STRUCTURE, ETC., OP PLEISTOPHORA PERIPLANETiM. 615

Observations on the Structure and Life-historyof Pleistophora periplanetae, Lutz

and Splendore.

\V. S. Pcrrin, B.A.,Sliuttlewortli Research Student of Gonville and Caius College,

Cambridge.

With Plates 37 and 38.

CONTENTS.PAGE

1. Introduction . . . . . . 615

2. Material and Methods . . . . . 616

3. Structure of the Vegetative Stages of P l e i s t o p h o r a pe r ip lane tce 619

i. Auto-infective Methods of Reproduction . . . 620

5. Spore Formation . . . . . 621

6. Classification of Parasite and Discussion of Results . . 625

7. Note on a hitherto undescribed Parasite (?) of B l a t t a o r i e n t a l i s 628

8. Literature . . . . . . 6 2 9

9. Description of Plate . . . . . 6 3 0

THE subject of this research, which is still in progress, isa Myxosporidian parasite of the ordinary black cockroachPeriplaneta orientalis, found in bakehouses, collegekitchens, and similar places. The presence of this parasitein the cockroach was first recorded by Schaudinn, who states(6) that the spores " eines Nosema" occur in the fascesof cockroaches examined by him, and emphasises theirsimilarity to the spores of Bacillus biitschlii, parasitic inthe alimentary tract of Periplaneta orientalis. He de-scribes the presence of a polar capsule in these spores, and

616 W. S. PERRIN.

further states that, under the highest magnifications thespirally wound filament of the polar capsule is often quiteplainly discernible.1

Lutz and Splendore (8) describe the presence of the sporesof a Myxosporidian in the Malpighiau tubules and neigh-bouring gut of the Brazilian cockroach Periplanetaamericana, and refer them to the genus Nosema and addthe species name " periplanetse." The slight descriptionsgiven by Schaudinn and Lutz and Splendore of the sporesthey find in Periplaneta orientalis and Per iplanetaamericana respectively, fit, as far as they go, the sporesof the Myxosporidian which is the subject of the observationsrecorded in this paper. These observations show, however,that the parasite investigated belongs to the genus Pleisto-phora, a conclusion impossible to arrive at from a know-ledge of the unstained spores alone.

In spite of the fact that all attempts to infect Periplanetaamericana with the parasite by feeding them upon theinfected fasces of Periplaneta orientalis have beenunsuccessful, it is possible that the parasite is identical withthat described by Lutz and Splendore. I therefore proposeto retain the specific name p eriplanetae, while referring theparasite to its proper genus Pleistophora. The reasonsfor adopting this course will be entered into later, after theaccount of the life-history has been given.

MATERIAL AND METHODS.

Pleistophora periplanetas lives in the lumen of theMalpighian tubules of Periplaneta orientalis, of whichit is the sole inhabitant. A few trophozoites are generally tobe found also in the ileum together with a larger number ofpansporoblasts containing ripe spores, and the ripe isolated

1 The polar capsule is a structure occurring in the spores of the Myxo-sporidia, containing a 61ament which is extruded under the stimulus of thedigestive juices, when the spore reaches the alimentary canal of its host.The filament serves to attach the spore to the wall of the alimentary canal.

STRUCTURE, ETC., 01? PLEJSTOPBOBA PEBIPLAttETiE. 617

spores themselves. The parasite lives freely in the lumen ofthe alimentary canal and the Malpighian tubules. Examina-tion of the fresh tubule usually shows the trophozoites crowdingup against the sides of the passage, although they are fre-quently present in sufficient numbers to block the wholelumen completely. When the trophozoites are present inany number the cells of the tubule are comparatively clearand hyaline, the very numerous granules ordinarily presentin the cells of uninfected tubules being conspicuous by theirabsence.

Examination of the fresh tubules and sections, both trans-verse and longitudinal to them, has failed to reveal thepresence of intracellular stages. The parasite appears to bewholly extracellular, and, in this feature, differs from the restof the Cryptocystes, which are by definition cell parasites.The parasite is not apparently fatal to its host. Every adulbPe r ip l ane t a examined contained the parasite, and frequentlygreat numbers of it, without any apparent inconvenience.The reason of this is probably to be sought in the fact thatalthough the parasite may be present in very large numbersits presence is confined to comparatively few of the Malpighiantubules, nor do the tubules themselves, however stronglyinfected, appear to be destroyed.

