SOME OBSERVATIONS ON ACINETAEIA. 351

Some Observations on Acinetaria.

ByC. II. Martin, B.A.,

Demonstrator in Zoology at Glasgow University.

With Plates "VII and VIII.

Part I.—The " Tinctin-kbrper " of Acinetaria and the Conjuga-tion of Acineta papillifera.

CONTENTS.

PAGE

I . Introductory . . . . . 351I I . The "Tinctin-korper " of Acinetaria, with some Obser-

vations upon the Nuclear Changes of T o k o p h r y ae l o n g a t a . . . . 352

I I I . ^Reproduction and Conjugation of A c i n e t a p a p i l l i f e r a 357IV. Conclusions . . . . . 375

V. Literature . . . . . 3 7 6

I. INTRODUCTORY.

The observations described in this paper were begun on aculture of Tokophrya elongata, which was given to meat Munich by Professor Hertwig in the summer semester of1902. This culture was followed for a period of about sixmonths, when it was unfortunately lost, but as during thistime I had never seen any indication of conjugation, I thought

VOL. 53 , PART 2. NEW SERIES. 25

352 0. H. MARTIN.

it better to place the results I had obtained on one side untilthe occurrence of a more favourable opportunity.

During the early summer of 1907, whilst working in Norfolk,I found an Acinetarian which corresponded with Keppen'sAcineta papillifera, in great abundance on the stems ofCordylophora lacustris in Hickling Broad. During theeai'ly part of September au epidemic of conjugation set in,and although, owing to the pressure of other duties, I wasnot able to follow the stages in the living form as completelyas I should have wished, I was able to obtain enough fixedmaterial to work through the main nuclear changes in thisprocess. In the second part of this paper the life-history ofa new Acinetarian parasite Tachyblaston ephelotensisis described, and in a future pa.per I hope to deal more fullywith some general questions as i*egards the mechanism, of thetentacles, as well as the relations between the lageniformand proboscidiform individual in that remarkable animalOphryodendron.

I should like to take this opportunity of thanking ProfessorHertwig, to whom the inception of this woi'k is entirely due,for the great kindness he showed me at Munich, and ProfessorGraham Kerr for allowing me to work through my materialin the laboratory at Glasgow University.

I I . THE " TlNCTIN-KOBPEK" OP ACTNETARTA WITH SOME OBSEE-VATIONS UPON THE NUCLEAR CHANGES OF TOKOPHBYAELONGATA.

Before proceeding to give an account of the conjugation ofAcineta papillifera, it will be necessary to give someaccount of those isolated masses of chromatin, the " Tinctin-korper" of Plate, which may occur in all free-living Acine-tarians, and which enormously increase the difficulties ofwork upon the nuclear changes during conjugation.

Plate in 1886 first drew attention to certain rounded bodiesoccurring in the cytoplasm of Dendrocornetes which were

SOME OBSERVATIONS ON ACINETARIA. 353

not blackened by osmic acid, and which were stained bysafranin, and also, though less brilliantly, by carmine.

He called these bodies " Tincfcin-korper," and noted thatin some individuals they seem to be altogether absent.

When present they are usually more or less rounded bodiesmeasuring up to "006 mm., but occasionally they assume" eine wurst-formige unregelinassig langliche Gestalt undeireichen dann auch eine viel betractlichere Grosse."

At first he was inclined to believe that these bodies wereequivalent to the micronuclei of the Ciliates, but he concludesby stating that the present state of our knowledge " liisst esrathsamer erscheinen sie vor der Hand nur als eigenartigeProdukte des Stoffwechsels anzusehen."

Schneider also observed these structures in Sty loco m etes,but was inclined to believe that the " Tinctin-korper " arethe products of the degeneration of the old macronucleusafter conjugation.

This view is also upheld by Biitschli (in 1889) " Ich haltediese Ansicht fur recht wahrscheinlich um so mehr, als wirja auch bei den Oiliaten erfuhren, dass die Fragmente desAlten Ma. N. haiifig sehr lange erhalten bleiben, und beider Theilung auf die Nachkonimen ubergehen konnen, wie esfur die Tinctinkorper der Dendrocometinen gilt."

Sand (1901) in his monograph puts forward the view(p. 31) that "lorsqu'un Acin&tien suce une proie, le sarcode decelle-ci, dtis son entree dans le cytoplasma du TentaculifSres'amasse en spheres de memes dimensions que les spheres detinctine; comme le cytoplasma mort est treis colorable, et quedu reste, les spheres contiennent, outre le cytoplasma le noyaude la proie, elles se colorent fortement par les teinturesnucleaii-es."

Personally I believe that under the name of " Tinctin-korper " chromatic granules which may originate in one ofthree distinct ways are confused—those derived

(1) From the ingested nucleus of the prey,(2) From the degenerating macronucleus after conjugation,(3) Possibly to a very slight extent in some cases from

354 0. H. MARTIN.

fragments of the macronuclens thrown out in connection withthe formation of a digestive ferment.

Of these three modes of origin the first is by far the mostcommon since conjugating Acinetaria are relatively quiterare. But it would still seem necessary to retain the generalterm "Tinctin-korper" since practically these bodies areonly distinguishable when the details of the past life of theindividual under examination are known.

Tokophrya (Biitschli) elongata (Clap, et Lachm.) is amore or less cylindrical Acinetan provided with a short stalkand with tentacles scattered in rather indefinite groups overthe surface of the body.

It is an exceedingly easy form to cultivate, as it feedsreadily upon almost all Ciliates, and appears to have fewenemies.

My cultures were kept in watch-glasses, to the bottoms ofwhich the young Acinetarians fixed themselves by their stalks;they were generally fed upon Stentor, but when the supplyof Steutor was short they seemed to thrive equally well onParamecia .

When the Stentor came in contact with the tentacles ofthe Acinetan, after some short struggles the cilia of the formerceased to beat and the animal remained paralysed.

As the Acinetan was very much smaller than its prey thefirst attack was rarely fatal, but if, as generally happened ina healthy culture, a single Stentor was attacked by five orsix Acinetarians the whole animal would disappear at the endof a couple of hours.

To the details of this process and the mechanism of thetentacles I hope to return in a future paper in which I wishto describe certain feeding experiments made on Ephelotagemmipara.

Tokophrya elongata reproduces itself by the formationof internal hypotrichous ciliated buds, which escape through" the birth opening"—a lateral slit in the pellicule.

In a normal Tokophrya elongata the macronucleus isa band-shaped structure passing down the long axis of the

SOME OBSERVATIONS ON ACINETAE1A. 355

body, but in addition to this there are always scattered inthe cytoplasm a number of granules of chrornatin, the" Tinctin-korper."

There seem to me to be three lines of evidence showingthat the ordinary " Tinctin-korper " cannot be regarded asintegral parts of a fragmented rnacronucleus—

(1) From their behaviour during reproduction and con-jugation.

(2) From their behaviour during starvation and feeding.(3) From fcheir absence in parasitic forms such as Tachy-

blas ton ephelotensis to be described later.(1) There is no trace of a connecting thread between the

" Tinctin-korper" such as is present in the macronuclei ofS tentor and H olophry a, and there is no reunion to a singlemass previous to division, such as has been described forsome Hypotrichous Oiliates with a fragmented macronucleus.

In dividing Tokophrya the macronucleus becomes twistedupon itself in the region of the future bud, but in all thestages of division isolated "Tincfcin-korper " are to be foundboth in the cytoplasm of the mother and of the bud(PI. VIII, fig. 2).

In conjugating Acineta papi l l i fera at stages at whichthe macronucleus was still intact individuals were found inwhich the " Tinctin-korper " seemed to be collected in thelower part of the theca, quite apart from the macronucleus.

