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PROTOZOOLOGY

PROTOZOOLOGY

A MANUAL

FOR MEDICAL MEN, VETERINARIANS
AND ZOOLOGISTS

BY

C. M. WENYON

C.M.G.. C.B.E., M.B., B.S., B.Sc. (Lond.)

DIRECTOR-IN-CHIEF OF THE WELLCOME BUREAU OF SCIENTIFIC RESEARCH
FORMERLY PROTOZOOLOGIST TO THE LONDON SCHOOL OF TROPICAL MEDICINE

IN TWO VOLUMES
WITH 565 ILLUSTRATIONS AND !20 COLOURED PLATES

VOL. I

NEW YORK
WILLIAM WOOD AND COMPANY

MDCCCCXXVI

Printed in Great Britain

FELIX MESNIL

MY FORMER TEACHER

THIS BOOK IS DEDICATED AS A TOKEN OF

PERSONAL INDEBTEDNESS AND RESPECT FOR

HIS MANY VALUABLE CONTRIBUTIONS

TO OUR KNOWLEDGE OF

PARASITIC PROTOZOA

CORRIGENDA

3 41, Pig. 23, for Cothurina read Cothurinia.
276, Fig. 125, for hirudinella read hirundinella.
323, line 14, for raice read rajce.

474, line 2, for hirudinis read hiriindinis.

475, line 4 from bottom, for hirudinis read hirundinis.
588, line 1, for Sternotherus read Siernothcerus.

604, Fig. 247, for raice read ra/ce.

710, line 19, for Afeliis read Atehs.

722, inscription to Fig. 307, line 7, for sphendosa read sphcerul

806, line 29, for aspzc read aspi«.

983, line 5, for 25-30 read 26-30.

1116, Plate XX., for Sporozoan read Sporozoon.

PREFACE

The subject of Protozoology has, in recent years, shown a tendency to
become divided into two sections. In the one the student's attention
is directed chiefly to the study of free-living Protozoa, in the other to
parasitic forms, more especially those which give rise to disease in man
and domestic animals. Such a division, if it becomes absolute, cannot
lead to a clear understanding of the group as a whole, for it is evident
that without some knowledge of free-living Protozoa, from which they
have been undoubtedly evolved, a wrong conception of parasitic forms
will be obtained. As in other branches of science, specialization appears
to be inevitable if any advance is to be made, but however specialized
a student becomes, it is his duty to keep himself informed of any progress
made outside his particular field. Anyone who wishes to make an intel-
ligent study of parasitic Protozoa must be acquainted with the funda-
mental principles of general Protozoology, and, indeed, with those of
general Zoology, Physiology, and even other sciences. This is merely
another way of stating the well-recognized fact that all sciences are inter-
dependent. On this account the student of the Protozoa which are
pathogenic to man and domestic animals should have a sound knowledge
of other parasitic Protozoa, and at least a good working knowledge of
non-parasitic forms as well. Conversely, those who study free-living
Protozoa should have a clear conception of the parasitic forms, for the
extensive investigations of recent years have contributed so much to our
knowledge that in many respects they are better known than their free-
living relations, particularly as regards the completeness of their life-
histories and the probable course of their evolution.

In this manual the writer has attempted to present the subject of
Protozoology in such a light that it will be of use to the zoologist who
wishes to obtain information regarding the general principles of the subject
and detailed knowledge of parasitic forms, and to medical men and
veterinarians who are chiefly concerned with those Protozoa with which
they have professionally to deal.

The investigations of Smith and Kilborne on the parasite of Texas
fever of cattle and its transmission by ticks; those of Laveran, Grolgi,
Ross, and Grassi on malarial parasites of man and birds, and their
carriage by mosquitoes ; and the researches of Bruce, who demonstrated the

vii

viii PREFACE

trypanosome nature of the African cattle disease, nagana, and its con-
veyance by tsetse flies, opened an entirely new field of enquiry which has
led to a most extensive study of parasitic Protozoa. The thousands of
papers on the subject which have been published during the past twenty
or thirty years are scattered in numerous journals, many of which are
difficult to obtain by any worker, and impossible by those who are
stationed in parts of the world where good libraries are not available.
Many workers have spoken to the writer of the difficulties associated
with this separation from literature, and it has been largely a desire to
remove at least a part of these difficulties that has led him to undertake
the present work on the subject of Protozoology.

The book deals with all groups of parasitic Protozoa, as well as with
free-living forms, though the latter have been dealt with very briefly,
except in the case of those which are coprozoic in habit and may lead
to confusion with parasitic organisms. The part played by invertebrates
in the transmission of certain parasitic Protozoa of vertebrates necessitates
the examination of invertebrates in order to trace the life-history of any
parasite which may develop in them. As knowledge of the parasites
which are peculiar to these invertebrates is essential if errors are to be
avoided, they have accordingly received special attention.

In reviewing the extensive literature on the subject of Protozoology
it has been necessary to criticize many statements and claims which have
been made, but, in expressing his own views, the writer hopes that he
has explained clearly the reasons which have led him to their adoption,
and that he has treated fairly those records which appear to him to be
of doubtful value.

One of the chief difficulties associated with the production of a manual
like the present one is that hardly a week passes without the publication
of some paper of importance; but an earnest endeavour has been made
to incorporate all new and essential data as they appeared, so that as the
book goes to press in its final form a fair claim can be made that it is
as complete as it reasonably can be. Rapid advances are being made
in the elucidation of the methods of transmission of kala azar and Oriental
sore, and there is every prospect that very soon the sand fly will be in-
criminated definitely as the vector of one or both of these diseases. The
treatment of general paralysis by inducing in patients attacks of malaria
is leading indirectly to the discovery of many interesting facts regarding
the development of malarial parasites. The recently described method
of cultivation of intestinal amoebse is assisting in the solution of many
problems connected with the life-history of these organisms. Three
hitherto supposed coccidia of man have been shown to be nothing more
than parasites of edible fish which are passing casually through the human

PREFAGE ix

intestine. Observations such as these are continually producing changes
in our outlook, so that, however quickly a book is produced, it is bound
to be out of date in certain respects when it appears. Nevertheless, the
greater part of the information which will be found on its pages is well
established, and will be lasting, so that it is sincerely hoped that the
two volumes will provide a reliable record of our knowledge up to the
beginning of 1926.

As the study of spirochaetes is intimately related to that of the Pro-
tozoa, especially in connection with blood w^ork, a section is devoted to
their consideration, though it is definitely maintained that they are not
Protozoa.

Many Protozoa which have affinities with those which produce diseases
in man and domestic animals have been found in the blood of other
vertebrates and in the intestines of invertebrates. A worker who dis-
covers such an organism has considerable difficulty in ascertaining if it
has been previously noted. To meet this difficulty a host list of the
blood-inhabiting parasites of vertebrates and one of the flagellates of
invertebrates have been compiled, and it is hoped they will be useful
references.

As difficulties associated with nomenclature, the accuracy of which
is of such importance, are constantly occurring, the International Rules
of Zoological Nomenclature, which many workers have little opportunity
of consulting, have been included.

The practical side of Protozoology has been constantly kept in mind,
as well as the difficulties which beset the path of those engaged in its
study. A special section deals with methods of investigations. This
is not intended to be a complete account, but merely a guide for the use
of those who already have a working knowledge of laboratory technique.

Authorities for all statements made in the text have been given, and
the exact references will be found in the list of publications at the end
of the book. Practically all these have been consulted in the original,
and with very few exceptions every reference has been verified. The
greater part of this laborious work has been carried out by Miss I. M.
Bellis, Librarian to the Wellcome Bureau of Scientific Research, whose
knowledge of languages and scientific publications has been invaluable.
The writer is glad to have this opportunity of acknowledging his in-
debtedness to her for the great care she has taken with this and many
other parts of the work. The writer has constantly had the assistance of
Mr. Cecil Hoare, Protozoologist to the Wellcome Bureau of Scientific
Research. Many intricate questions have been discussed with him, and
his sound judgment, together with his careful and critical reading of
the proofs, has been a great asset. For many of the drawings, both

X PREFACE

black and white and coloured, the writer is much indebted to Mr. B.
Jobling, now on the staff of the Wellcome Bureau of Scientific Research.
His knowledge of biology combined with his artistic skill has enabled
accurate copies and many original drawings from preparations to be
produced. The writer's thanks are also due to his sister, Miss M. G.
Wenyon, Assistant Secretary to the Royal Society of Tropical Medicine
and Hygiene, who has read carefully the final proofs, and has been a
means of detecting errors which otherwise would have marred the pages.

The writer is indebted to Professor Nuttall, F.R.S., Quick Professor
of Biology and Director of the Molteno Institute of Cambridge ; Dr. A. G.
Bagshawe, C.M.G., Director of the Bureau of Hygiene and Tropical
Diseases ; Professor Warrington Yorke of the Liverpool School of Tropical
Medicine; Professor A. E. Boycott, F.R.S., of University College, London;
Lieut.-Col. W. P. MacArthur, D.S.O., O.B.E., of the Royal Army Medical
College; Dr. Keilin of the Molteno Institute, Cambridge; and the Councils
of the Royal Society of Tropical Medicine and Hygiene and the Royal
Institution for the loan of blocks. He is also indebted to Mr. Clifford
Dobell, F.R.S., for the use of his original diagrams of Aggregata eberthi
and his drawing of the cyst of Balantidium coli, and for permission to
reproduce figures from his publications.

Much assistance has been derived from many of the books on Proto-
zoology or one or other of its branches, particularly Doflein's Lehrhuch
der Protistenkunde, Laveran and Mesnil's Trypanosomes et Trypanoso-
miases, Laveran's Leishmanioses, Minchin's An Introduction to the Study
of the Protozoa, Dobell's The Amoehce Living in Man, Dobell and O'Connor's
The Intestinal Protozoa of Man, and many others; but of all the publica-
tions, apart from original articles, the careful reviews by Professor Mesnil
which have appeared regularly in the Bulletin de VInstitut Pasteur since
1902, and those by various writers in the Tropical Diseases Bulletin,
have been most helpful. Any worker who wishes to keep abreast of
the times cannot do better than to read one or both of these excellent
bulletins with regularity.

Finally, the writer wishes to express his thanks to Mr. N. B. Kinnear
of the British Museum, Natural History, for the trouble he has taken
in checking the host list of birds, and to all the many others who have
been ever ready to give him valuable assistance.

C. M. W.
London,
June, 1926.

CONTENTS TO VOL. I

PART I
genp:ral description of the protozoa

Organization and Life-History of the Protozoa
Typical Division of the Metazoan Nucleus:

Mitotic Division ------

Meiotic or Reducing Division - - - -

greneral morphology of the protozoa

Encystment amongst the Protozoa

The Protozoan Nucleus -----

Multiplication amongst the Protozoa

Syngamy amongst the Protozoa - - - -

Nuclear Division amongst the Protozoa -

Behaviour of Chromosomes during Syngamy

Blepharoplasts, Parabasals, and Kinetoplasts -

Physiology op the Protozoa ....

Life-History of Protozoa -----

Immunity in Protozoal Infections - - - -

Action op Drugs in Protozoal Infections

Status op the Protozoa in the Animal Kingdom

PAGE

1

11

15

18

46

49

62

71

90

108

115

123

133

138

150

152

PART II

SYSTEMATIC DESCRIPTION OF THE PROTOZOA, WITH SPECIAL

REFERENCE TO PARASITIC AND COPROZOIC FORMS - 153

Classification of the Protozoa
PHYLUM: PROTOZOA - - - -

A. SUB-PHYLUM . PL ASMODROM A
1. CLASS: RHIZOPODA

1. Order : AMCEBIDA

2. Order: HELIOZOA

3. Order : RADIOLARIA -

4. Order : FORAMINIFERA

5. Order : MYCETOZOA -

- 155

- 156

- 160

- 160

- 165

- 165

- 166

- 168

- 172

XB^T-^

Xll

CONTENTS TO VOL. I

Systematic Description of the Order Amcebida -

1. Family : amcebid.e -

Genus : Amoeba -
Genus : Hartmannella
Genus : Vahlkampfia
Gemis : Sappinia -

Amceb^ of Plants

Gemis : Pelomyxa
Genus : EntamcEba
Genus : Endamoeba
Genus : Endolimax
Genus : lodamoeba
Genus : Dientamoeba

Diagnosis of Intestinal Amceb^ of Man -----
Action of Drugs on Intestinal Amceb^ - . - . -

AmCEB^E cultivated from F^CES COPROZOIC AUCEBJE . - -

Statistics of Intestinal Amceb^ of Man -----

2. Family : paramcebid^

3. Family : dimastigamcebid^

4. Family : rhizomastigid^ -

II. GLASS : MASTIGOPHORA -----

coprozoic mastigophora -------

Invasion of Blood-Stream by Intestinal Mastigophora
Flagellates which may contaminate Blood and Organ Smears
Division op Mastigophora into Sub-Classes and Orders

\. SUB-CLASS : PhyXomsisWgma. -

2. SUB-CLASS : ZoomsisWgmdi - - - -

A. Monozoic Forms :

1. Order : PROTOMONADIDA -

(1) Sub-Onler : Eumonadea

(2) Sub-Order : Craspedomonadea

Systematic Description of Genera and Species of Sub-Order Eumonadea

1. Family : monadid^

A. Monadid;ie with One Flagelluni -
Genus : Oikomonas

PAGE

173

175

175
175
177
180

181
182
182
235
238
242
248

250
254
257
259

260
260
266

268

270
271

272
274

282
285

285
286

288

288
288
289
289

CONTENTS TO VOL. I xiii

PAGE

Blackhead of Tukkeys -_-__-_ 291

Genus : Craigia - - - 294

Genius : Rhizomastix - - 296

Gemis : Proleptomonas - - 297

B. Monadidoe with Two Flagella - 298
Genus : Heteromita - - 298
Genus : Dimastigamoeba - - 302
Genus : Spiromonas - - 302
Genus : Phyllomitus - - 305
Genus : Costia - - - 305

C. Monadidse with Three Flagella - 306
Genus : Enteromonas - - 306

D. Monadidae with Four Flagella - 308
Genus : Tetramitus - - 308

E. Monadidse with More than Four

Flagella - - - 311

2. Family : trtpanosomid^ - - 312

Relation of Various Types to One Another - - - - 312

Orientation and Origin of Different Types - - . . 316
Subdivision into Genera - - - - - - -318

Cytology of Trypanosomes and Allied Flagellates - - - 321

Method of Reproduction -----.. 336

SyNGAMY ------... 339

Encystation -------.. 34X

General Features of the Life-History ----- 342

Classification - - - - -- - - - 344

Systematic Description of Genera and Species - - - . 343

Genus : Leptomonas - - 348

Genus : Crithidia - - - 355

Genus : Herpetomonas - - 363

Other Members of the Genera Lejytomonas, Crithidia, and Herpetomonas - 369

Forms found in Body Cavity and Salivary Glands - - 370

Roubaud's Genus Cercoplasma - - - - - - 370

Roubaud's Genus Cystotrypanosoma - - - - - 372

Patton's Genus Bhynchoidomonas . - . . . 374

Chatton's Observations on the Trypanosomid^ of Drosophila - 377

Genus : Phytomonas - - 382

Inoculation of Insect Trypanosomid^ into Vertebrates - - 392

CONTENTS TO VOL. I

PAGt

Inoculation of Insect Trtpanosomid^ into Invertebrates - - 395

Genus : Leishmania - - 396

Genus : Trypanosoma - - 442

Methods of distinguishing Trypanosomes- . . _ _ 444

Classification of Trypanosomes -.--.. 456

Curative Action of Drugs and Sera in Trypanosomiasis - - - 459

Systematic Description of Species ------ 463

Group A. Trypanosomes which develop in the Posterior Station

IN THE Invertebrate ----- 463

I. Trypanosomes of Eodents, Cheiroptera, Insectivora, Eden-
tata, Carnivora, and Monkeys - - - 463

(fl) Trypanosomes of Rodents - - . - 453

(6) Trypanosomes of Cheiroptera - - - - 479

(c) Trypanosomes of Insectivora - - - - 482

(d) Trypanosomes of Edentata . - - - 482

(e) Trypanosomes of Carnivora - - - . 433
(/) Trypanosomes of Monkeys - - - . 433

Genus : Endotrypanum - - 485

II. Trypanosome of Man in South America - - - 486

III. Non-Pathogenic Trypanosomes transmitted by Species of
Tabanus, Melojyhagns, or Other Blood-Sucking Arthro-

PODA - - - - - - - - 498

Trypanosomes of Cattle ----- 498

Trypanosomes of Sheep . - - - . 502

Trypanosomes of Antelope ----- 507

Group B. Trypanosomes which develop in the Anterior Station
IN the Invertebrate, or have become secondarily
adapted to Direct Passage from Vertebrate to

Vertebrate - - - - - - - 507

I. Pathogenic Trypanosomes transmitted by Blood-Sucking

Arthropoda ------- 507

General Remarks on Pathogenic Trypanosomes - - - 507

Relation to Game - - - - - - - - 508

Mechanism of Infection - - - - - - - 511

Identification of Trypanosomes in Tsetse Flies - - - 515
Experimentally proved Vectors of Pathogenic Trypanosomes of

Africa - - - - - - - - -517

CONTENTS TO VOL. I xv

PAGE

Passage of Trypanosomes from Parent to Offspring - - - 518

Trypanosomes as Filter Passers ------ 520

Classification of Pathogenic Trypanosomes - - - - 521

1. Pathogenic Trypanosomes transmitted by Species of

Glossina - - - - - -524

(a) Trypanosomes which develop in the Stomach,

Proboscis, and Salivary Glands of Tsetse Flies

— Polymorphic Trypanosomes - - - 524

(b) Trypanosomes which develop in the Stomach

AND Proboscis of Tsetse Flies — Monomorphic
Trypanosomes without Flagella - - 552

(c) Trypanosomes which develop only in the

Proboscis of Tsetse Flies — Monomorphic Try-
panosomes PROVIDED WITH FlAGELLA - - 559

2. Pathogenic Trypanosomes transmitted by Species of

Tabanus OR Other Blood-Sucking Arthropoda —
Monomorphic Trypanosomes provided avith Flagella 565

II. Pathogenic Trypanosomes passing directly from Verte-
brates TO Vertebrates - . - - _ 574

III. Trypanosomes of Birds - - - - - - 577

IV. Trypanosomes of Land Keptiles, including Crocodiles - 581

V. Trypanosomes of Aquatic Vertebrates transmitted by
Leeches :

1. Trypanosomes of Aquatic Eeptiles - - - 585

2. Trypanosomes of Amphibia - - . - 538

3. Trypanosomes of Fish - - - . . 599

3. Family : bodonid.e - - 607

4. Family: prowazekellid.e - 611

5. Family : embadomonadid^ - 615

6. Family : ciiilomastigid^ - - 620

7. Family : cercomonadid^ - - 629

A. Cercomonadidse with One An-

terior Flagellum:

Genus : Cercomonas - - 629

B. CercomouadidiE with Two An-
• terior Flagella:

Gemis : Trimitus - - - 633

C. Cercomonadidse witli Three An-

terior Flagella:

Genus : Tricercomonas - - 634

8. Family : cryptobiid^ - - 636

CONTENTS TO VOL I.

9. Family :

TRICHOMONADID^

Genus

; Trichomonas

Genus ,

• Gigantomonas

Genus .

.- Ditrichomonas

Genus .

; Eutrichomastix

Genus

; Janickiella

Genus :

Trichomitus

Genus .

• Devescovina

Gemis .

.• Foaina -

Genus .

• Retortamonas

Genus .

• Protrichomonas

Genus .

■ Polymastix

Genus .

