PSEUDOSPORA VOLV00IS, CIENKWSKl, 213

Pseudospora volvocis, Cienkowski.

ByMuriel Robertson,

Zoological Laboratory, University of Glasgow.

With Plate 12.

I HATE adopted the name " Pseudospora volvocis " for theprofcozoon here discussed, as the two most obvious phasescorrespond with the creature described under that name byCienkowski.

Pseudospora was noticed amongst Volvox, supplied byMr. T. Bolton to the Cambridge laboratory and, later on, toGlasgow. As the creature showed features of considerableinterest and the knowledge of its life history appeared to bevery imperfect, it seemed advisable to make it the subject ofspecial investigation.

I wish here to acknowledge my great indebtedness toProfessor Graham Kerr. This paper, which was begun athis instigation, would certainly never have been completedbut for his kind supervision and guidance.

All the processes described were followed out on the livingspecimens and checked by stained preparations. The investi-gations were made for the most part on material mounted onslides under cover-slips supported with wax. It was impos-sible, however, to keep slides of this description under obser-vation for more than twelve to eighteen hours, as after thatperiod the Pseudospora invariably died. Cultures in watchglasses, even when kept in a damp chamber, were not alto-

214 MURIEL ROBERTSON.

gether satisfactoi'y, partly owing to the Volvox not thrivingin the small volume of water, partly on account of the inroadsof bacteria. I finally found that Pseudospora could be got tolive quite normally for a week to a fortnight on slides with acircular moat hollowed out round the part where the objectto be observed was laid. Spirogyra was placed in the moat,and the whole covered with a supported coverslip. The edgesof the coverslip were then sealed up with vaseline.

The preserved preparations, with very few exceptions,wei-e stained under the covei-slip and mounted in balsam.The stains used were : Ehrlich's hfematoxylin, iron hsema-toxylin (Heidenhain's), borax carmin, picrocarmin, safranin,and Romanowski's stain. Paracarmin was tried, but nevergave good results. Orange G., methylene blue, and eosinwere also not, as a rule, veiy successful.

Mitosis was followed out on corrosive material stainedwith Ehrlich's heematoxylin and checked by osmic materialstained with Ehrlich's haematoxylin and picrocarmin. Forthe observations on the development of spheres, Romanowski,safranin> and Ehrlich's heeniatoxylin were used, the bestresults being obtained with Rornanowski, which proved, how-ever, to be an exceedingly difficult stain to use.

Pseudospora volvocis was first named by Cienkowski in1865 (' Archiv f. mikr. Anat.', vol. i, p. 214) ; he described aflagellate and an amoeboid form, and also a definite double-walled cyst, surrounded by a gelatinous veil. As will appearlater, I have been unable to find this encysted form. Biitschliin Bronn's ' Klassen und Ordnungen,' 1883, places Pseudo-spora amongst the Isomastigoda, but does not describe itslife history.

Zopf, in his work on ' Die Schleimpilze,' mentions itunder the name of Diplophysalis volvocis, and refers toCienkowski's paper. The next reference is in Klebs' 'Flagel-laten Studien,' 1892, where he points out the ambiguous sys-tematic position of the genus. The group to which Pseudo-spora belongs seems from the literature somewhat neglectedsince Zopf's work on c Die Schleimpilze.'

PSEUDOSPOBA VOLVOOIS, OIENKOWSKI. 2 1 5

PSEUDOSPORA VOLVOCIS, ClENKOWSKI.

There are three adult forms: A. an amoeboid form;B. a flagellate form; and C. a radial form, with very finepseudopodia. Cienkowski's paper describes only the first twoof these forms.

