On the Physiology of Amoeboid Movement.*

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 contractility. 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 proportionally to the same scale so that the value at io°C. was approximately 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 completely 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

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. In the experiment quoted the Amoeba required three to four minutes to recover the normal velocity after the temperature had been raised to 17.6° C, that is 2.50 below the optimum. One of the factors upon which amoeboid activity depends is therefore some substance or structural arrangement of substances which is rapidly destroyed near and above the optimum temperature. If this substance is partially destroyed or is present in less than the normal amount, the velocity of the Amoeba is proportionately lower, though otherwise the behaviour is normal. This is shown in fig. 2. In that experiment, immediately after the Amoeba had been paralysed by being heated to 26.30 C, the temperature was lowered to I5.8°C, and the Amoeba allowed to recover. Thirty minutes later it had only partially recovered and the velocity was proportionately below the normal for that temperature (point 16). On now raising the temperature the relative increase in velocity was about the same as in the normal Amoeba (point 17), but the velocity was still below its normal value for this temperature. It might be said that the Amoeba was following the normal temperature curve on a reduced scale, the reduction being proportional to the amount of the substance or structure that had yet to be re-formed by recovery.

4. Extrapolation of the Velocity: Temperature Curve. It has been shown that the fall in velocity above the optimum is due simply to a destructive effect. In the absence of this effect the velocity should continue to rise above the actual optimum in continuation of the lower, normal part of the velocity : temperature curve. If allowance could be made for the destructive effect we could thus extrapolate the normal part of the velocity : temperature curve. This would be of great value because the limited range over which the normal curve extends makes it difficult to compare with the temperature curves of other physiological processes. VOL. 1.—NO. 4. 525 a M C. F. A. Pantin The destructive effect may be allowed for approximately in the following way. Assume that an Amoeba moves with a velocity, V10, at 10° C. Let the temperature be raised above the optimum to T° C. for a certain time. If there were no destructive effect the velocity at T° would be VT, the velocity having continued to rise with the temperature as in the normal part of the curve. But, owing to the destructive effect acting during the time that the temperature is T°, the observed velocity at the end of the time will be V1,., which is less than

VT. The ratio ryr will be related to the amount of destruction. v T Let the temperature be now suddenly lowered again to io° C. The amount of the destruction is unchanged so that before recovery the velocity will be V\o, which is less than V10, such that fTT-is also related to the amount of destruction. We have v 10 seen (fig. 2, points 16 and 17) that the velocity of a partially recovered Amoeba varies with the temperature as it does in the normal Amoeba, only on a proportionately lower scale. Hence both -^ and —• are related to the same amount of V T V 10 destruction in the same way.

V1 VT = ^ji x V10 (all three of which can be observed). v 10 This equation should allow us to extrapolate the normal part of the velocity: temperature curve. Unfortunately certain experimental difficulties render VT difficult to estimate with accuracy. The velocity V10 should be measured immediately the temperature is reduced to io°. But it is found that the velocity does not reach its minimum value until a few minutes after the temperature has fallen. This effect, though perhaps partly instrumental, is probably due to the relatively slow rate at which certain factors {e.g. viscosity) fall to the normal value for the temperature. However, the time required to reach the minimum is small compared with the total period of recovery, and if V1^ be taken to be the 5*6 The Physiology of Amoeboid Movement minimum velocity after cooling, the equation will still hold approximately. In order to find the mean value of VT for several experiments, the velocities should all be reduced proportionally so that the velocity at io° C, (Vi0), is i.o. The relative value of VT is then given:—

Relative VT = ^L i.o. * 10

Table II. gives the mean value of VT for type B Amoebae V1 from six determinations of ^— at three different temperatures. V

TABLE I \.~Type B Amoebae.

Temperature raised Range of Variation to T° for ten minutes. Mean VT = ¥£-. * 10 v 10

T = 17-9 "C. 176 1-32 to 2-08 20-1 2-OI i-55 » 2-95 21-6 2-7 i-5 .. 3-2

The range of variation is very great, so that the values cannot be considered to be very accurate ; but the results when plotted (fig 3, points A, B, C) do indicate that, were it not for the destructive effect of the high temperature, the normal rise of velocity with temperature would continue.

