DURING the summer of 1938, while examining material collected from

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1 Amoeba taylorae n.sp. 375 By CATHERINE HAYES, S.N.D., B.Sc, PH.D. (From the Research Department of Notre Dame College, and the Zoology Department, Glasgow University) SUMMARY In material collected from Tannoch Loch, Dunbartonshire, a large free-living, hitherto undescribed amoeba was found. Individual specimens vary in length from 420 to 500/x and in width from 70 to 140/x. Viewed over a black background this amoeba looks dense, white, and opaque, while in transmitted light it has a dusky, almost black, hue, due to the presence in the endoplasm of a large number of small crystals uniform in size, slender and pointed at both ends. There is always a uroid at the hinder end surrounded by bits of debris. The one large nucleus is without a karyosome but has masses of chromatin scattered through the nuclear substance. As a rule there is only one large contractile vacuole, but occasionally a few small ones may be seen at the hinder end. A certain number of nutritive spheres are always present. The author considers this amoeba to be a new species and names it Amoeba taylorae. SOURCE OF THE MATERIAL DURING the summer of 1938, while examining material collected from Tannoch Loch, Dunbartonshire, Scotland, and its outflow stream at Milngavie, I came across a large amoeba which even at a mere glance appeared to differ widely from A.proteus, A. dubia, A. villosa, and A. nobilis, all of which had previously been identified from this source by Sister Monica Taylor and all of which were known to me. A large number of A. villosa, a considerable number of A. nobilis, and a few A. proteus were present in the particular lot of material, but there was not the slightest difficulty in distinguishing the new amoeba, for under the low power of the Greenough binocular over a black background it looked dense, white, and opaque in contrast with the somewhat translucent appearance of the other amoebae. When placed on a slide and viewed under an 18 mm. or 4-4 mm. objective it was equally distinct, being of a dusky, almost black, hue. As will be explained later, these appearances are due to the presence in the cytoplasm of a very great number of minute crystals. Throughout the summer of 1938 a few specimens of the amoeba could be found in every lot of material collected from this particular source. In 1939 I have records of its occurrence in March, April, July, and October, while it was absent from what little material I was able to collect during 1940, 1941, and In February 1943 I had one specimen from material collected on the 2nd and one from material collected on the 10th. One individual was seen in the spring of DESCRIPTION OF THE AMOEBA This creature, one of the larger free-living, freshwater amoebae, has often to be freed from adhering debris with a pair of fine needles. If sometimes it [Quarterly Journal of Microscopical Science, Vol. 96, part 3, pp , 1955.]

2 376 Hayes Amoeba taylorae n.sp. should happen to be spread out more or less fan-like when found, it begins rapidly to assume a very characteristic shape when placed on a slide. The shape referred to is elongated, narrow at the hinder end, and gradually widening out in front into a single broad, flat, directive pseudopodium (fig. 1, A). The tip of this pseudopodium often bifurcates (fig. 1, B), sometimes re- FIG. 1. A, a living A. taylorae, showing the uroid, contractile vacuole, nucleus, a great number of small crystals in the endoplasm, and a considerable number of nutritive spheres. B, outline of A. taylorae, to show debris attached to the uroid, the bifurcation of the anterior pseudopodium, and the direction (shown by arrows) of the cytoplasmic stream. maining divided for a considerable time, sometimes returning to the original condition almost at once, while less often the whole of the cytoplasm flows into one limb, the other meantime disappearing, thus restoring the more normal monopodial condition. As a rule there is not more than this one large pseudopodium, there being very little tendency to form lateral pseudopodia. The average length of the amoeba when it has assumed this very usual shape is about 420/x, though this varies greatly from individual to individual, as I have had specimens measuring nearly 500/i. My records show the width to vary from about 70 to 140ju..

