The observations in the preceding chapters on the general movements of the surface layer of amebas will afford a sufficient basis for an inquiry into the nature of this layer. The mere demonstration of the existence of this layer is, of course, interesting enough, for a number of contradictory statements by various students of the amebas are satisfactorily cleared up by these observations. But the problem of ameboid movement affects other organisms besides amebas, and since the movement of the surface layer is so intimately associated with ameboid movement, it becomes of more than ordinary interest to learn something of the nature and composition of this layer.

In the first place the property of carrying particles toward the anterior end of amebas does not appear to be of any advantage. That is, whatever the movements of the outer layer may be, the ameba does not appear to be better off when particles are carried forward than when none are carried, for such particles are very small and almost without exception devoid of food value. The particles are masses of debris which accidentally adhere to the ameba, and the ameba makes no visible effort to make such particles adhere, nor to get rid of them. The ameba seems to be quite indifferent to the presence of such particles.

On the other hand, as Schaeffer (’17) has pointed out, the capacity for transporting particles cannot but be looked upon as a hindrance to locomotion. As has been stated, the surface film moves in the same direction as the ameba. Whenever the surface film comes against a solid object, it pushes against the object, and nullifies to a certain, though small, extent the energy expended in moving forward. And it will be seen without further argument, of course, that the energy involved in carrying particles forward is not only itself lost but consumes an appreciable part of the energy available for forward movement. This fact, together with the universal occurrence of this phenomenon among amebas indicates beyond question that it is intimately associated with ameboid movement as it is ordinarily understood in amebas, and that it is almost certainly a “necessary” physical consequence of the more fundamental physical processes involved in the movement of amebas.

That the third layer moves in the same general direction as the ameba has already been mentioned. The direction of a moving particle is however not necessarily parallel with the stream of endoplasm below. In a retracting pseudopod that lies nearly parallel to and by the side of the main advancing pseudopod, the particles on the far side and near the base frequently move across the pseudopod at an angle (and therefore also across the endoplasmic stream), and up the active pseudopod on the near side. This shows conclusively that the direction of flowing endoplasm by itself has no direct connection with the direction of flow of the surface layer.

To say that the particles carried by the surface layer bring up at the anterior ends of pseudopods or of the ameba when in clavate shape, admits of further qualification. The advancing edge is not a straight line but an arc, and the sides near the advancing edge are building at a slower rate than the extreme tip. The most rapid formation of ectoplasm is at that point of the ameba that is farthest ahead. At this point all the ectoplasm to be made is still to be made, but as one passes back along the side of the pseudopod more and more ectoplasm is encountered and less and less remains to be made. There is therefore a gradient in the rate and in the amount of ectoplasm formed as one passes back from the forward end of the longitudinal axis of the pseudopod along the side. This is especially the case with certain amebas like Amoeba discoides, A. laureata and others in which the pseudopods are more nearly cylindrical. In such amebas as A. proteus and A. verrucosa, the factor of ridge formation complicates to some extent the longitudinal gradient of ectoplasm formation. But in spite of these specific differences, the general statement still holds that the rate of ectoplasm formation at the extreme anterior end is higher than anywhere else in the ameba, and that the rate gradually falls to zero as the nearly straight and parallel sides of the pseudopod or ameba, as the case may be, are approached.

Now we have seen that if a particle becomes attached to the outer layer of such an ameba as discoides, which has nearly symmetrical pseudopods, at some considerable distance from the tip of the pseudopod, it moves forward until the tip of the pseudopod is reached. It does not tend to come to rest near the tip of the pseudopod, where the rate of ectoplasm formation is much higher than at the sides of the pseudopod, though not as high as at the tip, but it moves on until the tip is reached. That is, the movement of particles on the surface film is toward that small area at the extreme anterior end where the rate of ectoplasm formation is highest.

In such an ameba as verrucosa, however, the highest rate of ectoplasm formation would be, not at a small circular area, but a very narrow strip along the anterior edge; for the rate of ectoplasm formation over a considerable portion of the width of the anterior end of the ameba is practically the same, according to observation. Consequently we do not find particles which are attached to the outer layer tending to move to a point lying on the longitudinal axis, but their paths are found to be straight and parallel with the longitudinal axis, if headed toward any point over a considerable stretch of the anterior edge on either side of the longitudinal axis.

