Studies on the cytology of the hypotrichous infusoria. The relation of structure to regeneration

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STUDIES OK THE CiTOLOGX-.QP TJffi;. HYPOTRICHOUS INFUSORIA THE RELATION OP STRUCTURE-. TO REGENERATION.

by Uverett Lassiter Bishop, Jr.

A dissertation submitted In partial fulfillment of the require­ ments for the degree of Doctor of Philosophy, in the Department of Zoology, in the Graduate College of the State University of Iowa. May, 1942

P roQ u est Num ber: 10311080

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TABLE OF CONTESTS

Introduction - - - - - - - - - - - - -

1

Methods

4

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- - - - - - - - - - - - -

Observations - - -

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Discussion - - - - - - - - - - - - - - 2 2 Conclusions

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Literature Cited - - - - - - - - - - - 4 0 Table I Table I I Flat@s

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--45 46—51

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INTRODUCTION The phenomenon of regeneration and the mechanisms underlying this process give an experimental approach to the study of certain of the morphogenetic processes occurring within cells and organisms, and so offer a method by which information may be obtained about the normal*

The funda­

mental organization which exists in all parts of an organism, and permits any part (with certain limitations) to reorganize itself and regenerate into a complete organism homologous with the original, can best be studied by experiments on the regeneration of organisms. The Protozoa, especially the larger forms, offer very suitable material for studies of this type, due to the absence of physiological and morphological factors which exist between cells of higher organisms.

The hypotrichous

Infusoria were chosen as subjects for this Investigation for several reasonst

the morphology, 11fe-history, and

cytology are perhaps as well-toown as are those of any group of the Protozoa;

cultural methods are simple;

the

localization of easily recognizable zones of pellicular structures facilitates orientation and recognition of fragments• 1,

The Problem. An attempt is here made to find those factors

which control, direct and influence the processes of reorganization and regeneration, and the limitations of

-2-

eaeh factor.

Th© relationships of the nuclear components

and various morphological and physiological factors to regeneration have been recorded and analysed and compared with the results of other workers in the field* The us© of ultra-centrifugation as a method for fragmentation differs radically from methods used by other investigators, which have been mainly by the actual cutting of the organisms.

By cutting, it is possible only to

isolate those fragments which contain the inclusions normal to that region, and the separation of nuclei from central regions of large size is almost impossible.

With the ultra-

centrifuge, fragments of virtually any size may be obtained from any region of the organism, containing all, part or none of the nuclear complex*

Also, through the use of a

medium of high specific gravity, it is possible to obtain measurements of the relative density of the various components of the organism. 2.

Previous Work* Investigators have used several methods for

obtaining fragments whose regeneration can be observed and conclusions drawn.

Cutting has been the most prevalent

method for protozoological Investigations, though some have used micro-pipettes for withdrawing various parts of the organism (Taylor and Farber, 1924).

The use of chemicals

for Inducing the loss of the pellicle permits the study of the regeneration of this structure (Nadler, 1929, on Blepharisma).

Mechanical agitation causes a breakdown of

structure with a subsequent regeneration, and has been utilized by a few workers (King and Beams, 1941).

One

Investigator (Tittler, 1958) used electric currents as a tool for producing fragmentation of tTroleptus, followed by regenerative processes.

Tartar (1940) noted an apparatus

for mass cutting of paramecia by whirling razor blades. The ultra-centrifuge has been used extensively for regeneration and growth studies of developing eggs of invertebrates (Harvey, 1939) and for studies on recovery and macronuclear structure in Paramecium (King and Beams, 1937).

The present work Is, however, the first to utilize

ultra-centrifugation as a method for fragmentation of Protozoa, In order to study th© regenerative processes as related to various structures and functions within the organisms. 3.

Acknowledgments, I wish to express my gratitude to Professor

H. W. Beams for suggesting the problem, and to Professor Beams and Professor K* L. King for their advice and criticism during the course of this Investigation.

I am

also Indebted to my wife, Irene B. Bishop, for assistance in making the photographs which accompany this report.

METHODS The organisms used in this work Include Qxytrlcha fallax, Stylonychia sp*, Euplotes patella, and Uroleptus sp. Most of the detailed study of regeneration Is from Qxytrlcha fallax;

the other forms were used only to obtain comparative

Information.

Identification of these species was by us© of

the taxonomic key of Xahl (1935). i*

Descriptions of Organisms. Qxytrlcha fallax is a hypotrichous ciliate, oval

in shape as seen from the ventral or dorsal surface, and in section Is dome-shaped.

The motor organelles (cirri) are

segregated and arranged in definite groups on the ventral surface (text fig. 1), which makes for ready identification of the fragments.

The nuclear content consists of a

bipartite macronucleus and two (occasionally three) micro■ j nuclei. The macronuclear bodies lie in the midplane of the organism, on© in the anterior, the other in the posterior half.

One micronucleus is loosely imbedded in the periphery

of each macronucleus, except at the time of division or conjugation.

The organism averages 125 to 150 microns In

length, and Is about one-half as wide as long.

Variation

in size Is common in this species, due to differences in cultural conditions (Gregory, 1923).

Occasional giant forms

were encountered in older cultures, ranging In size up to 350 microns long, but these were atypical as to structure and motor organelles.

This gigantism Is probably due to

cannibalism, as has been shown to occur In several other Protosea (Giese and Alden, 1958)*

T m

4

-

J3

17,

FIGURES

macronucieas

JT \l3\\J0 IS \ M

JO

i

i Text figure 1*

2.

Seml-dlagraim&atie ventral view of Gxytricha fallax* Cirri are numbered to correspond with those of text figure 2* Diagram of cirri regeneration Dembowska, 1925.

field, after

Th© anterior end is much more highly differentiated than is th© posterior*

The adoral and oral cirri are

numerous and form a conspicuous row or membranelle•

The

oral groove passes from the left side of the anterior end to the center of the ventral surface* membrane

An undulating

Is present, lying near the posterior

of the oral

median portion

groove, but Is inconspicuous and usually

—c>—

difficult to observe In the living animal*

This organism

Is Identified by th© number, arrangement, and grouping of th© cirri and by th© presence of three relatively short caudal cirri originating from the dorsal surface of th© posterior end (text fig* 1)*

For a more detailed descrip­

tion of this species, see Gregory (1923), Kahl (1935), or Lund (1935). The other organisms used in this study were utilised in .an attempt to see if the results obtained with Qxytrlcha would ,apply to other hypotrlchs*

They have all

been Intensively studied and are well described by previous workers*

Stylonychia (Dembowska, 1925, 1938) Is very

similar to Qxytrlcha in structure and in functions, and gives similar result© as regards regenerative phenomena* Fuplotes (Hammond, 1957;

Turner, 1930) differs from the

above organisms in having only one elongated macronucleus and one micronucleus and in having fewer groups of cirri* The anterior end is more specialized in Euplotes, and the animal is tougher, as evidenced by the necessity for an Increased centrifugal force required to fragment the organism* XJroleptus (Galkins, 1921;

Tittler, 1938) is a hypotrlchous

ciliate varying only slightly from Qxytrlcha and Stylonychia in the number and arrangement of the ventral and marginal cirri and in nuclear organization*

This form has the

macronucleus in several parts and micronuelel varying in number from two to six*

Here, the fact that both macro-

nuclei and micronuclei are very numerous makes th© obtaining

7

of enucleate fragments difficult. 2*

Cultural Methods . Cultures of all foras were made in small stender

dishes containing approximately ten ec* of medium. used included split pea soup (Baker, 1926);

Media

timothy hay,

split peas and whole wheat grain medium (Turner, 1950);

and

modifications of the latter using rice grains, alfalfa or both in place of th© hay or wheat.

