On Pelomyxa Viridis, Sp. N., and on the Vesi- Cular Nature of Protoplasm

ON PELOMYXA VIRIDIS. 357

On Pelomyxa viridis, sp. n., and on the Vesi- cular Nature of Protoplasm. By AlfreJ Glblb* Bourne, D,Sc.(Lond,), C.M.Z.S., F.li.S., fellow of University College, London; Fellow of Madras University ; Professor of Biology in the Presidency College, Madras.

With Plate XXVIII.

MY assistant, M.R.Ry. A. Sambasivan, B.A. (Madras), during the course of an exhaustive examination of the fauna of a small tank (pond) in the neighbourhood of the Presidency College discovered and drew my attention to numerous green " egg"like " bodies, of about -fa inch in diameter, which were to be seen on the surface of the finely divided mud. These bodies proved upon examination to be Rhizopods be- longing to the remarkable genus Pelomyxa, and forming a new species of that genus, but differing in some important particulars from all hitherto described Rhizopods. I propose to call the species P. viridis. The species is peculiarly interesting not only on account of its great size (it is larger than any known form of the Lobosa), but also on account of the presence of chlorophyll and symbiotic bacteria,1 and the assistance which this renders in the study of its protoplasm. 1 The rod-like bodies here regarded as bacteria were described as crystals of unknown composition by Greeff in his account of Pelomyxa palustris. Leidy describes them as exhibiting transverse striations. The symbiosis of bacteria and amoeboid Protozoa has a special importance at the present moment in connection with MetschnikolPs doctrine of Phagocytosis. 858 ALFRED GIBBS BOURNE. Habitat and General Description. I have only found P. viridis in one tank, and this a tank which has not been known to dry up for many years. When the mud from this tank is placed in a dish with a little water and allowed to stand, several individuals of P. viridis may generally be found close to the surface of the mud ; if these are removed and the mud stirred up and again allowed to stand, it is not usual to find many other specimens. So that I believe that this Bhizopod lives close to the surface of the mud. I have never seen it crawling on the sides of the dish or on any other object; nor have I ever seen it much spread out while in the mud, but when mounted on a slide with a cover-glass it readily spreads itself out and becomes very flat, and soon commences to exhibit active amoeboid movements. Pig. 1 represents a mounted specimen, partially spread out and magnified about four diameters. It is difficult to convey any idea of the size to which such an organism may attain, as so much depends upon the extent to which it is spread out. I have seen specimens so much spread out that they would average as much as £ inch in diameter. The specimens are of very varying sizes, but I have never found very small specimens ; I have never, in fact, been able to find a specimen by the aid of the microscope which I had not previously picked out with the naked eye, although I have searched many slides of the mud for this purpose. It may be that small individuals are to be found at some other time of year, or it may be that I have not, in spite of my search, happened to alight upon a small specimen. "When viewed by reflected light individuals vary a good deal in colour, from the rich transparent green of a gelatinous lichen to that of a faded leaf. I presume that the chlorophyll undergoes some degeneration. I have never found any approach to the red colour found in Euglena or Haemato- coccus. There is often a whitish, more opaque-looking spot, such as has been shown in the middle of fig. 1. This is caused by an aggregation of sand particles, and it is often more clearly defined than in my figure. When seen by transmitted light ON PELOMTXA VIEIDIS. 359 with a low power snch regions naturally appear dark, but with polarised light they appear as bright spots, owing to the doubly refracting character of the sand particles. A number of these particles is always present, but at times their number is so enormous that there is almost as much sand as protoplasm. I have watched an individual so filled collect the greater number of these particles at one spot, and then simply pour them out from the protoplasm. An individual may thus get rid of hundreds of these particles in a minute or two, but the process of collecting them together preparatory to their extrusion is slow. I believe that there is a periodical whole- sale extrusion of these particles. These particles have all the appearance of sand ; they are of very varying size and of very irregular shape, and are insoluble in all ordinary reagents; they are crystalline in nature, and, as stated above, are doubly refracting. The presence of these particles has not, I fancy, any special significance, they occur in great numbers in the mud and are taken in with the food ; the animal, indeed, seems to exercise no discretion as to what it takes in. They become collected together as a mechanical result of slight movements, and wheu the animal makes some more extensive movements are thrown out in great numbers.

