SOME OBSERVATIONS ON A KJiAV GRT5GARINE. 2 6 1

Some Observations on a New Gregarine (Meta-mera schubergi nov. gen., nov. spec).

ByII. Lyiidliurst Duke, B.A., B.C.Cmitab.,

W i t h P la tes 15 and 16.

C O N T E N T S .

P A G E

In t roduct ion . . . . . . 261Material and Methods . . . . . 263St ruc tu re of t he Trophozotte . . . . 266Cyst-formation and Development of the Spores . . 270Discussion of Some Special Po in t s in the Life-cycle . . 278Diagnosis of M e t a m e r a s c h u b e r g i ' . . . 282Li te ra tu re References . . . . . 282Explanat ion of P l a t e s . . • . . • 284

I N T R O D U C T I O N .

WHILE working at Heidelberg in 1906, under ProfessorsBiitschli and Schuberg, the latter kindly called my attentionto a new species of gregarine in the gut of G-lossosiphoniacomplanata L. (Olepsine sexoculata), and suggested itsfurther investigation. The preceding summer, while busiedwith a recently discovered coccidium occurring in the leechHerpobdella atomaria Car. (=Nephelis vulgaris),Professor Schuberg turned his attention to Glossosiphoniacomplanata, which occurs in company with Herpobdellain the Neckar and occasional ponds in the Heidelberg district.Deeming it probable that two forms so alike in habit audenvironment might harbour the same parasites, he dissected

262 H. LYiNDHUEST DUKK.

several specimens of this leech, and, though the results werein the main negative, he found several animals infected witha species of gregarine. Reference to the literature proved,the parasite to be identical with a species briefly mentionedby Bolsius in 1895 (2), and the subject of a more detailedbut still fragmentary paper in 1896 (3). Beyond a super-ficial study carried on incidentally during his work on theG los sos iphon ia Bolsius seems to have paid no furtherattention to the parasite, which remained unnoticed until1900, when Castle (5), in an exhaustive treatise on theN. American Rhynchobdellidte and their parasites, mentionshaving observed, the gregarine seen by Bolsius in about halt'the specimens of Clepsine e l o n g a t a which he examined.He adds, however, that he only finds the animals in thestomach diverticula, and never in the intestine or crop, asindicated, by Bolsius in his diagrams. Castle also mentionsencysted protozoa which he found in C. fusca, and suggeststhe possibility of their relationship to the form in Gr. coin-p l a n a t a . The cysts he found in tlie muscle-layers of thebody-wall, so that they probably have nothing to do withthe gregarine in question.

Lube (14) quotes the parasite as having been mentionedby Bolsius, and suggests that it probably belongs to thetricystid gregarines.

The gregaiine is thus a new and previously undescribedform, for which I propose the name Me tarn era s c h u b e r g i . 1

In the preparation of the sections and the study of theliving auimal, during the last few weeks of my stay in Heidel-berg, Professor Schuberg assisted me most kindly in everyway in his power; and it is due solely to him that I was ableto obtain Bolsius' principal pamphlet. My thanks are alsodue to Geheimrat Prof. J3iitschli, whose practical suggestionsI found of the greatest value.

1 The form which appears most closely allied us regards structure ofthe trophozoite is Ech inomera . A study of the life-history, however,has revealed points of difference which seem to warrant the creating ofa new cjenns for the form under consideration.

SOME OBSEEVATJONS ON A NEW GJIEGARINE. 263

By the kindness of Professor Sedgwick, who allowed me afree hand in the laboratory of the Imperial College of Science,S. Kensington, I was able eventually to complete my study ofthe sections. And in this connection I must express myindebtedness to Mr. C. C. Dobell, who is at present lecturingat the College. His unrivalled knowledge of protozoan life-history and technique has always been most generouslyplaced at my disposal, and has proved of the greatest value inthe preparation of this paper.

MATEKIAL AND METHODS.

The leech which serves as host to M e t a i n e r a s c l i ube rg iis G l o s s o s i p h o n i a complan a t a Linn. A few specimensof H e m i c l e p s i s m a r g i n a t a were also found infected. Theleeches live under stones in shallow water—running by pre-ference—though I have found them in smaller numbers in stillpools. The material was collected at Heidelberg from theshallows left by the summer frill of the Neckar in the neigh-bourhood of the electric power station, below the new bridge,and also from the opposite bank, along the wall separatingthe skating rink from the river itself. The leeches are fairlycommon, and may be found clinging firmly to the under-sideof stones at the water's edge, especially in the numerous lumpsof red sandstone which litter the shore everywhere.

Recently I examined some specimens of G-lossosiphoniac o m p l a n a t a sent me from the neighbourhood of Cambridge,and found them well infected.1 These latter were obtainedin January, when the leeches are hard to find owing to thescanty vegetation in the ponds in winter. In all the speci-mens I examined from this source I only obtained one cyst,and that a very small and early one.

The leeches can be kept for an indefinite period iu a good-sized glass jar, provided the water be aerated by passingbubbles of air through it. Food is not necessary, though a

1 For this I have to thank Mr. Harding, and also for Ins kindness inassisting me to determine the species.

264 . H. r-YNDHUBST DUKJS.

fevy small water-snails are much appreciated. Owing to thetransparent nature of the integument in G-lossosiphonia,the parasites are visible in the living leech; and if the latter beforcibly pressed between two slides provided with wax corners,and examined under a low magnification, the gregarines maysometimes be detected in the stomach diverticula and intes-tine. Unfortunately, however, this method of diagnosis isby no means infallible, as the numerous pigment-cells with,their clear nuclei look very like gregarines, and renderaccurate observation impossible. The gregarines occur inthe hindermost stomach diverticula and the intestine, just asindicated by Bolsius in his diagram. The cysts are found inthe same regions of the alimentary canal, but are especiallynumerous in the intestine.

Examination of sections shows that cysts can develop asfar as the sporoblast stage in the intestinal canal of the host,though they are often expelled with the faeces at a muchearlier stage in development.

