I Have adopted the name “Pseudospora volvocis” for the protozoon here discussed, as the two most obvious phases correspond with the creature described under that name by Cienkowski.
Pseudospora was noticed amongst Volvox, supplied by Mr. T. Bolton to the Cambridge laboratory and, later on, to Glasgow. As the creature showed features of considerable interest and the knowledge of its life history appeared to be very imperfect, it seemed advisable to make it the subject of special investigation.
I wish here to acknowledge my great indebtedness to Professor Graham Kerr. This paper, which was begun at his instigation, would certainly never have been completed but for his kind supervision and guidance.
All the processes described were followed out on the living specimens and checked by stained preparations. The investigations were made for the most part on material mounted on slides under cover-slips supported with wax. It was impossible, however, to keep slides of this description under observation for more than twelve to eighteen hours, as after that period the Pseudospora invariably died. Cultures in watch glasses, even when kept in a damp chamber, were not altogether satisfactory, partly owing to the Volvox not thriving in the small volume of water, partly on account of the inroads of bacteria. I finally found that Pseudospora could be got to live quite normally for a week to a fortnight on slides with a circular moat hollowed out round the part where the object to be observed was laid. Spirogyra was placed in the moat, and the whole covered with a supported coverslip. The edges of the coverslip were then sealed up with vaseline.
The preserved preparations, with very few exceptions, were stained under the coverslip and mounted in balsam. The stains used were : Ehrlich’s haematoxylin, iron haematoxylin (Heidenhain’s), borax carmin, picrocarmin, safranin, and Romanowski’s stain. Paracarmin was tried, but never gave good results. Orange G., methylene blue, and eosin were also not, as a rule, very successful.
Mitosis was followed out on corrosive material stained with Ehrlich’s hæmatoxylin and checked by osmic material stained with Ehrlich’s hæmatoxylin and picrocarmin. For the observations on the development of spheres, Romanowski, safranin> and Ehrlich’s hæmatoxylin were used, the best results being obtained with Romanowski, which proved, however, to be an exceedingly difficult stain to use.
Pseudospora volvocis was first named by Cienkowski in 1865 (‘Archiv f. mikr. Anat.’, vol. i, p. 214) ; he described a flagellate and an amoeboid form, and also a definite doublewalled cyst, surrounded by a gelatinous veil. As will appear later, I have been unable to find this encysted form. Bütschli in Broiin’s ‘Klassen und Ordnungen,’ 1883, places Pseudospora amongst the Isomastigoda, but does not describe its life history.
Zopf, in his work on ‘Die Schleimpilze,’ mentions it under the name of Diplophysalis volvocis, and refers to Cienkowski’s paper. The next reference is in Klebs’ ‘Flagellaten Studien,’ 1892, where he points out the ambiguous systematic position of the genus. The group to which Pseudospora belongs seems from the literature somewhat neglected since Zopf’s work on ‘Die Schleimpilze.’
Pseudospora Volvocis, Clenkowski
There are three adult forms : A. an amoeboid form ; B. a flagellate form ; and C. a radial form, with very fine pseudopodia. Oienkowski’s paper describes only the first two of these forms.
A. Amoeboid form. Size, ·012 mm. to ·03 mm. Structure
Ectoplasm.—In the amoeboid Pseudospora (fig. 1) there is a narrow band of not very markedly differentiated ectoplasm forming a comparatively firm outer layer which is capable of being prolonged from time to time into pseudopodial processes. The shape of the creature is very changeable and inconstant. The pseudopodia, which are sometimes very long, vary considerably in shape ; they can be extended from apparently any part of the animal, and, though seen to branch, do not anastomose. The pseudopodia are frequently prolonged into either very fine processes, in which case they are often arranged in bunches, as in fig. 2, or else are simply broad at the base and pointed at the end. Occasionally, however, blunt pseudopodia are met with. The various forms of pseudopodia merge into one another, and are capable of changing shape with considerable rapidity. The longer and more slender processes are in some specimens occasionally bent backwards and forwards after the fashion of flagella, but the movement is fitful and slow.