• Hexamastix

Genus :

• Cochlosoma

10. Family :

DINENYMPHID^ ■

PAGE

646

648
670
670
671
675
676
677
677
677
679
680
681
681

2. Order: HYPERMASTIGIDA - - - 682

3. Order : CYSTOFLAGELLATA - - 684

B. Diplozoic Forms :

4. Order : DIPLOMONADIDA - - - 684

Genus : Hexamita - - 684

Genus : Giardia - - - 691

Genus : Trepomonas - - 711

(\ Polyzoic Forms :

5. Order : POLYMONADIDA - - - 714
Frequency or Intestinal Flagellate Infections of Man - - 714

III. Oi:^*S.S'. CNIDOSPORIDIA - - - - - 716

Order ; MYXOSPORIDIIDA - - - 718

Subdivision of the Myxosporidiida - - 724

Detailed Description of Certain Species - 728

Order : MICROSPORIDIIDA - - - 734

Subdivision of the Microsporidiida - ' - 737

Detailed Description of Certain Genera and

Species . . . . . 740

Certain Microsporidiida of Blood-Sucking

Arthropoda and Neinatoda - - 748

Supposed Microsporidiida in Rabies and En-
cephalitis of Rabbits . . . 754

Order : ACTINOMYXIDIIDA - - - 756

Parasites of Undetermined Position . . . . . 750

SARCOSPORIDTA .---... 76O

GLOBIDIUM - - - - - - . . 769

HAPLOSPORIDIA - - - - - - - 773

RHINOSPORIDIUM - - - - - - - 776

PART I
GENERAL DESCRIPTION OF THE PROTOZOA

PROTOZOOLOGY >. ^:,; x

PART I

GENERAL DESCRIPTION OF THE PROTOZOA

ORGANIZATION AND LIFE-HISTORY OF THE PROTOZOA.

During the latter part of the seventeenth century Antoni van
Leeuwenhoek (1632-1723), working with a simple microscope, investigated
free-living Protozoa and studied the parasitic forms in the intestine of
frogs. He also found that he himself was infected with one of these
organisms, which, as Dobell (1920) has pointed out, was probably the
well-known Giardia intestinalis. The great Dutch microscopist thus not
only discovered free-living Protozoa, but was the first to study parasitic
forms, and he can be justly regarded as the father of Protozoology and
of its more specialized branch. Medical Protozoology. Since Leeuwen-
hoek's day an ever-increasing number of investigators, availing themselves
of the experiences of those who had gone before them and of the steady
improvement in the microscope, have brought to light an enormous
assemblage of minute living creatures, many of which, like the bacteria,
were quite beyond the scope of the simple magnifying apparatus used by
Leeuwenhoek and other early workers. These minute organisms absorb
nourishment and grow, and finally, as in higher animals, reproduce by
detaching portions of their bodies to form those of their offspring, while
any remaining portion dies. It may be that the entire body of the parent
is used up in the production of progeny, or only a small portion of it, as
in higher animals, but in either case, extending from parent to offspring,
there is a continuity which entitles all living creatures to be regarded as
immortal in that a j^ortion at least of the living matter is handed on from
one generation to another, unless accidental death prevents reproduction.
The fact that all the complex mechanisms of life are concentrated in
these minute portions of living matter has led observers to seek in them .
an explanation of the phenomena of life in general. A single organism
may be kept under observation for the whole of its individual existence,
and the visible changes undergone by it during its life, which is terminated
by its final production of offspring, may be actually followed under the
microscope. It seems evident that beyond the scope of the microscope
there exist organisms, or stages of development of visible organisms,

3

4 OEGANIZATION AND LIFE-HISTORY OF PROTOZOA

which cannot be seen — the ultra-microscopic viruses. Dark field illumina-
tion has done much to facilitate the study of these forms, but, as yet,
the exact nature of the numerous minute objects which it has revealed
in every fluid, and which are in constant motion (Brownian movement),
has not been satisfactorily determined, so that at present it is in many
cases impossible to decide whether they are actually living organisms or
granules of inanimate material.

The study of microscopic organisms has revealed the fact that, in
their method of nutrition, some of them resemble plants and others
animals. On the basis of this physiological distinction it has been the
custom to regard them as belonging to one of two main groups — the
Protophyta and the Protozoa. The study of the former has been relegated
to the botanist, and that of the latter to the zoologist. Though some of
these organisms show undoubted affinities with the algse and higher
plants and others with animals, there exists a miscellaneous assemblage
of indeterminate forms which cannot be placed legitimately in either
group. Accordingly, it is safer to regard them all as belonging to one
large group, the Protista, the study of which is known as Protistology,
as first suggested by Haeckel (1866). Without being able to define
accurately the limits of either group, it is nevertheless convenient to
regard the Protista as comprising the two subdivisions of the Protozoa
and the Protophyta. In the case of the former, nutrition is effected by
the ingestion of preformed proteid material, either as solid particles or
in solution. The Protophyta, on the other hand, nourish themselves
like plants on comparatively simple chemical compounds, and when
possessing chlorophyll or some similar substance, make use of the carbonic
acid of the liquid in which they live. Very frequently they secrete around
themselves capsules of cellulose. A typical Protist consists of a small
portion of cytoplasm and a nucleus which contains as its most essential
constituent a substance called chromatin. The contents of the nucleus
are separated from the cytoplasm by a nuclear membrane. Other bodies
may be present in the cytoplasm, but these, at least as definite visible
structures, are not essential to life.

Amongst the existing Protista the most primitive forms are possibly
the bacteria, spirochsetes, and allied organisms, which in many cases do
not appear to possess definitely constituted nuclei, though granules of
a substance which some observers have identified with chromatin are
present in the cytoplasm. Alexeieff (1924fl) maintains that it is not
chromatin, and that this substaiice is absent from bacteria. These
forms, however, are in most cases so minute that accurate information
regarding their cytological structure and life-histories is difficult to obtain.
It can, at any rate, be safely affirmed that those Protista which are most

CELL THEOEY 5

highly developed and most complex in structure possess definite nuclei,
and the small particle of cytoplasm with its included nucleus of which
the body of each is composed is regarded by most biologists as a cell on
account of its resemblance to the cells of higher animals and plants.

The term "cell" was first introduced for the cellulose capsule or wall
which encloses the portions of cytoplasm of which the higher plants are
built up. It was later realized that the wall itself was merely a sup-
porting structure, and that the cytoplasm within it was in reality the
living material. Accordingly, the term "cell" was then applied, not to
the cell wall, but to its cytoplasmic contents. The latter consists typically
of a small mass of cytoplasm containing a single nucleus. When it was
discovered that the tissues of higher animals were also built up of similar
elements or units, the term "cell" was applied to them also. It soon
became evident that, in the case of many microscopic organisms, the entire
body consisted of a similar mass of cytoplasm containing a nucleus, and
the resemblance of these to the cells of higher animals and plants gave
rise to the view that these organisms were single cells, and the distinction
between unicellular and multicellular animals was drawn. This con-
ception, which was first clearly expounded by Schwann (1839), has been
generally accepted, though Dobell (1911) considers it erroneous. He
believes that an amoeba, for instance, is as much an entire organism as
one of the higher animals, and that though the latter may be regarded
as being multicellular, as a result of the division of its cytoplasm and
nucleus into cells, the former should be considered as a non-cellular
organism, and not a unicellular one, since it corresponds, not to any
single cell, but to all the cells which compose the body of the multi-
cellular organism.

When it is realized that amongst the numerous cells which compose the
body of one of these higher animals there are many wandering cells, such
as macrophages, which behave in all essential respects like amoebae, in that
they form pseudopodia, ingest solid proteid material of various kinds, and
multiply by fission, it is difficult to resist the conviction that such a cell has
a definite claim to be regarded as an individual organism like an amoeba
itself. Furthermore, it has been clearly demonstrated that very minute
portions of the tissues, consisting of groups of cells, or even single cells of
higher animals, can be artificially cultivated, and that they will live and
multiply indefinitely provided they are given a continuous supply of suit-
able nutriment. From these culture experiments it seems clear that the
cell, which forms but a part of the entire multicellular animal, is capable of
nourishing itself and reproducing as a single organism. Another illustration
of the power of independent existence and multiplication of isolated cells
of multicellular animals is seen in malignant disease. In this condition

6 ORGANIZATION AND LIFE-HISTORY OF PROTOZOA

certain cells acquire the power of continuous and rapid multiplication,
so much so that they become to all intents and purposes parasites, which
bring about the death of their host. These cells can be inoculated from
animal to animal indefinitely, and in them they will continue to multiply,
just as trypanosomes do in successive passages in experimental animals.

An ovum, according to the non-cellular view, is a non-cellular indi-
vidual, which at once becomes cellular when segmentation occurs. The
cells, each of which gives rise to only part of the individual which will
normally develop from the ovum, are nevertheless potential individuals
themselves, as is demonstrated by the fact that if the cells are separated
from one another artificially, as in the well-known experiments with sea-
urchin eggs, each is capable of giving rise to a complete embryo.

It seems evident that the cells of higher animals are capable of
independent life provided the proper environment exists. Under
natural conditions all the cells of the body contribute to the production
of this environment, which is so delicately balanced that separated and
isolated cells invariably die unless the proper environment is present or
is artificially provided, as in the culture experiments just mentioned.
If the environment necessary for the continued life of cells in the body
can be kept constant, the cells will survive and reproduce indefinitely,
but if some of the cells fail to fulfil their part in the production of this
environment, the other cells will suffer and death will result. It may be
said that any single cell of a Metazoon is living in a condition of symbiosis
with all the other cells. Without entering further into the discussion,
for purposes of this work it is sufficient to follow the more orthodox view
and to regard the Protista as unicellular organisms, or single cells which
still lead a completely independent existence, and the multicellular
organisms as groups of cells which work together for a common end. The
latter have become so completely interdependent that their power of
separate existence has been largely lost. Yet in many features, such as
their structure, mode of life and method of reproduction, nuclear division
and syngamy, they retain the unmistakable characteristics of their uni-
cellular ancestors. It must not be supposed that the ancestors of either
the multicellular or unicellular organisms any longer exist. The primitive
forms from which they may be supposed to have originated have probably
long since disappeared in the course of evolution. The Protista of the
present day, as well as the individual cells of higher animals and plants,
have undoubtedly evolved along different lines and acquired certain charac-
teristics which their common ancestors did not possess. Biologists are
nevertheless justified in still regarding the portion of cytoplasm with its
nucleus as a cell, whether it occurs amongst the Protista or the Metazoa
and Metaphyta, in spite of the fact that the cells of each group may now

CELL KEPRODUCTION 7

possess distinctive features of their own. The cell may be justly regarded
as an individual, whether it is one of the Protista or only part of the body
of a multicellular organism. Tn the latter case it must be admitted
that a number of individuals have remained united as a colony to form
a single larger individual. Of the cells of the latter, only certain ones are
destined for reproduction, as in the case of spores of Cnidosporidia,
where a group of cells is formed by division from a single cell, and of
these only one is a reproductive cell, the others dying after fulfilling other
functions. A single soldier or a regiment of soldiers may both be units
in the military sense, but the soldiers composing the regiment, though
sacrificing their individuality to some extent for the good of the individual
regiment, are as much individuals as the single soldier.

It has been clearly demonstrated that a Protozoon quickly dies if
deprived of its nucleus, and there is little doubt that the cells of higher
animals are similarly dependent on their nuclei. A single unicellular
organism may be divided into several portions, but though those which
do not contain the nucleus may exhibit movements and survive for some
time, they ultimately die, whereas any nucleated portion may re-form
itself into an entire individual which is able to continue its existence.
It is evident the nucleus plays a very important part in the life, and
metabolism of the cell. The Protozoan cell does not differ from other
cells in its capacity to absorb and digest food, and grow and increase in
size. It is able to perform spontaneous movements as a result of con-
tractions of its cytoplasm, though these are reduced to a minimum in
some cases. Finally, the cell is able to multiply, usually by a process
of binary fission, but sometimes by a process of multiple fission. In
binary fission the single nucleus divides into two parts, and this is followed
by division of the entire cell into two daughter cells. Usually, these
are approximately equal in size {equal binary fission), but it may happen
that one daughter individual is larger than the other {unequal binary
fission). When the difference in size is marked, it appears as if a small
daughter individual is separated from a much larger parent which retains
its original form, and the process is spoken of as budding or gemmation.
In the case of multi'ple fission or multiple segmentation, after the first
division of the nucleus the body of the organism does not immediately
divide, but the two daughter nuclei again divide to form four nuclei, and
these may again divide to give rise to eight. After a number of nuclei
have been thus produced by repeated divisions, the body of the organism
segments into, or more accurately buds off, a number of portions cor-
responding to the number of nuclei. This method of multiple fission of
cells, which more correctly should be called multiple gemmation, occurs
in higher animals as well as in the Protozoa, amongst which it is seen

8 ORGANIZATIOX AXD LIFE-HISTORY OF PROTOZOA

typically in the parasitic Sporozoa, and is known as schizogony. Usually
there is a residue which does not participate in the formation of the buds:
it is discarded as a residual body which quickly disintegrates.

During the life-history of many cells a sexual process occurs from
time to time. The advantages gained from such a process, which is
called synga7Mj, are far from being clearly understood. In its simplest
form it consists in the complete union of two cells and fusion of their
nuclei. The uniting cells are known as gmnefes, and the single cell resulting
from the union is a zygote. The zygote proceeds to multiply by binary
or multiple fission.

The process of syngamy must be distinguished from another type of
union which sometimes occurs. Two or more cells may fuse to produce
a multinucleate cytoplasmic body known as a plasmodiiitn. In this

/ ■ .- -

■■^..^' ^ \ ,,*: * /

If ^

A B

Fig. 1. — Diagram of Cells. (Original.)

A. Metazoan cell. The cytoplasm contains a centrosome and a nucleus with a nucleolus.

B. Protozoan cell {Entamoeba). The cytoplasm, differentiated into ectoplasm and endoplasm,

contains a nucleus with central karyosome and numerous food vacuoles. No centrosome is
visible.

manner plasmodia containing many hundreds of nuclei may be formed.
The nuclei show no tendency to unite with one another, as they do in
syngamy, and after the plasmodial phase has existed for some time
segmentation into. uninucleate cells takes place.

The typical cell, wherever it occurs, consists of the two essential parts
— cytoplasm and nucleus (Fig. 1). Each of these is a mixture of
substances of highly complex chemical constitution, the reactions of which
produce the phenomena characteristic of living matter. The cytoplasm
appears to be made up of at least two substances, one of which is suspended
in the other in the form of an emulsion. The nucleus, which is limited
by a nuclear membrane, consists of a substance called nuclear sap, which
occupies interstices in a more solid material. The latter, when viewed in

STRUCTURE OF CELL AND NUCLEUS 9

optical section, has the appearance of a network, and is known as the
linin network, of which the nuclear membrane may be regarded as a special
development. Upon this network, and on the nuclear membrane in the
form of granules or larger masses, is arranged another substance, the
chromatin, which has a strong affinity for certain stains. It is generally
regarded as the most important constituent of the nucleus, and this is
borne out by the fact that nuclear division takes place by an elaborate
process known as 7nitosis, which results in an equal sharing of the chromatin
between the daughter nuclei. In the nucleus of the Metazoan cell there
is usually present a conspicuous body known as the nucleolus. It is devoid
of chromatin, and when nuclear division takes place it passes to one of
the daughter nuclei, the other daughter nucleus forming a new nucleolus.
A very similar body exists in the nuclei of certain Protozoa (Opalina),
and it passes to one of the daughter nuclei when division takes place. In
other Protozoa, as, for instance, in Karyolysus and Hepatozoon, a similarly
achromatic body divides at nuclear division, each daughter nucleus re-
ceiving half (Fig. 35). When such a body occupies a central position in
a Protozoan nucleus it is known as a haryosome, and it has been generally
assumed that it is composed largely of chromatin. It is becoming in-
creasingly evident, however, that the karyosome may be actually devoid
of chromatin, and the supposition that in certain nuclei the entire chro-
matin may be concentrated in the karyosome is a very doubtful one. The
nucleus is often regarded as consisting of two substances — the achromatic
and the chromatic material. The achromatic material, including the
nuclear membrane, linin network, nuclear sap, and other bodies (karyo-
some, nucleolus) which are sometimes present, undoubtedly comprise
several distinct substances, some of which, at any rate, are able to give
rise to chromatin, for the quantity of chromatin in the nucleus varies from
time to time, and increases with its growth. Ahother important constituent
of the cell, which as a rule only becomes visible during nuclear division,
is the centrosome (Fig. 1, A). It is commonly present in the cells of
Metazoa, but it is not so frequently seen in the Protozoan cell. Repro-
duction of a cell by binary fission or multiple segmentation is always
preceded by division of the centrosome, if one is present, followed by
division of the nucleus, which in most cases takes place by mitosis. It
is during nuclear division that the nature of many of the constituents of
the nucleus first comes to light, and for this reason it will be necessary to
consider mitosis, as it occurs typically in the Metazoan cell. During
mitosis there are formed, mainly out of the chromatin, certain bodies
known as chromosomes, which are constant in number for each species of
animal, the same number appearing at each succeeding nuclear division.
There is some evidence that in the resting, or more accurately the non-

10 ORGAXIZATIOX AND LIFE-HISTORY OF PROTOZOA

dividing nucleus, though the chromosomes are no longer visible as
individual units, they still exist as separate entities. During syngamy,
when two gametes unite and their nuclei fuse, the chromosomes of the two
uniting nuclei enter the zygote nucleus, so that, unless a reduction is made
in the number of chromosomes, at each succeeding union the chromosome
number would be doubled. Usually the number of chromosomes in the
gamete nuclei is only half that of the nuclei of other cells of the body, and
the process by which this reduction is brought about is known as the
reducing division, or meiosis.

Though in the vast majority of cases it is recognized that the nuclei
of daughter cells are the products of division of the nucleus of a parent
cell, it is supposed that occasionally amongst the Protozoa nuclei may be
formed from extra-nuclear chromatin granules which appear in the

A B

Fig. 2. — Formation of Nuclei from the Chromidial Body in Arcella vulgaris

( X ca. 300). (After E. Hertwig, 1899.)

A. Normal individual with two nuclei and mass of chromidial substance.

B. The chromidial substance is breaking up and nuclei are being formed from the fragments.

cytoplasm (Fig. 2). It seems to be an undoubted fact that chromatin
material in the form of granules may leave the nucleus and take up a
position in the cytoplasm. This has been described as taking place, not
only in Metazoan cells, but also in the Protozoa. Such granules of
chromatin, which occur in the cytoplasm, are known as cTiromidia. It
is not, however, an easy matter to determine the true nature of granules
which occur in the cytoplasm, and it has not infrequently happened that
identical granules or material have been described as chromatin by one
observer, and as some other substance by another. There seems little
doubt that both in the case of Metazoan cells and Protozoa, chromidia
do not occur so frequently as some have supposed. When the question
of the origin of nuclei from these chromidia is considered there is still

CHROMIDIA 11

greater uncertainty. Some observers believe that the chromatin granules
or chromidia in the cytoplasm may, under certain conditions, arrange
themselves in groups, each of which becomes transformed into a nucleus.
It is difficult to avoid the impression that most, if not all, of the records
of nuclei arising, as it were, by crystallization of chromidia are the result
of misinterpretations, and that the appearances on which the conclusions
have been based might be accounted for in another and more probable
manner. In all cases in which accurate and continuous observation of
reproducing cells has been possible, daughter nuclei have been found to
arise only by division of pre-existing parent nuclei. A classical instance
of this kind is seen in Arcella vulgaris, a binucleate shelled amcBba
(Figs. 2 and 79). Like many other shelled amoebae, in addition to the
true nuclei, Arcella vulgaris contains a mass of material which, on account
of its affinity for certain chromatin stains, is supposed to be of chromidia!
nature, and is called the chromidial body. It was claimed by Richard
Hertwig (1899) and other observers that at certain phases of development
the two existing nuclei degenerate and disappear, and that numerous
secondary nuclei are formed from the chromidial body. Schirch (1914) has,
however, shown that in some cases, at least, the numerous nuclei which
are present result from repeated divisions of the two which occur in the
normal individual. It seems not improbable that the so-called chromidial
body of Arcella and its allies is not really of chromatin nature, but consists
of a special material which may be concerned with the development of
the shell, which is a characteristic feature of these shelled amoebae.

