Ciliates, -with a Description of the Maturation Phenomena in ......Ciliates, -with a Description of...

Cytological Observations on GymnostomatousCiliates, -with a Description of the MaturationPhenomena in Diploid and Tetraploid Formsof Chilodon uncinatus .

By

Mary Stuart MacDougall.

With Plates 23-33.

SOME form of supporting structure is found around the oralopening in many species of ciliates. This varies from a fewtrichites around the mouth, as in S p a t h i d i u m s p a t h u l a ,or a simple tube of trichites, as in P r o r o d o n t e r e s , to thevery complicated apparatus found in various species ofC h i l o d o n and N a s s u 1 a. The early investigators designatedthis structure the pharyngeal basket or ' Eeusenapparat '.There are many references to this organelle, either of a descrip-tive or speculative nature, particularly as to its function.Kent (1881) gives a partial account of its behaviour in divisionand eneystment, and there is an incomplete account byEnriques (1908) of its behaviour in division and conjugation.It seems desirable, therefore, to make an extended study ofthe structure in order to determine its origin, if possible,and its behaviour in the complete life-cycle. This naturallyled to a study of the basket during division, conjugation, andeneystment phases, and the material obtained for this purposewas found to be favourable for the further study of nucleiduring division and conjugation.

MATERIAL.

The material upon which the observations were madeconsisted of several holotrichous ciliates, C h i l o d o n un -c i n a t u s (Ehrenberg), C h l a m y d o d o n m n e m o s y n e , and

NO. 275 B b

862 MARY STUART MACDOUGALL

Prorodon teres . The major part of the work was doneon Chilodon uncina tus . Prorodon teres was usedfor cutting experiments, and for a study of the behaviour ofthe basket during encystment.

The original culture of Chilodon uncinatus came fromone individual isolated from a mass-culture given me byDr. E. C. Rhodes, of Emory University, Georgia, where theinvestigation was started. The cultures were kept fromNovember 1922 until June 1923 when, in transferring themfrom Georgia to Woods Hole, Mass., they were lost. Othermaterial was obtained at Woods Hole and at Van CortlandtPark, New York.

The Prorodon teres and Chlamydodon mnemo-syne material was obtained from School House Pond andKnowlton's Ditch, Woods Hole, Mass.

METHODS AND TECHNIQUE.

C h i l o d o n u n c i n a t u s is cultivated in a dilute Locke-eggsolution made up as recommended by Hogue (1922), i.e. byadding two eggs to 400 c.c. of Locke's solution,1 boiling overa water-bath for fifteen minutes, and filtering through a cottonfilter. It is then sterilized in an Arnold sterilizer. The bestresults were obtained by using one part Locke-egg solution toten parts Locke's solution and ten parts distilled water. If thecultures became too turbid with bacteria, more distilled waterwas added. They were kept in small aquaria covered with glassplates (the animals grow and divide rapidly in this medium, andan unlimited supply of material may be obtained by its use).

Various fixing fluids were used, the best results being obtainedwith hot Schaudinn's fluid. Other fixatives used were Flem-ming's strong solution, chrom-acetic-osmic acid, Bouin's picro-formol solution, Hermann's platino-aceto-osmic mixture,Benda's fluid—a modified Plemming, and picro-mercuricsolution, as recommended by Yocom (1918).

Haidenhain's iron haeniotoxylin stain, long method, gave1 NaCl. 0-9 per cent,, CaCL 0-025 per cent., KC1 0-042 per cent.,

NaHCO3 0-020 per cent., Dextrin 0-25 per cent., H2O 100 c.c.

GYMNOSTOMATOUS CILIATES 363

the best results in studying chromosomes and the pharyngealbasket. Counterstaining with acid fuchsia was useful instudying pronuclei, and in studying developing rnacronuclei,as these stages take the haemotoxylin stain faintly. Borrel'sstain gave very good differentiation—the nuclear elementsstaining bright red, the pharyngeal basket green, and thecytoplasm blue. Mallory's triple stain was found to be usefulin studying sections of P r o r o d o n , the basket staining abright red. Plemming's triple stain, safranin and light green,and the Benda stain were also used, but gave no results ofvalue.

Leitz 2 mm. apochromatic objective, with 12x oculars (monobjective binocular microscope), was used in all cases exceptin the study of the somatic division and the studies on cilia,for which a Zeiss 1-5 objective and 12x oculars were used.A four-hundred-watt Spencer microscope lamp was used forcritical stages. The drawings were made Avith the aid of thecamera lucida.

ACKNOWLEDGEMENTS.

The work herewith presented was suggested by Pro-fessor G. N. Calkins, of Columbia University, in the summerof 1922. The work was begun at Agnes Scott College,Decatur, Georgia, in the winter of 1922, and continued, underProfessor Calkins's supervision, at Woods Hole in the summerof 1923, and at Columbia University since that time. Muchof the success of the investigation is due to Professor Calkins'sadvice and helpful criticism. The writer wishes to express toProfessor E. B. Wilson her appreciation for many helpfulsuggestions, and for a friendly interest in the progress of thework. She is indebted to Professor E. C. Ehodes, of EmoryUniversity, for Chi lodon material, and to Professor E. H.Bowen, who kindly gave her a bibliography of species ofChi lodon .

STRUCTURE OF C H I L O D O N U N C I N A T U S .

Chi lodon u n c i n a t u s (probably Gruber's cu rv i -dentis) is a holotrichous ciliate belonging to the family

Bb2


Chlainydodontidae. It was mentioned by Ehrenberg in 1835,and described by him in 1838 as follows :

' Body depressed, oblong, rounded on both ends, the anteriorright side hooked. . . . It is very transparent, and I could notdistinguish the series of body cilia, though I do not doubttheir existence. I counted only eight teeth in the mouth.A large round gland, and four to six stomachs were clear.I noticed it first on April 13, 1835.'

