The Genus Stomatophora, Drzewecki.

By

B. L. Bhatia, M.Sc, F.Z.S., F.R.M.S.,

Government College, Lahore, India.

With Plate 23.

INTRODUCTORY.

THE investigations on which this paper is based were largelycarried on at the Zoological Laboratory of the King's College,London, while I was, working there during the session 1920-1.A large number of genera and species of Monocystid parasitesof earthworms have already been described. Still, it wasbelieved that the study of the less well-known parasites, orof parasites from earthworms which had not previously beeninvestigated, might yield interesting results, and be helpfulin throwing light on the mutual relationship of the variousfamilies and genera of the Gregarine parasites of the Annelids.It was with this object that I started examining specimens ofP h e r e t i m a r o d e r i c e n s i s (Grtibe) obtained from theLily-house in the Eoyal Botanic Gardens at Kew. I feltinterested in this worm as it belongs to a genus the species ofwhich are rare in Europe, being only found in green-housesof the Botanical Gardens at various places, but are commonlyfound at Lahore and in other parts of India. I thought there-fore, that I should be able to continue my investigations onthe parasites of these species of P h e r e t i m a and variousother earthworms on my return to India. Specimens ofP h . r o d e r i c e n s i s were almost always found to be infectedwith a parasite which, though referable to the genus S t o m a t o -p h o r a (Drzewecki), did not agree with the previously describedspecies of that genus, and which, after careful comparisonwith the specimens of the other species, I have described in

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this paper as new and named St . s imp lex . On one occasiona single specimen of P h . r o d e r i c e n s i s was found to con-tain specimens of S t . c o r o n a t a (Hesse), which Hesse haspreviously described from this earthworm.

Since my return to India in July 1921 I have continued towork on the Gregarine parasites of earthworms, and have hadthe opportunity of examining preparations from the contentsof the seminal vesicles of the following earthworms :

P h e r e t i m a b a r b a d e n s i s (Beddard).P h e r e t i m a h e t e r o e h a e t a (Mchlsn.).P h e r e t i m a p o s t h u m a (L. Vaill.).A l lo lobophora foe t ida (Savig.).Al lo lobophora ca l ig inosa (Savig.).

Of these only P h . barbaclensis (Beddard) has been foundto contain both S t . c o r o n a t a (Hesse) and St . d iadem a(Hesse). In the course of these investigations, beside S toma-t o p h o r a species of several other genera, some of which arenew, have been studied, and the observations on these will becommunicated in subsequent papers.

As mentioned above, the earlier part of this work was carriedout in the Department of Zoology, King's College, London.I was working under the direct supervision of Dr. D. L. Mac-kinnon, and I am deeply indebted to her for guidance andvaluable suggestions and criticisms during the progress of thework. I am also much indebted to Professor. Arthur Dendy,F.B.S., for the encouragement and help I received at all timeswhile I was working in his laboratories. My grateful thanksare also due to Professor J. H. Ashworth, F.E.S., and Dr.J. Stephenson, of Edinburgh University, for looking throughthe manuscript of this paper and making some useful sugges-tions, and to the latter also for specific identifications of theworms.

HISTORICAL.

In 1904 Hesse described a new species of Monocys t ia ,Monocys t i s c o r o n a t a (4), from seminal vesicles ofP h e r e t i m a spp . and gave the following brief diagnosis :

THE GENUS STQMATOPHORA 483

' M o n o c y s t i s c o r o n a t a nov. sp., is a remarkableMonocystid distinguished by the presence at the anterior poleof a petaloid sucker with radiating ribs, and by the sporocystsbeing of a form intermediate between those of Gregarina andof Monocystis.'

Drzewecki, studying the same form, described it in 1907 (3)as possessing a mouth and a peristome, also an anal opening,and affirmed that the organism ingested the spermatozoa of thehost as solid food-stuff. He created a new genus S t o m a t o -p h o r a for the reception of the species, and was even temptedto consider S t . c o r o n a t a as the first link betM'een Mono-cystids and parasitic Infusoria, as for example P r o t o p h r y ao v i c o l a (Kofoid), which is a transition form between theparasitic 0 p a 1 i n a e and free living A s p i r o t r i c h a.

In 1909 Hesse, in his monograph on the Monocystids ofOligochetes (5), published a detailed study of the form showingthat the alleged mouth and peristome were simply parts ofa sucker-like organ. He further stated that in spite of numerousdetailed investigations he had not found any anal opening toexist, and had not seen the parasite absorb solid nourishmentby the mouth or by the rest of the surface of the body. Accord-ing to him, the sucker seemed to be wholly comparable instructure and function to the various epimerites indicated inother Monocystids. He, however, considered it desirable tokeep the genus S t o m a t o p h o r a for the Gregarine with asucker found in P h e r e t i m a. In the same work he describedanother species found in the seminal vesicles of P h . h a w a -y a n a (Eosa) and another unidentified species of P h e r e t i m a ,and named it S t o m a t o p h o r a d i a d e m a. Since that dateno further work appears to have been published on this remark-able and highly interesting genus.

TECHNIQUE.

After killing a worm it was dissected under 0-75 per cent, saltsolution. A seminal vesicle was removed to a slide and teasedin the same solution. The contents were spread out into a smearand examined for the existence of the trophozoites or other

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stages of the parasites. If the worm was thus found to beinfected, the various stages were examined alive, and moresmears made from the remaining seminal vesicles for permanentpreparations. The contents should be spread out into thinsmears, and before the latter have begun to dry up immersed inthe fixative. I generally employed Schaudinn's fluid (sublimatealcohol) as a fixative followed by staining with Heidenhain'siron haematoxylin or Dobell's iron haematein. Besides thesmears the parasites were also examined in sections of theseminal vesicles stained by the same methods or after fixationwith Bouin, staining with borax carmine and picro-indigocarmine. For studying the spores, smears were stained withcarbol fuchsin.

MORPHOLOGY OF THE SUCKER.

The sucker, being the characteristic structure which distin-guishes the organisms belonging to this genus, deserves to betreated in detail. In the diagnosis of S t . c o r o n a t a , Hesse(5, p. 162) remarks that in the anterior part of the parasitethere exists a kind of radiate sucker (' ventouse ', literally acupping-glass) formed as a hollow sphere presenting the formof a cast of a melon with projecting ribs ; the external covering(' plafond ', literally ceiling) of this cavity, constituted solelyof hyaline ectoplasm, has the form of a petaloid crown. Lateron (pp. 164-7) he gives a complete description of the organ,and also discusses its homology and functions.

It would seem from his description and from his diagrammaticfigures lxxvii and Ixxviii, that the petaloid crown has noexistence apart from the sucker, that in fact the petals arenothing but epicytal folds (with a thin layer of hyaline sarco-cyte) forming the wall of the cavity, which is seen when thesucker is viewed end on ; and, further, that when, as sometimeshappens, a certain quantity of endoplasm penetrates theepicytal folds, the collar of petals is no longer seen. I haveexamined a large number of films containing specimens ofS t . c o r o n a t a from P h . b a r b a d e n s i s found at Lahore,in which I have invariably found the typical appearance of the

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sucker and the petaloid crown described above. I have alsoexamined large numbers of S t . s i m p l e x s p . n o v . (fordescription please refer to p. 491 et seq.) from P h . r o d e r i -c ens is (Griibe) obtained from the Lily-house in Kew Gardens,in which I have found a typical sucker but with no trace ofthe petaloid crown. I Avould fain believe that these latter alsobelonged to S t . c o r o n a t a in which the petals had not beenfixed and stained owing to the cause hinted above, but thereare sufficient well-marked differences in other respects to justifythe two forms being regarded as distinct. Then again inS t . d i a d e m a , as described by Hesse himself (his figs. 66-8)from P h . h a w a y a n a (Rosa) and which we also come acrossin P h . b a r b a d e n s i s (Beddard) at Lahore (p. 503 et seq.and figs. 26, 28), there is no petaloid crown. In this latterspecies the body of the parasite is cut up into lobes by furrowswhich are seen to run outwards over the surface of the bodyfrom the denticulations on the circumference of the sucker,in continuation with the epicytal striations radiating fromthe dark central spot of the sucker.

