The Neuromotor System of Euplotes patella during Binary ......The essential features of the...

The Neuromotor System of Euplotes patelladuring Binary Fission and Conjugation.

By

Datus M. Hammond,

Department of Zoology, University of California, Berkeley.

With Plates 21-22 and 11 Text-figures.

CONTENTS.

PAGEINTRODUCTION . . . . . . . . . . 507METHODS 508

Cultures, p . 508. Observation of Living Organisms, p . 509. Demon-stration of the Peripheral Polygonal System, p . 510. Demonstra-tion of the Neuromotor System, p . 511.

STRUCTURE OF THE NEUROMOTOR SYSTEM . . . . . 512

The Neuromotorium, p. 515. Cirri and Membranelles, p . 515. Cyto-pharynx, p . 520. Peripheral Polygonal System, p. 521. BasalApparatus of the Bristles, p . 522.

ASEXUAL REPRODUCTION . . . . . . . . 526

Peristome, p . 528. Development of Cirri, p . 529. Resorption ofCirri, p . 532. Differentiation of Fibrils, p . 532. Multiplicationof Bristles, p . 533. Peripheral Polygonal System, p . 537.

SEXUAL REPRODUCTION . . . . . . . . 541

Reorganization of the Gamete, p . 542. Reorganization of theZygote, p . 545.

DISCUSSION 546

SUMMARY . . . . . . . . . . . 552

R E F E R E N C E S . . . . . . . . . . 553

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

THERE is a life-cycle in the Protozoa, which is comparable tothat of the Metazoa (see Kofoid, 1923). This life-cycle beginswith the zygote, which undergoes successively periods ofcleavage, differentiation, asexual reproduction, senescence, andfinally death. Although the nuclear aspects of the life-cycle ofseveral ciliates have been worked out, there is little detailedknowledge concerning plasmic structures during the different

NO. 316 L 1

508 DATUS M. HAMMOND

stages of the cycle. Stein (1859) discovered that there is a moreor less complete loss of cytoplasmic structure during asexualreproduction in the complex ciliates in the order Hypotricha,and Wallengren (1901) worked this out more completely.Engelmann (1862) showed that a similar change occurs duringsexual reproduction in this group, and Maupas (1889) confirmedand extended his observations. However, our knowledge con-cerning the extent and manner of these cytoplasmic changes indifferent stages of the life-cycle, and their relationship to thenuclear phenomena, is still incomplete.

The hypotrichous ciliate E u p l o t e s p a t e l l a Ehrenbergprovides admirable material for the study of this problem. Ithas a highly differentiated body, with a well-developed neuro-motor system, consisting of complex locomotor organelles, co-ordinated by an integrated system of fibrils, as shown by Yocom(1918) and Taylor (1920). Also, the nuclear phenomena duringthe entire life-cycle have been thoroughly studied (Turner,1930). The same variety as that studied by Yocom and byTurner was used as the subject of this investigation. Thisvariety is easily recognized by the deep peristomial groove withits wide extension under the frontal field, and by the deep inden-tation in the macronucleus near the left anterior side (Text-fig. 2).

The purpose of this investigation was to determine to whatextent and in what manner the structure of the neuromotorsystem changes during the different stages of the life-cycle ofthe organism, and how these changes are related to the basicphases of the life-cycle, such as asexual reproduction, and thehaploid and diploid conditions at the time of sexual reproduction.

The writer wishes to express appreciation to Dr. C. A. Kofoidfor the use of his library, and for his many helpful suggestionsduring the course of the investigation; also to Dr. Harold Kirbyfor his helpful criticisms of the work.

METHODS.

C u l t u r e s .The organism was cultured by a modification of Turner's

(1930) method. Timothy hay and wheat grains in standardsolution (Chalkley, 1930) provided the most satisfactory nutrient

EUPLOTBS PATELLA 509

medium. Five grammes of timothy hay and 3 grains of wheatwere added to 50 c.c. of this solution at boiling-point, andallowed to boil for one minute. When cool, this was added to250 c.c. of the solution in a round refrigerator jar. After stand-ing for one day the culture was started by inoculation withE u p l o t e s from a previous culture. Cultures made from grainsof wheat also provided a good growth of the organisms. Inthese cultures, 10 grains of wheat were substituted for thetimothy hay and wheat in the procedure already given. TheE u p l o t e s were obtained originally from Thornhill Pond, afreshwater pool in the vicinity of East Oakland. Variousorganisms were used as food for the E u p l o t e s . Chilo-monas provided the most abundant growth, but Asp id i scagave better results, for the E u p l o t e s feeding on it were morefree from food masses which obscure cytological details.

Since specimens undergoing binary fission were of especialinterest in this study, cultures of from three to ten days in ageprovided most of the material. In cultures of this age a largepercentage of the organisms were undergoing fission. For thestudy of conjugation cultures three weeks to one month inage were used. The organisms were very abundant in thecultures at the time epidemics of conjugation began.

O b s e r v a t i o n of L iv ing Organ i sms .Organisms were studied in hanging drops over depression

slides, and under cover slips supported by vaseline rings. SinceE u p l o t e s p a t e l l a is extremely active, study of the livingorganisms entails some means of retarding movement. This wasaccomplished by reducing the amount of water in the hangingdrop to such an extent that surface tension held the organisms inplace. With the organisms under cover slips movement wasretarded by several methods, of which those suggested by Lund(1935), namely, the use of sodium amytal as a narcotic agent,and keeping the organisms stationary by applying the coverslip closely to the slide were found most useful. Gum arabic asa means of increasing the viscosity of the solution and thusretarding movement was found to be unsuccessful, because theorganisms became abnormal in appearance.


Several vital dyes were used, including cresyl blue, Janusgreen B, Bismarck brown, acid fuchsin, methylene blue, toluidinblue, and neutral red. Acid fuchsin in a 1 per cent, solution, assuggested by Cole (1934), was useful for studying the structureand movement of the ciliary apparatus of the cytopharynx.Neutral red in a dilution of 1-10,000 was the best vital stain forstudy of the rodlets at the bases of the bristles, membranelles,and cirri.

One per cent. NiSO4 and Ni(N03)2were employed as suggestedby Gelei (1935) in order to differentiate the parts of the cyto-pharynx, but the results were unsatisfactory.

In order to ascertain the mechanics of feeding and to studythe path of food particles in the endoplasm, organisms wereobserved in a medium containing finely ground indigo andcarmine. The particles of indigo were taken in readily by theorganisms, while carmine was rejected. As an aid in observationof the process of ingestion the action of the organelles wasretarded with sodium amytal.

Microdissection methods were employed in an attempt todetermine experimentally whether the so-called 'sensorybristles' are really sensitive to tactile stimuli, but this wasfound to be too difficult on account of the small dimensions ofthese bristles.

D e m o n s t r a t i o n of t h e P e r i p h e r a l P o l y g o n a lsys tem.The silver impregnation methods of Klein (1926), Yabroff

(1928), Gelei (1934 a), Lund (1933), and Turner (1933) wereused. The silver-osmic acid-formalin method of Gelei (1928)gave the best results in differentiating the rodlets at the base ofthe bristles. These could not be demonstrated by the osmicacid-toluidin blue process of Gelei (1926-7). A modification ofthe mercuric chloride-Golgi solution-silver method of Gelei(1934 a) was the most advantageous in demonstrating the poly-gonal network. This method as used consists of the followingsteps:

(1) Fixation for one minute in concentrated aqueous mercuricchloride, to which are added, just before use, two drops of a


3 per cent, potassium-bichromate solution, and one drop of a1 per cent, osmic acid solution per c.c. of mercuric chloride.

(2) Wash twice in standard solution.(3) Place in a 2 per cent, silver-nitrate solution and expose

to bright sunlight until the organisms reach the proper stage ofdarkening. This must be ascertained under the microscope.Uniform exposure of the organisms is accomplished by circulat-ing air through the solution as suggested by Lund (1933).

(4) Wash several times in distilled water.(5) Dehydrate rapidly, clear in xylol, and mount in balsam.The organisms were handled in centrifuge tubes. This method

produces uniformly good results, and has the advantage overKlein's method that the organisms are not distorted by drying.

D e m o n s t r a t i o n of t he N e u r o m o t o r S y s t e m .For whole mounts and sections Schaudinn's, Bouin's, Flem-

ming's, Yocom's, Champy's, and Zenker's fixing fluids were used.Champy's and Flemming's fluids were used cold, and the othershot. Schaudinn's fluid gave the best results for differentiationof fibrils, while Champy's fluid was the most useful in fixationof material for study of the basal apparatus of the bristles.The organisms were fixed in centrifuge tubes, then for wholemounts were mounted on slides from 70 per cent, alcohol afterfixation in Schaudinn's, Bouin's, Yocom's, or Zenker's fluids.In this way the distortion and uneven results of fixation oncover slips was avoided. The material fixed in Flemming's andChampy's fluids was handled in centrifuge tubes throughout,because of the difficulty which was experienced in making theorganisms adhere to the slides.

Stains used were Heidenhain's iron alum-haematoxylin,Mallory's triple connective tissue stain, and toluidin blue.Heidenhain's haematoxylin gave the best results with the shortmethod, using the mordant 6-12 hours and the stain half anhour, both cold. Alcoholic haematoxylin as recommended byDobell (1914), except that he used haematein instead of haema-toxylin, gave excellent results. This method differentiated thebasal structures of the locomotor organelles to better advantagethan that using aqueous haematoxylin.


For sectioning the individual organism method used by J. P.Christenson (unpublished paper) was employed. This consistsof staining the organisms with eosin in 95 per cent, alcohol, andimpregnating in paraffin in a Syracuse watch glass. Only asmall number of organisms were placed in each dish, and thesewere uniformly distributed over the bottom of the dish. Aftercooling of the paraffin and removal from the dish, the organismsmay be seen under a dissecting microscope and the blocks ofparaffin cut so that each block contains only one or a feworganisms, whose orientation is known. This method involvesmore precision and less waste than mass methods of sectioning.

Kolatchev's osmic acid method, as given by Bowen (1928),was employed for the demonstration of mitochondria and Golgimaterial.

STRUCTURE OF THE NEUROMOTOR SYSTEM.

In order to understand the changes in structure of the neuro-motor system during the different stages in the life-cycle, itstypical structure must first be considered.

The essential features of the neuromotor apparatus, asdescribed by Yocom (1918), are five fibrils running anteriorlyfrom the bases of the anal cirri and converging near the anteriorend of the body, where the resulting single fibril is attached toa motorium. Another fibril, the membranelle fibril, runs an-teriorly from the motorium along one side of the adoral zone ofmembranelles and connects with a lattice-work structure inthe anterior lip. Yocom interpreted this fibrillar system asfunctioning in the co-ordination of the anal cirri with themembranelles. He also described fine fibrils attached to thebases of all the remaining cirri. These fibrils were found infrom 1-3 groups of from 4-6 fibrils each, radiating out in differentdirections from the bases of each of the frontal and ventral cirri.Only one group was seen to be attached to each of the marginalcirri. These fibrils were considered as a dissociated part of theneuromotor apparatus which functions in conveying stimuli tothe cirri, but which has not yet developed the function of co-ordination. Yocom noticed that the cirri are composed of cilia,the basal granules of which are arranged irregularly in round

EUPLOTES PATELLA 513

motX ad. memh

TEXT-FIG. 1.