Confusion with tissue elements or with any other membersof the singularly rich and varied fauna harboured by thecockroach is impossible on account of the very characteristicappearance of the trophozoites, more particularly when theyare stained with Giemsa's eosin-azur solution. As beforementioned, every adult cockroach examined contained theparasite in larger or smaller numbers. The very youngestindividuals still possessing the light brown tint characteristicof the infant cockroach were alone found to be uninfected.Spores occur regularly in the faeces.

For examination of the living plasinodia the Malpighiantubules were freed from adhering fat-body, cut in small piecesand examined in normal salt solution. Much of the structurerevealed in the stained preparation can be made out in the

618 W. S. PBERIN.

living cell. The addition of small quantities of vital stainingreagents, neutral-red, brillant-cresyl-blau and methylene bluefailed to disclose any further details of structure. Formaking pennanenfc preparations of the parasite, films fixedand stained in various ways were found to be alone admissible.Sections of 2 ju thickness cut in paraffin were found to be ofbut little value.

Films were prepared in two ways:1. The Malpighian tubules were cut up into small pieces,

which were spread over a coverslip. The coverslip was thenallowed to dry, immersed in absolute alcohol for ten minutes,dried, and stained with one of GHemsa's modifications of theRomonowsky-Nocht stain. The best results were given byleaving the films over night in a mixture of an aqueous solu-tion of eosin and azur I [ freshly prepared according toGriemsa's well-known recipe.1 The coverslips were washedwith tap-water after staining, dipped for a moment in absolutealcohol to prevent overstnining, washed again with tap-water,dried, and mounted in cedar wood oil.

When a preparation was required immediately, Giemsa'sready prepared eosin-azur solution was employed. Thespecially powerful mixture of azur I, azur II, and eosin usedby Schaudinn for the Spirochaete of syphilis was found to staintoo deeply and was discarded.

The above method of fixation and staining, though appar-ently drastic, gives excellent results. Careful comparisonwith the living material and preparations fixed with osmicvapour shows that the trophozoites themselves are but verylittle deformed in general outline, frequently not at all, whileno other method of staining differentiates nucleus and proto-plasm so sharply (see figs, on left hand side of plate).

The method has an additional advantage in tliat thenuclei stain differently. This point will be referred to later.Nuclei of the cells of the Malpighian tubules upon the otherhand are completely spoilt by this method (see tig. 15). The

1 Ten parts of a solution of 1 gr. eosin B.A. in 1000 c.c. water is addedto 1 part of a solution of -8 gr. Azur II (Griibler) in 1000 c.c. of water.

STRUCTURE, ETC., OP FLEISTOPHORA PERIPLANEQ.1.®. 6 1 0

reason for this is to be found in the different shape of theplasmodia and the nuclei. The plasmodia are thin and gene-rally leaf-like, while the nuclei are spherical, and henceundergo great deformation in drying on to the glass.

2. The Malpighian tubules were cat up in a very small dropof filtered egg-white, and fixed either by exposure to osmicvapour or by immersion in boiling corrosive sublimate andabsolute alcohol in the proportions 2 : 1 . After osmic thecoverglass was washed in water, after corrosive sublimate inwater plus iodine, to precipitate the mercury, the film in eachcase being either first hardened by passing through thealcohols in grades of 10 per cent., or stained straightway inDelafield's hasrnatoxylin. The excess of stain was then washedout iu acid alcohol, the film being finally mounted in xylol,or, preferably, alcohol, Canada balsam. Films fixed by theabove method were also stained with Heidenhain's iron-hasmatoxylin. Delafield's haematoxylin gave, however, betterresults. Fixation with osmic vapour and corrosive sublimategave practically identical results. The network of the nucleiof the cells oE the Malpighian tubule is somewhat finer withosmic than with sublimate, but the difference is very slight.

STRUCTURE OP THE VEGETATIVE STAGES OF PLEISTOPHOKA

PEB1PLASBTJ3.

The trophozoites are amoeboid nucleated masses of proto-plasm varying from uninucleated specks of protoplasm,fig. 1, from 2—3 fx in diameter to masses measuring in somecases 30/x by 55 JU, and containing 60 or more nuclei (fig. 13).The protoplasm may be homogeneous and hyaliue (figs. 1—6),or exhibit a marked foam-like structure, produced by thepresence of a large number of small vacuoles (figs. 10, 35).Vacuoles of every size are of frequent occurrence. Theprotoplasm of the smallest trophozoites possessing only a fewnuclei is, as a rule, hyaline, that of the larger inultinucleate

620 W. S.

ones sometimes hyaline (fig. 20) and sometimes foamy (fig.35). Granules appeal* to be absent, but large masses of amucoid-looking substance are occasionally enclosed by thetrophozoites.