I agree with Hess's interpretation of the sucker itself. Fromthe anterior aspect it is seen in stained preparations as a darklystained circumference of a circle, with still more deeply staineddenticulations on it, and a central dark point from whichdarkly stained lines may (PL 23, fig. 10) or may not (PI. 23,fig. 23) be seen to be running outwards crossing the circle andreaching the denticulations on the circumference of the circle.This appearance is undoubtedly due to the fact that the suckeris like a hollow sphere with a cap cut off from the distal pole;the other pole of the cavity is elevated like the bottom ofa bottle, and meridional lines running from this proximal poleto the rim of the cup, project inwards from the wall of thecavity. In preparations stained with iron haematoxylin, therim of the cup is seen as the stained circumference, the lowerelevated pole as the more deeply stained spot, the meridionalprojections as stained lines diverging radially from the centre,and the upper ends of these projections as the denticulationson the circumference. The lower elevated pole is, of course,

486 B. L. BHATIA

the mucron. It is seen as a small circular area and not merelyas a point or dot. This mucron is a conical projection of theepicyte containing the clear hyaline sarcocyte. Sometimesthe entocyte also runs into it and forms the dark central core(PI. 23, fig. 18). It is for this reason that this central spot isrecognized sometimes as a clear ring-like area (PI. 23, fig. 23)or a dark circular spot (PI. 23, fig. 10), according as there isa core of entocyte in its anterior or not. This clear ring-likearea seen in some preparations does undoubtedly give theappearance of a mouth of the organism, but of course is nothingbut an appearance (cf. also Hesse's figs. 47, 48, 66, and 67,and Drzewecki's figs. 31, 38, 49, and 58). The meridional ridgesare simple projecting folds of epicyte, which are seen runningacross the circle or not, according as they are more or lessbulging into the interior of the cavity, their upper terminationsbeing seen in either case as the denticulations on the circum-ference of the circle.

Now to turn to the collar of petals surrounding the suckerin S t . c o r o n a t a though not in S t . s i m p l e x and S t .d i a d e m a. The petals vary in number in different specimensfrom eight to nineteen, and vary in size amongst themselves(PI. 23, figs. 22, 23). They are flattened plate-like structures,not unlike the blades of an electric fan, with their narrowinner ends abutting on the circumference of the sucker. Theydo not take up the stain well, and appear in preparationsstained with iron haematoxylin as colourless and non-granular,but the lines between the adjacent petals are stained bluish.These lines are seen to end in the denticulations on the circum-ference of the sucker, that is to say the petals are as many innumber as the denticulations, or in other words the same innumber as the meridional ridges projecting into the anterior ofthe cavity of the sucker. The petals represent a well-developedcrown of sarcocyte in the region of the body immediatelysurrounding the sucker and not the side-walls of the suckeritself. The area comes to be marked off into distinct petalsby specially marked epicytal striations spreading outwards overthe body starting from the denticulations on the rim of the

THE GENUS STOMATOPHORA 487

sucker. The petals themselves are often marked by numerousepicytal striations which are only finer than those which markoff the petals from one another. I am able to come to thisconclusion as the result of (1) certain observations I was luckyenough to make on a living specimen, in which the petalsactually came.to be distinctly seen where nothing but epicytalstriations existed before, (2) comparison of the state of thingsas seen in the three species of S t o m a t o p h o r a , and (3) com-parison with the very similar epimeritic structure as seen incertain cases of E h y n c o c y s t i s p i l o s a . We shall considerthese facts and arguments in the following paragraphs.

A trophozoite of S t . c o r o n a t a was found from the seminalvesicles of P h . r o d e r i c e n s i s (Griibe) from the Lily-housein Kew Gardens on March 7, 1921, although the worms pre-viously obtained from the same place had always shownspecimens of S t . s i m p l e x . The organism in the fullyextended condition measured 147/n in length, and 105/* in itsgreatest width about the middle of its body (PI. 23, fig. 21).The anterior end was somewhat flattened and the posteriorpart of the body was considerably narrower, measuring nearthe posterior end only 63p across, and was rounded at theextremity. The trophozoite showed gradual movements accom-panied by a certain amount of alteration in form (compare theoutline of fig. 22, PI. 23, which represents the same organismdrawn a quarter of an hour later). The epicyte covering thehyaline sarc'ocyte was thin, and showed longitudinal wrinklesor furrows over the middle of the body. The endoplasmincluded coarse paramylum granules, which were so numerousand close together that almost the whole of the body of thegregarine was opaque, except the middle part near the anteriorend which appeared as a clear white area. A little way behindthis clear white area, at the moment of contraction, when thegranules in the endoplasm would flow backwards from thisarea, would be seen the sucker. When first recognized thesucker was seen as a clear circle, with a granular round body atits centre (mucron). The circumference of the circle showeda number of dark points (denticulations), and a large number

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of extremely fine lines were seen to radiate outwards from itin all directions. The petals were not observable, and theposition of the sucker was also puzzling, as it appeared to liein a position corresponding to the ventral sucker of a liver fluke.

About a quarter of an hour later, however, some of thepetals began to be seen distinctly (PL 23, fig. 22). Three on theright side of the body and two on the left side were seen clearly.Each of these petals had its outer margin broader than theinner, and the outer margin was distinctly flattened againstthe epicyte covering it. These five petals were opaque andcoarsely granular like the rest of the body, due no doubt tothe extension of the endoplasm under the sarcocyte in thisregion. The clear area just behind the anterior extremity ofthe body was next seen to be occupied by three petals, resem-bling the others in outline, but differing from them in beingwhite and hyaline, on account of endoplasm not extendinginto them. The outer ends of these anteriorly directed petalsreached the anterior margin of the body. The radial linesseparating the petals were seen to run inwards and end in thedenticulations on the circumference of the circle alreadyreferred to. In addition to these more prominent lines separat-ing the petals, the surface of the petals was crossed by numerousfine radiating lines which have been mentioned above. Theoutlines of the petals arising from the remaining portion of thecircumference could not be made out, but the fine radiatinglines were clearly seen, and the number of these petals and theirplace of origin from the circumference could now be inferredfrom the number of denticulations on the circle, which wereplaced at equal distances on it. In other words these remainingpetals were still in the process of formation.

The view that the petals represent a specially differentiatedregion of the body surrounding the sucker is also supportedby a comparative view of the three species of S t o m a t o -p h o r a. In S t . d i a d e m a (PL 23, figs. 26, 28) the epicytalstriations radiating from the centre of the sucker are continuedbeyond its circumference, and run across the whole surface ofthe body of the parasite. The more prominent ones among

THE GENUS STOMATOPHORA 489

these are deepened into meridional furrows which cut up thebody into a number of irregular lobes. The petals, so to say,extend over the whole of the body, but the lines between thepetals being deeper the body appears to be divided into anumber of lobes. Next in S t . c o r o n a t a (PL 23, figs. 22, 23)the petals extend over a certain area immediately surroundingthe sucker, and thus the petaloid crown appears to form aparapet round the sucker. But in certain cases, as describedby Hesse himself (see his fig. 40), the lines separating the petalsextend beyond them, radiating over a considerable part of thesurface of the body. Lastly, in S t . s i m p l e x the crown ofpetals is altogether absent. It is not at all uncommon to findamong other genera of Monocystids closely related speciespossessing more or less marked epicytal striations or none atall, as for example M o n o c y s t i s s t r i a t a and M. a g i l i s ,or R h y n c o c y s t i s p o r r e c t a and E . pi 1 o s a .