TYPICAL DIVISION OF THE METAZOAN NUCLEUS.

1. Mitotic Division.

The Protozoan nuclei divide in a variety of ways, and it is probable that
amongst them the more primitive types of nuclear division will be found.
There is every transition between what is little more than a simple con-
striction of the nucleus into two parts {amitotic division) and the elaborate
method of division known as mitosis or haryokiyiesis, in which chromosomes
are formed and divided in such a manner that the chromatin of the nucleus
is equally distributed to the daughter nuclei. The division of nuclei by
mitosis occurs most typically in the cells of higher animals and plants,
and it was in their cells that the details of the process were first elucidated.
The terms employed for the different structures and the various stages
which occur were first applied to their nuclei, and were used subsequently
for the corresponding stages which occur during the division of Protozoan
nuclei. Mitosis in its typical form is characterized by the formation
from the chromatin and achromatic material of the nucleus of a number

12 DIVISION OF METAZOAN NUCLEUS

of usually elongate structures called chromosomes, each of which splits
longitudinally into two daughter chromosomes, one of which passes into
each daughter nucleus. This division and separation of chromosomes is
associated with the formation of the achromatic figure which arises in con-
nection with a structure called the centrosome situated in the cytoplasm
outside the nucleus. The whole process can be regarded as talcing place
in a number of stages known as the prophase, metaphase, anaphase, and
telophase (Fig. 3).

PROPHASE. — The centrosome, which is a spherical structure at the
centre of which is a deeply staining granule, the centriole, divides into two
parts which separate from one another. As they separate, the two
daughter centrosomes remain connected by fibres which are arranged as
a spindle, the spindle fibres, while similar fibres radiate into the cytoplasm
from the centrosomes (Fig. 3, B and C). Each centrosome with its
radiating fibres constitutes the aster. Within the nucleus the linin net-
work becomes arranged in what has been supposed to be a long coiled
thread in which the chromatin granules are embedded. This thread is
known as the spireme. Structures such as nucleoli and karyosomes may
break up and disappear, and any chromatin they contain becomes arranged
in granular form with the rest of the chromatin of the nucleus upon the
spireme. Finally, the nuclear membrane disappears, while the spireme
segments into a number of chromosomes (Fig. 3, C). It seems probable
that the conception of the spireme as a single long coiled thread is not
correct, and that from its first appearance it consists of a number of long,
intercoiled, separate segments which become distinct as they contract to
form the chromosomes, the name given to the separate parts into which the
spireme was supposed to divide. With disappearance of the nuclear
membrane the separate chromosomes, each of which can often be seen to
consist of two closely united parallel threads, arrange themselves in a
looped fashion round the equator of the spindle, and in the plane of this
equator in such a manner that the bend of each loop is directed towards
the centre and the two ends away from it (Fig. 3, D and E). The chromo-
somes, which have become shorter and thicker at the equator of the spindle,
form the equatorial plate.

METAPHASE — The chromosomes, which are now arranged as the
equatorial plate, and each of which may consist of two closely apposed
parallel structures, divide longitudinally into daughter chromosomes,
which commence to move towards the pole of the spindle (Fig. 3, E).

ANAPHASE.^The daughter chromosomes now separate completely
into two groups at the poles of the spindle. The natural interpretation
that they are drawn there by the action of the fibres of the spindle to which

MITOSIS

13

they are attached does not appear to be a satisfactory explanation of their
movements (Fig. 3, F).

Fig. 3. — Diagram of Nuclear Division by Mitosis. (After Agar, 1920.

A. Resting nucleus with centrosome.

B. Early prophase with dividing centrosome.

C. Middle stage of projjhase: appearance of spindle and dividing chromosomes.

D. Late prophase.

E. Metaphase with divided chromosomes as equatorial plate.

F. Anaphase: separation of daughter chromosomes.

G. Telojihase: aggregation of chromosomes.

H. Completion of nuclear and cell division and reconstructed daughter nuclei.

TELOPHASE. — The spindle fibres gradually disappear, the nuclear
membrane re-forms around the chromosomes, which gradually become
transformed into the linin network and chromatin characteristic of the

14 DIVISION OF METAZOAX NUCLEUS

original nucleus (Fig. 3, G and H). The centrosome remains outside the
nucleus, the fibres of the aster becoming no longer visible.

The centrosome appears to be the ruling factor in the process, and
the appearance of the aster and spindle fibres can be interpreted as visible
indications of some force which is being exerted. It must be remembered,
however, that in the mitotic division of the nuclei of the higher plants,
as also that of many Protozoa, though all the stages of mitosis seen in the
animal cell occur, definite visible centrosomes are not present. The
fibres of the aster and spindle radiate from an apparently structureless
area, which may be regarded as a potential centrosome. An important
fact to be noted is that for any particular species the number of chromo-
somes present in the nucleus of any cell of the body is constant. In the
much studied cells of Ascaris ntegalocephala, of which there are two
varieties, the number of chromosomes is two or four respectively. In
man it is twenty-two, while in other animals it may be much higher
than this. Each species of animal has thus a definite chromosome
number.

The chromosomes which are formed in any nucleus are not necessarily
all alike in size or form. It is often found that they can be grouped in
pairs, the members of each pair resembling one another more closely than
those of other pairs. The members of each pair are known as homologous
chromosotnes. During the progress of mitotic division the chromosomes
are at first elongate structures, but there is a tendency for them to shorten,
so that at the stage when the equatorial plate is formed they may be
roughly spherical. Though these alterations in size take place, all the
chromosomes are similarly aft'ected. Their relative size and shape remain
the same, so that the homologous pairs can still be recognized.

During the telophase, when the chromosomes of the daughter nuclei
are becoming transformed to reproduce the structure of the resting nucleus,
it can sometimes be seen that the chromatin and achromatic material
of each chromosome is occupied in reconstructing a j^articular portion
of the nucleus. When chromosomes are re-formed at the next nuclear
division, the material in each portion concentrates again into a chromo-
some. In these cases it appears as if there is a permanent separation of
the constituents of each chromosome, even when the nucleus is in the
resting condition. This has given rise to the doctrine of the continuity
of chromosomes, which supposes that each chromosome is a permanent
structure, which, though changing its form, is present as an individual
unit even during the period when the nucleus is not dividing. The proof
of this, however, is exceedingly difficult to obtain, and it must be regarded
at present as little more than a plausible theory.

The chromosomes themselves are not homogeneous bodies, but consist

MEIOSIS 15

of a number of small granules of chromatin of varying size, tlie chromo-
meres, embedded in an achromatic matrix. Very frequently homologous
chromosomes resemble one another very closely as regards the arrange-
ment and variations in size of the chromomeres which they contain.

2. Meiotic or Reducing Division.

A sexual process or syngamy, which consists in the union of two cells
together with fusion of their nuclei, occurs in higher animals and plants,
and it was amongst them that the nuclear changes associated with the
process were first studied. Attention has been drawn to the fact that
the chromosome number for each individual species is constant, so that
it must be evident that, if the nuclei of two cells unite, the number of
chromosomes in the resulting zygote nucleus, which is known as the
synkarion, would be double the usual number. This increase in number
does not actually occur, for the nuclei of the uniting cells or gametes
contain only half the number of chromosomes possessed by other cells.
The reduction is brought about by a special type of mitotic division of
the nucleus during the formation of the gametes (Fig. 4). When the
chromosomes arrange themselves on the spindle fibres as the equatorial
plate, instead of splitting into daughter chromosomes as in ordinary
mitosis, they become separated into two groups, one of each pair of
homologous chromosomes passing to each group (Fig. 4, C and D). In
this way the daughter nuclei contain half the number of chromosomes
possessed by the parent nucleus. The reduction in the number of chromo-
somes in the nuclei of the gametes is effected either at the last cell division
which gives rise to gametes, or at the one immediately preceding it. The
process is known as meiosis, and the nuclear division the meiotic division
or reducing division. When the gametes unite and their nuclei fuse, the
synkarion therefore contains the usual number of chromosomes. The
gamete with half the number of chromosomes is said to be haploid as
regards its chromosomes, while the original cell from which the gametes
were derived and the zygote resulting from their union, which contain
both chromosomes of each homologous pair, are said to be diploid.

Amongst the higher animals, as also frequently amongst the Protozoa,
the gametes can be distinguished as male and female. The former, in
the vast majority of cases, are smaller than the latter, so that the gametes
can be distinguished as 7nicroga?netes and macrogametes. The micro-
gamete of a Metazoon is known as a spermatozoon and the macrogamete
as an ovum. The microgametes are derived from a large number of cells
called spermatogonia, which, like all the other cells of the body, contain
the normal or diploid number of chromosomes. One of these cells in-

16

DIVISION OF METAZOAN NUCLEUS

creases in size and becomes the prmiary spermatocyte. By division two
secondary spermatocytes are produced, and each of these again divides,
giving rise to four spermatids, which become directly transformed into
microgametes or spermatozoa. It is during the first or second of these
two divisions that meiosis occurs and the number of chromosomes is
reduced. When it occurs it is seen that, as the chromosomes arrange

Fig. 4. — Diagram of Meiosis or Reducing Division of a Nucleus with Four
Chromosomes. (Original.)

A. Showing two pairs (dotted and lined) of homologous chromosomes and commencing formation

of spindle.
B and C. Syndesisor conjugation of homologous chromosomes.
D and E. Separation of the conjugated homologous chromosomes.
F. Formation of nuclei, each with half the original number of chromosomes; one of each pair of

homologous chromosomes has entered each nucleus.
In ordinary mitosis the chromosomes at C, instead of separating, would divide, so that two pairs

of homologous chromosomes would pass to each daughter nucleus.

themselves at the equator of the spindle, the individuals of each pair
of homologous chromosomes are closely applied to one another, so that
at first inspection it might be thought that only half the number were
present. This approximation of the chromosomes of each pair is known
as the conjugation of the chromosomes or syndesis, and it is supposed that

MEIOSIS 17

exchange of material takes place between them. As division of the nucleus
proceeds, separation of the conjugating chromosomes occurs, and the
two chromosomes of each pair pass to opposite poles of the spindle. It
will be seen, therefore, that in this division there has been no splitting of
the individual chromosomes as occurs in ordinary mitosis, but merely
a separation of two chromosomes which have come together temporarily
in syndesis. The number of chromosomes in the daughter nuclei are
thus half the original number. If the reduction division occurs at the
division of the primary spermatocyte, then the division of the nucleus
of the secondary spermatocyte is not a reducing one, the chromosomes
splitting longitudinally in the usual manner, so that the number is main-
tained. If the reduction occurs at the division of the secondary sperma-
tocyte, then the division of the nucleus of the primary spermatocyte is
of the ordinary type. In any case, the spermatids which become sper-
matozoa or gametes have half or the haploid number of chromosomes.

In the case of the female cell similar changes occur. Cells called
oogonia grow into primary oocytes. A primary oocyte divides to give
rise to two cells, which are, however, unequal in size. The large one is
the secondary oocyte, and the small one the^^rs^ polar body. The secondary
oocyte divides into two cells, which are again unequal in size. One of
these is the ovum, and the other the second polar body. The first polar
body, which corresponds to a secondary oocyte, may itself divide into
two cells. The nuclear changes which occur in these divisions are similar
to those which occur during the divisions of the spermatocytes described
above, so that the number of chromosomes in the ovum is half the number
present in the o5gonia. There is this difference, however: Whereas each
primary spermatocyte gives rise to four spermatozoa, each primary oocyte
gives rise to one large ovum and two small polar bodies, or three if the
first polar body divides. By this arrangement the cytoplasmic part of
the ovum is increased at the expense of that of the polar bodies, which do
not proceed further to develop. Another difference between the ovum
and spermatozoon is that the centrosome of the ovum has disappeared,
though that of the latter has persisted.

When conjugation or syngamy occurs, the nucleus of the microgamete
or spermatozoon unites with that of the macrogamete or ovum by a
process known as karyogamy to produce the nucleus or synkarion of the
zygote, which again has the diploid number of chromosomes arranged
in homologous pairs. The centrosome of the microgamete becomes the
centrosome of the zygote.

The pairs of homologous chromosomes of the zygote can be recognized
through all the subsequent divisions of the cell down to the moment
when the new adult individual again produces spermatocytes or oocytes.

I 2

18 MORPHOLOGY OF PROTOZOA

One chromosome of each pair was originally derived from the spermato-
zoon and the other from the ovum, and the two, or at least their descen-
dants, have remained distinct during all the subsequent divisions of the
nuclei. When the reducing division or meiosis occurs, the conjugation
of the individuals of each pair of chromosomes takes place, and it is
supposed that at this moment there is interchange of material between
them, and that transmission of hereditary characters is accomplished.

It will be shown below that amongst the Protozoa the production of
gametes may be associated with similar changes in the nuclei, the gametes
possessing half or the haploid number of chromosomes. On the other
hand, cases are known in which no reduction in the number of chromo-
somes takes place during gamete formation. It results that the zygote
contains double or the diploid number of chromosomes. In these cases
the first division of the zygote nucleus is a reducing division, the two
daughter nuclei again having the haploid number. In the one case the
reduction affects the gametes and occurs before syngamy, while in the
other it affects the two daughter cells, resulting from division of the
zygote, and occurs after syngamy.

GENERAL MORPHOLOGY OF THE PROTOZOA.

Of the Protozoa there are a very large number of genera and species,
some of which are free-living forms, while others lead a protected existence
within the bodies of higher animals. The latter have undoubtedly been
derived from the former, and have become modified to such an extent in
adaptation to their hosts that, generally speaking, they are no longer able
to live apart from them. As practically every higher animal is liable to
harbour in its body one or more Protozoa, it is evident that the number
of parasitic species is very large indeed. It should be remembered,
however, that to understand properly the parasitic forms the study of the
free-living Protozoa should not be neglected.

It is customary to regard parasites in general as degenerate organisms,
but though it is true they may have lost many of the organs possessed by
their free-living ancestors, they may have developed others in their place,
and reveal the same degree of adaptation to their environment as free-
living forms. Though a parasite may have lost certain structures which
it no longer requires, it digests its food, grows, and reproduces with all
the complexity exhibited by those which still possess them. It seems
incorrect to regard as in any sense degenerate an organism which is so
completely adapted to its environment as are the majority of parasites.
In fact it might be legitimately argued that if an organism retained
structures for which it had no further use, this would indicate a loss of

SHAPE AND SIZE OF BODY 19

adaptability to environment which in itself should be regarded as a sign
of degeneration.

SHAPE AND SIZE OF THE BODY.— The Protozoa vary considerably
in size, some of them being easily detected with the naked eye. Many
of the ciliates and gregarines can be seen as white specks or elongate
filaments, while certain multinucleate amoeboid organisms may be several
centimetres in diameter. The majority of Protozoa, however, are so small
that they cannot be seen without magnification. The adult individuals
of any species may vary considerably in size amongst themselves, and
there may be marked differences in size between the mature and immature
stages of development.

Protozoa may be of almost any conceivable shape, and the exact form
of the body may be regarded as a direct adaptation to their mode of life
and environment. When living in fluid media, unless the shape is deter-
mined by a relatively tough outer membrane or a skeletal support, there
is a tendency for the organism to assume the spherical form. Amoebae,
in which an outer membrane is entirely absent or represented by an
exceedingly fine pellicle, are spherical unless temporary contractions of
the cytoplasm or pressure of any body against which they come in contact
or over which they are moving overcomes the physical forces to which
they are subject (Fig. 5). So soon as relaxation occurs the spherical
form is resumed. In the majority of Protozoa the body is definitely
elongated even in a condition of repose, and it is evident that this form
is retained without any effort on the part of the organism itself. This
is due in most cases to the development of an elastic outer layer of cyto-
plasm, which retains its shape unless this is temporarily altered by pressure
or the contractions of the cytoplasm (Fig. 6). This outer layer of the
cytoplasm or periplast may attain a high degree of complexity. It may
be so tough, as in many of the Mastigophora and Ciliata, that the shape
of the body is practically constant.

In the case of certain Mastigophora, like Cercomonos, which are adapted
to a creeping mode of existence as well as a swimming one, the body is
constantly changing its shape when it is moving over a surface, with a
tendency to the assumption of an elongate form during progression
through a fluid (Fig. 7). In the majority of Mastigophora and Ciliata
which swim through liquids the body is elongated, and may even have
a spiral form, when movement is associated with revolution about the
longitudinal axis (Figs. 143 and 509). Certain Mastigophora and Ciliata
which lead a swimming existence as well as a creeping one upon the
surface of various objects are frequently flattened dorso-ventrally. In the
swimming forms there is a tendency for one end of the organism to be
more pointed than the other. Certain Protozoa become permanently

20

MORPHOLOGY OF PROTOZOA

attached to objects by means of filaments, and in such cases a cone-
shape is developed, the filament of attachment arising from the apex of
the cone (Fig. 19). Amongst truly parasitic Protozoa the body may be
a motionless'sphere, as in the growing phases of coccidia within the cyto-
plasm of cells; on the other hand, those which live in fluids in the body
spaces and are endowed with powers of
active movement, like free-living forms,
vary considerably in shape.

Amongst the Rhizopoda the body is
usually either globular or irregular in

-Cfi

Fig. 5. — Amceba proteus ( x 200).
(After Leidy, 1879.)

Fig. 6.
1,000).

—Eiiglena viridis ( x ca.
(After Doflein, 1916.]

C, Contractile vacuole ; Ch, chromato-
phores; R, reservoir; S, stigma.

shape, and there is no differentiation between an anterior and posterior
end or a dorsal or ventral surface (Fig. 5). In certain forms, however,
the body is protected by a shell, through an aperture in which pseudopodia
are extruded for purposes of locomotion and capture of food. In such
forms, of which Arcella and Difflugia are examples, it is possible to con-

SHAPE AND SIZE OF BODY

21

sider the aperture which is applied to the surface over whicli the organism
is moving as ventral in position, so that a dorsal and a ventral surface
can be distinguished (Fig. 8). Many Mastigophora are definitely elongate,

Fig. 7. — Cercomonas longicauda ( x 2000) : Changes in Shape undergone by
A Single Individual during Twenty Minutes' Observation. (Original.)

and locomotion takes place in the direction of the flagellate end (Fig. 6).
In these it is evidently possible to distinguish an anterior from a posterior
end. When a mouth aperture or cytostome is present, it is usually near
the anterior end, but slightly to one
side of the terminal flagella. The
surface nearest to which the cytostome
lies may be regarded as the ventral
surface, in which case it becomes
possible definitely to orientate the
organism. In the case of such a
flagellate as Trichomonas (Fig. 26) it
is legitimate to speak of the anterior
flagellated extremity of the body, the
posterior extremity through which the
axostyle protrudes, the ventral surface
^near which the cytostome is placed,
and the dorsal surface which is provided with the undulating membrane
and its basal fibre. This orientation becomes complicated to a certain
extent by the fact that a torsion or twisting of the body towards a spiral

Pig. 8. — Difflugia constricta : A Shelled
Rhizopod from Pond Water
( X 660). (Original.)