Ventral view of E u p l o t e s p a t e l l a showing structure of neuro-motor system. Fixed in Schaudinn's fluid and stained in alcoholichaematoxylin. X 890. ana.cir.fib., fibril of anal cirrus; ad.memb.,membranelles of anterior adoral zone; end.cil., endoral cilia;memb.jib., membranelle fibril; mot., motorium; postphar.fib., post-pharyngeal fibril; postphar.mem., postpharyngeal membrane;rad.fib., radiating fibrils; sub.memb., suboral membranelles;vewt.menib., membranelles of ventral adoral zone.


basal plates. The membranelles were seen to be composed oftwo parallel rows of fused cilia.

Taylor (1920) confirmed Yocom's interpretation of the func-tion of the neuromotor apparatus by experimental methods.Also, he discovered the presence of fibre plates beneath the basalplates of the cirri and membranelles, to which the fibril or fibrilsare attached.

Klein (1926) in an undetermined species of E u p l o t e sdescribed a superficial network of lines to which he gave thename ' Silberliniensystem', because of its selectivity for silverin the silver impregnation method. This was found to bearranged in a pattern of regular polygons on the dorsal surface,and of irregular polygons on the ventral surface. For this reasonthe term peripheral polygonal system will be used to designatethese structures. The lines were found to connect the bases ofthe bristles, cirri, and membranelles. Klein supposed this net-work to be the means of co-ordination, rather than the neuro-motor system of Yocom, which he considered to be supporting innature. However, more recently, Klein (1932) modified thisviewpoint to include the neuromotor system with the systemof co-ordination as a conductor of internal impulses to the loco-motor organelles, while the polygonal system receives externalstimuli and effects the co-ordinated working of these organelles.

Jacobsen (1931) concluded that both the silverline system andthe neuromotor apparatus are supporting in function.

Gelei (1929 a) with his osmic acid-toluidin blue and silver-osmic acid-formalin methods described an ectoplasmic 'neuro-plasm' which he considered to be a stimulus-conducting struc-ture. Gelei (1929 b) supposed that the neuroplasm togetherwith the silverline system provides for co-ordination betweenthe locomotor organelles, with the assistance of the neuromotorapparatus. At first (1929 a) he regarded the 'silverline system'as being of a skeletal nature, but he later (1934 V) concludedthat it may be also neuroid.

Turner (1933) demonstrated the polygonal system in Eu -p l o t e s p a t e l l a . He came to the conclusion that this systemconducts sensory stimuli received from the bristles to the neuro-motor system, which acts as a motor conductor system. Turner


was unable to demonstrate the motorium, but otherwise con-firmed Yocom's description of the neuromotor system.

In the present study the neuromotor system was found tofollow the description of Yocom (1918) in its essential features.However, the location of the motorium and the finer structureof the cirri, membranelles, and cytopharynx was found to differin some respects from his account.

The N e u r o m o t o r i u m .

The motorium is located differently than Avas described byYocom (1918). The fibril formed by the convergence of the analfibrils continues its straight course anteriorly beyond the placeat which the motorium is located according to Yocom. Thispoint is immediately to the left of the most anterior frontal cirrus.The fibril passes just to the left of the base of this cirrus andcontinues anteriorly to the edge of the frontal area.

The beginning of the membranelle zone is immediately dorsalto the anterior margin of this horizontal frontal area, on whichthe cirri are located. In favourably stained preparations in sideview a vertically placed bilobed body is present in the regionbetween the anterior margin of the frontal area and the mem-branelle zone (Text-fig. 1). The fibril from the anal cirri is con-nected to this body on its ventral side and the membranellefibril is attached on the dorsal side. This is probably themotorium described by Yocom (1918). Its vertical position andits location at the edge of the frontal area where destaining isrelatively rapid makes the demonstration of the motorium verydifficult. However, there is not a direct connexion between thefibril from the anal cirri and the membranelle fibril as describedby Turner (1933). In side view the bilobed body is seen tointervene between these two fibrils.

Cirr i and M e m b r a n e l l e s .

The arrangement of the eighteen cirri into seven frontal, twoventral, five anal, and four marginal cirri as given by Taylor(1920) will be followed. For convenience in description those ofeach group will be numbered from left to right as indicated inText-fig. 2. The basal plates of the anal cirri have a rectangular


mic-memb.

md B V ; J

cuirec-

vent.br—-

uentar.—ci/rrod-

suhmemb. -cytophaz—

ana. c/r

rn ac •

mat cit

TEXT-FIG. 2.

Ventral view, showing arrangement of cirri and relationship of therodlets to the cirri and membranelles. Silver-osmio acid-formalinmethod, supplemented by observations on living organisms stainedwith neutral red. Ventral view. X 700. ana.cir., anal cirri; dr.rod.,group of rodlets at base of cirrus; cytophar., cytopharynx; cyt.rec,cytostomal recess; fr.cir., frontal cirrus; mac, macronucleus;mar.cir., marginal cirrus; intmb.rod., rodlets at base of mem-branelles; mtc, micronucleus; ros.vent.br., rosettes of ventral rowof bristles; sub.memb., suboral membranelles; vent.cir., ventralcirrus.

shape as indicated by Taylor (1920). However, the basal platesof the frontal and ventral cirri are rhomboidal to square inshape rather than round (Text-fig. 1). At least two and mostoften three sides of the basal plates of these cirri are straight.

BUPLOTES PATELLA 517

The bases of the marginal cirri are very difficult to observebecause of their slanting position on specimens lying flat onthe slide. These cirri have irregular square to oval bases.

The basal granules of the cirri are arranged in definite straightrows, contrary to what was found by Yocom. This arrangementconsists of primary rows and secondary rows approximatelyperpendicular to the primary rows, resulting in the appearanceof a grating. The primary rows are oriented in a longitudinaldirection in the anal cirri and in an oblique direction in thefrontal and ventral cirri, with the anterior ends of the rowspointing toward the middle of the body (Text-fig. 1). In themarginal cirri the primary rows of basal granules are in generalperpendicular to the margin of the body. There are nine to tenrows of basal granules in the anal cirri and six to seven in theother cirri. A similar arrangement of the basal granules instraight rows was described in E u p l o t e s w o r c e s t e r i byGriffin (1910 a). He found only three to four rows in each cirrus,with a maximum of fourteen granules in a row according to hisfig. 8, p. 300. The number of basal granules in the rows inE u p l o t e s p a t e l l a could not be determined, but it is muchgreater than Griffin found in E u p l o t e s w o r c e s t e r i .

Studied in relationship to the rows of basal granules of thecirri the arrangement of the fibrils of the cirri is given addedsignificance. These fibrils have a definite orientation with respectto the direction of the rows of basal granules of the cirrus towhich they are attached. In the left ventral cirrus, for example,three groups of fibrils extend out in different directions from thebase of the cirrus (Text-fig. 1). One group proceeds anteriorly,another posteriorly, and the third in a transverse directiontoward the left side of the body. The fibrils of the anterior andposterior groups are parallel with the primary rows of basalgranules of the cirrus, while the transverse fibrils are parallelwith the secondary rows of these granules. This close correla-tion in direction of fibrils with the rows of basal granules doesnot hold true for all cirri. In some cases the fibrils can be seento attach to the ends of rows of basal granules. Usually three tofive fibrils make up each group. For some cirri there are twogroups of fibrils and for the marginal cirri only one group.


According to Yocom's description the anal cirri are suppliedwith only the main longitudinal fibrils, one for each cirrus, butin critically stained preparations transverse fibrils can also be

mewb,£v- - mac.

-anpeifmic.

an. fitcitf

-on. litmarxiii

-h-anaa'r.fern,mac.

- anri.maf-cif.

TEXT-ITO. 3.

Reorganization of zygote. Schaudinn's, alcoholic haematoxylin.Ventral view. X 700. ana.cir., anal cirri; an.fr.cir., rudiment offrontal cirrus; an.lft.mar.cir., rudiment of left marginal cirrus;an.per., rudiment of posterior portion of peristome; an.rt.mar.dr.,rudiment of right marginal cirrus; mac, macronucleus; memb.,membranelles; mic., micronucleus; rem.mac, remnant of oldmacronucleus.

seen. These extend laterally from each anal cirrus, althoughthose attached to the first cirrus are most noticeable.

These fibrils of the frontal, ventral, and marginal cirri extendmuch farther from the bases of the cirri to which they are


attached than was described by Yocom. No connexion couldbe demonstrated at the distal ends except in one case, where thefrontal cirri one and two are connected by these fibrils.

The membranelles consist of three rows of cilia rather thantwo, as has hitherto been described. In the part of the zone ofmembranelles which is on the ventral side of the body, this thirdrow is incomplete in that it extends only part of the way acrossthe width of the zone. In that part of this region which lies ina frontal plane the third row consists of about 7-10 cilia (fig. 10,PI. 21; Text-fig. 1). The membranelles which extend across theanterior end of the body have three complete rows. The transi-tion between these membranelles and those with an incompletethircj»row is abrupt, and occurs at the place where the zone ofmembranelles turns around the left anterior end of the body.This third row was first seen in material prepared by the silver-osmic acid-formalin method, but it can be plainly distinguishedin the living organism as well.

Kahl (1932) found a difference in number of lamellae in themembranelles in different parts of the adoral zone in S t e i n i a.In this species nine to ten of the membranelles near the cyto-pharynx have two lamellae, while the remainder have three.

The arrangement of the basal granules of both the cirri andthe membranelles in rows, and the relationship of the radiatingfibrils to these, may be explained by supposing that the complexprotozoan E u p 1 o t e s with its composite organelles arose froma simple form in which the cilia were distributed in slightlyspiral longitudinal rows over the entire surface of the body.According to this interpretation, the rows of cilia in the cirri areregarded as being derived from the longitudinal rows of theonce evenly distributed cilia. Griffin (1910 a) regarded eachcirrus of E u p l o t e s w o r c e s t e r i as having been derivedfrom several rows of cilia of a simpler form. The oblique direc-tion of the rows in the frontal and ventral cirri is indicative ofa fundamental spiral organization, which is also expressed inthe spiral arrangement of the adoral zone of membranelles.