The appearance and structure of the sucker and the sur-rounding petaloid crown recall to our mind the very similarepimeritic structures possessed by M. s t r i a t a (Hesse) (hisfigs, xxxv and 32) and a certain variety of R. p i 1 o s a (Cuenot)figured by Hesse (fig. 22). In both these animals the anteriorpart of the body is excavated by a shallow cavity from thebottom of which projects the hyaloplasmic mucron. The layerof sarcocyte is spread over the edges of this cavity and the ribsdescribe regular meridional curves. But in both these formsit is stated that in the adult condition the cavity disappearsand the anterior extremity of the body forms a projection.The mucron is usually described as being very little developedin S t o m a t o p h o r a , but I have sometimes seen it projectingfrom the middle of the anterior extremity of the body, whichwas seen to be flattened (PL 23, fig. 13).

Nor is the possession of a sucker a permanent feature in thelife of S t o m a t o p h o r a . According to Hesse, in S t .c o r o n a t a the parasite always possesses a sucker whileinside the blastophore, the same persisting sometimes fora rather long period in the free form; most often, however,it atrophies rapidly. In studying the different stages of growth

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in S t . s i m p l e x I have sometimes seen a clear circular areanear the anterior extremity in very young individuals, but havenever seen a well-formed sucker in the young trophozoites.For a time in the growth of the parasite the sucker is welldeveloped and of a normal form (PI. 23, fig. 10), but later on,as the free trophozoite grows older, various alterations takeplace. The greater portion of the hyaline sarcocyte mayrecede towards the sides of the body, and the mucron withthe granular endoplasm advancing into it as its core may beseen projecting from the middle of the anterior extremity(PI. 23, fig. 13). Or, again, the mucron may be altogetherwithdrawn into the body, and the anterior extremity of thebody may present a more or less deep crateriform excavation(PI. 23, fig. 12) or a shallow saucer-like depression marked withdenticulations (PI. 23, fig. 11). Lastly, a small clear area nearthe anterior extremity may be the only indication of the formerpresence of the sucker, and the anterior margin of the bodymay sometimes be undulated (PI. 23, fig. 14) or even smooth.So ultimately the sucker nearly always disappears before asso-ciation takes place.

I have never come across forms in which the sucker waselevated on a striated cylindrical stalk, or formed a striatedplate with undulated borders, as shown by Hesse in his figs. 45,47, and 48.

As regards the function of the sucker, it is clear that the organis used like a true sucker for fixing the parasite to the wall ofthe seminal vesicle of the host, as also for filling itself withnutritive substances. These substances penetrate, however,osmotically into the body as in all other Sporozoa and not assolid particles through any so-called ' mouth ', of which, likeHesse, I have not been able to find any trace.

There is no doubt that both in structure and function thesucker of S t o m a t o p h o r a is strictly comparable to thevarious epimeritic structures met with in some other Mono-cystids and the epimerites of the Polycystids, but differs fromthe latter in not possessing a fixed form, and in not beingabandoned inside an epithelial cell of the host.

THE GENUS STOMATOPHOBA 491

S t o m a t o p h o r a s i m p l e x , s p . n o v .

I have often found the vesiculae seminales of the specimensof P h . r o d e r i c e n s i s (Griibe) obtained from the Lily-housein Kew Gardens, infected with this parasite, which differsfrom the other species of the genus by the absence of the petalssurrounding the sucker, or other meridional striations orfurrows over the body. On account of this peculiarity I havegiven the name S t . s i m p l e x to this new species.

DIAGNOSIS.—Monocystid with a spherical, oval, or ellipticalbody, reaching up to 55^ in length. The body is not markedwith any furrows or epicytal striations. At the anterior endis a sucker, in the form of a circular cup with a central pro-jecting portion from which meridional ribs radiate outwardsto the rim of the sucker. Nucleus which may be placed inany part of the body is spherical and possesses one largerounded caryosonie surrounded by a clear area and a peri-pheral zone of fine chromatine granules. Movements wellmarked but slow.

Cysts usually elliptical, reaching 68 to S4 ̂ by 48 to 75 fi.Spores navicular with the extremities flattened ; irregularlycrowded together in the cyst and not sticking together end toend to form chains. The extreme dimensions of the spores are8-5/x to 13-6/x in length by 5/x to 6-8/a in width.

HOST.—Ph. r o d e r i c e n s i s (Griibe).I have examined the seminal vesicles from a large number of

specimens of P h . r o d e r i c e n s i s from Kew Gardens duringthe months of January to June, and practically every onewas infected.

The trophozoites when examined alive in 0-75 per cent,salt solution show characteristic gregarinoid writhing move-ment. The body has a distinct cuticle, the endoplasm beingfinely granular and laden with a large number of refrigentgranules which were carried about passively by the move-ments of the cytoplasm. The nucleus is a clear round bodywith a central caryosome, and is also passively carried about.The anterior end of the body is flattened and broad, and the

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posterior end usually narrower and rounded. One individualwhen fully extended measured 50-4/x in length and had amaximum width of 29-4/x. Movements bring about importantchanges in form, the same organism being seen at intervals toassume the form of an elongated cylinder, an urn, a vase, ora club (PI. 23, figs. 1-3). Sometimes there is a central con-striction (PI. 23, fig. 3). The slow and gentle movement of thebody is rendered obvious by the streaming movement of thedark refractile granules with which it is seen to be studded.The granules move now in one direction and now in quite acontrary one. So from these movements alone it would bedifficult to infer which is the anterior and which the posteriorend of the organism. But the anterior end is distinctly widerand shows indications of a depression. The important changesin form assumed by the parasite can also be observed in stainedfilms (cf. PI. 23, figs. 10-14).

The E a r l y S t ages .—I have not been able to find thefree sporozoites nor to determine how they get into the seminalvesicles. In fact, so far as is known to me, no one has yetbeen able to follow their migration and determine their modeof penetrating the seminal vesicles in the case of any Mono-cystid. In the youngest stages that I have observed theparasite was seen already lodged within the sperm-follicle.In spite of a careful examination of a large series of prepara-tions, both smears and sections of seminal vesicles, fixed withSchaudinn's fluid or Flemming's solution without acetic acidand stained with iron haematoxylin, I could not find anyparasites actually in the act of penetrating the sperm-follicleor the blastophore as described by Hesse for S t . c o r o n a t a .

On one occasion while examining fresh material I founda young trophozoite surrounded by cells of a sperm-follicle.The parasite was quite spherical (PI. 23, fig. 4) and measured10-2/x across. The cytoplasm was finely granular and a roundedcentrally placed nucleus was plainly visible. The nucleusshowed a shining dot-like caryosome which was slightlyeccentric in position. The chromatin grains were adherentto the nuclear membrane mostly on one side. In the stained

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preparations also similar young individuals were studied.One of them is represented in PI. 23, fig. 5, and is seen to becovered over with spermatozoa. It is spherical and measures10-2ja across. The cytoplasm is compact and finely granular.The nucleus measures 1-7/* in diameter and shows a distinctlythick nuclear membrane, a simple slightly eccentric caryosornesurrounded by a clear zone, round which fine chromatingranules are arranged in a ring. The distribution of thechromatin granules is quite unequal, only a few of them placedagainst the nuclear membrane on the side of the caryosorne,the rest being arranged on the opposite side, forming a crescentand recalling the ' nucleoleuring ' of Drzewecki. The cytoplasmalso contains a few deeply stained irregular rod-shaped bodies,and on one side a clear circular area containing a single deeplystained granule (cf. Hesse's figs. 43, 44, 55, and 70). Hessesometimes found this granule to be connected by a filamentwith the caryosome, and thought it looked like a centrosome.He also suggested that perhaps it had to do with the beginningof the formation of the sucker, but, as he says, this cannot beaffirmed until one is able to follow its transformation up to thetime when the sucker appears. In these young stages thereis, however, no other indication of a sucker, and I have nevercome across a well-forrned sucker in the young trophozoites,as is illustrated by Hesse in his fig. 193 for S t . c o r o n a t ain parasites of 8/x to 10/x diameter. In S t . s i m p l e x there isno well-formed sucker in elongated, ellipsoidal trophozoites ofeven 17/i in length (PI. 23, fig. 6). Considering the fact thatthe adult trophozoite of S t . s i m p l e x is very much smallerthan the full-grown trophozoites of S t . c o r o n a t a , it isclear that the development of the sucker must be taking placevery much later than in S t . c o r o n a t a . The parasite repre-sented in PI. 23, fig. 6, had a maximum width of 10-2/x and wascovered by spermatozoa. The cytoplasm was finely granular,contained a few chromatoid bodies, and the clear circular areawith a deeply staining granule referred to above. The nucleusof the body situated in the posterior half measured 3-4/u indiameter and contained a caryosome measuring 1-7/* across.