In many cases differentiation of the trophozoite into adenser, more deeply staining, external region or ectoplasm,and an internal, more fluid, and vacuolated region or endo-plasm is well marked (figs. 25, 34, 35). Pseudopodia arefrequent, and generally lobose and rounded (tig. 34), thoughelongated and pointed pseudopodia also occur. The pseudo-podia are, as a rule, formed from the ectoplasm alone, andprobably serve to attach the trophozoites to the surface of thecells of the Malpighian tubules.

The nuclei are of two kinds, one which stains a bright redwith Giemsa and feebly with basmatoxylin, and one whichstains a deep purplish red with Giemsa and very deeply withheematoxylin (figs. 9,10, 31, 35). The deeply-staining nucleiare frequently contiguous to a small area of protoplasmstaining bright blue (fig. 10). They appear to be composedof degenerating substance, since the residual nuclei of thepansporoblasts, which are sometimes at first bright red (fig.14), stain a deep purple as the spores mature and their owndegeneration becomes more advanced. The nuclei, whichstain a bright red with Giemsa, are generally reticular,though not infrequently compact. In fig. 11 the nuclei arecompact, in fig, 13 reticular. Division of the nuclei appearsto be effected by simple fission (fig. 4).

AUTO-INFECTIVE METHODS OP KEPKODUCTION.

Multiplication of the trophozoites within the host takesplace in four ways :

(1) The young uuinucleate trophozoite may divide amito-tically into two daughter cells (figs. 50 and 51).

(2) The nuclei of a young trophozoite may multiplyamitotically, and proceed to the periphery of the cell (figs.

STBUCTUKE, ETC., Of PLEISTOPBOKA PERIPLANEM. 621

16 to 18, 36), while the protoplasm aggregates itself aroundeach nucleus, the trophozoite dividing finally into a numberof spores (fig. 19). A process similar to this has beenobserved by Donein in the case of Glugea lophii. It is amethod of reproduction which, in P l e i s t o p h o r a p e r i -planette, strongly recalls the schizogony of the Telosporidin,e. g. the breaking up of the malarial parasite into a numberof small sporozoites within the red blood-corpuscle.

(3) A multinucleate trophozoite may divide into two by aprocess of simple fission (fig. 11).

(4) Portions of a large multinucleate trophozoite mayseparate themselves from the parent cell and commence anindependent existence (figs. 13, 20, 35). The piece buddedoff may have one or several nuclei, and it seems that thebuds are all either uninucleate or multinucleate exclusively(figs. 13 and 20), a trophozoite never apparently producingbuds with one and many nuclei at the same time. A similarprocess to the above, of a multinucleate cell breaking up intomultinucleate fragments, has been styled plasmotomy byDonein. Division of the trophozoite into two fragmentshe calls simple; division into more than two fragments,multiple plasmotomy. The production of uninucleated swarmspores from a trophozoite by budding does not appear tohave been hitherto recorded amoug the Myxosporidia. It isa noteworthy fact that no dark purple nuclei are to be foundin trophozoites undergoing the above processes of reproduc-tion, and it seems likely, more particularly in view of theabsence of pseudopodia, that the trophozoites containingthese nuclei are about to form resting spores. This poiutwill be referred to later (page 622).

SPOKE FOBMATION.

All the above methods of reproduction subserve auto-infection, but resting spores are also produced which bringabout the infection of fresh hosts. These resting spores

622 W. S. PBBR1N.

possess a spore-coat or shell which is highly resistent to theaction of reagents. The spores gain the exterior in the faecesof the cockroach. Doflein calls the production of nakedswarm spores for auto-infection, and resting coated spores forinfection of fresh hosts "multiplicative" and "propagative"methods of reproduction respectively. Among the Myxo-sporidia it is usual as a preliminary to sporulation for thetrophozoite to produce a number of internal buds, which giverise to a varying number of spores, the production of sporescontinuing within the trophozoite without any cessation ofits activity or growth. These internal buds haye been styledby Gurley pansporoblasts. In P le i s tophora periplanetas,however, no such formation of pansporoblasts occurs, thewhole of the trophozoite simply withdrawing its pseudopodia,rounding itself off and producing spores. As, however, theclassification of the Myxosporidia is largely based on thefeatures exhibited by the pansporoblast, the number of sporesit produces, etc., it seems advisable to regard the trophozoiteof P l e i s t opho ra periplanefcse as producing one pansporo-blast, although the point at which trophozoite ceases to betrophozoite and becomes pansporoblast may not be easy todefine.