(2) Their Behaviour during Starvation andFeeding.—During starvation there is a tendency for the" Tinctin-korper" to disappear, though even in the mostcomplete cases (PI. VIII, figs. 3 and 4) some remains were pre-sent. In PI. VIII, fig. 4, which represented the last stage ofstarvation, the individual measured 25/u in length and 18/uin breadth, a normal well-fed Tokophrya from the parentculture measuring 143 /j. by 58 /x. The tentacles and most ofthe cytoplasm had disappeared, bnt the nucleus seemed quitehealthy staining rather more readily than in the well-fedforms.

In preparations made a quarter and half an hour after

356 C. H. MARTIN.

feeding slightly-starved Tokophrya upon Stentor the" Tinctin-korper " showed no inai'ked increase over the con-trol forin, and it is interesting to note that up to this stagethe nuclei of the Stentor , although slightly swollen, had notbeen ingested. But iu individuals which had ingested thenuclei of the prey euoruious masses of " Tinctin-korper"were found resulting in such an appearance as is figured inPI. VIII, fig. 1, in which the whole of the cytoplasm isblocked with "Tinctin-korper."

It is remarkable how little attention has been hitherto paidto the possible presence of ingested chromatin in Protozoa,although it is evident that unless the process of digestion isextremely rapid, these ingested masses may quite easily forma considerable source of error in the description of the nucleusof any holozoic protozoon.

As far as I am aware the only careful description of theappearance and behaviour of ingested chromatin in a Proto-zoon is to be found in Schaudinn's account of Tricho-sphasriutn sieboldii. Schaudinn (p. 81) found that inTrichosphseriuni the nuclei eight lioui-s after ingestion werenot greatly changed; in later stages the linin is dissolved,and the chromatin sinks to the lower side of the nucleus."Das Chromatin wird nun auch allmahlich gelost, und niinmthierbei meist Kugelgestalt an. Es schien mir, als ob hierbeiseine Farbbai-keit zunimrab was vielleicht darauf beruht, dassbei der Verdauung ein nicht farbbarer Theil seiner substanzfriiher gelost wird, wahrend die farbbarer Theilchen dichterzusainrnengedraugt werden und daher in ihrer Gresainmtheitdunkler gefarbt erscheinen."

As regards the details of this process of digestion inAcinetai-ia, the earlier stages are passed through whilst thenucleus is still lying in the cytoplasm of its prey. But in theearly stages of the degeneration of the macronucleus of aconjugating form quite analogous early stages of digestionare to be found in the cytoplasm of the Acinetarian.

The later stages of the digestion as figured by Schaudinn

SOME OBSERVATIONS ON ACINETAE1A. 357

have an absolutely identical appearance to the " Tinctin-korper " of the Acinetaria.

(3) In a parasitic form, Tachyblasbon ephelotensis,which I describe in the second part of this paper, I couldnever find any trace of "Tinctiu-korper."

It is interesting to note that in this case the nucleus of thehost was never ingested by the parasite.

From a consideration of these points it will, I think,become evident that, in the great majority of cases, thescattered chi'omatin granules found in Acinetaria can onlybe regarded as the partially digested nucleus of their prey.

III. THE CONJUGATION OF ACINETA PAIMLIIPERA.

Without attempting to give a history of our knowledge ofconjugation in the Acinetaria (which has already been fullydone by Biitschli in his great work on Protozoa as regardsthe earlier pei-iod), it will be necessary to give a shortaccount of the work done by later observers of this process,and more particularly of Keppen's woi'k upon Acinetapapillifera, the intrinsic merits of which have almostentirely been overlooked, owing largely, no doubt, to thefact that it was written in Russiau. Keppen, in his paperpublished in the ' Memoires de la Societe des Naturalistes dela Nouvelle Russie/ Odesse, T. 13, 1888, states that he hadnot been able to follow the whole process of conjugation, ofwhich he could only find some stages, but that the little hehad seen had led him to believe that conjugation in thisgroup, as in the Infusoria, was connected with a reformationof the nucleus, the new macronucleus being formed from thedivision of the micronucleus. He described in some detailthe breaking down of the macronucleus, and in the laterstages he saw amongst the degenerating masses of the oldmacronucleus lightly-staining bodies, which he regarded, onthe analogy of Ciliata, as the division products of the micro-nuclei. Keppen figures four stages in conjugation, of which

358 0. H. MARTIN.

fig. 50 shows the macronuclei drawn out into long strandsand two micronuclear spindles, and fig. 31 shows an Acinetawith a new light-staining nucleus surrounded by the smallspherical products of the degeneration of the old nucleus.

At the time at which Keppen wrote the part played by themicronucleus in the conjugation of ciliates was not thoroughlyunderstood, but it is to him we owe the first definite recogni-tion of a micronuclear spindle in an acinetarian.

At the date at which Biitschli wrote his account of theSuctoria in ' Bronn's Thierreich' there were numerous iso-lated references to conjugation amongst Acinetaria to befound in the literature of this group. But as the onlyobservers—Plate and Schneider—(with the exception ofKeppen vide supra) who had followed the internal changesconnected with this process, denied the existence of a micro-nucleus, it will be seen that much remained to be done.Biitschli himself remarks, p. 1917:—"Die vorstehendenErwagungen zeigten, dass Copulationen nicht tnit geniigen-deu Sicherheit erwiesen sind. Dagegen istfiir die Dendroco-metinen sicher, dass ihre conjugation im Wesentlichen wiedie partielle der Oiliaten verlauft, woraus wohl geschlossenwerden darf, die's gelten auch fur die iibrigen, uicht genaueruntersuchten Conjugation."

In Maupas' paper, " Le Kajeunissement Karyogamiquechez les Cilies" (1889), he mentions without figures theresults he had obtained in the conjugation of two acine-tarians Podophrya fixa and Podophrya cyclopum. Inboth these forms there is only a single micronncleus, and inPodophrya cyclopum there is apparently complete fusionwithout the previous existence of any differentiation betweenthe two gametes.

Maupas describes the conjugation of Podophrya fixa,which, as in the case I describe, is partial, in the followingwords (p. 385) :—" Chez la Podophrya fixa j'ai observe lesstades A, B, C and H Pendant ce dernier stade je n'ai jainaisvu qu'un seul nouveau corps nucieaire et un seul' micro-nucleus. Je ne serais done pas etonne que la seconde

SOME OBSERVATIONS ON AC1NBTAEIA. 359

division de la nucleus mixte de copulation, correspondanfc anstade Gr, ne se produise pas chez cette espece. L'ancienNoyau se desorgaaise et disparait."

As regards the structure of the micron ucle us, Maupas onlystates (p. 386) that it is " beaucoup plus tenu que chez lesCilieSj.et sa substance se colore fort peu par les tincturesmicrochemiques. De la, avec d'autres causes qa'il seraittrop long d'enumerer, ici les grandes difficultes de la mise enevidence. Biles sont, en effet, si grandes, que je considerel'6tude d'une de les conjugations d'acinetiens, comrne une desrecherches les plus penibles qu'un micrographe puisse entre-prendre."

The next account of conjugation in Acinetaria is to befound in Rene Sand's ' Jfifcude Monographique sur le Groupedes Infusoires Tentaculiferes, 1901/ and I do not think thatifc can be regarded in auy way as marking an advance in ourknowledge of the process in this gi'oup, mainly, apparently,on account of the author's obsession by the theory that theAcinetaria are descended from the Helioza.