A. Amoeboid form. Size, "012 mm. to "03 mm.Structure .

Ectoplasm.—In the amoeboid Pseudospora (fig. 1) thereis a narrow band of not very markedly differentiated ecto-plasm forming a comparatively firm outer layer which, iscapable of being prolonged from time to time into pseudo-podial processes. The shape of the creature is very changeableand inconstant. The pseudopodia, which are sometimes verylong, vary considerably in shape; they can be extended fromapparently any part of the animal, and, though seen to branch,do not anastomose. The pseudopodia are frequently pro-longed into either very fine processes, in which case they areoften arranged in bunches, as in fig. 2, or else are simplybroad at the base and pointed at the end. Occasionally, how-ever, blunt pseudopodia are met with. The various forms ofpseudopodia merge into one another, and are capable ofchanging shape with considerable rapidity. The longer andmore slender processes are in some specimens occasionallybent backwards and forwards after the fashion of flagella,but the movement is fitful and slow.

Endoplasm.—The endoplasm alters greatly in characteraccording to the exact condition of the animal. In very youngspecimens, and in individuals which have been in the free-swimming state for a considerable time, the protoplasm pre-sents a very homogeneous and hyaline appearance. If nofood has been ingested for some time, the endoplasm is usuallysomewhat grey in colour, with highly refractive granules,apparently of stored-up food material; the number and sizeof these are quite inconstant. After feeding, however,

VOL. 49 , PART 1. NEW SERIES. 16

216 MURIEL EOBERTSON.

many large green and brown masses are to be seen in theendoplasm. The green particles are the still undigestedVolvox individuals, the brown, those which are in process ofdigestion. Definite food vacuoles do not seem, as a rule,to be formed in the amoeboid Pseudospora, that is to say, theoutline of the vacuole is so close to the food particle as to beindistinguishable. Sometimes the creatures are so denselyfilled with food particles as to appear quite green.

Very bright spherical particles, which are, as a rule, to beseen in Pseudospora individuals, which have sojourned forsome time in the Volvox colonies, appeared, upon treatmentwith osmic acid, to be globules of some fatty substance. Twoor three contractile vacuoles are present; these are, however,not very conspicuous in either the amoeboid or the flagellateforms.

Nucleus.—The resting nucleus (figs. 1 and 2) measuresabout "0046 mm. to -0057 mm. in diameter. It is a single, well-defined body lying in the centre of the creature. It isbounded by a fine membrane staining with chromatin stains.Inside the membrane lies a deeply-staining spherical body orkaryosome surrounded by a very definite clear space. Inpreserved specimens the karysome stains with Romanowski'sstain, safranin, Ehrlich's heematoxylin, Heidenhain's ironhaematoxylin, borax carmin, and picrocarmin. The chromatinlies diffused through the karyosome, which presents, in theresting state, an almost homogeneous appearance. Thekaryosome is produced into fine rays, which pass to thenuclear membrane, these stain somewhat less intensely thanthe karysome.

B. F l age l l a t e form. Size, "012 mm. to '03 mm.

The flagellate Pseudospora is an oval, oblong or pear-shaped creature bearing two flagella at one end (fig. 3);these are usually equal in length, though in many cases oneis shorter than the other. The flagella are comparativelythick and do not taper at the end; they are two or three

PSEUDOSPORA VOLVOOIS, OIBNKOWSKr. 217

times the length of the individual. A slight depression is insome cases to be seen at the point of insertion of the flagella.This is, however, not by any means a constant feature. Inthe pear-shaped individuals there is often a blunt processabout one third of the way from the flagellate end. Theectoplasm, endoplasm, and contractile vacuoles show nospecial features. The nucleus is identical with that describedin the amoeboid form, and is situated immediately behind theinsertion of the flagella.

M e t h o d of swimming.—These flagellate forms swimwith one flagellum dragged behind—in the cases where theflagella are unequal the longer one—tbe other is lashed outin front. The movement of the front flagellum varies slightly;usually the flagellum starts from a position in a straight linewith the longitudinal axis of the animal, it is rapidly lashedto one side, and then slowly returns to its original position.This may be repeated either first on one side and then on theother or over and over again in the same direction. Whenthe flagellum is used in the latter way the creature tends toswim in a circle, the flagellum which is dragged behindcorrecting this to a certain extent. The flagellum is some-times passed round the individual, causing it to revolve roundits longitudinal axis. Occasionally both flagella are usedwith a very rapid vibratile movement.