Structure of the Protoplasm. Viewed with an ordinary high power of the microscope the main mass of the organism appears of a green colour, while at the periphery, from time to time, perfectly colourless regions may be seen. One's first impression is that there is a green endoplasm and a colourless exoplasm (figs. 2 and 3). With the same magnifying power, in addition to the various protoplasmic contents described elsewhere, what appear to be granules may be recognised abundantly distributed through the greater mass of the protoplasm. The colourless peripheral regions above mentioned are usually devoid of these granules. The granules all prove upon further examination to be bacteria, and but for their presence the protoplasm itself would appear VOL. XXXII, PAET III. NEW SER. A A 360 ALFRED GIBBS BOTTKNE. absolutely non-granular. Exceedingly careful and repeated observations with a Powell and Lealand's -fa inch oil immersion objective, Abbe's sub-stage condenser, and Engelmann's dark chamber, which last is invaluable in the glare usually pre- valent here, have enabled me to ascertain with great accuracy and clearness the real structure of the body substance. The protoplasm is throughout a perfectly colourless and apparently homogeneous substance, and this substance, except in the peripherally placed portions which flow out from time to time to be sooner or later reabsorbed into the central mass, is densely packed with spherules of a semi-fluid stroma, impregnated with what I have ascertained to be chlorophyll, and to these chlorophyll-bearing spherules the green colour of the organism is due. In these spherules we have not to do with chlorophyll- corpuscles, as they exhibit no phenomena of division, and are, moreover, more fluid than chlorophyll-corpuscles. Their fluid character often becomes very evident after the death of the organism, when some of them may be usually observed to run together into larger masses. Where these spherules are packed closely together they assume the form of regular polyhedrons. They seem about as fluid as the stroma of human red blood-corpuscles. I have come to the conclusion that they correspond to the vacuoles or, as they are better termed, to distinguish them from other vacuoles, vesicles, which occur in so many specimens of protoplasm and give to such specimens a vesicular character. The fact that they contain in P. viridis a substance im- preguated with chlorophyll, a state of things hitherto un- observed, has enabled me to throw some new light upon their nature and upon the organization of the body substance. The word protoplasm is used above in the sense in which Biitschli1 uses the word plasma. It designates the substance which Leydig speaks of as the spongioplasma, as distinguished from hyaloplasma (chylema, Strasburger). In other words, I consider that the substance within the vesicles is to be 1 Bronn, 'Protozoa,' p. 1392. ON PELOMYXA VIRIDIS. 361 regarded as an entoplastic product of the protoplasmic activity rather than as any portion of the protoplasm itself. The chlorophyllogenous stroma occupying the vesicles of P. viridis is no more entitled to be regarded as a portion of the protoplasm than are chlorophyll bodies or oil-globules, or any other structures ordinarily spoken of as protoplasmic contents. It is, of course, the presence of the chlorophyll which leads me to this conclusion. If I am correct in regarding these chlorophyllogenous structures as the homologues of the vesicles found in other protoplasms, the condition of P. viridis lends strong support to Biitschli's theory that the plasma is the substance which forms the envelopes of these vesicles only, or, as Butschli has termed it, the substance forming the scaffolding of a honeycomb (Substanz des Wabengerusts), and does not include their contents (Wabeninhalt), The structure of P. viridis is, on the other hand, rather opposed to Biitschli's most recent views with regard to protoplasmic movements. If I understand rightly from the abstract available to me, Butschli believes that a movement starts by the bursting of some vesicle, a phenomenon which would naturally escape observation in ordinary protoplasm, but of which, if it is constantly occurring, I should surely have seen something in a protoplasm whose vesicular contents are brightly coloured. With regard to the homology which I have assumed to exist between the vesicles of ordinary protoplasm and those of P. viridis with their chlorophyllogenous contents, it is quite clear, in the first place, that these are the structures which render the protoplasm of P. viridis vesicular. There exist, it is true, in addition to them a number of vacuoles of varying sizes, but of these there are after all. comparatively few, and if they alone were present the protoplasm could not be called vesicular. There is no evidence that these vacuoles contain any substance other than water. We can undoubtedly distinguish in P. viridis between two varieties of non-contractile fluid-containing spaces (nicht contractile Fliissig-Keitsraume), the vacuoles containing water and the vesicles having chlorophyllogenous contents. 