As a matter of fact I am inclined to regard the productionof the deeply staining purple nuclei (figs. 9,10, 12) as indica-tive of incipient sporulation for the following reasons :

(1) As mentioned above these nuclei never occur in tropho-zoites, which may be inferred to be actively metabolic, e. g.trophozoites in any of the stages of multiplicative repro-duction.

(2) The presence of these nuclei is associated with anabsence of pseudopodia and a general appearance, as thoughthe trophozoite were rounding itself off to form a pansporo-blast (figs. 9, 10).

(3) Apart from the spores it produces, the pansporoblastcontains a considerable amount of protoplasm and a number ofnuclei, which are not used in the production of spores. Thesenuclei are left behind, and, during the later stages of sporu-

STRUCTURE, ETC., OF PLEISTOPHORA PER1PLANETJE. 623

lation nearly always, and during the earlier stages usually,stain exactly the same tint as the above-mentioned purplishnuclei. Occasionally, it is true, these residual nuclei of thepansporoblast remain of a bright red tint even when sporu-lation is comparatively far advanced. This is, however,exceptional.

The first stage, then, of sporulation consists in the roundingoff of the trophozoite to form a pansporoblast, and the appear-ance of nuclei of two different kinds, the nuclei of the sporo-blasts, and the residual nuclei, which disappear with theremains of the protoplasm of the pausporoblast. The pan-sporoblast (figs. 14, 30—33) is usually a more or less ovalbody, occasionally oblong and rounded at the ends. It isalmost completely filled with developing spores, all roughlyin the same stage of development. The interstices betweenthe spores are occupied by protoplasm and nuclei, whichultimately disappear. The number of spores produced variesfrom three or four (fig. 33) to forty or more. From twentyto twenty-five is about the usual number. Each spore isformed from an oval mass of protoplasm—the sporoblast,—which rather exceeds in size the spore it produces. Instained preparations the sporoblast appears to lie in avacuole. This vacuole is not, however, evident in the livingcell, and is probably an artifact produced by shrinkage ofthe protoplasm during preparation. It is most marked inpreparations fixed with osinic vapour and sublimate alcohol,although it also occurs in preparations fixed by drying ou tothe slide and immersing in absolute alcohol (figs. 14, 30—32).The number of sporoblasts and residual nuclei is roughly thesame. The two kinds of nuclei present, however, considerabledifferences in appearance and staining power. The residualnuclei are compact and have a sharply-defined outline fromthe first. With Delafield's hasmatoxylin they stain a verydeep blue; with Giemsa, as mentioned above, usually a deeppurple. The nucleus of the sporoblast stains differentlyaccording to its state of development. In the earliest stages(figs. 14, 30, 38, 39) it is in the form of a sparse reticulum or

624 W. S. PEERlN.

a number of discrete particles. With Giemsa it stains brightred, with liEematoxyliD, however, but faiutly, in marked con-trast to the residual nuclei (figs. 30—33). Later, the nucleuscondenses to form a sphere containing a central particle(fig. 31). This sphere stains faintly at first, but as it con-tracts the staining becomes much deeper (figs. 32, 40—42).The now compact and deeply staining nucleus shifts itsposition from the centre to the end of the spore, iti corre-spondence with the development of a vacuole with highlyrefractive contents.

Figs. 41—43 illustrate the gradual development of thisvacuole and the shifting of the nucleus, which now dividesinto two, the daughter nuclei moving to the centre of thespore (figs. 44—47). Fig. 47 indicates that the two nucleiare once more dividing. Later stages than the above havenot been satisfactorily stained owing to the very resisteutnature of the spore coat. While these nuclear changes havebeen taking place the spore has been surrounding itself witha spore coat or shell. This, as shown in fig. 49, consists oftwo halves meeting in a median suture. It is presumablythrough this suture that the sporoplasm when infecting a newhost makes its exit. The spore in its final form is a flatoblong structure rounded at the ends (figs. 48, 49), varyingfrom 5 /.t—6 JU in length, and 2"5ju—3 ^ in breadth. In theliving state the sporoplasm is finely granular, and at one endof the spore a small highly-refractive globule is present. Therelation of the polar capsule, which is almost certainly present,to the vacuole described above and the refractive globule Ihave, as yet, been unable to determine. Application of the usualreagents—ether, concentrated sulphuric, nitric, and hydro-chloric acids, glycerin, iodine, and boiling water, etc.—hashitherto failed to procure extrusion of the filament of the cap-sule. Experiments now in progress will, I hope, throw furtherlight upon this point. The spore coat is unaffected by theabove-mentioned reagents, and also by 'eau de Javelle' (sodiumhypochlorite). Attempts to pi-oduce the emergence of thesporoplasm from the spore by treating the feeces with the

STBTJCTUBG, ETC., OP PLRTSTOPHORA PERIPLA.NEM. 625

digestive juices and contents of the alimentary canal of othercockroaches have been as yet unsuccessful.