The shell is strengthened by adherent grain
of sand.

22 MORPHOLOGY OF PROTOZOA

form may occur. Thus in Trichomonas itself the undulating membrane
takes a slightly spiral course round the body, though its general tendency
is to be on the dorsal surface. Amongst the Ciliata this differentiation may
be carried to a high degree of complexity. In a few forms such as Prorodon
teres (Fig. 24) the cytostome is at the extreme anterior end of the body, and
the cilia pass in longitudinal rows from it to the posterior end. Though it
is possible in these cases to distinguish an anterior and posterior end, there
is actually no dorsal or ventral surface. In other forms the cytostome
has moved from its terminal position, and it at once becomes possible to
regard the surface on which the cvtostome is situated as the ventral one

VJJ.

m^f-^^

m

Fig. 9. — Stylonychia mytilus : Side and Ventral Views ( x ca. 250).
(From Lang, 1901, Slightly Modified.)

The side view shows the ciliate resting on a surface by means of the foot-like cirri formed by
fusion of groups of cilia. The dorsal cilia are few in number. There is a central contractile
vacuole with two excretory canals leading to it.

The ventral view shows the macronucleus in division and two daughter micronuclei. The
V-shaped peristome is bordered on its outer edge by a row of membranes passing round the
anterior end of the ciliate and leading to the cytostome at the apex of the V. A row of cilia
borders the other edge of the peristome, within which is a longitudinal membrane. The
contractile vacuole and parts of the canal are seen as clear areas.

(Fig. 14). In the majority of the free-living Ciliata there are definite
dorsal and ventral surfaces. These are most conspicuous in those forms
which lead a creeping mode of life, owing to the loss of cilia on the
dorsal surface, and the development of cirri and membranelles, through
the fusion of groups of cilia, on the ventral surface (Fig. 9). The cyto-
stome is on the ventral surface; it is not median in position, but displaced
to one side. In the case of attached forms such as Vorticella (Fig. 19)

POLYMORPHISM 23

the ciliated area in which the cytostome lies may be regarded as the ventral
surface, and the filament of attachment as a development from the apex
of the cone-shaped dorsal surface.

POLYMORPHISM.— It has to be recognized that amongst the Protozoa
variations in the shape and form of the body occur at different
stages of development. Such a variation is not a characteristic feature
of the Rhizopoda, for the smallest individuals of any species have essen-
tially the same body form as the fully-grown larger ones. Amongst the
Dimastigamoebidse at certain stages of development one or more flagella are
formed. Though it is purely an arbitrary matter whether the flagellate
stage is considered to be the adult form or not, these amoebae are definitely
polymorphic (Figs. 119 and 120). As all Rhizopoda, however, are able
to encyst under certain conditions, the encysted stage has to be recognized
as another form in which any particular amoeba may occur. Amongst
the free-swimming Ciliata, again, the smallest individuals differ little
except in size from the fully -grown largest forms.

Protozoa which only show a slight degree of variation in body form
during their life-cycle are termed monomorphic, to distinguish them from
polymorphic forms, which show this to a marked degree. This poly-
morphism is well illustrated by the development of the Suctoria (Fig. 532).
Amongst these Protozoa th e attached adult buds off a small ciliated embryo,
which, after leading a free-swimming existence for a time, finally attaches
itself, loses its cilia, and grows into the adult, which is provided with
sucking tentacles. As the ciliated stage is only of a temporary nature,
and is small when compared with the tentacled stage, it is regarded as the
embryo. Amongst the Sporozoa there is a high degree of polymorphism
associated with their complicated cycles of development. In the case of
the malarial parasites, for instance, the organism passes through a constant
series of changes of form (Fig. 391). The minute amoeboid organism
within the red cell grows into the schizont, which breaks up into elongate
merozoites, which again become amoeboid forms in other cells. Some
merozoites develop into gametocytes of two types, which can be distin-
guished from the schizonts. In the mosquito the gametocytes change
in character and produce elongate vermicular zygotes, which pass through
the stomach wall and develop into oocysts, which again produce a large
number of minute sickle-shaped sporozoites, which differ in character from
the merozoites. In this case, as in other Sporozoa, there is a high degree
of polymorphism, as exhibited by a constant series of changes in the size
and form of the body. Very frequently there is a polymorphism associated
with the occurrence of a sexual process and the formation of gametes.
In the gregarines the gametes which unite may be exactly alike, in which
case the process is known as one of isogamy. On the other hand, in certain

24 MORPHOLOGY OF PROTOZOA

gregarines the uniting cells difier from one another (anisogamy), so that
there is a degree of polymorphism as regards the character of the gametes
(Fig. 482). It may happen that the individual which gives rise to gametes
of one type differs from that which gives rise to gametes of the other type.
This differentiation may extend further back in the life-history, so that
it is possible to recognize two distinct types of reproducing individual,
each with its particular characters (Fig. 341). The individuals of one
series may eventually, after a period of multiplication, give rise to gametes
of one type, while those of the other series give rise to gametes of another
type. In such cases it might be supposed that one was dealing with two
distinct organisms, each reproducing its kind. The fact that the gametes
produced by the one unite with those produced by the other proves that
the two series belong to one polymorphic species. This condition is known
as one of sexual dimorphistn, a term which is also employed in a more
general sense to indicate the occurrence of individuals of any species
which can be distinguished as male and female.

Though all these variations in form, which occur as a result of growth,
complicated life-cycles or the sexual process, are examples of polymorphism,
the term is often employed in a more restricted sense. When it has been
decided which stage of the organism is to be regarded as the adult form,
it may be found that the adults resemble one another very closeh^, in which
case the organism is said to be monomorphic. Thus, in the case of
trypanosomes the commonly observed forms in the blood of an animal
may vary very slightly. In these cases the organism is termed a mono-
morphic trypanosome, examples of which are Trypanosoyna evansi and
T. congolense (Figs. 227 and 234). In other cases, as, for instance, Try-
panosoma brucei, it may be possible to distinguish in the blood of an animal
several distinct types — long thin, intermediate, and stumpy trypanosomes
— and forms with or without free flagella (Fig. 225). On this account
T. brucei is regarded as a polymorphic trypanosome. If, however, the
whole life-cycle in the vertebrate and invertebrate hosts of such a form as
T. lewisi, which at certain phases appears monomorphic, is taken into
consideration, it will be found to exhibit as great a degree of variation as
in the polymorphic trypanosomes (Fig. 197).

It seems clear, therefore, that the term " polymorphism " is incapable of
exact definition. Strictly speaking, no Protozoan is monomorphic, while
all are polymorphic. Those which are considered monomorphic show
only a slight degree of variation, while those which are polymorphic show
the variations, but to a greater extent. Any organism may be regarded
as polymorphic because it differs at different stages of its growth and life-
history, or it may be considered as polymorphic because the individuals
which have all reached any particular stage do not resemble one another

RACES 25

very closely. Human beings may be regarded as polymorphic because
the child differs from the adult, or they may be considered polymorphic
because the adults differ amongst themselves. It is in the latter sense that
the term is commonly employed in connection with trypanosomes. It
must be recognized, however, that the trypanosomes which are regarded
as being polymorphic may not all be in the same stage of development.
There is evidence which points to the fact that the shorter stumpy forms
of T. brucei or T. gambiense are the result of growth from the long slender
forms which are present in the blood at the same time (Figs. 222 and 225).

RACES. — Amongst Protozoa, as amongst human beings, there occur
different races of one and the same species. The individuals of one race
differ from those of another in size, shape, rate of multiplication, and other
characters. Each race breeds true to its type to a large extent, so that
even after long periods of multiplication the same differences are observed
in the resulting progeny. On this account it often becomes a matter of
difficulty to decide whether two different forms are merely races of one
species or are actually different species. Thus, in the case of Entarnoeha
histolytica there appear to be several races which can be distinguished from
one another by the average size of the cysts they produce (Fig. 10).

Many researches have been conducted on the race question in species
oi Paramecium, Difflugia, and other Protozoa, especially by Jennings. It
has been observed that the characters of any particular race tend to remain
constant, so that there is considerable difficulty in understanding how these
races arose in the first instance. Evidence has, however, been obtained by
Jennings (1916) in the case of Difflugia corona and by Middleton (1915)
for Stylony cilia, which proves that after long periods of multiplication
definite inheritable variations do occur in the descendants of a single
individual, and this quite apart from any sexual process. It therefore
seems probable that if the observations were continued for a sufficient length
of time, it would be possible to separate from the descendants of a single
individual various races which would be as distinct from one another as
the naturally occurring races. If this were not so, it would be difficult to
understand how evolution could take place at all.

A practical point which arises from the knowledge which has been
acquired regarding races of Protozoa is that the separation of species,
on account of comparatively slight variations in size, is a very questionable
procedure. The literature dealing with parasitic Protozoa contains
numerous instances of the establishment of new species merely because
the dimensions differed slightly from those of a form previously described.

Another type of race peculiarity occurs amongst the Ciliata. It was
shown by Dawson (1919) that Oxytricha kymenostoma, which normally has
both a macronucleus and a micronucleus, may occasionally have the

26

MORPHOLOGY OF PROTOZOA

macronucleus alone. Such an amicronucleate race was cultivated by him
for several years, during which regular multiplication by fission took place.
Abortive attempts at conjugation appeared to be made, but the process
was never completed. Landis (1920) has studied a similar race oi Para-
mecium caudatum, and Patten (1921) one of Didinium nasutum, while
Woodruff (1921a) has described amicronucleate races of OxytricJia fallax
and TJrostyla gra?idis.

P^IG.

10. — Cysts of E. histolytica from Three Distincx Races ( x 2,200).
(After Wenyon and O'Connor, 1917.)

1-3. Race with exceptionally large cysts

7-9. Race with small cvsts

4-6. Race with usual type of cyst.

CYTOPLASM.— The cytoplasm of the Protozoan cell does not differ
in any essential respect from that of cells of multicellular animals. As to
the nature of its minute structure many theories have been advanced.
That which seems to be most satisfactory is Biitschli's view that cytoplasm
is of the nature of an emulsion consisting of at least two substances,
one of which in the form of minute globules is suspended in the other,
which forms the septa between the globules. In optical section, the

CYTOPLASM 27

substance between the globules has the appearance of a network of fibres.
Embedded in these apparent fibres or septa are granules of various kinds
and sizes. The cytoplasm commonly contains vacuoles, which are
spherical spaces containing a material which is more fluid than the con-
stituents of the cytoplasm itself. Very frequently within the vacuoles
are food particles which the organism has ingested. In such cases the
vacuoles are known as food vacuoles or digestive vacuoles, and into them
are secreted acid ferments capable of transforming the food into substances
suitable for assimilation by the cytoplasm. The products of digestion are
gradually absorbed into the cytoplasm, and any residue is got rid of by the
vacuole approaching the surface of the body and discharging its contents
into the medium in which the organism is living. The vacuole is then
no longer visible. In the majority of free-living Protozoa there are one
or more vacuoles, which are known as contractile vacuoles or pulsating
vacuoles. Such a vacuole is near the surface of the body, and when fully
formed contains a clear fluid. By a sudden contraction the contents of
the vacuole are discharged through the surface of the body, and the vacuole
disappears. Very soon, however, a minute vacuole reappears at the same
spot. It gradually increases in size owing to the flow of liquid into it,
sometimes along definite channels. When it has attained its full size,
expulsion of the contents again takes place. These vacuoles appear to be of
an excretory nature, and the intervals between the contractions vary with
the temperature and other conditions. For some reason not clearly under-
stood, contractile vacuoles are frequently absent in parasitic Protozoa.

Within the cytoplasm of many Protozoa there occur various structures
which are to be regarded as secretions of a skeletal nature. In the
Heliozoa, for instance, radially arranged rod-like supports for the pseudo-
podia are formed (Fig. 75), while in many of the Radiolaria complicated
fenestrated shells of a spherical or other shape are secreted in the cyto-
plasm (Fig. 78). These internal structures are not to be regarded as part
of the cytoplasm itself, but bear the same relation to it as the external
shells and coverings, which are sometimes formed around the organism
for protective purposes of a permanent or temporary nature.

A very noticeable feature of the cytoplasm of Protozoa is its differ-
entiation into an ectoplasm and an endoplasm. The former is of tougher
consistency and more hyaline than the endoplasm, and forms a superficial
layer of varying thickness enclosing the more liquid and granular endo-
plasm. The endoplasm, even when the organism is at rest, appears to be
constantly streaming in various directions. The different vacuoles and
bodies, and even the nucleus itself, are constantly changing position as a
result of the currents in the endoplasm. It is in the endoplasm that the
various vacuoles and internal skeletal structures occur, while the ectoplasm

28 MORPHOLOGY OF PROTOZOA

may become highly differentiated. A tough covering to the body, which
may be elaborately marked, is often developed from the ectoplasm, while
it is from this layer that the various permanent organs of locomotion such
as flagella and cilia originate. The ectoplasm also secretes the various
external coverings, such as shells and cysts. In the simpler Protozoa,
like the amoeba? and flagellates, the ectoplasm is merely a thin layer of
clear cytoplasm surrounding the endoplasm. It appears to be only
slightly more resistant than the endoplasm. In the more highly organized
ciliates and gregarines the ectoplasm is highly developed, and itself consists
of several distinct layers. It is a resistant membrane which enables the
organism to retain its shape. In any case, the most superficial layer of the
ectoplasm forms a delicate limiting membrane, the periplast. The surface
of the ectoplasm may be perfectly smooth, or it may be raised into a series
of longitudinal ridges. In other cases it is roughened, or may even develop
a series of symmetrical markings. In the amcebse, many of the simpler
flagellates, and many parasitic protozoa, the ectoplasm forms a complete
layer over the surface of the body, and when solid food is ingested this is
taken in at any part of the body. A particle comes in contact with the
ectoplasm which is gradually raised up round it, and finally closes over
it, so that the object, together with a certain quantity of liquid, is included
in a vacuole which sinks into the endoplasm. In other cases the solid food
particles are ingested in a similar manner at one particular spot on the
body surface. This occurs typically in certain flagellates, where solid food
appears to be ingested only at the base of the flagellum. In other flagel-
lates at this point there is a small excavation or pit in the ectoplasm into
which solid food is taken (Figs. 26 and 33). At the bottom of this pit the
food particle sinks into the endoplasm, and is included in a vacuole. This
depression is frequently of a permanent nature. In association with it
there may be special developments of the organs of locomotion which
create currents in the medium, so that food particles are directed into it.
In Chilomastix one of the flagella lies in a groove, at the posterior end of
which food particles enter the cytoplasm (Fig. 69). The opening in the
ectoplasm, which sometimes is capable of being opened and closed, is known
as the cytostome, while the funnel-shaped pit or tube leading from it to the
endoplasm is the oesophagus or cytopharynx.

As already pointed out, the residue from the digestion of food material
within the food vacuoles is discharged through the surface of the body.
This may occur at any point on the body surface, but in the Ciliata there
may be a permanent opening in the ectoplasm, the cytopyge, which, how-
ever, is usually only visible when a food vacuole discharges its contents
at the posterior end of the body (Fig. 512).

In some ciliates the cytostome is a simple opening on the surface of the

CYTOPLASMIC INCLUSIONS 29

body, but the region round the cytostome (peristome) may be modified
in various ways. There may be a ciliated groove leading to the cytostome
(Fig. 70), or a disc-like area upon which cilia are arranged in a spiral manner
(adoral zone of cilia) may be developed. These cilia are often continuous
with others within the cytopharynx. In the Peritrichida, like Vorticella
and Carchesium, the area round the cytostome is sunk in the form of a
funnel-shaped depression, the vestibulum, the opening of which may be
completely closed by contractions of the cytoplasm. Within the ves-
tibulum is found the cytostome itself, while the food vacuoles and con-
tractile vacuole also discharge their contents into it (Fig. 528).

CYTOPLASMIC INCLUSIONS.— In association with the ingestion of food
and metabolism, granules, globules, and crystals of various kinds may
appear in the endoplasm. These are quite distinct from the partially
digested food in the food vacuoles, though they result from food meta-
bolism. Many Protozoa having affinities with the plants and possessing
chlorophyll are able to form starch, which occurs in the cytoplasm as
characteristic starch granules. They are commonly present in Euglena
and other similar forms. Another substance allied to starch is known as
paramylum. Fat globules are seen especially amongst the Radiolaria
within the inner capsule. They also occur in the marine flagellate
Noctiluca, and it has been suggested that they assist these organisms to
float. Doflein (1910) has noted that, in old cultures of Trypanosoma
rotatorium the flagellates may contain droplets of fat. Another substance
which is of common occurrence in the cytoplasm is glycogen, or a closely
allied substance which was called paraglycogen by Biitschli. These have
a strong affinity for iodine, which colours them an intense brown. Glycogen
is present in gregarines, certain ciliates, and very commonly in the encysted
forms of amoebae and flagellates (Plate II., p, 250). The iodophilic body
which occurs in the encysted stage of lodamceha hiUschlii has given rise to
its generic name. A substance which is of wide distribution amongst
the Protozoa is volutin. It is usually seen in living organisms as globules
of a greenish refractile material which takes a yellow colour in iodine.
Owing to the fact that it may stain deeply with chromatin stains, it has
often been regarded as chromatin. Some observers maintain that it is
actually a forerunner of chromatin. Volutin is often present in the
cytoplasm of trypanosomes and other flagellates, and appears as dark
red granules when they are stained with Romanowsky stains. It commonly
occurs in hsemogregarines and many Sporozoa, as also in amoebae and
ciliates. A substance which may be allied to volutin is seen in the
chromatoid bodies which are present in the cysts of some intestinal amcebae.
They occur so frequently in the encysted forms of Entamoeba histolytica
in the form of bars that they are highly characteristic of this species

30 MORPHOLOGY OF PROTOZOA

(Fig. 96). They are less often seen in the encysted stages of Entamoeba
coli. Like the glycogenic or iodophilic body in the encysted form of
lodamoeba hutschlii, they disappear in the course of a few weeks after
escape of the cysts from the intestine, apparently serving as a supply of
nourishment for the enclosed amoebae.

Another type of cytoplasmic inclusion is the chromatophore, which is
characteristic of many plant-like flagellates grouped amongst the Phyto-
mastigina (Fig, 130). These are bodies which contain various pigments
known as chromatophyll. When green it is called chlorophyll, and when
red haematochrome. As in plants, these bodies enable the organism to
utilize the carbonic acid of the medium in which they live. It has been
show^n that the chromatophores multiply by fission in the cytoplasm, as
also do certain granules known as pyrenoids which may be present in the
chromatophores. It has been surmised that the chromatophores may be
symbiotic organisms living in the cytoplasm.

In the process of ingesting solid food many Protozoa actually ingest
other forms, or even their own species, either in the free or encysted
condition (Fig. 99). The writer has seen a large vacuole in Entamoeba
muris of the mouse filled with actively motile Triclioynonas. The in-
testinal amoebae of man frequently ingest the encysted forms of other
intestinal Protozoa. In many cases the ingested organisms are killed
and digested, but this is not always the case. Instances are known in which
amoebae and even ciliates may have their cytoplasm riddled with vacuoles
in which smaller amoebae or flagellates occur. These may eventually
escape apparently unharmed by their stay in the cytoplasm of another
organism. Protozoa are also liable to invasion by bacteria. Such a
condition approaches, and may actually be, one of parasitism. Instances
of true parasitism are seen in the invasion of the body of Parameciimi by
the Suctorian Sphcerophrya pusilla (Fig. 534), and of various intestinal
flagellates and amoebae by Sphcerita, a vegetable organism which often
resembles a cluster of large cocci (Fig. Ill, 4). The inclusion of smaller
organisms within the cytoplasm of larger ones has always to be remembered,
especially when a process of multiplication by the production of daughter
individuals within the cytoplasm of a parent is considered. The nuclei
of such forms may be mistaken for nuclei belonging to the host. A method
of reproduction of Pelomyxa palustris, a large multinucleated amoeba,
by the production of flagellated forms within vacuoles in its own cytoplasm
has been described by Schirch (1914). It seems not improbable that this
was an instance in which an amoeba had ingested, but failed to kill, a number
of flagellates which were present in the medium. Doflein (1916) mentions
an instance in which the cytoplasm of a ciliate, Stentor ca'rulevs, included
numerous small flagellate organisms.