In sections just above the anus no cysts were to be seen.This part of the gut was almost occluded by a mass ofcephalonts and some sporonts of a peculiarly blunt outline.The leech from which these sections were cut had previouslyevacuated fasces containing a few very early cysts amonga greater number in which sporoblasts could be distinguished,As many as ten cysts have been counted in one section.

To obtain the gregarine, the infected leeches were partiallydried on blotting-paper and the under-surface opened bythree incisions—two parallel and close to the margins, andone at right angles to the long axis of the animal, at aboutthe junction of the middle and anterior thirds. The flap oftissue was then carefully turned backwards towards the analsucker, the animal being placed in a watch-glass containingnormal saline solution. The gut-contents were thus emptiedinto the saline, together with connective tissue, which is ofno account. By the aid of a hand-lens the gregarines couldnow be seen sticking to the bottom of the glass, or still fixedto fragments of the host-tissue. These latter are useful in

SOME OBSERVATIONS ON A NEW GBEGARINE. 2 6 5

studying the structure of the epimente, as this organ, in thecourse of the teasing out, is very easily torn away, leavingdecapitated individuals which may be confused with truesporonts. By gentle coaxing with a pipette the gregarinescan be freed from the bottom of the watch-glass and trans-ferred to a slide for further handling.

Preparations in to to were made originally under a cover-slip provided with wax feet, and the various reagents drawnthrough with blotting-paper. In this way, by fixing thegregarines with alcohol and glacial acetic acid (9 : 1), a largenumber of animals may be treated under one cover-slip, whichis an obvious advantage. More recently I made some pre-parations by fixing the selected gregarines in a watch-glasswith picro-acetic acid (3 : 1) and adding the various fluids bymeans of a pipette and eventually picking out and mountingthe stained gregarines under a low magnification. I considerthe former method of treatment the more satisfactory andcertainly less laborious. As stains for these preparations Iused Grenadier's alcoholic carmine solution and Schuberg'smodification of Mayer's acid carmine. This latter solution,being acid.in reaction and not neutral, has the power ofpenetrating the cuticle, and in employing it the preparationsmust be very rapidly washed through with | ' per cent,solution of HCl to prevent precipitation of the carmine duringthe further treatment with the alcohols. Leeches destinedfor sections were fixed either in Gilson's fluid or in the above-mentioned alcohol and acetic mixture. Gilson's fluid should actfor two or three hours, and the sublimate constituent be mostcarefully washed out with iodine-alcohol or a solution of KIin 75 per cent, alcohol. As staining reagents heematoxylin(Delafield's) and eosin, safranin, and Heidenhain's iron-hsema-toxyliu were employed. Owing to the paucity of material,the laborious expedient of applying both methods in successionon the same preparation had to be employed. It was foundthat hasmatoxylin and eosin were satisfactory for the cepha-lonts and sporonts, but gave very incomplete1 and misleadingresults with the nuclear changes of the encysted forms, which

266 H. LYNITHUBST 'DUKE.

were defined much more distinctly with the iron-hrematoxylinmethod. All tissues were embedded in paraffin, with chloro-form as the intermediary fluid.

Cu l tu re of the cysts.—To obtain the ripe spores thecysts wex'e simply placed in the moist chamber, where, in thecourse oE seven or eight days, the spores were developed.The cysts were either placed simply on a slide in a drop ofNeckar water or under a cover-slip provided with wax feet.The cysts dehisced by simple rupture after about seven oreight days. Cysts placed in normal NaCl solution in themoist chamber did not develop successfully.

STRUCTURE OP THE TROPHOZOITK.

The body is divided by septa into epi-, proto-, anddeutomerite, .and is elongated in form (figs. 1-6). Someindividuals have a more thick-set appearance than others,especially in the extreme hinder end of the gut, where thegregarines are often crowded together. The animal measuresabout 150/x by 45/u. At the posterior end of the dentomeritethere are often present indications of further subdivision ofthe body, and occasionally as many as three complete segmentsare seen (fig. 4). This segmentation is not confined togregarines of any peculiar build, being present in both longand short forms, and it varies in the degree of developmentof the segments. It was present in about a third of thegregarines examined alive in Heidelberg, and is also verydistinct in the preparations of these animals made tit the time.The Cambridge gregarines also showed segmentation, thoughit was distinctly less in evidence, both in the- living animaland in carmine preparations of it. It appears to vary greatly—from the very faintest indication to quite definite septa.It must be stated in this connection that no segmentedgregarines were seen in the sections of the infected leeches,though constantly found in preparations made by teasing outthe host-tissues. This compels one to consider the possibilityof injury during extraction being the cause of this segmenta-

SOME OBSKBVA.WONS ON A • NKW-GREGAUCHE. 267

tion, although the stained preparations do not in the leastdegree support this suggestion.

The epimerite is a dome-shaped structnre. I t is providedwith short club-like processes, recalling those of E c h i n o -mera , but often branched, arranged in a dense ring aroundthe line of junetion with the protomerite, and also on the roofof the dome (figs. 4 and 5). These latter processes aremarkedly shorter than those of the ring, and decrease in sizeas the apex of the epimerite is approached. The processesare perforated at their somewhat clubbed ends by smallpores, clearly to be seen in the freshly mounted livinggregarine by the aid of a T^- in. oil-immersion lens. Judgingfrom analogy with such forms as E c h i u o m e r a and P t e r o -c e p h a l u s (Nina) , and also from the appeai*ance seen insections across the point of fixation to the host, there is nodoubt that fine pseudopodia are protruded through thesepores, which fix the gregarine to the intestinal mucous mem-brane of the host. The fixing apparatus is by no means easyto identify, as, owing to the unavoidable roughness of tliedissection, the gregarines are rudely torn from their moorings,and almost invariably carry away with them a crown-likefringe—derived from the host-cells—which surrounds theepimerite in the zone of the processes, and obscures alldetails of its structure (fig. 3).