CLASSIFICATION OF PAEASITE AND DISCUSSION OF RESULTS.

With the above facts of the life history of P l e i s t o p h o r aper ip lanetas to hand it is possible to classify the parasiteaccurately. From the minuteness of the spores, the fact thatthe pansporoblast produces more than two spores, and theinvisibility of the polar capsule in the fresh state, the parasitebelongs to the sub-order Crypfcocystes of the Myxosporidiaand the family Glugeidas, which possesses the characters ofthe sub-order. In the fact that the spores are not peai1-shaped, but oblong with rounded ends, and that the parasiteis extracellular it differs, however, from the i-est of theCryptocystes. It belongs to the section Oligosporogenea(Doflein), because the trophozoite produces a single pansporo-blast, and the production of numerous spores by the pan-sporoblast relegates it to the genus P le i s tophora . It isthis formation by the trophozoite of a single pansporoblastinstead of a number of them, which removes the parasite fromthe genus Nosema, section Polysporogenea, to the genusPle i s tophora , section Oligosporogenea. To sum up, theclassification of the parasite is as follows :Class.—Sporozoa.

Sub-class.—NEOSPOEIDIA.1

Order.—Myxosporidia.Sub-order.—C ryp tocys t e s .

Family.—G1 u g ei d EB.Section.—0 l igosporogenea .

Genus.—Pleistophora, Gurley, 1893.Species.—Periplanetse, Lutz and

Splendore, 1903.1 Reasons, based on the life-history of Pleistophora periplatietse, for

objecting to this method of subdivision of the Sporozoa by Schaudinn aregiven on page 626.

626 W. S. PBERIN.

There are several points of interest offered by the abovescattered details of the life history of P le i s tophora pe r i -planetas, both with respect to the Sporozoa in general, andthe Myxosporidiu in particular. In the first place it is seenthat there are practically two very definite phases in the lifehistory, a schizogonous phase, characterised by almost exces-sive multiplication with a view to auto-infection, and a sporo-gonous phase characterised by the cessation of growth andtrophic activity and the formation of resting spores. Thetwo phases are sharply marked off from one another and cer-tain morphological differences are presented by the tropho-zoites of the two phases. In this feature, i. e. the sharpseparation of schizogonous and sporogonous phases from oneanother, P l e i s t o p h o r a per iplanet te resembles The lo-hania mulleri , a Myxosporidian belonging to the closelyallied genus Thelohania, described by Stempell. Stempelldistinguishes two kinds of trophozoites of Thelohaniatniilleri—•" meronts," which multiply by a simple form ofdivision, and "sporonts," larger elements, which produceresting spores. No such difference of size marks out thetrophozoites characteristic of the multiplicative and propaga-tive phases of P l e i s tophora periplanetEe, but the markeddifference in behaviour of the two forms of trophozoites, and,perhaps, the appearance of the purple nuclei in the tropho-zoites about to sporulate, constitute an equally distinctivedifference.

The occurrence of sporulation at the end of the schizogonousphase by no means agrees with Schaudiun's division of theSporozoa into Telosporidia, i. e. Sporozoa in which sporeformation occurs at the end of the trophic phase, andNeosporidia comprising the Sarcosporidia and Myxosporidia,in which spore formation continues during the trophic phase.As far as the point in the life history, at which spore formationoccurs, is concerned, P le i s tophora pe r ip lane t t e is inexactly the same position as a Haemosporidian or aOoccidiau.In each case a trophic phase characterised by vigorous multi-plication is succeeded by a resting phase characterised by the

STRUCTURE, ETC., OE PLEISTOFHOBA PERIPLANET2E. 627

production of resting spores. As Stempell remarks,Schaudinn's grouping of the Myxosporidia and Sarcosporidiatogether as a sub-class separate from the rest of the Sporozoa,is probably well-grounded, but the difference between thetwo sub-classes must be expressed in other terms than theperiod in the life history at which sporulation occurs. Theevidence afforded by the disporous Phsenocystes, in which thetrophozoite produces only one pansporoblast, which gives riseto two spores, while the residual protoplasm ultimately diesoff, and the conversion of the trophozoites of The lohan iaand G-urleya into a single pansporoblast, support this view.