Sand considered that the micronucleus is a centrosoiue,and that the so-called conjugation is a plastogamy with twomain aims (p. 101) :

(1) Une renovation, un rajeunissement.(2) Un processus de nutrition et de conservation ayaut

pour but d'egaliser, de'moyenniser' leur situation nutritive.His figures of the process do not show the nuclei at all,

and apparently the whole of the observations, as apart fromthe conclusions which he formed, are contained in the follow-ing sentences (p. 99) :

"Nous avons toujours trouve les deux individus placessornmet contre sommet, leur axes etant dans le prolongeuientl'un de l'autre; les tentacules etaient retractes; les conju-gaisons etaient tres rares; Jes deux individus, au stadeobserve, etaient separes par une meuibi'aue, les noyauxetaient fragmented et les cytoplasmas semblaient appeles adevoir se melanger malgre la presence d'uue membrane quiles separait."

360 C. H. MARTIN.

"II etait visible que la conjugaison avait lieu entre unindividu riche, et un individu pauvre en tinctine."

In 1902 Hickson and Wadsworth published a detailedaccount of the conjugation of what was at one time regardedas a very aberrant Suctorian Dendrocometes paradoxus,owing to the peculiar structure of the tentacular arms. Theyshowed that the conjugation in this form was quite analogousto the process which occurred in the Ciliata. They foundthat there were normally three micronuclei, which undergotwo successive divisions. Of the products of these divisionsone divided again to form the male and female pronuclei.After cross-fertilisation the cleavage nucleus divides agaiutwice in succession; there appears to be some doubt as tosome of the later stages in the formation of the new nuclei,but normally two micronuclei degenerate; one becomes thenew micronucleus, and the other develops into the macro-nucleus. In other cases the new macronucleus is formed bythe fusion of two microuuclei, and in still other cases thethree micronuclei divide again, the later stages in this formof evolution of the new nuclei not being followed. Thereare, however, some points in their paper, more especially asregards the part played by the macronucleus during thisprocess, and also their general conclusions on the morphologyof the cell body in Infusoria, to which it will be necessary toreturn after describing the conjugation of Acinefca papilli-fera.

Methods. — As fixatives, Flemining's weak solution,Schaudinu's mixture, and corrosive-acetic Avere chiefly used,of which the weak Flemmiug solution seemed most suitable.

The preparations were stained either in alum-carmine oriron ha^inatoxylin.

The material fixed in the osmic mixture was treated withpicro-carmine before staining in alum-carmine.

ACINETA PAPILLIFJEEA (Keppen).—This species was firstdescribed by Keppeu in his paper in the ' Menioires de laSociete des Naturalistes de la Nouvelle Russie Odesse'

SOME OBSERVATIONS ON A01NETABIA. 361

(1888); he separated it from Acineta tuberosa, whichit somewhat closely resembles owing to the presence ofcharacteristic "valves" near the junction of the theca withthe stalk, and to the fact that the body of the animal isusually not attached to the base of the theca. Keppenfound it in both fresh and saltwater in the neighbourhood ofOdessa.

I first found this acinetarian in June, 1907, growing ingreat abundance on the hydrocaulus of Oordylophoralacustris, in Hickling Broad, the water of which is slightlybrackish. It seemed to be feeding mainly on Stentor.which was at this time very abundant in the broad. Asregards the main features of its structure, I have nothingmaterial to add to Keppen's account, except as regards therelation of the theca to the body of the Acineta, and also asregards some details in the process of reproduction.

The " valves."—I always found three of these structuresat the junction of the theca and the stalk, presenting undera low power an appearance corresponding with Keppen'sfigures, but in the interpretation of this appearance I shouldfeel inclined to differ from Keppen.

Keppen regards these structures as three parallel platescutting off the cavity of the stalk from that of the theca. Ithink that this appearance is merely the optical expression ofa joint consisting of a short tube overlapping the ends of thestalk and of the theca.

These structures are unfortunately very difficult to makeout in permanent preparations, but i arrived at this inter-pretation from an examination of the living animal before Ihad seen Keppen's paper, and it is, I think, confirmed bylongitudinal sections (cf. text-figui'e 1). This interpretationwould explain the frequeut occurrence of individuals whichhave bent laterally at the junctiou of the stalk and thetheca, so that the apical surface faces the base of the stalk.This bending is in some cases, I believe, purely passive, butI have often seen animals turn in this way by wrappingtheir tentacles around the stalk, and so pulling themselves

362 0. H. MAETISJ.

around. Oases in which either the stalk or the theca itselfis bent are much more uncommon.

TEXT-3?IGUIIB 1.—Section showing relation of theca and stalk inAcineta papillifera.

Cytoplasm.—The body of Acineta papillifera is pro-longed anteriorly and laterally into two lobes from which thetwo bundles of tentacles arise. In the individuals fromHickling broad there were almost always two vacuoles lyingside by side. Of these, one appeared to be a true contractilevacuole with a period of about one minute, whereas theother appeared to act as a reservoir, maintaining a constantsize about a quarter of that of the full contractile vacuole.

In more or less starved forms the cytoplasm is quitehyaline, but in better fed forms the protoplasm may becomevery opaque owing to the presence of certain bodies with anaffinity for nuclear stains. These bodies are apparently an-alogous with the Tinctin-korper of Plate VIII, the originof which I have already dealt with in the case of Toko-phrya elongata.

The macronucleus is generally an oval structure lyingmore or less centrally in the cytoplasm. Generally in wholestained preparations numerous spherical dark areas are to beseen resembling the so-called " Binnen-korper" of theInfusoria. In section these structures, as in the case of

SOME OBSERVATIONS ON ACINBTARIA. 363

some Infusoria (Biitschli, loc. cit., p. 1510) and Dendro-cometes (Hicksou), are found to consist merely of localthickenings in the mesh of the nuclear network, and there-fore resemble karyosomes rather than true nucleoli.

The micronucleus in the resting stage lies in a depressionof the macronucleus, and consists of a membrane surroundinga clear area, in the centre of which lies a mass of feeblystaining chromatin granules.

Reproduction.—As regards reproduction, the processwhich Keppen termed external budding is referred to at theend of the section upon conjugation. The only method ofreproduction observed in the Acineta papillifera fromHickling was by the formation of single internal ciliatedbuds.

Keppen describes in addition to this for his form fromOdessa, a process of multiple budding, but the figures thathe gives are not convincing. One figure seems to me toshow quite clearly that one so-called bud is simply a smallindividual with a fully developed stalk focussed through thelarge individual. The main features of the formation of theinternal bud correspond with Biitschli's description of theformation of the internal buds in Tokophrya quadri-part i ta . A small depression is formed on the apical sur-face of the acinetaria.n, which gradually widens so as to cutout a portion anterior to the macronucleus, the future bud.

The free bud has a roughly cylindrical but slightly flat-tened shape (text-figs. 2 and 3). On its physiologicallyventral surface it is covered with numerous rows of cilia.(Keppen speaks of 6—11 rows of cilia, but in the form fromHickling broad they'seemed more numerous.)

There is a contractile vacuole on the left side near theanterior end, with a small reserve vacuole near it. The ovalmacronucleus lies near the centre of the body, and in favour-able cases a micronucleus can be seen lying in a depressionof the nuclear membrane. At first, after its escape, theembryo moves rather slowly, but a little later it movesrapidly through the water in characteristic sinuons curves.

364 0. H. MARTIN.

QE

TEXT-I?IGURE 2.—Escaping ciliated bud of A. papillifera.C.E. Ciliated bud. Ma. Macronucleus.

TEXT-PIG. 3. TEXT-FIG. 4.

TEXT-FIGURE 3.—Free ciliated bud of A. papil l i fera. (Zeiss2 mm., apochr., com p. oc. 6.)

TEXT-FIGURE 4.—Later stage of free bud.

SOMK OBSERVATIONS ON ACIN.ETAR1A. 365

In an embryo (text-fig. 4) which left its mother at 10.45 p.m.,the movements became very slow at about midnight, and itwas fixed at 2 a.m. At this period the cilia had not yetdisappeared, but the commencement of the stalk was alreadyto be seen.