When kept under observation for some time—say an houror so—in NaCl solutiou, a curious phenomenon ensues. Justat the line of junction between the protomerite and epimeritea bubble-like vacuole appears, which gradually increases insize, and carries with it the fringe of host tissue with theembedded processes till they sit like a crown on its upperpole, sometimes symmetrically, sometimes displaced to oneside. Having reached a diameter about equal to that of theprotomerite the vacuole bursts, and the gregarine is suddenlydeprived of its epimerite (fig. 2). This vacuole formation hasbeen seen by Leger and Duboscq to occur-in P y x i n i a (14),and in my opinion has a probable bearing on the mootedquestion regarding the fate of the gregarine epimerite, in the

268 H. LYNDHURST DUKE.

transition from cephalont to sporont. Frenzel (14) believedthe epimerite to be absorbed in a manner'similar to theassimilation of a tadpole's tail. He found among numerouscephalonts with large epimerites individuals with but a minuteprojection from the protomerite, and he regarded this as ascene in the gradual absorption of the epimerite. The suddendisappearance he regarded as pathological, and due tochanges in the surrounding medium. My own observationspoint to the same conclusion. The vacuole formation quotedabove is plainly due to plasmoptysis, which can be followedunder the microscope from its eai-liest onset to the burstingof the bubble. Further, when the gregarines were examinedin a special solution of egg-albumen, NaCl and camphor, asprepared by Professor Butschli, the vacuole formation wasconsiderably delayed; a fact explicable on the ground thatthe solution more nearly resembles the natural environmentof the gregarine.

The behaviour of the finger-shaped processes also points tothe epimerite being absorbed rather than directly thrown offwhen the cephalont becomes free. In gregarines which arenormally lying free in the gut the processes are never to beseen (figs. 1 and 6). The epimerite is still present, but. theprocesses have been withdrawn during the process of separa-tion from the mucous membrane ; just as they are absorbedin Bchinomera when the cephalont becomes free in thegut (17). This applies to all the free-lying specimens seenin sections, and to a solitary living form which, togetherwith several cysts and some faeces, was pressed out throughthe anus during examination of a leech between two slides(fig- 1)-

In the living sporont (fig. 1) the extreme anterior end ofthe animal is quite transparent and devoid of granules, a fewof which, separate from the main endoplasmic mass of theepimerite, may be seen showing Brownian movement alongits anterior border. After some time the whole granularbody of the gregarine appears to shrink back somewhat intothe cuticular sheath which envelopes it, and this clear area

SOME OBSERVATIONS ON A NEW GREGARINE. 2 6 9

enlarges proportionally until almost the whole of the conicalknob which forms the epimerite is clear of granules. Duringthis process all three divisions of the endoplasm are still quitedistinct. By the time this stage has been reached osmosisasserts itself, and the vacuole formation mentioned abovecommences (fig. 2). In sections, however, the free-lyingsporonts all show a curious thickening of the extreme anteriorend of the epimerite, which behaves towards stains in thesame way as the rest of the cuticle, being, in fact, a thickeningof the latter anteriorly (fig. 6). It seems a feasible explana-tion of this structure to say that it represents the cuticularconstituents of the numerous processes of the epimerite, whichhave been retracted on the animal becoming free. It mayhere be mentioned that Liihe (14), in his review of thegregarines generally, pronounces in favour of the casting offof the epimerite as the typical wa.y in which the cephalontsbecome free.

The nucleus lies in the deutomerite. It consists of a nuclearmembrane enclosing a clear ground substance, in which lie alarge vacuolated karyosome and a number of masses ofchromatic substance (fig. 7). The specimens from whichfigs. 3 and 4 were drawn were very faintly stained owing toexcessive washing out, bat some other preparations stainedwith Grenadier's carmine confirm the appearances seen insections, especially as regards the vacuolated nature of thekaryosome. The nuclear area is about 18/j, in diameter; thekaryosome measures about 8 /u, and as a rule contains onevery large vacuole and several small ones. The largechromatin masses are scattered irregularly throughout thenucleus, and are of varying shape. The nuclear membraneis well marked, and in common with the karyosome and thechromatin masses stains deeply with both Delafield's haama-toxylin and Heidenhain's iron-hEematoxylin. The groundsubstance takes on a very faint blue tinge with iron-hEema-toxylin. In some of the sections the karyosome has yieldedalmost completely to the differentiating iron alum, and appearsgrey by contrast with the black chromatin masses. In

VOL. 5 5 , PART 2 . NEW SERIES. 18

270 H. LYNDHURST DUKE.

these cases its vacuolated structure is very plain (fig. 7). Asa rule, however, the karyosome shows very deeply stained inthe adult nucleus. Besides the nucleus there are usually tobe seen scattered throughout the body patches of a substancewhich stains deeply with chromatin stains. These patcheshave been described by Berndt (1) and others, and are espe-cially numerous in the protomerite. Comes (7) lias recentlyshown that these appearances in Stenophora are probablydue to metabolic products, and are not nuclear. There arealso deeply stained granules in connection with the epimeritoprocesses in sections stained with iron-hasmatoxylin, asdescribed by Scliellack in Bchinomera hispida (17).

CYST-FOKMATJON AND DEVELOPMENT OF THE STORES.

The act of association of two animals to form a cyst hasnot been observed in the living animals. As indicated above,in the sporont the epimerite tends to become less prominent,while a pad of cuticle forms anteriorly. Simultaneously withthis shortening of the long axis of the body the protomeriteincreases in breadth and bulges, particularly around theedges of the apical cuticular pad. From sections it wouldseem that the two animals come together with their epi-merites in contact. A ring of cuticle now arises around thebase of the terminal pad in one animal. Into the cup formedby this ring the cuticular pad of the other gregarine isinserted, while external to, and dovetailing with the ring ofthe cup, a similar ring of cuticle arises in the second animal(fig. 37). In very young cysts in which the nuclei of thetwo animals are still unaltered the above arrangement ofthe parts is very clear; but as development proceeds theseptum of cuticle dividing the encysted sporonts becomesincreasingly irregular. In this region in the earlier cyststhere are patches of deeply stained material suggestive ofmembrane, which are probably the remains of the cuticle ofthe contiguous epimerites (fig. 13).

