In the preceding chapters we have discussed the streaming of the endoplasm in various representative species of ameba, and its transformation into ectoplasm at the anterior end. We have observed that the details of streaming are not quite the same for any two species of ameba, and that in consequence the character of locomotion also is specific for every ameba. All the observations prove that movement in ameba is always associated with streaming, and streaming (in locomotion) with ectoplasm formation. It follows therefore that the form of movement observed in amebas depends invariably upon the streaming of the endoplasm accompanied by the formation of ectoplasm.

There is however another element which, although it appears to be a consequence of ectoplasm formation, must nevertheless be included in any account of ameboid movement because of the light it is bound to shed on the physical processes concerned in streaming. This element is the thin outer layer which separates the water in which the ameba lives from the ectoplasm. It is the properties of this layer to which we may now direct our attention.

That such a layer exists was indicated by observations of BÜtschli (’92) and Blochmann (’94), as already mentioned; but neither of these authors stated definitely whether they considered a third layer actually to exist or whether the ectoplasm as such moved forward. Jennings (’04), as has been pointed out, concluded that no third layer exists and that the particles clinging to the outsides of amebas, which are carried toward the anterior end, are carried by the ectoplasm. Gruber (’12) concluded however that an outer layer exists, composed of gelatinous substance, which moves ahead at about the same rate as the ectoplasm (p. 373). According to Gruber’s view the outer layer is a permanently differentiated layer of material. Schaeffer (’17), on the contrary calls it a layer of protoplasm, which moves forward faster than the forward advance of the ameba.

It is a very simple matter to demonstrate the existence of this layer. Although any insoluble non-toxic substance of low specific gravity such as carmine or soot, when reduced to very small particles and mixed with the water in which the amebas to be examined live, will cling to the outside of the ameba so that the movement of the outer layer can be observed; in my experience the best as well as the most convenient substance to use is the dried flocculent colloidal sediment from ameba cultures, rubbed to powder with the ball of the finger. This powder swells up in water into flocculent masses which are large for their weight and do not show such active Brownian movement as particles of carmine or india ink, and they consequently adhere more easily to the ameba. Moreover no foreign substances are thereby introduced into the water.

Of the more common species of amebas, those with the firmer ectoplasms are the most favorable for studying the movements of the outer layer. We may therefore first take up several observations on Amoeba sphaeronucleosus (Figure 13). This ameba resembles the more common A. verrucosa. It is about 120 microns long and is usually of an oval shape in locomotion. It is more active and less disturbed by jars than verrucosa.

Figure 14 represents a sphaeronucleosus with a small particle attached to the middle of the upper surface of the ameba. As the ameba moves forward, shown by successive outlines, the particle likewise moves forward, but, as will be observed, at a more rapid rate. Measuring the distance from particle outline 1 to 4, and from ameba outline 1 to 4, it is seen that the rate of movement of the particle compares with the rate of movement of the ameba as 2.48 to 1.

Particles lying near the side do not move forward as rapidly as those lying in the middle. Figure 15 shows two particles, one of which, a, lying near the middle of the ameba, moved 2.6 times as fast as the ameba advanced in the region of the particle; while particle b moved only 1.9 as fast as the ameba in front of the particle. The speed ratio of particle a to particle b was as 1.26 to 1.

Figure 16 shows a particle lying still more to the side than in the preceding figure. In the first six stages the particle moved 1.85 times as fast as the ameba. The particle then came to the edge. From stage 7 to 10 the particle moved more slowly than the ameba. At stage 11 the particle had come to lie in the posterior half of the ameba, where the tendency of the surface layer is to travel toward the middle of the upper surface. In stage 12 the particle had gotten away from the edge of the ameba and already shows a gain in speed. From stage 13 to 16 the particle moved again about 1.83 times as fast as the ameba. But at stage 16 the edge was reached with a consequent decrease in speed of the particle.

The direction of the path described by a particle carried on the back of an ameba depends upon what part of the ameba is most rapidly forming ectoplasm. That is, the particle tends to

move toward that part of the anterior edge that is advancing most rapidly. Figures 17 and 18 illustrate this point. Figure 17 shows an ameba with two particles on its back, and with an unequally advancing anterior edge. Particle a moved more rapidly than b because: (1) it was moving away from a more rapidly receding posterior region; (2) the right anterior edge was advancing more rapidly than the left anterior edge; (3) the particle was nearer the anterior edge. The rapidly advancing right edge in stage 4 accounts for the veering of the particle a to the right. The more rapid advance of b from stage 3 to 5 is due to the remoteness of the anterior right edge, which, because of its nearness to particle a pulls on it to a much greater extent than on particle b. That is to say, when a particle lies somewhere between two rapidly growing regions on the anterior edge, leading in different directions, that particle is attracted to the edge less rapidly than a particle lying immediately back of either advancing region. As may readily be observed each change in speed or direction of movement of the particle b finds its explanation in the amount and location of ectoplasm formation at the time. Large particles like a do not so readily reflect changes in the direction of pull of the surface layer.