LOCOMOTOR AND PREHENSILE ORGANS 31

ORGANS EMPLOYED IN LOCOMOTION AND CAPTURE OF FOOD. -The

simplest organs which are used for purposes of locomotion are the
pseudopodia, characteristic of the Rhizopoda or amoebae (Fig. 5). They
are simply processes of cytoplasm which are formed from the surface of
the body. A small elevation of the ectoplasm occurs at any point, and
this gradually increases in size till the endoplasm also takes part in its
formation. When it has reached a certain size it may be withdrawn
gradually, and another formed in some other direction. On the other hand,
it may increase steadily in size till the whole body of the organism flows
into it. In this manner, by the regular production of pseudopodia, an
amceba may move from one spot to another. It is by means of pseudo-
podia passed around any object that food particles are ingested. The
movements and changes in shape associated with the formation and with-
drawal of pseudopodia are termed amoeboid movements, w^hich are exhibited
typically by the amoebae. Certain flagellates as well as Sporozoa, such as
the malarial parasites, may also move in this manner. The pseudopodia
may be blunt finger-like processes of a lobose type, or they may be relatively
long, thin, and tapering, and of a filose type. The long narrow filose
pseudopodia may remain separate from one another, or they may become
united by lateral anastomoses, so that an organism possessing many of
them appears to be surrounded by a fine network of cytoplasm, as in the
Foraminifera (Fig. 72). In the case of the Heliozoa and Radiolaria, the
filose pseudopodia are more permanent structures, known as axopodia,
and are supported by radially arranged axial rods secreted by the endo-
plasm, or formed as outgrowths from the central granule (Fig. 51).

Flagella and cilia are more permanent organs of locomotion. The
former are characteristic of the Mastigophora, and the latter of the
Ciliophora. They are long, narrow, whip-like processes which are capable
of performing vindulating or lashing movements, which cause currents in
the medium and enable the organism to progress through it. A single
flagellum has essentially the same structure as a cilium, though it is usually
larger, and is capable of more violent lashing movements. Generally
speaking, the small number of flagella possessed by a flagellate fulfils
the functions of the large number of cilia possessed by a ciliate. A
flagellum, as pointed out by Alexeieff (191 le), consists of an axial filament,
for which the term axoneme, suggested to the writer by Colonel A. Alcock,
will be employed, and a thin sheath of cytoplasm (Fig. 157). The axoneme
itself takes origin in a minute granule, the blepharoplast, which is situated
in the cytoplasm, and sometimes upon the surface of the nuclear mem-
brane. The axoneme passes to the surface of the body, and there, acquiring
a thin sheath of cytoplasm, becomes the flagellum. There can thus be
distinguished an intracytoplasmic portion of the axoneme and a flagellar

32

MORPHOLOGY OF PROTOZOA

portion. For the former the name rhizoplast is often employed. When
an organism is developing a flagellum, a blepharoplast first becomes
apparent in the cytoplasm, and an axoneme is formed as an outgrowth
from it. When the surface of the body is reached, increase in length still
takes place, the axoneme pushing out a thin covering of cytoplasm. It is
probable that the axial rod of an axopodium is a homologue of the axoneme
of a flagellum.

The flagella of the Mastigophora vary in number. In the typical forms
they are not numerous. There may be only a single one, or as many as
eight. They arise most usually from the anterior end of the body, and
are directed forwards. By their lashing movements they propel the
organism through the medium. In some instances certain flagella arise
from the posterior end of the body, and are directed backwards. Thus,
in Hexamita two of the eight flagella are posterior in position, but their
axonemes can be traced through the cytoplasm to the anteriorly situated

blepharoplasts (Fig. 288). In other
cases, as in Tricercomonas and Cerco-
monas, the axonemes of the posterior
flagellum can be traced over the surface
of the body to the anterior end, where it
enters the cytoplasm and passes to the
blepharoplast (Figs. 259 and 261). In
the flagellates of the genera Trypano-
plasma and Trichomonas such a back-
wardly directed axoneme is adherent to,
or embedded in, the margin of a thin
band of cytoplasm, the undulating mem-
brane (Figs. 26 and 151). In other cases, such as Bodo, one flagellum is
directed backwards, and acts as a trailing flagellum without being attached
to the surface of the body (Figs. 21 and 33). In the case of the trypano-
somes, the blepharoplast occupies an unusual position at the posterior
end of the body. The axoneme arising from it is directed forwards, and
passes over the surface of the body or along the margin of an undulating
membrane as far as the anterior end of the body, where it either terminates
or becomes a flagellum (Fig. 28, B).

All the flagella possessed by a flagellate may be uniform as regards
length and thickness when they fulfil the same function. Frequently,
however, variations occur. In the case of E^nbadomorias, one of the two
flagella, which organisms of this genus possess, is associated with the
cytostome, and is much thicker and shorter, and performs more regular
undulating movements than the anteriorly directed one (Fig. 11).
Flagella are employed not only for purposes of progression, but also for

Fig. \\.~Enibadomonas sp. from
Culture of Intestinal Con-
tents OF Testudo argentina
( X ca. 1,500). (Original.)

LOCOMOTOR AND PREHENSILE ORGANS

33

the capture of food. A cytostome, when present, is always near the point
of origin of the flagella, one of which may be specially modified in con-
nection with the cytostome. Thus, in Chilomastix one flagellum is
permanently within the cytostomal groove, where it functions by creating
currents which assist in the capture of food (Fig. 69). The thicker of the
two flagella possessed by Emhadomonas has a similar function (Fig. 11).
As already remarked, in typical flagellates the flagella are few in
number, but there occur certain forms which possess many flagella.

Fig.

12. — Parajcenia grassii ( x 1,500).
(After Janicki, 1915.)

Fig. 13. — Holomastigoldes hertwigi

( X 320). (After Hartmann,
1910.)

These are the Hypermastigida, which occur chiefly as intestinal parasites
of white ants (Figs. 12 and 13), They stand in this respect as a connecting
link between the Mastigophora and the Ciliophora, with both of which
groups observers have classed them. Though the possession of flagella is a
characteristic feature of the Mastigophora, it must be remembered that
these organs of locomotion are not peculiar to this group. Certain forms
which are classed with the Rhizopoda, and which are amoeboid organisms,
may have flagella at certain stages of development. Similarly, amongst
the Sporozoa the microgametes are commonly supplied with one or two
I. 3

34

MORPHOLOGY OF PROTOZOA

flagella, which enable them to move about in search of the macrogametes
(Fig. 337).

As noted above, the cilia which characterize the tiliophora resemble
small flagella. They have a similar structure, and their axial fibres take
origin in minute granules situated in the ectoplasm. It seems reasonable
to suppose that the axial fibres and the basal granules of cilia are homo-
logous with the axonemes and blepharoplasts of flagella. A single ciliate
possesses a large number of cilia, which exhibit more regularity and
co-ordination in their movements than the flagella of one of the Mastigo-
phora. In some ciliates the body is covered uniformly with cilia, which,
however, are usually arranged in longitudinal
rows (Fig. 14). In other cases the cilia are
limited to special regions of the body. The
cilia may be fairly uniform in length, but fre-
quently those on the extremities of the body
and those which surround the cytostome are
slightly longer than the others. Cilia are often
continued into the cytopharynx. Sometimes,
as in Cyclidium and other forms, one posterior
cilium is much larger than the others, and forms
a kind of tail or caudal process which has very
much the same size and structure as a flagellum
(Fig. 500). Several adjacent cilia may fuse
together to form stout processes known as cirri.
These are seen typically on the ventral surface
of those ciliates (Hypotrichida) which lead a
creeping mode of existence (Fig. 9). They
function as supporting structures or legs. In
some cases, again, rows of cilia may unite to
form membranes. This occurs frequently in
the cytopharynx of certain ciliates, such as
Paramecium, Pleuionema, and others (Fig. 70).
These membranes, or membranelles as the smp.ll
ones are often named, are distinct from the undulating membranes
of Mastigophora (trypanosomes), which are thin ridges of ectoplasm,
and are not formed by the fusion of rows of cilia. The cilia on the
peristome region near the cytostome may differ little from those on other
parts of the body. On the other hand, they may be considerably
modified in character and arrangement. In many forms they are
arranged as a spiral to form the adoral zone of cilia, which are continuous
with those in the cytopharynx. The spiral may be a left-handed spiral
or a right-handed one. It may consist of only a single turn or part of one.

FiG. 14. — BaJaritidiuni ento-

ZOOn FROM THE KeOTUM

OF THE Frog ( x 650).
(Original.)

The longitudinal rows of cilia on
the surface of the body are
represented by dots.

LOCOMOTOR AND PREHENSILE ORGANS

35

or there may be as many as five complete turns. The spiral may be com-
pared with a portion of a fiat watch-spring, the cytostome being situated
at the outer end of the spiral, which lies on the peristome area in front of
the cytostome. The cilia composing the spiral generally consist of several

Fig. 15. — Various Species (jf Suctoria. (After Saville Kent. 1880-1882.)

(a) Sp^cerophrija magim feeding on ciliates ( x .300).
(c) Tokophrijalemnarum {X 100).

(h) Acinetagmndis (X 100).
(d) Discophri/a elongata (X 1.30).

parallel rows, and those of adjacent rows may unite in such a way as to
form a series of spirally arranged, fiat, tongue-like processes or mem-
branelles (Fig. 511). Within the cytostome the cilia may fuse to form one
or more membranes parallel to the axis of the cytopharynx. The general

36

MORPHOLOGY OF PROTOZOA

structure and arrangement of the cilia on the body of ciliates and the
modifications undergone by those associated with the cytostome are
features of importance in the classification and determination of the
species and genera of the Ciliata, just as the number and characters of
the flagella are of importance in the classification of the Mastigophora.

Amongst the Suctoria, which in their youngest stages are provided
with cilia, special organs for use in nutrition are developed in the adults

Pid. 16. Monosiga coiisociatum from Hay Infusion ( x 2,000). (Original.)

1-7. Free and attached individuals of varying size. 8 and 9. Encysted forms.

CFW. 15). These are known as tentacles, and each is a tubular process
of cytoplasm terminating in a disc-like sucker. The latter is applied to
food material, which is taken into the body by a sucking process. It is the
possession of these sucking tentacles which has given rise to the names
Suctoria and Tentaculifera, by which these forms are known.

Another type of structure which probably has to do with the capture
of food is the thin collar which is developed in certain Mastigophora
(Fig. 16). The cytoplasm at the anterior region of the body is raised

LOCOMOTOR AND PREHENSILE ORGANS 37

into a thin cylindrical collar or cuff round the flagelliim. The collared
forms frequently possess attachment filaments, simple or branched, and
often cup-like loricge. The collared forms are generally known as the
Choanoflagellata. Similar flagellated collar cells are found in the group
of Metazoa to which the sponges belong. In many cases it appears that
the collar is not a cylinder, but a cuff with overlapping edges.

A peculiar modification of the ectoplasm which facilitates locomotion
occurs in gregarines. These organisms are able to glide over a surface
without exhibiting any movements of contraction of the body by reason
of longitudinal ridges of ectoplasm between which a quantity of mucoid
material can be rapidly excreted. The excretion of this tenacious material

Fig. 17. — Codonosiga allioides : A Colony of Collared Flagellates on a
Branched Filament ( x 320). (From Lang, 1901, after Kent.)

causes the organism to be pushed forwards without any apparent move-
ments of the body. Similar gliding movements are often exhibited by the
merozoites or sporozoites of the Sporozoa. In the case of certain amoebae
such a gliding movement appears to be the result of constant streaming
of the cytoplasm from behind forwards, w^hile the ectoplasm in contact
with the surface remains stationary, very much as a bag of water can
be pushed along the surface of a table.

ORGANS OF ATTACHMENT.— Though the majority of the Protozoa
are free-living organisms, certain forms are able to attach themselves
temporarily or permanently to objects.

Amongst the Mastigophora there are many pedunculated forms. The
posterior extremity of the body is developed into a filament, by means

38

MORPHOLOGY OF PROTOZOA

of which fixation to various objects is brought about (Fig. 18). Such
forms are more or less permanently attached. By longitudinal division
of the attached flagellate and the continued development of the filament

Fic;. 18. — Various Attached Flagellates. (1, From Lang, 1901, after Kent;
2, From Lemmermann, 1914, after Kent; 3, After Doflein, 1916.)

1. PoUceca dickotoma {X 1,000). 2. Codonosiga botrijtis {x 1,200).

3. Amphimonas (jlohosa ( x 1,500).

from the posterior end of the body complicated branched filaments are
developed (Fig. 17). Sometimes the end of the branch is continued
round the organism as a cup-like expansion or lorica, in which it lives

ORGANS OF ATTACHMENT

39

(Fig. 18, i). Similarly, amongst the Ciliata filaments of attachment, either
simple or branched, may be developed. In some cases, as, for example,
Vorticella, the filament contains a contractile thread, by means of which
it can be suddenly coiled up in a spiral manner and the ciliate withdrawn
when it is subject to adverse stimuli (Fig. 19).

Amongst parasitic Protozoa, many gregarines are provided with
special organs of attachment. The young organism which develops from
the sporozoite is at first intracellular, but as growth occurs it leaves the
host cell, to which, however, it remains attached by a process known as
the epunerite (Fig. 20). This structure is developed in various ways,
and may be compared to the organ of attachment of tape-worms. It

Fig. 19. — VoHicella nehulifera : A Group of

Stalked Ciliates attached to an Object

( X 200). (From Lang. 1901, after
d'Udekem.)

1. Contractile vacuole; 2, daughter individual with
circlet of cilia; .3, dividing form; 4, conjugation.

m

Fig. 20. — A Cephaijne Gre-
GARINE {Corycella armata)
( X ca. 300), showing
Epimerite, Protomerite,

AND DeUTOMERITE. (AfTER

Leger, 1892.)

may be a simple swollen body embedded in the cytoplasm of the cell,
and connected with the parasite by a kind of neck, or there may be de-
veloped from it a series of filaments or roots which anchor the parasite
to the cell. In some cases a large sucker-like process is applied to the
surface of cells, and from it a series of filaments pass into the cells or
between adjacent cells. In other cases the epimerite is supplied with a

40 MORPHOLOGY OF PROTOZOA

series of small spines. After growth of the gregarine is complete, the
epimerite is detached (Figs. 481 and 485).

Many Mastigophora are able to attach themselves temporarily to
objects. This is generally effected by a flagellum, as in species of Bodo
(Fig. 21), but some forms, like Oiko-
monas, can become fixed by a pseudo-
podium-like process developed from the
posterior end of the body. In the case
of trypanosomes and their allies attach-
ment to cells is an important feature of
development in the invertebrate host.
In the intestine, proboscis, or salivary
gland of insects in which development
is taking place, large numbers of the
flagellates may be attached to the sur-
face of the cells, and as longitudinal
division may take place while they are

Fig. 21. — Bodo saltans: A number of In-
dividuals ATTACHED TO A MaSS OF

Debris by the Trailing Flagella
(x 1,000). (Original.)

Fig. 22. — Stentor cceruleus ( x 146).
(Original Drawing from Life
by b. jobling.)

attached, the surface of the cells may become completely covered with
attached organisms. In this process, what usually happens is that the
flagellum disappears, attachment being effected by the tip of the axoneme
(Fig. 150).

In some Protozoa there is a sucker-like development of the surface

SKELETAL OR SUPPORTING STRUCTURES

41

of the body which enables the organism to attach itself temporarily.
In the case of Giardia {Lamblia) the ventral surface develops a large
sucking disc, by means of which the flagellate is able to attach itself to
the surface of the intestinal cells (Fig. 291). Amongst the Ciliata Stentor,
which is conical in shape, is able to fix itself to objects by pseudopodium-
like processes at its tapering posterior end (Fig. 22).

SKELETAL OR SUPPORTING STRUCTURES.— It has already been
pointed out that some Protozoa are able to build for themselves pro-
tective external coverings. Amongst
the Rhizopoda these are seen typically
amongst the Foraminifera and Radio-
laria. The shells may be strengthened
by the adhesion of granules of sand,
spicules, or other material. In the
Foraminifera the shells are external
coverings, the pseudopodia being pro-

FlG. 23. — CiLIATES WITH LORIC.E AND

Opercula which Close the Orifice
WHEN Retraction Occurs ( x 250).
(From Lankester, 1903, after Kent
AND Wright.)
1. CotJiurinaaffinis. 2. Cothurinavalvala.

Fig. 24.
(From

teres ( x 660).
1912, after

Schewiakoff, 1896.)

N, Macronucleus ; n, micronucleus ; o,
mouth; ces., oesophagus with rod-like
supports; f.v., food vacuoles; c.v., con-
tractile vacuole; al, alveolar layer; st,
meridional rows of cilia; «., anal opening.

truded through an opening as a snail emerges from its shell (Fig. 8).
In the Radiolaria the skeletal supports are more complicated, and
consist of spherical or asymmetrically formed fenestrated shells,
strengthened by various radially or tangentially arranged spicules
embedded in the cytoplasm (Fig. 78). The cup-like loricae found
amongst the Mastigophora (Fig. 18, i) and Ciliata (Fig. 23) may be

42

M0RPH0L0C4Y OF PROTOZOA

regarded as external skeletons or supports. These various structures

are secreted by the cytoplasm, from which they are separate. In all

Protozoa which have a distinctive body form it is the rigidity of the

ectoplasm which enables the organism to retain its shape. In certain

cases, what may be regarded as

modifications of the cytoplasm are

developed for purposes of support.

Thus, in certain Ciliata, as, for

instance, Prorodon, the anteriorly

placed cytostome leads to a cyto-

pharynx which is supported by a

series of longitudinally arranged

rods (Figs. 24 and 25). These rods,

or trichites, can be drawn apart and

the cytostome opened by radially

arranged contractile fibres attached

to each rod. In connection with the

cytostome of certain Mastigophora,

such as Chilomastix, the margin of

the cytostomal groove which leads

to the cytostome is supported and

rendered rigid by special fibres

(Fig. 69). In Trichomonas, again.

Fig. 25. — Section through Cyto-
stome OF Prorodon teres, showing
Supporting Rods ( x ca. 600). (From
MiNCHiN, 1912, after Maier.)

N, Nucleus; R, rods; ;«.?-. and m.r.',
myonemes.

Fig. 26. — Trichomonas {Pentatricho-
monas) from the Human intestine
( X 3,200). (After Kofoid and
SwEZY, 1924.)

the line of attachment of the undulating membrane is supported by a
special basal fibre which takes origin in a blepharoplast, and appears to
function by keeping the membrane stretched to its full extent (Fig. 26).