Following this idea farther, the radiating fibrils are consideredas derivatives of the interciliary fibrils of the cilia of the simpleancestor. In each cirrus those fibrils in the groups parallel with


the primary rows of basal granules are derived from fibrilsconnecting the cilia of the same rows, while the fibrils parallelwith the secondary rows of basal granules are derived from thetransverse fibrils. Since these fibrils are not so numerous as arethe rows of basal granules of the cirrus, either loss or fusion offibrils may have occurred, resulting in a reduction in number. Inseveral cases groups of two to three fibrils are fused into a singlefibril at their distal ends, indicating how such a reduction innumber may have occurred.

Although this interpretation is entirely speculative it suppliesan explanation of the arrangement of the basal granules inuniform rows and the constant relationship of the fibrils tothese rows.

C y t o p h a r y n x .

The cytopharynx of E u p l o t e s p a t e l l a has not beenadequately described. Yocom (1918) noted that the adoral zoneof membranelles extends along the left wall of the cytopharynx.He also noted that the right wall of the cytopharynx is ciliated,but did not describe the arrangement of the cilia.

In the present study the cytopharynx was found to correspondin structure with the description given for E u p l o t e s wor-c e s t e r i by Griffin (1910 a). The cytopharynx of E u p l o t e sp a t e l l a is much shorter and less curved than is the case inE u p l o t e s w o r c e s t e r i , but the ciliary apparatus is similar.This consists of the adoral zone of membranelles on the left-posterior wall, several oblique rows of endoral cilia on the dorsalwall, and suboral membranelles on the right wall of the cyto-pharynx (Text-fig. 1). An undulating membrane or ridge ofprotoplasm on the right wall of the cytopharynx was seen inliving specimens stained with acid fuchsin. This structure isevidently located along one side of the suboral membranelles.The suboral membranelles are connected by a longitudinal fibrilwith the membranelle fibril. Another fibril extends into theendoplasm from the end of the membranelle fibril. There is aslight enlargement where these three fibrils are joined.

The endoplasm in a circular area immediately adjacent to thecytopharynx is sharply demarcated from the surrounding cyto-


plasm. This area appears to be delimited by a circular fibrilattached at one end to the cytopharynx. In sections this is seento be a continuous membrane lying close to the ectoplasm ofthe ventral surface. This membrane has an incomplete dorsalboundary, and thus is in the shape of a partially enclosed sac.

In the feeding experiments the food particles were seen tofollow along the deep groove at the right side of the peristomialfield. Fairly large particles up to 15 microns were ingested. Assoon as a large particle entered the endoplasm, streaming move-ments carried it away from the cytopharynx along a definitecourse. This course followed the circumference of the mem-branous sac in an anti-clockwise direction. The particles wereseen to make from one-fourth to one or more revolutions alongthis course. Plasmosis occurred only at the time of ingestionof particles and only in the area within the membrane. Nodefinite food vacuoles were seen.

The food masses are received into the membrane as they arefirst ingested. Processes of digestion are evidently started inthis partially enclosed sac. The large dorsal opening allows thefood particles to leave the sac and circulate freely in the endo-plasm during the later stages of digestion.

P e r i p h e r a l P o l y g o n a l S y s t e m .

The arrangement of the polygonal system agrees in generalwith the description given by Turner (1933). The polygonallines, however, do not come into direct contact with the basalgranules of the bristles, as will be described later, nor is thereany uniform connexion with the basal plates of the cirri. Themembranelles are, however, directly connected with the poly-gonal lines at the ends of the membranelle plates (Text-fig. 1).The median membranelle fibril described by Turner (1938) iscoincident with the central row of rodlets of the membranellezone.

The polygonal lines in E u p l o t e s p a t e l l a are pellicularstructures rather than subpellicular as described by Turner(1933). The position of the lines in the pellicle was demonstratedin the modification of the wet silver method, in the silver-osmicacid-formalin method, and in preparations of material fixed in


Schaudinn's fluid and stained with the alcoholic haematoxylinmethod. The latter preparations show the polygonal lines onlywhen the organisms have been allowed to become partly dry inmounting on the slides after fixation. The clearness of the linesis proportional to the degree of drying which the organismsunderwent in mounting. In the organisms which became moredry the lines show up as definite ridges on the surface of theorganism. This supports the interpretation that the polygonalsystem is pellicular in position.

The diagram of Turner (fig. 2, p. 56, 1933) supports this viewif the structureless superficial layer labelled ectoplasm is inter-preted as the pellicle, and the deeper layer labelled endoplasmas the ectoplasm.

B a s a l A p p a r a t u s of t h e B r i s t l e s .

The so-called sensory bristles occur in longitudinal rows alongthe surface of the body. There are nine rows of these on thedorsal surface and one on the ventral surface, near the leftmargin of the body. The bases of these bristles are surroundedby elongate granules arranged in rosettes (fig. 7, PL 22). Theserows of bristles are coincident with the sides of ridges along thesurface, which may or may not be evident, and thus the numberof rows is the same as the number of ridges. The bristles are notrigid, nor are they vibratile, but they do show slight movements,which may be passive.

In the silver preparations these bristles are seen to be con-nected directly by longitudinal fibrils and indirectly by crossfibrils (Text-fig. 5), as described by Klein in E u p l o t e sh a r p a (1926) and by Turner in E u p l o t e s p a t e l l a (1933).Each bristle arises from a basal granule which forms part ofa complex basal apparatus. In E u p l o t e s p a t e l l a thisconsists of a depression in the ectoplasm in the form of an in-verted cone, of which the basal granule forms the apex (Text-fig. 7). Toward the surface the membrane lining the depressionends in a ring, which connects on either side with the longitu-dinal fibril of the polygonal network (Text-figs. 5 and 7). Sur-rounding the depression is a group of rod-like bodies arrangedin rosette fashion. There are usually six of these for each basal


TEXT-FIGS. 4-11.

All figures, except 7, 9, 10, and 11, x 700.Fig. 4.—Rudiment (Anlage) of right marginal cirrus for anterior

daughter developing in row of bristles in region of multiplication.Champy's fluid, Heidenhain's haematoxylin.

Fig. 5.—Portion of peripheral polygonal system of dorsal surface.Bach ring surrounds a depression in which the basal granule of abristle lies. Wet silver method.

Fig. 6.—Sketch showing the relation of the peripheral polygonalsystem to the membranelle zone. Wet silver method.

Fig. 7.—Diagrammatic representation of the structure of the basalapparatus of a bristle. The basal granule is at the bottom of adepression surrounded by rodlets, two of which are shown. Thering which encircles the depression is connected on either side withthe peripheral polygonal system.

Fig. 8.—Sketch of a portion of the ventral surface of an organism inan early stage of binary fission, showing the networks of the newperipheral polygonal system and their relation to the old polygonalsystem and rudiments of the cirri.

Figs. 9, 10, 11.—Diagrams illustrating successive stages in thefission of a basal granule of a bristle.—Bristle omitted.

NO. 316 M m


granule, although there may be more or less. These are tiltedat an angle toward the surface, so that the outer end of each ispointed toward the surface and toward the centre of the depres-sion. Thus the appearance of the group is similar to that of thepoles of a wigwam. The outer ends of the rodlets are locatedat the boundary between ectoplasm and pellicle, but each onemakes a tiny bulge on the surface. This accounts for an earlydescription of them (Stokes, 1885) as minute prominencesarranged in a stellate cluster. These rodlets can be demonstratedin the living organism with or without vital dyes and are welldifferentiated with Gelei's silver-osmic acid-formalin method.They are not mitochondria or Golgi material because they donot stain with methods for differentiation of these.

This agrees with the description of the basal apparatus ofthe bristle as given by Gelei (1933) for an undetermined speciesof E u p l o t e s , except that the depression is cone-shapedinstead of cylindrical and is not filled with protoplasmicmaterial. Also, the neuroplasm connecting the bases of thebristles in the same longitudinal row was not seen. Gelei (1929 a)has named the depression ' Sensucylinder' and the rodlets' Sensucysten', but since these terms imply a function which hasnot been demonstrated they will not be used here. Jacobsen(1931) saw that the bristle arises from a depression in thesurface of the body. She considered that the edge of the pelliclesurrounding this depression is coincident with the silverline ringdescribed by Klein. Turner (1933) described the bristles andthe rosettes surrounding the bases of these, but did not see thedepression or ring.

Basal structures without bristles occur on the ventral surfacein connexion with the membranelles and cirri (Text-fig. 2).Their occurrence on the ventral surface has been described byGelei (1929 a) for an undetermined species of E u p l o t e s , andGriffin (1910a) in E u p l o t e s w o r c e s t e r i saw the bristlesas well as the rosettes in these locations. Each basal apparatuspresent at the bases of the cirri and membranelles in E u p l o t e sp a t e l l a appears to consist only of the rodlets, ring, and thecentral depression. There are two to three groups of rodletsat the base of each cirrus. They always have a definite location


with regard to the cirrus at whose base they lie. Where there areonly two groups of rodlets they are placed on opposite sides ofthe cirrus. They are always placed near the corners of the basalplates of the cirri and along the side of the primary rows ofgranules. Thus, they have an antero-posterior relationship withregard to the frontal cirri one, two, four, five, and seven, anoblique position with regard to frontal cirri three and six, anda lateral position at the base of the ventral cirri. They areplaced near the anterior and posterior ends of the anal cirri.The groups of rodlets at the bases of the marginal cirri areparallel with the margin of the body.

There are three rows of groups of rodlets along the adoralzone of membranelles. This results from the location of onegroup near the centre and one near either end of each mem-branelle plate. For those membranelle plates occurring on theventral surface these groups occur just posterior to each plate.These continue anteriorly along the frontal portion of the zone(figs. 8 and 9, PI. 22), and posteriorly into the pharyngeal por-tion of the zone. There is also a group of rodlets at the anteriorend of the suboral membranelles.

These rosettes have been seen by many investigators. Eees(1881) observed rows of very small vesicle-like structures alongthe dorsal ridges of E u p l o t e s l o n g i p e s . He suggestedthese might be of the nature of contractile vacuoles. Stokes(1885) saw the rodlets in E u p l o t e s c a r i n a t u s andE u p l o t e s p l u m i p e s and described them as variable minutedots or oblong elevations marking the surface of the body.Minkiewicz (1901) considered the rosettes in E u p l o t e sv a n n u s to be crystalline in nature. Griffin (1910a) appliedmicrochemical tests to the structures in E u p l o t e s wor-c e s t e r i and on the basis of these determined that they wereof the nature of reserve fatty food substances. Gelei (1929 a,1934&) confirmed the fatty nature of the bodies but supposedthat they are used in amplifying the sensory stimuli receivedby the bristles. Jacobsen (1931) follows this interpretation ofthe significance of the rosettes. Gelei further suggests that thegroups of rodlets at the bases of the cirri receive their stimulifrom the cirri and are thus rheoceptive sensory organs. This


view is not supported by the position of the rodlets with relationto the cirri, because the rodlets are not in the direction of strokein the case of the ventral cirri, frontal cirri three and six, and someof the marginal cirri.