362 ALFRED GIBBS BOURNE. The vesicles vary very little in size, being always about E 0*0 0 inch in diameter. The vacuoles, on the other hand, vary greatly in size, some being almost as small as the vesicles, while others attain five or six times that diameter. No distinction has, as far as I am aware, been hitherto drawn between vacuole and vesicle in any so-called " vesicular" or " vacuolised" protoplasm. Biitschli,1 writing ten years ago, says of non-contractile vacuoles, " Seltener hingegen wird ihre Zahl so betrachtlich, dass das sie trennende Plasma nur noch ein Maschenwerk von Scheidewanden zwischen ihnen herstellt, das Plasma eine schaumige oder alveolare Beschaffenheit annimmt. Ein derartiges Verhalten begegnet uns z. b. gewohnlich bei Pelomyxa, auch bei gewissen Amb'ben tritt ahnliches mehr oder weniger deutlich hervor." Of Biitschli's most recent views lean only judge from M. Yves Delage's2 note on the models of protoplasm with which Butschli has been recently experimenting, and if I understand rightly in, for instance, the soap and xylol model, the plasma (Biitschli) is represented by the soap and the chylema (Stras- burger) by the xylol. So that the contents of both vacuole and vesicle would be termed chylema, but there is no word as to the possibility of the chylema being other than a single substance. Now, in P. viridis, two distinct substances take the place of the xylol of Biitschli's model, the contents of the vacuoles and those of the vesicles, and were it not for the presence of the chlorophyll in one of them these would not be optically distinguishable one from the other. Two species of Pelomyxa have been hitherto described, P. palustris, Greef,3 and P. villosa (Amoeba villosa), Leidy.* The protoplasm of both these species has been de- 1 Bronn, ' Protozoa,' p. 101. 3 ' Arch. Zool. Experimentale,' 1889. 3 'Arch. f. mikr. Anatomie,' Bd. x, 1874. 4 'Fresh-water Rhizopods of North America,' Washington, 1879. ON PELOMtfXA VIRTDIS. 363 scribed as highly vesicular, and Gruber,1 in speakiug of P. villosa, which he found in Germany, says, " Zunachst fallen die zahlreichen Fliissigkeitsvacuolen ins Auge, welcher den grossten Bestandtheil des Korpers ausmachen und dem- selben ein Schaumiges Aussehen verleihen. Diese Vacuolen, die von wechselnden Umfange Sind, liegen eingebettet in dem homogenen Plasma, welches tnehr oder weniger feine Scheidewander zwischen ihnen bildet, ahnlich eine Intercellu- larsubstanz zwischen den einzelnen Zellen eines Gewebes." I think it probable that in these forms we have both vacuoles and vesicles, but as the vesicles are filled with colourless substance no distinction has been drawn between them. It will be interesting to re-examine these forms to ascertain if from the relative size and frequency any distinction can be drawn. The importance of the question lies in this, that the vesicles have probably an intimate relation to the structure of the protoplasm, and possibly to the production of amoeboid movements; while the vacuoles vary from time to time in size and number, and have nothing to do with the ultimate structure of the protoplasm. Fig. 6 represents a fragment of the protoplasm in a specimen which was deeply stained with osmic acid. At x is seen pro- jecting at the surface a small portion of the hyaline protoplasm, devoid of all contents, stained brown and rendered granular by the osmic acid. The rest of the drawing shows the vesicles once occupied by the chlorophyllogenous substance bounded by the protoplasm. The chlorophyll has been dissolved, but whether any substance now remains in the vesicles I cannot determine; there is nothing which is stained by osmic acid. With all other fixing reagents which I have used the vesicular structure disappears. The protoplasm of P. viridis appears then to be perfectly homogeneous, and small portions of it may at times be observed at the periphery of the organism free from all contents, but the great mass of it forms a mere scaffolding for the numerous vesicles, and is, moreover, densely packed with bacteria 1 ' Zeit. f. w, Zoo!.,' Bd. xli, p. 189. 364 ALFRED GIBBS BOURN Jl. ('' crystals " of Greef), to say nothing of its various other contents. The vesicles contain a fluid substance impregnated with chlorophyll. The vesicles and the bacteria are to be regarded as bodies contained in the protoplasm, and the latter may flow out leaving all its contents behind. When the protoplasm does flow out in this way some of the bacteria soon follow, and may then be seen to start an active movement; and if the outflow continues, the superficial vesicles leave the central mass and may be seen isolated in the hyaline protoplasm (fig. 5).