All the evidence that is at hand therefore points to the conclusion that the direction of movement of the surface film in a moving ameba is toward that point where ectoplasm is formed most rapidly.

But where do the particles come from? At exactly what regions of the ameba do they start to travel toward the anterior ends of the ameba? In sphaeronucleosus and its congeners, it is very difficult to determine just when the particles begin to move toward the forward edge. Particles near the posterior end on the upper surface of these amebas moved forward slowly, much more slowly than particles near the middle. Sometimes particles near the posterior end seem to be motionless for some time, but the incessant though slow kneading process going on at the posterior end makes accurate observation difficult. Only in a general way it may be stated that particles begin their forward march at or near the posterior end. In amebas that habitually form pseudopods more accurate information can be obtained.

In proteus or discoides, for example, projecting pseudopods are often suddenly stopped and retracted, with a resultant change of an anterior to a posterior end. Particles attached to the outer surface on such pseudopods move toward the anterior end, of course, as long as the pseudopod is building, in the manner described in the preceding pages. But when the endoplasmic stream is arrested, the forward movement of the particle likewise stops. When the endoplasm starts to flow back into the main body of the ameba, the particle also starts moving back; but there is a period of a few seconds after the endoplasmic stream is reversed during which the particle remains quiet. And when it does start in to move, it moves only slowly. Within a few seconds, however, the average speed of movement is attained. This is true of particles located some distance away from the tip of the pseudopod. If the particle has reached the tip of the pseudopod before reversal of the endoplasmic stream takes place, the particle often remains at the tip until the pseudopod is almost completely withdrawn into the main body of the ameba (Figure 26, p. 60). At other times such a particle becomes displaced, presumably by irregular retraction of the tip of the pseudopod, and finds itself at the side of the pseudopod. When this happens it moves slowly toward the main body of the ameba, but faster than the tip of the pseudopod does.

It frequently happens, especially in annulata, but also in proteus and other forms with many pseudopods, that when an advancing pseudopod is about to be withdrawn, there intervenes a stage where the endoplasm in the distal part moves away from the ameba, while that in the proximal part moves toward the ameba, with a neutral or motionless zone between. In such case a particle on the distal end moves slowly toward the tip while a particle in the proximal region moves toward the base of the pseudopod. Particles over the neutral zone are motionless. In these cases, however, changes in the direction and speed of the ectoplasmic stream are too frequent and the relative strengths of the distal and proximal currents too variable, to enable one to secure very accurate data by means of camera lucida drawings (a kinematograph is essential for this purpose), so no figures of the speed of movement of such particles are given. Nevertheless the general results of the observations are as stated. It might be added that in some cases the neutral zone for the particles attached to the surface did not coincide exactly with the neutral zone of the endoplasm, but was located a little further distally.

From these observations it appears that a rough index of the direction of movement of the surface film is the direction of the streaming of the endoplasm; and that the surface layer moves away from regions where ectoplasm is in the process of being converted into endoplasm. Since a particle attached to the surface may remain for some time at the tip of a retracting pseudopod, while one that is attached to the sides of a pseudopod moves toward its base, it appears that the speed of the moving surface film is not directly correlated to the rate of transformation of ectoplasm into endoplasm. The slower speed of particles near the posterior end points also in this direction. The formation of ectoplasm at the anterior end seems therefore to be much more intimately connected with the movement of the surface film than the destruction of the ectoplasm, though it is not yet clear that the liquefaction of the ectoplasm is altogether without effect.

Now as to the speed with which the surface film moves. The foregoing illustrations and figures show that the particles attached to a sphaeronucleosus on the upper surface move from 2.5 to 3.6 times as fast as the ameba (Figure 19) while particles attached to a discoides move only from 1.2 to 2 times as fast as the ameba moves. In proteus the speed of the particles is still slower, because of the longitudinal ridge-like waves of protoplasm which are continually being thrown out. In this species it frequently happens that because of the numerous ridges, the ameba moves faster than the particles attached to the outer surface; but this is to be looked upon as a mechanical complication, not as indicating a difference in the nature of the surface layer.