The pea soup medium

gave very rich cultures in which, however, th© organisms did not continue for more than one week at a high rate of division*

By making new cultures every four or five days,

mass cultures were kept continuously at peak concentration for use in the experiments, and cyclical variations were thus eliminated.

Cultures using other media were never as

thick but were used in order to provide a stock supply for reinoculation of th© pea soup cultures. 3.

Methods of Fragment at ion. The ultra-centrifuge, used for many biological

studies by Beams (1937), Harvey (1939), King and Beams (1937) and many other workers, was utilized here as a means for mass fragmentation of th© organisms.

About two or three cc.

of a rich culture of the organism to be studied were placed in the rotor of the air turbine ultra-centrifuge, which was operated for five to ten minutes at pressures varying from fifteen to twenty-five pounds.

The most satisfactory

time was five to seven minutes at a force of approximately

«*s**

117,600 times gravity, since this combination gave a large proportion of fragments which were not so badly torn or so small as to make observations difficult or the percentage of regenerating organisms low. Organisms were also fragmented by mechanical agitation (placed in a tube and shaken by hand for four or five minutes), but this method did not give as consistent results as did the centrifugal force method. Stratification of the organisms for the determination of the relative densities of various inclu­ sions was done by replacing the medium with a five per cent starch or gum solution.

This served to hold the organisms

away from the sides of the rotor during centrifugation, and resulted in the layering of inclusions without any breaking of th© pellicle (figs. 3, 19). j4.

Isolation Methods » Immediately after centrifuging, a drop of the

culture was removed from the rotor to a slide, and the various fragments isolated with a micro-pipette to individual hanging drop slides.

Upon isolation the frag­

ment was examined under th© high power to determine the portion of th© organism of which the fragment was a part, and also to check on the isolation.

A rapid camera lucida sketch

was made and th© slid© laid aside for a time.

Since it was

found that many ©nucleate fragments tend to disappear from the medium after about on© hour, no Isolations were mad© after that time.

Th© remainder of the centrifuged animals

-9-

were fixed, after varying Intervals of time, with Schaudinn* s or formalin acetic alcohol fixatives, and stained with Peulgen* s nucleal reaction count ©retained with fast green, or with iron alum hematoxylin and eosin. Periodic examinations were mad© of the isolated fragments to determine the existence, extent and duration of regeneration and the presence or absence of nuclear material*

As soon as regeneration was seen to b© under

way or when disintegration had begun, the organism was fixed and stained with modified Noland* s stain (1928) to which methyl green was added, or was fixed with formalin acetic alcohol and stained by th© FeuXgen technique*

The

temporary stain was found to b© adequate in determining both the nuclear content and the condition, number and regenera­ tion of the motor organelles*

Some organisms were permitted

to regenerate completely and divide, giving rise in some cases to amicronucleate lines.

A few individuals of such

lines were fixed and stained at intervals to determine and verify the nuclear condition* It was found that organisms which did not show any evidences of regeneration within two hours following centrifugation died, although they occasionally lived for as long as two days without regenerating*

Therefore, such

cases were watched especially carefully and were fixed as soon as the beginning of disintegration could be detected. All data were kept on mimeographed sheets containing a diagrammatic outline of the normal organism.

The portion from which the fragment was derived was marked on this diagram to permit a comparison between the various experiments#

Du© to the regularity of the arrangement of

the cirri in groups and within the groups, the delimitation of the fragment could be made with reasonable accuracy and checked after fixation and staining with the modified Holand.' s stain.

11

OBSERVATIONS fh©se observations are divided into several groups 5 nuclei and regeneration, else and regeneration, pellicular structures and their influence upon fragmentation, division and regeneration, and the reformation of the motor organelles* Some of the experiments give data on two or more of the above groups, although many show evidence relating only to one*

The interrelationships of conjugation and regeneration

have not been investigated in this work, since no case of conjugation has been observed in the cultures during a period of over two years.

Turner (1941) gives a survey of

conjugation processes in Protozoa, and Galkins (1921) and others have found that cutting during conjugation {in those cases in which the injury is not severe) does not upset the conjugation process, and that regeneration takes place during the course of reorganization* Regeneration in Qxytrlcha follows, in general, a plan which is similar to the reorganization accompanying division, conjugation or starvation (Dembowska, 1925, 1926, 1938j

Sokoloff, 1924).

Immediately after injury, the

pellicle in the region of the break tends to close over, and the endoplasm in contact with the medium forms a semi—permeable surface membrane through which no protoplasm passes.

This occurs whether regeneration follows or not*

In those cases in which regeneration occurs, the pellicle is reformed In the zone covered by th© surface membrane,

becoming continuous with fch© edges of the original pellicle* A zone of cirri buds {^Begenerationsfelde11 of Dembowska, 1925} fonas in or near the middle oX the ventral surface {text fig. 2;

rigs* 11, 12, 15, 17, 20).

At the same time,

th© bands oX oral and adoral cirri and the marginal cirri are replaced by smaller, more proportionate structures*

The

cirri of* the regeneration field migrate to their final positions with respect to the size of the newly regenerating organism and grow to normal size with the growth of the organism.

Th© oral groove forms, or becomes proportionately

smaller, as the oral and adoral cirri reform*

This entire

process lasts from twelve to twenty-four hours, varying with th© extent of th© injury, but its onset (best seen by the formation of th© regeneration field) occurs within two hours fo 1lowing fr agmenta t ion * XX present, th© nuclei are usually thrown to one side or end of the fragment and the macronuclei and micro­ nuclei are separated*

Th© nuclei soon migrate to a central

position in the fragment and usually have taken such a position within one to two hours after fragmentation.

In

those cases in which the organism is centrifuged but not fragmented (through the use of a medium of high specific gravity), the macronuclei are always displaced to th© centrifugal end but soon recover their normal position. Th© mleronuclel, which are thrown Into the centripetal half, return to a position close to but no longer embedded In the maeronuclei.

This condition Is, however, restored following

13-

the next division. In fragments containing only one macronucleus, the normal double macronucleate condition is restored during the period of growth and repair by simple division of the existing macronuclear body, usually within twelve hours after fragmentation.