Although Ehrenberg mentions the number of trichites inthe basket as eight, his figures show more. The usual numberfor the species is ten to twelve. The body is convex on thedorsal side and flat on the ventral side.

Perty (1852) refers to Ehrenberg's description, and adds thathe saw the animal in longitudinal (s i c) division.

Stein (1863) considered this an immature form of C h i l o d o nc u c u l l u l u s . Enriques (1908) points out that although hecarried cultures of C h i l o d o n u n c i n a t u s , descended froma single individual, for a long time, no specimens of C h i l o d o nc u c u l l u l u s appeared in his cultures. I can confirm thisobservation. Maupas (1883) considered C h i l o d o n u n -c i n a t u s and C h i l o d o n c u c u l l u l u s as distinct on accountof the differences in the structure of the pharyngeal apparatusand in the nuclei.

In the living animal, in the hyaline border, irregular rows ofgranules, as described by Enriques, are seen (fig. 1, PI. 23).If the animals are killed with fixing fluids containing osmicacid, these granules are seen to be scattered through the thickerportion. They stain with Janus green, and take the othermitochondria stains. In the anterior portion, on the animal'sleft side (right if viewed from the ventral surface), is a smallvesicle containing granules, usually four in number (fig. 1,PI. 23). These have been seen to divide during the stages ofconjugation and division. Although no function has beenfound for them, their position leads one to infer that they areconnected with the neuromotor apparatus, the details ofwhich have not been made out as yet. Further work alongthis line is planned.

GYMNOSTOMATOUS OILIATES 365

Cilia.—Maier (1903) first described the ciliation of Chi lo -don u n c i n a t u s where the cilia are confined to the ventralsurface. In the anterior end, on the left side, there is a zonealong which a band of stronger cilia leads from the projectionto the mouth. From this zone five rows of cilia take theirorigin, pass over the anterior end, and down the right side, theoutermost row extending half-way down the body, the othersending at different points near the posterior end, the innermostrow extending almost completely around the posterior end. Onthe left-hand side (right in the figure) four rows take their origin atthe ' zone '. These are of different lengths, the outside row beingvery short, and the inside row extending nearly to the posteriorend. Two other rows begin about half-way down, and extendto the neighbourhood of the posterior end (figs. 1 and 2, PI. 23).

C o n t r a c t i l e Vacuoles.—There are usually two con-tractile vacuoles, one near the anterior end on the right side,and one on the left side near the posterior end (fig. 1, PI. 23).Other vacuoles are sometimes present, but these two seem to becharacteristic.

The Macronuc leus .—The macronucleus is situated ina vesicle at the posterior end. It is a spherical mass of granules,having in the centre an endosome containing a kinetic element,which has been designated by Calkins as the endobasal body.Nagler (1911) described this endobasal body in C h i l o d o nin both the macronucleus and the micronucleus. Enriques(1908) gives an incomplete account of this structure in themacronucleus. I can confirm the work of both these observersin every respect.

The endosome, if the long method of staining with Haiden-hain's iron haemotoxylin be used, is shown to be composed of aflat plate of plastin, overlaid with a network of chromatin,and with an endobasal body in the centre. There is a distinctradiation of threads from the endobasal body to the peripheryof the endosome (figs. 1 and 2, PL 23).

The Micronucleus .—The micronucleus is posterior tothe macronucleus. After ordinary staining, it appears to behomogeneous, but if the long method of staining with iron


haemotoxylin be used, and destaining is carried far enough, itis found to be a core of plastin, overlaid with a network ofchromatin, and containing an endobasal body. Counter-staining with acid fuchsin helps to bring out the details of thisnetwork. The endobasal body is not always easy to find(figs. 1 and 2, PI. 23).

The Oral B a s k e t or ' Eeusenappa ra t ' .—The oralbasket in 0 h i 1 o d o n is in the anterior end and about in themedian line of the body. It extends from the mouth openingto the region of the macronucleus. The number of trichites isusually ten ; specimens with eight and twelve have been seen.The trichites are distinct in the anterior end, and merge intoa smooth wall at the posterior end, which, in side view, has theshape of a ' twice-wound horn '. Kiernik (1909) pointed outthat it is only in the resting condition that the end is coiled, asit becomes more or less straightened during feeding. Theinner part, a funnel-shaped tube, with the mouth in theanterior end, extends only a short way back, seeming to fusewith the outer wall.

Bergh (1896) states that the oral basket of Nassu la willdissolve in 2 per cent, acetic acid, but that the trichites aroundthe mouth of S p a t h i d i u m are not affected by acetic acid.Acetic and hydrochloric acids of various strengths, and alsodilute sodium chloride and potassium hydroxide, were tried onthe Chi lodon basket. They seemed to have little effect, ifany. Animals killed with picro-mercuric solution (Yocom, 1918)show the basket swollen or ' exploded ', though each of theingredients of the fixative, tried separately, had no effect.

It was found, however, that the basket could be digested outvery quickly with artificial gastric juice (0-2 per cent. HC1,0-1 per cent, pepsin in 100 c.c. of water). Animals fixed oncover-glasses, treated with artificial gastric juice for a fewseconds, then stained with iron haemotoxylin, show only a spacewhere the basket was, or, at best, the ghost of a basket, therest of the animal appearing almost normal. The chemicalnature of the basket is thorefore protein. The elements fromwhich the basket is formed were not determined.


C h i l o d o n u n c i n a t u s varies considerably in size, accord-ing to abundance of food, and the age of the culture medium.It measures from 18-25/A in length.