A slightly later stage of the development is seen in text-fig. 5, in which the stalk had almost attained its definitivelength, and the cilia have disappeared. At a still later stagethe tentacles make their appearance scattered irregularly

\—Mcu.

J-St.

TEXT-FIGURE 5.—A. papil l ifera soon after attachment, show-ing disappearance of cilia and development of stalk. (Zeiss 2 mm.apoclir., comp. oc. 6.) Ma. Macrouucleus. St. Stalk.

over the surface of the body, and the theca develops as acup surrounding the body at its junction with the stalk.

Finally, the superfluous tentacles are withdrawn, and theanimal becomes compressed laterally, assuming the shape ofthe fully developed form, the theca growing up to surroundthe whole body with the exception of a slit at the apicalextremity, through which the lateral lobes bearing the tenta-cles protrude, and the ciliated embryos escape.

Conjugation.—As regards the early details of conjuga-tion, there is no doubt that they agree with Maupas's generalscheme for Ciliates (Scheme I).

In preparations of the later stages the small size of theniicronuclei, and the large number of chromatin-masses

366 0. H. MARTIN.

scattered irregularly throughout the cytoplasm render itdifficult to attain to absolute certainty as to the mode of

SCHEME I.—General scheme to illustrate the behaviour of themicronuclei of Ciliat.es in relation to conjugation—after Maupas.Microuuclei small black dots. Degenerating micronuclei smallcircles. New macroniiclei large circles.

development of the new micro- and macro-nucleus. At first,I believed the normal process of reconstruction involved the

II . III.SCHEMES I I and III.—Diagrammatic schemes to illustrate the

two possible modes of nuclear reconstruction after conjugation inA. pap ill i fera.

growth of two of the four micronuclei into macronuclei,and their subsequent fusion (Scheme II). Later, however, I

SOME OBSERVATIONS ON ACFNETARIA. 367

became more inclined to the view that normally one micro-nucleus develops into the new macronucleus, one remains asthe new micronucieus and two degenerate (Scheme III).

It is quite possible that both these methods of reconstruc-tion occur normally since Prandtl has shown recently in avery careful work on conjugation in Didinium nasutum,

TEXT-FIGURE 6.—A. papillifern, first stage of conjugation,showing the prolongations (C.P.) by which contact between con-jugating individuals is effected. Ma. Macrouuclei. (Zeiss 2 mm.apochr., comp. oc. 6.)

that here the nuclear apparatus may be restored by one outof no less than five different methods, and Hickson hasdescribed three alternative methods in Dendrocometes.

The first indication of conjugation was found by ProfessorMinchin among specimens collected and examined on August29th, and the majority of the individuals fixed at this periodwere found on examination to be early stages A—E. I have

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368 0. H. MARTIN.

no evidence as to the period occupied by the entire processof conjugation, but the pair figured in PI. VII, fig. 8, whichwere found on fixation to have reached the final stage in thereconstruction of the nuclei, were followed for forty-six hours,but it is possible that this period is largely influenced byexternal conditions, e. g. temperature. Contact is usuallyeffected between two conjugants by the prolongation of theapical lobes, the prolongations usually shortening as conjuga-tion proceeds (text-fig. 6). More rarely the apical lobe ofone conjugant may come in contact with the side of the otherconjugant (PI. VII, fig. 8), and a still more rare conjugation ispurely latei-al. In these latter cases the thin wall of thetheca of one or both conjugants is dissolved away, so as toallow the contact between the cytoplasm of the twoindividuals.

Stage A.—The first internal indication of conjugation isafforded by the macronucleus. In PL VII, fig. 1, the macro-nucleus of the individual labelled A still shows the charac-teristic appearance of the resting nucleus with large darkly-staining areas, the pseudo-nucleoli. The micronucleus isunchanged, and closely applied to the macronucleus. Thetentacles are partly withdrawn.

In B the macronucleus has already taken on the coarselyfibrillar appearance which is absolutely chai-acteristic of thestages of its degeneration during conjugation. The micro-nucleus has left its original position near the macronucleus,and is preparing for the first division.

Stage B (PL VII, fig. 2).—The macronuclei of both indivi-duals have become elongated, and the fibrillar appearance ismore marked. The dividing micronucleus in the left-handindividual is covered by the macronucleus. The appearanceof the micronucleus in the right-hand form seems to presentsome analogy to the so-called " Sichel" stage in the firstdivision of the micronucleus in conjugating Parameciadescribed by Hertwig, and more recently by Hamburger.

Stage C.—Both individuals of the conjugation shown in

SOME OBSERVATIONS ON ACINETARIA. 369

PI. VII, fig. 3, contained two tnicronuclear spindles, whichwere slightly smaller than those of the fii'st division.

Stage D.—This stage is, I believe, represented by PI. VII,fig. 4. In each individual there is one inicronuclear spindlenear the line separating the two individuals, which wouldgive rise to the male and female pronuclei. Of the othermicronuclei, two in process of degeneration lie near the con-tractile vacuole, and the third is probably concealed by thelnacronucleus.

Stage E.—This stage is shown in PI. VII, fig. 5, and thedetails of the male and female pronuclei under a slightlyhigher power in PI. VII, fig. 6.

At this stage the degenerating macronuclei have becomeenormously drawn out, and as their length is so great, theyare frequently thrown into coils which may abut on the parti-tion dividing the conjugation, and readily give the appearanceof a macronuclear conjugation. It is to such appearances asthis that I think Hickson's account of macronuclear conjuga-tion in Dendrocometes paradoxus must be attributed,and as Hickson seems inclined to attach much importance tothis process (vide p. 349 : "I am prepared, however, to gofurther than Sand, and regard the presence of the meganucleiin the conjugation processes not only as evidence of theirrelation to the intei*change taking place in the cytoplasm,but as evidence of the necessity of the interchange of mole-cules of the substance of the meganucleus itself ") it will benecessary to examine in some detail the evidence on whichthis meganuclear conjugation rests. This appeal's to me tobe contained in the two following passages :

(1) Page 3B1. " At some time during the last thi'eestages (H, J, K) the old meganucleus becomes very large,and is bent on itself in the form of a loop or horse-shoe.One extremity of this figure passes into the conjugativeprocess, and approaching the limiting membrane, traversesit and fuses with the corresponding extremity of themeganucleus in the other individual. The exact phaseat which this meganuclear conjugation takes place seems

370 0. H. MARTIN.

to vary considerably; all that can be said at present isthat, as far as my experience goes, it usually occursbetween stages I and K."

(2) Page 342. " In my preparations of Dendro-cometes I have at least three cases in which the mega-nuclei actually touch, but a considerable number inwhich they approach one another very closely in theconjugation processes. That the junction is not merelycasual contact, but actual organic connection, is provedby the preparation which is represented in PL 18, fig. 11.Here there is no sign of any boundary between the twonuclei, and the chromatin granules are fixed in such amanner as to suggest very forcibly that during life theywere flowing from one side to another."

As at this stage of conjugation the macronuclei are rapidlydegenerating, Hicksou, in his attempt to show that this con-tact of the macronuclei is of fundamental importance inconjugation, puts forward the theory that " there is noinconsistency in the view that after the disappearance of theold meganucleus its nucleoplasm is still living iu a modifiedform diffused through the cytoplasm."

The latter stages in the degeneration of the inacronucleusin a conjugating aciuetarian are almost precisely similar tothe stages figured by Schaudinn in the digestion of thenucleus of a Trichosphserium sieboldi which has beendevoured by one of its congeners, and to the stages which Ihope to describe in the digestion of the nucleus of S ten torby Tokophrya, and I believe that it is quite as justifiableto speak of the nucleoplasm still living in a modified formdiffused through the cytoplasm in the one case as in the other.