The function of the rosettes is obscure. That they amplifythe stimuli which might be received by the bristles is improbablebecause they do not come into direct contact with the basalgranule or bristle. They are apparently connected in some waywith the function of the bristle.

The function of the bristles is also obscure. It has generallybeen assumed that they possess a sensory function (Griffin,1910 o; Gelei, 1929 a; Turner, 1933), since they are not used forpropulsion. Until experimental evidence is obtained the viewthat the bristles are sensory in nature seems most plausible.

The bristles are generally considered to have arisen by atransformation of functional cilia. This view has been suggestedby Johnson (1893), Wallengren (1901), and Klein (1932). Gelei(1933) has advanced a different interpretation. He supposesthat the bristles are not derived from cilia, but are outgrowthsfrom the end of a sensory neuroid structure, similar to theNebenkorn in P a r a m e c i u m. This view cannot be supportedby a consideration of the structure in E u p l o t e s p a t e l l a ,since no fibrillar continuation of the basal granule of the bristlehas been demonstrated. Furthermore, the structure of the mem-branelles, cirri, and bristles is comparable as regards thepossession of basal granules and basal groups of rodlets. Thecirri and bristles are associated in ontogeny, as will be broughtout later. Therefore, the weight of evidence is in favour of thederivation of the bristles by a modification of cilia.

The body of E u p l o t e s p a t e l l a is thus seen to be relativelycomplicated. There are several kinds of ciliary organelles whichare co-ordinated in their action by the fibrillar system. Thebehaviour of these differentiations during binary fission willnext be considered.

ASEXUAL BEPKODUCTION.

It has long been known that the common method of repro-duction in ciliates is binary fission or asexual reproduction.


Stein (1859) observed one stage in fission of E u p l o t e sp a t e l l a and several stages in E u p l o t e s c h a r on. He wasthe first investigator to note the interesting phenomenon ofcomplete resorption of the entire set of old cirri and its replace-ment by two sets of new cirri, during binary fission. Mobius(1887) described the process of binary fission in E u p l o t e sh a r p a. He supposed that the set of old cirri of the parent ishalved, and only enough new cirri are formed to make up thedeficiency in the two daughters. Schuberg (1899) investigatedbinary fission in E u p l o t e s p a t e l l a , but observed only a fewstages and consequently was not able to give a completedescription of the process. Wallengren (1901) made a thoroughstudy of binary fission in E u p l o t e s h a r p a , and Griffin(1910) gave a detailed account of the process in E u p l o t e sw o r c e s t e r i . Yocom (1918) briefly described binary fission inE u p l o t e s p a t e l l a , and Turner (1930) made a detailed studyof the nuclear phenomena in asexual and sexual reproductionin this species.

It has been shown by Turner (1930) that the first evidenceof approaching fission is the appearance of bands composed oflightly and darkly staining areas on the ends of the macro-nucleus. These bands gradually move along the macronucleusuntil they meet at the centre. This process Avas interpreted byTurner (1930) as a reorganization of the material in the macro-nucleus, resulting in a rejuvenation of the organism. In laterstages the micronucleus undergoes a mitotic division and thecondensed macronucleus is split into two parts. Each daughterreceives one macronucleus and one micronucleus.

The dedifferentiation and redifferentiation of the nuclei isfollowed by similar processes in the cytoplasm, as will be shownlater. Before the parent organism divides into the two daughtersthere are extensive changes in the cytoplasmic structure, in-volving the appearance of a new peristome for the posteriordaughter; the differentiation of two complete sets of new cirri,one for each daughter, and the dedifferentiation of the old set;the development of a number of new bristles in each row; andthe reorganization of the polygonal system over almost theentire surface of the body.


P e r i s t o m e .

The first cytoplasmic change to occur during binary fissionis the appearance of the rudiment of a new peristome as a depres-sion in the ectoplasm immediately posterior and to the left ofthe old peristome (fig. 11, PL 22). This becomes visible soonafter the reorganization of the macronucleus begins. A patchof cilia appears in this depression, and these cilia becomearranged in rows, which are the beginning of the membranelles.The depression deepens into a pocket, which extends firstposteriorly, then anteriorly, and the membranelles develop alongthe floor and side of this pocket. The development of the peri-stome has been described by Griffin (1910b) and, since thedevelopment in E u p l o t e s p a t e l l a agrees with this descrip-tion, it will not be described in detail here. As soon as thestructure of the individual membranelles can be made out itis seen that the organization of these is similar to that in thefully developed organism. The third row is complete in theanterior one-third of the zone, and incomplete in the remainingportion.

There is apparently no change in structure in the old peristomeduring binary fission. This agrees with Yocom's (1918) andTurner's (1930) observations. There is likewise no visiblereorganization in the old peristome during binary fission inE u p l o t e s w o r c e s t e r i (Griffin, 1910b), or in S t e n t o r(Hetherington, 1932; Schwartz, 1935).

On the other hand, Wallengren (1901) noted a gradual re-placement of the old adoral zone in E u p l o t e s h a r p a , anda similar process has recently been observed during binaryfission in U r o n y c h i a (Taylor, 1928) and D i o p h r y s(Summers, 1935). A reorganization of the adoral zone duringbinary fission has also been reported in several heterotrichousciliates including B l e p h a r i s m a u n d u l a n s (Calkins, 1912),and B u r s a r i a t r u n c a t e l l a (Schmahl, 1926).

It is possible that a gradual reorganization of the adoral zonemight occur before or after binary fission in the forms in whichthe old peristome is retained without visible change of structureby the anterior daughter.


D e v e l o p m e n t of C i r r i .

Although the development of the cirri has been thoroughlydescribed for E u p l o t e s h a r p a by Wallengren (1901) andfor E u p l o t e s w o r c e s t e r i by Griffin (19106), there has beenno adequate study of this process in E u p l o t e s p a t e l l a .Maupas (1889) gave a brief description of the origin of the newcirri during conjugation, while Schuberg (1899) and Yocom(1918) have given general accounts of this process during binaryfission.

The first indication of the new cirri can be made out soonafter the appearance of the rudiment (Anlage) of the new peri-stome as a series of ten slit-like depressions along the middle ofthe ventral surface. These depressions are clearer in appearancethan the surrounding ectoplasm and they result from a resorp-tion of the pellicle and a change in the appearance of theectoplasm at these points.

The depressions are arranged in two approximately transverserows which consist of five depressions each. One row, whichis directly anterior to the other five, gives rise to cirri whichbecome a part of the anterior daughter, while the posterior rowgives rise to cirri which become a part of the posterior daughter(fig. 11, PI. 22). The depressions do not appear simultaneously,as in E u p l o t e s w o r c e s t e r i , but those on the left appearfirst, as in E u p l o t e s h a r p a .

These depressions, which are at first narrow slits, becomebroader and longer. Beginnings of cirri appear along the floorof each. In each of the three depressions to the left three cirriarise, and in each of the remaining two depressions two rudi-ments (Anlagen) appear, making a total of thirteen rudimentsof cirri for each daughter (fig. 4, PL 22; Text-fig. 8). Accordingto Yocom (1918) and Turner (1930) only the last depression tothe right has two rudiments and all the rest have three rudiments,making a total of fourteen cirri arising out of the five depres-sions, as is the case in E u p l o t e s h a r p a (Wallengren, 1901)and E u p l o t e s w o r c e s t e r i (Griffin, 19106). This accountis incorrect for there are only nine anteroventral cirri inE u p l o t e s p a t e l l a as compared with ten in the same group


in E u p l o t e s h a r p a and E u p l o t e s w o r c e s t e r i , andthis difference develops from the absence of the third rudimentin the second depression from the right in E u p l o t e s p a t e l l a .

The posterior rudiment in each depression becomes an analcirrus. The anterior rudiments in the two depressions to theright develop into ventral cirri, and all the remaining rudi-ments become frontal cirri. Of these the two anterior rudimentsin the middle develop into frontal cirri three and six; the twoanterior rudiments in the second depression from the left giverise to frontal cirri two and five; and the two anterior rudimentsin the first depression to the left develop into frontal cirri oneand seven. As development occurs the cirri migrate to theirdefinitive positions, and at about the time these definitive loca-tions are reached the old cirri are resorbed. Since the migrationof the cirri agrees with that described for E u p l o t e s h a r p aby Wallengren (1901) the details of this process will not beconsidered here.

Soon after the two rows of depressions become visible arudiment appears on the median wall of the pocket in which thenew peristome is developing. A corresponding rudiment of acirrus arises on the median wall of the old peristomial field inthe region of frontal cirrus seven. These rudiments developinto frontal cirrus four for the anterior and posterior daughterrespectively. This cirrus arises in the peristomial field ratherthan on the frontal field as in E u p l o t e s w o r c e s t e r i(Griffin, 19106). Wallengren (1901) describes this cirrus asarising in E u p l o t e s h a r p a 'auf dem protoplasmatischenTheil der Unterlippe des alten Peristome'.

The left marginal cirri arise in two depressions to the left ofthe peristome (fig. 5, PI. 21). These were seen by Schuberg(1899), but he was unable to determine how many rudiments ofcirri were present in each depression, and he did not follow theirdevelopment. Wallengren (1901) found in E u p l o t e s h a r p atwo rudiments in each depression and saw that the posterior pairgive rise to the left marginal cirri of the posterior daughter,while the anterior pair form the left marginal cirri of the anteriordaughter. The development of the left marginal cirri inE u p l o t e s p a t e l l a agrees with this description. The rudi-


ments of these cirri appear slightly later than the groups ofdepressions already described.

The origin of the right marginal cirri has not been describedfor E u p l o t e s p a t e l l a , and only incompletely for Eu-p l o t e s w o r c e s t e r i and E u p l o t e s h a r p a . In both ofthe latter species the right marginal cirri were described asoriginating upon or near the margins of the daughters' bodiesnear their definitive location at the time of constriction of theparent into the two daughters.

By using material fixed in Champy's or Plemming's fluids thewriter was able to trace the origin of these cirri to a muchearlier stage in binary fission. In organisms in which the rudi-ments of the cirri have just appeared and before there is anysign of the constriction between the two daughters it is possibleto identify the rudiments of the right marginal cirri. These havea remarkable origin, in that they arise on the dorsal surfacealong the rows of bristles, and only at about the time of separa-tion of the daughters do they assume their definitive ventralpositions. The rudiments of the right marginal cirri for theanterior daughter arise near the centre of each of the last tworows of bristles to the right (fig. 3, PI. 21), and those for theposterior daughter appear in the same two rows near theposterior end.

The first indications of these rudiments appear as minutedeeply staining areas immediately posterior or anterior to abasal granule of a bristle (Text-fig. 4). These increase in size,and, during constriction, the plane of division passes immedi-ately posterior to the right marginal cirri for the anteriordaughter. By this process these cirri reach their definitivepositions on the ventral surface. The right marginal cirri forthe posterior daughter reach their ventral positions by a migra-tion around the posterior margin of the body. This movementis closely associated with the replacement of the polygonalsystem as will be shown later.