Enriques (1908) showed the diploid number of chromosomesin C h i l o d o n u n c i n a t u s to be four, and the haploidnumber two. This I have confirmed, but there was someconfusion in results in the present investigation until it wasfound that on some of the slides, made from Georgia pure-linecultures, there were animals with eight chromosomes insteadof four. All of the slides made from these cultures show animalswith four chromosomes except those made during the lastmonth (May 1923), and these show no animals with fourchromosomes, eight being invariably present. These slideshave a great number of conjugating pairs on them and it hasbeen possible to work out the complete story of division andconjugation from them. As all the animals in these culturescame fron one individual, an exconjugant, isolated from amass-culture, and since the cultures were kept covered andcontamination by cysts was hardly possible the animal withthe tetraploid number of chromosomes must have been amutation. The slides show that after it appeared it becamedominant, there being no animals showing four chromosomesin the cultures the last month of their existence. I shall here-after refer to the animal with four chromosomes as animal A,and to the animal with eight chromosomes as animal B.

A comparison of animal B with animal A shows that thereis no difference except as to size, animal B measuring from about32 to 44/x. As fig. 2, PI. 23 shows, the nuclei are situated inthe posterior end ; there is the same number of trichites in thebaskets, usually ten, and the baskets have the same shape ; therows of cilia are arranged just the same as they are in animal A,and the position of the vacuoles is the same (figs. 1 and '2, PI. 23).

DIVISION OF C H I L O D O N U N C I N A T U S .

The B a s k e t and Cilia.—Kent (1881) pointed outthat ' the basket is destroyed and formed in duplicate intransverse and longitudinal division ', the longitudinal division


here mentioned evidently being conjugation. Bnriques (1908)showed that the basket is destroyed and formed anew indivision and conjugation. He did not, however, follow the fullhistory and fate of this structure.

The first sign of the basket is a slight imagination, both inthe anterior and posterior ends (fig. 14, PI. 24). This is thefirst visible evidence of the cells that are to result, and it is easierto observe the early stages of differentiation in the posteriorthan in the anterior end because the presence here of the oldbasket obscures things a little. The first part of the basket tobe differentiated in the cortex is the extreme tip of the insidefunnel, the mouth (fig. 14, PL 24). Then the tips of the trichites,hardly more than granules at first, appear (fig. 15 a, PI. 25),and the two rows of oral cilia come in just above the basket(fig. 15 b, PI. 25). Even before the appearance of trichites,the rows of ventral cilia are shown to be broken, and theybegin to turn in. The manner in which the cilia grow in aroundthe basket in the posterior end is shown in figs. 15 b, 16, and17, PI. 25. Whether all of the cilia are formed anew at divisioncannot be definitely stated.

The basket grows backward rapidly, and the rows of ciliaare completed. The old basket moves to one side at an earlystage (fig. 15 b, PI. 25), and, by the time division is completed,is entirely absorbed. It does not break up into a formlessmass, but seems to fade out or dissolve away, retaining to thelast some semblance of its former shape. In the anterior endthe basket is seen to arise in s i t u , while in the posterior endit migrates forward as it grows, the cilia growing forward atthe same time.

R e g e n e r a t i o n Experiments .—Enriques (1908) sug-gested that the presence of the old basket may be necessaryfor the formation of the new ones, or that part of it may benecessary to regenerate the new ones. In order to test thishypothesis experiments to remove the old basket were planned.

Ch i lodon u n c i n a t u s is not suited for cutting experi-ments, both on account of size and on account of the position ofthe basket, the posterior end being too near the macronucleus.


P r o r o d o n , of the order Holotrichida, sub-order Gymno-stomina, family Enchelinidae, has a relatively simple basketlocated at the extreme anterior end. The animal is large, andeasily handled under the low power of a binocular microscope.

One animal at a time was isolated in a small culture-dish ina very small drop of water, the anterior end then being cutoff with a glass microdissection needle or a very small, sharpscalpel. Both pieces of the animal were left in the dish, anda drop of spring-water, or filtered pond-water (sterilized), added.The survivors were killed at different intervals and stainedwith haemotoxylin.

Almost invariably the anterior end died in a short time. If thecut was made at about the centre of the animal, just abovethe nucleus, the anterior end swam around a few hours—sixhours being the longest time observed.

The time for regeneration of the posterior half of the animalvaried, probably on account of the age of the animal and theextent of injury. At the end of from four to six hours a ringappears where the future mouth is to be located. The tips ofthe trichites appear, as in division ; the basket grows backward,and in 18-24 hours the anterior end is completely regenerated.

If the animal is cut at about the time of division, but beforesigns of it are apparent, in addition to the regeneration of theanterior end with its basket another basket is formed over atthe side. This is probably the basket belonging to what wouldbe the posterior daughter-cell if the animal were allowed todivide in the normal manner.

From the above, it is shown that the presence of the oldbasket is not necessary for the formation of the two newbaskets at division.

The Macronucleus .—About the time the micronucleusdivides the chromatin network of the endosome breaks up intogranules, and at that time it is hard to distinguish the endo-basal body from the other chromatin granules (figs. 4 and 5,PI. 23). In favourable preparations, especially after its division,however, the two bodies may be found connected by a thread,a centrodesmus (figs. 6 and 7, PI. 23 ; fig. 8, PI. 24). In


later stages, after the other granules have disappeared, theendobasal body may be found in a vesicle, but greatly reducedin size (fig. 9, PI. 24). In daughter-cells which have justseparated, it can be found without difficulty. Here theendosome is in the shape of an exclamation point, and theendobasal body is seen in a vesicle a little anterior to the centre(fig. 10, PI. 24).

The granules of chromatin tend to run together and forma solid felt-like mass around the endosome. At cell division,this endosome divides into two equal parts (fig. 9, PI. 24).