C. R a d i a l form. Size, -012 mm. to '02 mm.

The radial Pseudospora (fig. 4) differs considerably in itsexternal features from the two previously described forms.It is normally a spherical creature with fine radial pseudo-podia; these spring most frequently from all parts of theanimal, though they are at times confined to certain parts.The pseudopodia are sometimes three or four times thelength of the diameter of the creature, and are not alwaysequally fine.

While moving about either in the Volvox colony, or in thefree condition, the creature frequently temporarily adopts aspindle shape (fig. 5) with a number of long pseudopodia at

218 MURIEL ROBERTSON.

each end, the pseudopodia elsewhere being usually, but notinvariably, withdrawn.

Occasionally the radial Pseudospora in the colony assumesa semi-amoeboid appearance, the pseudopodia being for themost part withdrawn. In this condition it could not be dis-tinguished from an individual in the amoeboid phase, whichpossesses fine pseudopodia arranged in bunches, were it notthat here the amoeboid shape is very transitory, the creaturessoon again becoming radial.

The protoplasm of the radial form is on the whole morehomogeneous and translucent than that of either the flagel-late or the amoeboid form ; the contractile vacuoles are verylarge and conspicuous; four, and even five, are to be seen irithe creature at one time. Occasionally they arise very nearthe surface as in a heliozoon, causing a temporary protuber-ance, which disappears when the vacuole bursts.

In the radial phase the food particles are often containedin large vacuoles. The nucleus corresponds with that alreadydescribed, but is usually somewhat smaller in size; its aver-age diameter is -0034 mm. The radial form is more passivethan either of the other two forms; in the free state it floatsmuch as an Actinosphjerium does or creeps in the manneralready described. It attacks and leaves the colony withoutlosing the radial character. It ingests Volvox individualseither by engulfing them bodily, or by passing them down abroad pseudopodium, or by drawing them towards itself bytwo adjacent pseudopodia. The radial form merges into theflagellate and amoeboid forms, from which it differs merely inshape and method of moving. It is, however, so far as myexperience goes, always the predominant form in a culturewhere the Volvox are not moving. If the culture continuesto be fed on Volvox in this condition the flagellate forms dis-appear entirely. The radial Pseudospora divides by fission,often without withdrawing the pseudopodia.1

*I observed at different times a number of Pseudospora individuals—usuallybut not always of the radial type—which presented a peculiar and very evenlygranular appearance.. Upon being watched for some time the Pseudospora

PSBUDOSPOEA VOLVOOISj OIENKOWSKI. 219

Some Pseudospora, usually transparent radial individuals,adopt a peculiar amoeboid form which has eruptive lobopodpseudopodia. I have never seen this except in cultures wherethe radial form predominated, and then only when most of theVolvox had been destroyed. It is quite possible that this ismerely a pathological form due, perhaps, to the protoplasmbecoming more fluid. This form occurs very rarely, but hasbeen seen too often to be passed over without mention.

• LIFE HISTORY AND HABITS.

The amoeboid Pseudospora (A) may conveniently be takenas the starting point of the life history. This form is foundin the Volvox colony, it creeps about on the outside, finallyboring its way into the interior. The creature feeds uponthe Volvox individuals either by surrounding them with broadprotoplasmic processes or by engulSng them bodily. Some-times a long pseudopodium is seen to surround a Volvox cellwhich is at some distance from the main part of the body;the food particle is then either digested at the end of thepseudopodium or is passed along it into the interior of theanimal.

Pseudospora individuals collect in masses round the young

broke up, setting free the granules which now moved rapidly about. Inman; cases the granules were in very active motion inside the Pseudosporafor a considerable time before it disintegrated. When seen with the ordinarypowers of the microscope, I took this process to be some form of sporeformation. On referring to Dallinger and Drysdale's work on the life historyof Monads, I found that the process of spore formation there describedappeared to be very similar to what I had observed in Pseudospora. Underthe three-millimeter immersion objective, the granules appeared rod-shaped,and were seen to move in straight lines; while progressing they turned slowlyon their longitudinal axes.