I feel inclined, until further evidence is adduced, to regardthe appearance figured by Hickson as abnormal, the forceswhich tend to elongate the macronuclei in the degeneratingstages having carried the process too far, and breakingthrough the partition dividing the two conjugants led to theapparent macronuclear conjugation. In both the conjugants

SOME OBSERVATIONS ON AOINETABIA. 371

in fig. 5 the remains of three degenerating micronuclei areto be seen.

In PI. VII, fig. 6, the conjugating processes of the samepair are seen under a higher magnification. In the right-handform the spindle of the fused male and female pronuclei isalready formed. In the left-hand form the female pronucleusis lying close to the partition separating the two conjugants,whilst the male micronucleus is in a depression between thetwo conjugating forms.

This stage seems to be comparable with that figured byHickson for Dendrocometes, PI. 17, fig. 10, and showsthat here, as iu Dendrocometes, in contradistinction to thestate of affairs found in Paramecia, the male and femalepronuclei fuse not in the spindle, but in the resting stage.

It is also interesting to find that here, as in the case ofconjugation in Didiniuni nasutum described by Prandtl,the male pronucleus seems to be considerably smaller thanthe female pronucleus.

The later stages iu conjugation become exceedingly diffi-cult to follow, as the chromatin of the macronucleus seems tobe dissolved out of the achromatic network, and to be scat-tered through the cytoplasm iu the form of darkly-staining,irregularly spherical blocks.

Normally, the zygote nucleus formed by the fusion of themale and female pronucleus appears to divide twice in suc-cession, and of the products of this division two degenerate—one becoming the new micronucleus, and one the newmacronncleus.

In PI. VII, fig. 7, the achromatic network of the oldmacronucleus (Ma.F.) is still to be seen, though nearly all thechromatin appears to have been dissolved out, and to lie inirregular blocks (Chr.) in the cytoplasm.

Two of the division products of the zygote nucleus are tobe seen as faintly staining vacuolar bodies, both closelyapplied to a mass of chromatin (Mi.). This relation suggeststhe absorption of the dissolved chromatin by the growingnucleus, a process to which Hamburger has drawn attention

372 0. H. MARTIN.

in her recent account of conjugation in Parainecium. In thiscase probably the new macronucleus would have resultedfrom the fusion of these two bodies, the new micronucleusbeing probably covered by some part of the old macro-nucleus.

I can find no evidence in my prepai^ations in support ofHickson's view that the feeble-staining capacity of the deve-loping macronucleus is due to the fact that " at this stageeither the whole or the greater part of the chromatin in itsmodified form passes into the surrounding cytoplasm, leavingthe new meganucleus perfectly clear and homogeneous" (p.345), a process which Hickson compares to the extrusion ofchromatin in the egg cells of some Metazoa. I am moreinclined to believe that this primary loss of staining poweron the part of the developing macronucleus is to be explainedby a simple increase of size, which is compensated for at alater stage by the absorption of the chromatin from theremains of the old macronucleus.

The final stage in the reformation of the nuclei is shownin PI. "VII, iig. 8. This pair of conjugants was examined atintervals from 10.45 p.m. on August 31st to 8.10 p.m. onSeptember 2nd. Unfortunately, nothing could be made ofthe micronuclear changes in the living animal. During thelast five hours the tentacles, which had been previouslyshortened, commenced to elongate, and when the individualswere killed they had almost reached their normal length.

From other observations on living material I am led tobelieve that the tentacles are not usually withdrawn duringthe early period of conjugation up to the stage F or Gr;during the much longer period associated with the finaldisappearance of the old macronucleus and the developmentof the new one the tentacles may become much shortened,and only attain their normal length after the reconstructionof the macronucleus, and shortly before the separation of theconjugants.

In the final stage the macronucleus has a very characteristicappearance of a lightly-staiuing loose mesh, with scattered

SOME OBSERVATIONS ON ACJNETAB1A. 373

chromatin granules, and all traces of the old macronucleushave disappeared (PI. VII, fig. 8).

External budding.—It will be now necessary to refer toa process which Keppen has termed " external budding,"and which he describes as the development of a lateral out-growth, which may be followed by disappearance of thetentacles. He found that this outgrowth may change itsshape and size considerably, and after a period of ten totwelve hours the animal may return to its original shape. Insome very rare cases the outgrowth may break off, but hewas never able to determine the fate of the bud so formed.In several cases he observed the nucleus carried to theboundary between the acinetariau and the so-called bud, andunder these conditions he found appearances of fragmenta-tion of the nucleus, though there was nothing to suggest anormal division.

In one case (fig. 47) he found a specimen of Acinetapapil l i fera with a spade-like body containing a nucleus,and covered with moving cilia attached near the tentacles.This body, owing to its resemblance in shape, he was inclinedto compare to the outgrowths described above. The bodyremained in contact with the acinetarian for some time, untilthey were finally both lost.

These structures are probably identical with those whichFraipont had previously described as " diverticules genera-teurs" in acinetan division, and which, according to him,were not to be regarded as external buds, but as structuresout of which endogenous ciliated buds were to be developed.This theory was based on a single observation in which hefound a ciliated bud in contact with a fixed form.

I believe that Keppen has confounded under this term twoquite distinct phenomena—

(1) The formation of long conjugation processes in indi-viduals which by reason of their position cannot come intocontact with another individual ripe for conjugation.

(2) Cases of conjugation between a fixed form and a free-swimming ciliated embryo.

374 0. H. MARTIN.

(1) It is interesting to notice that both Fraipont andKeppen examined individuals during a conjugation epidemic.

During the conjugation epidemic which I had the oppor-tunity of observing I found, especially in material fixedduring the commencement of the period, numerous examplesof these long processes. The early stages of their formationare analogous to those of the formation of the conjugatingprocesses, as may be seen by a comparison of text-fig. 6 andPI. VII, fig. 9. I was never able to see an instance in whichone of these so-called buds became free, but in severalexamples from later material I have found evidence ofchange in the nucleus (PI. VII, fig. 9).

Although I have not been able to follow out these stagesin detail, the appearance of the macronucleus is absolutelyidentical with the fibrillar stage which occurs previous to thefragmentation in normal conjugation.

I am inclined to believe that under certain circumstances,e.g. the absence of mature neighbours within range, a pro-cess of parthenogenesis occurs similar to that described byHertwig for Paramecium axirelia (p. 224), and by meansof which Prandtl later has explained the behaviour of thethird individual in cases of triple conjugation in Didiniuni.

It would seem that the chances against two neighbouringacinetaria exhibiting at the same time the three generallyaccepted symptoms of conjugation, viz. (1) sexual maturity,(2) distant relationship, (3) starvation, would be far moreremote than the chance of meeting of two free swimminginfusoria. And accordingly it is not surprising that inpreparations made during a conjugation epidemic suchappearances of presumable parthenogenesis are fairly fre-quent.

(2) Conjugation between a fixed individual and afree ciliated bud.

It is under this heading that I feel inclined to placeKeppen's figure (49). I believe that this process is rare,

SOME OBSERVATIONS ON ACINETARIA. 375

and, in fact, I was only once able (text-fig. 7) to follow theprocess in the living individual; but if the cilia, as is the casein the normal bud, disappear at the end of a couple of hours,i\i might be very difficult to distinguish the later stages ofprocess (2) from those of process (1) (text-figs. 0 and 7).

-Me.

J—Ma,.

TEXT-riGUKE 7.—A. papil l i fera, conjugation between a freeciliated bud and a fixed form. Ma., Me. Macronucleus. Mi.? Mictonucleus. St. Stalk. (Zeiss 2 mm. apoclir., comp. oc. 6.)