Protoplasmic Movements, Pseudopodia. Specimens of P. viridis will often remain for a long time absolutely quiescent; in such a condition the animal is, if not flattened by a cover-glass, fairly spherical in shape, and the green vesicles extend close up to the periphery. They always remain embedded in the protoplasm, so that they do not actually come to the surface j and, indeed, there is at the surface something more than the mere envelopes of the most superficially placed vesiculae, or else the margin of an optical section would present a sinuous curve, the sinuosities of which corresponded to a row of vesicles, which is not the case. In specimens where movements are taking place these movements are usually very active, and most so at the periphery, although they are by no means confined to that region. In this matter P. viridis agrees with other species of Pelomyxa.1 Long-continued movements often take place without the protrusion of any pseudopodia. Owing to its great size, P. viridis, and, indeed, other species of Pelomyxa, move about from place to place, at any rate while under observation, less frequently than smaller amoeboid organisms. When the movements, which result neither in the protrusion of a well-marked pseudopodium nor iu a translation of the organism, are taking place, the margin of an optical section consists of a hyaline layer, the movements in which have a wave-like, undulating appearance. It looks as though the 1 Bronn, 'Protozoa,' p. 97, foot-note. ON PELOMYXA VIBID1S. 365 hyaline protoplasm protruded at some one spot, leaving all its contents behind, and then spread out laterally, while a sharply defined line seems to mark the original boundary; this line afterwards disappears, giving one at first the impression that the protoplasm, which has, so to speak, overflowed the original boundary but remained in contact with it, is separated from the rest of the protoplasm, except at the spot whence it actually protruded, by a sort of pellicle, which is subsequently dissolved. Gruber1 has figured such an appearance for P. villosa, and says, " Wenn an einem ausgetretenen Protoplasmafortsatz die Vacuolen und die Kornchen einige Zeit durch eine scharfe Linie von der Sarkode getrennb bleiben, so beruht dies darauf dass das Pseudopodium durch Zusammenfliessen des Plasmas ueber diese Stelle hinentstand, und dass die feine peripherische Schicht von Protoplasma, welche vorher die Grenze gegen das umgebende Medium bildet, und welche jetzt von der Masse des Pseudopodiums umflossen wird noch eine Zeit lang er- halten bleibt;" and regards this as further evidence of the existence of a relatively hard cuticular layer or pellicle formed by the action of the water, a view which the same author put forward on a previous occasion.3 I cannot accept this interpretation of the facts. I do not believe that the hyaline protoplasm is protruded at one spot only, and that there is lateral displacement of its particles; the undulating appearance is chiefly caused, like other wave movements, by extrusions and retractions in a radial direc- tion. The appearance of the boundary line may be explained by careful focussing, the line being at some slightly different level from the protruding protoplasm, and its disappearance being due to a subsequent protrusion at that particular level. I am not at all sure how far the existence of any such pellicle as Gruber describes is in accordance with the views as to the relation of protoplasm to imbibition water.3 If the density of the surrounding medium is increased, as 1 ' Zeit. f. w. Zool.,' fid. xli, 1884, pi. xiii, fig. 2, and p. 190. 3 'Zeit. f. w. Zool./ Bd. xxxvi, 1882, pp. 457—469. 3 See Engelmann on " Protoplasmic Movement," in this Journal, vol. xxiv, 1884, p. 390. 