5. Experiments with Type A Amoebae. The relation of velocity to temperature was determined for type A Amoebae. Fig. 4 shows a mean curve obtained from five different Amoebae. This Amoeba moves much more irregularly than type B, especially at temperatures above 180 C, when fresh pseudopodia are continually thrown out. The curve is therefore not of the same order of accuracy as that of type B, but it shows that temperature affects these two very different Amoebae in a similar manner. The range of activity of type A Amoebae extends both above and below the range of type B. S27 C. F. A. Pantin

d The Velocity at Normal Temperatures. Fig. 3 shows that the velocity of a type B Amoeba is almost a linear function of the centigrade temperature. This recalls the observation of Knowlton and Starling10 that the

3-0

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2-0

20 30°C FIG. 4.—The relation to temperature of ciliary activity (from Gray), and of velocity of type A and type B Amoebae. All three curves reduced to scale (velocity at io°= i-o). variation of the rate of beat with temperature in the mammalian heart is almost linear below the optimum. In the case of the heart, Clark8 has since shown that the variation of rate increases more rapidly with the temperature than a linear function. This is also true of amoeboid movement; the velocity increases rather more rapidly for each successive temperature interval. 5*8 The Physiology of Amoeboid Movement The apparent straightness of the curve is probably only due to the short range of temperature over which it extends. The variation with temperature is really closely similar to that of many other biological processes. Fig. 4 shows the mean curves for type A and type B Amoebae plotted on the same scale as the curve obtained by Gray* for the mechanical activity of cilia. The similarity is brought out more strongly by Table III., which gives the mean observed velocities for type B Amoebae and the temperature coefficients calculated from them by interpolation : except near o°C. the temperature coefficients agree well with those for ciliary activity.

TABLE III.—Type B Amoebae.

Velocities reduced proportionally to the Value i-oo at 10° C.

Mean Temp. Mean Vd. Mean Temp. Mean VeL Interpolated Temperature Coefficients.

-3-i° C. 0-00 15-1° C. 1-48 Temp. Amoeba Cilia t -i-9 O-OI 17-4 1-68 Qio. Qio. o-o 013 20-2 177 + 2-5 032 22-O 1-57 0 to 5 I6'9 3-52 4.9 0'53 23-O 0-82 5 .. !° 3'i9 3-oo 7-6 0-77 .24-2 o-3S 10 „ 15 2-31 2-37 9.9 0-96 257 o-oo 15 „ 2O* 2-04 2-25 12.6 I-2I 20-0* 2-1

* Extrapolated value. t Calculated from Gray's figures.0 It cannot be concluded at once that temperature affects the mechanism of amoeboid activity in the same way as most other biological processes, and that since the temperature coefficient lies between 2 and 3, that the activity is controlled by a chemical reaction. Apart from the criticisms of the validity of applying Van't Hoffs law given by Krogh,11 Lucas,18 and others,10'1 it is not permissible to argue that because the velocity has a temperature coefficient of the value required by this law, that amoeboid activity depends upon a simple chemical reaction. If any biological process varies with the temperature, all the implica- tions of this variation must be considered. We must therefore determine what relations the activity might have to the velocity. VOL. 1.—NO. 4. 529 C. F. A. Pantin