In view of Schaudinn's recent work on Trypanosoma the bacteria-likeappearance of the particles was not in itself sufficient ground for attributingthe phenomenon to the agency of parasites. Einally, however, after searchingcarefully through the cultures where these individuals were seen, I found thatthe rod-shaped bodies entered from the outside and multiplied so as toform a dense mass absorbing the protoplasm of the Pseudospora. Theyare, beyond doubt, parasitic bacteria.

220 MURIEL ROBERTSON.

daughter colonies ; they are often to be seen lying in groupsclosely resembling the destroyed daughter colonies. Some-times the Pseudospora attacks a segment as large as itself,slowly absorbing it or even creeping into its interior if it isvery large (fig. 6).

As a rule the Pseudospora begins to feed at once uponarriving in the colony. One individual which I observedingested no less than fifteen Volvox cells in two and a halfhours. Nevertheless, starved or very young specimens willoften lie in a colony for some time (six to seven hours)before beginning to feed.

When well established in the colony, Pseudospora dividesabout once in every twenty-four hours (at temperatures6°—16° C ) . When about to divide the creature withdrawsits pseudopodia and becomes spherical in shape, and the foodparticles become arranged in a band round the centre. Aconstriction then appears and the animal divides in two, thedaughter individuals usually lying in close proximity forsome time after they are quite separate. Finally the creaturesput out pseudopodia and creep actively away. The timeelapsing between the withdrawing of the pseudopodia and thedivision of the animal varied between three quarters of anhour and an hour and a half in the specimens which Iobserved.

The first preparations for division of the nucleus (figs. 7-11)occur, just before the animal rounds itself off, in the breakingdown of the nuclear membrane. The whole nucleus seemsnevertheless to be still quite separate and clearly definedfrom the general cytoplasm. The chromatin at this stagebegins to gather together into irregular masses, giving thekaryosome a somewhat mottled appearance. The wholenucleus now increases in size and the rays become indistinct.The achromatic part of the karyosome can still be distin-guished, while the chromatin seems to have segregated outfrom it, forming a number of small masses lying towards theequator of the nucleus. Careful examination of the nucleusat this stage (fig. 7) shows a roughly oval structure with

PSEUDOSPORA VOLVOCIS, C1ENKOWSKI. 221

faintly staining granules towards the periphery; these arepossibly derived from the rays. Towards the equator of theoval lies the chromatin separated out from the karyosome.

In the next stage (fig. 8) the nucleus shows the completelydeveloped spindle. There is now no sign of the faintly stain-ing granules described in the prophase. The spindle seemsto be formed entirely from the achromatic intra-nuclearelements. The chromatin has now formed separate chromo-somes, which appear to be rod-shaped, though from theirsmall size and highly refringent character it is difficult tomake certain of this; they are arranged round the equatorof the spindle. The chi-omosornes now move apart (fig. 9 a),leaving the central fibres exposed. Finally the space occupiedby these fibres is nipped across (fig. 9 b), and the two nucleiare completely separated. Each nucleus soon shows thekaryosome (fig. 10), somewhat irregular in shape, mainlycomposed of deeply staining masses of chromatin, which ap-pear to surround the remains of the achromatic spindle. Therays are not, as a rule, visible at this stage. Shortly after-wards the whole animal divides (fig. 11). This frequentlyoccurs before the nuclei have quite reached the resting state.

After a time the Pseudospora leaves the colony (fig. 12) andswims away in the flagellate condition. If, as is often thecase, the amceboid form has retained its flagella, it now with-draws its pseudopodia and becomes oval or oblong. In thecase of the non-fiagellate individuals, the flagella can some-times be seen to develop gradually from fine elongatedpseudopodia; more often they seem to arise directly withoutany obvious pseudopodial stage. The shape of the animal isoften very irregular for some time after the flagella are formed;finally, however, the creature completely withdraws its pseudo-podia. The nucleus always comes to lie directly behind theinsertion of the flagella.

