. The lower sealed dish has inlets for circulating water into each of the two separated chambers which serve as water jackets for the observation dish above. Water at controlled temperatures was circulated into each of these chambers, thus generating a temperature gradient of various steepness across the midline when different temperatures were chosen. At 22°C room temperature, operating at temperature pairs between 15 and 40°C (range within which protoplasm streams) with water driven by peristaltic pumps at 150 ml/min, the temperature of the exit water measured by a thermometer is less than I°C different from the incoming water. In determining optimal temperature, a narrow temperature pair was chosen which raised the accuracy to within +_ I°C. In the observation dish, a wet Millipore filter was laid on top of another sheet of wet Whatman filter to provide moisture and support for the plasmodium. Individuals tested responded to the temperature gradient by moving away from 19°C and toward 29°C (Fig. 2a-d). This migration was due neither to growth nor to a following-up event of a previous physiological programmed migration pattern such as the directional growth of the advancing edge of the plasmodium. The thermotactic behavior was studied in the absence of growth medium and completed in 2 hr, about one-sixth of the doubling time (12 hr) of the organism in nutrient medium. The plasmodium, when grown on solid support, does grow by expansion and filters containing plasmodium lifted from an advancing edge may contain a predetermined migration pattern. But we found that rotating the filter of plasmodium in any direction has no effect on the thermotactic response. Filters supporting plasmodium having no directional bias have been obtained by lifting covered filters and placing new filters in the clearing zone to allow the plasmodium to migrate in from all directions. The thermotactic response of these organisms were equal to those which were directionally biased by growth.
B
C
D
E
Fig. 2. Migration of four individual plasmodia in a temperature gradient, a,b,c,d are photographs at 0,45,60, and 90 min after a gradient of 10°C is applied. The left side is 19°C and the right side is 29°C. In e, the migration of the organism between two extreme temperatures at the end of 2 hr (22°C on the left and 37°C on the right; 29°C is the preferred temperature, see text and Fig. 3).
A
o
© ,-]
~]
502
TSO AND MANSOUR
There is a lag (approx 30 rain) in the behavioral response to the stimulus which is probably due to one or both of the following reasons: (a) injury and/or (b)shock (anaesthetic) reaction (Seifriz and Epstein, 1941) when the specimen was transferred to the observation dish. The possibility that the organism requires a latent period to express the response when started from resting condition seems unlikely since a small intact plasmodium atop a larger filter support exhibits no lag in thermotaxis. When the temperature gradient is reversed, the organism reverses by flowing out from the once posterior edge without making a turn around. This further supports the idea that thermotaxis is independent of previous migration pattern. The manner in which the reversion is made is identical to that observed in a chemotactic response (Coman, 1940). Any part of the organism can be the migrating front. Unlike galvanotaxis (Anderson, 1951), the thermotactic movement is not due to a unilateral inhibition but to an actual directed movement in response to the temperature gradient. This is demonstrated in Fig. 2d, by the gradual flowing out of the plasmodium from the 19°C (cold side) and is not simply a cessation of movement on that side. Not only did it migrate away from the cold side, but also from the hot side (Fig. 3, Expts 1, 3, 6, 10). When both temperatures are at extremes, the individuals tend to move laterally and prefer to remain in the intermediate temperature zone (Fig. 2e) suggesting that there is an optimal temperature in which the organism prefers to stay. When the temperature difference is small, the individuals ignore the gradient, resulting in having 50% of the individuals tested go either direction (Fig. 3, Expts 5, 7, 9). By choosing different temperature pairs, the sensitivity of the organism detected by this method is 3°C in the 15-35°C temperature range (Fig. 3, Expts 4-9). The preferred temperature was determined to be 29 + I°C (Fig. 3, Expts 8, 10-12). Will the magnitude of the cold stimulus be equal to that of the hot stimulus? We chose temperatures on either side of 29°C and found 8 degrees in the hot side balanced 4 degrees in the cold side, thus confining the organism to lateral migration. (Fig. 2e) Light inhibits growth and promotes sporulation in Physarum polycephalum (Gray, 1938). However, in the dark or in fluroescent light, the thermotactic behavior is unaffected by light (Fig. 3, Expt 2). Starved as well as nourished organisms exhibit equal thermotaxis (Fig. 3, Expt 2), indicating that the detection mechanism of the thermal stimulus does not link to the metabolic state. Temperature adaptation and thermoregulation are known to occur in higher organisms (Wessels, 1968). However, P. polycephalum grown at 15, 20, 22, 25, and 30°C showed the same temperature preference. Apparently, this behavior is independent of the growth history of the organism. We have not looked into the possibility of temperature regulation in P. polycephalum.