7. The Rate of doing Work. A possible way in which amoeboid activity might be conceived to occur would be by means of some chemical reaction which directly supplied the energy necessary for the activity. The rate at which the Amoeba did work would then be proportional to the velocity of the chemical reaction— provided the efficiency was constant. In this case it is not the effect of temperature on the velocity, but on the rate of doing work, with which the effect of temperature on other biological processes must be compared. Part of the work done by an Amoeba will be external and part internal. It has been shown15 that continuous movement of a limax Amoeba is effected by the streaming of the fluid endoplasm forward through a tube of ectoplasm. The rate at which internal work is done is therefore approximately proportional to the speed of streaming of the endoplasm multiplied by the resistance. But the resistance itself varies directly as the speed of streaming, v, and as the viscosity, n, so that:— Rate of doing internal work =z/V And since the velocity of progression, V, must be proportional to the speed of the endoplasmic stream :— Rate of doing internal work °=V\* Compared with the internal work, the external work is probably negligible. The method of locomotion of a limax Amoeba is such that frictional resistance is minimal. Moreover, the velocity of an Amoeba to which large pieces of debris become attached remains unchanged, although the resistance, and therefore the external work, must be greatly increased. But viscosity is the factor which makes it almost certain that the internal work completely outweighs the external. For the endoplasm is a viscous fluid flowing through a tube of very narrow bore, and—a fact perhaps not generally realised—the absolute viscosity of even "fluid" protoplasm is enormous compared with that of water. * This general formula also holds for " rolling " Amoebae, e.g. A. verrucosa. 53° The Physiology of Amoeboid Movement A simple experiment illustrates this. A very little vaseline is put on the bottom of a small dish filled with water. With a fine pipette a few small Amoebae are placed by the side of the vaseline, so that they may be examined together by a f-in. objective. If the vaseline is teazed with a needle it will be seen that, under the microscope, it appears to be quite fluid : a thread of thickness comparable to that of an Amoeba if pulled out rapidly rounds off into droplets. If the protoplasm of the Amoebae is teazed in the same way, the impression is gained that the viscosity of the more fluid Amoebae {e.g. type A) is of the same order as that of vaseline, whereas the viscosity of the more solid Amoebae {e.g. type B) is much greater. It must be remembered that the protoplasm is bounded by a more solid ectoplasm (Seifriz18 and others), but even allowing for this it is difficult to avoid the conclusion that the protoplasm of the more solid Amoebae is at least as viscous as vaseline. Again egg albumen is much more viscous than water,8 but it appears to be far more fluid than vaseline although solid albumen is precipitated at the air interface. A value for the viscosity of vaseline is not available to the writer, but that of "Mobiloil BB," a heavy lubricating oil, is 9.47 c.g.s. units at 20° C. compared with 0.0101 units for water.6 Assuming vaseline to have a viscosity of this order, we arrive at the conclusion that the absolute viscosity of protoplasm may be at least 1000 times that of water. This is corroborated by Seifriz's18 estimate that the viscosity of the endoplasm of echinoderm eggs is of the same order as that of concentrated glycerine {i.e. about 1500 times as viscous as water at io° C). For these reasons it is probable that almost the whole work done during amoeboid activity is internal, and that therefore :—• Rate of doing work The velocity, V, is known, but we do not know the relative viscosity, n- The viscosity varies with the temperature, and to compare the rates of doing work at different temperatures we must know this relative variation. On account of the practical difficulties this cannot be determined directly for Amoeba, but the writer determined the relative changes in viscosity for C. F. A. Pantin Nereis eggs,16 by Heilbrunn's method.8 The relative values are given in Table IV. TABLE IV. Viscosity of Nereis Eggs. Temperature. . . —07° 10° 20° 30° Relative viscosity. . 1-95 t-oo 071 057 Near oc C. the viscosity rises far more rapidly than it does in the case of water. The protein solutions used by Chick* show the same effect, the rise being more rapid with increasing concentration of protein. If we assume that the viscosity varies relatively with the temperature in the protoplasm of Amoeba as it does in the protoplasm of Nereis eggs, we can, by interpolation, find i. Since we know the velocity, V2^ can be determined (Table V.).

TABLE v. Temperature and the Rate of doing Work.

Temperature. Rate of doing Work Temperature. Qio « vv (by Interpolation).

0-0° C. 0-03 o° to 5° •39-3 4.9 037 5 .. IO 60 9.9 0-92 10 „ 15 3-6 15-1 i-8o 15 » 20 3-1 20-0 3-14

The very high and variable temperature coefficient of the rate of doing work does not resemble that found in most biological processes. We are probably justified in concluding that the rate of amoeboid activity does not depend directly upon the velocity of some chemical reaction from which the energy of activity is derived.

