CHAPTER VIIExperiments on the Surface Layer of the Ameba

Figure 12. Illustrating the high degree of elasticity in the pseudopods ofBiomyxa vagans. Inaandbare shown two stages of a small section of the pseudopodial network, which remained unchanged while a small lump of protoplasm (near the arrow) moved rapidly up and down the slender pseudopod. Movement along the whole length of the pseudopod occupied about half a second. Just exactly what the movement was due to could not be determined, but the distance between the forks in the pseudopod did not change, nor did the thickness of the protoplasmic strand on which the protoplasmic lump moved change noticeably.

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.

Figure 13.Amoeba sphaeronucleosus.In locomotion. Note the nucleus, contractile vacuole, ectoplasmic ridges. This ameba is not known to form pseudopods. Length, 120 microns.

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 onAmoeba sphaeronucleosus(Figure 13). This ameba resembles the more commonA. 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 thanverrucosa.

Figure 14represents asphaeronucleosuswith a small particle attached to the middle of the upper surface of the ameba. As theameba 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.

Figure 14. Illustrating the movement of a particle on the upper surface layer of anAmoeba sphaeronucleosus. Length of the ameba, 120 microns.

Figure 15. AnAmoeba sphaeronucleosuswith two particles attached to its upper surface film, one in the middle and one at the side.amoved 2.6 times as fast as the ameba whileb, lying nearer the side, moved only 1.9 times as fast as the ameba. Length, 100 microns.

Particles lying near the side do not move forward as rapidly as those lying in the middle.Figure 15shows 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 particlebmoved only 1.9 as fast as the ameba in front of theparticle. The speed ratio of particleato particlebwas as 1.26 to 1.

Figure 16. Illustrating more rapid movement of the surface film in the middle ofAmoeba sphaeronucleosusthan near the edge. The vertical lines connecting the particle with the ameba outlines were drawn only for convenience of reference. Length of ameba, 120 microns.

Figure 16shows 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

Figure 17. Illustrating the different speeds with which particles move when attached to the surface film of anAmoeba sphaeronucleosus, depending upon their location. Particleamoved 3.5 times as fast as the ameba andb2.7 times as fast. Length of ameba, 110 microns.

move toward that part of the anterior edge that is advancing most rapidly. Figures 17 and 18 illustrate this point.Figure 17shows an ameba with two particles on its back, and with an unequally advancing anterior edge. Particleamoved more rapidly thanbbecause: (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 particleato the right. The more rapid advance ofbfrom 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 particleb. That is to say, when a particle lies somewherebetweentwo 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 particlebfinds its explanation in the amount and location of ectoplasm formation at the time. Large particles likeado not so readily reflect changes in the direction of pull of the surface layer.

The rapid rate of movement of particlea—3.5 times as fast as the ameba—finds its explanation in an actively advancing anterior edge that was unusually wide. Particlebmoved 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. Illustrating the effect on the path of a particle attached to the surface film of anAmoeba sphaeronucleosuswhen the ameba changes its direction of movement. From stages 3 to 5 the ameba veered to the right, also the particle. From stages 6 to 9 the ameba turned sharply to the left, and this change of direction was reflected in the movement of the particle. Length of the ameba, about 120 microns.

Figure 18shows 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.

Figure 19. Illustrating the rapid movement of the upper surface of anAmoeba sphaeronucleosusunder the most favorable conditions. The particle moved 3.56 times as fast as the ameba. Length of the ameba, 130 microns.

The movement of particles on the under side of anAmoeba sphaeronucleosusdepends 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 differentfrom 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.

Figure 20.Amoeba sphaeronucleosus.a, the under side of the ameba. The part of the ameba attached to the substratum is stippled. Particles attached to the surface film atxmoved slowly forward.b, the under side of the ameba, showing the attached parts stippled. The particles suspended in the water atxmoved slowly forward with the ameba.c, a cross section of an ameba of shape shown inb, showing the ridges on the surface. Length of the ameba, about 100 microns.

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

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 asproteusare found in a limax or clavate shape. One of the most favorable of the large amebas in this respect isdiscoides. It is frequently found in clavate shape and it possesses the further advantage in being nearly cylindrical incross 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 indiscoidesin the clavate shape, holds likewise for free pseudopods extended into the water out of contact with a solid support (Figure 22).