Endoplasm.—The endoplasm alters greatly in character according to the exact condition of the animal. In very young specimens, and in individuals which have been in the free-swimming state for a considerable time, the protoplasm presents a very homogeneous and hyaline appearance. If no food has been ingested for some time, the endoplasm is usually somewhat grey in colour, with highly refractive granules, apparently of stored-up food material ; the number and size of these are quite inconstant. After feeding, however, many large green and brown masses are to be seen in the endoplasm. The green particles are the still undigested Volvox individuals, the brown, those which are in process of digestion. Definite food vacuoles do not seem, as a rule, to be formed in the amoeboid Pseudospora, that is to say, the outline of the vacuole is so close to the food particle as to be indistinguishable. Sometimes the creatures are so densely filled with food particles as to appear quite green.
Very bright spherical particles, which are, as a rule, to be seen in Pseudospora individuals, which have sojourned for some time in the Volvox colonies, appeared, upon treatment with osmic acid, to be globules of some fatty substance. Two or three contractile vacuoles are present; these are, however, not very conspicuous in either the amœboid or the flagellate forms.
Nucleus.—The resting nucleus (figs. 1 and 2) measures about ·0046 mm. to ·0057 mm. in diameter. It is a single, well-defined body lying in the centre of the creature. It is bounded by a fine membrane staining with chromatin stains. Inside the membrane lies a deeply-staining spherical body or karyosome surrounded by a very definite clear space. In preserved specimens the karysome stains with Romanowski’s stain, safranin, Ehrlich’s haematoxylin, Heidenhain’s iron haematoxylin, borax carmin, and picrocarmin. The chromatin lies diffused through the karyosome, which presents, in the resting state, an almost homogeneous appearance. The karyosome is produced into fine rays, which pass to the nuclear membrane, these stain somewhat less intensely than the karysome.
B. Flagellate form. Size, ·012 mm. to ·03 mm
The flagellate Pseudospora is an oval, oblong or pearshaped creature bearing two flagella at one end (fig. 3) ; these are usually equal in length, though in many cases one is shorter than the other. The flagella are comparatively thick and do not taper at the end ; they are two or three times the length of the individual. A slight depression is in some cases to be seen at the point of insertion of the flagella. This is, however, not by any means a constant feature. In the pear-shaped individuals there is often a blunt process about one third of the way from the flagellate end. The ectoplasm, endoplasm, and contractile vacuoles show no special features. The nucleus is identical with that described in the amoeboid form, and is situated immediately behind the insertion of the flagella.
Method of swimming.—These flagellate forms swim with one flagellum dragged behind—in the cases where the flagella are unequal the longer one—the other is lashed out in front. The movement of the front flagellum varies slightly; usually the flagellum starts from a position in a straight line with the longitudinal axis of the animal, it is rapidly lashed to one side, and then slowly returns to its original position. This may be repeated either first on one side and then on the other or over and over again in the same direction. When the flagellum is used in the latter way the creature tends to swim in a circle, the flagellum which is dragged behind correcting this to a certain extent. The flagellum is sometimes passed round the individual, causing it to revolve round its longitudinal axis. Occasionally both flagella are used with a very rapid vibratile movement.
C. Radial form. Size, ·012 mm. to ·02 mm
The radial Pseudospora (fig. 4) differs considerably in its external features from the two previously described forms. It is normally a spherical creature with fine radial pseudopodia; these spring most frequently from all parts of the animal, though they are at times confined to certain parts. The pseudopodia are sometimes three or four times the length of the diameter of the creature, and are not always equally fine.
While moving about either in the Volvox colony, or in the free condition, the creature frequently temporarily adopts a spindle shape (fig. 5) with a number of long pseudopodia at each end, the pseudopodia elsewhere being usually, but not invariably, withdrawn.
Occasionally the radial Pseudospora in the colony assumes a semi-amoeboid appearance, the pseudopodia being for the most part withdrawn. In this condition it could not be distinguished from an individual in the amoeboid phase, which possesses fine pseudopodia arranged in bunches, were it not that here the amoeboid shape is very transitory, the creatures soon again becoming radial.
The protoplasm of the radial form is on the whole more homogeneous and translucent than that of either the flagellate or the amoeboid form ; the contractile vacuoles are very large and conspicuous; four, and even five, are to be seen in the creature at one time. Occasionally they arise very near the surface as in a heliozoon, causing a temporary protuberance, which disappears when the vacuole bursts.