& The Rate of Change of State. The movement of a limax Amoeba must now be re-examined to determine what other factor might control the variation of activity with temperature. In the former paper16 it was shown that during continuous locomotion the protoplasm of a limax Amoeba moves forward 53* The Physiology of Amoeboid Movement as the endoplasmic stream from the hind to the anterior end, through the tube formed by the surrounding ectoplasm. The endoplasmic stream turns outwards at the anterior end, and by forming ectoplasm at the sides of this region, continuously adds to the ectoplasmic tube. This tube is continuously undergoing contraction as fast as it is formed, and is finally resorbed into the endoplasmic stream at the hind end. From this it is evident that during continuous amoeboid movement any "element" of protoplasm undergoes a more or less periodic change of state: it first moves forward as the endoplasmic sol, then reaches the anterior end and is incorporated in the ectoplasmic gel; it then moves towards the hind end in the contracting ectoplasm and is finally resorbed into the endoplasmic stream. The velocity of locomotion of the Amoeba is directly related to the time required for the complete period of this change of state; for the velocity with which an "element" moves along the endoplasmic stream, and the velocity with which it passes towards the hind end in the contracting tube of ectoplasm are both directly proportional to the velocity of locomotion. It is possible to consider, therefore, that the controlling factor in the velocity of amoeboid movement is the rate at which the protoplasm can effect this change of state from sol (endoplasm) to gel (ectoplasm), and vice versa. It is not suggested that protoplasm consists of a large number of "elements" each undergoing a rhythmic change, sol^=gel, in a regular succession. It is merely pointed out that in continuous amoeboid movement the protoplasm is undergoing a periodic change of state ; that the rate at which this change of state takes place is proportional to the velocity of locomotion, and that the rate at which it can occur may be the controlling factor in amoeboid activity. The assumption that the rate of change of state is the controlling factor in amoeboid activity gives a rational explanation of the fact that it is the variation of the velocity of an Amoeba with temperature, and not that of the rate of doing work, that runs parallel to the effect of temperature on other biological processes. This is clearly shown, in the case of ciliary activity in 533 C. F. A. Pantin Table III., and also in Table VI. which gives the temperature coefficients of some other processes for comparison.

TABLE VI.

0° to 10°. 5° to 15°. 10° to S0°.

Q 10 velocity of Amoeba .... 7-33 271 2-17 „ rate of beat of frog's heart,3 37 z-54 2-27 „ „ Terrapin heart,10 2-2 17 IO2 3-5 „ „ Ceriodaphnia heart, 2-5 „ rate of conduction in nerve,11 2-OI i-79 „ latent period of nerve," 3-34 3-51

The assumption also points to a possible explanation of the high temperature coefficient near o° C. Near that temperature the Amoeba becomes shorter and broader. The result is that for a given rate of change of state the velocity is much lower than if the animal were in the normal elongated form. Perhaps this change of form is the result of a great rise in viscosity, since the short broad form offers much less resistance to the streaming endoplasm.