The method of leaving the Volvox is very constant; thecreature approaches the periphery of the colony and piercesthrough the jelly by means of pseudopodia. The protoplasmthen flows into the pseudopodia until the creature is hour-

222 MOEIEL ROBERTSON.

glass-shaped; finally it slips out, keeping the hour-glass shapeuntil almost quite free. While in the free swimming state thecreature is capable of becoming amoeboid and of again recover-ing its original shape without withdrawing the flagella.

Well-fed flagellate individuals can be seen to divide in thefree state by transverse fission, but I have never seen thisoccur twice in succession in a specimen out of the colony, norhave I ever been able to observe it in creatures which hadbeen without food for some time. Cold, or prolonged lack offood, causes the animal to withdraw its flagella, round itselfoff, and sink to the bottom of the pond, but I have neverobserved the formation of a very definite cyst.l

The flagellate Pseudospora soon attacks another colony, butnot as a general rule so long as it contains green food particles.If water containing free-swimming individuals is put into atube with healthy Volvox the first to attack are those whichstill contain brown particles. Those specimens, curiouslyenough, which are quite transparent owing to the absenceof food particles, take from twelve to twenty-four hours, orsometimes even longer, before attacking the Volvox colonies.Pseudospora are often seen to attack both Eudorina andPandorina even when Volvos are present, and on some occa-sions when starved they ingested small green algae, but I havenever seen Spirogyra or other filamentous algse attacked. Astarved Pseudospora sometimes ingests another Pseudosporawhich is densely filled with green food particles. This appearsto be merely a process of feeding and to have no connectionwith either conjugation or association.

I have never observed the formation of a true plasmodium,but temporary fusion of the protoplasm only may occur be-

' I have been uuable to find the double-walled cyst described by Cienkowski.The rouuded-off Pseudospora appears to possess a more definite covering thanthe flagellate or amoeboid individuals, but nothing that in my opinion wouldjustify the term cyst.

These rounded-off individuals disintegrate at once upon the drying up of thewater, so far as my experience goes. This may, however, be due to theprocess having taken place too rapidly.

PSEUDOSPOBA VOLVOC1S, ClENKOWSKI. 223

tween two or more individuals. In the cases I observed thecreatures separated after about fifteen to thirty minutes. Theprocess is very rarely to be seen, and occurs more frequentlywithin the Volvox than in the free state. This might possiblybe a step towards the formation of plasmodia. Protomonasand Protomyxa, nearly allied genera, form plasmodia, but theyare not known to occur in either Vampyrella or Pseudosporaaculeata. I have, however, not sufficient evidence to drawany conclusions as to the meaning of this phenomenon. ;

Pseudospora, when attacking the Volvox, attaches itselffirmly to' the colony by pseudopodia; these are extendedapparently indifferently from the non-flagellate end of theci'eature or else from either side.

When newly arrived in the Volvox or while still on the out-side the animal is very sensitive to any change of conditions;for instance, rise of temperature, evaporation of the water, orstoppage of the motion of the Volvox will cause the creature toleave the colony; this is to be seen even in starved individuals.

The processes above described—alternation of the flagellateand amoeboid condition and reproduction by fission—continuefor some time (fourteen to twenty-one days) and then a differentform of reproduction appears.

GrAMETOGENESJS AMD CONJUGATION.

In the amoeboid Pseudospora there are developed spheresof a clear greyish appearance. The number of these to befound in a single individual varies; in one culture one- andtwo-sphered forms greatly predominated, in another I foundindividuals with three or four and on one occasion with eightspheres. The individual in which the spheres arise does notform any kind of cyst. The pseudopodia are not withdrawn jin some cases the flagella persist, and movement and feedingmay still go on after the spheres are a considerable size.Finally, the protoplasmic body surrounding the sphere dis-integrates, but the time at which this occurs varies greatly inrelation to the state of development of the sphere. The pro-

224 MURIEL ROBERTSON.

cess of sphere formation occurs both in the colony and in thefree condition. These spheres are destined to give rise to thegametes.