Another structure which also occurs in Trichomonaf! and allied forms
is the axostyle. This is a stift rod which commences in the blepharoplasts,

.SKELETAL OR SUPPORTIXG STRUCTURES

43

and passes through the centre of the body to protrude with a sharp
point at the posterior end. It is a structure which has little affinity for
stains, and its function and origin are not properly understood. Some-
times the flagellates are seen attached to debris by the pointed extremity
of the axostyle, but this is possibly only accidental. It is, perhaps, best
to regard the organ as skeletal in nature. Not infrequently, as explained
above, some of the axonemes which arise from the blepharoplasts at the

(:

Fig. 27. — Trichomonas vaginalis, showing tendency of Axostyle to Split into
A Series of Fibrils ( x ca. 2,000). (After Reuling, 1921.)

anterior end of the body, instead of becoming free flagella at the anterior
end, pass backwards through the cytoplasm to become free flagella at
other parts of the body surface. This condition is well seen in Hexamita
and Giardia (Figs. 288 and 291). It is customary to speak of the intra-
cytoplasmic portions of the axoneme in these flagellates as axostyles,
but this is clearly a misapplication of the term, for there is no evidence
that the axostyle of Trichomonas has any real homology with an axoneme,
though Kofoid and Swezy (19L5) have suggested that it represents an

44

MORPHOLOGY OF PROTOZOA

intracytoplasmic flagellum. The axostyle usually appears as a clear
homogeneous structure, but sometimes a fibre has been described as
passing along its central axis, while Reuling (1921) has noted that the
axostyle of Trichomonas vaginalis may sometimes split into four separate
fibrils which originate in the blepharoplasts. He regards the axostyle as
composed of four united fibres (Fig. 27).

MYONEMES.— It may be accepted that one of the characteristics of
cytoplasm is its power of spontaneous movement. In many Rhizopoda
and Mastigophora there are no visible structures which will account for
this movement, which involves a relatively large expenditure of energy.
In many Protozoa, however, special con-
tractile fibres are developed. An instance
in point is the axoneme of a flagellum, which
by its contractions causes the flagellum to per-
form its lashing movements. Similarly, the

A B

Fig. 28. — Myonemes in Gregaeine and Trypanosome. (From Minchin, 1912,

AFTER Schneider and Minchin.)

A. Clepsidrina nmnieri. B. Trypamsoma perccB ( x 2,000).

contractile fibres in the filaments of attachment of certain ciliates, like
Vorticella, enable the organisms to withdraw themselves suddenly (Fig. 19).
In many of the larger trypanosomes, gregarines, and ciliates there are
developed in the ectoplasm a series of fibres of a contractile nature known
as myonemes (Fig. 28). These run in various directions, and by their
contractions the organisms are able to perform movements of flexion and
extension. They not infrequently give rise to a longitudinal marking of the
surface of the body. A common type of movement seen typically in gre-
garines and merozoites of Sporozoa is the formation of rings of constriction,
which pass as peristaltic waves along the body. In certain ciliates para-

MYONEMES— EXTRUSION FILAMENTS

45

sitic in the rumen of cattle, such as some of the complicated forms like
Diplodinium (Fig. 520), the anterior region of the body is highly developed,
while in association with this there is a complicated system of contractile
fibres which enables the organisms to withdraw the whole anterior ciliated
region of the body into a def)ression, which becomes closed over it. In
a similar manner the ciliated peristomal region of Vorticella and its allies
can be suddenly retracted or withdrawn. The curious elongate ciliate
Spirostomum is well supplied with
longitudinal myonemes, which enable
it to retract suddenly to the globular
form when stimulated (Fig. 509).

The presence of these myonemes
often renders it extremely difficult to
obtain satisfactorily fixed specimens
in the fully expanded condition, as
stimulation of the fixing fluid causes
immediate contraction of the myonemes,
and consequent rounding up of the body.

EXTRUSION FILAMENTS.— In some
Protozoa special structures occur which,
on stimulation, have the property of
discharging filaments of varying length.
These may be protective or aggressive in
function or serve the purpose of fixation.

As organs of protection they are
known as trichocysts, and are found
amongst the Ciliata such as Parame-
cium, Prorodon, Dileptus, and many
other forms. They appear as minute
ovoid bodies embedded in the ectoplasm
(Fig. 29). From the blunt end there arises a fine process which extends
as far as the pellicle or outer layer of the ectoplasm. When stimulated,
the fine process is ejected as a tapering filament. Several explanations
of the sudden formation of the filament have been suggested. One is
that a very rapidly coagulating fluid is discharged. Whether this is the
correct explanation or not, it does not appear that the filament as such
exists in the trichocyst before it is visible externally. A larger organ
with a similar function is the Nessel's capsule or nematocyst. It is
present in Epistylis, and is arranged in pairs (Fig. 529).

Another type of filament which can be suddenly discharged occurs
in the Cnidosporidia (Fig. 30). In this group the resistant cysts or spores
are provided with one or more polar capsules from which long filaments.

Fig. 29. — Trichocysts as seen in
Sections of Paramecium cauda-
tum ( X ca. 1,500). (From Minchin,

1912, AFTER MaIER.)

A. Body Surface.

B. Mouth and oesophagus.

T. Trichocysts; /.t'.,food vacuoles; M.m.,
undulating membrane formed of fused
cilia in the oesophagus .

4G ENCYSTMEXT OF THE PROTOZOA

sometimes fifty or a hundred times as long as the spore itself, can be
extruded. The spores of the Cnidosporida are developed in a complicated
manner from a group of cells, some of which form the polar capsules.
These possess a tough outer covering within which the coiled-up filament

Fig. 30. — Dark Field View of Spore of Nosema apis with Extruded Polar
Filament ( x 1,200). (After Kudo, 1921.)

can be seen. It is supposed that the filament is inverted in the capsule,
and that it is discharged by pressure from within, just as an inverted
finger can be everted by blowing into a glove (Fig. 312).

ENCYSTMENT AMONGST THE PROTOZOA.

The majority of Protozoa under certain conditions which are generally
adverse to their continued existence, or in anticipation of such conditions,
are able to enclose themselves in resistant capsules of varying degrees of
impermeability. Encystment is effected by the secretion from the surface
of the body of a substance which quickly hardens in the medium. In
the majority of instances, while this is taking place, the organism, which
has become contracted to a spherical form, revolves slowly, so that fresh
secreted material is applied regularly to the layer already formed. These
capsules are known as cysts, and are generally composed of a clear, hyaline,
transparent substance. In free-living Protozoa which live in water
encystment takes place when the medium is drying up, and there is danger
of desiccation. In this manner complete drying is prevented, and survival
for long periods may occur in conditions under which it would be im-
possible for the exposed organism to live. It has been shown that the
sand of the desert exposed to the tropical sun contains encysted Protozoa,
which emerge from their cysts when brought into more favourable sur-
roundings. The cysts are usually perfectly smooth on their outer surface,
but sometimes they are roughened by the formation of tubercles or other
markings. Very frequently, after the resistant cyst has been formed
there is secreted a membranous inner lining to the cyst. In such cases
one can distinguish a resistant r/>/r//.s7 from a more delicate cndocyst.
Sometimes cysts are pfovided with })ores, several of which are present

PROTECTIVE AND REPRODUCTIVE CYSTS 47

in those of Dimastig amoeba gruberi (Fig. 120). To prevent drying of the
contents of the cyst, such pores are closed by plugs of some material
formed by the cytoplasm. They probably facilitate emergence from the
cyst.

Those Protozoa which are able to contract during life to the spherical
form produce spherical cysts, but others become encysted without changing
their shape to any extent. Thus, the species of Giardia produces ovoid
cysts, while species of Chilomastix and Embadomonas cysts which are pear-
shaped (Figs. 293, 255 and 256). The cysts (oocysts) which are formed
round the zygotes of various species of Eimeria are frequently ovoid in
shape, while those which form round the zygotes of the Gregarinina are
generally spindle-shaped (Figs. 350 and 483). Though the majority of
Protozoa form cysts at some stage of their development, there are some
forms in which cysts have never been observed.

The behaviour of the organism within the cyst varies considerably.
In many cases the cysts are purely protective in nature, the organism
remaining unchanged in the cyst till circumstances again become favour-
able to a free existence. The encysted organism escapes from the cyst
by its gradual dissolution, or through special pores when these are present.
In other cases multiplication takes place within the cyst. In the case of
Entamoeba coli, for instance, the nucleus of the encysted amoeba divides
repeatedly to produce eight nuclei (Fig. 101). Within the oocysts of
coccidia and gregarines there are produced a varying number of daughter
individuals known as sporozoites (Fig. 337). Similarly, within the oocysts
of the malarial parasites on the stomach of the mosquito there are de-
veloped very large numbers of sporozoites (Fig. 391). Within the cyst
of Giardia there are produced two daughter flagellates, while in that of
Prowazekella lacertw as many as sixty-four daughter flagellates are formed
(Fig. 253). Amongst the ciliates, when cysts are formed, they are usually
purely protective in nature, but in some cases, at least, reproduction
within the cyst takes place. Thus, the various species of Colpoda appear
to reproduce only in the encysted condition. The ciliate becomes spherical,
and by constant rotation forms a spherical cyst. Within it division into
two and then into four daughter ciliates occurs. The cyst is then ruptured
and the four young ciliates emerge. They then grow into the adult form,
when the process is repeated (Fig. 38).

Cyst formation is a very characteristic feature of parasitic Protozoa.
Having adapted themselves to life within another organism, their powers
of survival under external conditions have been largely lost, and it is b^
means of their encysted stages that they are able to pass from one host
to another. It thus arises that whenever an organism passes from one
host to another in such a manner that exposure to external conditions

48 ENCYSTMENT OF THE PEOTOZOA

must occur, it is the encysted forms which render such a transference to
a new host possible. In the case of the intestinal amoebse, though both
free and encysted forms escape from the body, it is only the encysted
stages which are able to carry infection to a new host. Even if direct
transference of unencysted stages occurred, these would, in all probability,
be killed by the digestive fluids of the stomach, which the encysted stages
can withstand. In the large group of insect flagellates (Trypanosomidse),
again, it is by means of minute encysted stages passed in the faeces that
another insect is infected (Fig. 164).

When encystment is about to occur, very frequently changes take
place in the encysting organism. The cytoplasm is freed from all food
residues, and in consequence becomes much clearer. Not infrequently
the cytoplasm becomes charged with food reserve material, such as
glycogen. Sometimes, as in the case of Entamoeba coli and Entamoeba
histolytica, in preparation for encystment special forms of the amoeba
which are smaller than the ordinary free individuals are produced (Figs.
96 and 100). These forms, which are preparing for encystment, are known
as precystic forms. Amongst the Sporozoa encystment is associated
with a sexual process. In the case of the gregarines two individuals
become enclosed in a cyst (gametocyst), within which each gives rise to a
number of gametes (Fig. 465). The gametes unite in pairs, and the
zygote thus produced itself becomes encysted in the oocyst, within which
it divides into a number of sporozoites. In the case of the coccidia, the
zygote is encysted in the oocyst which is formed either before or after
syngamy has taken place (Fig. 337). Within the oocyst the zygote divides
into a number of sporoblasts, which in their turn become encysted in
sporocysts. Inside the sporocysts the sporoblasts divide into sporozoites.

A special type of cyst is produced by the Cnidosporidia. These are
provided with one or more polar capsules from which long filaments can
be rapidly extruded. They serve the purpose of anchoring the cysts in
the intestine, while the wall is opened for the liberation of the enclosed
organism (Fig. 311).

For a long time the resistant encysted stages of the Cnidosporidia,
coccidia, and gregarines were known as psorosperms, a name introduced
by Johannes Miiller (1841) for the spores of Myxosporidiida. The spindle-
shaped oocysts of gregarines were frequently referred to as pseudo-
navicellse, a name first used by von Siebold (1839).

The production of secondary cysts within the primary one occurs
occasionally in other groups, as in the ciliates of the genus Colpoda. A
ciliate may become encysted and undergo a diminution in size within the
cyst, and then form a second cyst. The process may even be repeated
again, giving rise to three concentric cysts. As stated above, the different

PROTECTIVE AND REPRODUCTIVE CYSTS 49

species of Colpoda multiply within cysts. The four daughter ciliates
usually rupture the cyst and escape (Fig. 38). On the other hand, they
may each become encysted within the primary cyst. The process of
encystment was probably first developed for purely protective purposes,
but various reproductive processes have become associated with it. It
must be remembered, however, that encystment is not essential to either
of these processes, as they frequently occur quite apart from any encyst-
ment whatever.

In the majority of cases, when once formed, a cyst undergoes no change
in size or shape, though it may gradually increase in thickness. The
cysts of parasitic forms are ruptured or dissolved by the action of the
digestive fluids of a new host. In some cysts, however, there are formed
special pores which are kept closed by a plug of material which is more
easily dissolved than the rest of the cyst. Amongst the Sporozoa such
a pore in the oocyst is termed a tnicropyle, and through it the daughter
individuals which have been formed within the cyst emerge (Fig. 337).

It sometimes happens that with growth of its contents the cyst in-
creases in size after it is first formed. This process is well illustrated by
the oocyst of the malarial parasite, which increases enormously in size on
the stomach wall of the mosquito (Fig. 391). A similar growth also
occurs in the case of the oocysts of the hsemogregarines {Hejpatozoon), and
the cysts of the flagellate (Prowazekella lacertw), which is parasitic in the
intestine of lizards (Figs. 253 and 254). It will be evident that if the
thickness of the cyst is to be maintained, there must be constant addition
to it of fresh material secreted by the enclosed organism.

The cysts produced by any particular species of Protozoon are usually
fairly uniform in size, and possess distinctive features. On this account
their characters are of great importance for purposes of identification and
classification.

THE PROTOZOAN NUCLEUS.

GENERAL FEATURES. — The nucleus, which is an organized structure
containing chromatin, is the most important constituent of any Protozoan
cell, as, indeed, it is of all cells. It has been shown that the nucleus is
essential to the continuation of life, for individuals which have been
deprived of their nuclei, though they may survive and move about for
some time, quickly degenerate and die, while portions of the cytoplasm,
if they contain nuclei, often survive, regenerate, and continue their
existence. It seems probable that all the activities of the cell are governed
and regulated by the nucleus, which also appears to be mainly responsible
for the transmission of hereditary character.

I. 4

50 PROTOZOAN NUCLEUS

In some Protista, as, for instance, the bacteria and spiroclisetes, it
appears that there is no definite structure which can be called a nucleus,
though a granular material usually identified with chromatin is presumed
to fulfil the functions of the organized nucleus. AlexeiefE (1924a), however,
maintains that the granules are not chromatin, but mitochondria. It is
an exceedingly difficult matter to give a precise definition of the term
nucleus, though every biologist knows that it is a definite circumscribed
structure containing chromatin, and that it behaves in a well-recognized
manner. The fact that it divides when cell division takes place is one
of its most important features, but there are other structures in the
cytoplasm which behave in a similar manner. The one feature which is
not shared by other bodies in the cell is that during the sexual process
the nucleus, or one of its descendants, is able to unite with another
nucleus. In other words, a nucleus is potentially capable of karyogamy.

The majority of Protozoa possess but a single nucleus, except during
the process of multiplication, when two or more may be present. Some
forms, however, possess two nuclei during- the greater part of their life-
history, and are therefore binucleate, while others, again, have many
nuclei and are multinucleate. Such binucleate and multinucleate forms
may be regarded as individuals in which the nucleus has divided prepara-
tory to division of the body, which for some reason or another has been
delayed. In the binucleate and multinucleate individuals the nuclei are
easily recognized as being of one type. It sometimes happens that when
active multiplication by binary fission is taking place, the rate of division
of the nucleus exceeds that of division of the cytoplasm, so that temporary
multinucleate stages occur. In the case of Trypanoso7na brucei and other
pathogenic trypanosomes in laboratory animals, when active multiplication
is proceeding, individuals with four or even a larger number of nuclei
may be seen (Fig. 160). In such cases, however, the condition is quickly
rectified by repeated divisions of the cytoplasm without further division
of the nucleus. In most, if not in all, cases there arrives a period in the
life-history of multinucleate forms when division of the body takes place
and uninucleate individuals are produced. This is well seen in Opalina
ranarum of the intestine of the frog, where the organism is multinucleate
during the greater part of its life-history, but in the spring uninucleate
individuals are produced (Figs. 448 and 449).

Amongst Euciliata there exists a special type of binuclearity- These
Protozoa usually possess two nuclei, which differ from one another not
only in size and structure, but also in function. The larger one of the two
is known as the macronucleus, and the other as the micronvcleus (Fig. 70).
In ordinary division of the organism both nuclei divide, and when the
body is split into two parts the two daughter individuals each have two

BINUCLEARITY 51

nuclei. At certain stages in the life-history the macronucleus disin-
tegrates and disappears, while the micronucleus divides into two parts,
one of which becomes a new macronucleus. This process of regeneration
of the macronucleus occurs most usually in association with the process
of conjugation, but may also occur during the course of the ordinary
asexual multiplication, when it is known as endomixis. The fact that the
macronucleus is formed from one of the products of division of the micro-
nucleus is the primary reason why the macronucleus is regarded as
a nucleus at all. Furthermore, since the macronucleus disappears during
conjugation, and takes no part in the process, it is assumed that the micro-
nucleus is essentially the sexual nucleus, and that the macronucleus is
vegetative in function, and governs the metabolism and activities of the
cell at other times. Though this may be the case, the absolute proof is
difficult to obtain. Apart from the fact that it is small in relation to the
size of the body, the micronucleus behaves in every way during the whole
life of the ciliate as does the nucleus of an organism, such as a flagellate
or an amoeba, which possesses no macronucleus. There is little direct
evidence that the micronucleus of a ciliate is controlling the metabolism
and activities of the cell to a less extent than is the single nucleus of such
an organism as an amoeba.

It is clear that the macronucleus plays an important part in the economy
of the cell, and it is equally clear that it is of nuclear origin, but it does
not seem clear that because of its existence the functions of the micronucleus
are suppressed or supplanted while it is present. The view which main-
tains that the micronucleus is purely passive during the asexual life of the
organism, and only, so to speak, wakes up to activity during conjugation,
while the metabolism of the cell at other times is controlled by the macro-
nucleus, has given rise to the conception of two kinds of chromatin, the
one sexual or generative in function and the other vegetative. Amongst
the Euciliata the two kinds of chromatin are presumed to be separated in
different nuclei, while in other cases the same two elements are supposed
to coexist in the single nucleus. It is thought that dviring the sexual pro-
cess it is the generative chromatin that functions, the vegetative chromatin
having been largely got rid of by so-called reduction or maturation pro-
cesses. At other times it is the vegetative chromatin which is active,
while the generative chromatin, though still present in the nucleus, is
passive.

In this connection it is necessary to recall the fact that in the Mastigo-
phora the flagella take origin from a structure called the blepharoplast. ■ In
its simplest form this consists of a minute homogeneous granule, which
appears to be little more than a thickening of that end of the axoneme
which is in the cytoplasm. In certain stages of development of some

52 PROTOZOAX NUCLEUS

flagellates the flagella are lost, and a non-flagellate stage is developed.
When the flagellate stage is resumed, a new axoneme is developed as an
outgrowth from the blepharoplast, which may or may not have persisted.

fe'

f

..J

1 t ;V

¥

O '

"^■M'i

t

)

o

.P:

■i S 6

Fig. 31. — Parypolytoyna satura, to snow the Origin of the Blepharoplast

FROM THE KaRYOSOME OF THE NUCLEUS ( X 2,600). (AfTER JaMESON, 1914.)

1. Adult flagellate shortly before division.

2. First division completed: two daughter individuals with the old blepharoplasts and flagella.

3. Second division: reconstruction of nuclei and division of body into four.

4. Second division completed: new blepharoplasts are budding off from the karj^osome in the

upper and left-hand individuals, while the right-hand individual retains the old blepharo-
plasts.