The dorsal location of the rudiments of the right marginalcirri may account for their being overlooked by the earlierinvestigators. The differentiation of these rudiments along therow of bristles indicates a possible kinship between the cirri


and bristles. This will be further discussed in connexion withthe multiplication of the bristles.

K e s o r p t i o n of Ci r r i .

The dedifferentiation of the cirri was studied in organisms inthe living condition. Since the marginal cirri project beyond themargin of the body, and thus have the greatest visibility, theywere found most suitable for this study. In the cases observedthe two laterally placed marginal cirri on either side were thefirst of these to be resorbed. This occurred about a half-hourbefore separation of the two daughters. The median marginalcirri were resorbed later. Usually the left one of these dis-appeared at about the time of separation of the daughters, whilethe right one persisted five to thirty minutes after separation.Thus, the posterior daughter can be recognized for some timeafter fission by the presence of a supernumerary marginal cirrus.

The resorption of a cirrus is preceded by sudden cessation ofmovement of this cirrus. Immediately after it stops beatinga decrease in size occurs. The fimbriation which Taylor (1928)noted in the dedifferentiation of cirri in U r o n y c h i a , andWallengren (1901) observed in E u p l o t e s h a r p a , was notseen. The cirrus decreases in diameter as well as in length.This process occurs rapidly and is completed with the dis-appearance of the cirrus within two minutes of the time itbecomes motionless. The cirrus is drawn in the direction of thefibrils at its base as dedifferentiation occurs. Thus, it is pulledout of its former position, which the corresponding new cirrusthen occupies.

D i f f e r e n t i a t i o n of F i b r i l s .

The fibrils of the anal cirri are first visible as heavier linesdirectly beneath the basal plates and parallel with the rows ofbasal granules of the cirri to which they are attached. Theyappear first beneath the second and third cirri from the left side,followed by the first, then the fourth, and finally the fifth. Thefibrils begin to develop at about the same time in the anteriorand posterior daughters.

In a later stage in binary fission, the fibrils are extended


anteriorly from the basal plates and are seen to run superficiallyto the old fibrils. They are thus ectoplasmic in origin. Thisextension continues until the fibrils from the anterior set of cirrireach the anterior end of the organism. At this time the oldset of fibrils is resorbed and the membranelle fibril becomesconnected with the new set.

The fibrils from the posterior set of anal cirri are compressedinto a single fibril at the time of constriction. At this time theconnexion with the membranelle fibril is effected, completingthe integration of the neuromotor apparatus of the posteriordaughter. The origin of the motorium was not determined.

The fibrils of the frontal and ventral cirri also develop fromthe basal plates of the cirri. When they first appear, these fibrilsare more closely in line with the rows of basal granules of thecirri to which they are attached than in the fully developedorganism.

M u l t i p l i c a t i o n of B r i s t l e s .

Until recent times there has been no detailed knowledge con-cerning the manner in which the bristles are increased in numberduring binary fission. Sterki (1878) suggested that at least a partof the bristles are formed anew during binary fission, since thedaughters have as many bristles as the parent. Wallengren(1901) studied the origin of the dorsal bristles in U r o n y c h i a .He found that, in a stage in which the new ventral and marginalcirri have begun to develop, clear lines appear on the dorsalsurface to the left of the longitudinal furrows in which the oldbristles are located. Soon after, small, closely set, cilia-like,actively moving structures appear in this line. He supposedthe old bristles to be resorbed, but did not follow this process.

Gelei (1929 a, 1933,1984 b) has conducted intensive investiga-tions on the subject of the origin, structure, and function of thebristles. He studied the multiplication of these in an undeter-mined species of E u p l o t e s , and found that only the bristlesin the central part of each row take part in multiplication, andthat from the basal structure of each of these several newbristles arise.

Klein (1932) supposed that the new bristles are derived from


the polygonal system during binary fission. Gelei (1934&) hasshown that this is not ordinarily the case in the species ofE u p 1 o t e s with which he was working.

The two methods used by Gelei, and in addition materialfixed in Champy's and in Flemming's fluids and stained inHeidenhain's haematoxylin, were used by the writer in studyingthe multiplication of the bristles. The results obtained agree ingeneral with those of Gelei.

In E u p l o t e s p a t e l l a only the central bristles in each roware involved in the process of multiplication. This begins soonafter the rudiments of the left marginal cirri have appeared onthe ventral surface. The basal granules of about the threecentral bristles in each row divide, so that two granules insteadof one can be plainly seen at the bases of each of these bristles(Text-fig. 9). The adjacent basal granules anteriorly and pos-teriorly are then involved until five to eight basal granules ineach row are undergoing fission (figs. 1 and 2, PI. 21). Thisfission continues until up to as many as six new basal granulesare produced from each old basal granule involved (Text-figs.10 and 11). Those basal granules on the anterior and posteriormargins of the area of multiplication give rise to fewer newgranules, sometimes as few as one, while those in the centre ofthis area may give rise to as many as six new ones. Thus thereis a gradient of regeneration, with the highest point at the centreof the organism, and decreasing both anteriorly and pos-teriorly.

In the process of fission of a basal granule there are tubularoutgrowths extending anteriorly and posteriorly from the basalapparatus of each bristle involved (Text-figs. 10 and 11). Thesedevelop soon after the first fission of the basal granule has takenplace. The daughter granules move out within this tubularmembranous process and, since the interior of these outgrowthsappears relatively clear with the stains used, the new granulescan be followed through all the stages of their multiplication.The new granules give rise to the new bristles and it is probablethat the accessory parts of the basal apparatus such as the coneare derived from the membranous process (fig. 3, PI. 21). Theorigin of the rodlets was not determined. They were seen to be


present around the bases of the new basal granules at aboutthe time that these gave rise to the new bristles.

Gelei (1934 b) did not see the division of the basal granulesand supposed that the new granules arise by differentiation fromthe membranous outgrowths. By the methods he used theseprocesses appeared solid, and thus masked the origin of the newbasal granules.

In the form studied by Gelei an average of seven basalapparatuses in each row participated in multiplication, and fourto five new basal granules arose from each of these. In Eu-p l o t e s p a t e l l a fewer basal structures take part, but eachone produces more new basal granules.

There is no dedifferentiation of bristles during binary fission.This conclusion is supported by the observation of differentstages of the process from the earliest indication of multiplica-tion until the new bristles are fully formed. The old bristleswere seen to be present throughout this process until theycould no longer be distinguished from the new bristles inter-polated between them. Also, the bristles outside the zone ofmultiplication showed no evidence of resorption or replace-ment.

Another line of evidence which supports the assumption thatthe bristles are not resorbed is supplied by a numerical studyof the new bristles as compared to those in the completedorganism. There are about as many new bristles produced ineach row as were present in the row before fission. Thus, if thenumber is to be kept uniform, resorption must not occur. Gelei(1934 b) described dedifferentiation of the bristles in the regionof multiplication in the species of E u p l o t e s with which heworked, but the writer found no evidence of this process.

The bristles anterior and posterior to the region of multiplica-tion are received without any change in structure by the anteriorand posterior daughters respectively. There is a redistributionof the new bristles along the rows so that a more or less uniformspacing results.

Indications of division of individual bristles independently ofthe remainder have been noted. In several organisms at a stagein which the rudiment of a new peristome was just detectable,


and long before the beginning of multiplication of bristles asdescribed above, two bristles so closely placed as to be almostcontiguous were seen.

This might be interpreted as a process of reorganization and,if so, would indicate how the reorganization of the bristlesoccurs. At any rate, a reorganization of the bristles might takeplace during stages which have not been observed.

The origin of the right marginal cirri along the rows of bristlesis worthy of further notice here. The anterior set of rudimentsof the cirri occur, as previously mentioned, along the last tworows of bristles to the right and near the centre of the region ofmultiplication. In the early stages of the multiplication of thebristles no indication of the rudiments of the cirri can be seen. Thebasal granules of the bristles in the rows in which rudimentswill appear undergo fission, as do the basal granules in the otherrows. The first indication of a rudiment is a more deeplystaining area which is usually immediately posterior to an oldbasal granule, and just anterior to the centre of the region ofmultiplication (Text-fig. 4). This darkened area has at first thesame dimensions as the membranous outgrowth from the basalapparatus and is possibly derived from it, or from the productsof division of the basal granule. Later, the area becomes greaterin diameter, and sinks beneath the level of the dividing basalgranules. All the new basal granules which occupied the areabetween the two old basal granules wherein the rudiment arosedisappear.

The rudiments of the right marginal cirri for the posteriordaughter arise slightly earlier than those for the anteriordaughter. They appear just posterior to the last bristles in thesame two rows wherein the rudiments for the anterior daughterdevelop. As this location is outside the region of multiplica-tion the appearance of the rudiments is somewhat different thanis the case for the more anterior rudiments. However, here also,the first indication of the rudiment is a deeply staining splinter-like area immediately posterior to the bristle, and in a direct linewith the row of bristles. The development is similar to thatalready described for the rudiments of the anterior marginalcirri. It is possible that the rudiment arises as an outgrowth


from the basal granules of the last bristle in the row involved,but this could not be determined.

The behaviour of the bristles during asexual reproduction isvery different from that of the cirri. While two sets of cirri areformed entirely anew and the old set of cirri is dedifferentiated,the new bristles are formed from multiplication of the previousstructures, and there is apparently no dedifferentiation of thebristles. This difference may be associated with the fact thatthe bristles are not vibratile and thus would not become wornout or injured so readily as the actively beating cirri. However,the bristles may also be replaced during stages of the life-cyclewhich have not come under observation during this study.

P e r i p h e r a l P o l y g o n a l S y s t e m .

Klein (1928) studied a stage in binary fission of an undeter-mined species of E u p l o t e s with especial reference to theso-called silverline or polygonal system, but confined his obser-vations to the development of the peristome. According to Klein,the polygonal lines have the power to reproduce themselves, togrow, and to form new locomotor organelles. Gelei (19846) hasdescribed in detail the behaviour of the polygonal system onthe dorsal surface of an undetermined species of E u p l o t e sduring binary fission. According to this description a newsystem of polygons arises in connexion with the multiplicationof the bristles. A row of meshes appears on either side of eachrow of developing bristles and spreads out laterally until themeshes of adjacent rows meet, entirely replacing the old poly-gonal system in the middle region of the body. These newmeshes grow out from the already differentiated basal apparatusesor from the longitudinal connexions between these. This isdirectly opposite to Klein's view of the origin of these structures.

The results of Gelei on the origin of the polygonal system onthe dorsal surface were in general confirmed by the presentstudy. Klein's view that the polygonal system produces thebasal granules and thus indirectly the bristles cannot be sup-ported because the basal granules arise by fission from previousbasal granules. The new polygonal system arises after fissionof the granules is completed. The exact sources and points of


origin of this system have not been determined. The new meshesarise along the rows of developing bristles and spread laterallyfrom these, as Gelei (1934 b) has described.