Behaviour of the nucleus prepara tory to the

SOME OBSERVATIONS ON A NEW GREGABINE. 271

format ion of t h e f i rs t two d a u g h t e r - n u c l e i . —Although the material which I was able to collect wasvery limited, I was fortunate in obtaining one leech veryheavily infected. In the intestine of this animal I foundnumerous cysts, and also an enormous number of adultgregarines mostly fixed to the gut-wall. A study of thesesections has revealed several phases of the first division ofthe nucleus, though to elaborate all the stages is impossiblewithout further examples, which I hope shortly to procure.In order, therefore, to make the most of this limited material,I employed first haematoxylin (Delafield's) and eosin, andthen after decolorisation with acid alcohol, re-stained byHeidenhain's method. This latter method revealed numerousimportant facts quite indiscernible with the original staining.My thanks are due to Dr. Pembrey, of Guy's Hospital, whovery kindly provided me with all the necessary apparatus forstaining.

For some time at any rate after a definite cyst-wall hasformed, the nuclei of the encysted gregarines remain appa-rently unaltered. Then the chromatin masses begin to frag-ment, with the result that ehromidia are formed within thelimits of the nuclear membrane. Simultaneously, this mem-brane becomes increasingly thin, and the karyosome throwsout masses of substance from its interior, becoming in con-sequence markedly reduced in size. These masses are moreor less spherical and of distinct outline'; they stain verydeeply, showing black with iron-hsematoxylin. Theirnumberand size vary greatly (figs. 9-14). At times one large massis present, almost equal in size to the original karyosome; atothers, numbers of small masses are seen. The actual processof extrusion of one of these masses is shown in fig. 36. Aftertheir extrusion, the main karyosome-relic shows a blue colourwith hasmatoxylin and eosin, as contrasted with the morepurple hue shown by the intact karyosome and the chromatinmasses of the trophozojte nucleus. The extruded masses onthe other hand behave throughout, as regards stains, likethe chromatin masses. After the fragmentation of the

272 H. LYNDHUKST DUKE.

chromatin masses and the breaking up of: the karyosomehave proceeded for some time, a new structure appears inthe nucleus. In close proximity to the main karyosomeresidue, which is seen lying near the periphery of the nucleus,an ill-defined mass appears which takes up nuclear stainsvery definitely. The earliest appearance of this mass is shownin fig. 9 before the chromidia formation has progressed veryfar. A. slightly later stage is shown in figs. 10 and 11, wherethe nuclear area presents a homogeneous appearance, withoutany signs of the.chromidial elements being discernible, whilethe neighbourhood of the mainkai-yosome residue is occupiedby a somewhat elongated mass, showing faint longitudinalstriation (fig. 11). The relative size of this mass, which I willcall the " achromatic mass," * is shown in figs. 9, 10,11. It willbe noticed that the various products of the karyosome are inclose connection with it.

At this stage, the absence in my preparations of anystructures distinguishable as definite chromosomes or cen-trosomes is to be emphasised. The achromatic mass stainsdeeply with iron-htematoxylin, but yields to the differentiatingiron-alum before the karyosome and its products becomedecolorised.

The next stage in the division represented is shown in figs.12 and 13. The achromatic mass has increased in bulk anddefinition, and has become more drawn out. The striation isvery marked, and for the first time in the course of the divisionthe true chromosome element appears. At each pole of: theachromatic mass, which is now distinguishable as a truespindle, there is a small black mass of chromatin; whileconverging towards this mass, like the ribs of a basket, areseen deeply stained streaks of granules of chromatin, arrangedupon the spindle-fibres and obviously en rou te for the re-spective poles of the figure. It may here, again, be seenthat the spindle stains very deeply with chromatin stains, and

1 I call this structure the " achromatic mass " because of its function—as seen in its later development—and not on account of its stainingproperties.

SOME OBSEEVATION8 ON A NEW GREGARINE. 273

it is only on very thorough differentiation that the chromo-somes are rendered visible. The spindle fibres appear tomerge with the terminal chromatin mass. Distal to this thereis no true astral arrangement visible.

Each terminal chromatic aggregation now gives place toa. definite vesicular structure, situated at the poles of thespindle and forming the centre of a definite astral radiation(figs. 14 and 15). Simultaneously with the appearance of thevesicle, the chromatin streaks and granules disappear fromthe spindle, so that the more definite the terminal vesicle, thefewer the chromosomes on the spindle. Fig. 12 shows a ring-like arrangement of the terminal chromatin aggregation atone pole of. the spindle (a), while fig. 15 shows a true polarvesicle containing definite granules of chromatin, in oneinstance arranged indiscriminately around the circumference,in the other accumulated at one point upon it. These vesiclesare the points upon which the very definite spindle-fibresconverge, and measure from 1£—2J /.i across. In figs. 14 and15 it will be noticed, firstly, that—apart from the granuleswithin the vesicles aud the karyosome products—there arepractically no other discrete chromatin elements to be seen ;secondly, that some of the spindle-fibres plainly run downinto the midst of the nuclear area and the karyosome remnants,where these latter are not already lying on the spindle. In fig.15 will be seen, lying close to the large irregular karyosomeresidue, a collection of deeply stained granules, which areconnected with the karyosome and with each other by deeplystained strands. They have probably been receutly thrownout from the karyosome, which is much distorted from itsoriginal spherical shape.

The latest stage of the first division represented among myslides was unfortunately injured before anything more than arough drawing had been made of its structure (fig. 16). Itrepresented the spindle very much drawn out, just before thefinal separation of the two daughter-nuclei. There was ateach pole a well-marked vesicle, containing numerous granulesof chromatin, and distal to this vesicle was a mass of achro-

274 ' H. LYNDHURST DUKE.

inatic substance, sliowing within it a granule of deeplystained substance.' The figure was very- suggestive of thestate of affairs seen in fig. 18 a and b; with, however, a singlepolar granule. The sparsity of material unfortunately rendersa complete account of the first division phenomena out of thequestion. From a careful study of the slides at my disposalI suggest the following as the more striking points, thesignificance of which I shall revert to later on (see p. 278).f i r s t l y , the depth to which the spindle proper stains withboth Delafield's and Heidenhain's bsernatoxylin: secondly,the proximity of the karyosome to the origin of the achro-matic mass, and, later on, the very definite spindle-fibresrunning down in among the kai'yosome remnants and the siteof the old nucleus : t h i rd ly , the absence of regular chromo-somes such as can at any stage be outlined or counted withanything approaching certainty: four th ly , the vesicles atthe poles of the later spindles, which form the centres of-definite astral figures. The nature of these vesicles it isdifficult- to decide. Are they centrosomes or incipientdaughtei*-nuclei ? As will be seen later, the daughter-nucleiare strikingly vesicular; and the fact that, if these vesiclesare considered as centrosomes pure and simple, there are noother defined chromatic elements in the spindle figure, seemsto indicate their being early stages of the daughter-nuclei.This being the case, the centrosome must be sought either inone of the granules on the circumference of the vesicle, ordistal to the latter. On this point, though tempted to anexplanation, I dare not base a theory upon a drawing sodiagrammatic as fig. 16.