3 Hayes Amoeba taylorae n.sp. 377 At the hinder end there is always a well-marked uroid (fig. 1, A and B), -which never appears elsewhere in this amoeba, a fact differentiating it from A. villosa in which a uroid may appear at any point of the ectoplasm. A considerable amount of debris, consisting of small pieces of decayed vegetable matter, long strips of Oscillatoria, diatoms, &c, is generally carried with the uroid, and it often looks as if most of this debris consists of the waste indigestible parts of the food (fig. 1, B). In accordance with the fixed position of the uroid at its posterior end, the amoeba never, so to speak, flows backwards; that is, the posterior end never becomes directive and anterior. When about to change direction the amoeba does so by letting the anterior tip of the pseudopodium flow to one side or the other or by pushing out a large lateral pseudopodium into which all the cytoplasm flows; thus the direction may change through a right angle, but the original uroid persists and is still posterior. The cytoplasm and its inclusions The cytoplasm consists of endoplasm and ectoplasm, the latter being very clear and fairly broad, especially at the anterior end where it pours out in wave-like fashion as the creature flows along. The endoplasm is somewhat coarse and granular, but it has a rapid, easy flow, especially down the central region of the body. This is indicated by arrows in fig. 1, B. There are no ectoplasmic folds and therefore the amoeba does not belong to the genus or sub-genus Chaos, which is characterized by such folds (Schaeffer, 1926), but to Metachaos, as do A. discoides and A. kerrii. The uroid consists of fine ray-like prolongations of the ectoplasm (fig.i, A and B). Embedded in the cytoplasm are: 1. The contractile vacuole There is one large contractile vacuole which occasionally may grow to a great size, 70, 80, or even 90/11. It is generally situated at the hinder end of the amoeba near the uroid, and is always, even when it is well forward, behind the nucleus. In other large amoebae, e.g. A.proteus, A. discoides, A. lescherae, the contractile vacuole is often alongside, under, overlying, or anterior to the nucleus, as both these structures are carried along in the cytoplasmic stream. The contractile vacuole always bursts near the uroid, and does so very gently; and I have noticed on several occasions that it does not disappear entirely, a very minute droplet remaining to initiate the next vacuole. Although there is only one large contractile vacuole, it often happens that a number of small vacuoles lie beneath the uroid near the large vacuole; these may burst separately and vanish, or one or other of them may increase in size and become the main contractile vacuole. Surrounding the contractile vacuole is a well-defined layer of material of unknown composition and function, which stains slightly with ordinary nuclear dyes. In A. proteus, A. discoides, and A. lescherae (Taylor and Hayes, 1944) this layer is in the form of blocks resembling in appearance the chromatin blocks of the nucleus.

4 cytoplasm, has an average diameter of about 30/i, though individual nuclei may be somewhat less or considerably greater in diameter (fig. 2, A and B). Its shape seems to be that of a biconvex lens and hence it appears circular when viewed from above and elliptical when seen in side view. The ground-substance of the nucleus is coarse and gives the impression of being viscid and stiff, while the chromatin masses are large and conspicuous and, as there is no karyosome present, they are scattered evenly through the nuclear substance, only occasionally appearing as a definite layer underlying, cytoplasm ':,*&? "' _ v.yvv'<vr '-W^ nuclear, membrane ; ' e.?, s~- tf K^ layer of...chromatin blocks C= in ^ chromatin blocks Scale of figs. A and B FIG. 2. A, nucleus of ordinary size, with very thin nuclear membrane and chromatin blocks scattered throughout the nuclear substance, B, a very large nucleus with only a trace of the nuclear membrane, but with a distinct layer of chromatin blocks round the periphery. Fig. 2 was drawn from specimens fixed in Bouin's fluid and stained in Ehrlich's haematoxylin and light green. A Zeiss apochromat 2 mm. oil immersion objective was used. the nuclear membrane (figs. 1, A, and 2, B). Fixed and stained preparations reveal the nuclear membrane to be thin (fig. 2, A). The nucleus is often observed at the posterior end of the creature, whence it may move slowly forward to about the middle of the body; here it generally remains stationary while the cytoplasm flows past it rapidly, thus restoring it to its original posterior position. The slow movement of the nucleus through the rapid cytoplasmic stream and its ability to remain at rest in this stream is indicative of its viscid and rather solid nature. The nuclei of A. proteus, A. discoides (Hayes, 1938), A. lescherae (Taylor and Hayes, 1944), and A. kerrii (Taylor, 1947), which appear to be more fluid, are carried along rapidly in the cytoplasmic stream and rarely remain stationary. I have not been able to procure a sufficient number of specimens to work out the nuclear division. 3. Nutritive spheres A certain number of nutritive spheres are always present (fig. 1, A). Generally these are small, but in two or three individuals examined the spheres