366 ALFBEJD GIBBS BOURNE. by the addition of a 1 to 2 per cent, solution of common salt, the protoplasm shrinks, owing to withdrawal of imbibition water. This might, of course, take place through the pellicle, but this is not probable, as such a layer must be impermeable, or difficultly permeable, to water, if it have the function of protecting the central protoplasm from the action of water; and, in any case, when a sudden shrinking took place, one might expect to see some crenation of the surface, such as is exhibited by a human red corpuscle when it is exposed to a medium denser than its own plasma. No such crenation takes place; but, on the other hand, some portions of the protoplasm usually stick to the cover-glass and become torn off, while some of the protoplasmic contents escape into the water and are carried away by the current. I have at times observed a similar phenomenon when a long pseudopodium was quickly withdrawn. A specimen of P. viridis, which has been moving about under a cover-glass, habitually leaves some portions of itself sticking to the glass. Appearances, such as are shown in fig. 2, constantly occur where a pseudopodium is retreating, and are due to the sticking of the superficial protoplasm to the glass. I have also constantly seen villi, such as G-ruber1 has figured for P. villosa, formed in the same manner. Such phenomena seem to me to render the existence of any pellicle unlikely. Moreover, as will be described below, when a specimen of P. viridis is torn into pieces with needles the water seems to penetrate and cause almost instant disintegration. Now, if the protoplasm of these forms is in the habit of forming a pellicle directly any new surface is exposed to the water, why does it not do so in this case ? It appears more probable that so long as the protoplasm is alive the amount of imbibition water which can be taken in is regulated, and that in the instance above cited the protoplasm is killed by a shock caused by the teasing, so that excess of water can at once penetrate and cause disintegration. The pseudopodia are usually very blunt and lobose, and 1 ' Zeit. f. w. Zool.,' Bd. sli, pi. xiii, fig. 3. ON PELOMYXA VIRIDIS. 367 resemble those figured for P. palustris. When one of these large pseudopodia is about to be extruded the movements affect very deep-lying portions of the protoplasm; a wave-like bulging occurs, which gradually pushes out in a radial direc- tion ; an active current passes outward along its centre; all the protoplasmic contents in the neighbourhood are carried outwards and, having arrived at the extremity, pass back again in the peripheral portions of the pseudopodium, which thus narrows while it is elongating. The whole process gives one strongly the impression that the impulse for the extrusion is central and deep-lying in its origin. It is not that a small pseudopodium is gradually increased in size, but the mass of protoplasm extruded is actually greatest at first and then gradually diminishes. Even when fully extruded the backward peripheral currents still continue, especially in the proximal portion of the pseudopodium, which thus becomes flask-shaped, joined to the main portion of the organism by a narrow neck. All this time no peripherally placed colourless layer appears, the vesicles and other protoplasmic contents being pushed on up to the surface; but when this pseudopodium commences to retreat the contents pass backward faster than the protoplasm itself, and a colourless superficial layer is left. Fig. 3 represents the distal extremity of a pseudopodium which has just commenced to retreat. If this withdrawal is rapidly continued some of the colourless protoplasm will be left sticking to the glass. Besides these large pseudopodia, small ones are often found which seem to take their origin in the superficial layer. In these latter the hyaline protoplasm is first protruded, and into this a number of the bacteria appear to burst out, followed almost at once by some of the vesicles, which often separate a good deal from one another aud appear as so many isolated spherical green droplets; these are sometimes followed by the other protoplasmic contents (fig. 5). These pseudopodia are never prolonged to any extent. The rate at which the large pseudopodia are protruded is 368 ALFRED GIBBS BOURNE. considerable. I have found it to be about 075 mm. a minute. Engelmann1 states that a velocity of 0*5 mm. a minute is sometimes attained by an Amoeba, and may be con- sidered as exceptionally rapid. I have made my observations at the ordinary temperature of the room; but this is about 30° C. at this time of year, and is thus not far below the optimum temperature for protoplasmic movement.