The rapid rate of movement of particle a—3.5 times as fast as the ameba—finds its explanation in an actively advancing anterior edge that was unusually wide. Particle b moved at a slower rate, 2.7 to 1. It started from near the posterior edge where it moved comparatively slowly for a short distance.

Figure 18 shows more pronounced changes in the direction taken by a particle attached to the back of an ameba. The change in direction at stage 6 was caused by a wave of ectoplasm thrown out at the left side, and cessation of movement at the anterior edge. At 7 a small wave was thrown out at the anterior edge and a large wave on the left. At stages 8 and 9 the direction of the particle was again a response to the waves of ectoplasm thrown out at the left anterior edge, which thus became the anterior end.

The movement of particles on the under side of an Amoeba sphaeronucleosus depends upon what part of the ameba is attached to the substratum. Where the ameba is attached there is of course no movement of the surface layer and the particles remain stationary. In an ameba attached as shown in figure 20, a, there was a very slow movement of particles forward near the middle of the attached region (x), but whether this was related to the movement of the outer layer of the upper surface was not determined. The movement of these particles was considerably slower than the movement of the ameba. In another ameba attached at the anterior and posterior ends (Figure 20, b) no movement of particles on the under side could be discerned. The small particles showing Brownian movement, with the surrounding water, are dragged along as a mass. This movement is purely mechanical, and is what would be expected on purely physical grounds, when a more or less cup-shaped object is moved along in water in close contact with a flat surface. Such particles as have become attached to the surface layer on the under side of the ameba, because of their slower movement than that of the ameba, eventually bring up at the sides near the posterior end, as the ameba moves along. From here they are carried forward in the manner already described. Thus there comes about a “rotation” of particles adhering to an ameba as described by Jennings (’04) and Dellinger (’06), though the explanation is different from that given by Jennings (l. c.) as we shall see further on. No case of a similar rotation of larger particles which had sunk into the ectoplasm, as described by Jennings (’04, p. 142), has come under my observation.

The movement of the surface layer in A. verrucosa is quite like that of sphaeronucleosus. Figure 21 shows a group of three particles carried by a verrucosa while changing its direction of locomotion. The particles changed position with regard to each other and they moved at different speeds. Particles a, b, c, moved respectively 2.40, 3.26, 2.85 times as fast as the ameba advanced. Other experiments indicate that the outer layer of verrucosa moves at about the same speed, compared with the speed of the ameba, as that of sphaeronucleosus.

Amebas with so-called limax-shaped bodies do not possess surface layers that carry particles forward with the same speed as those amebas with broad bodies. It is only occasionally that large amebas such as proteus are found in a limax or clavate shape. One of the most favorable of the large amebas in this respect is discoides. It is frequently found in clavate shape and it possesses the further advantage in being nearly cylindrical in cross section. It is also more in the habit of loping along the surface in the manner described by Dellinger (’06, p. 57) so that what is observed to take place in discoides in the clavate shape, holds likewise for free pseudopods extended into the water out of contact with a solid support (Figure 22).

In figure 23 is shown a clavate discoides with a small particle attached to its side. The particle moved forward until it came to lie at the anterior edge, 10. The speed of the particle from 1 to 10 was 1.36 times as fast as that of the ameba, a much slower rate than was observed in sphaeronucleosus. At 6 a new pseudopod was projected for a short distance, thus increasing the amount of new ectoplasm forming in proportion to that of the whole ameba. This change was reflected in the increased speed of the particle, which moved 1.64 times as fast as the ameba from 5 to 6. At 10 the anterior end again spread out and again the particle moved faster—twice as fast as the ameba from 9 to 10. Stages 11, 12, 13 are added to show that the particles do not tend to go to the under surface but remain at or very near the tip. The slight irregularity of the waves of hyaloplasm pushed out at the anterior end accounts for the changing position of the particle after it has reached the anterior edge. The particle remained at the edge of the advancing ameba for several minutes after the stage drawn at 13.

