ON THE PHYSIOLOGY OF AMOEBOID MOVEMENT.* II.—THE EFFECT OF TEMPERATURE. BY C. F. A. PANTIN. (Assistant Physiologist at the Marine Biological Laboratory, Plymouth.) IT was shown in the first paper of this series1S that amoeboid activity was affected by certain changes in the conditions of the medium in the same way as certain other forms of con- tractility. This suggested that some fundamental mechanism of contractility was similar in all these cases. Like the majority of biological processes, contractility is affected in a characteristic manner by temperature, and if there really is a fundamental similarity between amoeboid and other forms of contractility, the effect of temperature should be similar in both cases. i. Material, Methods, etc. Marine Amoebae, obtained from the laboratory tanks, were used for the experiments. A full description of the Amoebae, their habitat and mode of progression, has been given in a previous paper.16 The Amoebae were of the " Umax " form, that is, they progressed by the continuous protrusion of a single anterior pseudopodium. Two species were used, one relatively "fluid" (type A), and one with relatively "solid," consistent, protoplasm (type B). In the absence of external stimuli the Amoebae tend to move in a straight line. And if the conditions of the medium are constant the velocity of an individual Amoeba is constant to within from i per cent, to 5 per cent, for at least twenty-four hours. This holds true even if the conditions of the medium have been varied and then brought back to the original state, provided the variation has not been great enough to damage the organism. • Received May 21st, 1924. C. F. A. Pantin In these, as in the previous experiments, the velocity of progression was used as a measure of amoeboid activity. A "Ghost-micrometer"4 was placed at such a distance from the microscope that the lines appeared to be 25 M apart, and the velocity was measured by timing the Amoeba over a given number of divisions with a stop-watch. Harrington and Learning7 have stated that the conditions of lighting affect certain Amoebae. A half-watt frosted electric lamp was therefore used as illuminant in all the experiments. The temperature was controlled by means of a specially constructed warm stage. A cell containing the Amoeba was H FIG. 1.—Diagram of Warm Stage. The Amoeba is put in the cell A, the top and bottom of which are sealed with thin glass coverslips. The cell contains about 3 cc of sea water. Water at a constant temperature flows through the inner chamber B at a slow rate. The temperature is registered by a thermometer F. The lid, D, is sealed on to the inner chamber with paraffin wax, P. The inner chamber is protected by an outer chamber, G. The lid, the inner and the outer chambers are all provided with glass windows (E, C, and H). K represents the microscope objective (i inch). completely immersed in a chamber through which flowed a stream of water at the desired temperature. A thermometer entering the chamber registered the temperature to TV C. Details of the construction of the warm stage are given in fig. i. Amoeboid activity is influenced by factors other than temperature {e.g. PH): the medium employed was always natural sea water at a constant PH which varied in different experiments from PH 7.8 to PH 8.2. 2. The Relation of Velocity to Temperature. The usual procedure adopted was to determine the velocity at about 15° C, and then to lower the temperature by successive intervals to the critical point at which activity was inhibited. 520 The Physiology of Amoeboid Movement The temperature was then raised by intervals until movement ceased owing to the destructive effect of the high temperature upon the protoplasm. The velocity was measured five to ten minutes after each change of temperature to allow the Amoeba to become acclimatised to the new conditions. However, the velocity usually became constant in about one minute, unless the temperature was close to either critical point at which all movement ceased. Since the warm stage itself takes from a half to one minute to attain a constant temperature after each change, acclimatisation is probably rapid except near the critical temperature. The velocity of both kinds of Amoebae studied varies markedly with the temperature. Fig. 2 shows a typical curve obtained from a type B Amoeba. The experiment was commenced at I5.2°C. ; the temperature was then lowered successively (through points 2 to 6) to — 2.00 C, and then raised. From just above o°C. to i5°C. the velocity is approximately proportional to the centigrade temperature. For this Amoeba there is a distinct "lag" in the velocity