494 B. h. BHATIA.

The nuclear membrane was, however, not lefinite ; the caryo-some was surrounded by a clear area, outsH: which was a lightlystained achromatic network.

It is remarkable that sometimes equally small trophozoitesare found free and uncovered with spermatozoa in the contentsof the seminal vesicles of the host. One such, which measures10-2/n and has a maximum width of 5-1 /x, is represented inPL 23. fig. 7. It shows a distinct epicyte, a thin layer of sarco-cyte, and close-set finely granular endoplasm. The latterincludes a number of deeply stained granules situated in theanterior part of the body, in the neighbourhood of the nucleus.Situated near the anterior end are two clear, circular areas eachcontaining a deeply stained granule. If the clear area at allcorresponds to the sucker as believed by Hesse, it is verydifficult to explain the occurrence of two such areas in a singlespecimen. The nucleus has the characteristic appearance.It is spherical, has a distinct nuclear membrane, and a deeplystained central round caryosome surrounded by very fineehromatin granules.

I have occasionally come across instances of early associa-tion between young trophozoites, either not surrounded bya cyst or while still contained in a blastophoric envelope sup-porting the spermatozoa. In one case (PI. 23, fig. 8) the youngtrophozoites are only 10-2/x in length, and the maximum widthof the two together is 11-9/x. The organisms are closelyadhering, and each contains a nucleus of the usual type, withthe caryosome placed eccentrically. In another instance theneogamous ' association had led to a complete fusion of the

bodies, only the nuclei remaining separate and thus givingthe appearance of a binucleate trophozoite (PI. 23, fig. 9).The nuclei are rather peculiar in this case, and perhaps indicateimpending change. The nuclear membrane in each is indistinct,and the caryosome is large and shows a vacuolated structure,with certain parts of it stained more deeply than the rest.A few chromatoid bodies are also situated in the cytoplasmon one side. It is probable that the cysts containing theneogamous individuals, and clothed over by spermatozoa, are

THE GENUS STOMATOPHORA 495

due to the association of two parasites inhabiting a commoncytophore.

A d u l t T r o p h o z o i t e.—The adult trophozoites ofS t o m a t o p h o r a are not difficult to recognize from thoseof the other genera of Monocystids on account of the posses-sion of a characteristic sucker-like epimeritic structure. Aswill be remarked later, however, this sucker is sometimes notwell developed or is in process of disappearance, and thespecimens are then likely to be confounded with those ofM o n o c y s t i s .

The trophozoites when lying free in the seminal vesiclesare much smaller in dimensions than those of S t . c o r o n a t a .They are on an average 45/xx26/x, but specimens have beenfound to attain a maximum size of 54-4/J. in length by 35-7/xin width. In S t . c o r o n a t a the parasites do not on anaverage exceed 80/^x60/*, but exceptionally large tropho-zoites may reach a size of lSO ẑ in length by 130^. in width.The general outline of the trophozoites is much like that inS t . c o r o n a t a , and considerable diversity in form is some-times met with. The movements of the living parasite and thechanges of form brought about thereby have been describedabove. In fixed and stained specimens the parasite may havevarious forms (PI. 23, figs. 10-14).

Both in living specimens and in stained preparations itis found that the ectoplasm is very much reduced. There isalways a well-marked epicyte and a feebly developed layer ofsarcocyte. The sucker is formed almost entirely of ectoplasm,except the dark central projection of it which corresponds toa niucron and which may have a core of endoplasm prolongedinto it (PI. 23, figs. 10 and 13). The appearance and detailedstructure of the sucker has been described above (pp. 484 to 490).It may be noted, however, that the sucker in S t . s i m p l e xis not surrounded by a crown of petals, as in St c o r o n a t a .Nor are the epicytal striations continued from the sucker overthe rest of the body of the parasite, marking the latter off intoa number of lobes as in S t . d i a d e m a. The various altera-tions in shape of the sucker which this parasite exhibits during

496 ' B. L. BHATIA

its period of growth are also described elsewhere (p. 490), andreference should be made to PI. 23, figs. 11-14, for a clear indica-tion of such alterations. I have not been able to observefibrils of myocyte, which must be very poorly developed, ifat all.

The endoplasm is formed as usual by an alveolar cytoplasm,in which there are often various reserve granules and chroma-toid bodies. The cytoplasmic granules are exceedingly fineand form an alveolar framework. In the alveoli of this frame-work can be seen large ellipsoidal, spherical, or irregular-shapedgranules of paramylum, also occasionally darkly stainedgranules or rod-like chromidial particles.

The nucleus is not fixed in position (PI. 23, figs. 10, 13). Itis moved about in the body by the contractions of the parasite,along with the paramylum bodies and the cytoplasmic granules.The nucleus is spherical and surrounded by a nuclear mem-brane which stains darkly with iron haernatoxylin. Inside thenucleus is contained a spherical, deeply staining caryosome.The caryosome, which is eccentrically placed in the nuclei of theyoung trophozoites, usually becomes central in the nucleusof the adult trophozoite. It is surrounded by a clear area,and outside this fine chromatin grains are dispersed all overthe rest of the nucleus on an exceedingly delicate linin network,which is not always obvious (PI. 23, fig. 10). The caryosome isgenerally seen to be a compact body, but sometimes it isvacuolated as seen in PI. 23, fig. 11. I have never seen a verydark-staining little granule which, according to Hesse, is almostalways stuck on the surface of the caryosome in the nucleusof S t . c o r o n a t a , and which is homologized by him withthe caryosome described by Leger in S t y l o r h y n c h u s .But for this small difference the nucleus is almost identical inits structure with that of S t . c o r o n a t a . But, as will beseen from the following table, the relative dimensions of theparasite, nucleus, and the caryosome differ markedly fromthose in S t . c o r o n a t a and M. a g i l i s as given by Hesseon pp. 83 and 175 of his monograph.

THE GENUS STOMATOPHOHA 497

Body oj S . simples Nucleus.170/x by lO-2/i . . . . 3-4,1.28-9,1 by 16-1/13 7 - 4 / i b y 2 2 1 M39-1/i by 35-5/J.40-8/i by 25-5,140-8/i by 27 -2 /*42-5/ iby 18-7/x42-5/i by 28-9 /i44-8 / iby 18-7 JH47-6/1 by 34-0/i54-4,1 by 35-7/1

8-5/x6-8/i6-8/»6-8 ti8-5/i6-8/i8-5/t6-8/x7-4,t8-5/i

Caryosome.1-7/t2-0/i2-o/il-7/i3-4/i2-7/13-4/i2-5/i2-5/il-5/il-7/i

It will be seen that in all the above individuals the sizes of thenucleus and the caryosomes are smaller than those of corre-sponding-sized specimens of S t . c o r o n a t a .