IV. CONCLUSIONS.

(1) The Tinctin-korper of Acinetaria are generally frag-ments of the ingested nucleus of their prey.

(2) Conjugation in Acineta papill ifera agrees in allessentials with the process occurring in the Ciliate Infusoria,and that described by Hickson for Dendrocometes para-dox us.

It is possible that in those cases in which a fixed formcannot come into contact with another mature individual, areorganisation of the nuclei may be effected associated with

376 C. H. MAKTIN.

eitlier (a) conjugation with a free swimming ciliate bud, or(b) a process of parthenogenesis associated with the forma-tion of the so-called " external buds " of Keppen.

LlTBEATOBE.

BALBIANI.—"Les Acine'tiens," ' J. Mic. Paris,' 18S7-S.

BUTSCHLI, O.—" Protozoen," 'Bronn's Tliierreicii.'

GLAPAIIEDE et LACHMANN.—'Etudes sur les Infusoires et ltliizopodes/

Geneve, 185S-1861.

DELAGE et HEROUAKD.—'Trait6 de Zoologie concrete,' T. i, Paris, 1896.

EISMOND.—'' Zur Frage iiber der Saugmechanismus bei Suctorien," 'Zool.Anzeig.,' 1S90.

ENTZ.—" Ueber Infusorien des Golfes von Neapel," ' Mitt. Zool. Slat.,' 1884.FKAIPONT.—" Recherches sur les Acinetiens," ' Bulletin de l'Academie Beige,'

T. xlv, 1878.HAMBURGER.—"Die Conjugation von Paramecium bursaria ," ' Archiv

fiir Protist,' T. iv, 1904.

HARTOG, M.—"Notes on Suctoria,' ' Archiv fiir Protist,' T. i.

HEKTWIG, R.—" Uber die Konjugation der Infusorien," ' Abb. d. Kgl. bay.Akad. d. Wiss.,' Bd. vii, Abt. i, 1889.

" Uber Kernteilung, B-ichtungskorperbildung und Befruchtung vonActinospbcerium eichorni i ," ' Abh. d. Kgl. bayr. Akad. d. Wiss.,'Bd. xix, Abt. iii, 1898."Die Protozoen und die Zelltheorie," ' Archiv fiir Protist.,* T. i,

1902.

'Uber das Wechselverhaltniss von Kern und Protoplasma,' Miinchen(Lehmann), 1903.

"Uber Physiologische Degeneration bei Actinospluerium eich-ornii," "Haeckel's festsclirift,' Jena, 1904.

HICKSON, S. J .—"Dendrocometes paradoxus ," ' Q . J. M.S. / vol.45,1902.

ISCHIKAWA. —" Ueber eiue in Misaki vorkommende Art von Ephelota,"

'Japan Coll. Sci. Imp.,' vol. x.

KENT, S.—'Manual of the Infusoria/ 1881-2.

KEPPEN.—' Mem. de la Society des naturalistes de la Nouvelle Russie,Oddessa,' T. 13, 1888.

SOME OBSERVATIONS ON ACINETARIA. 377

LTJHE.—"Befruchtungsvorgang bei Protozoen," ' Schr. Piiys. Ok. Ges.Konigsberger,' 43 Jahrg., 1902.

DAUPAS.—" Contributions a l'etude des Acinetiens," ' Arcbiv de Zoo!.

Exper.,' vol. ix.'La Rajeunissement Karyogamique chez les Cilies," 'Aroliiv de

Zool. Exper.,' ii serie, T. vii, 1884.PIATE.—" Untersucliung einiger an den Kiemblattern des Gammarus

pulex, lebenden Ektoparasiten," ' Zeit. f. w. Zool.,' vol. xliii, 18S6.

" Studien iiber Protozoen," 'Zool. Jalirbuch,' T. iii, 1SS8.

PiiANDTL, HANS.—"Die Konjugation von Didinium nasutum," 'ArcliivMir Protist.,' vol. vii, 1906.

SAND.—' Etudes Monographiques sur le groupe des Infusoires Tentaculiferes,'Brussels, 1901.

SCHADDINS.—'Tricliosphceriuni sieboldii, ' Berlin, 1S99.

SCHNEIDER.—'Tablettes Zoologiques,' T. i, 18S6.

VEESLUYS.—" tiber die Konjugation des Infusorien," ' Zool. Centralblatt,'1906.

378 C. H. MARTIN.

Part II.—The Life Cycle of Tachyblaston ephelotensis(Gen. et spec, nov.), with a possible identification of

Acinetopsis rara, Eobin.

CONTENTS.PAGE

I . Introductory . . . . . 378I I . The Budding of E p b e l o t a gem mi p a r a . . 379

I I I . T a c h y b l a s t o n e p h e l o t e n s i s . . . 380IV. Literature . . . . . 3S7V. Explanation of Plates, illustrating Parts I and II . 3SS

I. INTRODUCTORY.

In this par t I wish to describe a new Acinetarian parasite—Tachyblaston ephelotensis (Nov. Gen. Nov. Spec),which I found during a visit to Naples in May, 1908. Ishould like to take this opportunity of thanking the staff ofthe Aquarium for their kindness.

The early observers of parasitic Acinetaria regarded theseanimals as the embryoes of their host, and it was not until"1860 that Balbiani put forward the view that the so-calledembryoes were really parasites. The truth of this theorywas first shown by Metchnikoff in his early work onSphasrophrya paramosciorum in the 'Archiv furAnatomie und Physiologie' in 1864, to which he again refersin his classical work ' Lecons sur la Pathologic Comparee derinflamniation' (Paris, 1892). Metchnikoff showed that inthis case the internal parasite divided, giving rise totentacled buds, which, after developing cilia swam off andinfected another host. It had frequently been suggestedthat certain rounded bodies found in Acinetaria were to beregarded as parasites, but this view has, as far as I amaware, never been confirmed, and Biitschli says in his account

SOME OBSERVATIONS ON AOINETARIA. 379

of the Snctoria :—" Im Kapitel uber die freie Knospung (s. p.1894) wurde schon betont, dass gewisse angebliche Knospeneiniger Arten moglicherweise kleine parasitische oder com-mensalistische Suctorien sind, welche auf grossei'en leben.Ebenso fanden wir es nahezu, wenn nicht ganz gewiss, dassendosphaerenartige Suctorien in grosseren Arten ihres eigenenStammes schmarotzen."

METHODS. — My material was fixed either with weakFlemming or corrosive acetic. Of these two methods theFlemming seemed to give far the most faithful cytoplasrnicfixation. The Flemming preparations, after washing inwater, were treated with a dilute solution of H3O2 in 70 percent, alcohol, and then stained either in alum carmine orMayer's haemalum.

II. THE BUDDING OF BPHELOTA GEMMIPAEA.

Before proceeding to give a detailed account of the some-what complicated life-history of Tachyblaston ephelo-tensis, it will be necessary first to refer to the structure ofthe macronucleus and the mode of reproduction of its hostBphelota gemmipava, as it will only then become apparenthow it is possible to distinguish the various stages in thelife-history of the host from that of the parasite.

The macronucleus of Bphelota is generally described as ahorse-shoe shaped structure lying in the horizontal plane ofthe animal, and giving rise to a varying number of branches,especially towards the apical surface, on which the buds areformed. The reproduction of Bphelota (Podophrj'a)gemmipava has been described by Richard Hertwig, whoseresults have been confirmed in all essentials by the laterworkers upon this form. The buds first make their appear-ance as small swellings i*ound the apical pole, varying innumber according to the size of the budding individual from1 to 12. These small swellings slowly enlarge until theybecome elliptical bodies flattened along one surface, the

380 C. H. MARTIN.

future ventral surface along which, at a later stage, a deepciliated groove is formed. A branch of the macronuclenspasses into each bud and becomes twisted upon itself inrather a complicated manner, so that the macronucleus ofthe young embryo at quite an early stage is already a twistedband (text-figs. 8, 9).