Nuclei. The number of nuclei present is enormous. I have en- deavoured to estimate the number on stained preparations. I have made these preparations in various ways; one of the most satisfactory is to fix and harden on the slide with chromic acid (2 per cent, solution), followed by water and alcohol of increasing strengths; stain with picro-carmine, wash, and again treat with alcohol of increasing strengths; dehydrate, clarify, and mount in Canada balsam. I put down the number of nuclei present in a large individual at 10,000. They vary a little in size, but average, when living, about 0'03 mm. in diameter. It has struck me that there may be some connection between the bulk of nuclear matter and the bulk of protoplasm connected with it. I calculate that in P. viridis all the nuclei taken together occupy -gij of the total bulk of the organism. I have not many data at hand for comparison with this calculation, but in a mammalian ovarian ovum the nucleus occupies about ^ of the bulk of the ovum. So that 10,000 nuclei in P. viridis give relatively about the same bulk of nuclear matter as is found in a mammalian ovum. The nuclei closely resemble the nuclei of other species of Pelomyxa. In a fresh condition they are spherical, and consist of a nuclear membrane containing a perfectly clear and transparent fluid nuclear substance, in which are seen from nine to twelve highly refracting spherical nucleoli (fig. 7"). 1 L. c, p. 379. ON PELOMTXA. VIRIDIS. 369 I have never seen these enlarged or containing anything like a vacuole, a condition described by Greef for P. palustris; nor have I ever seen anything in the protoplasm of P. viridis resembling the " glanz-korper " of that author, or which might be nucleoli escaped from a nucleus. I have frequently seen nuclei extruded from the protoplasm, usually along with food debris or sand particles; when so extruded they remain for some time unacted upon by the water, but in time the water seems to penetrate the nuclear membrane, and the nucleoli exhibit Brownian movements, and after some time come out into the water (fig. 9) ; but although I have watched them for a long time I have not observed them to undergo any change. I have been unable by the use of any preservative or staining process to make these nucleoli behave as does the single nucleolus of so many Amoebae. This latter when treated with osmic, chromic, or picric acids, and subsequently with picro- carmine or alum carmine, stains deeply, while the nuclear substance remains clear and transparent, almost unstained. But when any one of these acids or even acetic acid is allowed to act upon the nuclei of P. viridis, the nuclear substance becomes opaque and granular, and the nucleoli disappear from view on account of the now opaque character of the nuclear substance. When stained, either by osmic acid or by picro- carmine or alum carmine (preceded by fixing and hardening reagents), and mounted in Canada balsam, they appear as quite different bodies from the fresh nuclei. To such an extent is this the case, that until I had actually watched a single nucleus throughout the entire process I was not satis- fied that the bodies which I had taken to be nuclei in the fresh state were actually such bodies. What really happens is, according to the views of Pfitzner and others, that the chromatin substance, which is something distinct altogether from the nucleoli, is brought into view by the staining process, and obscures to a certain extent the nucleoli. The nucleus stains diffusely all over, while very numerous minute granules appear to stain more deeply. 370 ALFRED GIBBS BOURNE. I have even after most careful examination of specimens, mounted whole and of sections, been unable to find any cases of nuclear division. Other Protoplasmic Contents. I have already spoken of the chlorophyllogenous substance, the permanent vacuoles, and the sand particles. In addition to these, I have observed in many individuals small globular refractive bodies, about i6*00 inch in diameter, which, from the fact that they are blackened by osmic acid, I suspect to be of a fatty nature. They are much more numerous at some times than at others. I have on two or three occasions found all the specimens then examined crowded with large (about -%fa inch in diameter) disc or lens-shaped, fairly transparent, but not highly refracting bodies, which, from the fact that all these specimens had been feeding on some special substance, I believe to be nutritive bodies of some kind. I have very little doubt from the appearance of the debris that the food on these occasions had been Spirogyra filaments, but I was never fortunate enough to see them in the protoplasm in an undigested state. The protoplasm in all these individuals was simply crammed with the food debris. It is probable that these specimens had got hold of some Spirogyra, of which there was a little on the surface of the mud, and that it had proved a very easily digest- ible food, and that a great deal of nutriment had become stored up in the protoplasm. The bodies under discussion stain a rich deep colour with iodine, but do not lose the colour on warming. They are probably some amyloid substance. P. viridis takes in plenty of solid food of all kinds, Daph- nise, Ostracods, Diatoms, Naids, &c. I have also found specimens which had taken in two or three or more plants of what I believe to beWolffia arrhiza (one of the Lemnacese), and which swarms in most of the tanks in Madras, and is by- the-bye often eaten, curiously enough, in enormous quantities by the tank frogs. ON PBLOMTXA VIEIDTS. 