Another point of interest in the life-history of P le i s to -phora pe r ip lane t t e is afforded by the existence of theresiduary nuclei,1 which, together with the protoplasm of thepansporoblast, die off, while sporulation is being effected.In the remaining members of the Cryptocystes and all thePhaenocystes, with the exception of the Disporea—also "free"parasites—no such separation of residuary nuclei occur. Inthe Disporea two residuary nuclei are lefb behind to die off,one for each spore of the pansporoblast. The meaning ofthese residuary nuclei is obscure. At first sight it wouldappear that the residuary nuclei and protoplasm are togetherhomologous with that part of the trophozoite of the Disporeawhich remains after the separation off of the pansporoblast,but if this is the case, what represents this residuary mass ofnuclear material and protoplasm in the remainder of theOligosporogenea, including Thelohania mul ler i , Grurleya,etc., in which the trophozoite is converted bodily into thepansporoblast with no residuum ?

It is significant that in The lohan ia mulleri , while thespore in its final form possesses two nuclei just like P le i s to-phora periplanetas , these two nuclei increase to four innumber while lying in the gut of a new host (Gammarus)before germination. Stempell regards this process as one of.nuclear reduction, preliminary to conjugation with the amoe-boid contents of another spore. In view of the above fact it

1 See spore formation, p. 621.

628 "W. S. PERRIN.

seems possible that the residuary nuclei of the pansporoblastof P le i s tophora per iplauetfe represent reduction bodies,and that in the case of Thelohania miilleri (and possiblyother Oligosporogenea) the extrusion of these reductionbodies takes place later on from the spore itself. It isperhaps somewhat premature to attempt any homologisationsor explanation of the meaning of these residuary nuclei inthe present undeveloped condition of this branch of Protozoanresearch, more particularly in view of the fact that in nosingle case has conjugation been recorded in the case of aMyxosporidian, and in no single case has the development ofan adult trophozoite from the sporoplasm of a resting sporebeen traced. In view, however, of the rapidly increasinguumber of cases in which conjugation has been shown totake place among the Protozoa, e.g. Actinosphserium, itseems very improbable that the Myxosporidia will prove tobe an exception to what is being shown to be apparently therule among even the most unspecialised forms of life. Thepresence of these residuary nuclei and Stempell's observa-tions on the spore of The lohan ia miilleri are certainlysuggestive of a preparation for conjugation.

Among the bacteria themselves, a class of organism whichhas been regarded till lately as in no case exhibiting aconjugatory process among its members, conjugation in amodified form has been shown to take place.1 The existenceof the above-mentioned gap in our knowledge of the Myxo-sporidian life-history, and the silence of all observers on thesubject of conjugation among the Myxosporidia are pheno-mena probably not unconnected with each other.

NOTE ON A HITHERTO TJNDESCRIBED PARASITE (?) OF

PEKir-LANETA OKIENTALIS.

In the course of these present researches certain peculiarstructures, which occurred in many of the stained prepara-

1 Scliaudinu lias recently (1902) shown that a kind of conjugation occurs inBacillus butschlii parasitic in the ileum of Peri planet a ameticana.

STRUCTURE, ETC., OP PLEISTOPHORA PERIPLANEM. 629

tions of the Malpighian tubules, attracted attention. Severalof these structures are represented in figs. 21—24, and 37.They present an appearance similar to the trophozoites andspores of P l e i s t o p h o r a p e r i p l a n e t s e upon a smaller scale,and stain in similar manner with the same reagents. Thetrophozoites (figs. 21—23) are, however, of a more delicatestructure than the trophozoites of P l e i s t o p h o r a p e r i -planetas, the nuclei being much smaller and more compact,while the protoplasm is also more delicate. The spores(figs. 22 and 37) are very much smaller, measuring not morethan 1 [x—2JU in length and about "5JU iu breadth, instead of5-5/x x 3fi as in P l e i s t o p h o r a p e r i p l a n e t r e . No inter-mediate stages between these forms and P l e i s t o p h o r ap e r i p l a n e t s e have as yet been observed, and I am inclinedto think that these structures are the trophozoites and sporesof a new Myxosporidian parasite. The structures are, how-ever, in need of further investigation, and pending furtherresearch I have decided only to mention their occurrencewithout giving them a name. They may turn out to belongto the life-cycle of P l e i s t o p h o r a pe r i p l ane t s e , and indeedlittle beyond the statement that they are almost undoubtedlyorganised bodies is permissible, under the circumstances.

LITERATURE.

1. 1867. BALBIANI, G.—"Etudes sur la Maladie Psorospermique des Versa Soie," extrait du 'Journal de l'anatomie et de la phjsiologie,' de M.Ch. Robin.

2. 1894. GUKLEY, R. R.—'Bull. U.S. Fish Comm./ Rep. of Comm., 1892,pp. 65—298.