In E u p l o t e s p a t e l l a replacement of the polygonalsystem occurs over the entire dorsal surface, and is not limitedto the area of multiplication of the bristles, as indicated byGelei. The process goes on differently outside this region. Inthe extreme anterior and posterior parts of the body, eachbristle acts as a centre for development of a unit of the newsystem. The development in these areas proceeds later thanthat in the middle of the body, and is not noticeable until afterseparation of the daughters. In such a stage each bristle towardthe posterior end of a posterior daughter is surrounded by acircle with a longitudinal partition, in the centre of which is thebristle. This circle widens during development until it comesin contact with those anterior and posterior to it. A similarreplacement occurs in the anterior end of the anterior daughter.When this process is completed, an entirely new dorsal poly-gonal system results.

The networks are completely formed in miniature at the timewhen they can first be distinguished, and development con-sists merely in enlargement of the meshes and integration of theunits. The old system is resorbed as the new networks spreadout. The integration of the meshes of adjacent rows of granulesinvolves a fusion of the lateral walls of the approaching meshes,since only a single line can be seen at these points in the com-pleted system. Since the new polygonal system originates in somany independent loci a delicate co-ordination, both as to timeand space, is involved during its development.

Other than Klein's description of a stage in the developmentof the new peristome, no work has been done on the behaviourof the polygonal system on the ventral surface of E u p l o t e sduring binary fission. This process is of considerable interest,in that it throws light on the differentiation of the cirri.

The first indication of a new polygonal system on the ventralsurface appears as a fine network on the floor of the depressionin the ectoplasm which will develop into the new peristome.According to Klein (1928) the appearance of the rudiment of


the new peristome is the first indication of approaching binaryfission. Turner (1930) pointed out that the first of the changeswhich constitute binary fission is the appearance of the re-organization bands in the macronucleus. This latter viewpointis upheld in the writer's study of many dividing organisms, inwhich the reorganization bands invariably appeared before therudiment of the peristome.

The rudiment of the zone of membranelles appears along theside of the depression. It is visible, however, as early as the finenetwork, and thus there is no reason to follow the belief of Kleinthat the membranelles arise from the network.

The network covering the walls and floor of the depressionspreads out over the ventral surface surrounding the openingof the depression. In this way the frontal cirrus four, whicharises within the depression, is carried to its definitive positionon the frontal field.

Other parts of the new polygonal system on the ventral sur-face appear as networks of fine lines in each of the depressionswherein the rudiments of the cirri arise (figs. 5 and 6, PI. 21;Text-fig. 8). Including the depressions in which the left marginalcirri arise, there are twelve separate areas covered by these finemeshes. These become visible at about the time when the rudi-ments of the cirri can first be made out. Delicate networks canbe seen covering the entire surface of the depressions except theareas occupied by the rudiments of the cirri.

As the rudiments of the cirri develop, the originally smallareas covered by the networks of fine lines increase in extent,especially in a longitudinal direction. The networks are appa-rently completely formed, except for size, when they firstbecome visible, and development consists only in enlargementof the meshes as the areas which they cover expand. As thisspreading occurs, there is a dedifferentiation of the old poly-gonal system at the edges of the areas, so that the surfaceoccupied by the new system increases at the expense of thatoccupied by the old. This process continues until there is ameeting of adjacent new networks, including the one arisingin the depression in which the new peristome develops, whichthen form an integrated new system covering the entire ventral

NO. 316 N n


surface except the old peristomial field, and connecting with thenew system of the dorsal surface.

This throws new light on the process of ^differentiation. Thedepressions in which the rudiments of the cirri arise are formedby a dedifferentiation of the pellicle and ectoplasm at thesepoints. Differentiation then occurs over this area, formingrudiments of cirri. The appearance of the fine networks in thedepressions around the rudiments indicates that a new pellicle,and new ectoplasm or superficial layer of ectoplasm, are alsodifferentiated at these loci.

As these redifferentiated areas gradually expand, dediffer-entiation of the old ectoplasm and pellicle with its polygonalsystem occurs at the edges of the areas. That expansion of theredifferentiated areas takes place by intussusception of newmaterial over the whole surface of the differentiated area, ratherthan by differentiation at the margin, is shown by the fact thatthe polygons are completely formed in miniature over the areabefore expansion occurs. So far as could be determined, no newmeshes were added at the edges. The growth in size of meshescan be explained only by assuming that the area is stretched orthat intussusception of new material occurs, increasing the sur-face without alteration of the pattern.

Since the groups of cirri arise in the centre of the expandingareas, each cirrus is carried to its definitive position by thespreading of those areas. A very precise co-ordination is requiredboth as to time and space in the development of the independentrudiments of the polygonal system into an integrated uniformpattern. The degree of perfection of this co-ordination is shownby the lack of any indication of multiple origin in the completedsystem. There are some indications, however, that the polygonallines are labile structures, thus allowing for adjustment as theseparate meshworks come together.

If, as was indicated above, the polygonal system is located inthe pellicle, the origin and development of this system showsthat the pellicle is not a lifeless secretion, but undergoes theprocesses of dedifferentiation and redifferentiation character-istic of such active structures as the cirri. Also, the develop-ment by a process of spreading out, while retaining the


pattern of structure, would not be possible with a secretedcovering.

It is evident that during asexual reproduction there must bea doubling of organelles if each daughter is to have a completeset. This doubling occurs differently in the different kinds ofcytoplasmic structures. By a series of fissions a limited numberof basal granules of bristles produce enough new bristles so thateach daughter will be provided with a complete set. One newperistome is formed independently of the old peristome, whichis retained by the anterior daughter. In both of these casesno apparent dedifferentiation of the structures of the parentorganism occurs. Two new complete sets of cirri and fibrillarsystems are formed independently of the old set, which under-goes resorption. The peripheral polygonal system is likewiseformed independently of the old system, which is replaced overthe entire surface pf the body with the exception of the oldperistomial field. The development of the polygonal system inconnexion with the parts of the neuromotor system indicatesa close relationship between these two systems.

SEXUAL EEPEODUCTION.

The occurrence of reorganization of the cirri and membranelleswas briefly described in E u p l o t e s c h a r o n by Engelmann(1862). He also noted that the exconjugants possess no cyto-stome until two to three days after separation. Maupas (1889)found a similar behaviour during conjugation in E u p l o t e sp a t e l l a . According to his description the exconjugants, inaddition to having no cytostome and posterior part of theadoral zone of membranelles, lacked one frontal cirrus of havinga complete set. This missing cirrus was found to arise at thesame time as the cytostome and remaining part of the mem-branelle zone, about eighty-five hours after separation of theconjugants at twenty-two degrees. The cytostome and itsciliary apparatus were seen to arise in a region separate from themembranelles already present.

Turner (1930) confirmed Maupas's description of the re-organization of the cirri and membranelles during conjugationin E u p l o t e s p a t e l l a , but did not notice the incomplete


condition of the set of cirri of the exconjugant. Also, he sup-posed that the cytostome and missing membranelles aregradually formed without having a separate origin as describedbyMaupas(1889).

Since the nuclear phenomena of conjugation were thoroughlyinvestigated by Turner, that aspect of conjugation was notconsidered in this study. In order to relate the cytoplasmicprocesses to the nuclear behaviour, a brief summary of this willbe given as worked out by Turner (1930).

The micronucleus undergoes a preliminary division and twomaturation divisions, forming eight nuclei, of which two undergoa post-meiotic division. Of the four products of this divisiontwo become pronuclei and two degenerate. Eeduction of theeight chromosomes occurs in the second maturation division,and the migrating haploid pronuclei, with four chromosomeseach, are mutually exchanged. The zygote nucleus dividestwice and, of the four products of these divisions, one becomesthe micronucleus, one develops into the macronucleus, and twodegenerate. The macronuclear rudiment joins with a reorganizedportion of the old macronucleus to form the new macronucleus.In the present study it was found that in addition to thereorganization during conjugation described by Maupas (1889)and Turner (1930) there is another reorganization which takesplace after the conjugants have separated. Thus there are tworeorganizations of the neuromotor system during sexual repro-duction. The first, or that of the gamete, begins while theconjugants are together, but before the pronuclei are exchanged;the second, or that of the zygote, occurs in the exconjugantsduring the development of the nuclei. Each of these reorganiza-tions is similar to that which occurs during binary fission, exceptthat only one neuromotor system is redifferentiated instead oftwo and that the peristome is replaced. This renewal of theperistome occurs partly in the reorganization of the gamete andpartly in the reorganization of the zygote, so that in this respectthe two reorganizations are complementary.

K e o r g a n i z a t i o n of t h e Gamete or C o n j u g a n t .Two organisms come together in conjugation in such a way


that the ventral surfaces of each are contiguous. A fusion ofthe two bodies occurs in a small triangular area in the leftanterior region of the peristomial field of each conjugant (fig. 13,PI. 22). At the edges of the area the peripheral polygonal linesof the two organisms become joined, making the polygonalsystems of the two conjugants continuous with one another inthis area. Klein (1929) observed a similar coalescence of theso-called silver-lines in an undetermined species of E u p l o t e s .This behaviour further supports the conception of the pellicleas a living substance.

Soon after the conjugants come together the posterior partof the peristome is resorbed, leaving intact only the anteriorportion. This resorption is peculiar in that the cytostome withits membranelles is first drawn into the endoplasm, where themembranelles may remain visible for as long as two days(fig. 12, PL 22). This phenomenon was noted by Maupas (1889).It is probable that the fusion of the bodies of the two conjugantsin the regions of the membranelles prevents their resorptionin the manner which is usual for the cirri.

The rudiment of the anterior portion of the new peristomearises as a depression in the ventral surface. Lining the floorof this depression is the rudiment of the anterior portion of thezone of membranelles. The development of this rudiment agreesin general with the description of Turner (1930), except that therudiment gives rise to only the anterior portion of the new peri-stome instead of the whole organelle as was stated by him.After separation of the conjugants the rudiment increases insize, moves anteriorly, and assumes a more transverse position.The replacement of the remnant of the old anterior portion ofthe peristome by the new one occurs about two hours afterseparation of the conjugants. The old membranelles are re-sorbed by dedifferentiation of the anterior lip region, whichstarts on the sides and progresses towards the middle. Themembranelles beat irregularly until the zone has disappeared.After replacement is completed the posterior portion of theperistome, including the cytostome, is still lacking.

The structure of the new portion of the zone of membranellesis similar to that of the fully developed organism, with the


membranelles across the anterior end each composed of threerows of cilia, and those on the ventral surface having each twocomplete and one incomplete rows.