obtained with a rising temperature; that is, the velocity at 10° is higher if approached from a high temperature than if approached from a low one. This "lag" was not always observed in other Amoebae. The rate of increase of velocity falls rapidly above 17.50 C, so that the velocity itself reaches a maximum at about 200 C. After this the velocity falls rapidly to zero at 260 C, though death does not occur rapidly (within ten minutes) until 30° C. These points are well shown in fig. 3, which shows the mean curve obtained from experiments on ten Amoebae. For each Amoeba the velocities measured were reduced proportion- ally to the same scale so that the value at io°C. was approxi- mately 1.00. The mean velocity was then taken for each 2.5° rise in temperature. Like the majority of biological processes, amoeboid activity is inhibited a few degrees below o° C. This inhibition is com- pletely reversible: even if the Amoeba has been kept for some time at - 30 C, yet on raising the temperature, recovery takes place within a few minutes and the Amoeba moves with the 5" C. F. A. Pantin speed characteristic of the temperature, except in cases which show the slight "lag" effect already described. Again, as in other processes, the activity rises with the temperature to an optimum, the position of which shows slight a 0 20 30C FlG. 2.—Variation of velocity with temperature for a single type B Amoeba. The numbers and arrows indicate the order in which the values were determined. individual variation, and above this optimum activity falls rapidly to zero. The fall in velocity above the optimum (figs. 2, 3, and 4) may be partly due to failure of the method. Above the 522 The Physiology of Amoeboid Movement optimum the Amoeba moves rather irregularly and tends to form lateral pseudopodia : with this loss of the limax form, the velocity is no longer so accurate a measure of the activity of the Amoeba. However, the optimum for amoeboid activity is of the same 20° 30°C. FlG. 3.—Mean variation of velocity with temperature for 10 type B Amoebae. Velocity reduced to scale (velocity at IO° = IJD). A, B, and C are extrapolated values referred to in the text character as the optima of other processes ; if the temperature be raised above it until activity is inhibited we find that the inhibition is only partially reversible, as in other processes {e.g. ciliary activity6). Type B Amoebae were found to require several hours to recover after inhibition at a temperature above the optimum, and type A Amoebae rarely, if ever, recovered after complete inhibition by heat. 5*3 C. F. A. Pantin The low temperature of the optimum is of interest; it is always about 20° C. for type B, and 22° to 2 5°C. for type A. This low optimum does not appear to be general for all cases of amoeboid activity, because McCutcheon M has shown that the optimum for human leucocytes occurs at 40° C, as might be expected. Moreover, it seems probable that many freshwater Amoebae can withstand temperatures actually above the death- point of these marine Amoebae. 3. The Fall in Velocity above the Optimum. The occurrence of an optimum activity suggests that above this point destructive forces begin to act on the mechanism. Gray6 has suggested that the fall in ciliary activity above the optimum temperature might be due to the destruction of an enzyme: the argument applies also to amoeboid activity, although in this case the optimum is very low. The following experiments show definitely that the fall in amoeboid activity is due to the destruction of some mechanism in the protoplasm. The velocity of a type B Amoeba was determined at 10° C.; the temperature was then suddenly raised and maintained at a higher value for ten minutes ; at the end of this time the temperature was suddenly lowered again to io° C, and the velocity of the Amoeba measured at intervals. At first the velocity was below its initial value at io° C, but recovery gradually took place. The times for recovery for different temperatures are given in Table I. The values were obtained successively with the same Amoeba. TABLE 1. Initial Velocity measured at 10° C. Temperature changed Time required for to T° C. for ten minutes. recovery at 10° C. T = 15-3° C. 0 minutes 17-6 3-4 ,. [optimum) 20-0 20 „ 21-9 70 „ 20-5 20 „ 21-3 32 22'5 about 240 „ These experiments definitely show that high temperatures tend to destroy something necessary for amoeboid activity, and that 524 The Physiology of Amoeboid Movement the amount of destruction increases very rapidly with the temperature above the optimum. They also show that a limited amount of destruction has taken place before ever the optimum is reached.