3. 1898. DOFLEIN, J.—' Zool. Jahrb.,' Bd. xi, " Studien zur Naturgeschichteder Protozoen : III. Uber die Mjxosporidien."

4. 1S99. DOFLEIN, J.—"Fortschritte auf dem Gebiete der Myxosporidien-kunde," 'Zool. Centralbl.,' vi Jahrg., Nos. 11, 12.

5. 1899. MRAZEK, Al.—' S. B. Bohm Ges.,' viii, 5 pp.

6. 1902. SCIIAUDINN, F.—'Archiv fur Protist./ 306— 343.

7. 1902. STJSMPELL, W.—' Zool. Jahrb. Abth. f. Anat.,' xvi, 2, pp. 235—272.

8. 1903. LUTZ and SPLENDOM.—' C. B. Bakt. Pk.' (1), xxxiii, pp. 150—157.

VOL. 49, PART 4. NEW SERIES. 46

630 W. S. PEBRIN.

9. 1904. WOODCOCK, H. M.—" Myxosporidia in Flat Fish," Herdman'sReport on the Lancashire Sea Fisheries Scient. Inv. f. 1903,' Trans.Biol. Soo. Liverp.,' vol. xviii, 1904.

10. 1904. WOODCOCK, H. M.—"Nole on a Remarkable Parasite of Plaiceaud Flounders," ibid.

11. 1901. STEMPELL, W.—"Uber Nosema anomalum (Monz)," 'Archivf. Protist.,' iv Bd., 1 Hft., 1904, p. 1.

EXPLANATION OF PLATES 37 & 38,

Illustrating Mr. W. S. Perrin's paper on "Observations onthe Structure and Life-history of Pleistophora peri-planetfe, Lutz and Splendore.

All the figures refer to Pleistophora periplanetae from the Malpighiantubules and gut of Periplaneta oricatalis with the exception of Figs. 21,22, 23, 24, and 37, which refer to another Myxosporidian parasite from thesame locality.

The figures are all drawn with the help of Zeiss's drawiug camera fromstained preparations. Those upon the left half of the plate are drawn andcoloured from preparations stained with Giemsa's eosin-azur. Nuclear struc-tures are in all cases tinged a bright red or purple and protoplasm a brightblue. The remaining figures are drawn from preparations staiued withDelafield's hsematoxylin, with the exception of Fig. 28, which was stained byHeidenhain's iron hsematoxylin method.

A Zeiss microscope with an apochromatic 3 mm. homogeneous oil immersionobjective (mm. ap. l'4O) and compensating eye-pieces 12 aud 18 was used inall cases.

Critical illumination with strong artiGcial light, Welsbach incandescent,etc., was employed.

Magnifications.—Figs. 1—32, 34—37, 50, 51: 1750/1..Figs. 33, 38—49 : 2700/1.

PLATE 37.

FIGS. 1 to 8.—Series of trophozoites of increasing size and with nucleiranging from one to eight in number. Fig. 4 shows a trophozoite with threenuclei, one of which is in process of division. The series illustrates thedevelopment of a multinucleated plasmodium from a uninucleated sporozoite.

FIG. 9.—Multinucleated trophozoite with reticular protoplasm and numer-ous vacuoles. The nuclei are of two kinds, the more numerous variety being

STRUCTURE, ETC., OF PLEISTOPHORA PERIPLANEM. 631

reticulated and coloured a bright red, the less numerous compact and of apurplish tint. In the middle of the trophozoite an aggregation of these purpleand probably degenerated nuclei is to be seen. The presence of these nucleiprobably indicates approaching sporulation.

FIG. 10.—Another trophozoite of similar character.

1?IG. 11.—An example of simple plasmotomy. A trophozoite is in the laststage of binary fission.

FIG. 12.—Small trophozoite showing the early occurrence of the purplenuclei.

FIG. 13.—Large trophozoites undergoing multiple plasmotomy, or, in otherwords, budding off small multinucleated masses of protoplasm, which willbecome independent trophozoites. One bud is already detached.

PIG. 14.—Pansporoblast at an early stage of development. Each of theoval sporoblasts appears to be lying in a vacuole. This appearance is pro-duced by the shrinkage which the protoplasm undergoes in preparation. Thenuclei of the sporoblasts are seen to be diffuse and reticular. The interstitialor residual nuclei retain in many cases the bright red tint of the normalnucleus. Two can be seen to be purplish. Fig. 30 represents a pansporoblastat a very slightly earlier stage.