5. New blepharoplasts on outer surface of nuclear membi'ane in three of the individuals, while the

left-hand individual retains the old blepharoplasts.
0. Stage shortly before release of four daughter flagellates: the left-hand individual has the old
blepharoplasts and flagella, while the others have new blepharoplasts and young flagella.

Some evidence has been produced by Jameson (1914) in the case of a
^SigeWa^te, Pai'apolytoma satura, and by Entz (1918) in the case oiPolytoma
uvella, that when the non-flagellated stages are about to develop flagella new

BLEPHAROPLAST AND PARABASAL

53

basal granules or blepharoplasts are developed from the nucleus or from its
karyosome (Fig. 31). In the case of Dimastigammha gruberi (Fig. 120), the
amoeboid phase of which develops fiagella under certain conditions, it was
stated by AlexeiefE {I9l2g) and Wilson (1916) that when this took place the
blepharoplasts of the two fiagella migrated into the cytoplasm from the
karyosome of the nucleus, with which they remained connected by a fibre.
As explained below (p. 263), the writer has been quite unable to observe
the origin of the blepharoplasts in this manner. It seems more probable
that the blepharoplasts are present in the cytoplasm, possibly on the outer
surface of the nuclear membrane, during the whole of the amoeboid phase

Fig. 32. — Devescovina striata ( x ca. 1,900).
A. Ordinary flagellate showiiag coiled paraba"^al.

(After Janicki, 1915.)
B. Dividing form.

of tlie organism, and that they move to the surface of the body when
fiagella are commencing to form. In many Mastigophora, in association
with the blepharoplast, is another structure to which Janicki (1911) has
given the name parabasal ; it stains intensely with certain stains (Figs. 32,
33, 67). In some cases — as, for instance, in trypanosomes and their
allies — it seems to be in actual union with the blepharoplast, and to form
with it a composite body, the hinetoplast (Fig. 157). There is no conclusive
evidence that the parabasal body is of nuclear origin, as some have supposed.
It is a well-established fact, however, that division of the organism is
preceded not only by division of the nucleus, but also by division of the
blepharoplast and parabasal as well, and it becomes a tempting hypothesis

54

PROTOZOAN NUCLEUS

to suppose that the nucleus and the kinetoplast of a flagellate represent
two nuclei, as do the micronucleus and the macronucleus of a ciliate.
Such an assumption has been made by Hartmann (1907) and others,
who regard the kinetoplasts as true nuclei, and the flagellates which possess
them as constituting a special group of the Mastigophora, the Binucleata.
As pointed out by Alexeieff (19176), there is no ground for this assumption,
and in order to avoid confusion he proposed the name kinetoplast in place
of kinetonucleus and other terms which implied a nuclear nature. It is

safer to regard the kinetoplast as a
distinct structure concerned with the
activities of the flagellum, even though
it divides when cell division occurs,
and possibly may have originated from
the nucleus in the first place. It might
equally be argued that though the
macronucleus of a ciliate has originated
from the micronucleus, and though it
multiplies by division during reproduc-
tion, it has ceased to be a nucleus in the
true meaning of the term, and has be-
come modified to serve some other pur-
pose, possibly in connection with the
development of large numbers of cilia -
It is worthy of note that the macro-
nvicleus does not divide like a true
nucleus, which in most cases, at least,
shows some indication of mitosis.
Against this view, however, can be
raised the argument that there occur
certain races of ciliates which possess
no micronuclei, though other races of
the same species have both micro- and
(p. :^5), Dawson (1919) discovered an
amicronucleate race of Oxytricha hyiiienostoma, a ciliate which normally
possesses both nuclei. The ciliate was kept in culture for several years, and
though possessing only a macronucleus, it reproduced regularly by fission.
ENDOMIXIS. — As already remarked, the macronucleus of a ciliate
degenerates during conjugation, and a new macronucleus is developed
from one of the products of division of the micronucleus. This re-
placement of the macronucleus from the micronucleus may occur at
times other than during conjugation. Wlien Paramecium aurelia
reproduces repeatedly by simple division over long periods at certain

\^-

Fig. 33. — Bodo caadatus Coprozoic
IN Human F.^ces ( x ca. 1.500).
(Original.)

I and 2. Forms showmg two blepharo-

plasts with associated parabasal.
3. Small individual. 4. Encysted form.

macronuclei. As noted above

ENDOMIXIS

55

intervals, the macronucleus degenerates, and is replaced from the
microniicleus as at conjugation. Woodruft' and Erdmann (1914) described
the process in Paramecium aurelia, and named it endomixis. P. aurelia
contains normally two micronuclei and one macronucleus (Fig. 34). When
endomixis occurs, the macronucleus disintegrates and is eventually
absorbed. The two micronuclei divide to form four, and these again to
form eight micronuclei. Of the four derived from each original micro-

<g5Vb6?Vb> C

Fig. 34. — Endomixis in Paramecium aurelia: Diagrammatic Representation
OF Nuclear Changes as described by Woodruff and Erdmann ,
1914. (After Jennings, 1920.)

A-B. Degeneration of macronucleus and first division of two micronuclei.

C-D. Second division of micronuclei and degeneration of .six of the daughter micronuclei.

E. Division of ciliate to produce two daughter individuals each with a single micronucleus.

F-G. Two divisions of micronuclei to give rise to four, two of which increase in size to become

macronuclei. H. Further division of micronuclei.

I. Division of ciliate to give rise to the normal type as at A.

nucleus, three degenerate, so that two micronuclei are left. The ciliate
now divides, giving rise to two ciliates, each with a single micronucleus. In
each of these daughter ciliates the micronucleus divides to form two, and
again to form four micronuclei. Two of these increase in size and become
macronuclei, while the other two divide to form four micronuclei. The
ciliate now has two macronuclei and four micronuclei. It now divides to
form two ciliates, each of which has two micronuclei and one macronucleus
as in the original form.

56 PROTOZOAN NUCLEUS

Endomixis has been demonstrated in several races of Paramecium
aurelia by Woodrufi and Erdmann, as well as in another species of the
same genus. Fermor (1913) claimed to have seen the same process in
Stylonychia, while Calkins (1915 and 1916) described it in Didinium and
Uroleptus. The meaning of endomixis is not clear. That it takes place
quite apart from unfavourable conditions has been noted by Woodruff
(1925), who also proved that certain races of Paramecium in which it did
not occur died out. All that can be stated is that for the satisfactory con-
tinuation of the functions of the macronucleus, whatever these may be, it
seems necessary in many cases that this structure be renewed from time
to time. Though this usually takes place during conjugation, it may
occur at other times. An exception to this rule is afforded by the
behaviour of the ciliate Spafhidium spathula (p. 132).

STRUCTURE OF THE NUCLEUS.— The nucleus of a Protozoon possesses
a nuclear membrane, which may be regarded as a special develop-
ment of the linin network of fibres or septa which traverse the enclosed
space (Fig. 1). The meshes of the network or spaces between the septa
are filled with a fluid substance known as nuclear sap. Distributed upon
the membrane or network as distinct granules or in one or more larger
masses is the chromatin material, while in most cases, somewhere on the
network, and most usually at or near the centre of the nucleus, is a body
known as the karyosome, which, on account of its affinity for certain stains,
has generally been regarded as consisting partly of chromatin and partly of
an achromatic substance {plastin). In some Protozoan nuclei the
karyosome does not seem to be present, but it appears in the nuclei of the
majority of forms. From what takes place in nuclear division, it appears that
the karyosome is composed mostly, if not entirely, of plastin material, and
that the chromatin of the nucleus is represented by the granules outside the
karyosome, for it is from them that the chromosomes are formed. Doflein
(1922), from a study of the nucleus of the flagellate Ochromonas granulans,
was led to believe that a true karyosome was devoid of chromatin, and
that during nuclear division it gave rise to the achromatic part of the
spindle, while the chromosomes were derived from the peripheral chromatin
which was situated outside the karyosome. On the other hand, Stern
(1924), from a study of the nuclear division of the Heliozoon Acanthocystis
aculeata, arrives at a conclusion which is the exact opposite of this. He
believes that the karyosome breaks up and gives rise to the chromosomes,
while the spindle is formed from the part of the nucleus between the
karyosome and the nuclear membrane.

Sometimes several masses of plastin occur in a single nucleus, but it
seems doubtful if these should all be regarded as karyosomes. It is often
assumed that an intranuclear centrosome, the centriole, is present in the

STRUCTURE OF NUCLEUS 57

nuclei of Protozoa. It is supposed to be embedded in the karyosome,
and only to become recognizable as centrosomic in nature during nuclear
division, when it divides into two parts which separate from one another,
though remaining connected for some time by a fibril, the centrodesmose.
As the daughter centrioles move apart they take up positions at the ends
of the elongating nucleus, while spindle fibres surrounding the centro-
desmose may form between them (Fig. 59). The chromatin of the nucleus
may form definite chromosomes, which arrange themselves as an equatorial
plate at the equator of the spindle. Two daughter plates are formed,
and these travel towards the centrioles at opposite poles of the nucleus.
When the nuclear membrane divides, the spindle fibres and centrodesmose
disappear, a new karyosome is formed around the centriole, and the
chromosomes break up into granules, which are distributed on the
nuclear membranes or linin network. Observers are, however, by no
means convinced that such a granule is a true centrosome, for in many
Protozoa undoubted centrosomes exist in the cytoplasm outside the
nuclei.

The size of the karyosome in proportion to that of the entire nucleus
varies considerably. In many nuclei, especially those of small size, it
has been the rule to regard the bulk of the chromatin as being aggregated
in the relatively large karyosome, and to suppose that little, if any, is
distributed upon the nuclear membrane or linin network. It is becoming
increasingly evident, however, that all nuclei contain some chromatin
on the linin network or membrane (peripheral chromatin). In other cases
the karyosome is relatively small, while definite chromatin granules occur
upon the nuclear membrane or the linin network. The nuclei of the first
type are often spoken of as of the karyosome type, but every transition
between the two types of nuclei occurs. This question as to whether a
centriole is always present or not is a very difficult one to decide, for the
statements regarding it are most conflicting. Some observers are able
to find centrioles in nearly every nucleus they examine, while other
equally competent observers fail to detect them. The difference of opinion
is to be accounted for by the fact that the centriole is merely a minute
granule, the nature of which can only be determined by its behaviour
during actual division of the nucleus. While division is taking place, there
are always numbers of granules in the nucleus. Many of these are chro-
matin granules, and as spindle fibres are often present while the nucleus
is dividing, it is easy to interpret any two granules and a connecting fibre
as centrioles and centrodesmose. It thus happens that it is more than
doubtful if most of the structures which have been described as centrioles
are actually of this nature. The mere presence of a central granule in
a karyosome of a resting nucleus appears to some observers to be sufficient

58 PROTOZOAN NUCLEUS

ground for calling it a centriole. Karyosomes do not always stain homo-
geneously, and may have a granular structure, while the appearance of
a central granule may be merely the result of irregular extraction of stain.
Before deciding as to the centriole nature of a granule, it is necessary to
trace its division and the separation of the two daughter centrioles, and
to observe the actual centrodesmose uniting them. Furthermore, when
spindle fibres are developed for mitotic division, the daughter centrioles.
will occupy the poles of the spindle. Such appearances must not be of
rare occurrence, but must be detected in the majority of dividing nuclei.
When a large number of dividing nuclei of any Protozoon such as an
amoeba are examined, it is a relatively easy matter to find isolated examples
of spindles which have granules at their poles, though the majority of
spindles may not show them. The occasional presence of such apical
granules does not justify the assumption that they are actually centrioles.
It is an undoubted fact that definite spindles may be formed within the
nuclei of Protozoa without there being any evidence of centrioles at the
poles, though it is not difficult for those who desire to see such structures
to convince themselves that granules, which they interpret as centrioles,
are present. The fact that during the mitotic nuclear division of the cells
of higher animals centrosomes are almost always present, and that un-
doubted centrosomes occur in some Protozoa, has undoubtedly led to many
structures being described as centrioles or centrosomes which have quite
another nature. In the present state of our knowledge it is impossible
to state that a centrosome or centriole is an essential constituent of all
Protozoan nuclei. Nevertheless, it cannot be supposed that all the
descriptions which have been given are erroneous. In the division of the
nucleus of Dimastig amoeba gruberi, which has a large central karyosome,
the latter structure elongates and becomes dumb-bell-shaped, and finally
divided into two parts. As these separate, they remain connected by
a fibre which can be shown in many cases to unite two granules which occur
in the two daughter karyosomes (Figs. 61, 4, and 120, 12). AVhether such
granules are to be regarded as intranuclear centrosomes (centrioles) is a
question more difficult to decide.

The true structure of a nucleus is that which it possesses in the normal
living cell. Fixing fluids and other reagents may considerably alter its
appearance, so that the greatest care has to be exercised in the interpreta-
tion of the structures seen in fixed material. Experience has shown that
certain fixing fluids, stains, and reagents produce better results than
others, and are reliable in giving accurate pictures of the true structure.
Nevertheless, the literature dealing with the Protozoa, especially the blood
parasites which have been largely studied in dried films, is full of erroneous
descriptions of nuclei. The dry blood film stained by Romanowsky stains,

STRUCTURE OF NUCLEUS 59

though it may give useful information as to the type of cell or parasite
present, is completely misleading when it comes to a consideration of the
minute nuclear structure. Descriptions of the characters of nuclei which
are based on preparations of this kind are not only worthless but misleading.

From the above description it will be realized that the nuclei of Protozoa
may be roughly divided into two classes: those in which there is a central
karyosome, and those in which no such karyosome is present. Compared
with the size of the nucleus, the karyosome may be a relatively small
structure, or it may occupy a large part of its bulk. As a type of nucleus
with small karyosome, that of Entamoeba histolytica will serve as an illus-
tration (Fig. 95). There is a definite nuclear membrane, on the inner
surface of which practically all the chromatin is arranged in the form of
small granules. At the centre of the nucleus is a small granule, the
karyosome, which presumably consists of plastin material and possibly
some chromatin. Surrounding the karyosome is a clear area, the limits of
which form a sphere (or ring in optical section) of fine granules. These do
not contain chromatin, but represent the inner limit of the linin network
which connects the sphere with the nuclear membrane. The linin network
appears to be free from chromatin. Dobell (1919), who has studied the
nuclear division in this amoeba, could obtain no evidence of the existence
of a centriole in the karyosome, though such a structure has been described
by Hartmann (1908-1913). During division of the nuclei within the cysts
the writer has seen forms which suggest the presence of a central granule
which divides (Fig. 57).

A type of nucleus in which a definite and relatively large karyosome is
present is of frequent occurrence. It is seen typically in trypanosomes,
many free-living amoebae, and other Protozoa (Figs. 48, 89, 224). These
karyosomes are comparatively large structures which are connected with
the nuclear membrane by the linin network. It is possible, though by
no means certain, that some of the chromatin of the nucleus may be con-
centrated in the karyosome, which stains intensely with certain nuclear
stains. The nuclear membrane and the linin network may have com-
paratively little chromatin, which in small nuclei, such as those of trypano-
somes, is difficult to detect. In many cases nuclei of this type are
described as possessing centrioles within the karyosomes. The large
karyosome may appear perfectly uniform and homogeneous, or it may
show indications in stained specimens of being composed of a varying
number of deeply staining bodies embedded in a more faintly staining
l)lastin matrix. The karyosome is often spherical in form, but it may be
irregular in shape. In some cases on the surface of the karyosome there
occur deeply staining granules, which may be chromatin, while the central
part consists of })lastin. Sometimes one or more vacuoles are present.

60 PROTOZOAN NUCLEUS

Between these two types of nuclei many intermediate forms are found,
and individual variations are of common occurrence. These variations
may affect the nuclear membrane, which may be exceedingly fine in some
forms and comparatively thick and dense in others. The arrangement
of the chromatin upon the membrane may be in the form of uniformly
distributed fine granules, or there may be coarse granules more irregularly
distributed, or most of the chromatin may be aggregated into a semi-
lunar mass planted on one side of the membrane.

The linin netw^ork itself may be in the form of a uniform mesh, or it
may consist of radially arranged strands. The meshes of the network
may contain granules other than chromatin or globules of an undetermined
nature. The minute structure of the nuclei is of considerable importance
in the differentiation of species.

Though it is not possible to draw a hard-and-fast line between those
nuclei which possess karyosomes and those which do not, there never-
theless exist certain nuclei in which there appears to be no tendency
towards the formation of a central structure. Amongst the gregarines,
for instance, certain individuals of a particular species may show a single
deeply staining body in the nucleus, or more than one, while in some
Protozoa there are a series of deeply staining bodies upon the nuclear
membrane, while the interior of the nucleus is occupied by a uniform
meshwork of fibrils. It appears impossible to speak of several bodies
in the nucleus as karyosomes, a term which is undoubtedly used by the
vast majority of zoologists, for the single more or less centrally placed
structure described above. Nuclei of the type which has no definite
karyosome may, however, contain a body which may or may not be
central in position, and which is regarded as devoid of chromatin, owing
to the fact that it does not stain intensely with chromatin stains. Such
a structure occurs, according to Metcalf (1909), in the nuclei of species
of Opalina, in which a deeply staining karyosome is not present. It
resembles. the nucleolus which is commonly found in the nuclei of the
cells of higher animals. Bodies of this type have also been described by
Reichenow (1921) in the nuclei of various stages of development of the
hsemogregarines of the genus Karyolysus. They are also present in the
nuclei of Hepatozoon balfouri, and, as in the case of Karyolysus, they divide
during nuclear division (Fig. 35). They are commonly present in the
nuclei of coccidia. They take no part in the formation of the spindle or
the chromosomes.

Another type of nucleus which has to be mentioned is the macro-
nucleus of the Euciliata. It has already been explained that these
Protozoa typically possess two nuclei — the micronucleus and the macro-
nucleus. The former is usually of the type which contains a central

STRUCTURE OF NUCLEUS , 61

karyosome, and when it divides it does so by mitosis, while the latter,
though developed from a nucleus like the micronucleus, has so changed
in appearance and structure that it seems doubtful if it should still be
regarded as a true nucleus (Fig. 37). It is sometimes spherical in form,
but it is more usually slightly elongated. It may be many times as long
as it is broad, and in such cases may have a beaded appearance, as in
Stentor and Spirostomvm (Figs. 22 and 509). It may even be irregularly
branched, as in certain Suctoria (Fig. 531). It consists of a dense material
impregnated with granules which become more evident during division.
Vacuoles are often present, while sometimes, as in the species of Colfoda,
the elongated macronucleus contains within it one or more deeply staining
bodies (Fig. 498). It is evident that the macronucleus differs in many
ways from the micronucleus and the nuclei of other Protozoa. During
division it does not behave as true nuclei do, and there seems to be
little change in its appearance, except for the greater clearness of its

#1 '0^ ^#^ ^^

Fig. 35. — Stages in the First Nuclear Division in the Schizont of Hepatozoon
balfouri, showing Division of the Karyosome, which appears to be entirely
devoid of Chromatin ( x 6,000). (Original.)

granules. It is generally assumed that the granules in the macronucleus
are chromatin, but, if this be so, there must have taken place a remarkable
increase in the chromatin during its formation and growth from the micro-
nucleus from which it was originally derived. It seems not impossible
that this material is not actually chromatin, but some other substance
which has been elaborated to fulfil a special function.