Similarly, the normal mioronuclear

condition is restored by mitotic division of the existing micronucleus.

This always occurs before any division of

the organism takes place.

Such a phenomenon Is correlated

with the fact that in many hypotrlchs th© integration between nuclear and cytoplasmic division is dislocated by one (sometimes two) whole nuclear divisional

that Is, th©

nuclear divisions precede by one cycle the division of the cytosome.

Young (1926) also finds that th© return to normal

nuclear balance by amitotic division of the macronucleus and mitotic division of the micronucleus occurs before division of the organism. Several instances of abnormal body shape and structure have been found.in mass cultures*

These Include

supernumerary cirri, duplication of cirri groups, irregular body shapes and the presence of a tail-like projection in the region of the caudal and anal cirri.

Several of these

were isolated, but no return to normal condition was found except In the case of the tall-like projection. degeneration and death resulted* due to one of several causes:

Instead,

Such abnormalities may be

cannibalism with tearing and

loss of part of the pellicle, abnormal division processes

-14-

including atypical nuclear distribution and cytoplasmic division, or degenerative changes of the individual* Prowazek (1904) describes similar abnormalities occurring in cultures of Btylonyohia mytllus and suggests that such findings might b© correlated with degenerative hyperplasma in metazoan tissues. i*

Hue lei and He genera t ion* It Is immediately apparent from examination of

the data that nuclear material must be present In order that regeneration and life may continue*

In this Investiga­

tion, in no instance did any sign of regeneration occur In the absence of nuclear material other than a slight rounding up and closing over of the region of Injury, as previously described. From a tabulation of all data on fragments (Table I) and their nuclear constitution, the following conclusions are made concerning th© influence of centrifugal forces upon these organisms:

the micronuclei and macronuclei

are In fairly close conjunction but are not attached to each other, as shown by th© fact that many (50*5$) of th© frag­ ments contain both of these nuclei and that in those cases in which the macronuclel are eliminated, th© micronuclei are usually thrown out also (21^);

the micronuclei are

usually thrown out in the smaller or centripetal fragment when the two types of nuclei are separated, while the macro­ nuclel remain in th© larger, more coherent, centrifugal fragment (26%)*

The above data are correlated with results

-15-

obtained giving th© relative densities or these two types of nuclei*

In the stratified organism, th© micronuclei

lie in the centripetal half, while th© macronuelei lie in the extreme centrifugal end, sometimes being thrown out completely. A*

Macronucleus»

With few exceptions, fragments

Qxytrlcha fallax show regeneration when all or a portion of th© maeronuclear component is present, with or without micronuclei, and show no regeneration when maeronuclear material is lacking.

The exceptions will be considered

under th© following topic, the relation of size to regeneration. Figures lO, 11, and 12 show the regeneration of a typical anterior fragment containing only a single macronucleus, while figures 13, 14, and 15 show the same phenomenon for a posterior fragment.

In twelve such cases,

the regenerated organism was permitted to divide and produce an amleronueleate line,

One of these lines was continued

for about six weeks, until sufficient organisms had accumulated for centrifugation and subsequent regeneration. In these cases, single organisms ware isolated from each line from time to time and stained to check on the nuclear content* The lack of mlcromxclear material makes no difference in the regenerative capacity, division rate, motility, or morphology of Qxytrlcha fallax*

Table I

summarises the results of the Influence of nuclei upon

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re gene rat ion in this form* As regards the other hypotriehs upon which correlating experiments were made:

Stylonychia gives

similar results to those cited above;

Euplotes, although

showing no regeneration when only maeronuclear material is present, is Injured by the Increased centrifugal force necessary to produce fragmentation with the loss of the micronucleus, and shows a low percentage of regeneration even when portions of both types of nuclei are present; Uroleptus shows an Irregularity In the regenerative capacity which may be correlated with the age of the organism (Calkins, 1921;

Tittier, 1938).

In some Instances, the appearance and size of the maeronuclear remnant show that It has been broken. Since no difference in the method, time and completeness of the regeneration process is noted in these instances, apparent­ ly th© quantity of maeronuclear material does not enter into the relation to regeneration or to cell vitality;

it is

the presence rather than the absolute amount of maeronuclear material that is important* B*

Micronucleus.

Although most of the fragments

containing only micronuclear material were small, a few fragments were large enough to exclude the size factor from consideration.

In these fragments no regeneration occurs

except for the rounding up and closure of the injury as previously noted*

They do not live any longer than similar

fragments containing no nuclear material whatsoever, nor do

-17

they show any variation in movement or structure differing from th© latter. 2*

Size and Regeneration. Several exceptions have been mentioned to the

regeneration of fragments containing maeronuclear material. These failures are due to two causes: small size of the fragment (fig. 7);

1) the extremely or 2) the exposure or

denudation of a large surface, in most instances by the longitudinal fragmentation of th© organism (fig. 16).

These

failures of regeneration are represented by fragments con­ taining both types of nuclei, those containing only macronuclear material or only micronuclear material or no nuclear material at all. The exact size relationship to regeneration in these hypotriehs has not been determined, since this varies with th© portion from which the fragment Is derived, and with th© extent of the Injury.

Thus, a fragment (containing

maeronuclear material) derived from th© anterior end may regenerate if it consists of as little as one-eighth of the normal organism, while a similar fragment from the posterior end must be at least one-sixth of the normal organism for regeneration to occur.

This effect seems to be correlated

with endoplasmic structure, and possibly with the surface or the ectoplasm! c volume of the fragments.

There may

also be a correlation with the structure and regeneration of the neuromotor apparatus, but this is as yet undetermined. In several cases (fig* 16) the fragmentation is

18

longitudinal, *and th© organism, due to its relatively rigid pellicle cannot bend around th© region of the break to close the wound*

As a result, a surface membrane is formed to

cover a large area, and, being more permeable than the pellicle, cannot withstand the osmotic pressures developed by th© imbibition of water and the fragment explodes.

Al~

though the speed of this disintegration is probably correlated with the presence or absence of the contractile vacuole, no data have been obtained on this factor. 3.

Pellicular Structure and Fragmentation. Xt is noted that although anterior fragments may

consist of any proportion of the original organism (figs. §,

posterior fragments never are more than one-half

the original size j

I* e., they never include any of the

anterior structures of the organism (figs. 8, 15, 14).

This

is due to th© relative rigidity and strength of the anterior half of th© animal, caused primarily by th© oral, adoral and neuromotor structures.

In fragmentation, if a break is

mad© through the anterior half of the organism, the forces necessary for this break cause a disintegration of th© more posterior portions, making it impossible for a posterior fragment to survive.

Only when the break Is through a

relatively fragile portion of the organism (posterior to the oral groove) can th© posterior portion of the organism remain viable and restore th© normal structure and function of th© individual. The majority of breaks (65$) occur through the

19

middle of the organism (Table II) passing through or in th© region of the ventral cirri.

This Is to be expected from

the physical forces acting upon a solid structure of this shape.