Development of the Spheres.—In tracing out the de-velopment of the spheres it is more convenient to considerfirst the case of a single-sphered individual and thereafter tonote the slight differences that occur in the cases where thereare more than one sphere. The sphere arises directly fromthe nucleus (cf, figs. 13-19). In the very earliest stage thenucleus differs only from the resting nucleus in that the rayshave become thicker and the membrane more distinct (fig.13a). Later the whole nucleus increases in size and thekaryosome assumes a somewhat eccentric position. The firstsigns of the sphere itself now begin to appear. The substancewithin the nucleus becomes differentiated—showing a differentstaining reaction, e. g. blue with Romanowski—to form a spheri-cal mass which fills almost the whole nuclear space. The raysnow appear as small rounded masses; some of these are withinthe sphere; the greater number, however, lie on the outside andseem rather to be connected with the membrane which at thisstage appears surrounding the sphere with its enclosed karyo-some (fig. 13 b) than with the sphere itself. The karyosomegradually moves further from the centre until it finally comesto lie quite outside the sphere (fig. 14 h). I t appears to takeno further part in the process. The position of the karyosomeat any one moment bears no exact relation to the size of thesphere. It is sometimes to be seen lying within a compara-tively large sphere, in other cases it is already on the outsidealthough the sphere occupies little more than the space ofthe original nucleus. The size of the sphere has, however, novery constant relation to its state of development.

In the stage shown in fig. 14 the small chromatin massesderived from the rays have decreased in number. Those onthe outside have as a rule disappeared, though in some casesthey can just be distinguished as very minute particles.Those inside the sphere now appear as definite sphericalmasses; in one case I could only count three of these.

PSEUDOSPORA VOLVOCIS, 01ENK0WSKI. 225

A later stage (fig. 15) shows a very considerable increasein the nuclear material; about eight to thirty nuclei largerin size than the original masses can be seen in the sphere. Iam unable to say how the increase in the number of nucleitakes place; this much, however, seems certain that all thenuclear material of the sphere is derived from the thickenedrays of the original nucleus. The sphere increases stillfurther in size (fig. 16) and the nuclei break up apparentlyinto minute particles, as for some time before segmentationbegins they can no longer be detected in my preparations.When the sphere has reached its full size, '007 mm.-'Oll mm.in diameter, segmentation occurs. A constriction appearswhich divides the sphere into two equal segments (figs.3 7-18). The parts, however, remain closely apposed to oneanother. Bach of these now divides. After this the divisionof the segments is not quite regular, and the spherical shapeis usually lost. Finally a very large number of segments areformed—in one case I counted a hundred and sixteen, and evenin very small single-sphered individuals I have never foundfewer than sixteen. The process of segmentation occupies asa rule from ten to thirty minutes. After the segmentationis 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 hasnot already broken down they pierce through it and escape.The segments set free are small oval or round uniflagellategametes, vai-ying in size from •00116-"00186 mm. The flagel-lum is thick (average length -0046 mm.) and slightly curled ;it arises from a point about half way from the anterior endand propels the animal forwards. Each gamete appears topossess a nucleus, that is to say, in stained specimens a spotthat stains more deeply than the rest of the creature can justbe discerned with the highest powers of the microscope.This probably is the nucleus, but I can say nothing as to itsstructure.

Shortly after becoming free the gametes fuse in pairs,forming zygotes with two uagella (fig. 20). Gametes arising

226 MURIEL KOBERTSON.

from the same sphere conjugate together, but I have neverseen this occur in all the spores from any one sphere; someindividuals were always seen to swim away singly. Incolonies where the spheres of several individuals had seg-mented, I observed conjugation of gametes at some distancefrom the place of segmentation of the spheres, but had nomeans of making sure that the conjugating individuals hadreally arisen from different Pseudospora. Conjugation onone occasion certainly took place between individuals fromdifferent 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 bylater observations, in which the gametes were equal. Onaccount of the small size of the nuclei and the difficulty ofobservation, I am unable to say anything about the fusion ofthe gamete nuclei.