5 Hayes Amoeba taylorae n.sp Crystals The most striking feature of this amoeba is the very great number of small crystals which it normally contains (fig. 1, A). As already mentioned, it is the presence of these crystals that causes the amoeba to appear opaque in reflected light and dark in transmitted light. Most of the crystals are slender and sharply pointed at both ends, but the points and edges of many of them are rounded so that they appear elliptical. As in the other amoebae, the crystals dissolve in the process of fixing and dehydrating. Food of the amoeba As far as I have been able to ascertain, this amoeba is not a diatom-feeder, though the material from which I have obtained it is exceedingly rich in many forms of diatoms and swarms with A villosa, a heavy diatom-feeder; it is often possible to count as many as 20 diatoms (of various varieties) in the cytoplasm of one individual. Flagellates and ciliates seem to be the principal food organisms. Occasionally, however, especially in the early part of the year, the amoeba's numerous food-vacuoles contain spherical bodies, golden brown in colour. This is also true of A. nobilis and A. villosa, and especially A. hugonis. After it was ascertained that these bodies were not encysted spores, the material was submitted to Professor E. G. Pringsheim, who identified the spherules for Sister Monica Taylor as consisting of a very hard core of a chemical substance that he could not identify, covered with a layer of carotene. Pringsheim and Horasse (1950) show that masses of pigment of varying sizes having the same composition as the eye-spot occur in a variety of E. gracilts. Pringsheim expressed the view that the cultures containing Euglena thus affected were not in a healthy condition. The question arises, how do the amoebae obtain these spherical bodies? Do they ingest the E. gracilis, partly digest it, and get rid of the carotene bodies by defaecation? Sister Monica Taylor (1952) could not decide the question for A. hugonis. She never found the golden spherules apart from the living animal. The fact of its not being a diatom-feeder is beneficial from the point of view of bringing the amoeba under laboratory culture conditions, as flagellate and ciliate food is much more easily produced all the year round in the laboratory than are diatoms. Life-history Not finding the amoeba in large numbers has prevented me from collecting any facts about its life-history, but I have had two or three small amoebae which might very well be its young stage. I hope to evolve a technique for keeping the amoeba under laboratory conditions and so be able to elucidate its life-history. DISCUSSION ON THE IDENTIFICATION OF THIS AMOEBA When consulting the literature with a view to the identification of the amoeba just described I was impressed by the resemblance between it and