The high proportions of anterior fragments (66$)

also testifies to the conclusion that th© anterior end is more coherent than is the posterior.

Breaks through the

anterior end comprise only seven per cent of th© total breaks, as compared with twenty-six per cent passing through the posterior portion*

Of these fragments including only a

part of the anterior end, only one and on©-half per cent were actually broken through the oral groove, the remainder passing just behind its termination,

of the posterior

breaks, sixty-five per cent were anterior fragments, correlated again with th© relative strengths of the zones mentioned above. 4*

Division and Begenerat ion. Several cases of fragmentation of organisms while

in division stages have been found (figs. 4, 9, 17, 20). In most cases, when maeronuclear material is retained, the organism discontinues th© division process and undergoes regeneration of th© normal body form and structure*

Th©

organism remains In th© vegetative state until the size of th© normal organism Is regained, and then proceeds through the process of division as do normal individuals.

One case

(figs* 17, 18) In which no pellicular area was lost but one macronucleus and some cytoplasm were thrown out, was In the middle stage of division when centrifuged.

This organism

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lost the new cirri fields which were forming, resorbed the new oral and adoral structures, filled out the cytokinetic constriction, reformed the normal vegetative nuclear structure/ and reformed a single set of motor organelles* After twelve hours, during which time it replaced its lost protoplasmic mass and underwent nuclear division, the organism divided normally. In a few cases, division of the fragment continued, producing a normal daughter and a non-viable fragment (figs. 20, 21).

The occurrence of such phenomena is due to

fragmentation without serious shifting or loss of the nuclear complex and with little disturbance of the preformed division plane, during the later stages of division. 5*

Regenerafc1on of the Motor Organelles* All eases In which the material permits the study

of this process show that this occurs in a manner described by Dembowska (1925) for Stylonychia*

A cirri regeneration

field is formed in the mid-region of th© fragment or organism, and as the new cirri migrate to their proper locations, the old cirri are resorbed*

Also, the adoral

and oral cirri form In their proper location In relation to the newly formed or reformed oral groove.

These cirri grow

out from the margin of the oral and adoral regions, replacing the old adoral and oral cirri as they are resorbed* In all except those organisms In which only a small portion of the pellicle Is lost, the entire motor apparatus Is regenerated following fragmentation according

» 2 1 l»

t© the plan outlined above*

The resulting structures are

all in correct proportion to the sice of th© newly re­ organized organism, and these then grow with th© organism to normal size*

Th© entire process of regeneration of

motor organelles is coincident with the resorption of the ©Id ones, with the result that there is little or no cessation of movement of the organism during this process. Feeding, however, Is Interrupted, as is apparent in the scarcity of food vacuoles in the newly reorganized animal* Figures 10, 11, and 12 show a typical example of regeneration of the motor organelles coincident with the resorption of those cirri remaining, in an anterior fragment containing maeronuclear material*

-22

DISCUSSION fh© mechanisms, controlling factors and the processes of regeneration are problems which, although investigated fairly extensively, give widely varying results under different experimental conditions and with organisms of various groups -

In general, the following principles

have been found to hold throughout the Protozoa (Balamuth, 1940a|

Calkins, 1941;

Summers, 1941)t

the capacity for

regeneration exists in all principal groups of free-living Protozoa (data on parasitic forms Incomplete);

regeneration

involves those processes which occur normally at some stage in the llfe-history of the organism such as at division, conjugation, starvation, eneystment, etc*;

there exists

some type of fundamental organization in all parts of the organism, which can and does initiate the reorganization phenomena upon stimulation from the environment (fragmenta­ tion, starvation, pH, cultural conditions, etc*) or from the organism itself (conjug&tlon, division, or eneystment); regeneration occurs only following such stimulation, never (or almost never) spontaneously;

nuclear material Is

necessary for the maintenance, reproduction and repair of the organism;

in most forms, a physiological gradient exists

in the regulation of regenerative processes* A comprehensive review of the processes of regeneration as exemplified by the Protozoa Is presented by Balamuth (1940a)*

He gives a complete review of the

literature, with a summary of th© results of the workers

In this field*

Also, Suborners {1941} gives a survey of

research on morphogenetic processes of Protozoa.

Since

these papers cover the field, no general survey of the literature will be attempted in this paper* 1.

Nuclei and Regeneration* Although their findings varied in the details of

the influence of nuclear material on the regenerative processes, even the earliest workers found that nucleus and a certain amount of cytoplasm were necessary for regeneration {clt* Summers, 1941)*

Several cases of form and shape

regeneration have been noted (Sokoloff, 1924;

Schwarts,

1935) in enucleate fragments, although the forms used were holotrichous and heterotrichous ciliates and what they observed may have been the closure of the wound by the adjacent pellicle, as was found to occur In all fragments of organisms used in the present investigation.

In

enucleate fragments, the occurrence of a slight regeneration may be explained by the fact that those nuclear Influences which are produced by the maeronucleus, and which cause the Initiation and completion of the processes of regeneration, are present in small amounts In the enucleate fragment; therefore they are able only to start the regeneration processes before becoming exhausted*

A similar condition

exists In the parthenogenetic merogony of invex^fcebrate eggs (Harvey, 1939).

Such development is best explained on

the basis that nuclear Influences remain after removal of the nuclei, and continue to control and direct developmental

-24-

and regenerative processes until exhausted* In the hypo trlchous dilates, two possible modifications of this pattern present themselves j

either

the amount of these nuclear influences low in the vegetative organism, being able only to cause the closure of the wound and form a surface membrane before disappearing;

or the

release of these Influences by the macronucleus occurs only after stimulation of some sort brings about some change In the internal relations of nucleus, cytoplasm and morpho­ logical and physiological factors of the organism. Much controversy exists in the literature as to the relative importance of macronucleus and micronucleus in the maintenance of vital functions.

However, it is generally

agreed that the macronucleus Is essential for continued life, division and regeneration, while the micronucleus, In many species, may be eliminated without fatal effects upon the organism {Balamuth, 1940a )• Taylor and Farber (1924) find that the removal of the mlcronucleus in Euplotes proves fatal to the organism, while in those cases in which the mlcronucleus Is removed and immediately replaced, the individuals continue their normal vegetative existence.

It Is suggested by these

workers that the micronucleus may function in the formation of enzymatic or formative substances which are necessary for the continued vitality of this species, in addition to its sexual or genetic function.

Moore (1924) finds that for

Spath!dlurn and Blepharisma both macronuclear and micronuclear

—25

materials are necessary for regeneration to occur*

How­

ever, she also finds that the micronucleus alone is unable to prevent disintegration,

Burnside (1929), by cutting

Stentor Into fragments containing varying amounts of nuclear material, finds that In later generations individuals of normal size result, whatever the proportion of nuclear material in the original fragment,

Causin (1931) gives

similar results * X>embowska (1925) finds that ©nucleate fragments of Stylonychia show no true regeneration and that the macro nuclei exert some influence upon th© formation of the regeneration fields of the cirri.