The Micronuc leus .—At the beginning of division themicronucleus moves out from its place in the macronucleusand the endobasal body divides (fig. 3, PI. 23). The micro-nucleus increases in size, and a ' parachute ' stage, as describedby Calkins (1919), may be observed in the better-stainedpreparations (fig. 11, PI. 24). This consists, in the early stages,of a network massed at one end, and the two halves of theendobasal body are connected by a thread, a centrodesmus.The spireme breaks up into granules, and these condense intoshallow dumb-bell chromosomes, four in the case of animal A(fig. 12, a, PL 24) and eight in the case of animal B (fig. 13, a,PI. 24). The nucleus is very small and compact, and thesplitting of the chromosomes has not yet been observed. Thelater stages, however, show four chromosomes at each end ofthe dividing nucleus in animal A (fig. 12, b, PI. 24) and eight inanimal B (fig. 13, b, PI. 24). These stages, while very hard tofind, have been clearly demonstrated. The daughter nucleiare connected by a thread for some time, but this is brokenbefore cell division is complete. After the nucleus divides,the daughter nuclei so6n reach the condition characteristic ofthe resting stage.

It was much easier to follow the stages of somatic divisionin animals with the tetraploid number of chromosomes thanin animals with the diploid number. In nearly every case thestages were first found in animal B and then checked up inanimal A.

E e c o n s t r u c t i o n . — W h e n the cell divides, the two


daughter-cells are a little over half the size they will become.The oral basket is full grown, and seems out of proportion tothe size of the cell (fig. 10, PI. 24). The micronucleus has aboutreached its resting state, but the granular part of the macro-nucleus is an almost solid mass in the shape of a horseshoe.Around the periphery of this felt-like mass are small granulesconnected by small indistinct threads (fig. 10, PI. 24). Thesegranules are present in the later stages of division (fig. 9, PI. 24).The mass of chromatin becomes vacuolated and breaks up(fig. 10, PI. 24), the new granular macronucleus now becomingdifferentiated. The endosome, which is elongated during theprocess of division, rounds up, and the nucleus assumes itscharacteristic appearance. Just how the granular masssurrounding the endosome is differentiated from the felt-likemass has not been made out.

THE CONJUGATION OF O H I L O D O N U N O I N A T U S .

B e h a v i o u r of the P h a r y n g e a l Basket.—At thebeginning of conjugation the animals assume characteristicpositions. The animals are drawn from the ventral surfacein the figures, and therefore the right and left sides appearreversed. When right and left are stated hereafter, actualright and left as seen from the dorsal side are meant.

The animal on the right side always appears larger than theone on the left. This is partly due to the fact that the anteriorend of the right-hand animal is placed over the left-handanimal, and that the right-hand animal remains almost ina normal position, while the one on the left is constantlychanging its position. This difference in position was firstpointed out by Maupas (1883), and later by Enriques (1908),who showed that the animals do not come together in just theway Maupas set forth. The oral cilia show that the protoplasmicbridge is not formed just at the mouth, but a little to the rightof it in the right gamete (fig. 19, PI. 26). The position of theoral cilia in the left gamete is not so easy to observe. Inthe early stages the left edge of the right-hand animal and theright edge of the left-hand animal come together. The basket


in the right-hand animal leaves the mouth, as shown by thecilia, and moves over to the edge. The basket in the left-handanimal turns around toward the right edge, the baskets seemto touch, and the protoplasmic bridge is formed. As no bridgeis formed before the baskets meet it seems that they mustplay some role in the formation of this protoplasmic structure,especially as they move away at the end of the first maturationdivision. If they do play any role it is probably only amechanical one.

The two old baskets keep their places at the point where theprotoplasmic bridge is formed until the close of the first matura-tion division, when they begin to move away (fig. 24, PI. 27).In the later stages they may be found in almost any positionin the cell (fig. 36, PI. 28), though the tendency is to pass to theposterior part of the cell. By the time conjugation has beencompleted both old baskets are completely absorbed. As indivision they do not break up into a granular mass, but are.gradually absorbed.

As has been described for the process of division, the firstsign of the new basket is a slight invagination, usually justabove the old macronucleus (fig. 36, PL 28). In C h i l o d o nthe old macronucleus undergoes very little change duringconjugation, and the new one is not formed until after theanimals separate. The first part of the basket to appear is thetip of the inside funnel, or the mouth, then the ring of trichites,and the two rows of oral cilia are differentiated as in division(fig. 18, PI. 25 ; fig. 34, PI. 28). In this case it is easy to followthe development of the two rows of cilia, as they are formed inthe cortex, considerably below the place they will eventuallyoccupy. The right-hand animal always shows the two oldrows of cilia plainly, just above the protoplasmic bridge. Theother member of the pair is so twisted, usually, that it is noteasy to see the two old rows of cilia, though the new basket,and its accompanying cilia, are always clearly seen (fig. 84,PL 28).

Before the formation of the new baskets the straight rowsof cilia on the left side of the right-hand animal are shown


leading to the protoplasmic bridge (fig. 25, PI. 27). Certainlysome of these cilia disappear and are formed anew (fig. 24,PI. 27). Shortly after the first signs of the new basket aremanifest, the rows of cilia ' turn in ' and grow around thebasket just as they do in division (fig. 18, PI. 25). The basketgrows backward, but as the cell grows it goes forward, carryingthe cilia with it. The whole anterior end seems, in a way,to be ' made over '. It has not been possible to follow all thecilia, but certainly, in addition to the oral cilia, some of thosein the anterior end disappear and are formed anew. At theclose of conjugation, 24-30 hours, the two new baskets arecompletely formed and in place, and the animals have theirventral sides placed against each other (fig. 38, PI. 29).