Proceeding to the further division of the daughter-nuclei,all uncertainty about the centrosome vanishes. In theearliest stages, where eight or nine nuclei are present in eachcyst (fig. 17 a,b, and c), the astral radiations are very marked,and the centrosome consists of a deeply stained mass at theperiphery of the nuclear vesicle, from which emanate thestriae. These, where they spring from the centrosome, areextremely obvious. In fig. 19 c and d, stained with hsema-

SOME OBSERVATIONS ON A NEW GKEGA.RINE. 27.5 .

toxylin and eosin, the centrosome is differentiated into a •faintly stained peripheral portion—the centrosphere—in thecentre of which is a black centriole; this also shows in fig. 20stained in-the same manner.

l a studying the various generations of daughter-nucleiseveral interesting points demand attention. They presentan infinite variety as regards the arrangement of theirchromatin. . Except when actually drawn out into a spindle .they are invariably vesicular in structure; and, in the greatmajority of cases, in the earlier stages at any rate, theycontain a distinct karyosome. This is of interest in that inE c h i n o m e r a h i sp ida , described by Schellack (17), wherethe karyosome invariably appears in the daughter-nuclei,its origin is referred to the unpaired chromosome of thisform, which chromosome thus has a function allotted to it.In Sty lorhy nchus , which also shows this phenomenon, thereii, however, no such unpaired chromosome (11). The fate oEthese daughter-karyosomes in M e t a i n e r a s c h u b e r g i is notcertain. The corresponding spindle figures do not show anytraces of karyosome fragments in their neighbourhood. Onthe other hand, in such stages as shown iu figs. 19 c aud d,where the nucleus is on the point of elongating into a spindle,the karyosome seems to be extruding part of its substance.If this is so, the process is one of immediate and completesolution, and not exactly parallel with the behaviour of theadult karyosome. It must be clearly understood that, as thefigures show, a karyosome cannot be always with certaintyidentified in these daughter-nuclei. There are always presentmasses of chromatic substance of varying sizes, and theirarrangement is at times such as to make the distinctiouimpossible. In the daughter spindle-figures, as with the first ,division, there is agaiu no definite chromosome formation.The chromatic elements are sometimes discernible as streaksand granules near the poles of the spindle ; sometimes thedeep black appearance of the spindle-fibres, alone present,suggests that these latter may be conveying chromatin invery minute particles. A constant feature of these young

276 . H. IiTNDHORSl1 DUKE.

spindles is a black mass of deeply staining matter at theextreme poles. In some early spindles shown in fig. 18 a, theearliest actual daughter spindle-stage to hand, this polarmass is seen as two adjacent granules or centrioles lying in adefinite centrosphere showing radiations. In fig. 18 & thesetwo grannies are connected by a deeply staining link. ThisI interpret as the early division of the centrosome, occurringalmost before the daughter-nuclei, which in the figs. 18a and bare distinguishable as faint vesicles, are free from their parentspindle. In this connection it is of interest to note that thedaughter-nuclei always appear provided with two centrosomes.I have not been able to discover any with a solitary centro-some. This is in keeping with the above suggestion as tothe early division of the centrosome in the history of eachdaughter-nucleus. As the daughter-nuclei become smallertheir division-figures become less complicated, while thechromatin becomes arranged as a single mass rather than asseparate particles. Some of the smallesb spindles still showoccasionally distiuct chromatin elements near their poles, butthe majority do not. There appear to be no definite astralrays distal to the terminal mass of chromatic substance(figs. 21 d, 23, and 24 b). Finally all traces of spindle-formation disappear, and the nuclei are reduced to meremasses of chromatin about 1 to 1"5 /j. in size. These arearranged on the periphery of masses of protoplasm, afterthe fashion of a typical so-called Pe r l ens t ad ium, and theprotoplasm soon becomes mammillated round each nucleuswith the formation of gametes (fig. 25).

That part of the protoplasm which does not take part inthe formation of the gametes—the Restkorper—containsa few nuclei which have not kept pace with the generaldivision (fig. 25 b). These laggard nuclei are present here andthere in all sections of the later daughter-divisions, and arenoticeable in that they are larger than their more numerouscompanions. Similar nuclei have been noticed by Leger andDuboscq in Hop lo rhynchus (13). Scattered throughoutthe later cysts are also seen a number of round clear bodies

SOME OBSERVATIONS ON A NEW GBKGABINE. 277

(fig. 25a) stained very faintly with iron-haematoxylin. They aremost obvious in cysts containing gametes or sporoblasts, andhave not been seen in the earlier cysts, at any rate in the sameform. Their size varies considerably, and they appear to beproducts of the original karyosome which have lost most oftheir staining properties, and which have become more obviousowing to the splitting up of the protoplasm entailed in gameteformation. The majority are rather too large to be referred tothe daughter-karyosomes. The main residue of the originalkaryosome is often to be found, deeply stained, in these latercysts.