Taylor (1928) makes no

attempt to follow or isolate the smaller enucleate fragments tlronyshia unclnata, being interested only in the re­ generation of the motor organelles of the nucleated portion, Reynolds (1932) finds that amicronucleat© Oxytrieha fallax show no variation from the pattern of regeneration of normal mieronucleate Oxytrieha*

She then cites data of merotomy

of Buplotes showing that no regeneration occurs in the absence of micronuclei, and concludes that in the amiero— nucleate race of Oxytrieha the micronucleus must be fused with the macronucleus.

Results obtained in the present

investigation coincide with her results but show in addition that amicronucleat© fragments from previously micronucleate Oxytrieha will undergo regeneration differing in no detail from that of fragments containing both types of nuclear material*

In Euplotea» the present data are Incomplete,

*420'

but seem to Indicate that the mleroxmeleua plays more of an active role In vegetative life or the organise that it does in Oxytrieha and stylonyebia*

Further work is necessary

before definite conclusions can be made concerning the relation of the nuclei to regenerative processes in Fuplotes, Schwarts {1935) finds evidence in the regenerat ion of Stentor that the micronucleiss is necessary for the com* plot Ion of regeneration and subsequent vitality, although amicronueleate fragments may regulate and regenerate external form*

In addition, he finds that the number of

maeronuclear segments is regulated to correspond, with the cell else rather than showing any constant number* Wittier (1938) f inds that both maaronuc1ear and mlcronuclear material are necessary for regeneration In Uroleptus mobilis* which Is supported by the evidence of the present investiga­ tion* Studies of amicronucleat* lines of Frotosoa show that the mlcronucl©us is unnecessary for continued life and reproduction In many foms*

Woodruff (1921) describes races

of amicronueXeate dilates and -suggests several possibilities as to the origin of such formss

transformation of all

micronuelel (synkaryon products) into macronuclei following division of the synkaryon after conjugation?

fusion of the

tro^oehromatin and 1dlo chroma tin with the result that the idl©chromatIn is unavailable for sexual processes; unequal distribution of mleromielei at the time of division* Reynolds (1932) presents data showing that amicronucleat©

-27

Oxytrieha do not differ in any respect from raicronucleat© organisms of the species* S o i m t b o m (1940) finds certain forms of Paramecium aurelia In which th© macronucleus is regenerated from the old maoronueleus during autogamy and conjugation, Instead of from the products of th© synkaryon.

In such forms, the presence

of micronuclear material is unnecessary for continued life since similar regenerations of macro nuclei from the old macronuclei occur at short intervals* Several workers deny the possibility of production of amicronucleat© races of Protozoa by experimental means* Calkins (1911) and Young (1922) describe regeneration occurring in amicronucleat© fragments of bronyehia when cut late in the Interdivisional period, although these regenerated Individuals died later without undergoing division*

Young

(1922) states that these Individuals starved to death, since food was neither digested nor assimilated.

Opposed to this

Idea I® that supported by th© evidence of a majority of investigators who find that digestion and assimilation I® under the control of the maeronucleus.

Taylor and Farber

(1924) show that amicronucleate Euplotes may divide, but death results in two or three days*

Moore (1924) states

that she could not produce amicronucleat© Spathldluta or Blepharisma and concludes that idiochromatin is essential for normal asexual reproduction in these forms*

Reynolds (1932)

concurs with other Investigators on Euplotes that experiment­ ally amlcronucleate animals are non-viable, and attempts to

-28-

generalize from this that all hypotrichs must retain the micronuclear material for continuation of life. A few Investigators have found that In certain species of Protozoa regeneration and continued life of amicronucleat© portions are possible.

King and Beams (1937)

present data showing that Paramecium rendered amicronucleat© by ultra—centrifuging could divide and regenerate, although no amieronucleate lines were established*

Balamuth (1940b)

states that amicronucleat© fragments of hicnophora undergo regeneration and reorganization, although no data are given on the subsequent history of th© fragments.

Schwartz (1935)

was abl© to produce amicronucleat© elones of Stentor.

Xn

the present investigation it is found that experimentally amicronucleat© fragments of Oxytrieha and Stylonychia undergo normal regeneration, division and asexual life cycles, amicronucleat© lines being produced and continued for several generations. Complete agreement is to be found that th© micronucleus alone does not change the vitality, regenerative ability, or actions of fragments from those characteristic of completely ©nucleate fragments*

Fortner (1933) after

expressing the macronucleus from an unidentified hypotrich, finds that the organism behaves like a totally enucleate fragment in lack of digestion, assimilation, lowered motility and ultimate death.

Schwartz (1935) agrees with this

conclusion from results obtained with fragments of Stentor» Moore (1924) finds that Spathldium and Blepharisma fragments

-29

containing only mieronuclear material are Incapable of main­ taining normal form and of giving rise to maeronuelear material* These results, varying widely with different species, give supportive evidence for several conclusions* It is immediately apparent that there exist macronuelesr types differing in degree of differentiation, from those which function both as manufacturing centers and as vital controlling centers, to those whose only function is the manufacture of substances necessary for initiation, continuation and maintenance of the processes of reorganiza­ tion, digestion and assimilation*

In the latter case, vital

controlling mechanisms are located in th© micronucleus * The former type has been termed by some (Moore, 1924; Reynolds, 1932) an amphinucleus.

It Is this type of macro-

nueleus which must be present in those forms which exhibit regeneration and life in the absence of micronuclear material*

In other words, the macronucleus in these forms

contains not only the materials necessary for the production of enzymes or format Ive substances required for th© abovementioned processes, but also a portion of the v idiochromatin” which is necessary for the maintenance of vital functions, retained during the differentiation of th© synkaryon products into macronuclel•

A very early stage In this

differentiation is seen in the conjugation of Anoplophrya (Collin, 1909), in which both the micronucloi and maeronuclei form wandering and stationary nuclei with subsequent exchange

•30

mid fusion*

It would be interesting to attempt regeneration

experiments upon amicronucleat© fragments of this organism. In those forms in which both maeronuclear and raicronucXear materials are necessary for continued vitality, th© macronucleus has progressed further in its differentia­ tion and specialization, until it no longer contains the wIdiochromatln^ material, necessitating the presence of micronuclei which contain such substances*

Such an

assumption permits the explanation of th© formation of an amicronucleate race in those forms which normally require the presence of mlcronuclei, since in these Instances the differentiation of th© macronucleus has not proceeded to its normal completion before the loss, by some means, of the mieronuclear content*

The macronucleus or portions of it

therefore takes over the functions of the mleronucleus in addition to Its own, and continues to reform and rejuvenate those parts which under normal conditions of differentiation would be controlled by the mleronucleus.