A s s o c i a t i o n , E n c y s t m e n t , and Gamete F o r m a -t ion .—Early associations sometimes take place and have beendescribed already (p. 494). Usually, however, association occursbetween equal-sized sporonts which have completed theirgrowth, and a common cyst formed round the two. Twosporonts in association measured about 50/u. in length each, andhad a maximum width of 29-4/1. The cyst-wall is thin and thegametocytes contract, leaving a clear space between the cyst-wall and themselves. The cysts are generally ellipsoidal, andI have never seen in this species fusiform cysts which are some-times met with in S t . c o r o n a t a . These cysts vary a littlein their dimensions, the usual size being 75 to 85/i. by 63 to68 jn. The measurements of a few are noted below :

68-0/1 by 48-0/x75-5/x by 62-9/a84-0/1 by 75- 6 /*

105-0/1 by 88-2/1

At the stage of gamete formation a marked difference in sizewas noticeable between the two gametocytes. No differencewas observed, however, in the dimensions of the gametes pro-duced by each.

The gametes are rounded bodies, each containing a sphericalnucleus with a deeply stained central caryosome. The nucleusis placed eccentrically near the side of the gamete.

498 B. L. BHAT1A

S p o r e s and Sporozo i tes .—Each zygote becomescovered, as usual, bjr a tough membrane or sporocyst. Ina mature cyst a large number of spores are found irregularlycrowded together. These are not arranged in rows, nor arethey seen sticking end to end, as described by Hesse forS t . c o r o n a t a . I have observed the cysts on numerousoccasions, in salt solution as also in stained preparations, butI have never found the spores sticking end to end in thisspecies.

In some cases the cysts are seen to be packed with sporesand no cystal residuum can be seen. In PL 23, fig. 18, is repre-sented a cyst which measured 84/* by 75-6/* and possesseda thick and granular cyst-wall, measuring as much as 2/x to4/4 thick towards one pole and gradually becoming thinnerand not well formed at the other pole. The contained sporo-blasts were mononucleate, the nucleus appearing as a shiningbody when unstained. Usually, however, the cyst-wall isquite thin, and more or less residual protoplasm is seen in addi-tion to the spores. In the cystal residuum are also seen acertain number of nuclei, which are probably gametic nucleiwhich did not arrive at maturity or did not undergo syngamy.The spores are of two distinct sizes, the larger somewhat moreelongated ones, and the smaller ones appearing to be morebulging or swollen out in the middle. The macrosporesmeasure 11 fx to 13-6/x in length and 5/A to 6-8/x in thickness,whereas the microspores measure 8-5/u. to 10-2fi. in lengthand 5-1 /x in thickness. There is, however, no very sharp distinc-tion in size between these two kinds. These dimensions showa marked difference from those recorded for the spores ofS t . c o r o n a t a by Hesse, and I have never come across anymicrospores measuring 7 JX by 3 /x. In fact, in no case have theybeen found to measure less than 8-5/x by 5-1/x. The spores aretruncated at the ends, and show clear button-shaped areasat the two poles. This is characteristic of the other knownspecies of the genus also. This flattening is believed to be dueto the tendency of the spores to stick together by their endsto form.chains, but, as remarked above, this has never been

THE GENUS STOMATOPHORA 499

ohserved in S t . s i m p l e x , and so far as my observations gois a rare circumstance even for S t . c o r o n a t a .

The development of the sporozoites within the spores is ofthe usual octozoic type. I have not been able to follow theachual division of the nuclei, but have frequently seen sporescontaining two, three, four, or eight nuclei (PI. 23, figs. 15-19).These nuclei do not remain in the equatorial plane as is generallydescribed for Monocystids, but are scattered over the granularsporoplasm, so that when the longitudinal cleavage of thesporoplasm takes place the resulting eight sporozoites do not-all have the nucleus in the middle. In some the nucleus wouldbe near the extremity of the sporozoite, as described forP y x i n i a r u b e c u l a . The dehiscence of the spores and theactual liberation of the sporozoites has not, however, beenobserved.

S t o m a t o p h o r a c o r o n a t a (Hesse).

Hesse found the organism very abundantly in the seminalvesicles of two exotic earthworms acclimatized in the green-houses of the Botanical Gardens at Grenoble, viz. P h.r o d e r i c e n s i s (Griibe) and P h . h a w a y a n a (Eosa). Asmentioned previously, while working on P h . r o d e r i c e n s i s(Griibe) obtained from the Lily-house in Kew Gardens, onlyonce did I come across a specimen, the seminal vesicles ofwhich contained parasites belonging to this species. I havefound, however, that specimens of P h . b a r b a d e n s i s(Beddard) so commonly found in Lahore (India) are generallyinfected with this species. As the result of my studies on thisform I would amend the diagnosis of the species, as given byHesse, in the following respects :

(1) The anterior part bears a sucker, formed as a sphericalcavity with a cap cut off from the upper pole, and with a centralmucron arising from the proximal pole as a projection into thecavity ; the sucker is surrounded by a crown of a variablenumber of petals formed of hyaline sarcocyte covered over byepicyte. (2) Cysts are generally spherical or ellipsoidal, some-times fusiform. (3) The spores may be sticking together by

500 B. L. BHATIA

their ends to form long chains or may be distinct and irregularlycrowded together in the cyst.

The adult trophozoites which are found lying free in theseminal vesicles of the hosts are usually oval or ellipsoidal inform. They are found to occur more abundantly during themonths of April, May, and June than in other seasons of theyear, so far as the earthworms examined in Lahore are con-cerned. Specimens of S t . c o r o n a t a usually attain a sizeof 38/x to 80/x in length, though one obtained from P h .r o d e r i c e n s i s from Kew measured as much as 147/J. inlength and had a maximum width of 105/x. Hesse says thatthe organisms may reach 180/x, but this appears to be the sizeof unusually large specimens and I have never come acrossanything like that. The animal exhibits active though byno means rapid movements of the body, thus causing altera-tions in form from moment to moment. The body is generallywidely expanded at the anterior end, so as to make it resemblea top or a flower vase. There may soon appear a sharp con-striction round the middle of the body, causing it to assumean hour-glass-like shape, and later it may acquire a moreregular outline again (PI. 23, fig. 20 a, b, c). In these movementsof the body, the contained paramylum grains and the nucleusalso participate, and are moved about from place to place.

The chief characteristic feature of the organism is the posses-sion of the sucker, which is an epimeritic structure situated atthe anterior pole as already described. I have not eorne acrossany specimen in which the sucker was lifted off from the bodyon an elongated or striated cylindrical stalk, or in which thesucker was everted, as was sometimes seen by Hesse (cf. hisfigs. 45, 47, and 48). However, I have seen specimens in whichthe sucker was clearly degenerate and showed irregular jaggedmargins, indicating the way in which the entire disappearanceof the sucker takes place in some free individuals prior toassociation. I have never found the sucker in very youngindividuals contained in the blastophore, nor persisting afterassociation has taken place, as stated by Hesse (5, p. 166).According to my observations, the sucker is well developed

THE GENUS STOMATOPHOKA 501

only in adult trophozoites lying free in the seminal vesicles,and on one occasion at least I have witnessed the actual appear-ance of the petals taking place, though of course the suckerwas present already. It is difficult to explain this late develop-ment of an epimeritic structure, and from Hesse's figs. 50, 72,and 73, it seems pretty certain that the sucker does make itsappearance as a rudiment in the early intrablastophoric phases,so I may probably have missed this appearance for want offavourable material.

I cannot, however, agree with Hesse when he makes thefollowing observation: ' For one thing the epimerite is notalways liable to fall off, very often association takes placebetween Gregarines which still have their suckers, that is,between cephalate individuals, which never happens among thePolycystids ' (5, p. 166). Hesse does not give any figure toillustrate this condition, and I have never come across anyassociates in which the sucker was still present. It would bevery unusual for an epimeritic structure to persist after associa-tion and cyst formation had occurred. Before the gameto-cytes associate, as a rule considerable alteration has taken placeat the anterior pole. The sucker has practically disappearedthough its previous existence is indicated by the anterior polebeing more or less flattened and sometimes even denticulated.