Shortly before the bud is set free the last strand connecting

TEXT-FIG 8. TEXT-FIG. 9.

TEXT-FIGURE 8.—Normal Ephelota with external buds. (Zeiss4 mm. apoclir., comp. oc. 4.)

TEXT-FIGURE 9.—Free ciliated bud of Ephelota. (Zeiss 2 mm.,apoclir., comp. oc. 4.)

its niacronucleus •with tliafc of t h e p a r e n t form is b rokenthrough.

III. TACHYBLASTON EPHBLOTENSIS.

At the beginning of May received large quantities ofbrown seaweed (Cystoseira) from Nisida, which weresimply covered by Ephelota gemmipara, visible to thenaked eye as minute yellow dots. In material brought onthe 6th May I was struck by the fact that some of theEphelota contained peculiarly refractile rounded bodies, andthat none of the Ephelota which presented this appearance

SOME OBSERVATIONS ON ACINETA1UA. 381

showed any trace of budding. Suspecting the presence of aparasite, I placed some of the infected foi-ms in a deep watch-glass, and I was able to follow all the stages in the life cycleof the parasite in the living form.

TEXT-FIGCnE 10.—Scheme of life-history of Tachy blastouephelotensis . (Zeiss 4 nun. apochr., comp. oc. 4.) A. Internalparasitic form. B. Ciliated bud. C. Recently fixed ciliated bud.D. Later stage of fixed bud showing stalk. E. So-called " Acine-topsis" stage. F. Two free tentacled buds creeping up the stalkof the Ephelota.

In order to avoid recapitulation, a diagram of the life cycleof tlie parasite is shown in text-fig. 10. The internal parasitegives l-ise by division to ciliated spores. These, after a short

382 C. H. MARTIN.

free life, fix and develop into a stalked form, which, onaccount of its great resemblance to a form previously de-scribed by Robin, may be termed the Acinetopsis stage.This gives l-ise, by repeated budding, to a large number ofpeculiar small buds, each of which is provided with a singlethick tentacle. The tentacled buds become free, and crawlby means of their tentacle up the stalk of an Ephelota, whichthey iufect.

In material brought on the succeeding days the ravages ofthe disease became more and more apparent, until in materialcollected on May 9th there was nothing to be seen except alarge number of bare stalks of Ephelota, a few encystedforms, and a large number of the empty thecEe of theparasite.

Iu material brought on May 12th I found, after muchseai*ch, a few encysted Ephelota and some young healthybudding forms ; these rapidly increased in number until thecollected weed was again covered with Ephelota. On May16th a second epidemic, which corresponded with the onedescribed, recurred. I propose to call this parasite Tachy-blaston ephelotensis in order to emphasise the extra-ordinary rapidity with which it infects large numbers ofEphelota.

THE INTERNAL STAGE OP THE PAKASITE.— The infectedEphelota are readily distinguished even under a low powerbinocular microscope (1) by the absence of external buds,(2) by the presence of peculiar rounded bodies in their cyto-plasm. In a fairly early stage of infection, shown in text-fig.11, which was drawn from a living specimen, there was asingle rounded body occupying the centre of the body of theEphelota. The nucleus of the parasite could be clearly seenas light area lying in the cytoplasm. The cytoplasm in this,as in all the other stages of the parasite, is clearly marked offfrom the cytoplasm of its host, by the peculiar small refrin-gent granules which it contains (PI. VIII, fig. 5).

At first I was inclined to attribute this appearance to thepresence of fat, which is very common in Acinetaria, but as

SOME OBSERVATIONS ON ACINETARIA. 388

I could never find any trace of blackening after treatmentwith osmic acid in preparations in which the host showedconsiderable blackening this view had to be abandoned; onthe other hand, in sections stained with hsematoxylic eosinthe granules seem to show considerable affinity for the eosinstain. I am inclined to regard the granules as reserve foodstuff, as their number seemed to diminish in starved forms.

TEXT-PIG. 11. TEXT-FIG. 12.

TEXT-FIGURE 11.—Early stage of infection of Ephelota byTaohyblaston, from the living animal. (Zeiss 16 mm. apoohr.jcomp. oc. 8.)

TEXT-FIGURE 12.—Stage three hours later than that shown intext.flg. 11. The tentacles of the host are not shown. (Zeiss1G mm. apoclir., comp. oc. S.)

About three hours later (text-fig. 12) the parasite had under-gone one equal division and showed traces of the formationof a secondary bud. It seemed, as a result of examining alarge number of preparations, that the first division is alwaysapproximately equal, but that this is followed by the forma-tion of a number of true buds which do not obtain their fullsize at once. By the next morning seven internal parasites,some of which were developing cilia, could be counted in theabove-mentioned specimen, and the cytoplasm of the hosthad become reduced to a thin shell surrounding the centralmass of the parasites. As the budding of the parasite pro-ceeds (PI. VIII, fig. 6), frequently two or three chains of buds

VOL. 53, PART 2. NEW SERIES. 27

384 0. H. MARTIN.

ai*e formed, radiating from a central mass, and each termi-nating in a ciliated bud. The nucleus in the internal parasiteis a rounded homogeneous mass, which can be readily distin-guished in stained preparations from the branched coarselygranular nucleus of its host. Under very high powers inmaterial fixed with Flemming's solution it could be seen thatthe nucleus of the internal parasite is also very finelygranular.

The ciliated spores (PI. VIII, fig. 7) are more or less ovalbodies, about "04 mm. long by -018 mm. broad, with a patchof long cilia on the right anterior area of the ventral surface.I saw no trace of the contractile vacuole in the living forms,but in most stained preparations a clear vacuole is present.The ciliated spores after a short free existence (about ten tofifteen minutes) settle down and begin to form a stalk(PL VIII, fig. 8). At this stage the whole animal becomessurrounded by a wall, the first trace of the theca, which ispresent, though not in so dense a condition, at a slightlylater stage, even over the apical surface. In the fully deve-loped stalked form (PI. VIII, fig. 9) the theca measures from•043 to '03 mm. long by '03 mm. broad. The stalk measuresfrom "02 to "03 mm. long, and is usually curved. In thisstage the animal resembles very closely a form which wasdescribed many years ago by Robin, and which has not beenseen since, Acinetopsis rara. Robin, in his account ofthis form, states that he only saw it three or four times inthe midst of Acinetaria on stalks of Sertularia. Hedesci-ibes it in the following words :

"C'est un animal long de 0*07 mm.—0-09 d'un tiers moinslarge, remplisant une 9oque ou theque en forme de verre a,pied qui supporte un tres grele pedoncle long d'un dixiemede millimetre.

" Corps uniformiment grenu, grisatre, avec une petite vesi-cule pulsatile, a surface libre plane, portant a son centre untentacule et x-etractile alternatement."

Eobin himself stated that he knew nothing of the reproduc-tion of this form, and later observers seem to have been

SOMB OBSBBVATIONS ON ACINJETARIA. 385

equally unfortunate, since Sand, in his monograph on theAcinetaria, says, " La classification de ce genre est provisoire,la mode de nutrition et de reproduction et n'ayant pas eteobserve."