The genetic status

of th© macronucleus In such instances is questionable, since fragments of th© macronucleus function as efficiently as does the entire normal complement and are able to reform and regenerate the lost portions, and in addition, division of the macronucleus Is amitotic (King and Beams, 1937)* Reynolds (1938) puts forth several Interesting theoretical observations concerning th© macronucleus— micronucleus relationship*

He concludes that the macro—

nucleus (trophochromatin) is the functional portion of the

—51

nuclear material and that the mleronucleus (idiochromatin) la only concerned with replacement of the macronucleus, and is potentially immortal* The structure and reorganization of the macronucleus of d i l a t e s and of th© hypotrichs in particular may have some connection with the problems of regeneration*

In general, a

reorganisation or purification process occurs within the macronucleus preceding or following division, and Is in the first case th© earliest evidence of the forthcoming division* Turner (1930) gives a complete description of this process *^n Euplotes*

Just preceding division, th© maeronueleus is

traversed by two reorganisation bands, on© starting at each end and passing to th© middle, where they fuse and disappear* The passage of th© bands Is associated with changes in structure and organization of the chromatin granules, th© final products being finer, more closely packed and more homogeneous*

Summers (1935) also finds that a reorganization

band passes along each macrormcleus in Stylonychia, Aspldisca and Biophrys*

After passage of the bands, the reorganized

nucleus contains more chromatin than before and achromatic bodies disappear*

His Interpretation is that this process

is a karyolysis and resynthesIs of maeronuclear materials. Reorganization bands as have been described are commonly found in Oxytrieha.

One band starts from the

peripheral end of each macronucleus and passes down th© nucleus to the proximal end, where it disappears*

The

chromatin material exhibits a morphological change as a

—32-

result of this passage, which is similar to that described by Turner {1930) and Summers (1935)*

In the normal Oxytrieha,

these bands begin to traverse the nuclei soon after the completion of division and complete their journey just before the onset of the next division*

The presence and

movement of the reorganize! ional bands is in no way different in the amicronuGleate forms* Although In normal individuals the bands move synchronously on the two macro nuclei, in many of the centri­ fuged organisms the bands are not in the same relative location on the two macronuclei*

Apparently the agitation

and centrifugal forces upset the regularity of the movement of these bands, causing one to move slower than the other, or causing one band to disappear entirely and a new band to form and start to traverse the nucleus*

A more complete

investigation of this phase of th© problem will be reported in the future. 2*

Size and Regeneration. Although several workers have investigated th©

relation of size to regeneration, no work has yet been done upon the hypotrichs*

It is found that there exists a

differential in regenerative capacity, depending upon th© region from which the fragment Is derived.

The failure of

smaller fragments to regenerate even though they include the necessary nuclear elements Is due apparently to the higher degree of specialization of the organism as a whole and to the relative area of exposed or denuded endoplasm in

-33—

proportion to the volume or the fragment (surfaee-volum© relationship)* Th© existence or a physiological axial gradient in many Proto&oa is shown by the results of several^ workers. Child (1914), using the effect or cyanide upon d i l a t e s of various groups, finds that in all cases the anterior end undergoes disintegration first, followed by a wave of disintegration which passes from th© anterior end toward the posterior*

He concludes that the axial gradient is the

dynamic basis of morphological and physiological polarity in the d i l a t e Infusoria as well as in other organisms.

In

a more recent publication, Child (1929) states that physiological correlation consists of relations of dominance or control and subordination between parts, which, are variable and changeable with growth and development.

That

such an area of dominance exists In the hypotrichs is shown by the movement, Irritability and regenerative ability of anterior as opposed to posterior fragments,

Anterior fragments

show little If any differences in the above functions from normal Individuals$

posterior fragments are slow, less

irritable, and initiate and undergo regeneration more slowly. 3*

Pellicular Structure and P ragmen tat ion, Sine© this is one of the first Investigations using

a method of fragmentation which would be influenced by such structural factors, little evidence bearing upon this problem is available from th© literature*

King and Beams (1941) find

that long-continued centrifugation of Paramecium In a medium

-34

©f high specific gravity results in the extrusion of the protoplasm, often leaving the empty pellicle intact#

In

the ease of Oxytrieha, centrifugal forces under such conditions result in the fragmentation of th© organism, which is stratified according to the relative densities of the various inclusions, into two, three or several fragments, none of which have thus far survived#

This type of fragmenta­

tion is similar to that achieved by Harvey (1939) with Ghae topt erus eggs # 4*

Division and Regeneration# Most workers find that the processes of division,

conjugation etc*, are nall or none® phenomena;

±*e* , once

started, they continue to completion regardless of internal or external changes, except in those instances in which the macronucleus is removed (Faure-pramiet, 1910; Moore, 1924)*

calkins, 1911;

Urostyla, when cut during the early stages of

division, reforms and reorganises into two more or less normal animals*

If cut during th© middle of the division

process, th© smaller fragment dies, while the larger reforms into a normal individual*

During the later stages of

division, only th© larger fragment survives and this proceeds through the completion of the division in the original plane of cleavage, producing a normal daughter and a small noa-viable fragment (Faure—Fremiet, 1910)*

XJronychia shows

less ability to regenerate throughout the division process, but division of th© large fragment continues in the original plane even when cut very early in the pi’ocess (Galkins, 1911)*

-35-

Conjugation la also round to bo an irreversible process by Galkins (1921}. The nucleo-cytoplasmic ratio is thought by many to be the cause of initiation of division, although others have found that there is no relation between nucleus, cytoplasmic volume and rate of division.

Woodruff {1913}

finds that th© nucleo-cytoplasmic ratio shows wide variation during the life cycle of Oxytrieha and concludes that this ratio is an incidental result rather than the cause of cell division. The present investigation finds that division is a reversible process vhen interrupted during the stages up to and including macronuclear elongation and early cytokinesis {fig. 17}.

This is In accordance with the

findings of Faure-Fremiet {1910).

Th© preformation of the

division plane also seems to be reversible in some cases {figs. 17, 18) while in others it persists (figs. 20, 21). 5.

Regeneration of the Motor Organelles. Dembowska (1925, 1926, 1938) gives a complete

account of the mode of regeneration or reorganization of th© motor organelles of several hypotrichs.

Th© formation of

the regeneration field (text fig. 2), the migration of the cirri buds and growth of these to normal size, simultaneously with the reformation of the adoral, oral and marginal structures, results in the complete proportional reformation of th© motor organelles. variation in this method.

The present investigation finds no The processes of dedifferentiation

-36-

and re&Ifferentiation of the motor organelles following fragmentation is similar to those accompanying division, conjugation, autogamy and ©ndomixis.

The experimental

initiation of motor organelle reorganization is found by Taylor (1928) to lie in the neuromotor apparatus, either in the fibrils themselves or in the terminations at the bases of the cirri, or in a disturbance by deep incision into the body.

Bembowska (1938) states that the reorganization

stimulus lies in the disturbance of the proportions of biological systems.