The gametocytes attach themselves by the anterior polesand begin to shorten in the antero-posterior axis, and widenin their transverse diameter. Thus when the cyst-wall has beensecreted the sporonts are apposed to one another by theiranterior ends, and the united length of the two is approxi-mately equal to the width of either of them. The cysts arethus generally spherical or slightly ellipsoidal. The cysts havean average diameter of 60 to 70//. by 50 to 60 /*. The cyst-wall is thin. As the Gregarines continue to contract after thecyst-wall has been secreted, there comes to be a large emptyspace between the associates and the cyst-wall, and this spaceis increased still further by the action of the fixatives. I havenever come across fusiform cysts, nor can I corroborate Hesse'sstatement that most often before secreting the cyst-wall the

502 B. L. BHATIA

Gregarines fold over their caudal region on the rest of the bodyin such a way that they assume a rounded form. His fig. Ixxxis far from convincing.

There is generally a small difference in size between thetwo gametocytes enclosed in a cyst (PI. 23, fig. 24). Themeasurements of the associates in a few of the gametocytesare recorded below :

Bigger gametocyle. Smaller gametocyte.(1) Longer diameter . . 40-5/i 37-8/u.

.Shorter diameter . . 27-0/x 22-5^(2) Longer diameter

Shorter diameter(3) Longer diameter

Shorter diameter

38-2/u31-5/1 27-0ji40-5/1 40-5 ix.26-1 PL 20-7/*

The dividing membrane between the associates is double,being formed merely by the cuticles of the two where they aretouching each other. The cytoplasm during this stage (PI. 28,tig. 24) is finely granular. Nuclear multiplication for the forma-tion of gametes takes place mitotically, but the mitoses arenot synchronous. The spindle as represented in PI. 23, fig. 24,is a short one, markedly swollen at the equator, and fourdaughter chromosomes are distinctly seen migrating towardseach pole. Beyond the difference in size of the gametocytes,there is no other difference in structure, in the process ofnuclear division, or the number of nuclei produced, whichmight be indicative of anisogamy. When gametes are formedno boundary line between the gametocytes is visible. Thegametes measure about 4-5p. by 3-6^ to 4 t̂ in diameter. Icannot confirm Hesse's statement that there is even slightanisogamy. The gametes are, generally speaking, roundedwith a large nucleus, though there are also present some whichare rather elongated with a proportionately smaller and densernucleus. But I am not in a position to say that one gametocyteforms those of one kind and the other of the other kind, orthat the actual fusion takes place between two of differentkinds. The outlines of the gametes are usually indistinct,although the nuclei are deeply stained and conspicuous.

The spores are of a form intermediate between the spores

THE GENUS STOMATOPHORA 503

of Monocys t i s which are spindle-shaped and pointed atboth ends, and those of the genus Grega r ina which arebarrel-shaped. They are spindle-shaped, but their ends aresomewhat drawn out and truncated, presenting button-shaped enlargements. They are generally found irregularlycrowded in the cyst and not sticking end to end to form chains.Only on one occasion did I find the spores sticking end to end,and forming short chains of three or four each, and not theneven forming long twisted necklaces as described by Hesse.The spores have fairly constant dimensions in any one cyst,though these dimensions vary from one cyst to another.Sometimes spores of two distinct kinds are met with in the samecyst, the macrospores measuring 9 to 11/x by 5 to 6/A, andmicrospores from 7 to 7-5 p by 3/x, but there are also sporeswith intermediate dimensions.

The spores are octo'zoic, as is usual with the Monocystids.Each contains eight sporozoites arranged round a centralsporal residuum. The nuclei of the sporozoites may be situatedin the centre of each or nearer one of the ends.

S t o m a t o p h o r a d i adema (Hesse).

Hesse (1909) described this species from the seminal vesiclesof P h . h a w a y a n a (Eosa) and another P h e r e t i m a sp .found in the green-houses of the Botanical Gardens at Grenoble.He considers it very rare, having come across it only twice.The seminal vesicles of the infected hosts contained a verysmall number of parasites, and so it was not possible for himto study the form in detail. Working at Lahore, we havecome across parasites of this species in smears prepared fromthe seminal vesicles of P h . b a r b a d e n s i s (Beddard) whichhad been prepared primarily with the object of studyingSt . c o r o n a t a , which is found in the same host. Prepara-tions from a number of worms show specimens of this parasite,and the number of parasites found and examined is also large.But much to my regret I have not been able to see them in theliving state before the preparations were made, which wasdoubtless due to their rarity.

504 B. L. BHATIA

S t . d i a d e m a has the form of a sphere which has beenflattened and compressed between its two poles. Hessedescribes the form of the body as a dome or a hemisphere,the summit of which is occupied by a sucker. He says furtherthat the height of the body is in general equal to half itsdiameter, except in the forms with very large diameter whichare strongly depressed. In fig. 68 he gives a profile view of theparasite which would seem to confirm the statement. I havenot been able to see this parasite in the living condition nor tostudy it in profile, and so am unable to affirm that the parasitehas not a considerable vertical height. But judging from thesmears, which are fixed in Schaudinn's fluid and stained iniron haematoxylin, it does certainly appear that the body isso much antero-posteriorly flattened or compressed that itmay be almost described as a plate or a disc cut up into anumber of lobes. Examining under the oil immersion, a slightmovement of the fine adjustment is sufficient to put theparasite entirely out of focus, which would not happen if itpossessed any considerable thickness. The average diameteris said to be 70 p, though the largest specimens found by Hessemeasured as much as 105/*. A specimen which measuredacross 67*5fi along one diameter and 58-5yu along another, isrepresented in PI. 23, fig. 26, and may be taken as a specimenof an average size. Even in this case the whole body can beclearly seen through without any vertical movement of thetube of the microscope.

The surface of the body of the parasite is excavated byfurrows radiating outwards from the circumference of thesucker, and the body is thus cut up into a variable numberof irregular lobes, some of which are considerably bigger insize than the others. In PI. 23, fig. 26, the body of the parasiteis divided into five lobes, of which three are of an approxi-mately equal size, one considerably bigger, and one considerablysmaller. In PI. 23, fig. 28, is represented an older specimenwhich is almost circular and measures 90/*. across. In thiscase the number of lobes is ten, of which two are bigger thanthe rest. This unequal size of the lobes imparts a curiousasymmetrical appearance to the parasite.

THE GKNUS STOMATOPHORA 505

The sucker occupies as usual the summit of the body, andowing to the flattening of the latter is seen in its centre. Itis a shallow circular cup-like depression with a central conicalprojection or mucron. The diameter of the aperture is describedby Hesse as equal to a third, sometimes even half of thediameter of the Gregarine, but in my preparations is seen tobe considerably less. The circular border of the aperture isintensely stained with iron haematoxylin, and so also area number of projections on it, and the central mucron. Anumber of faintly stained lines are seen to radiate from thecentral mucron, run across the wall of the sucker, and end inthe deeply stained projections on its circular border. Thefurrows separating the lobes also end in these same projections(PI. 28, fig. 28). Besides these furrows no other epicytal striationsare visible in fig. 28. But an interesting appearance is repre-sented in fig. 26. The circular aperture of the sucker is sur-rounded by a dark area, which in its turn is encircled by a cleararea Avhich has been left unstained. The sucker, as well as thedark and the clear areas encircling it, are marked by epicytalstriations many more in number than the furrows separatingthe lobes. Lastly, in a very much younger specimen (PI. 23,fig. 27), which measures only 31-5/x in diameter and is insidethe blastophoric capsule, there is no trace of a sucker orepicytal furrows whatever. It would seem, therefore, that,unlike the epimerite of Polycystid Gregarines, the sucker isnot present in the earlier stages of development in any of thespecies of S t o m a t o p h o r a ; that during the growth of thetrophozoite a simple cup-like sucker comes to be developed atfirst, by a flattening and inpushing taking place at the anteriorend of the body, the median projection or mucron being thuscarried to the bottom of the cup-like depression. This processwould seem to cause the epicytal striations to appear firston the surface of the sucker extending from the central mucronto certain definite points on the circular border of the aperture,and later extend beyond over smaller or larger portion of thebody. In S t . s i m p l e x these striae do not extend beyondthe region of the sucker; in S t . c o r o n a t a they extendover a small area of the body immediately surrounding the