o. Discussion and Conclusion. We have seen that the velocity of an Amoeba varies with the temperature in a similar manner to many other biological processes. The velocity is greatest at an optimum temperature above which some part of the mechanism is progressively destroyed. It is possible that this mechanism is of the nature of an enzyme, though the optimum is low. Were it not for this destruction, the velocity would continue to rise with the temperature as it does below the optimum. The rise in velocity with temperature is probably general for all forms of amoeboid activity, for it has been shown to occur in human leucocytes14 and to a certain extent in the leucocytes of Limulus.12 It is also interesting to note that the velocity of protoplasmic streaming appears to follow the same law, both in the Myxomycetes and in plant cells (Kanitz, pp. 87-889). At normal temperatures the temperature coefficient of the velocity appears to be in accordance with Van't Hoffs law. But examination of the mechanics of amoeboid movement 534 The Physiology of Amoeboid Movement shows that if the effect of temperature were due directly to the alteration of the velocity of a chemical reaction from which the energy of amoeboid movement was derived, then the rate of doing work and not the velocity of progression would vary as the velocity of this reaction. But calculation of the rate of doing work shows that its temperature coefficient is very high and very variable, and unlike that found in most biological processes. On the other hand, the rate of change of state (soK^gel) in the protoplasm is directly related to the velocity. If this is the factor which determines the rate of amoeboid activity we at once have an explanation of why the velocity of locomotion should vary with the temperature in the same way as other biological processes. This fact must be accounted for on any hypothesis concerning the nature of the controlling process in amoeboid activity. From this it is argued that the velocity of continuous amoeboid movement does not depend directly upon the velocity of some chemical reaction supplying the necessary energy, but that it probably does depend on the rate at which the protoplasm can change its state. Temperature then affects the rate of this change of state as it does the rate of most other biological processes. This does not preclude the possibility that the source of the energy of amoeboid movement is ultimately a chemical reaction. It is only implied that, if this is so, the rate of doing work is not proportional to the velocity of this reaction. We are therefore not dealing with the direct transformation of the energy of a simple chemical reaction into work done by the Amoeba. The rate of this transformation does not "set the pace" of amoeboid activity. The energy expended by an Amoeba in doing work may be derived directly from the tension developed in the contracting tube of ectoplasmic gel, perhaps also from the pressure of the swelling protoplasm at the anterior end of the animal.16 If the change of state (sol=?±:gel) of the protoplasm were itself brought about by a chemical reaction, the rate of change of state would depend on the velocity of the reaction. The rate of amoeboid activity itself would then ultimately depend upon 535 C. F. A. Pantin the velocity of the reaction, but only indirectly. The work done by an Amoeba would be derived indirectly from the chemical energy of the reaction by way of the potential energy acquired by the protoplasm in changing its state. Therefore there would be no reason to suppose that the rate of doing work should bear any direct relation to the velocity of the ultimate chemical reaction on which amoeboid activity depended. Finally we may inquire how far the temperature coefficient of the velocity of amoeboid activity indicates that the change of state in the protoplasm is itself brought about by a chemical reaction. Snyder20 points out that the temperature coefficients of chemical processes are not quite constant, and that Van't Hoff suggested that they should be corrected for viscosity. The writer has shown10 that if the temperature coefficients of biological processes could be corrected for viscosity changes, they would probably tend to become constant and of the magnitude characteristic of a chemical reaction. Moreover, correction for changes in viscosity makes approximate allowance for those very changes in the conditions of the protoplasm with temperature which have been considered to vitiate the comparison of the rate of biological processes with chemical reactions.11' *• 18 It is therefore probable that the rate of amoeboid activity is controlled by the rate at which protoplasm can change its state, and the rate of this change of state is in turn possibly controlled by a chemical reaction.

io. Summary. 1. The effect of temperature on the velocity of locomotion of two species of marine limax Amoebae has been determined. In both the velocity rises with the temperature. It is reversibly inhibited just below o° C. There is a low optimum temperature (type A, 22°C. to 250 C. ; type B, 20° C.) above which the velocity falls rapidly ; at higher temperatures activity is inhibited irreversibly. 2. Evidence is brought to show that the fall of velocity above the optimum is due to a destructive effect on the mechanism of amoeboid activity. It is shown that were this 536 The Physiology of Amoeboid Movement effect absent, the velocity would probably continue to rise with the temperature in a normal manner. 3. The temperature coefficient of the velocity is similar to that of ciliary activity and many other biological processes. 4. The rate of amoeboid activity is probably not controlled by the velocity of some simple chemical process the energy of which is directly converted into work done, because the temperature coefficient of the rate of doing work is high and variable and unlike that usually met with in biological processes. 5. The rate of amoeboid activity appears to be controlled by the rate at which the protoplasm changes its state (sol ^2= gel). This provides a rational explanation of the fact that it is the velocity and not the rate of doing work which varies with the temperature as do other biological processes. 6. In view of conclusions arrived at in another paper,16 it is possible that the value of the temperature coefficient indicates that the rate at which protoplasm can change its state is controlled by a chemical reaction.

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