In the radial phase the food particles are often contained in large vacuoles. The nucleus corresponds with that already described, but is usually somewhat smaller in size ; its average diameter is ‘0034 mm. The radial form is more passive than either of the other two forms; in the free state it floats much as an Actinosphærium does or creeps in the manner already described. It attacks and leaves the colony without losing the radial character. It ingests Volvox individuals either by engulfing them bodily, or by passing them down a broad pseudopodium, or by drawing them towards itself by two adjacent pseudopodia. The radial form merges into the flagellate and amoeboid forms, from which it differs merely in shape and method of moving. It is, however, so far as my experience goes, always the predominant form in a culture where the Volvox are not moving. If the culture continues to be fed on Volvox in this condition the flagellate forms disappear entirely. The radial Pseudospora divides by fission, often without withdrawing the pseudopodia.1
Some Pseudospora, usually transparent radial individuals, adopt a peculiar amoeboid form which has eruptive lobopod pseudopodia. I have never seen this except in cultures where the radial form predominated, and then only when most of the Volvox had been destroyed. It is quite possible that this is merely a pathological form due, perhaps, to the protoplasm becoming more fluid. This form occurs very rarely, but has been seen too often to be passed over without mention.
Life History and Habits
The amoeboid Pseudospora (A) may conveniently be taken as the starting point of the life history. This form is found in the Volvox colony, it creeps about on the outside, finally boring its way into the interior. The creature feeds upon the Volvox individuals either by surrounding them with broad protoplasmic processes or by engulfing them bodily. Sometimes a long pseudopodium is seen to surround a Volvox cell which is at some distance from the main part of the body ; the food particle is then either digested at the end of the pseudopodium or is passed along it into the interior of the animal.
Pseudospora individuals collect in masses round the young daughter colonies ; they are often to be seen lying in groups closely resembling the destroyed daughter colonies. Sometimes the Pseudospora attacks a segment as large as itself, slowly absorbing it or even creeping into its interior if it is very large (fig. 6).
As a rule the Pseudospora begins to feed at once upon arriving in the colony. One individual which I observed ingested no less than fifteen Volvox cells in two and a half hours. Nevertheless, starved or very young specimens will often lie in a colony for some time (six to seven hours) before beginning to feed.
When well established in the colony, Pseudospora divides about once in every twenty-four hours (at temperatures 6°—16° C.). When about to divide the creature withdraws its pseudopodia and becomes spherical in shape, and the food particles become arranged in a band round the centre. A constriction then appears and the animal divides in two, the daughter individuals usually lying in close proximity for some time after they are quite separate. Finally the creatures put out pseudopodia and creep actively away. The time elapsing between the withdrawing of the pseudopodia and the division of the animal varied between three quarters of an hour and an hour and a half in the specimens which I observed.
The first preparations for division of the nucleus (figs. 7-11) occur, just before the animal rounds itself off, in the breaking down of the nuclear membrane. The whole nucleus seems nevertheless to be still quite separate and clearly defined from the general cytoplasm. The chromatin at this stage begins to gather together into irregular masses, giving the karyosome a somewhat mottled appearance. The whole nucleus now increases in size and the rays become indistinct. The achromatic part of the karyosome can still be distinguished, while the chromatin seems to have segregated out from it, forming a number of small masses lying towards the equator of the nucleus. Careful examination of the nucleus at this stage (fig. 7) shows a roughly oval structure with faintly staining granules towards the periphery; these are possibly derived from the rays. Towards the equator of the oval lies the chromatin separated out from the karyosome.
In the next stage (fig. 8) the nucleus shows the completely developed spindle. There is now no sign of the faintly staining granules described in the prophase. The spindle seems to be formed entirely from the achromatic intra-nuclear elements. The chromatin has now formed separate chromosomes, which appear to be rod-shaped, though from their small size and highly refringent character it is difficult to make certain of this ; they are arranged round the equator of the spindle. The chromosomes now move apart (fig. 9 a), leaving the central fibres exposed. Finally the space occupied by these fibres is nipped across (fig. 9 b), and the two nuclei are completely separated. Each nucleus soon shows the karyosome (fig. 10), somewhat irregular in shape, mainly composed of deeply staining masses of chromatin, which appear to surround the remains of the achromatic spindle. The rays are not, as a rule, visible at this stage. Shortly afterwards the whole animal divides (fig. 11). This frequently occurs before the nuclei have quite reached the resting state.