As it seemed advisable to follow up this work on some otherform, another member of the same family, Ch lamydodonm n e m o s y n e , easily obtained at Woods Hole,.was used. Themouth of Ch lamydodon mnemosyne is in the anteriorend of the animal, a little to the right, on the ventral side.The basket is very large, and is composed of separate trichites,probably held together by some substance the structure ofwhich is not made out. While the work on this form has notbeen completed, it can be definitely stated that here, also, thebasket disappears completely during division and is absorbed,the two new baskets appearing and growing exactly as inCh i lodon ; only, in this case, there is no inner tube, so thatthe trichites appear as a ring and form at a later stage ofdivision. In conjugation the stages have been traced up tothe disappearance of the old basket, and, as far as observationshave been made, the behaviour is the same as in Chi lodon .

The Mic ronuc leus du r ing Conjugation.—Thepicture of the maturation processes presented by my material,especially with reference to the endobasal body and spiremestages, as well as the second maturation division, is so differentfrom that described by Enriques (1908) that full details arepresented herewith.

After the two animals are joined the first thing observed isthe division of the endobasal body in the micronucleus. If the


destaining is not carried far enough, the endobasal body is notdifferentiated from the mass of chromatin, and figures are seenlike those figured by Enriques. Examination of many pairs ofconjugants has demonstrated that the very early stages, asshown by Enriques, are incorrect. The chromatin is sometimesmassed at one end of the micronucleus (fig. 29, PI. 28), some-times in two masses, or sometimes it extends along the lengthof the micronucleus (fig. 33, PL 28), the mass being largerat one end, giving the picture presented by Enriques. As thenucleus increases in size a ' parachute ' stage (Calkins, 1919)is observed (fig. 20, PL 26 ; figs. 51 and 54, PL 31). Thespireme stages are much clearer in animal B than in animal A.This spireme breaks up into granules, and these seem tolengthen out into two long rows. .In some of the preparationsthese strands seem to split, four strands resulting (fig. 22, PI. 26).The granules arrange themselves on these strands (fig. 23,PI. 26), four rows in the case of animal A and eight rows inthe case of animal B. These granules condense into smalldumb-bell chromosomes which go on the spindle. In neithercase was the number of granules made out.

A very confusing element is observed. Besides the chromo-somes, there is plastin on the spindles in somewhat irregularlumps. Unless the preparations are very favourable thesesometimes stain heavily, giving all sorts of clumping figures.If destaining is carried far enough the chromosomes, sometimesimbedded in the plastin mass, are shown to be small dumb-bells.The chromosomes seem to split longitudinally, appearing beforeseparation as double (fig. 52, PL 31). The chromosomesmigrate to the poles, and there fuse in pairs forming ' diads '(fig. 25, PL 27 ; fig. 53, PL 31). They go into the resting stagein this state, spinning out, as shown in fig. 26, PL 27). Thefirst maturation division seems therefore to be equational.

The Second M a t u r a t i o n D iv i s ion , the E e d u c -t ion Division.—After the telophase of the first divisionthe daughter nuclei go into the resting stage for a short time.The endobasal body divides as before (fig. 29, PL 28) and thespireme is formed (fig. 28, PL 27), which condenses into


granules. The granular strands are not so long as in the firstmaturation division (fig. 30, PI. 28). The granules condenseinto four chromosomes in the case of animal A (fig. 32, PI. 28)and eight chromosomes in animal B (fig. 55, PI. 31). Theseare arranged on the spindle, and two go to the poles in animal A(fig. 32, PI. 28), while four go to the poles in animal B (fig. 56,PI. 32). The chromosomes are reduced to one-half the numberat this time, and the nuclei go into the resting stage again(fig. 56, PL 32, animal on the right hand, left in the figure).

The T h i r d M a t u r a t i o n Division.—The third divisionfollows the same general order as the two preceding divisions.The endobasal body divides and the spireme is formed. Thetwo strands of granules are short in this stage (fig. 57, PL 32).In animal A, two chromosomes are formed, and in animal B,four are formed. By this time, the old basket is completelyout of the way, and the nuclei move into the region of theprotoplasmic bridge. The division is apparently transverse(fig. 35, PL 28), as Enriques describes. After division of thechromosomes the pronuclei are formed, and the migratingnuclei go across the bridge (fig. 36, PL 28 ; figs. 59 and 60,PL 32) and reach the stationary nuclei. There is little, if any,difference between the migrating and the stationary nuclei(fig. 37, PL 29 ; fig. 61, PL 32). When the nuclei touch, themembranes dissolve, and the two nuclei come to lie side byside. These lengthen out, become granular, and fusion iscomplete. At this stage the animals separate (fig. 39, PL 29).

The A m p h i n u c 1 e u s.— After fusion of the pronuclei theprocess of reorganization begins. The granules condense intofour chromosomes in animal A, and into eight chromosomes inanimal B (fig. 41, PL 29 ; fig. 63, PL 33). These go on thespindles and divide longitudinally, apparently (fig. 42, PL 29).In the case of animal A, four go to each pole (figs. 43 and 44,PL 29), and in the case of animal B, eight go to each pole(fig. 63, PL 33). The nucleus then divides, forming the newmicronucleus and the new macronucleus (fig. 45, PL 29 ; fig. 46,PL 30 ; figs. 63, 64, 65, PL 33).