The new cirri become visible during the second maturationdivision. Their appearance is usually, but not always, precededby the development of reorganization bands in the macro-nucleus. The new cirri arise in exactly the same manner as inbinary fission, except that only one set develops instead of two,and the frontal cirrus four, which develops in the peristomialfield during binary fission, does not appear. This is associatedwith the fact that the portion of the peristome in which thecirrus ordinarily arises has not been redifferentiated at thisstage. As a result of this cirrus not developing the exconjuganthas only seventeen cirri instead of eighteen after the old set ofcirri has been resorbed. This agrees with the observation ofMaupas (1889). The incomplete condition of the cirri was notnoticed by Turner (1930), due to his incorrect observation ofthe numbers of cirri arising in the rows of depressions on theventral surface. Both the left and the right marginal cirri arisein the same localities as do those for the posterior daughter inbinary fission.

The development of the cirri and peristome is usually, butnot always, at the same stage in the two conjugants. Some-times the rudiments are noticeably further developed in one con-jugant than in the other. In these cases the reorganization bandsare farther along in the conjugant in which the cytoplasmicorganelles are further developed.

Eesorption of the cirri begins while the conjugants are stilltogether, and is completed within one hour after their separationoccurs.

Although no preparations by silver impregnation method oflate stages of conjugants were obtained, the replacement ofthe ventral polygonal network during conjugation was indicatedby the alcoholic haematoxylin preparations.

The bristles undergo no visible change in structure duringconjugation. That the bristles are not included in the reorganiza-tion process during conjugation may be correlated with the factthat they are not reorganized during binary fission, but merely


undergo a multiplication process so that there will be a sufficientnumber for both daughters.

R e o r g a n i z a t i o n of t he Z y g o t e .

When replacement of the cirri and anterior portion of theperistome is completed, the exconjugants are lacking frontalcirrus four and the posterior part of the peristome. About twoto three days after separation of the conjugants, the macro-nuclear rudiment, which until that time has a spherical shape,begins to elongate in a lateral direction. At this time the rudi-ment of the posterior part of the peristome appears as a depres-sion immediately posterior to the end of the incomplete zone ofmembranelles (fig. 14, PI. 22; Text-fig. 3). On the floor of thisdepression a series of rudiments of membranelles appear, themost anterior of which is contiguous with the most posterior ofthe membranelles already present. This rudiment of the pos-terior part of the zone of membranelles has nearly the samewidth as the fully formed anterior part of the zone, but thebases of the membranelles are crowded very close together. Thepocket gradually extends backward, lengthening the intervalbetween membranelles, and at the same time the cytopharynxand its ciliary apparatus are formed.

This agrees in general with Maupas's account of the origin ofthe posterior portion of the peristome, except that he describedthe rudiment as arising more posteriorly in the middle of theventral surface.

Soon after the appearance of the posterior portion of theperistome, the rudiments of an entire set of cirri appear in thesame manner as in the reorganization of the gamete (Text-fig. 3).The frontal cirrus four arises under the right margin of thedeveloping peristome, so that, when replacement is complete,there results an organism with an entire peristome and a fullset of cirri. So far as could be determined the bristles do notundergo any change of structure during this period. Im-mediately after the completion of this reorganization, and veryoften before the macronucleus has assumed its usual shape, theorganism undergoes a binary fission during which cytoplasmicreorganization proceeds as usual.


Maupas (1889) observed only a portion of the reorganizationof the zygote in E u p l o t e s p a t e l l a . The replacement ofthe cirri escaped his attention and he supposed that cirrus four,missing in the organization of the gamete, arises separatelyeither as a new formation, or from a division of a cirrus alreadypresent. However, the entire process of reorganization of thezygote, including replacement of the cirri, was described byMaupas (1889) in O n y c h o d r o m u s g r a n d i s , S t y l o n y -chia p u s t u l a t a , and O x y t r i c h a f a l l a x . He interpretedthe reorganization during conjugation as a simple process ofregeneration with the function of replacing the organelles, whichmight have been injured in the course of conjugation. Thesecond reorganization as found in O n y c h o d r o m u s , &c,was regarded as associated with the particular arrangement ofthe ciliary apparatus of the species in question.

In the present study it has been shown that the two periodsof reorganization during sexual reproduction have a definiterelationship to the phases of the life-cycle. The first reorganiza-tion takes place in the gamete, and is associated with thehaploid condition of the chromosomes. The second reorganiza-tion occurs in the zygote, and is associated with the restorationof the diploid condition resulting from fertilization.

Each phase of the life-cycle has its own organization. Someof the structures, such as the bristles, are apparently continuousfrom one organization to the next, but for the most part thedifferentiated structures in the organization are resorbed at theend of each phase. This periodic loss of differentiation may bea factor in preventing senescence of the organism.

DISCUSSION.

It has been shown that profound changes in protoplasmicorganization are correlated with both asexual and sexual repro-duction in E u p l o t e s p a t e l l a . During each of these periodsin the life-cycle there is a dedifferentiation and a ^differentiationof certain cytoplasmic structures, including the cirri, the fibrilsattached to the bases of these, and the peripheral polygonalsystem. This process occurs twice during sexual reproduction:once in the gamete, and once in the zygote. The peristome


undergoes this process of renewal during sexual reproduction,but there is no evidence of dedifferentiation in this structureduring asexual reproduction. The more complete reorganizationof the neuromotor system during sexual reproduction is asso-ciated with the more profound nuclear changes at this time.Dedifferentiation of the bristles was not seen at any time.

Unless some kind of renewal occurs, the peristome and bristlesmust be worn out or broken off in the course of time. The oldperistome is similarly retained during binary fission in severalciliates, notably S t e n t o r (Hetherington, 1932; Schwartz,1935). Schwartz (1935) described a periodic reorganization inS t e n t o r, during which there is a reorganization of the nucleiand a replacement of only the oral portion of the membranellezone. Thus the aboral portion of the membranelle zone isretained apparently unchanged both in the periodic reorganiza-tion and during binary fission. It has been shown that whenS t e n t o r is placed in a 0-5 per cent, solution of NaCl themembranelles are thrown off, but the basal structures are re-tained, and new membranelles grow forth from these (Prowazek,1904). Schwartz (1935) suggests that this may occur normallywhen the membranelles are in need of replacement. This sortof renewal might also take place in the membranelles and bristlesof E u p l o t e s .

Eeorganization of protoplasmic structure during asexualreproduction has been described in many other Protozoa. Taylor(1928) confirmed Wallengren's (1901) description of the replace-ment of the entire set of locomotor organelles in U r o n y c h i a .Summers (1935) noted a more or less complete cytoplasmicreorganization in D i o p h r y s , A s p i d i s c a l y n c e u s , andS t y l o n y c h i a . Poljansky (1934) noted a complete renewalof cytoplasmic organelles in B u r s a r i a t r u n c a t e l l a .MacDougall (1925) found that the cilia and trichites are entirelyrenewed in Ch i lodon u n c i n a t u s during binary fission.Lucas (1932) described a complete reorganization of the ciliaryapparatus in C y a t h o d i n i u m p i r i f o r m e .

Reorganization of cytoplasmic structure during conjugationhas also been described in many ciliates. In Ch i lodonu n c i n a t u s (MacDougall, 1925)a reorganization of the pharyn-


geal basket and some of the cilia takes place at this time. Tworeorganizations occurring during sexual reproduction have beennoted only in O n y c h o d r o m u s g r a n d i s , S t y l o n y c h i ap u s t u l a t a , and O x y t r i c h a f a l l ax (Maupas, 1889),although it is probable that other cases would be found if athorough study of other hypotrichous ciliates were made.

Keorganization of cytoplasmic structure may also occur atother periods during the life-cycle. Although encystment hasnot been observed in E u p l o t e s p a t e l l a , Garnjobst (1928)showed that a complete dedifferentiation of external organelles,accompanied by nuclear reorganization, occurs during the pro-cess of encystment in E u p l o t e s t a y l o r i .

It is known that a reorganization, similar to that which occursduring binary fission, takes place if the organism is mutilated.This has been proven for E u p l o t e s p a t e l l a (Taylor, 1923;Reynolds, 1932), E u p l o t e s v a n n u s , E u p l o t e s c h a r o n ,D i o p h r y s , A m p h i s i a (Dembowska, 1926), and U r o n y -ch ia (Calkins, 1911; Young, 1922; Taylor, 1928).

A periodic reorganization of certain of the structures inS t e n t o r (Schwartz, 1935) and C y a t h o d i n i u m p i r i f o r m e(Lucas, 1932) may occur without reference to any basic phaseof the life-cycle.

The manner of reorganization is different in the various formsdescribed. In some cases certain cytoplasmic structures persistthrough the stage of dedifferentiation and give rise to the newciliary apparatus during redifferentiation. Chatton and hiscollaborators have described the formation of the new ciliaryapparatus by division of the basal bodies of the cilia of the parentin Ch i lodon (1931 a) and other ciliates. A process of multiplefission of the basal granules of the cilia on either side of the planeof division has been described i n P a r a m e c i u m (Gelei, 1934d),which closely corresponds to the method of multiplication of thebristles in E u p l o t e s p a t e l l a .

Klein (1928, 1932) supposed that the new ciliary organellesare formed in s i t u from the so-called silver-line system. Aspointed out above, the rudiments of the ciliary organelles inE u p l o t e s p a t e l l a are not connected with the old polygonallines, and they arise either before or at the same time as the


new polygonal lines. Therefore the ciliary apparatus is notderived from the polygonal system. However, the differentiationof the polygonal system is closely associated with that of theneuromotor system, which is indicative of a relationship betweenthem.

MacLennan (1935) found a continuity in structure of theneuromotor system through the stage of dedifferentiation inI c h t h y o p h t h i r i u s . During redifferentiation the new ciliaryapparatus was found to develop from fibrils of the neuromotorsystem.

All the above cases involve some degree of visible cyto-plasmic structure persisting through the stage of dedifferentia-tion and giving rise to the new parts of the ciliary apparatusduring redifferentiation. In E u p l o t e s p a t e l l a the bristlesare the only structures derived from similar structures in theprevious organization.

In another group of organisms, especially the hypotrichousciliates, the new ciliary apparatus is derived from apparentlyundifferentiated cytoplasm. This group includes E u p l o t e sh a r p a (Wallengren, 1901), E u p l o t e s w o r c e s t e r i (Griffin,1910b), and U r o n y c h i a u n c i n a t a (Taylor, 1928). Theredifferentiation of the peristome, polygonal system, and cirriin E u p l o t e s p a t e l l a belongs to this category. In all theseforms the ciliary organelles arise in the ectoplasm, but an endo-plasmic origin of the ciliature has been described in C y a t h o -d i n i u m p i r i f o r m e (Lucas, 1932).

The regenerative activities are localized in a region at thefuture plane of constriction in E u p l o t e s p a t e l l a . This isshown by the appearance within this region of all the rudimentsof the various parts of the neuromotor system except the rightmarginal cirri, and the extreme posterior and anterior parts ofthe peripheral polygonal system. This localization is best shownin connexion with the multiplication of the bristles, which ismost active at the future plane of constriction and decreasesboth anteriorly and posteriorly from this region.