How is the difference in the speed of movement of the surface layer between sphaeronucleosus and discoides to be explained? There are no ridges to retard the movement of particles in discoides, while there are ridges in sphaeronucleosus, where the particles move on the average twice as fast as on discoides. In the first place the advancing edge, the edge where ectoplasm is being made, is proportionately much wider in sphaeronucleosus than in discoides as compared with the amount of surface back of it. Figures 23 and 24 show that the rate of movement of the surface film is directly proportional to the amount of new ectoplasm forming. In the second place, the greater part of the under surface in the forward half of sphaeronucleosus is attached to the substrate, so that the surface layer which flows toward the anterior end is derived almost wholly from the upper surface; while in discoides the whole surface in free pseudopods, and nearly the whole surface in attached amebas (cf. Dellinger’s observations described on p. 56) possesses mobile surface protoplasm. Observation of moving particles on these amebas proves this. Then again, the anterior edge of a sphaeronucleosus is not attached at the points farthest advanced, but the point of attachment is some distance back, as indicated in figure 20. The effect of this is to increase the amount of forming ectoplasm in proportion to the surface of the ameba from which surface protoplasm may be drawn. Still one other factor must be considered. As is well known sphaeronucleosus, verrucosa and their congeners possess longitudinal ridges on the upper surface which consist of ectoplasm, covered of course by the surface film. These ridges are formed near the anterior edge, not by wrinkling, but by the construction of new ectoplasm. Once formed, they remain until the ameba, so to speak, flows out from under them. That is, the ridges undergo comparatively slight changes until changed back into endoplasm at the posterior end of the ameba. As the ameba flows ahead the ridges are of course continually being added to or lengthened, by the conversion of some endoplasm into ectoplasm. The ridges may thus retain their identity for a long time although the substance composing them is changed every time the ameba moves the length of its body. It is clear, therefore, that there is more ectoplasm formed at the anterior end of a sphaeronucleosus than would be the case were the upper surface of the ameba plane; and the conclusion therefore is obvious that the formation of ridges, occurring as it does, chiefly at the anterior end, serves further to accelerate the forward movement of the surface film.

If the form of sphaeronucleosus were more regular than it is, the amount of ectoplasm in the process of forming at any given moment could be compared with a similar relation existing in discoides, to see whether these respective ratios were proportional to the speed of the moving surface films in the two amebas. As it is, the irregularity of form of sphaeronucleosus makes such computation subject to the possibility of considerable error. In discoides however the problem is comparatively simple. I therefore did not go into this matter extensively, but merely worked out the relations mentioned in one case, and I mention it here to illustrate the method rather than to record the result, which is not to be taken as very exact.

Since the movement of the surface film is obviously a surface phenomenon, only the surfaces of the amebas need to be taken into account. In Figure 28 is illustrated a discoides of such a shape as to allow a fairly accurate computation of its surface. Three outlines of the anterior end only are given; the rear portion of the ameba remained approximately the same size and shape in the three outlines. The cross lines at the anterior end divide the forming ectoplasm of the ameba from the formed. As will be noticed the cross lines are drawn through the intersections of two successive outlines. Computing the areas on both sides of the cross lines for the two outlines and averaging them, there is found a ratio of 1 to 10; one-eleventh of the total surface represents forming ectoplasm, and ten-elevenths formed ectoplasm. (One-twenty-second of the total surface was deducted for surface attached to the substratum.) Sphaeronucleosus stands in contrast with discoides for it is attached to the substratum over a much greater area and in consequence only a slight amount of surface is drawn from the under side. This ameba may therefore be regarded in this connection as of only one surface, the upper. That part of outline 1 in Figure 14 cut by outline 2 indicates, as in discoides, the region of forming ectoplasm, and the space between outlines 1 and 2 may be used as a basis of computation. New ectoplasm is formed in this zone and far enough back to include the tips of the longitudinal ridges, of which we have already spoken (Figure 13). The zone of forming ectoplasm would therefore be about twice as wide as the average width of the three zones between the successive outlines in the figure, and of approximately the same shape. On this basis, the surface occupied by forming ectoplasm is ¹/_5.8 of the total surface, and the ratio of formed to forming ectoplasm is 4.8 to 1.

(For the sake of completeness, a few factors whose values cannot easily be computed may be mentioned. 1. The anterior edge is not attached to the substratum at its farthest point, but at some little distance back of the edge, thus increasing the relative amount or forming ectoplasm; but this is offset by the surface of a part of the under side at the posterior end where the surface layer is active. 2. The ectoplasm composing the ridges, which must be added to the formed ectoplasm, would increase the ratio, though only slightly).