In the many sphered individuals the essential processes ofthe development of the spheres correspond with those alreadydescribed 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 precocioussegmentation of the single sphere, though I am unable to say-what is the cause. In other cases the nucleus appears todivide before the formation of spheres, and from each ofthese nuclei is formed one or more spheres. Occasionally inthese individuals only one of the nuclei becomes convertedinto a sphere, the other apparently disintegrating.

The cultures in which the process of sphere formation wasobserved were kept at an almost even temperature of11°-13° C. and well supplied with food material. In twocultures of uninfected Volvox which were inoculated withsphere-forming individuals it was found that Pseudospora•reproduced by fission for exactly fourteen days and thenagain formed spheres. In one culture this period repeateditself thrice in succession. In other cases the period variedfrom fourteen days to about twenty-one. The formation ofgametes is often nearly synchronous throughout a culture,

PSEUDOSPORA VOLVOCIS, CIENKOWSKI. 227

almost all the Pseudospora individuals breaking up withintwenty-four to forty-eight hours.

The zygote derived from the fusion of the two gametesafter a time withdraws the flagella and appears as a l-oundtransparent little creature, with a just discernible spot which,on'staining, appears as the nucleus. It now becomes amoeboidand creeps in to a Volvox individual, where it feeds and in-creases in size; it destroys the Volvox cell, and is to be seenlying in its place surrounded by the brownish-coloured debr i sof the chromatophore.

The small Pseudospora is usually spherical at this stage,and the protoplasm appears slightly granular. The creaturenow either becomes amoeboid again and invades another in-dividual, or puts out flagella of the type found in the adult.In either case the animal developes directly into the adult, thedevelopment of the flagella in the former case being merelypostponed for a time. The young flagellate individual becomesvery easily amoeboid without losing the flagella.

The zygotes appear to have some considerable power ofresisting unfavourable circumstauces. Thus on one occasionI was able to start a culture from sediment containing themin which there had been no Volvox for about three weeks.The individuals which first appeared were the small flagellateand amoeboid creatures just described; these gradually deve-loped into the normal individuals.

If immediately after the formation of gametes in a cultureof Pseudospora motionless Volvox be introduced, the zygotedevelopes into the radial form instead of the amoeboid or flagel-late. Here also, as in the amoeboid form, spheres are formed.

GENERAL CONSIDERATIONS.

The nucleus of Pseudospora seems to me to indicate a con-dition intermediate between the centro-nucleus described byKeuten and the metazoa-like nucleus of Actinosphasrium. Itis a centro-nucleus in so far as the spindle apparatus is intra-nuclear, but the formation of the spindle and chromosomesshows a marked advance upon such forms as Euglena.

228 MURIEL ROBERTSON.

I have made no attempt to reconcile the formation ofspheres as I observed it in Pseudospora with the sporocystsdescribed by Zopf in Polysporella, Pseudospora aculeata,Vampyrella, and other forms. The true test of their similaritywould lie in the relations of the nucleus in spore formation.

As to the behaviour of the karyosome in sphere formationHertwig (: Archiv f. Prot.,} vol. i, 1901) refers to a some-what analogous form of nuclear multiplication where " in einemgrossen, oft so gar riesigen Mutterkern zahlreiche Tochter-kernaulage eutvvickelt werden; welche in dass umgebendeProtoplasma heraustreten wahrend der Mutterkern zu grundegeht." In sphere formation we have an essentially similarprocess, only here the cell bodies for the daughter nuclei arenot derived directly from the maternal cytoplasm but fromprotoplasm built up within the original nuclear membrane.Part of the achromatic portion of the maternal nucleus appearsindeed to become converted directly into a mass of rapidlygrowing protoplasm.