That these reorganization processes occur at very definitetimes in the life-cycle of the organism may be correlated withthe physiological condition of the organism at these times.


Calkins (1911) and Young (1922) have shown in U r o n y c h i athat the power to regenerate is at a maximum at the time ofbinary fission, and at a minimum after division is completed.This was explained by suggesting that during its condensationthe macronucleus gives off a substance into the cytoplasm whichinfluences the development of new organelles (Calkins, 1911), orthat a substance dependent upon the presence of the micro-nucleus accumulates in the protoplasm up to the time of binaryfission, when it is used in the regeneration of organelles (Young,1922). That the macronucleus might give off an activatingsubstance is supported by the fact that the progressive re-organization of the macronucleus always precedes the changesin the visible structure of the cytoplasm i n E u p l o t e s p a t e l l a .

Child (1916) advanced the view that the processes of re-organization which accompany binary fission in the Protozoaresult in rejuvenescence. This tends to counteract senescence,which consists in a decrease in the metabolic rate and a pro-gressive accumulation of the more stable components of thesubstratum of the protoplasm during growth, development, anddifferentiation. Calkins (1933) has developed this viewpoint,and has shown that there are evidences of reorganization duringdivision in all the groups of the Protozoa. Taylor (1928)suggested how rejuvenation might be brought about by dis-persion of the molecular components of the formed structuresduring dedifferentiation and recombination of these duringredifferentiation, in which process the products which tend todecrease the metabolic rate may be eliminated.

More recently, Taylor (1935) has proposed the view that thereis a comparable return to a condition of minimal differentiationin binary fission, conjugation, encystment, and regeneration.This does not hold true for E u p l o t e s pa t e l l a , since re-organization in binary fission differs from that during conjuga-tion in having no replacement of the peristome. Thus Taylor'sconclusion that binary fission, as well as each of the other stagesin the life-cycle in which reorganization occurs, is comparableto the end of an old life-cycle, and the beginning of a new onein the Metazoa is not supported by the facts in E u p l o t e sp a t e l l a .


The structural changes in the protoplasm are closely relatedto the basic phases of the life-cycle. The zygote is formed atthe time of fertilization and inherits the body of the gametewith its cytoplasmic structures. The zygote then undergoesnuclear divisions, in which four products of the fertilizationnucleus are formed, followed by differentiation of nuclearmaterial into macronucleus and micronucleus. Coincident withthe differentiation of the nuclei there is a change in cytoplasmicstructure of the body, during which the organization of thegamete is replaced by an organization developed under theinfluence of the new combination of genetic material. Anyhereditary changes in the structure of the organism should beexpressed at this time.

The zygote then undergoes a period of asexual reproductions,during which no new nuclear material is introduced. Some ofthe cytoplasmic changes during binary fission may be explainedby the need of the doubling of organelles. This would includethe multiplication of bristles and origin of the new peristome.Changes such as the development of two sets of cirri may beexplained only partly on this basis. Wallengren (1901) supposedthat two entirely new sets of cirri were developed during binaryfission in order to produce a matched set, and to replace theold worn-out cirri. This explanation would not hold in Eu -p l o t e s p a t e l l a where the old membranelle zone is retained,resulting in an unmatched condition of the locomotor organelles.The dedifferentiation of structure during asexual reproductionmay serve the function of rejuvenation.

This periodic rejuvenation during asexual reproduction mighthelp to explain why certain species of Protozoa may continueto thrive in culture for an indefinite period of time without theoccurrence of sexual reproduction. A somewhat similar proto-plasmic reorganization occurs in the cells of Metazoa duringthe process of mitosis, as pointed out by Taylor (1928). Therejuvenation which may accompany this reorganization mighthelp to explain why tissues can be cultured indefinitely in theproper medium.

Under certain conditions not fully known the organism mayundergo sexual reproduction. During this process there is a


maturation of the micronuclei involving the reduction ofchromosomes from the diploid to the haploid number. A re-organization of cytoplasmic structure is associated with thischange in nuclear condition so that the haploid gamete acquiresa new cytoplasmic organization. By the process of fertilizationthe diploid condition is restored. The resulting zygote receivesa new set of cytoplasmic structures by means of the reorganiza-tion following fertilization.

The problem of endomixis was not touched in this study.Investigation of the protoplasmic reorganization during endo-mixis is needed to complete the problem.

SUMMARY.

1. The neuromotor system of E u p l o t e s p a t e l l a Ehren-berg was studied in different stages of the life-cycle, includingthe adult, and asexual and sexual reproduction.

2. In the adult the basal granules of all the cirri are arrangedin straight primary and secondary rows. The fibrils from thebases of the cirri are in general parallel with either the primaryor secondary rows of basal granules of the cirri to which theyare attached. This is interpreted as an indication of the evolu-tion of the organism from an ancestor with a simple neuromotorsystem in which the cilia were uniformly distributed over thesurface of the body in longitudinal, slightly spiral rows.

3. The basal apparatus of a bristle consists of a basal granuleat the bottom of a depression in the ectoplasm which is sur-rounded by a group of rodlets and ends at the surface in a ringconnected with the polygonal system. The polygonal or silver-line system is pellicular in position.

4. During asexual reproduction the entire set of cirri isresorbed and replaced by two new sets of cirri, one for eachdaughter. One frontal cirrus for the anterior daughter developsin the old peristomial field of the parent, while the correspondingcirrus for the posterior daughter develops in the new peristome.The right marginal cirri arise on the dorsal surface along therows of bristles and later move to the ventral surface. Thefibrils develop outward from the bases of the cirri.

5. During binary fission a new peristome arises in a depression


in the ectoplasm independently of the old peristome. The oldperistome remains in the anterior daughter without at the timeundergoing any visible change in structure.

6. The multiplication of the bristles is limited to a zone oneither side of the future plane of constriction. Multiplicationoccurs by a series of fissions of the basal granules in this area.Dedifferentiation of bristles was not seen.

7. The polygonal system is replaced over the entire surfaceof the body with the exception of the old peristomial field. Thenew system originates in many separate loci in connexion withthe bristles on the dorsal surface, and with the rudiments(Anlagen) of the cirri and peristome on the ventral surface.

8. During conjugation there are two successive reorganiza-tions of the neuromotor system, that of the 'gamete' (conjugant)and that of the zygote.

9. The first reorganization, that of the gamete, begins duringthe maturation divisions preparatory to fertilization. Duringthis reorganization the posterior portion of the peristome isresorbed and the remaining anterior portion is later replaced.The eighteen cirri are replaced by a set containing only seventeencirri.

10. The second reorganization, that of the zygote, occursduring the differentiation of the nuclei in the exconjugants. Itinvolves the completion of the peristome by differentiation ofthe missing posterior portion, and the replacement of the setof seventeen cirri by a complete set of eighteen cirri.

11. These protoplasmic reorganizations, invariably associatedwith both asexual and sexual reproduction, involve a structuraland presumably a physiological rejuvenation resulting in thepossibility of an indefinitely continued existence of the individualin the Protozoa. It may be that sexual reproduction is notnecessary to bring about this result, but further work on therelationship of endomixis to the problem is needed.

E E F E R E N C E S .

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EXPLANATION OF PLATES 21 AND 22.

All figures are microphotographs taken by Mr. J. E. Gullberg of theCinematographical Laboratory of the University of California.

PLATE 21.

Kg. 1.—Dorsal surface of E u p l o t e s p a t e l l a in stage of binaryfission, showing process of multiplication of bristles. Specimen fixed inChampy's fluid, stained in Heidenhain's haematoxylin. x 1,300.

Fig. 2.—Another specimen in about the same stage as that in fig. 1.The basal granules of the six central bristles are undergoing fission.Champy's, Heidenhain's haematoxylin. X 1,300.

BUPLOTES PATELLA 557

Fig. 3.—A later stage in binary fission. The new basal granules havegiven rise to new bristles. In the centre of the row of bristles is the rudi-ment of one of the marginal cirri (right) of the anterior daughter. Chanipy 's,Heidenhain's haematoxylin. x 1,300.

Fig. 4.—Ventral surface of organism in stage of binary fission. Cyto-pharynx near lower right (of organism). A portion of the peripheral poly-gonal system is visible posteriorly and to left of cytopharynx. In the tworows of five depressions, the rudiments of the new cirri can be seen. Wetsilver method, x 650.

Fig. 5.—A later stage in binary fission. New networks of the peripheralpolygonal system are visible in each depression. The rudiments of the cirriare more widely separated. The rudiments of the left marginal cirri forthe anterior daughter appear to the (organism's) left of cytopharynx. Wetsilver method, x 650.

Fig. 6.—Same aa fig. 4, at a higher magnification, showing detail of oneof the networks of the new polygonal system. X 1,650.

PLATE 22.

Fig. 7.—Dorsal surface o f E u p l o t e s p a t e l l a , showing rodlets whichsurround the depression wherein the basal granule of the bristle lies.Silver-osmic acid-formalin method. X 650.

Fig. 8.—Dorsal surface, showing groups of rodlets at the ends of themembranelle plates in the anterior portion of the adoral zone. Silver-osmicacid-formalin method. X 650.

Fig. 9.—Same as fig. 8, at a higher magnification, to show detail of groupsof rodlets. X 1,650.

Fig. 10.—Ventral surface, showing basal structure of membranelles.Incomplete row of cilia is visible at the (organism's) right side of eachmembranelle. Silver-osmic acid-formalin method. X 1,650.

Fig. 11.—Ventral surface of organism in stage of binary fission. Rudi-ment of new peristome is posterior and to the (organism's) left of the oldcytopharynx. Two depressions of each transverse row of five are visible,and three rudiments of cirri appear in each of these. Champy's, Heiden-hain's haematoxylin. X 650.

Fig. 12.—Organism during conjugation, showing posterior portion ofzone of membranelles in a rolled-up position inside the body. Macro-nucleus appears anteriorly. Schaudinn's, alcoholic haematoxylin. X 1,300.

Fig. 13.—Conjugants, showing triangular area of fusion of the twobodies. Membranelle zones, visible posterior to the area of fusion, have notyet been taken into the endoplasm. Wet silver method. X 1,300.

Fig. 14.—Reorganization of zygote. Macronuclear rudiment appears asa round, darldy staining body. The rudiment of the posterior portion ofthe peristome is visible as a crescentic structure anterior to remnant of oldmacronucleus. Rudiments of new cirri, and fibrils of the old anal cirri ap-pear at (organism's) left side. Schaudinn's, alcoholic haematoxylin. X 1,300.

. Journ. Micr. Sci Vol. 79, N. S., PI 21

D. M. Hammond

Quart. Journ. Micr. Sci. Vol. 79, N. S., PI 22

13D. M. Hammond

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