The resemblance of this animal to the fixed stage of Tach y-blaston is very striking, and this resemblance becomes evenmore significant when Robin's account of the formation of asecond kind of small bud in Ephelota is taken into con-sideration. Robin did not follow the relation between thenuclei of these buds and the Ephelota, and Biitschli(p. 1894) refused to admit that these structures could reallybe regarded as buds of the Ephelota. From the considera-tion of these points, I think it very probable that Robin's" Acinetopsis r a r a " is merely a stage in the life-historyof Tachyblaston ephelotensis. After the developmentof the theca the Tachyblaston starts budding very rapidly,the number of buds reaching twelve or fourteen. Each budis provided with a single thick tentacle, the size of which, incomparison with other Acinetaria, seems out of all proportionto the size of the body. The bud, in this fully-formed condi-tion with a contracted tentacle, has rather an elongated pearshape, the stalk of the pear being formed by the tentacle.In the centre of the body there is a large vacuolated nucleus ;at the opposite pole to the tentacle the bud is produced iuto acurious short tuft, which I am inclined to regard as an adhesiveorgan. (This bud seems to bear a curious superficial resem-blance to the genus Rhynceta, described by Zenker, fromthe appendages of a fresh-water Cyclops [PI. VIII, fig. 10]).If the living buds are watched carefully it will be found thatsome of them shoot out their tentacles, which, at the sametime, become very thin, to a length fully equal to that of thestalk and theca, about "07 mm. The tentacle then sways toand fro in a way that recalls the tentacle of Urnu la. If thetentacle comes in contact with a foreign body, e. g. the stalk ofan Ephelota,it retracts, and the bud is pulled out of its theca.Very frequently the bud remains for some time attachedto its tentacle, swaying in a curious pendulum-like manner.

386 0. H. MARTIN.

The body of the bud exhibits cui-ious englenoid changes ofshape, and in this way, with the aid of its tentacle, the budcan travel considerable distances up the stalk of theEphelota which it is about to infect. After penetrating itshost the tentacle seems to be completely withdrawn, and thebud seems to grow very rapidly to reach the size characteristicof the internal parasitic form (about "04 long by '03 broad),the life-cycle being thus completed.

The Effect of the Paras i te upon the Host.—Metchnikoff, in his ' Lectures on the Comparative Pathologyof Inflammation' (p. 27), has already put forward the viewas regards Sphasroplirya parameciorum, that "Pour semaintenir dans Finteriur du protoplasnia des iufusoires lesacinetiens doivent exercer quelque influence paralysaute surl'action digestive. II est probable que ces parasites secretentquelque substance toxique, parce que'on a vu souvent diversinfusoires tomber dans un etat de paralysie et ruourir a lasuite des attaques des acinetiens libres. En vegetant danTinterieur des infusoires, les acinetiens parasitiques provoquentune degenerescence du noyau, qui se fragmente en grainsronds."

As far as I could see from sections of infected Ephelota,the effect of the parasite was far more marked upon thecytoplasm than on the nucleus of its host, and it is only inlater stages of infection, when the cytoplasm has beenreduced to a thin shell surrounding the mass of the parasites,that one finds traces of degeneration in the nucleus, thegranules of chromatin running together to form darkly-staining lumps. It is, I thiuk, a fact of some importancethat here, whei'e there is no direct absorption of the chromatinof its prey, the parasitic Acinetarian is quite free from so-called " tinctin-korper " of Plate.

Systematic Position.—From the account given of theadult structure of the Tachyblaston it must, I think, beregarded as closely related to the family Urnulina, and not asin any way related to the parasitic Sphserophrya described by

SOME OBSERVATIONS ON ACINETARIA.. 387

Metchnikoff.1 I t is also noteworthy that we have here, as inthe case of many other parasitic animals, e.g. M o n t s t r i l l a ,a form the systematic position of which is quite indeter-minable from the parasitic stages of its development, butwhich shows quite characteristic featui'es in its later free-living stages. T a c h y b l a s t o n e p h e l o t e n s i s is also ofinterest as showing the beginning of a development of acomplicated life cycle in association with a parasitic methodof life in a group of Protozoa which are characteristicallyfree living, or, at any rate, purely external parasites. Start-ing from the Acinetopsis form, we have free-living tentaclebuds, which, passing into their host, give rise, by a processof division, to ciliated spores of large size, which in turndevelop on fixation into the Acinetopsis form from which westarted.

LITERATURE.

BALBIANI,—" Evolution des Microorganismes Parasites," ' C. K. Acad. deSciences/ 27 Aout, 1860.

BUTSCHLI, O.—" Protozoa," ' Bronn's Tliierreieh,' Leipzig, 1S89.HICKSON, S. J.—" Acinetaria," in 'Lankester's Zoology,' Oxford, 1903.KOEPFEN.—' Amoebophrya," ' Zool. Anzeig.,' vol. xvii, 1894.METCHNIKOFP, E.—" Ueber die Gattung Sphairophrya," ' Archiv fur Ana-

tomie und Physiologie,' 18G9.NEIIESIIJGIMEK.—"Die Mesozoen," p. 257, 'Zool. Centr.,' vol. xv, 1908.ROBIN.—" Memoire sur la Structure et la reproduction de quelques Infu-

soires," ' Journ. de 1'Anat. et de la Pbys.,' 1879.SAND.—' Etude Monographique sur le groupe des Infusoires Tentaculiferes,

Bruxelles, 1901.ZEKKBU.—" Beitrage zur Naturgeschichte der Infusorien," ' Archiv fur

Mikroscop. Anat.,' T. ii, 1866.1 The only other parasitic form which has been placed amongst the Acine-

taria is that curious animal Amoebophrya which occurs in certain lladiolaria.I have never been able to understand from the published accounts of thisanimal why it was ever placed in this group; and in a recent paper by Nere-sheimer in the ' Zoologisches Centralblatt' it is far more appropriately placedamongst the Mesozoa.

388 0. H. MARTIN.

EXPLANATION OF PLATES VII AND VIII,

Illustrating Mr. C. H. Martin's " Observations on Acine-taria."

PLATE VII.Acineta papi 11 ifera.

LETTERING.

Chr. Clu'Omatin. C.P. Conjugating process, c.v. Contractile vacuole.Ma. Macronueleus. Ma.F. Macronuclear framework. Mi. Micronucleus.Mi. <J • Male pronucleus. Mi. ? . Female pronucleus. Mi.Sn. Spindle offused male and female pronuclei.

All figures, except 6, drawn under 6 comp. oc. + 2 mm. Zeiss apochr.

FlG. 1.—Conjugation stage A.

PIG. 2. „ „ B.FIG. 3. „ „ C.FIG. 4. „ „ I).FIG. 5. „ „ E.FIG. 6. „ „ E, detail.

FIG. 7. „ „ F.FIG. 8. „ ,, H.FIG. 9.—So-called external bud.

PLATE VIII.

Tokophrya elongata .

FIG. 1.—Recently fed individual showing " Tinctin-korper." (6 comp. oc.+ 4 mm. apochr.)

FIG. 2.—Budding individual showing division of the macronucletis and"Tinctin-korper." The tentacles were not drawn. (6 comp. oc. + 4 mm.apochr.)

FIG. 3.—Starved individual. (6 comp. oc. + 2 mm. apochr.)FIG. 4.—Last stage of starvation. The pellicule is much wrinkled. The

tentacles, and practically the whole of the cytoplasm, have disappeared,(6 comp. oc. •+• 2 mm. apochr.)

SOME OBSERVATIONS ON AC1NETABIA. 389

Taoliy hlaston eplielotensis.

FIG. 5.—Early stage of infection, showing a single Tachyblaston (Ta.)lying in an Ephelota.

FIG. 6.—Late stage of infection, showing the formation of ciliated buds ofTacliyblaston.

FIG. 7.—Free ciliated bud of Tachyblaston. Ventral view.FIG. 8.—Recently fixed ciliated bud.FIG. 9.—So-called "Acinetopsis" stage.FIG. 10.—Wandering tentacled bud. "Rhynceta stage." (4 com p. oc.

+ 2 ram. apochr.)

3.

A K l.!i».-!l