NO. 271 L 1

506 B. L. BHATIA

sucker thus forming the crown of petals. As an exceptionalcircumstance, as would appear from Hesse's fig. 40, thestriations may extend beyond the crown of petals over someportion of the body. And lastly, in S t . d i a d e m a theyextend not only over the whole of the body but some of themdeepen and, involving the deep layers of the ectoplasm, causethe body to be cleft into lobes. This view is amply supportedif my figs. 10, 22, 23, Hesse's fig. 40, and my figs. 26 and 28are placed in a series and compared. So far as S t . d i a d e m ais concerned, it is clear from a comparison of my figs. 26 and28 (or Hesse's figs. 66 and G7) that as the parasite grows oldermore and more of the epicytal striations become replaced byfurrows, and thus the number of lobes goes on increasing.In Hesse's figures the number of lobes represented is fifteenand twenty-one respectively. In the full-grown condition(my fig. 28 or Hesse's fig. 66) the epicytal striations as apartfrom the furrows have practically disappeared. A comparisonof the sucker with very similar structures met with in theyoung stages of R h y n c h o c y s t i s p i l o s a and Mono-c y s t i s s t r i a t a (Hesse's figs. 22 and 23) is very suggestive,and would lend further support to the view set forth above.

The endoplasm is finely granular and very close-meshed.The nucleus, which is generally rounded though sometimesoval, is always eccentric in position, lying between the marginof the sucker and the edge of the body. It may be confinedto one lobe, or may be extending over an area covered by twoor more lobes. It contains a large deeply stained centralcaryosome surrounded by a clear zone and a large number offine chromatin granules dispersed over a linin network. Thecaryosome when examined under a high magnification is seento contain a dark central body surrounded by a number of verysmall vacuoles. The nucleus in an average-sized individualmeasures 13-5/u. in diameter and the caryosome 3-5 p. Theadults are usually free in the seminal vesicles of the host,and I have never found them covered by a thick layer of thephagocytes of the host, as stated by Hesse.

The cysts are most often spherical (PL 23, fig. 30) thoughthey may be sometimes ellipsoidal. Seminal vesicles of

THE CENUS STOMATO1JHOHA 507

Eli. barbadensis are commonly infected with bothSt. coronata and St. diadem a, and cysts of bothspecies are commonly found lying together in the same prepara-tion. There is a difference in size between such cysts. Thetrophozoites of St. diadem a are on an average considerablybigger than those of St. coronata , and it is safe to concludethat the distinctly larger cysts measuring 125/t to 170/u,belong to St. diadema and the smaller ones measuring00ix to 70ix to St. coronata . Hesse has given the dimen-sions of the cysts of St. diadema as 45n to 50n, but Ibelieve he must have mistaken some of the cysts of St .coronata for those of St. diadema. The spores containedin these distinctly larger cysts of St. diadema are easily dis-tinguishable from those of the two other species by their beingrather drawn out on both sides before coming to the truncatedend. The average size of these spores in 8 to 12 n by 3 to 4 n,and not 12 to 15 ju. by 5 to 6 fj. as given by Hesse.

SYSTEMATIC POSITION OF THE GENUS, WITH BEMABKB ONTHE CLASSIFICATION OF EUGREGARINAE.

Hesse, in his important memoir on the Monocystids of theOligochetes (1909), split up the old genus Monocystis andcreated a number of new genera on the basis of his work onnew or already known species, and recognized that two of thesegenera possess a true epimerite, which is in the form of a moreor less elongated cylindro-conical trunk in E h y n c h o c y s t i sand in the form of a sucker in S t o m a t o p h o r a.

In the family Doliocystidae an epimerite is present,and may attain a considerable size, as in Doliocystis(Monocystis) aphrodi tae (E.E.L.), without any septumdividing the rest of the body. Brasil has already advocatedthat this family should be transferred to the Monocystids.Besides this, certain other genera also of the Monocystids, viz.Lankes te r ia , some Gonospora as well as E h y n c h o-cystis and Stomatophora are known to possess a definiteepimerite, though not of the same character as that possessedby the Cephalina.

Minchin (7, 8) and Brasil (1) have discussed the primary

508 B. L. BHATIA

division of the Eugrogarines on the basis of the occurrence orabsence of an epimerito into Cephal ina and Acephal ina ;or, on the presence of a septum dividing the body into proto-merite and deutomerite or the absence of this septum, intoS e p t a t a and H a p l o c y t a . I think we should prefer thelatter division, which is also the earlier mode of classifying(Lankester, 1885), and I should like to suggest that theE u g r e g a r i n a e be divided as follows :

Bub-order I. SEPTATA (Lank.) (=Cepha l ina , exclud-ing Do l iocys t i dae , or Po lycys t i da sensus t r ic to) .

Sub-order II. HAPLOCYTA (Lank.) ( = A c e p h a l i n a +Dol iocys t idae and other primarily Dicystid forms).

Tribe I. DICYSTIDBA. Including all primarily Dicystidforms, that is, forms possessing a distinct epimerite,but with the body not divided into a protomerite anda deutomerite.

Tribe 2. MONOCYSTIDEA (Stein) or ACEPHALINA (Kolliker).Including all really Acephaline forms.

The advantages of the above scheme may be briefly recapi-tulated thus :

(1) Dol iocys t idae are brought into closer juxtapositionto Monocys t idea , a position to which they are clearlyentitled on account of their being non-septate parasites ofmarine Annelids, their sporocysts showing close affinity tothose of Monocystid genera, and the epimerite in the differentspecies of Dol iocys t i s being plastic and invaginable.

(2) The old group Cephal ina is purged of a family thatdid not show any natural affinity with the rest.

(3) Boorn is created for genera like L a n k e s t e r i a , Gono-spora , S t o m a t o p h o r a , and E h y n c h o c y s t i s , whichpossess more or less distinct epimerites but are otherwise relatedto Monocystids, in close proximity to the latter.

SUMMARY.

1. The genus S t o m a t o p h o r a is closely related to otherMonocystid parasites of earthworms, but differs from themin the possession at the anterior end of the body of a sucker-

THE GENUS fcSTOMATOPHORA 509

like epimeritic organ with a central niucron. This epinieriteof S t o m a t o p h o r a is capable of undergoing alterations inform during the life of the parasite, and it is not abandonedinside a cell of the host as it is in Cephalina. This differencein behaviour is undoubtedly associated with tho fact that thegrowing trophozoite is not fixed to a single epithelial cell, butlies surrounded by the cells in a cavity which contains abundantnutrient material which it can absorb.

2. Two species of the genus have been described beforeand a third is now added. In Stornatophora simplexsp. nov. the sucker is not surrounded by a crown of petals.

The following comparative table shows the chief charactersof the three species of the genus S tomatophora :

Epinierite.

Trophozoite.

Maximumsize.

Cysts.

Spores.

Size of spores.

Hosts.

St. n i m plex.An anterior suckerwith a central muc-ron and meridionalribs.

No crown of petalssurrounding thesucker.

Usually elliptical,08 to 84 ix by 48to 70 ii.

Navicular, but withthe extremitiesflattened.