The micronucleus soon reaches the resting stage, while the


macronucleus begins to enlarge (fig. 47, PI. 30). This enlarge-ment continues until the new macronucleus nearly fills thecell (fig. 48, PI. 30). The chromatin in the macronucleusis in the form of a broken spireme which takes the nuclearstain very faintly. It stains an intense red with acid fuchsin.The old macronucleus moves farther and farther forward,seeming to be pushed by the developing macronucleus. Itbecomes smaller in proportion as the new macronucleus becomeslarger. The endobasal body of the old macronucleus, beforethe conjugants separate, becomes ' spread ', and sends threadsto the periphery of the endosome (fig. 37, PL 29). It seems tobecome diffuse, though whether it, or any part of it, persists asthe endobasal body of the new macronucleus could not bedetermined. The new macronucleus begins to condense, thethreads become heavier, then break up into characteristicgranules. In the meantime the endosome has become differen-tiated, and takes its place in the centre of the mass of granules.The manner of the formation of the new endosome and theendobasal body has not yet been determined.

The exconjugants can always be recognized in a culture bytheir large size, and by the appearance of the macronucleus.

The Macronuc leus du r ing Conjugation.—At thebeginning of conjugation the macronucleus moves up from itsposition in the posterior end of the animal to about the middle.There is very little change in it until after the animals haveseparated. In favourably stained preparations the endosoinehas changed its appearance, in that the endobasal body hasbecome much ' spread ', and is connected with the peripheryby threads. The history of the macronucleus after conjugationis described above.

DISCUSSION.

There are many modifications, or adaptations, in the cyto-stome of the Protozoa for special modes of feeding. Exceptfor the trichites, or armature of the pharynx, these do not comewithin the scope of this paper. D i d i n i u m has a peculiartongue-like organ for capturing prey. This is strengthened by

GYMNOSTOMATOUS CIMATES 377

a series of fine rods. Spathidium has separate trichitesarranged around the mouth, while Prorodon teres hasa simple tube of trichites ; the basket of Chlamydodon(sp. ?) has a series of hooks attached to the anterior end, andthis has also been observed in one species of Chilodon.There are all gradations of complexity. Blockmann (1897)states that ' the distinction between the structure of themouth apparatus with Enchelydon, Pseudoprorodon,Spathidium, &c, on the one side, and Prorodon,N a s s n 1 a, and Chilodon on the other side, is already longknown '.

It seems clear that these structures relieve strain incidentto the ingestion of food. While Ohilodon uncinatus canlive and thrive upon bacteria, in its natural state it ingestsdiatoms and algae. One has only to bring in a wild culture andexamine the animals, to find them filled with diatoms and algae.There are many forms of Protozoa which feed upon algae anddiatoms, yet do not have a special armature around the oralgroove, but in them the matter of stress incident to feeding istaken care of some other way, e.g. in Frontonia , Gold-schmidt (1922) states that ' a series of sharp contractions inthe body-wall assists in relieving certain other tensionpoints ', &c.

In every case, so far observed, when the trichites are arrangedin the form of a basket the structure is extremely elastic. Thebasket of Prorodon can be expanded almost as widely asthe breadth of the body. In this genus, as shown by Maier(1902), the basket is seen to be operated by myonemes on eachside. Sections of Prorodon show this feature very well.No myonemes have been found attached to the basket inOhilodon uncina tus . Indeed, the structure moves fromits place at the mouth opening in division and conjugationstages, and can be found in almost any position in the cell.Both Chlamydodon and Chilodon ' scrape along ' thebottom of the dish with the basket when feeding, the basketsometimes a little protruded.

In cases where fixed specimens have been accidentallyNO 275 c c


crushed, the basket is pressed out of the body, retaining itsform, and showing that it is a formed body, a distinct organelle.

Origin of the T e t r a p l o i d Form.—Since the tetra-ploid form of Ohilodon u n c i n a t u s arose in a pure-lineculture, it is suggested that it arose in one of the followingways :

1. By the failure of the chromosomes to reduce their numberat the second maturation division.

2. By the division of the chromosomes without the divisionof the nucleus, either just after fertilization or later in somaticdivision.

Inasmuch as abnormalities are found, e. g. extra long indivi-duals with two nuclei, showing a division of the nucleus withoutcell-division, it is easy to suppose that the chromosomes coulddivide, and the division of the nucleus be suppressed in someAvay. Fig. 66, PI. 33, shows such an animal conjugating witha normal individual. The macronuclei show that this is nota case of an exconjugant conjugating again, as Enriques hasshown may sometimes occur.

R e d u c t i o n Divis ion in the Protozoa.—In mostof the cases where a complete investigation of the processes ofconjugation has been made, it has been found that the secondmaturation division is the division in which the number ofchromosomes is reduced. There are cases, however, wherethe number of chromosomes is reduced during the first matura-tion division, e.g. Gregory (1923) working on O x y t r i c h af a l l a x .

Jamieson (1920) found that in Dip locys t i s reduction inthe number of chromosomes occurs shortly after fertilization,and that the animal goes through its asexual cycle with thehaploid number of chromosomes, and Dobell (1915) hasdescribed the same phenomenon in the case of Aggrega t ae b e r t h i , a coccidian.

Kofoid (1923) in discussing Jamieson's work and reproduc-tion in some of the lower plants suggests that ' it gives usoccasion to consider whether or not sexual reproduction maynot have been elaborated gradually and independently within


the different groups of Protista, and subsequently in them andin the higher forms of life the diploid state has extended itsdomainmore and more throughout the life-cycle of the organism'.

This suggestion may possibly prove true as our know-ledge of complete life-cycles increases, but, to date, thereis little evidence that the haploid condition through theasexual cycle is widespread in the Protozoa. In the case ofCh i lodon u n c i n a t u s it has been proved beyond a reason-able doubt that the reduction in the number of chromosomestakes place at the second maturation division, and that the animalgoes through its asexual existence with the diploid number ofchromosomes. In the ciliates, it seems that no case has beenfound where the animal goes through its asexual existence withthe haploid number of chromosomes, though there are possiblyother cases than those described by Dobell and Jamieson inother groups of Protozoa.