The gametes are very like those described for L a n k e s -t e r i a ascidiaa by Siedlecki (18), and show no signs of sexualdifferentiation (fig. 26). Considering the fact that there isat no time iu the history of the encysted animals any differencein structure, and that the nuclear changes are practically co-incident, this isogamous type of gamete is what one wouldexpect. Conjugation has not been observed in the livinganimal, owing to my studies being interrupted by my departurefrom Heidelberg. Fig. 27 shows, however, what is practicallycertain to be a zygote. The gametes measure about 3JU, andare roughly circular in outline. Their nuclei consist of smallmasses of chromatin with no definite vesicular structure.The zygote measured over 4"5 n, and contained two distinctnuclei. Several cysts were found containing sporoblasts,(figs. 28 to 33). These are ovoid bodies measuring 6 /u by 4yu,and containing large vesicular nuclei. These sporoblasts gradu-ally acquire a spore coat, and grow iu size somewhat duringthe process (fig. 33), so that in a cyst of sporoblasts one ortwo may be detected with the outline of a formed spore (fig.34). The fully formed spore is shown in fig. 35. The nuclearchanges resulting in the formation of the sporozoiteshave notbeen made out, nor did I obtain a view of a free sporozoite.It was easily seen, however, in optical sections of the livingspores that eight sporozoites were arranged peripherallyaround a granular mass of residual protoplasm. The sporesmeasure 9 ju by 7 n, and are navicelliform, provided at eachend with a little peg-like projection (fig. 35).

278 H. LYNDHURST DUKE.

DISCUSSION OF SOME SPECIAL POINTS IN THE LIFE-CYCLE.

In the description of the trophozoite mention has beenmade of the traces of further segmentation shown occasionallyat the posterior end of the deutometrite in Me tameraschube rg i . The presence of segmentation in some gre-garines, apart from the three fundamental divisions of thebody, is a well-established fact, Leger (12) having describeda form, Tasniocystis , where this phenomenon is so wellmarked as to make the animal resemble a small cestode.Porospora (13) also shows a segmentation, which, however,appears to be somewhat different in nature, as the animal issaid to be capable of obliterating its segments merely bystretching itself out during movement.

In Metamera schnberg i the segmentation is alwaysconfined to the posterior end of the deutomerite, and is notconstantly present. In their full development these posteriorsepta appear in every way as definite as those of the anteriorpart of the gregarine; but in some animals, on the contrary,it requires the most careful focussing to demonstrate theirexistence. I am unable to explain the significance of thesesepta; whether they mark a certain period in the life-cycle orwhether they are due to some form of plasmolysis I cannotsay. They are, however, sufficiently often present to form astriking feature of this gregarine.

As regards the explanntion of the phenomena shown in thedivision of the nucleus, it is difficult to discover anything ofthe nature of a precedent in the current description of thisstage. The vacuoles described by Cuenot (6), Prowazek (16),and others, in close proximity to the sporout nucleus, or bySiedlecki (18) within the latter, have not been seen inMetamera s chube rg i . From the proximity of the com-mencing achromatic mass to the actively disintegi'atingkaryosome, I suggest that this latter body supplies material—more or less,1 it is impossible to say—which will assist inthe formation of the two daughter-nuclei. Another point, to

SOME OBSERVATIONS ON A NEW GJREGAltlNE. 279-

which attention has been frequently called, is the intensestaining capacity shown by the achromatic mass, both at itsfirst appearance and later in the fully formed spindles. Thisapplies equally to Delafield'shasmatoxylinand to Heidenhain'smethod, which latter is known to stain plastin-substancedarkly. Now the chromosome material, when first detected,is seen as streaks lying on the spindle-fibres near the poles;or, when the fibres-are seen in optical section, as a line ofcontiguous granules (figs. 12 and 13). No preparationshowing an equatorial arrangement of the chromosomes w;isobtained, although, of course, this does not prove the non-existence of snch a stage. Fig-. 12 shows some of thechromatiu streaks directly continuous with the well-markedterminal mass; and it is thus possible that this mass repre-sents a collection o£ chromatin which has been delivered bythe spindle-fibres. I suggest, therefore, that throughout thedivision the spindle-fibres are carrying chromatin in a formunrecognisable as discrete particles, until it undergoes con-densation towards the poles of' the figure. With theappearance of the vesicles the chromatin elements disappearfrom the spindle, leaving only the few scattered granules offigs. 12 and 15. These vesicles would thus appear to havebeen formed from the chromosomes- of the earlier stages,and supposing them to be indeed daughter-nuclei, it isconceivable that they go on growing at the expense ofchromatic substance still u,ncondeused in the spindle-fibres,until finally they become free as the first pair of daughter-nuclei. This theory would account for the staining propertiesof the spindle; and the absence, at the earliest stage of thedivision, of definite chromosomes.

As regards the origin of the chromatin of the daughter-nuclei, there is nothing upon which to dogmatise. We havethe fragmentation of the original chromatin masses, whichproceeds until the resultant particles are indistinguishable,and we have the breaking-up of the karyosome, both ofwhich might supply a souroe for the chromatin. That thischromatin is being in some way drawn up on to the spindle

280 H. LYNDBUHST DUKE.

from the debr i s of the old nucleus is obvious from figs. 14and 15.

Siedlecki, in his work on the karyosome of Ca ryo t ropha(19), reviewing the role played by this body in Coccidia,points out that while in some types the karyosome plays apurely vegetative part, in others it has definite responsibilitiesregarding the reproductive functions. The latter appears tobe the case in Metamera schuberg i . If, as I believeto be the case, the daughter-nuclei reform their karyosomes,may not these daughter-nuclei—which are, after the upheavalof the trophozoite nucleus during its first division, presumablysexual in nature—throw some light on the functions of thekaryosome ? If the latter be purely vegetative in function, •why should it recur in the daughter-nuclei, which, with their'two centrosoines, are plainly not in a vegetative condition?