In Pseudospora the nucleus is more specialised than in theform 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 serveto accentuate the close relationship between the achromaticpart of the nucleus and the protoplasm of the surroundingcell-body.

The tendency of recent work on the Protozoa appears to beto accentuate the importance of Doflein's main subdivisionPlasmadroma, and, on the other hand, to point towards thepossibly artificial character of the subdivisions of the Plasma-droma. Pseudospora with its amoeboid, heliozooid, and flagel-late phases accentuates this. It seems clear that tasonomicdistinctions resting on observations of anatomical featuresduring one phase of the life history are so unreliable as to bealmost worthless. Such characters are shown by the transfor-mation o£" species " of Amoeba into one another by slight modi-fication of the external conditions to be of the most superficialkind, being mere morphological reflections of surrounding

PSBUDOSPORA VOLVOCIS, C1ENK0WSKI. 229

conditions, and of no phylogenetic weight. In classifying theProtozoa it is essential to have regard to the whole lifehistory.

SUMMARY.

1. A Pseudospora individual may adopt three forms—anamoeboid, a flagellate, and a radial form. This last, at least,appears to be a direct reaction to external conditions.

2. A single nucleus is present. It is bounded by a mem-brane, which contains the karyosome surrounded by a clearspace ; fine rays pass from the karyosome to the membrane.

3. The nucleus divides by mitosis. The chromatin formschromosomes, which are apparently rod-shaped. The spindleappears to be formed from the achromatic intra-nuclear sub-stance.

4. Pseudospora reproduces by fission. After fourteen totwenty-one days gametes are formed.

5. Gametogenesis. The nucleus of the Pseudospora be-comes converted into a sphere, the nuclear substance of whichappears to be derived from the rays of the original cell nucleus.The karyosome is extruded from the sphere.

6. The sphere segments to form a large number of gametes.7. The gametes conjugate in pairs, forming zygotes, which

develop into the adult Pseudospora.

EXPLANATION OP PLATE 12,

Illustrating Miss M. Eobertson's paper, " On Pseudosporavolvocis."

The figures are drawn under Zeiss apochromatio homogeneous immersionobjective, three-millimetre focus and compensating eye-piece No. 12.

FIG. 1.—Amoeboid Pseudosporn, with short pseudopodia. n. Nucleus.k. Karjosome. c. s. Clear space round the karjosome. r. Kays passingfrom the karyosome to (m.) the Membrane. / . Pood particles.

FIG. 2.—Amoeboid Pseudospora, with fine pseudopodia arranged in bunches.FIG. 3.—Flagellate Pseudospora,

230 MDRIEL ROBERTSON.

FIG, 4.—Radial Pseudospora. c. v. Contractile vacuole. / Pood particlecontained in a vacuole.

FIG. 5.—Transitory spindle shape adopted by a radial Pseudospora.

FIG. 6.—Pseudospora attacking an uusegmented Parthenogonidium (p.).

FIG. 7.—Early stage of mit.osis. Tt. Karyosome. ch. Chrornatin cometogether in irregular masses.

FIG. 8.—sp. Spindle, eh. Chromosomes arranged in an equatorial plate.

FIG. 9 a.—Chromosomes at separate ends of the spindle, c.f. Central fibres.FIG. 9 h.—Nuclei reforming, c.f Central fibres being nipped across.FIG. 10.—Karyosomo reforming, ch. Masses of chromatin.FIG. 11.—Pseudospora immediately after division, n. Nucleus.FIG. 12.—Pseudospora leaving the colony.FIG. 13 a.—h. Karyosome. r. Thickened rays terminating at the nuclear

membrane. The protoplasm has shrunk a little away from the nucleus.FIG. 13 h.—h. 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 karyosomeis quite outside the sphere: the nuclear material has come together to formdefinite 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 membranewith small masses of chromatin which are greatly reduced in size.

Fia. 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, althoughthe protoplasmic body lias 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.

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