Approximately twice as much ectoplasm is therefore in the process of formation in sphaeronucleosus as in discoides when compared with the formed ectoplasm in the respective amebas, over which the surface film is active. This ratio corresponds very well with the rate of movement of the outer surface in these amebas, which as we have seen is about twice as fast in sphaeronucleosus as in discoides.

Where does the surface layer come from and what becomes of it after it arrives at the anterior end? It moves continually forward as long as the ameba moves forward. There would seem to be a tendency therefore for it to accumulate at the end of a free pseudopod in such a form as discoides, and even under ordinary conditions of locomotion where there is occasional attachment to the substratum by very short pseudopods, the surface layer is continually moving toward the anterior end on practically all sides. Every time, therefore, that the ameba moves a little less than its own length, there would accumulate at the tip of the ameba, if it were not removed, an amount of surface layer equivalent to that which covers the whole ameba. No such accumulation can be detected however, from which we infer that it is removed as fast as brought there. And the posterior region of the ameba, which is the main source of the surface film, does not become poorer in this material by reason of its continual flow forward, but new surface is made continually to take the place of that moving forward. This process of destruction and creation of surface is accordingly rapid during active locomotion;—a discoides, moving approximately once its length at room temperature in two minutes, destroying therefore the equivalent of its entire coat of surface in that time; while a sphaeronucleosus, moving once its length in two or three minutes, destroys all its surface every minute.

From what has been said thus far, it must be apparent that there is striking resemblance between the general movement of the surface layer of the ameba, and of a surface tension layer in a drop of fluid in which the tension is changed at some point. Let us now inquire briefly into this resemblance.

As is well known the surface of a liquid in contact with another liquid, solid or gas, with which it does not mix, behaves like a stretched membrane, so that when the tension is reduced at any point the surface layer moves away from that point. A good illustration of the effect of a decrease of surface tension is found in a drop of clove or other oil with which some substance that reduces the surface tension, such as alcohol or soap, is brought into contact at one side. If previously some dust particles have been placed on the surface of the oil drop, it will be easy to see that the surface of the oil moves to the opposite side from where the alcohol or soap solution touched the oil. In practice it is a very simple matter to lower the surface tension of a drop of fluid as described, so as to show the movement of particles on the surface. Almost any liquid may be used for this purpose. But it is comparatively very difficult to increase the surface tension at some point of a drop of fluid in such a way as to cause particles on the surface to move toward that point. The principle underlying the movement of the surface film in both cases is however exactly the same; so, although it would be more desirable to compare the surface movements in a drop of fluid in which the surface tension is increased at some point, because this is what happens in an ameba during locomotion, we shall nevertheless find it necessary to consider a drop of fluid in which the surface tension has been lowered. The application of the illustration is readily made.

When the surface tension of a drop of fluid is lowered by bringing into contact with it some other substance that possesses this power, the surface rushes away at great speed in all directions from the point where the tension is lowered, because usually the tension is reduced very considerably. In this surface movement it is found that new surface is made where the tension is lowered and old surface is destroyed, that is, pulled into the interior over a large part of the surface opposite to where the tension is lowered. The speed of the surface movement is most rapid near the point where the tension is lowered and becomes gradually slower as the opposite side of the drop is approached, where there is no movement. This variation in speed of the moving surface seems to be due largely to the small area in which the tension is lowered as compared with the whole surface of the drop.

In the ameba the conditions are reversed. The surface layer moves toward a point with increasing speed, instead of away from a point. In both the ameba and the drop the greatest speed is attained near the small area where the change in surface tension occurs.

The behavior of large and heavy particles on the surface of a drop of fluid and on an ameba are similar. A heavy particle as of sand, or a small glass rod, laid on the ameba, is not moved by the surface layer. It forms an island of surface matter around which the moving surface layer flows. Precisely the same thing happens in surface layer movements in inanimate fluids.

Again in point of thinness there is no disagreement so far as microscopic observation goes. Neither the surface film on an ameba nor the surface film on a fluid can be directly observed microscopically to be different from the fluid below it. The surface layer is, as is generally believed, of molecular dimensions, and its thickness is beyond the limits of vision. Unless some special means is discovered therefore for making visible the surface film, such as a process of staining, it may be impossible to ascertain its ultimate structure directly, for it overlies a mass of heterogeneous fluid whose composition is constantly changing.