THERMOTAXIS TERMINAL TEMPERATURE (°C)
EXPT.
1 2•
503
4/4
0/4
0/4
,el
-"- 4/4
0/4
0/4
4/4
4/4
2/4 ''=r-'4=~ 2/4 10
4/4 •
11
2/4~2/4
12
0/4
2/4"=z"--'-'-I~ 2/4
Fig. 3. Direction of plasmodium movement studied at different temperature pairs and different conditions. The two ends of each bar indicate the pair of temperature chosen. The ratio indicates the fraction of individuals migrating in the designated direction scored at the end of 2-hr testing period. (-~-~)Means approaching random and undirected migration. (, ) Means migration as directed by the arrow. ( ~ff ) Means away from two extreme temperatures and migrate laterally as in Fig. 2e. *Identical results were observed from four luminated and four starved samples.
Reports from previous investigators have indicated that the growth rate and protoplasmic streaming rate of the plasmodium are influenced by temperature (Daniel and Baldwin, 1964; Kamiya, 1953; Lomagin, Berstam, and Zheleznjak, 1972). Kamiya observed that the motility of the organism as monitored by rate of streaming is faster at higher temperature, studied up to 25°C (Kamiya, 1953). Recently, Lomagin et al. (1972) reported on the adverse effect of high temperature to P. polycephalum (Lomagin, Bernstam, and Zheleznjak, 1972). The plasmodium stopped protoplasmic streaming and formed sphemles above 38°C. We have confirmed the results by allowing plasmodial inocula to migrate at different temperatures and measured the rate of the area covered by the spreading plasmodium. We found the motility reached a maximum at 30°C and then decreased rapidly until it stopped at 35°C. Daniel and Baldwin (1964) compared the doubling time of the organism at 21.5, 24.0, and 27.5 °, and found that it is shortest at 27.5°C. We have extended this study over a temperature range of 15-37°C and found that it grows fastest in the temperature range 25-300C. Probably a temperature of 29+ I°C is the combined optimum for various physiological processes. Encystment and sporulation are mechanisms for the organism to survive
504
TSO AND MANSOUR
adverse conditions. A p p a r e n t l y t h e r m o t a x i s is a m o r e sensitive and i m m e d i a t e process enabling P. polycephalum to avoid unfavorable t e m p e r a t u r e s .
REFERENCES Anderson, J. D. (1951). Galvanotaxis of slime mold. J. Gen. Physiol. 35, 1-16. Carlile, M. J. (1970). Nutrition and chemotaxis in the Myxomycete Physarum polycephalum: the effect of carbohydrates on the plasmodium. J. Gen. Microbiol. 63, 221-226. Coman, D. R. (1940). Additional observations on positive and negative chemotaxis. Arch. Pathol. 29, 220-228. Daniel, J. W. and Baldwin, W. W. (1964). Methods of culture for plasmodial Myxomyceres. In D. M. Prescott (Ed.), "Methods in Cell Physiology," Vol. I, pp. 9-41. New York: Academic Press, Inc. Gray, W. D. (1938). The effect of light on the fruiting of Myxomycetes. Amer. J. Bot. 25, 511-522. Kamiya, N. (1950). The protoplasmic Flow in the Myxomycete plasmodium as revealed by a volumetric analysis. Protoplasma 39, 344-356. Kamiya, N. (1953). The motive force responsible for protoplasmic streaming in the Myxomycete plasmodium. Ann. Repts. Sci. Works Osaka Univ. 1,53-83. Lomagin, A. G., Bernstam, V. A. and Zheleznjak, L. I. (1972). The influence of nutrition on the thermoresistance of Physarum polycephalum. Tsitologia (USSR) 14, 463-471. Seifriz, W. (1943). Protoplasmic streaming. Bot. Rev. 9, 49-123. Seifriz, W. and N. Epstein. (1941). Shock anesthesia in Myxomycetes. Biodynamica 67, 191-197. Wessels, N. K. (Ed.) (1968). "Vertebrate Adaptations," Chap. 50 San Francisco: Freeman & Co.