6 380 Hayes Amoeba taylorae n.sp. an amoeba identified in Kansas by M. Anthony Payne (1930) as A.granulosa. A sub-title to her paper records the fact that this is the third record of the appearance of A. granulosa, i.e. 'Griiber's Rare Ameba'. Gruber's record was made in 1885, and he gave the name to a small amoeba ('von ungefahr 0,03 mm. Durchmesser'), of which he gives very few details. The second record of this amoeba was that of Penard in A glance at Gruber's figure was sufficient to convince me that the amoeba which I have described is not Gruber's 'Rare Ameba'. Penard's (1902) account is much fuller than Gruber's (indeed, I fail to see how Penard could have thought that the amoeba he described was identical with Gruber's A. granulosa), and many points, such as the size, the character of the crystals, and the general appearance of the amoeba caused by these crystals are in accordance with my description, but he states that his amoeba has a spherical nucleus and large pseudopodia in different parts of the body, while he makes no mention of the presence of a uroid. Payne's specimens were smaller than Penard's, yet she agrees with him on other details except that she says that few lateral pseudopodia are formed during locomotion and emphatically adds that a 'uroid is not present and the ameba never carries debris'. Both these authors figure their amoeba and, while the general appearance and in particular the anterior portion of Payne's fig. 5 does resemble my amoeba, the posterior portion of all her figures and the whole of Penard's figure differ from my findings. Cash (1905) describes an amoeba from Hale Moss, Cheshire, which he calls A. proteus var. granulosa, but from his figures it is quite evident that my amoeba bears little resemblance to it and even less to Leidy's (1879) fig. 4, pi. I, which Cash considers to be the same species as his. Payne considers that Cash's figures were drawn from A. proteus or A. discoides and in this I agree with her, especially in view of the fact that under certain physiological conditions both these amoebae may be very much packed with crystals. The 'mulberry-shaped caudal extremity', which Cash refers to and figures, is not a uroid but a temporary condition of the ectoplasm, such as may often be seen in all the large amoebae with which I am acquainted. To sum up, although resembling A. granulosa in some respects, the amoeba described in this paper differs from it in having a biconvex nucleus, a permanent posterior uroid, and a great tendency to the monopodial condition when creeping. In view of these outstanding characteristics and believing that it has not hitherto been recorded, I propose to call the present species Amoeba taylorae, in dedication to Sister Monica Taylor, who has devoted the research of her lifetime to the genus Amoeba, its species, habits, and culture. DIAGNOSIS This amoeba was found in a freshwater loch to which there was a constant inflow of clean water and a similar outflow. The loch was also well stocked with various kinds of water weeds. In size the amoeba may reach 500/x long by 140p. at the broadest part. The shape is generally elongated and monopodial,

7 Hayes Amoeba taylorae n.sp. 381 the anterior end being broader than the rest of the body. The posterior end always possesses a uroid which generally has debris attached to it. The ectoplasm is fairly voluminous, especially at the anterior tip. The endoplasm is packed with small, slender-pointed crystals and hence is very dark in colour. There is one large contractile vacuole, which generally occupies a posterior position; occasionally there may be a few small vacuoles near the uroid. The one large nucleus (about30/x in diameter) is biconvex; it has no karyosome but blocks of chromatin are scattered through it. Sometimes a layer of chromatin blocks lines the thin nuclear membrane. There are no longitudinal folds in the ectoplasm; in this it resembles A. discoides and A. kerrii and differs from A. proteus and A. lescherae. It belongs to Schaeffer's group Metachaos. I consider it a new species and name it Amoeba taylorae. REFERENCES CASH, J., The British freshwater rhizopods and Heliozoa, 1, London (Ray Society). GROBER, A., 'Studien iiber Amoben.' Zeit. wiss. Zool., 41, 186. HAYES, C, Quart. J. micr. Sci., 80, 459. LEIDY, J., Freshwater rhizopods of North America. Washington (Government Printing Office). PAYNE, M. A., 'Metachaos granulosa in Kansas; third recorded appearance of Griiber's rare Ameba.' Kansas (University Science Bulletin). PENARD, E., Faune rhizopodique du Bassin du Letnan. Geneve (Libraire de l'lnstitut). PRINGSHEIM, E. G., and HORASSE, R., Arch. Zool. exp. gen., 86, 499. SCHAEFFER, A. A., 'Taxonomy of the Amoebas.' Publ. Carnegie Inst., 24, No TAYLOR, M., and HAYES, C, Quart J. micr. Sci., 84, Ibid., 88, Ibid., 93, 427.