371 The food sometimes lies in food vacuoles, while at other times the surface of the food particles is actually in contact with the protoplasm. Occasionally I have observed, when a large piece has lain in a large vacuole, pseudopodia protruded from the vacuolar wall and reflected over the surface of the food particle.

Physiology. I have tried but without success to demonstrate the activity of the chlorophyll. The animal does not readily allow of much experiment, as it almost always dies after any manipula- tion. Treatment with iodine has, moreover, failed to demonstrate the presence of starch. The enormous number of the symbiotic bacteria is correlated doubtless with the activity of the chlorophyll. I have tried by putting them together under a cover-glass to get two individuals to fuse together, but without success. On the other hand, I have often observed two pseudopodia of the same individual form and fuse together at their distal extremities, so as to leave for a time a hole right through the animal. Further, I have several times cut a specimen in half with a sharp scalpel, and kept the two halves living for some time, and by placing the two halves close together have induced them to fuse together again. I have also teased specimens into fragments with a pair of needles, but in that case the fragments have at once commenced to die. No information which we at present possess seems to me to explain in any way why the halves of a divided individual or the peripheral extremities of long pseudopodia protruded by any one individual should freely reunite, while separate individuals will do nothing of the kind. Gulliver's Views.—Since writing the above my attention has been drawn to a note on the minute structure of Pelo- myxa palustris by Dr. G. Gulliver.1 The author believes, in the first place, that the exoplasm is permanently differentiated 1 'Journal of the Royal Microscopical Society,' 1888, p. 11. 372 ALFRED GIBBS BOURNE. from the endoplasm; and in the second place, that the " endoplasm " is composed of a number of cells. Both these are very startling views, and while I feel sure that the conclu- sions are erroneous, I have observed certain phenomena which may have given rise to them. Gulliver writes, " In the process of hardening this layer (the exoplasm) readily separates from the subjacent softer endoplasm." I have noticed in hardening specimens in a watch-glass for section-cutting, and also under the cover-glass for subsequent staining and mounting as whole preparations, that the peripheral portion which is first attacked by the hardening reagent often becomes separated from the still living and shrinking central mass, another layer is then attacked, and so the whole structure may harden in irregular layers. The hardened portions break away with extreme readiness from the unhardened ones. Very curious appearances are sometimes thus produced, but I am convinced that the phenomenon is a mere accident in the hardening process. The examination of living specimens can leave no doubt as to the absence of any permanent differ- entiation of the body substance into exoplasm and endoplasm. With regard to the structures which Gulliver takes to be cells building up the "endoplasm," and which Lankester has sug- gested may be swarm-spores, I have formed a less definite opinion. In a living specimen one can see no such structure, but in hardened specimens one does undoubtedly find small rounded portions of the protoplasm lying in and yet separated from the rest of the protoplasm. These bodies have, however, no definite and constant characters, they differ considerably in size, sometimes include one nucleus, sometimes more than one, and frequently none at all. It is difficult to detail the precise appearances which lead me to the conclusion; but I am much inclined to think that we have here merely an acci- dental rounding off of portions of the protoplasm, which takes place during the hardening process. I have seen similar phenomena in specimens which have died from having been kept mounted for a long time under a cover-glass. The body ON PELOMYXA VIEIDIS. 373 substance in these large Pelomyxse forms a considerable mass, and this breaks up very readily after death into small droplets. I find nothing in the structure of P. viridis which would lead one to suppose that these organisms have any affinities, other than those usually assigned to them. Pelomyxa belongs, as Butschli says, to the family Amoebsealobosa. In spite of repeated endeavours I am unable to throw any light upon the reproductive processes which may obtain in Pelomyxa.