In another observation the effect of a narrowing of the advancing tip of the ameba is shown very well. In figure 24 the ameba was advancing with a broad anterior end, as shown at 1 and 2. From 2 to 4, the region where new ectoplasm was made, narrowed down very considerably. These changes in the width of the anterior end are reflected, as in Figure 17 by a decrease in the relative speed of the moving particle. Thus the particle moved 1.75 times as fast as the ameba from 1 to 2 while from 2 to 4 the particle moved only 1.27 times as fast as the ameba.

The movement of the third layer in proteus is difficult to study owing to the formation continually of ridges, as explained on page 20. Even in clavate shaped amebas, waves of protoplasm are pushed out on the sides and on the tip with consequent formation of ectoplasm, so that the ameba grows in width slowly at the same time that it grows in length. A typical shape of a proteus in clavate form is slightly tapering toward the anterior end. This shape is maintained by gradual extension of the sides of the anterior half or two-thirds of the ameba as it moves along. These conditions are just the reverse of what was seen to be the case in sphaeronucleosus and verrucosa, where the anterior edge was wider than any other part of the body. But discoides, although free from the ridges and grooves characteristic of proteus, frequently has an anterior edge that is narrower than any part of the body, thus necessitating extension of the sides as the ameba moves forward.

Let us now see what is the effect of ridge formation upon the movement of the surface layer. Figure 25 shows a proteus and a narrow anterior end in proteus with two pseudopods and a particle attached to the side of the ameba at 1. Both pseudopods advanced until stage 4 was reached, but the particle was not appreciably deflected from an approximately straight path by the small pseudopod at the other side of the ameba. Reference to the figure shows that the particle travelled much faster while the pseudopod on the side was extending than after it began to retract. The particle moved 1.43 times as fast as the ameba from 1 to 4. But from 4 to 7 the particle moved only 1.06 times as fast as the ameba.

In the earlier stages the outer layer was pulled toward the tip of both pseudopods, in the later stages only toward one, and in this lies the explanation for a more rapid movement of the particles in the earlier, and a slower movement in the later stages. This effect was also observed in discoides, but the fact that the particle in the later stages moved only very little faster than the ameba is due to a narrow anterior edge and to the formation of ectoplasm in the ridges over the surface of the ameba. The effect of ridge formation on the movement of particles attached to the surface film is well seen when an ameba has two forward moving regions opposite each other. Under such conditions particles located equidistant or nearly so between such regions, move only very slowly or not at all, the pull upon the film being nearly or quite equal. In a similar manner the ridges which are constantly forming on a proteus are continually competing with the anterior end in their pull upon the surface layer, thus preventing rapid forward movement.

Figure 26 shows that the surface layer flows away from the tip of a retracting pseudopod that is located near the anterior end. The particle moves slowly until the body of the ameba is reached, when movement becomes more rapid, 8, 9. This proves that the third layer moves away from the retracting parts of an ameba, no matter how large the total area of these parts may be in proportion to the area of new surface that is being made. But whether the speed of the moving third layer changes in correspondence with a larger or a smaller ratio between building and retracting ectoplasm has not been ascertained.

Figure 27 shows that the relative positions of particles attached to the surface layer may readily change while the ameba deploys its psuedopods. Three particles marked a, b, c and connected

by a line for convenience of reference, were in the position indicated at 1 when the forward end of the ameba occupied the position indicated by outline 1. As the ameba moved forward the particle c gained slightly on a and b for no ascertainable reason, unless it was on account of the projection of the large pseudopod on the opposite side. At stage 2 a new pseudopod was started on the right, which at stage 3 had grown to large size while streaming in the original pseudopod was arrested. At stage 3 particles a and b retained the same position they had in stage 2, except for a slight turning to the right. Particle c however moved across the base of the original pseudopod and on to the middle of the new pseudopod. At stage 4 a and b had again only slightly moved to the right of the position they occupied in stages 2 and 3, while c moved rapidly toward the tip of the new pseudopod. The new pseudopod was then retracted and at stage 5 the particles had begun to move back toward the main body of the ameba. Particles a and b now gained considerably on c because they were located further away from the tip of the retracting pseudopod. Particles a and b were drawn to the middle of the retracting pseudopod because of the continuous enlargement of the large pseudopod on the right, below, through which the ameba moved on.

The most important feature of this observation is the change in the position of the particle c with respect to that of a and b. The latter particles retained their relative positions with very slight, if any, change, while c swung around a and b nearly 180°, and at the same time changed the distance very greatly between itself and the other particles. Moreover, b, at stage 5 led the procession of particles, while at stage 1, a led. No further demonstration is necessary to show that the surface layer is distinctly fluid and dynamic, and not at all such a static structure as an elastic permanent skin, as Jennings (’04) and Rhumbler (’14) maintained.