SUMMARY OF OBSEBVATIONS.

1. C h i l o d o n u n c i n a t u s , Ehrenberg, is a ciliate belong-ing to the family Chlamydodontidae.

2. In division, the old pharyngeal basket is entirely destroyedand two new baskets are formed, one in the anterior and onein the posterior part of the cell.

3. In conjugation, the old baskets move to the place wherethe protoplasmic bridge is formed, and remain there untilthe close of the first maturation division. They then movetoward the posterior part of the cell, and the new basketsappear just above the micronuclei. By the time conjugationis completed, the old baskets have been completely absorbed,and the new baskets are fully formed and in place.

4. In P r o r o d o n cysts, the basket is destroyed and formedanew.

5. Observations on C h l a m y d o d o n m n e m o s y n e showthat the behaviour of the basket in division and in conjugationis the same as in C h i l o d o n .

6. The basket in C h i l o d o n is quickly digested by artificialgastric juice, showing it to be protein in nature.

c c 2


7. If the basket be cut off in P r o r o d o n , it is completelyregenerated in twenty-four hours, showing that the old basketis not necessary for the formation of new ones.

8. The oral cilia are destroyed and formed anew both inconjugation and in division.

9. In pure-line cultures, an animal having a tetraploid ordouble the number of chromosomes normal for C h i l o d o nu n c i n a t u s appeared, the other animals dying out after itsappearance. This is designated animal B below, while theanimal with the diploid number of chromosomes is designatedanimal A.

10. The macronucleus is a spherical mass of granules, havingin its centre an endosome containing an endobasal body. Theendosome is overlaid by a network of chromatin. In division,the endobasal body divides, the chromatin network of theendosome breaks up, the endosome elongates, the granulesaround the endosome fuse and form a felt-like ring, whichdivides into two equal parts.

11. The micronucleus consists of a core of plastin overlaidwith a network of chromatin, and containing an endobasalbody. At the beginning of cell-division, the micronucleus movesfrom its place in the macronucleus, the endobasal body divides,and a ' parachute ' stage follows. The spireme breaks up intogranules, the granules fuse into chromosomes, four in the caseof animal A and eight in the case of animal B. The chromo-somes split, four going to the poles in animal A and eight goingto the poles in animal B. The nucleus then divides and thedaughter nuclei assume their resting aspect.

12. There are three maturation divisions during conjugation.In the first, the endobasal body divides, the spireme is of the' parachute ' type, the spireme breaks up into granules, thegranules condense into chromosomes, four in the case ofanimal A and eight in the case of animal B. The chromosomessplit, four going to the poles in the case of animal A and eightin the case of animal B. The chromosomes spin out, and a shortresting stage occurs.

13. The second maturation division follows the same orderas the first except that the chromosomes do not split, but two


whole chromosomes go to the poles in animal A while fourwhole chromosomes go to the poles in animal B. The numberof chromosomes is reduced to one-half.

14. The third maturation division follows the same order asthe other two. Two chromosomes are formed in animal A andfour in animal B. Division of chromosomes is transverse.One pronucleus from each animal migrates across the bridge, andcomes to lie close to the stationary nucleus. The membranedissolves and the nuclei become granular. At this stage, theanimals separate.

15. The amphinucleus forms a spindle immediately afterfusion. In animal A, four chromosomes condense and inanimal B eight. These split, and four go to the poles in thefirst case and eight go to the poles in the second case. Thenucleus divides, forming the future micronucleus and macro-nucleus.

16. The micronucleus reaches the resting stage, while themacronucleus enlarges until it nearly fills the cell. It thencondenses to normal size, the endosome is differentiated, andthe micronucleus moves to its place in the posterior end. Theold macronucleus is gradually absorbed during the enlargementof the new macronucleus.

17. It is suggested that the tetraploid form arose from thediploid form in one of the following ways :

(1) By failure of the chromosomes to reduce in number atthe second maturation division, the pronuclei formingafter the first maturation division.

(2) By division of the chromosomes without division of themicronucleus.

LITERATURE CITED.

Bergh, R. S. (1896).—" tjber Stiitzfasern in Zellsubstauz einiger Infu-sorien ", ' Anat. Heft', Bd. 7, pp. 103-12.

Blookmann, F. (1897).—' Trichitenapparat und Reusenapparat ", ' Zool.Anzeig.', Bd. 20, p. 133.

Calkins, Gary N. (1919).—" Uroleptus raobilis, Englin. 1, History ofNuclei during Division and Conjugation ", ' Journal of Exp. Zool.',1919, vol. 27, pp. 293-355.

382 MARY STUAET MACDOUGALL

Dolflein, F. (1916).—' Lehrb. d. Protozoenkunde', 4th ed., p. 64;p. 1118.

Ehrenberg, C. G. (1835).—" Zursatze zur Erkenntniss grosser organAusbildung in den kleinsten tMerohen Organismen", ' Abh. Akademiedev Wissenschaften', Berlin, p. 164.

(1838).—'Die Infusionsthierchen als vollkommene Organismen',Leipzig.

Enriques, P. (1908).—" Die Conjugation und sexuelle Differenzierung derInfusorien", ' Archiv fur Protistenkunde', Bd. 12, 1908, pp. 213-74.

Goldsmith, William M. (1922).—" The Process of Ingestion in the CiliateFrontonia " , ' Journal of Exp. Zool.', vol. 36, pp. 33-44.

Gregory, Louise H. (1923).—" The conjugation of Oxytricha fallax ",' Journal of Morphology ', vol. 37, pp. 555-81.