After a time the Pseudospora leaves the colony (fig. 12) and swims away in the flagellate condition. If, as is often the case, the amceboid form has retained its flagella, it now withdraws its pseudopodia and becomes oval or oblong. In the case of the non-flagellate individuals, the flagella can sometimes be seen to develop gradually from fine elongated pseudopodia ; more often they seem to arise directly without any obvious pseudopodial stage. The shape of the animal is often very irregular for some time after the flagella are formed; finally, however, the creature completely withdraws its pseudopodia. The nucleus always comes to lie directly behind the insertion of the flagella.
The method of leaving the Volvox is very constant ; the creature approaches the periphery of the colony and pierces through the jelly by means of pseudopodia. The protoplasm then flows into the pseudopodia until the creature is hour glass-shaped ; finally it slips out, keeping the hour-glass shape until almost quite free. While in the free swimming state the creature is capable of becoming amoeboid and of again recovering its original shape without withdrawing the flagella.
Well-fed flagellate individuals can be seen to divide in the free state by transverse fission, but I have never seen this occur twice in succession in a specimen out of the colony, nor have I ever been able to observe it in creatures which had been without food for some time. Cold, or prolonged lack of food, causes the animal to withdraw its flagella, round itself off, and sink to the bottom of the pond, but I have never observed the formation of a very definite cyst.1
The flagellate Pseudospora soon attacks another colony, but not as a general rule so long as it contains green food particles. If water containing free-swimming individuals is put into a tube with healthy Volvox the first to attack are those which still contain brown particles. Those specimens, curiously enough, which are quite transparent owing to the absence of food particles, take from twelve to twenty-four hours, or sometimes even longer, before attacking the Volvox colonies. Pseudospora are often seen to attack both Eudorina and Pandorina even when Volvox are present, and on some occasions when starved they ingested small green algæ, but I have never seen Spirogyra or other filamentous algæ attacked. A starved Pseudospora sometimes ingests another Pseudospora which is densely filled with green food particles. This appears to be merely a process of feeding and to have no connection with either conjugation or association.
I have never observed the formation of a true plasmodium, but temporary fusion of the protoplasm only may occur between two or more individuals. In the cases I observed the creatures separated after about fifteen to thirty minutes. The process is very rarely to be seen, and occurs more frequently within the Volvox than in the free state. This might possibly be a step towards the formation of plasmodia. Protomonas and Protomyxa, nearly allied genera, form plasmodia, but they are not known to occur in either Vampyrella or Pseudospora aculeata. I have, however, not sufficient evidence to draw any conclusions as to the meaning of this phenomenon.
Pseudospora, when attacking the Volvox, attaches itself firmly to the colony by pseudopodia; these are extended apparently indifferently from the non-flagellate end of the creature or else from either side.
When newly arrived in the Volvox or while still on the outside the animal is very sensitive to any change of conditions ; for instance, rise of temperature, evaporation of the water, or stoppage of the motion of the Volvox will cause the creature to leave the colony; this is to be seen even in starved individuals.
The processes above described—alternation of the flagellate and amoeboid condition and reproduction by fission—continue for some time (fourteen to twenty-one days) and then a different form of reproduction appears.
Gametogenesis and Conjugation
In the amoeboid Pseudospora there are developed spheres of a clear greyish appearance. The number of these to be found in a single individual varies ; in one culture one- and two-sphered forms greatly predominated, in another I found individuals with three or four and on one occasion with eight spheres. The individual in which the spheres arise does not form any kind of cyst. The pseudopodia are not withdrawn; in some cases the flagella persist, and movement and feeding may still go on after the spheres are a considerable size. Finally, the protoplasmic body surrounding the sphere disintegrates, but the time at which this occurs varies greatly in relation to the state of development of the sphere. The process of sphere formation occurs both in the colony and in the free condition. These spheres are destined to give rise to the gametes.