In the face of the facts it is certainly a reasonable sugges-tion that the original karyosome consists of two elements atleast. The one of these is thrown out at the first division of .the nucleus, and is of no further use in the formation of thedaughter-nuclei; the other is of vital importance in thepropagation of the species, as realised in the sexual gumetes.lu the daughter-karyosomes only one of these componentspersists—i. e. that part essential to nuclear division; theother part—for which, in the active reproductive processesnow proceeding no need remains—is not represented. Thus,in the daughter-spindles no karyosome remnants are seen.This is hardly the place for a discussion on the binuclearityhypotheses, so ably dealt with by Dobell (8), but the above-mentioned differentiation of the karyosome constituents issufficiently suggestive. On the one hand, the vegetative andreproductive elements of Goldschmidt's theory may be seenin the original karyosome residue and the so-to-speak moreintense daughter-karyosome respectively. On the other hand,one is equally justified in assuming that the karyosomeresidue merely represents elements whose life is over andwhose functions are exhausted, while the perpetuatedremainder persists in the daughter-karyosomes, which are

SOME OBSERVATIONS OX A NEW GEEGABTNE. 281

thus thoroughly equipped for their part in the ceremony ofdivision.

It will be noticed that, except in fifr, 15, where the vesiclesattain their maximum development, there is no true striationshown distal to the polar aggregation; in other words,although the spindle-fibres are throughout very distinct, thecentrosome elem.ent is not. This, again, suggests a bearingon the origin oE the centrosome. On the one hand, as Dobell(8) points out, we have a binucleate condition held as thestarting-point in the development of the centrosome; on theother there are observers, such as E,. Hertwig, who believethe centrosome to be a specialisation of the central spindle, sothat the spindle in the Protozoa is equivalent to centrosome +spindle of the Metazoa. Without wishing to claim originalityfor the suggestion, I may say that the first division figures ofM e t a m e r a s c h u b e r g i have all along pointed forcibly to amost interesting lack of differentiation and specialisationbetween the various constituents. The chromatin is notmarked off in the form o£ distinct chromosomes, nor are thecentrosomes—assuming my interpretation of the figures to becorrect—distinguishable as such. The three elements, chro-matin, spindle, and centrosome, act in concert in the formationof the first two daughter-nuclei, and it is difficult to say whereone begins and the other ends. I suggest, therefore, thatthe evidence afforded by M e t a m e r a s c h u b e r g i tends tosupport Siedlecki's view, expressed in connection with hisw o r k o n C a r y o t r o p h a (8), that "we have in a protozoan cell

but a single and simple nuclear apparatus beforeus/ ' and not a binuclear arrangement.

In conclusion, with reference to the apparent isogamyshown by this gregarine, it will be noticed that we haveanother apparent exception to what Leger (13) deems thegeneral rule in gregarines, i. e. anisogamy. In this connec-tion the recent work of Brasil (4) and Hoffmann (10) onMonocys t i s , which had previously been considered isoga-mous, is interesting. The work of the latter emphasises thefutility of drawing eo-nclusions from stained preparations.

282 >T. LYXDHTJRST DUKE.

He showed that a very definite anisogamy was visible in theliving cysts, whicli, however, became much less marked inthe process of fixing and staining. This may be so inM e t a m e r a s c h u b e r g i , but, considering isogamy as themore primitive condition, it is possible that this gregarine,whose first spindle suggests a phase in the evolution ofkaryokinesis, may also exhibit true isogamy.

I hope in the spring to renew my acquaintance with thisspecies, and to be able to complete its life-history.

DIAGNOSIS OF METAMERA SCHUBERGI N.G., N.SP.

A cephaline gregarine belonging to the family Dactylo-phoridte (Leger).1 Trophozoite ca. 150 /j. by 45/x. Ephneritesubconical, with apex excentrically placed, and surroundedby numerous branched, digitiform appendages. The deuto-merite sometimes (not always) shows a secondary septationinto one to three segments in the region posterior to thenucleus. Conjugation isogarnous, no sexual differentiationbeing observable at any stage in the life-cycle. Cyst dehisc-ing by simple rupture. Spores navicelliform, containingeight sporozoites, and measuring 9/i by 7 fx.

Hosts: Grlossosiphonia c o m p l a u a t a (Heidelberg andCambridge) and H e m i c l e p s i s m a r g i n a t a (Heidelberg).

GUY'S HOSPITAL,LONDOU, S.E.,

February, 1910.

LITERATURE.

1. Berndt, A.—" Beitrag zur Kenntnis der im Darnie der Larve vouTenebrio molitor lebenden Gregarinen," 'Arch. Protistenk.,'Bd. i, 1902.

2. Bolsius, H.—' Aim. Soc. Brnxelles,' vol. xix, 1S95.3. "Un parasite de ]a Glossiplionia sexoculata," 'Mem.

Acad. Lincei,' vol. xi, 1896.

1 See Minchin (15).

SOME OBSERVATIONS ON A NEW GKEGA.K1NE. 283

4. Brasil, L.—" Nouvelles recherches sur la reproduction des Grega-rines monocystidees," 'Arch. Zool. Expei-.,' iv, No. 4,1905.

5. Castle, W. E.—" Some North American Freshwater Rhynchob-dellidse and their Parasites—VI Parasites,".' Bull. Mus. Comp.Zool.,' Harvard, vol. xxxvi, 1900.

6. Cnenot, L.—" Recherches sur revolution des Gregarines," ' Arch.Biol.,' T. xvii, 1901.

7. Comes, L.—" Untersuclmngen iiber den Chromidialapparat derGregarinen," ' Arch. Protistenk.,' Bd. x, 1907.

8. Dobell, O. C.—" Chromidia and the Binnclearity Hypotheses,"' Quart. Journ. Micr. Sci.,' vol. 53, 1909.

9. Doflein, F.—' Lehrbuch der Protozoenkunde,' Jena, 1909.10. Hoffmann, R.—"Uber Fortpflanzungsei'scheinimgen von Mono-

cystideen des Lumbr icus agricola," ' Arch. Protistenk.,'Bd.xiii, 1908.

11. Lcger. L.—"La reproduction sexuee chez les S ty lo rhynchus , "'Arch. Protistenk.,' Bd. viii, 1904.

12. "Etude sur Tseniocystis mira Lcger, Grcgarine meta-merique," ' Arch. Protistenk.,' Bd. vii, 1906.

13. and Duboscq, O.—" Etudes sur la sexualite chez les Grega-rines," ' Arch. Protistenk.,' Bd. xvii, 1909.