Gruber, A. (1883).—" Beobachtungen an Chilodon ourvidentis, nov. sp.",Festschrift 56, ' Vers. deutsch. Naturf. und Arzte ', Freiburg, pp. 38-48.

Hogue, M. J. (1921).—" Waskia intestinalis ; its cultivation and cystformation ", ' Journal American Medical Association', July 9, 1921,vol. 77, pp. 112-13.

Jamieson, A. Pringle (1920).—" The Chromosome Cycle in Gregaries, withSpecial Reference to Diploeystis Schneideri", ' Quart. Journ. Micro.Sci.', vol. 64, part 2.

Kiernik, E. (1909).—" Chilodon hexastichus, nov. sp.", ' Bull. Intern.Acad. 8c. Cracovie ', pp. 75-119.

Kofoid, C. A. (1923).—' The Life-Cycle of the Protozoa", ' Science',April, 1923, vol. 57, p. 406.

Maupas, E. (1883).—" Contribution a Fetude morphologique et anatomiquedes Infusoires cilies ", ' Arch. Zool. exp.' (2) v. 1, pp. 427-664.

Maier, H. N.—" t)ber den fineren Bau des Wimperapparate der In-fusorien ", ' Archiv fiir Protistenkunde ', Bd. 2, pp. 73-179.

Nagler, Kurt (1911).—" Caryosom und Centriol beim Teilungsvorgang vonChilodon uncinatus ", ibid., Bd. 24, pp. 142-8.

Perty, M. (1852).—' Zur Kentniss Kleinster Lebensformen', Bern.Stein (1859).—' Dev Organismus der lufusionsthiere '.Woodruff, L. L., and Spencer, Hope (1922).—" Studies on Spathidium

spathula ", ' Journal of Exp. Zool.1, vol. 35, pp. 189-202.

EXPLANATION OF PLATES 23-33.

PLATE 23.

All figures drawn from ventral view.Fig. l.:—Chilodon u n c i n a t u s . Form with four chromosomes.

Animal A.Fig. 2.—Chilodon u n c i n a t u s . Form with eight chromosomes.

Animal B.


Fig. 3.—Beginning of division; division of the endobasal body in themicronucleus; enlargement of the endobasal body in the macronucleus.

Figs. 4, 5, 6, 7.—Stages in the division of the macronucleus.

PLATE 24.

Figs. 8 and 9.—Stages in the division of the macronucleus.Fig. 10.—The macronucleus just after the division of the cell.Fig. 11.—Division of the endobasal body and spireme stage of the

micronucleus.Fig. 12.—Early metaphase and telophase, showing the four chromo-

somes in animal A.Fig. 13.—Early metaphase and telophase, showing the eight chromo-

somes in animal B.Fig. 14.—Development of the pharyngeal basket. Tip of the mouth is

present.

PLATE 25.

Figs. 15 a, 156, 16, 17.—Stages in the development of the pharyngealbasket and oral cilia.

Fig. 18 and fig. 36, PI. 28).—Development of the new baskets and oralcilia during conjugation.

PLATE 26.

Fig. 19.—Conjugation of animal A. Division of the endobasal body inthe micronucleus.

Fig. 20.—Later stage. Chromatin is massed at one end.Fig. 21.—Chromatin breaks up into two strands of granules.Fig. 22.—Chromatin in four strands of granules.Fig. 23.—Beginning of condensation of granules to form chromosomes.

PLATE 27.

Fig. 24.—Four chromosomes on spindle.Fig. 25.—Telophase of the first maturation division.Figs. 26 and 27.—Later stages.Fig. 28.—Early stage in the second maturation division. Spiremes.

PLATE 28.

Fig. 29.—Later stage in second maturation division. The endobasalbody has divided.

Fig. 30.—Four strands in granules.Fig. 31.—Condensation of granules into chromosomes.Fig. 32.—Two chromosomes move to each pole. The reduction of the

number of chromosomes to two.Fig. 33.—Beginning of the third maturation division.


Fig. 34.—Nuclei move to the protoplasmic bridge.Fig. 35.—Division of the chromosomes.Fig. 36.—Migration of the pronuclei.

PLATE 29.

Fig. 37.—Fusion of the migrating and stationary nuclei to form theamphinucleus.

Fig. 38.—Lengthening of the amphinucleus.Fig. 39.—Breaking up of the chromatin into two strands of granules.Fig. 40.—Later stage.Figs. 41 and 42.—Metaphase.Figs. 43, 44, 45.—Telophase.

PLATE 30.

Fig. 46.—Division of the amphinucleus to form the new micronucleusand the new macronucleus.

Fig. 47.—Enlargement of the new macronucleus.Fig. 48.—Later stage.Fig. 49.—Contraction of the macronucleus. The appearance of the

endosome.Fig. 50.—Later stage.

PLATE 31.

Fig. 61.—Conjugation of animal B. Spireme stage of the first matura-tion division.

Figs. 52 and 53.—Metaphase and telophase of the first maturation division.Fig. 54.—The second maturation division. Spireme stage.Fig. 55.—Late prophase, showing eight chromosomes.

PLATE 32.

Fig. 56.—Four chromosomes migrate to the poles. The reduction ofthe number of chromosomes to four.

Figs. 57 and 58.—Early stages in the third maturation division.Fig. 59.—Migration of the pronuclei.Kg. 60.—Later stage.Fig. 61.—Fusion of the migrating and stationary pronuclei to form the

amphinucleus.PLATE 33.

Fig. 62.—Granule stage of the amphinucleus.Fig. 63.—Eight chromosomes at each pole, telophase.Fig. 64.—Division of the amphinucleus to form the new macronucleus

and the new micronucleus.Fig. 65.—The new micronucleus and macronucleus.Fig. 66.—Abnormal form with two nuclei conjugating with normal

animal.

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