Figure 21. Illustrating the similarity of the movement of the surface layer ofAmoeba verrucosawith that ofA. sphaeronucleosus. A group of three particles, connected by dotted lines for reference, change their relative positions as the ameba (verrucosa) changes its direction of movement. Length of the ameba, 150 microns.

Figure 22. Illustrating the movements of anAmoeba proteus, after Dellinger. Atcin stage 2 a pseudopod is projected which fastens itself to the substratum as shown atc, 3, whilea, 2, is pulled loose. In 4 another pseudopod is projected which fastens itself atd. The ameba is not in contact with the substratum at all points on its under side.

In figure 23 is shown a clavatediscoideswith 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 insphaeronucleosus. 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 againthe 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.

Figure 23. Showing the movement of a particle on the surface layer of anAmoeba discoides. The particle remained on the anterior end of the ameba for several minutes after stage 13. The ameba was about 320 microns long.

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 inFigure 17by 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.

Figure 24. Showing the effect of a narrow anterior end on the rate of movement of the surface. Length of the ameba, about 320 microns.

The movement of the third layer inproteusis 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 aproteusin 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 insphaeronucleosusandverrucosa, where the anterior edge was wider than any other part of the body. Butdiscoides, although free from the ridges and grooves characteristic ofproteus, 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 25shows aproteusand a narrow anterior end inproteuswith 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 toretract. 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.

Figure 25.Amoeba proteus.Rate of movement of the surface layer as compared with the rate of movement of the ameba. The pseudopod on the right was extended to stage 5; from then on it was retracted, as indicated by the outlines. Length of the ameba, 400 microns.

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 indiscoides, 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 aproteusare continually competing with the anterior endin their pull upon the surface layer, thus preventing rapid forward movement.

Figure 26. Showing the comparative rate of movement of the surface film over the retracting parts of the ameba. In figures 2 to 8 only a part of the ameba is shown. Length of the ameba, 500 microns.

Figure 26shows 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 27shows that the relative positions of particles attached to the surface layer may readily change while the ameba deploys its psuedopods. Three particles markeda,b,cand connected

Figure 27. A part of anAmoeba proteusillustrating what is perhaps the most characteristic quality of the surface layer of amebas, its fluid nature. Three particles,a,b,c, were moving forward along an actively growing pseudopod. In stage 2, particlesbandchad arrived nearly at the tip of the pseudopod. A pseudopod was then thrown out on the right, which resulted in the movement ofain the same direction, whilebandcremained nearly stationary. Later on this pseudopod was retracted.bandcwere drawn back toward the main body of the ameba whilecremained behind, moving only very slowly. Thus the relative positions of these particles was completely changed.

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 particlecgained slightly onaandbfor 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 sizewhile streaming in the original pseudopod was arrested. At stage 3 particlesaandbretained the same position they had in stage 2, except for a slight turning to the right. Particlechowever moved across the base of the original pseudopod and on to the middle of the new pseudopod. At stage 4aandbhad again only slightly moved to the right of the position they occupied in stages 2 and 3, whilecmoved 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. Particlesaandbnow gained considerably oncbecause they were located further away from the tip of the retracting pseudopod. Particlesaandbwere 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 particlecwith respect to that ofaandb. The latter particles retained their relative positions with very slight, if any, change, whilecswung aroundaandbnearly 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,aled. 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.

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 indicatesbeyond 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 likeAmoeba discoides,A. laureataand others in which the pseudopods are more nearly cylindrical. In such amebas asA. proteusandA. 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 asverrucosa, 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? Insphaeronucleosusand 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.

Inproteusordiscoides, 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 inannulata, but also inproteusand 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. Neverthelessthe 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 asphaeronucleosuson the upper surface move from 2.5 to 3.6 times as fast as the ameba (Figure 19) while particles attached to adiscoidesmove only from 1.2 to 2 times as fast as the ameba moves. Inproteusthe 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 betweensphaeronucleosusanddiscoidesto be explained? There are no ridges to retard the movement of particles indiscoides, while there are ridges insphaeronucleosus, where the particles move on the average twice as fast as ondiscoides. In the first place the advancing edge, the edge where ectoplasm isbeing made, is proportionately much wider insphaeronucleosusthan indiscoidesas 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 ofsphaeronucleosusis attached to the substrate, so that the surface layer which flows toward the anterior end is derived almost wholly from the upper surface; while indiscoidesthe 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 asphaeronucleosusis 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 knownsphaeronucleosus, verrucosaand 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 asphaeronucleosusthan 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 ofsphaeronucleosuswere 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 indiscoides, 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 ofsphaeronucleosusmakes such computation subject to the possibility of considerable error. Indiscoideshowever 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.