8-ij to 13-0 )x by H to0-8 ii.

Ph. roderiuensis(Griibe).

St. corvnata.As in St. simplex.

A well-devolopedpetaloid crown ofsarcocyte surround-ing the sucker,marked by epicytalstriations.

180 ix by 130 ix;average measure-ments 80 ix by 00 ix,thus usually smal-ler than St . d i a -d c m a.

Generally spherical,or slightlv ellip-soidal, 70"to mixby 00 to 00 ix.

Navicular, extremi-ties presenting abutton-like flatten-ing ; sometimessticking end to endto form chains.

9 to 11 jx by 5 to (i ftand 7 to 7-5 ft by 3/u.

Ph. rodericensis(Griibe),

Ph. h a w a y a n a(Rosa), and

Ph. b a r b a d e n s i s(Beddard).

Si. (Italic in-a .As in St. simplex.

The whoJc bodymarked by furrowsand divided intoa number of irre-gular lobes.

105 a.

Spherical. 130 ix to170 ix.

Navicular. extremi-ties rather drawnout and truncatedat the ends.

8 to 12/i by 3 to

P h . h a w a y a n a(Rosa), and

Ph. barbadensis(Beddard).

510 B. L. BHAT1A

'3. A revision of the classification of Eugregarinae issuggested.

REFERENCES TO LITERATURE.

1. Brasil, L.—" Le genre Doliocystis (Leger) ", ' C.B. Acad. Sc. Paris',torn, cxlvi, 1908.

2. Drzewecki, W.—" t)ber vegetative Vorgiingc iin Kern und Plasma* der Gregarincn des Regenwurmhodens", 'Arch, fiir Protisten-

kunde ', Bd. iii, 1904.3. " t)ber vegetative Vorgange im Kern und Plasma der Gregarinen

des Regenwurmhodens. II. iStomatophora coronata, nov. gen.",ibid., Bd. x, 1907.

4. Hesse,E.—" Monocystidee nouvelle des Pherethna " , ' Bulletin mensueldc l'Association franchise pour l'avancement des sciences ', no. 9,1904.

5. " Contribution a 1'etude des Monocystidees des Oligoehctes",' Arch. Zool. exp. et gen.' (5), torn, iii, 1909.

6. Lankester, E. B.—"Protozoa", ' Encyclopaedia Britannica', vol. xix,9th ed., 1885.

7. Minchin, E. A.—' Sporozoa in Lankester's Zoology', part i, secondfascicle, 1903.

8. ' An introduction to the study of Protozoa ', 1912.

EXPLANATION OF PLATE 23.

Illustrating Mr. B. L. Bhatia's paper on ' The genusStomatophora '.

Figs. 1 to 4, and 15, 16, 18, 20-3 are free-hand sketches oforganisms examined alive in 0-75 per cent, salt solution. Theremaining figures are from films fixed in corrosive-sublimatealcohol and stained by the iron-haematoxylin method or byDobell's iron haematein, and examined with a Zeiss microscopeusually with a 2 mm. achromatic oil-immersion objective andno. 2 eye-piece.

Fig. 1.—Trophozoite showing characteristic gregarinoid movements.Cytoplasm is finely granular and laden with refringent granules, x 600.

Fig. 2.—Trophozoite showing a clear epinieritic area near the anteriorend. x 1,200.

Fig. 3.—Trophozoite showing a constriction of the body, x 600.Fig. 4.—Young trophozoite seen in a spermatic follicle, x 620.Fig. 5.—Young trophozoite surrounded by developing spermatozoa.

THE ODNUS STOMATOPH0.RA 511

The nucleus shows a distinct membrane, a single, slightly eccentric caryosome, and fine chromatin granules, x 1,470.

Fig. 6.—Young trophozoite surrounded by fully developed spermatozoa.The nucleus shows a clear ring-like area surrounding the caryosome,outside which is the linin network with very fine chromatin granules.The nuclear membrane is not definite. A few chromatoid bodies are foundscattered in the cytoplasm, x 940.

Fig. 7.—Small free trophozoite, showing the characteristic nucleus, somechromatoid bodies, and two small granules each of which is surroundedby a clear circular area, x 2,840.

Fig. 8.—Neogamous association of two young trophozoites still sur-rounded by spermatozoa, x 2,250.

Fig. 9.—Young trophozoite covered over with spermatozoa, and con-taining two nuclei each with a distinct caryosome, but with no definitenuclear membrane.

Fig. 10.—A typical full-grown free trophozoite showing the charac-teristic sucker which in this species is not surrounded by a petaloidcrown, x 1,000.

Fig. 11.-—A full-grown free trophozoite, showing a shallow cup-likesucker, with a denticulated margin, x 1,000.

Fig. 12.—Trophozoite showing a crateriform sucker, with a distinctprojection in its middle, x 1,000.

Fig. 13.—Trophozoite showing a flattened anterior end, with a distinctmucron projecting in its centre. The mucron consists of hyalin sarco-cyte covered over by epicyte, and has dark central core of entocyte runninginto it. X 1,000.

Fig. 14.—Trophozoite with its anterior end flattened and wavy, x 1,000.Fig. 15.—Spore appearing to contain a single, shining, rounded nucleus

in its centre, x 1,000.Fig. 16.—Isolated spore showing two nuclei. The spore is distinctly

swollen out in the middle, and at each pole has a flattened button-likeenlargement,which is seen to be clear and blight when accurately focused,x 1,500.

Fig. 17.—A spore containing eight nuclei, very greatly enlarged. Thesporocyst is drawn out at each pole and distinctly truncated. All thenuclei are not situated in the same plane and not arranged equatorially-X 2,000.

Fig. 18.—Cyst full of spores. The cyst-wall is thick and granular. Thespores are navicular, but truncated and thickened at the two ends, x 440.

Fig. 10.—Spore, x 1,500.

Figs. 20-5 .—Stomatophora c o r o n a t a .Fig. 20.—Showing alterations of form in the living organism.Fig. 21.—An unusually large trophozoite showing the sucker with

512 B. L. BHATIA

a distinct central mucron. The sucker is surrounded by an area markedby epicytal striations but not marked by a crown of petals, x 435.

Fig. 22.—The same organism as above, sketched after a quarter of anhour, when a number of petals had become distinctly marked off. x 435.

Fig. 23.—A full-grown trophozoite of the usual size showing the suckerwith a crown of petals. The nucleus shows characteristic structure.X500.

Fig. 23 a.—Nucleus greatly enlarged, showing a central vacuolatedcaryosome surrounded by a clear area, outside which is the peripheralohromatin, dispersed on a linin network.

Fig. 23 b.—Sucker greatly enlarged, showing a crown of nineteen petalsmarked by epicytal striations.

Fig. 24.—Cyst showing two zooids in association, each containing asingle nucleus. The nucleus in one of them is showing mitotic division,four chromosomes are seen towards each pole, x 500.

Fig. 25.—Cyst containing a large number of gametes mostly in the peri-pheral part of the cyst, x 500.

Figs. 26-31.—Stomatophora d i a d e m a.Fig. 26.—A full-grown free trophozoite, showing central sucker and the

body divided into five irregular lobes. The nucleus shows characteristicstructure, x 475.

Fig. 27.—A young trophozoite still surrounded by spermatozoa, x 500.Fig. 28.—A full-grown trophozoite showing the central sucker and the

body cleft into ten lobes. The nucleus is situated at the junction of twolobes, which are thus seen to be only incompletely divided off from oneanother, x 500.

Fig. 20.—A cyst containing two gametocytes in association, x 220.Fig. 30.—A cyst full of spores showing cystal residuum in the middle.

X 430.Fig. 31.—A few isolated spores, showing the bulging in the middle and

the aporocyst drawn out and truncated at both poles, x 1,500.

Quart. Journ. Micr. Sci Vol. 68, N. &, PI. 23