It seems to follow from what is observed of the surface tension layers of the fluids of physics that such layers must be of the same constitution as the body of the fluid over which the layer is formed, although, as is well known, the proportion of the ingredients in the surface layer is different from that in the body of the fluid. Now since the resemblance between the surface layer of an ameba and a surface layer on a drop of fluid has thus far been found to be complete, it is pertinent at this point to discuss Gruber’s (’12) suggestion that the movement of particles forward on an ameba is due to the forward movement of an inert layer of mucus or gelatinous material secreted by the ameba.

To begin with, observation does not support Gruber’s suggestion. No such layer can be seen. Such a layer, since it is shown to persist for several minutes at least, should remain after an ameba bursts, under experimental conditions, but no such remains can be seen. Its existence should be demonstrable by the use of dyes, but the evidence is negative. Indeed there is not any direct evidence that can be brought in support of the suggestion that this surface layer is gelatinous in composition. Moreover, as we have seen, the layer on the ameba that carries particles forward seems to be destroyed at the anterior end, for in what other way would particles remain at the anterior end after being brought there? But the supposition that a gelatinous layer might be drawn into the interior at the anterior end is also negatived by observation, for no very small particles clinging to or imbedded in the surface substance are ever drawn into the ameba, as would almost certainly be the case if the substance composing the layer were gelatinous. And as to supposing that this layer, if gelatinous, might behave essentially as a surface tension layer and therefore be drawn in at the anterior end of the ameba, this is contrary to the experience of physics; for the physical nature of the ameba would make it impossible for the ameba to have a surface layer of gelatinous matter. There do not seem to exist any grounds therefore for supposing that the outermost layer of an ameba, the layer that carries particles as described in the preceding pages, can consist of an inert substance as Gruber suggests.[4]

From these considerations, then it appears that all the evidence available, both direct and indirect, points to the conclusion that the behavior of the surface layer on the ameba resembles in general and in detail the behavior of a surface tension layer in an inert drop of fluid, and that we must regard the surface layer on the ameba as a true surface tension layer. This layer is therefore a dynamic layer, containing free energy, and capable of performing work. It is physiologically distinct from ectoplasm, as ectoplasm is distinct physiologically from endoplasm. But the distinctive properties which the surface layer possesses are functions of its position. These properties clearly indicate that its constitution is protoplasmic, corresponding to the fluid parts of the internal protoplasm.

The surface layer of the ameba is probably identical with what is commonly called the plasma membrane or semi-permeable membrane as postulated by Overton (’07). The peculiar structure supposed to be possessed by plasma membranes are held to be due chiefly to surface forces. The fact that the surface layer of the ameba is continually being destroyed and re-created during locomotion does not support the view that the plasma membrane is of inert composition, as for example, lipoidal, as has been suggested. The observations, on the contrary, confirm HÖber’s (’11) view that the plasma membranes generally are living structures. But it may be regarded as certain that if lipoids are present in the protoplasm of the ameba, these substances, according to the principle of Willard Gibbs, will be found in higher concentration in the surface film than in the body of the ameba.

Perhaps the most important question that arises in connection with the surface layer of the ameba is: What causes it to move in the manner described? But we can do little more than ask the question. It has been seen that the surface film moves toward an area of increased tension rather than from an area where the tension has been lowered. However, since we are completely in the dark respecting the composition of the surface layer or of the fluid parts of the ameba, it is exceedingly hazardous to venture an explanation. If the surface layer should have its tension lowered by a concentration of lipoids in it, we would be faced by the necessity of explaining their removal at the anterior end. If we turn to electrical causes we meet again with great difficulties. An ameba moves with the electric current, when a current is passed through the water. The surface layer under these conditions behaves normally, as may be inferred from Jennings’ (’04) figure on page 198. That is, the current controls the direction of the movement of the ameba, with the current leaving the ameba at the point of highest surface tension. This is contrary to the action of the mercuric capillary electrometer, in which the mercury column also moves with the current, but because of lowered surface tension where the current leaves the mercury. The conditions surrounding these cases are so different however, that very little can be gained by setting them in contrast to each other.