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, im- pregnated 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 scaffold- ing 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.

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