In division, the formation of a

division plane divides the organism into two physiological Individuals;

in regeneration, the organism is reduced In

size mechanically|

in humger reorganization, reduction in

size Is at the expense of the endoplasm, thus changing the proportions of the cell.

Balamuth (1940b) finds that injury

to the fibrillar system In the adoral zone of X.lcnophora induces reorganization, while excisions not injuring this fibrillar system do not. Taylor (1941) gives a comprehensive survey of the structure and function of the neuromotor system In various dilates..

Lund (1935) describes the neuromotor system of

Oxytrieha as consisting of fibrils coordinating the adoral membranelles, th© basal fibril of the undulating membrane, the cytostom&l fibrils, the post-esophageal fibrils and perhaps the anal fibrils.

The frontal,- ventral and marginal

cirri are not concerned in this system.

Evidence from

fragm©^®-^!011 lu the present study show that In general the

-37

above conclusions are correct, and In addition, that a coordinating mechanism exists between the individual elrrl of each group, whether connected to the neuromotor apparatus or not.

In the normal organism, each group of cirri shows

coordinated waves of movement as well as some degree of coordination between th© various cirri groups.

The center

of coordination lies in the anterior portion of th© organism, apparently In th© region of the posterior part of th© oral groove, since if this portion is removed, coordina­ tion between the remaining cirri groups is lost.

Removal of

this region has no effect upon the coordination of the cirri within th© groups.

CONCLUSIONS A method for obtaining mas® fragmentation of Protozoa by use of the air turbine ultra~eentrifug© is described* Advantages of this method are:

obtaining of large

numbers of fragments containing all, part or none of the nuclear complex, and consisting of any proportion of the original organism*

i Begeneration of those hypotrichous d ilates investigated follows the plan of regeneration described by Bembowska (1925). The micronucleus is not concerned In the vegetative life of oxytrieha, Stylonychia and possibly TJroleptus ♦

The

data on Euplotes Is Incomplete, but seems to Indicate that in this form the mleronucleus does perform some vegetative function* The anterior part of these hypotrichs is more unified, has better survival and undergoes regeneration in a more organized manner than does th© posterior part* Division is a reversible phenomenon when Interrupted in the earlier stages by fragmentation or displacement of th© nuclei;

If Interrupted later it proceeds to

completion, regardless of extrinsic factors. Nuclear and cytoplasmic divisions are displaced on© whole division from each other in many hypotrichs*

If

th© normal nuclear content is upset, th© nuclear balance Is reformed by amitotic division of the macronucleus and mitotic division of the micronucleus before any

—59

division occurs.

Tills constancy of nuclear division

preceding eytosomal division by one or two cycles is an inherited character * due to unknown causes* 7.

An axial gradient exists in the regenerative capacity or the hypotrichous di l a t e s *

8*

In Oxytrieha and Styl onychia. amicronucleate lines have been produced varying In no observable detail from the normal organism In structure, function or regenerative capacity*

40

LITERATURE CITED Baker, w* B. 1926* Studies In the Ilf© history of Euglena. I. Euglena agilis. Biol* Bull. 51: 321-362. Balamuth, William 1940a. Regeneration In Protozoa: a problem of morphogenesis. Quart. Kev. Biol. 15: 290-337. 1940b. On the role of* the fibrillar system In the regeneration of* d i l a t e Protozoa. Anat. Record 78 (Suppl.)s 180. Beams, H. W. 1937. The air turbine ultracentrlfuge, together with some results upon ultracentrifuging the eggs of Fucus serratus. Jour. Mar. Biol. Assoc. United

TEb&rsrr svi'-eea.

Burnside, L. H. 1929. Relation of body size to nuclear size In Stentor eoeruleus. Jour, exper. Zool. 54: 473-483. Calkins, G. N . 1911. Regeneration and cell division in Uronychla. Jour, exper. Zool. lO; 95-116. 1921. Uroleptus mobllls Engelm. IV* Effect of cutting during conjugation'.' Jour, exper. Zool. 34: 449-470. 1941. Chapter I. General considerations, in Protozoa in Biological Research, edited by Calkins and Summers, Hew ¥orkt Child, C. M. 1914. The axial gradient in clliate Infusoria. Biol. Bull* 26: 36-54. 1929. Physiological dominance and physiological Isolation in development and reconstitution. Roux1 Arch. f. Entw. der Org. 117: 21-66. Collin, Bernard 1909. La eonjugaison d*Anopiophrya branchiarum (Stein). (A. circulans Balbiani). Arch. ZooI. exper. et gen. ser 5 , 1 : 345-388. Dembowska, W. S. 1925. Studlen uber die Regeneration von stylonychia mytilus* Arch. f. Mikro. Anat. u. Entw. 104: 185-209 1926* Studies on the regeneration of Protozoa. II. Regeneration of the ciliary apparatus In some marine Hypotrlcha. Jour, exper. Zool. 43: 485-504. 1938. Korperreorg&nlzation von Stylonychia raytilus belm Hungern. Arch. f. Protistenk. 91: 89-105.

-41

F&ure-Fremlet, E. 1910. La division de l yUrostyla grandis. Experiences do merotomi©* Bull. Sci. Fr. et Belg. 44: 215—219. Fortner, Hans 1933. Uber Kernresektion be! ©inem Hypotrichen (nov. sp?), Arch. f. Protistenk, 81: 284-307. Giese, a . C. and H, H, Alden 1938. Cannibalism and giant formation in Stylonychia. Jour, ©xper. Zool. 78: 117-134. Gregory, h. H. 1923. The conjugation of Oxytrieha fallax. Morphol. 37: 555-581.

Jour.

Hammond, D. M. 1937. Th© neuro-motor system of Euplotes patella during binary fission and conjugation! Quart. Jour. Micro. S c l , 79: 507-557. Harvey, R, B. 1939! Development of half-eggs of Chaetopterus pergamentaceus with special reference to ,nr parthenogenetlc merogony. Biol. Bull. 76: 385-404. Kahl, A. 1935.

Wimpertlere oder Cillata (Infusoria), in Die Tierwelt Beutschlands, edited by F. Dahl.

King, R. L. and H. W, Beams 1937. Th© effect of ultra-centrifuging on Paramecium, with special reference to recovery and maeronuclear reorganization. Jour. Morphol. 61: 27-49, 1941. Some effects of mechanical agitation of Paramecium caudatum. Jour* Morphol. 68: 149-159. Lund, E. E. 1935. Th© neuromotor system of Oxytrieha. Morphol. 58 : 257-276.

Jour.

Moore, E. L. 1924. Regeneration at various phases In the life history of Spathidium spathula and Blepharisma undulans. Jour. ©xper. Zool. 39: 249—316* — Nadler, E. J. 1929. Notes on th© loss and regeneration of the pellicle In Blepharisma undulans. Biol. Bull. 56 : 327-330. Noland, L. E. 1928* A combined fixative and stain for demonstrating flagella and cilia In temporary mounts * Selene© 67: 535.