Development of the Spheres.—In tracing out the development of the spheres it is more convenient to consider first the case of a single-sphered individual and thereafter to note the slight differences that occur in the cases where there are more than one sphere. The sphere arises directly from the nucleus (cf. figs. 13-19). In the very earliest stage the nucleus differs only from the resting nucleus in that the rays have become thicker and the membrane more distinct (fig. 13a). Later the whole nucleus increases in size and the karyosome assumes a somewhat eccentric position. The first signs of the sphere itself now begin to appear. The substance within the nucleus becomes differentiated—showing a different staining reaction, e. g. blue with Romanowski—to form a spherical mass which fills almost the whole nuclear space. The rays now appear as small rounded masses ; some of these are within the sphere ; the greater number, however, lie on the outside and seem rather to be connected with the membrane which at this stage appears surrounding the sphere with its enclosed karyosome (fig. 13 b than with the sphere itself. The karyosome gradually moves further from the centre until it finally comes to lie quite outside the sphere (fig. 14k). It appears to take no further part in the process. The position of the karyosome at any one moment bears no exact relation to the size of the sphere. It is sometimes to be seen lying within a comparatively large sphere, in other cases it is already on the outside although the sphere occupies little more than the space of the original nucleus. The size of the sphere has, however, no very constant relation to its state of development.
In the stage shown in fig. 14 the small chromatin masses derived from the rays have decreased in number. Those on the outside have as a rule disappeared, though in some cases they can just be distinguished as very minute particles. Those inside the sphere now appear as definite spherical masses ; in one case I could only count three of these.
A later stage (fig. 15) shows a very considerable increase in the nuclear material; about eight to thirty nuclei larger in size than the original masses can be seen in the sphere. I am unable to say how the increase in the number of nuclei takes place ; this much, however, seems certain that all the nuclear material of the sphere is derived from the thickened rays of the original nucleus. The sphere increases still further in size (fig. 16) and the nuclei break up apparently into minute particles, as for some time before segmentation begins they can no longer be detected in my preparations. When the sphere has reached its full size, ·007 mm. ·011 mm. in diameter, segmentation occurs. A constriction appears which divides the sphere into two equal segments (figs. 17-18). The parts, however, remain closely apposed to one another. Each of these now divides. After this the division of the segments is not quite regular, and the spherical shape is usually lost. Finally a very large number of segments are formed—in one case I counted a hundred and sixteen, and even in very small single-sphered individuals I have never found fewer than sixteen. The process of segmentation occupies as a rule from ten to thirty minutes. After the segmentation is complete (fig. 19) the segments lie motionless for a while, and then move a little apart before actually becoming motile. If the protoplasmic body which surrounded the sphere has not already broken down they pierce through it and escape. The segments set free are small oval or round uniflagellate gametes, varying in size from ·00116-·00186 mm. The flagellum is thick (average length ·0046 mm.) and slightly curled ; it arises from a point about half way from the anterior end and propels the animal forwards. Each gamete appears to possess a nucleus, that is to say, in stained specimens a spot that stains more deeply than the rest of the creature can just be discerned with the highest powers of the microscope. This probably is the nucleus, but I can say nothing as to its structure.
Shortly after becoming free the gametes fuse in pairs, forming zygotes with two flagella (fig. 20). Gametes arising from the same sphere conjugate together, but I have never seen this occur in all the spores from any one sphere ; some individuals were always seen to swim away singly. In colonies where the spheres of several individuals had segmented, I observed conjugation of gametes at some distance from the place of segmentation of the spheres, but had no means of making sure that the conjugating individuals had really arisen from different Pseudospora. Conjugation on one occasion certainly took place between individuals from different spheres which had arisen in the same Pseudospora. In this case the conjugating gametes were unequal in size, but that this was of no special significance was shown by later observations, in which the gametes were equal. On account of the small size of the nuclei and the difficulty of observation, I am unable to say anything about the fusion of the gamete nuclei.
In the many sphered individuals the essential processes of the development of the spheres correspond with those already described in the case of the single-sphered Pseudospora. Two or three spheres may arise inside the same nuclear space (fig. 21) ; this possibly is to be regarded as a precocious segmentation of the single sphere, though I am unable to say -what is the cause. In other cases the nucleus appears to divide before the formation of spheres, and from each of these nuclei is formed one or more spheres. Occasionally in these individuals only one of the nuclei becomes converted into a sphere, the other apparently disintegrating.
The cultures in which the process of sphere formation was observed were kept at an almost even temperature of 11°-13° C. and well supplied with food material. In two cultures of uninfected Volvox which were inoculated with sphere-forming individuals it was found that Pseudospora reproduced by fission for exactly fourteen days and then again formed spheres. In one culture this period repeated itself thrice in succession. In other cases the period varied from fourteen days to about twenty-one. The formation of gametes is often nearly synchronous throughout a culture, almost all the Pseudospora individuals breaking up within twenty-four to forty-eight hours.