14. Ltihe, M.—"Bau und Entwicklung der Gregarinen," 'Arch.Protistenk.,' Bd. iv, 1904.

15. Minchin, E. A.—" Sporozoa," ' Lankester's Treatise on Zoology,'pt. 1, faso. 2, 1903.

16. Prowazek, S.—" Zur Entwicklung der Gregarinen,"'Arch. Protis-tenk.,' Bd. i, 1902.

17. Schellack, 0.—" Uber die Entwicklung und Fortpflanzung vonEchinomera hispida (A. Schn.)," 'Arch. Protistenk.," Bd. ir,1907.

13. Siedlecki, M.—"Uber die geschlechtliche Vermehrung der Mono-cys t i s ascidite (R. Lank.)," ' Bull. Internat. Acad. Sci. Cracow,'1899.

19. . "Tiber die Bedeutung des Karyosoms," 'Bull. Intemat.Acad. Sci., Cracow,' 1905.

284 H. LYNDHURST DUKE.

EXPLANATION OF PLATES 15 AND 16,

Illustrating Mr. H. Lyndhurst Duke's paper on " SomeObservations on a New Gregarine (Metamera schu-bergi noy. gen., nov. spec.)."

PLATE 15.[Figs. 1 and 2 were drawn from living animal, figs. 3 and 4 from

preparations fixed with alcohol and acetic acid a.nd stained withSehuberg's modification of Mayer's acid carmine. Pigs. 5, 6, and 16are diagrammatic. Figs. 7-18 were fixed with Gilson's fluid and stainedwith Heidenhains iron-hrematoxylin. All these figures were drawnwith Zeiss oc. 6, obj. 2 mm. apochromatic. Figs. 9, 10, 11, and 17, 18are to scale at magnification of 2000. Figs. 12. 13, 14 are drawn ona slightly smaller scale.]

Fig. 1.—Living sporont expressed through aims of leech.Fig. 2.—Same sporont as Fig. 1, showing bubble-formation.Fig. 3.—Cephalont with epimerite embedded in fragment of host-

tissue.Fig. 4.—Showing optical section of epimevite.Fig. 5.—Diagram of structure of epimerite, etc.Fig. 6.—Diagram of sporont with cuticnlar pad on epimerite.Fig. 7.—Nucleus of trophozoite.Fig. 8.—Nucleus showing fragmentation of chromatin masses and

extrusion process of karyosome.Fig. 9.—Sporont nucleus showing earliest appearance of the " achro-

matic mass," with fragmentation of the karyosome.Figs. 10 and 11.—Successive sections of another nucleus showing

slightly later stage than fig. 9.These three figures (9, 10 and 11) are drawn from same cyst.Fig. 12.—First division of sporont nucleus showing at (a) the ring

arrangement beginning at the pole; also the streaks of chromatin andthe spindle-fibres in optical section. The two poles are respectively atthe extreme upper and lower focus. One of the chromatin streaks isseen running into the polar aggregation.

Fig. 13.—A.n early cyst, containing two associated individuals, withremains of epimerites seen at the centre. Nuclei at stage of firstdivision. In upper animal the polar aggregation and the chromatinstreaks are very marked. (Combined from two successive sections.)

SOME OBSERVATIONS ON A NEW GREGARINE. 285

Fig. 14.—Fii-st division of the sporont nucleus at a. somewhat laterstage than figs. 12 and 13. Shows polar vesicles more distinct. Alsothe distinct fibres running down into neighbourhood of original nucleusand karyosome.

Fig. 15.—First division of sporont nucleus at a later stage than fig. 14.Vesicles fully formed and fibres running down towards karyosome.The vesicles here shown were 6/J apart, lying respectively at top andbottom focus.

Fig. 16.—Diagram of first spindle just before final separation of first"two daughter-nuclei.

Fig. 17, a, b and c.—Earliest stage of daughter-nuclei, eight or ninein cyst.

a. Shows centrosomes connected by a thick band.6. Shows chromatin bunched as an early spindle figure,c. Shows karyosome.

All from same cyst.Fig. 18.—Somewhat later daughter-nuclei at end of division.

a. Shows two centrioles at each pole; also one daughter-vesicle.(The section has not passed thi-ough the left vesicle.)

6. Shows division of the centriole with poorly developed daughter-vesicle. (The vesicle at the right end of the figure lies outsidethe plane of this section, and is therefore not seen.)

e. Shows a separated daughter-vesicle.All from same cyst.

PLATE 16.

[Figs. 21-34 and 36 were fixed with Gilson's fluid and stained withHeidenhain's iron-hsematoxylin. Figs. 19 and 20 were stained withDelafield's hsematoxylin and eosin. Figs. 19-34 were drawn atmagnification of 2000. Fig. 35 is not to scale, being relatively toolarge.]

Fig. 19 (a-e).—From same cyst. Somewhat later daughter-nuclei.All show karyosomes. c and d show early stage of spindles, and thekaryosomes in a state of activity.

Fig. 20.—Showing differentiation of centrosome into centriole, andcentrosphere in a daughter-nucleus of same stage as fig. 19.

Fig. 21.— Similar daughter-nuclei showing karyosomes : also corre-sponding spindle.

Figs. 22 and 23.—Later stages of daughter-nuclei, mostly showingkaryosomes; also corresponding spindles.

VOL. 55j PART 2.—NEW SEEIES. 19

286 H. LYNDHUEST DUKE.

Fig. 24.—Smaller daughter-nuclei and spindles.Fig. 25.—Shows the Pe r l ens t ad ium, with a single free gamete.

Notice the clear karyosome remnants (o), and the residual nuclei (6).

Fig. 26—Gametes.Fig. 27.—A '/.ygote with two unfused nuclei.

Figs. 28-32.—Sporoblasts. Figs. 29 and 31 show these in transversesection.

Fig. 33.—Shows a sporoblast assuming shape of spore.Fig. 34.—Shows a spore coat in process of developing.Fig. 35.—Fully formed spore, with sporozoites in optical section.Fig. 36.—Shows the karyosome in the act of extruding some of its

substance.

Fig. 37.—Diagram to show method of apposition of associating;sporonts in a cyst.

1.

a,a.

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1617c.

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