Figure 28. A clavateAmoeba discoides, showing the amount of ectoplasm that is constantly being made at the anterior end. Length of the ameba, 310 microns.

Since the movement of the surface film is obviously a surface phenomenon, only the surfaces of the amebas need to be taken into account. InFigure 28is illustrated adiscoidesof 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 surfaceattached to the substratum.)Sphaeronucleosusstands in contrast withdiscoidesfor 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 inFigure 14cut by outline 2 indicates, as indiscoides, 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 is1/5.8of 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 insphaeronucleosusas indiscoideswhen 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 insphaeronucleosusas indiscoides.

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 asdiscoides, and even under ordinary conditions of locomotion where there is occasional attachmentto 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;—adiscoides, 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 asphaeronucleosus, 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 toincreasethe surface tension at somepoint 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 movestowarda 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 surfacelayer 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 layerof 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 theplasma membraneor 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 theprinciple 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.

The observations recorded in the preceding two chapters, while they do not tell us anything about the direct cause of the movement of the surface layer, nevertheless indicate clearly enough that the area where ectoplasm is made is the area toward which the surface film flows. There is no question therefore of the intimate relation between the transformation of endoplasm into ectoplasm and the movement of the surface layer.

The apparent absence of movement in the surface film of the pseudopods ofDifflugia(Schaeffer, ’17) and the definitely proved absence of movement in the surface layer in the foraminiferanBiomyxaand myxomycete plasmodia, where no ectoplasm is formed in the manner observed in amebas, also indicates a causal relation between movement of the surface layer and ectoplasm formation. The relation moreover seems to be a necessary one for the movement of the surface layer is contrary to the processes involved in locomotion. In other words, from the standpoint of the ameba, it is a “necessary evil,” so far as locomotion is concerned.

The transformation of endoplasm into ectoplasm is unfortunately not understood, though from what we know in a general way of the behavior of colloidal solutions it seems to be a surface tension effect due to (or accompanying) a change of phase. Something akin to gelation occurs as Kite (’13) has shown. It is a problem in the chemistry of colloids. But the structure or composition of the protoplasm is complex and practically unknown, and it is quite open to criticism whether analogies from the behavior of pure solutions of colloids, such as gelatin, afford any real basis for an explanation.

Although a knowledge of the movements of the surface layer is interesting enough by itself, it will achieve its true importance only when it can be related to other processes in the ameba in acausal manner. It does not at present give us any greater insight into the ultimate cause of ameboid movement, although it is clear that an important step in this direction has been taken. But no theory of ameboid movement can be accepted that demands conditions in the ameba that are contrary to those described in the preceding chapters, in connection with the surface layer. From this point of view therefore the discovery of the true nature of the outside surface of the ameba is of importance, for it widens to a very considerable extent the observational basis with which any theory of ameboid movement must conform. Since the properties of the outer layer are here described in detail for the first time, it becomes necessary to enquire to what extent the more commonly held theories of ameboid movement conform with the observed behavior of the surface film. Although the surface tension theory was the first detailed theory proposed toward an explanation of ameboid movement, I shall discuss Jennings’ (’04) observations on the movements of the ameba first, because a great part of his work deals with the movements of the surface film, although he did not recognize it as distinct from the ectoplasm in its movements.

It is generally recognized that Dellinger’s (’06) work proved that Jennings’ conception of the ectoplasm as a permanent skin in which the ameba rolls along, is probably inadequate for such amebas asproteus; though singularly enough it is still supposed thatverrucosaand its congeners move in the way described by Jennings (Hyman, ’17, p. 83).