The zygote derived from the fusion of the two gametes after a time withdraws the flagella and appears as a round transparent little creature, with a just discernible spot which, on’staining, appears as the nucleus. It now becomes amoeboid and creeps into a Volvox individual, where it feeds and increases in size ; it destroys the Volvox cell, and is to be seen lying in its place surrounded by the brownish-coloured débris of the chromatophore.
The small Pseudospora is usually spherical at this stage, and the protoplasm appears slightly granular. The creature now either becomes amoeboid again and invades another individual, or puts out flagella of the type found in the adult. In either case the animal developes directly into the adult, the development of the flagella in the former case being merely postponed for a time. The young flagellate individual becomes very easily amoeboid without losing the flagella.
The zygotes appear to have some considerable power of resisting unfavourable circumstances. Thus on one occasion I was able to start a culture from sediment containing them in which there had been no Volvox for about three weeks. The individuals which first appeared were the small flagellate and amoeboid creatures just described ; these gradually developed into the normal individuals.
If immediately after the formation of gametes in a culture of Pseudospora motionless Volvox be introduced, the zygote developes into the radial form instead of the amoeboid or flagellate. Here also, as in the amoeboid form, spheres are formed.
The nucleus of Pseudospora seems to me to indicate a condition intermediate between the centro-nucleus described by Keuten and the metazoa-like nucleus of Actinosphærium. It is a centro-nucleus in so far as the spindle apparatus is intranuclear, but the formation of the spindle and chromosomes shows a marked advance upon such forms as Euglena.
I have made no attempt to reconcile the formation of spheres as I observed it in Pseudospora with the sporocysts described by Zopf in Polysporella, Pseudospora aculeata, Vampyrella, and other forms. The true test of their similarity would lie in the relations of the nucleus in spore formation.
As to the behaviour of the karyosome in sphere formation Hertwig Archiv f. Prot.,’ vol. i, 1901) refers to a somewhat analogous form of nuclear multiplication where “in einem grossen, oft so gar riesigen Mutterkern zahlreiche Tochter-kernaulage entvvickelt werden, welche in dass umgebende Protoplasma heraustreten wahrend der Mutterkern zu grunde geht.” In sphere formation we have an essentially similar process, only here the cell bodies for the daughter nuclei are not derived directly from the maternal cytoplasm but from protoplasm built up within the original nuclear membrane. Part of the achromatic portion of the maternal nucleus appears indeed to become converted directly into a mass of rapidly growing protoplasm.
In Pseudospora the nucleus is more specialised than in the form described by Hertwig, and a definite part, the karyosome, is ejected when the nuclei of the spheres are formed.
The phenomena of sphere formation in Pseudospora serve to accentuate the close relationship between the achromatic part of the nucleus and the protoplasm of the surrounding cell-body.
The tendency of recent work on the Protozoa appears to be to accentuate the importance of Doflein’s main subdivision Plasmadroma, and, on the other hand, to point towards the possibly artificial character of the subdivisions of the Plasmadroma. Pseudospora with its amoeboid, heliozooid, and flagellate phases accentuates this. It seems clear that taxonomic distinctions resting on observations of anatomical features during one phase of the life history are so unreliable as to be almost worthless. Such characters are shown by the transformation of “species “of Amoeba into one another by slight modification of the external conditions to be of the most superficial kind, being mere morphological reflections of surrounding conditions, and of no phylogenetic weight. In classifying the Protozoa it is essential to have regard to the whole life history.
A Pseudospora individual may adopt three forms—an amoeboid, a flagellate, and a radial form. This last, at least, appears to be a direct reaction to external conditions.
A single nucleus is present. It is bounded by a membrane, which contains the karyosome surrounded by a clear space ; fine rays pass from the karyosome to the membrane.
The nucleus divides by mitosis. The chromatin forms chromosomes, which are apparently rod-shaped. The spindle appears to be formed from the achromatic intra-nuclear substance.
Pseudospora reproduces by fission. After fourteen to twenty-one days gametes are formed.
Gametogenesis. The nucleus of the Pseudospora becomes converted into a sphere, the nuclear substance of which appears to be derived from the rays of the original cell nucleus. The karyosome is extruded from the sphere.