EXPLANATION OF PLATE XXVIII,

Illustrating Professor Alfred Gibbs Bourne's paper on "Pelomyxa viridis."

FIG. 1.—An entire specimen, partially spread out under a cover-glass, magnified about four diameters. In the left-hand lower corner is a Daplinia shell, which the animal had just extruded. The opacity of the central region is caused by a great accumulation of mud or sand particles. FIG. 2.—Extremity of a blunt pseudopodium, which is retreating. Small droplets of the colourless non-granular protoplasm are being left sticking to the slide ; some of these will become detached as the pseudopodium retreats further. The magnification is not sufficient to allow of the nuclei and vesicles being distinguished, but a few of the larger vacuoles, v. »., are seen. FIG. 3.—A long thin pseudopodium in the act of retreating, drawn to an even smaller scale than the preceding figure. FIG. 4.—Extremity of a blunt pseudopodium, more highly magnified. The existence of the peripheral layer of colourless protoplasm indicates that the pseudopodium is not being actively protruded, while its small extent and comparative regularity shows that the pseudopodium is not being rapidly withdrawn, n. n. Nuclei. /. v. Food vacuole, containing food ddbris. s, Naid seta. m. m. Sand or mud particles, v. v. Vacuoles. The small circles which are shown all over the figure, except in the colourless peripheral portion, represent the vesicles with their chiorophyllogenous contents. In places 374 ALFBED GIBBS BOURNE. where, as over the food vacuole, marked f. »., there is only a single layer of these vesicles, the green coloration is very faint. FIG. 5.—Portion of the periphery of an individual where there has just been a slight outflow of colourless protoplasm, and which will be withdrawn without the protrusion of any pseudopodium. The bacteria (? crystals) seen coming out into the protruded protoplasm exhibit active (? Brownian) movements. The green bodies represent the vesicles; two of these are seen coming out into the protruded protoplasm. FIG. 6.—View of a region precisely similar to that drawn in the preceding figure, in an individual which has been treated with osmic acid. The colourless protoplasm has stained; while the vesicular contents, now no longer green, are not to be seen. It is seldom that the vesicular structure is so well seen; usually, upon the application of reagents, the vesicles seem to collapse, and so no such network as is here shown can be seen. FIG. 7-—View of a small portion of the central region of an individual, showing the same structures as in Fig. 4, with the addition of the bacteria. Three nuclei are drawn with their contained nucleoli. FIG. 8.—A nucleus with an unusually large number of nucleoli. FIG. 9.—A nucleus which has been extruded with food debris, and which has swollen under the action of the water, while its nucleoli have burst out. N.B.—Figs. 5—9 are drawn to about the same scale as the line A B at the bottom of the plate, which represents x$s§ of an inch. A-t:Av<™. Tc/JWlHS.rf/ mil..

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