—42 —

Reynolds, J* M. 1938* The significance of macronucleus and mleronucleus* Biol* Gen* 14 i 328-333* Reynolds, M* E* C« 1932* Regeneration in an amicronucleat© infusorian* Jour* exper* Zool* 62s 327-361. Schwartz, Viktor 1935* Versuch© uber Regeneration und Kemdirnerphismus toei Stentor coeruleus Eherenb* Arch, f • Protistenk* 85 s IOT-13’ 9.------- ~ Sokoloff, B* 1924* Das Regen©rationsproblem bei Protozoen. Protistenk* 47s 143-252*

Arch* f *

Sonnebom, T. M. 1940* The relation of maeronuclear regeneration in Paramecium aurella to maeronuclear structure, ami to sis,r anS~genetic determination. Anat. Record 78 (Suppl.)s 53-54. Stammers, F. M* 1935, The division and reorganization of th© macronuclei of Aapidisca lynceus Muller, DIophrys appendiculata Stelh, and Stylonychia pustulata EhrV Arch* ?! Pro 11st enk .^"85: 1 y 3-2q 8 * 1941. Chapter XVI. The Protozoa In connection with morphogenetic pi^oblems. In Protozoa in Biological Research, edited by calkins and Summers* New York. Tartar, Vane® 1940. Nuclear reactions in Paramecium. 78 (Suppl.)* 109. ~

Anat. Record

Taylor, C. V. 1928. Protoplasmic reorganization In Uronychla uncinata sp. nov. during binary fission and” regeheratIon* Physiol* Zool* Is 1-25. 1941. Chapter IV* Fibrillar systems in dilates, in Protozoa in Biological Research, edited by Calkins and Summers. N©w York. Taylor, c* V* end W. Purber 1924. Fatal effects of th© removal of th© mleronucleus in Euplotes * Univ. Calif* Pub. Zool. 28 s 131-144*

Tittier, I. A* 1938*

Regeneration and reorganization in uroleptua mobllls following injury by induced electric currents, Biol. Bull. 75s 533-541.

Turner, J t P# 1930 ! Division and conjugation In Euplotes patella Ehr., with special reference to the nuclear phenomena. Univ. Calif* pub. Zool. 33s 193-258. Chapter XII# Fertilization In Protozoa, in 1941 Protozoa In Biological Research, edited by calkins and Summers. New York. Woodruff, L. L. 1913, Cell size, nuclear size, and the nueleo cytoplasmic relation during th© life cf a pedigreed race of Oxytrieha fall ax. Jour# exper. Zool. 15 s 1-22. 1921 '"irr'Mcrohucleater"''1and amicronucleat© races of Infusoria. Jour, ©xper* Zool* 34s 329-337.

.

Young, 0. 33 1922 » A contribution to th© morphology and physiology of the genus Uronychla. Jour* exper. Zool. 36s ---- ----353-390. Nuclear regeneration in Stylonychia mytilus. 1926 Biol. Bull. 51s 163-165.

TABLE I Th© Relation of Hualear Content to Regeneration Macro and micro

Macro

micro

non®

total

Anterior fragments

98

46

3

28

172

regenerated

98

46

0

0

144

30

81

4

29

84

29

21

0

0

50

6*

0

5

25

4

0

0

12

11

0

0

115

11

0

0

109

Posterior fragments regenerated Irreg* fragments

14*

regenerated

8

Loss of cytoplasm

104**

regenerated

*)

**)

98

Failure to regenerate du© to exposure of large area, In most cases by th© longitudinal fragmentation or ©mall si&e of fragment* Irregularities found In mass cultures included. of these have shown any regeneration.

Hon©

table

xi

Relation of Structure to Fragmentation Region of Break

Anterior frags*

Posterior frags.

Total

Thru oral groove

1.5^

0.0$

1*5$

Thru anterior end

7.5

0.0

7.5

Thru middle of org.

40.0

25.0

65.0

Thru poet* portion

17.0

9.0

26.0

Totals

.

66 00

34.Off

100.00

PLATE 1 Photomicrograph® of fixed and stained organism® {Oxytrieha fallax) magnification llOOx. Fig. 1*

Organism in division; two dividing maeronuclel and four dividing micronuclel.

2*

N o m & l organism, showing reorganisation planes of macrDxmclei.

3.

Stratified organism. Macronuolei in extreme centrifugal portion; micronuclel (one visible) near ©enter of organism.

4.

Anterior fragment. micronuclel.

5.

Anterior fragment, immediately after centrifugation. Two maeronuclel (one protruding through break) and no micronuclel. Oral groove complete.

6.

Anterior fragment, one hour after fragmentation. Posterior end rounded, regeneration field formed. One macro nucleus and no micronuclel.

V.

Small fragment with one macronucleus And two micronuclel. Degeneration due to small slse.

B.

Posterior fragment, one hour after centrifugation. Anterior end rounded, cirri regeneration field forming, one macronucleus, no micro nuclei.

9.

organism in division. Has lost cytoplasm, but la continuing the division process. One-half hour after centrifugation.

Macronucleus In division}

two

PLATE I.

PLATE XX Camera lucida drawings of stained and of living organisms (Qxytrloha fallax). x 2015#

Fig* 10*

Anterior fragment, immediately after centrifugation. Region of break is closed by a surface membrane.

11*

Anterior fragment, one-half hour after centrifuga­ tion. Break is roundea, regeneration field formed.

12*

Anterior fragment, on® hour after centrifugation. Hew cirri passing to final positions. Resorption of old cirri Is starting, and oral and adoral cirri are being replaced.

15, 14. Posterior fragments, immediately after centrifugation. Break closed by a membrane. 15.

Posterior fragment, one-half hour after centrifuga­ tion. Cirri regeneration field formingj new oral and adoral structures forming. Animal Is swollen, apparently by imbibition of water.

16.

Longitudinal fragmentation of organism, ten minutes after fragmentation* This organism started formation of a regeneration field, but died, apparently from the exposure of a large unprotected surface area.

PTjA ; j. ’£. II.

/J

PLATE III

Fig* 17*

Organism in division when centrifuged} lost cytoplasm and macronuclear body# Immediately after centrifugation* Regeneration fields formed during division process evident; one dividing macronuoleus In posterior portion#

18*

Same organism a© in figure 17, four hours after centrifugation* Organism has returned to vegetative state* Ten hours later, this organism underwent normal division*

19*

Stratified organism* Macronuclei in posterior (centrifugal) end; micronuclei in centripetal portion* Centripetal end contains lipoidal and vacuolar materials*

20*

Organism In division when centrifuged} posterior end broken* Immediately after centrifugation* On© macronucleus In division and two mieronuelel present in anterior portion; regeneration field present and cirri beginning migration*

21*

Same organism as in figure 20, thirty minutes after centrifugation* Anterior organism has reformed cirri; posterior fragment shows little change* One hour after centrifugation this organism divided in the original plane, resulting in on© normal organism (anterior) and a small fragment (posterior) which degenerated*