Jennings (’04) describes the movements of amebas, bothproteusandverrucosa“types,” as follows:

“In an advancing Amoeba substance flows forward on the upper surface, rolls over at the anterior edge, coming into contact with the substratum, then remaining quiet until the body of the ameba has passed over it. It then moves upward at the posterior end, and forward again on the upper surface, continuing in rotation as long as the ameba continues to progress. The motion of the upper surface is congruent with that of the endosarc, the two forming a single stream (p. 148).

“We have demonstrated above, for Amoeba at least, that the forward movement is not confined to a thin outer layer, but extendsfrom the outer surface to the endosarc; in other words that the outer surface moves in continuity with the internal substance (p. 150).

“There is no regular transformation of endosarc into ectosarc at the anterior end. On the contrary the ectosarc here retains its continuity unbroken, moving across the anterior end in the same manner as across other parts of the body. In the same way the ectosarc is not regularly transformed into endosarc in the hinder part of the body.... Such transformation is by no means a regular accompaniment of locomotion” (p. 174).

According to Jennings, locomotion is aided by the projection of waves of hyaloplasm at the anterior edge, “an active movement of the protoplasm of a sort which has not been physically explained.” These waves, attaching themselves to the substratum, enable the ameba to pull itself along by a rolling movement as described in the quotation above.

As to the rate of movement of the outer surface as compared with that of the endoplasm, Jennings concluded:

“The direction of movement of particles on the outer surface is the same as that of the underlying particles of endosarc. The rate is also about the same as for the endosarc, though often, or perhaps usually, the outer particles move a little more slowly than those in the endosarc” (p. 142).

In view of the observations recorded in the preceding pages it is clear that Jennings’ statement that substance after moving forward on the upper surface, rolls over the anterior edge is quite erroneous. The attached particles, if heavy, may do so, but the surface film itself does not. It is, on the contrary, taken into the interior at the anterior edge.

The statement that the movement of the outer surface is congruent with that of the ectoplasm can likewise not be substantiated by observation, as has been demonstrated in the preceding pages. It is difficult to distinguish between the ectoplasm and the surface layer in such amebas assphaeronucleosusandverrucosa, for there are no large crystals or other bodies which get caught in the ectoplasm as it is formed from endoplasm at the anterior end. But attentive observation will demonstrate very definitely that the ectoplasm here is stationary to the same degree as inproteus.The stationary properties of the ectoplasm are however not properly a matter for discussion; for five minutes’ observation of aproteus,discoides,annulata, particularly alaureata, under 300 diameters magnification, will convince anyone that the ectoplasm is stationary while the surface film, with attached particles, moves over it. No one can possibly come to any other conclusion. Jennings’ conclusion was due undoubtedly to an error of observation.

Jennings’ statement that the rate of movement of the outer surface is the same as that of the endoplasm (p. 142) when taken in connection with his other statement that the ectoplasm is a more or less permanent skin, presents a mechanical impossibility; for unless the outer surface movestwice as fastas the endoplasm, no rolling movement would be possible. Several of Jennings’ figures (especially Figures 38, 39, and 41) indicate in fact that he conceived of the outer surface as moving faster than the ameba advances, or that the upper surface movesover the amebaas the ameba moves over the substrate. Jennings’ theory requires that the surface layer move twice as fast as the ameba advances. Hyman (’17) also makes a similar mistake in referring to the rate of movement of the outer surface (p. 85).

Lest the discussion of this point be suspected of being merely verbalistic, it should be recalled that the surface layer ofproteusoften moves at about thesame rateas the ameba; that the surface layer ofdiscoidesmoves abouttwice as fastas the ameba; that the surface layer ofverrucosaandsphaeronucleosusmoves aboutthree times as fastas the ameba; and that the ectoplasm does not move at all. It is of course incumbent on one to discuss what is stated; one is not at liberty to select one of several possible interpretations.

To illustrate this point graphically so as to avoid as far as possible future confusionFigure 29is appended. Inais shown a particle traveling on an ameba at the same rate of speed as the ameba; atbis shown a particle that moves twice as fast as the ameba; atcthe attached heavy particle does not move at all. For the sake of completenessd,Figure 29, is added here. It illustrates the backward moving ectoplasm in an ameba that is suspended in a jelly medium that prevents the ameba from sinking to the bottom. It must be admitted that in thus consideringthe rate of movement of the various tissues of the ameba from a single standpoint, a point outside of the ameba, little room is left for confusion.

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