The sphere segments to form a large number of gametes.
The gametes conjugate in pairs, forming zygotes, which develop into the adult Pseudospora.
Explanation of Plate 12,
Illustrating Miss M. Robertson’s paper, “On Pseudospora volvocis.”
The figures are drawn under Zeiss apochromatic homogeneous immersion objective, three-millimetre focus and compensating eye-piece No. 12.
Fig. 1.—Amoeboid Pseudospora, with short pseudopodia, n. Nucleus. k. Karyosome, c. s. Clear space round the karyosome, r. Rays passing from the karyosome to (ra.) the Membrane, f. Food particles.
Fig. 2.—Amoeboid Pseudospora, with fine pseudopodia arranged in bunches.
Fig. 3.—Flagellate Pseudospora,
Fig. 4.—Radial Pseudospora. c. v. Contractile vacuole, f. Food particle contained in a vacuole.
Fig. 5.—Transitory spindle shape adopted by a radial Pseudospora.
Fig. 6.—Pseudospora attacking an uusegmented Parthenogonidium (y;.).
Fig. 7.—Early stage of mitosis, ft. Karyosome, ch. Chromatin come together in irregular masses.
Fig. 8.—sp. Spindle, ch. Chromosomes arranged in an equatorial plate.
Fig. 9 a.—Chromosomes at separate ends of the spindle, c.f. Central fibres.
Fig. 9 b.—Nuclei reforming, c.f. Central fibres being nipped across.
Fig. 10.—Karyosome reforming, ch. Masses of chromatin.
Fig. 11.—Pseudospora immediately after division. n. Nucleus.
Fig. 12.—Pseudospora leaving the colony.
Fig. 13 a.—k. Karyosome, r. Thickened rays terminating at the nuclear membrane. The protoplasm has shrunk a little away from the nucleus.
Fig. 13 b.—k. Karyosome, ch. 1. Chromatin masses on the outside of the sphere, ch. 2. Chromatin masses inside the sphere, sph. Sphere, m. Membrane.
Fig. 14.—ft. Karyosome. n. Nuclear masses in the sphere : the karyosome is quite outside the sphere: the nuclear material has come together to form definite masses.
Fig. 15.—The karyosome is outside the sphere, which has increased in size, definite nuclei are to be seen in the sphere, m. Last vestige of membrane with small masses of chromatin which are greatly reduced in size.
Fig. 16.—Sphere shortly before segmentation.
Fig. 17 a.—Segmentation of sphere, two segments formed, protoplasmic body has not yet broken up.
FigS. 17 b and 18.—Segmentation becoming irregular.
Fig. 19.—Individual in which the gametes are already formed, although the protoplasmic body has not yet disintegrated.
Fig. 20 a.—Gametes.
Fig. 20 b.—Zygote with two flagella.
Fig. 21.—Pseudospora with three spheres arising from one necleus. k. Karyosome.
1 observed at different times a number of Pseudospora individuals—usually but not always of the radial type—which presented a peculiar and very evenly granular appearance.. Upon being watched for some time the Pseudospora broke up, setting free the granules which now moved rapidly about. In many cases the granules were in very active motion inside the Pseudospora for a considerable time before it disintegrated. When seen with the ordinary powers of the microscope, I took this process to be some form of spore formation. On referring to Dallinger and Drysdale’s work on the life history of Monads, I found that the process of spore formation there described appeared to be very similar to what I had observed in Pseudospora. Under the three-millimeter immersion objective, the granules appeared rod-shaped, and were seen to move in straight lines ; while progressing they turned slowly on their longitudinal axes.
In view of Schaudinn’s recent work on Trypanosoma the bacteria-like appearance of the particles was not in itself sufficient ground for attributing the phenomenon to the agency of parasites. Pinaily, however, after searching carefully through the cultures where these individuals were seen, I found that the rod-shaped bodies entered from the outside and multiplied so as to form a dense mass absorbing the protoplasm of the Pseudospora. They are, beyond doubt, parasitic bacteria.
I have been unable to find the double-walled cyst described by Cienkowski. The rounded-off Pseudospora appears to possess a more definite covering than the flagellate or amoeboid individuals, but nothing that in my opinion would justify the term cyst.
These rounded-off individuals disintegrate at once upon the drying up of the water, so far as my experience goes. This may, however, be due to the process having taken place too rapidly.