FIG. 15.—Nucleus of cell of Malpigliian tubule fixed by drying and showingthe red colour assumed by nuclei, when stained by Giemsa. The figure shouldbe compared with Fig. 27, which represents a similar nucleus fixed, however,with corrosive sublimate and alcohol, and stained with Delafield's hsema-toxylin.

FIG. 16.—Multiple amitosis; trophozoite about to break up into foursporozoites.

FIG. 17,—Similar figure, but nuclei more numerous.

FIG. 18.—Later stages of same process.FIG. 19.—Group of sporozoites just after division.EIG. 20.—Large trophozoite budding off unenuclealed spores.

FIG. 21.—Trophozoite of hitherto undescribed Myxosporidian with thesame habitat as Pleistophora periplanetse. The nuclei are compactand much smaller than the nuclei of the Pleistophora.

FIG. 22.—Trophozoite and group of spores of the same Myxosporidian.Several of the nuclei undergoing binary fission.

FIG. 23.—Trophozoite of same.

PIG. 24.—Trophozoite of same, the nuclei being reticulated and surroundedby a vacuole.

FIGS. 50 and 51.—Small trophozoites dividing into two.

632 W. S. PBRRIN.

PLATE 38.

5.—Trophozoite of Pleistophora periplanetse showing elongatednon-nucleated pseudopodium of ectoplasm. Sublimate alcohol and Delafield.

FIG. 26.—Trophozoite giving off bud.

FIG. 27.—Nucleus of cell of Malpighian tubule, fixed with hot sublimatealcohol, stained Delafield. This figure, with Fig. 15, is inserted for thepurpose of indicating the size of Pleistophora peri planet as—the nucleusmeasures 16 /i in diameter—and showing the drastic effects of fixation bydrying on such a spherical body as a tissue nucleus (see Fig. 15 as comparedwith Fig. 27). The leaf-like trophozoites are, upon the contrary, well pre-served by this method of fixation.

FIG. 28.—Trophozoite fixed with osmic acid vapour and deeply stainedwith iron hsematoxylin.

FIG. 29.—Trophozoite showing the mucoid masses which occasionallyoccur in the trophozoites.

FIG. 30.—Pansporoblast with sporoblasts containing a nucleus in the formof scattered dots or a reticulum. Stage is slightly antecedent to Fig. 14.The residual nuclei are seen as deeply stained masses between the sporo-blasts.

FIG. 31.—Pansporoblast at a later stage. The nuclei of the sporoblastsare now in the form of a sphere containiug a central particle.

FIG. 32.—Still later stage. The nuclei are still more condensed and thespore coats are beginning to be formed.

FIG. 33.—Small pansporoblast with four spores. Stage much later than inFig. 32. A thick spore coat is present and two nuclei, one each side of thecentral vacuole, are to be seen. Magnification 2700/1.

FIG. 34.—Trophozoite in an early stage of multiple plasmotomy.FIG. 35.—Later stage in same process. One bud is just free.FIG. 36.—Multiple amitosis ; see also Figs. 16—19.FIG. 37.—Spores of hitherto undescribed Myxosporidian fixed with sub-

limate alcohol and stained Delafield's hsmatoxylin.

FIGS. 38 to 47.—Series of figures showing the changes in the nucleus inthe development of the spore from the sporoblast.

Fig. 38.—Nucleus of the sporoblast as a number of discrete particles.

Fig. 39.—Nucleus somewhat more condensed than in Fig. 38. Particlesfewer and larger.

Fig. 40.—Nucleus of spore in the form of a ring of four chromosomes.Fig. 41.—Similar stage. A small vacuole is to be seen in the aporoplasmFig. 42.—Later stage. Vacuole increased in size.

STRUCTURE, ETC., OF PLEISTOPHOKA PEE]PLANETM. 633

Fig. 43.—Vacuole occupying nearly the whole of the length of the spore.Nucleus situated at one end.

Fig. 44.—Nucleus divided into two.Figs. 45, 46, and 47.—Showing change in position of two daughter

nuclei from end of spore to its middle. The vacuole has increased inits refractive power. Fig. 47 represents the latest stage of the spore tostain satisfactorily. The two nuclei show indications of division.

FIG. 48.—Spore in about the same condition as in Fig. 47> seen from theside.

FIG. 49.—Ripe spore showing the suture between the two halves of theshell or spore coat.

For Figs. 50 and 51 see Plate 37.

Sci.Vol. U9J.SM.3l

1.

3 3 .

3 4 . 3 5

3 6 . I I3 9. 40 . 41. 42. 43.

WS Parria,46?

Magnification,, Figs. 25~32, 34-37. '7<>°/,.33, 38 - 4-9. "°?,.

48 W 49.̂' London.