In this connection it will be necessary to refer again to a theory which
was suggested by Schaudinn, and subsequently elaborated by Goldschmidt
and others. According to this theory the Protozoan nucleus is constructed
of two fundamentally different parts, which in the Euciliata are separated
in two distinct nuclei. The one part consists of vegetative material which
controls nutrition, movement, and other vegetative functions, while the
other is composed of generative material which takes part in the syn-
gamic process. This theory has been extended to the chromatin itself,
which is supposed to be of two kinds, the one idiochromatin, which takes
part in syngamy, and is responsible for the transmission of hereditary

62 MULTIPLICATION OF PROTOZOA

characters, and the other vegetative chromatin, which has to do with
the vegetative functions. Amongst the Plasmodroma and the Proto-
ciliata both kinds of chromatin are contained in one nucleus, and it is
supposed that the extrusion of chromatin material from the nuclei of
gametes, which has been described as taking place in certain instances, is
an expulsion of the vegetative chromatin in preparation for syngamy.
If this explanation is the correct one, it has to be admitted that after
syngamy the vegetative chromatin can be re-formed from the generative
chromatin, as illustrated by the formation of new macronuclei from the
micronuclei after syngamy in the Euciliata. The theory depends very
largely on an exact definition of what is and what is not chromatin, and
a correct interpretation of the various parts of the nucleus, about which
at the present time there is considerable difference of opinion. Dobell (1 925)
has described a condition of binuclearity in Aggregata (see p. 873).

MULTIPLICATION AMONGST THE PROTOZOA.

Multiplication takes place by a process of binary fission or gemmation
in which an organism divides into two daughter organisms after division
of the nucleus, or by a process of multiple segmentation, which is generally
known as schizogony amongst the Sporozoa, where it occurs most typically,
after a number of nuclei have been formed by repeated divisions.

BINARY FISSION. — The process of binary fission may give rise to
daughter forms which are equal in size (equal binary fissions), or to forms
w^hich are unequal in size (unequal binary fission). When there is a
marked difference in size between the two, the process is known as budding
or gemmation, a method of multiplication which is seen typically amongst
the attached Euciliata (Peritrichida and Suctoria), where a large form buds
off a small ciliated embryo which does not itself reproduce till it has grown
to the adult form.

In the case of amoebae which have globular bodies, binary fission is
effected by the body becoming elongated and a constriction forming
around the middle of the body (Fig. 36). This deepens till the amoeba
is divided into two parts. The daughter forms may not divide again till
they have grown to the size of the parent. On the other hand, they may
divide before growth is complete, with the result that increasingly small
individuals are produced. If they divide only after they have grown to
a size larger than that of the parent, then larger forms are gradually
produced. In the case of the amoebae it is evidently impossible to state
that division takes place in any one plane, except that it occurs in a plane
at right angles to the axis occupied by the elongate dividing nucleus.

Directly it becomes possible to orientate an organism, and state that
it possesses an anterior and posterior end and a dorsal and a ventral

BINARY FISSION

63

surface, it is found that the plane of division is uniform in the different
groups. Thus amongst the Mastigophora, which have an anterior flagel-
lated end of the body, it is found that in binary fission the body splits
longitudinally from before backwards. In those forms in which a cyto-
stome is present, as in Chilomastix, in which a dorsal and ventral surface
can be distinguished, it is found that a new cytostome is formed near the
original one, and if this is also regarded as being on the ventral surface
then the body splits longitudinally from before backwards and in a dorso-
ventral plane which passes between the cytostomes. In actual division,
however, the body often becomes so distorted that it may be difficult to

Fig. 36. — Successive Stages in Binary Fission of Amceba jtoli/ podia ( x 250).
(From Lang, 1901. after Sciiulze, 1875, modified.)

distinguish the dorsal and ventral surface, though the plane of division
may still be regarded as in this direction. In flagellates such as Tricho-
monas and the trypanosomes, which possess undulating membranes,
division is more complicated (Figs. 160 and 271). A new axoneme grows
out from the free half of the divided blepharoplast and passes along the
border of the membrane. The membrane then divides between the two
axonemes, but the point up to which the membrane has divided at any
stage is always a short distance behind the end of the new axoneme.
When the new axoneme has reached the end of the membrane the division
of the membrane is completed, and the two undulating membranes, each

64

MULTIPLICATION OF PROTOZOA

with its axoneme, are formed. The body of the flagellate then divides
longitudinally from before backwards in a dorso-ventral plane between
the two dorsal membranes. Owing to the blepharoplasts being situated
at the anterior end of the body in Trichomonas and some other flagellates,
the membrane divides from before backwards in these forms. In the
trypanosomes, however, the blepharoplast is situated at the posterior end

of the flagellate, and the membrane

divides from behind forwards. In
either case, the body itself divides
from before backwards after the mem-
brane has completed its division.

Multiplication by binary fission
occurs also amongst the Opalinata
and C-iliata, but division is trans-
verse and not longitudinal, as in the
Mastigophora (Fig. 37). A ciliate
may develop a new cytostome at
some distance behind the first one,
and after division of both the macro-
nucleus and micronucleus the body
divides transversely or at right angles
to the longitudinal axis. It often
happens that, as a result of this
division, the character of the daughter
forms differs from the parent in the
relative size of the cytostome. As
the body of the new individual is
developed from the post-cytostomal
region of the parent, it follows that
the daughter forms will have a cyto-
stome which is relatively longer when
compared with the total length of the
body. In Paramecium the cytostome
of the parent takes up a position at
the centre of the body, and is divided
into two cytostomes of equal or unequal length, after which the body
divides transversely between the two.

Binary fission, when it occurs amongst the Rhizopoda, Mastigophora,
or Ciliophora, usually gives rise to individuals which are roughly equal in
size; but not infrequently, as, for instance, in Trypanosoma lewisi, a large
trypanosome will divide in such a manner as to give rise to one large form
and one which is very much smaller (Fig. 197, 5). The process is repeated

Fig. 37. — Binary Fission of Para-
mecium aurelia ( x ca. 500). (After
Lang, 1901.)

1 and 4, New contractile vacuole; 2 and 6,
dividing macronucleus ; 3 and 5, anterior
and posterior contractile vacuoles, which
will become the posterior vacuoles of the
daughter forms ; 7. new cytostome formed
as bud from original cytostome; 8 and 10,
mitotic division of two micronuclei.

SCHIZOGONY

65

by the large form, which apparently has ceased to grow, so that eventually
its entire cytoplasm is used up in the production of a number of small
forms. At each division there is a nearer approach to equal binary
fission. It is evident that such a method of division approaches a budding
process.

Binary fission usually occurs in the free-living state, and as the division
is taking place the organism may be actively motile. Amongst the
Rhizopoda, the amoebae are frequently perfectly quiescent while binary
fission is proceeding. In some cases, binary fission takes place in the
encysted condition. This appears to be the normal method of multiplica-
tion of species of Colpoda. The organism secretes a cyst in which it

Fic. 38. — Colpoda steini : Multiplication of a Single Indivibual during a
Period of Seven Hours' Observation ( x 650). (Original.)

A. Ciliate about to encyst. B. Encysted ciliate.

C. Division into two completed: commencing division into four.

D. Four daughter ciliates in cyst.

E-G. Escape of ciliates through rupture in cyst wall.
H. Crumpled cyst after escape of ciliates.

divides into two, each of which again divides (Fig. 38). The four daughter
ciliates then rupture the cyst and swim away. Similar divisions within
cysts occur amongst the Rhizopoda and Mastigophora (Fig. 143).

SCHIZOGONY. — By this term is understood a method of multiplica-
tion which occurs typically amongst the Sporozoa (Fig. 39). As the
organism is growing, repeated divisions of the nucleus and daughter
nuclei take place, till finally there may be present a large number of nuclei
in a single mass of cytoplasm. The number of nuclei produced varies
considerably, and may be as few as four or as many as a hundred or more.
The nuclei arrange themselves on the surface of the cytoplasm, which
becomes raised into a series of elevations, into each of which a nucleus

I. 5

66

MULTIPLICATION OF PKOTOZOA

passes. When the requisite quantity of cytoplasm has been raised into
the elevation this is divided off by a constriction, and the daughter forms,
termed merozoites, are produced. These grow into adults, which may
again reproduce by schizogony. It is often supposed that the multi-
nucleate adult, which is termed a schizont, suddenly segments into a

Fig. 39. — Hepatozoon cams : Developmental Stages in the Spleen of a
Bagdad Dog ( x 2,000). (Original.)

1. Young schizont in mononuclear cell. 2. Slightly older schizont.

3. Section of an older schizont \\ith anumber of nuclei.

4. Section of a schizont in w Inch merozoites are commencing to form by a budding process.
.5. More advanced stage of budding process as seen in section of mature schizont.

G. Merozoites and residual body after schizogony : the merozoites are gametocytes which enter

the mononuclear cells of the blood-stream.
7. Section of stage with eight large merozoites which are probably destined to become schizonts

again. 8. Stage similar to that depicted at 7.

number of merozoites, as in the case of malarial parasites. It appears,
however, that in all cases the merozoites, whether few in number or more
numerous, are formed as small buds at the surface of the schizont, as
described above. A variable quantity of the cytoplasm is unused in the
formation of the merozoites. This residual cytoplasm, within which may
be found a certain number of unused degenerate nuclei of varying size,

SCHIZOGONY AND SPOROGONY 67

and any other material to be discarded, such as pigment, is known as the
residual body. It takes no further part in the life of the organism, and
after separation of the merozoites quickly disintegrates (Fig. 39, 6).

Amongst the Sporozoa, after syngamy has taken place, the zygote
divides by a process which is essentially the same as schizogony. This is
termed sporogony, and it gives rise to sporozoites, which differ in size and
shape from the merozoites. The sporozoites arise from the multinucleate
zygote, which may have increased considerably in size and is called the
sporont, by a budding process which is very similar to that by which the
merozoites are formed (Fig. 455). The term " sporogony " is generally
extended to include the whole phase of the developmental cycle from the
beginning of the production of gametes or gametocytes to the formation
of sporozoites from the sporont after syngamy has taken place. To
distinguish the other phase of development during which schizogony
occurs repeatedly without the intervention of a sexual process, it has
been termed agamogony, and the various stages (merozoites and schizonts)
agamonts. The growing agamont is often termed a trophozoite.

During the formation of merozoites and sporozoites it not infrequently
happens that the number of nuclei present is so large that the surface
of the cytoplasm is insufficient to accommodate them all. By a process
of vacuolation of the cytoplasm the available surface is increased. The
vacuoles may open into one another, so that the cytoplasm is reduced to
the condition of a coarse network. In this way the available surface
upon which nuclei can take up their position is increased, so that the
merozoites or sporozoites can be budded off in the usual manner. A
typical instance of this increase in surface occurs during the formation of
sporozoites in the oocysts of the malarial parasites on the stomach of
mosquitoes, as also during schizogony of Aggregata eherthi and other
Sporozoa (Figs. 377 and 391).

A method of schizogony which occurs amongst the piroplasmata must
be mentioned. In these parasites the number of daughter forms produced
are two or four, which are described as arising from the parent by a budding
process, in contrast to the supposed segmentation of the schizont of the
malarial parasites. As already explained, the merozoites of malarial
parasites are not produced from the parent by a sudden splitting of the
body between the nuclei, but by the formation of buds from its surface,
as occurs generally amongst the Sporozoa. The piroplasmata are no
exception to this rule. In some species {Babesia canis) the buds are
usually two in number, but may be four (Fig. 417). In others (B. equi)
there are usually four buds, as in Plasmodium minasense (Fig. 416 and
Plate XVII., 6-15, p. 982). The bud commences as a small cytoplasmic
elevation on the surface of a rounded parasite. It gradually increases in

68 MULTIPLICATION OF PROTOZOA

size at the expense of the cytoplasm of the parent. It is difficult to
understand why an organism which is to produce only two daughter forms
should do so by a budding process instead of by a simple binary fission
into two parts. It seems possible that it is a condition which has evolved
from one in which a larger number of merozoites were originally produced,
as in typical schizogony.

When a schizont is in process of producing merozoites or a sporont
sporozoites, the schizont or sporont may first divide into a number of
intermediate bodies which actually produce the merozoites or sporozoites.
In the case of the coccidium Caryotropha mesnili, when about sixteen
nuclei are present in the schizont, it divides into sixteen portions which
have been called cytomeres or agametoblasts (Fig. 375). The nuclei of
these undergo further divisions, and finally merozoites are budded from
their surfaces. A similar method of multiplication occurs in Klossiella
cobayce and other forms (Fig. 449). Similarly, during sporogony the zygote,
instead of dividing directly into sporozoites, may first produce a number
of sporoblasts, which give rise to the sporozoites. In the coccidia sporogony
takes place within the oocyst which has formed around the zygote, and
it frequently happens that the sporoblasts secrete around themselves
secondary cysts or sporocysts, within which the sporozoites are finally
produced (Fig. 337).

Attention has already been called to the fact that occasionally, amongst
flagellates which normally multiply by binary fission, the rate of division
of the nuclei may exceed that of the cytoplasm during very rapid multi-
plication, so that stages are reached in which an abnormal number of
nuclei are present (Fig. 142). The excessive nuclear multiplication,
however, comes to an end, and the body divides repeatedly till a number
of normal uninucleate forms are produced. In some cases such multi-
nucleate stages occur normally in the developmental process. Thus, in
the course of the development of Trypanosoma lewisi in the flea, the
trypanosomes taken up from the rat enter the cells lining the stomach,
and there grow into large bodies which possess as many as sixteen nuclei,
kinetoplasts, and flagella (Fig. 200). The " sphere," as it is called, then
divides into a corresponding number of trypanosomes. Such a method
of multiplication is really one of delayed division of the cytoplasm,
and must be distinguished from true schizogony. It seems probable
that the final division of the " sphere " takes place by repeated binary
fissions.

During the process of schizogony the merozoites produced by any
particular organism vary as regards size and numbers. In certain cases
the variations are at a minimum, as, for instance, amongst the human
malarial parasites. Plasmodium malaricB of quartan malarial fever pro-

SCHIZOGONY AND SPOROGONY 69

duces nearly always eight merozoites, and these vary little in size (Plate
XIII., p. 934). ^ijnilsiTly, Plasmodium vivax of benign tertian fever pro-
duces, as a rule, sixteen, but departures from this number are not uncom-
mon (Plate XII,, 16-18, p. 926). Amongst other Sporozoa, however, greater
variations occur, as will be described below. In some cases it has been
supposed that the schizogony was of two types- — the one giving rise to a
small number of large merozoites, and the other to a large number of
smaller ones (Fig. 39, 6 and 7). It was supposed that this represented
a sexual dimorphism, one line ending in gametocytes of the female sex,
and the other in gametocytes of the male sex. More careful study of such
cases has thrown doubt on these conclusions, and has tended to show that
every transition, both as regards numbers and size, occurs between the two
types. Thus, in the case of Adelina dimidiata, a coccidium of the centipede,
the merozoites produced by a schizont vary in number from four to
sixteen, as demonstrated by Schellack (1913). As a rule, when the number
is large the merozoites are small, and vice versa. In Hepatozoon canis
(Fig. 39) the number of merozoites produced may be only four, or it may
exceed a hundred. In this case it appears that with successive schizogony
the number of merozoites produced increases, while their size diminishes,
till finally there are formed a large number of small ones which enter the
leucocytes and become gametocytes. It has thus to be remembered that
in any individual species the merozoites produced at schizogony may vary
considerably, both in number and size.

In the case of sporozoites which are produced from the zygote by a
process similar to schizogony, the number and size is much more constant.
Thus the zygotes of coccidia belonging to the genus Eimeria invariably
produce eight sporozoites which are contained in pairs in four sporocysts
(Fig. 337). In other cases, as, for instance, in the genera Barrouxia and
Aggregata, though the number of sporocysts produced by the zygotes of
any particular species may vary considerably, the number of sporozoites
in the sporocysts is constant (Fig. 376). On account of its uniformity
the type of sporogony is of greater value for purposes of identification
and classification than are the forms observed at schizogony.

GEMMATION OR BUDDING.— By this method of reproduction is to
be understood one in which an organism, after its nucleus has divided,
instead of splitting into two equal or nearly equal parts, divides very
unequally, so that one daughter form is very much smaller than the other.
The condition is one of extreme unequal binary fission. It is usual to
regard the large form as a parent individual, and the small one as a daughter.
The process has been described as occurring in free-living amoebae, and the
unequal divisions seen in Tnjpanosoma lewisi, which has already been
referred to, may be regarded as an instance of gemmation (Fig. 197). It

70

MULTIPLICATION OF PROTOZOA

occurs, however, most typically amongst the Euciliata in the attached
Peritrichida like Vorticella, and amongst the Siictoria. In many species of
Vorticella and allied forms the body divides into two equal parts, so that
two equal-sized individuals are attached to the end of a single stalk. One
of these may escape and, attaching itself, develop a new stalk, or it may
remain attached, and the two individuals may form new stalks, so that
eventually a complicated system of dichotomous branches is produced.
The division, though apparently longitudinal, is really transverse, as will
be evident if it is remembered that the organisms are attached to the
stalks by their dorsal surfaces. In some cases the division of the body is
unequal, so that a very small individual is separated from a large one.
These small forms are provided with circlets of cilia, by means of which

Fig. 40. — Ephelota gemrnipara in Gemmation {xca. 350). (After Collin, 1912.)

1. Section of an entire organism, showing method of budding of the macronucleus to form nuclei
of buds. 2. Surface view of budding individual.

they swim away, and ultimately conjugate with one of the larger attached
forms (Fig. 44).

In the Suctoria buds are formed, either from the surface of the body
or in cup-like depressions. In Ephelota, studied by Hertw^g (1876), the
nucleus becomes much branched, and as buds are formed on the surface of
the body, portions of the macronucleus enter each bud. The buds are finally
separated as ciliated embryos (Fig. 40). In other cases, as in Tokophrya
and Choanophrya, there occurs a process of internal budding (Fig. 532).
A depression is formed in the cytoplasm, and the margins of this close to
include a space which communicates with the exterior by a pore. A bud
is formed from the surface of the cytoplasm within this space. A ciliated
embryo is detached, and eventually escapes through the pore.

Though the daughter individuals formed at binary fission may be so

GEMMATION 71

unequal in size that the process is regarded as one of budding or gemmation,
the nucleus of the bud arises by equal division of the nucleus of the parent,
so that the large and small daughter forms have their nuclei of equal size.
A method of formation of the nuclei of buds from chromidia has been
described as occurring in certain Protozoa. Thus, in the case of Entamoeba
histolytica, Schaudinn (1903) supposed that granules of chromatin occurred
in the cytoplasm outside the nucleus. These granules were supposed to
collect in groups at the surface of the organism, become organized into
nuclei, and enter the buds which were forming. Such a process certainly
does not occur in E. histolytica. Another instance in which nuclei have
been described as arising in this manner is that of Arcella vulgaris re-
ferred to above (Fig. 2).

SYNGAMY AMONGST THE PROTOZOA.

As m the higher animals and plants, at certain phases of development,
two cells unite and their nuclei fuse, so amongst the Protozoa a similar
process may occur. This is generally known as a sexual process, or
syngamy. It may take place in one of two ways: either two individuals,
which are known as gatnetes, unite by fusion of their cytoplasm, followed
by union, or haryogamy, of their nuclei; or two individuals become incom-
pletely united, and part of the nucleus of each passes over into the other
individual to unite with its nucleus. After this transference of nuclear
material the individuals separate. The process in which two individuals
unite completely is known as copulation, while that in which interchange
of nuclear material between two temporarily associated individuals takes
place is called conjugation. The two processes are not essentially different
from one another, for it may be considered that in conjugation each of
the two associated individuals really produces two gametes, one of which
is large and contains all the cytoplasm and a nucleus, while the other is
small and consists of a nucleus only. The small gamete produced by one
individual unites with the large gamete produced by the other. It is,
however, convenient to distinguish the process of copulation from that of
conjugation, as the latter is the characteristic method of syngamy amongst
the Euciliata.

COPULATION. — This process consists in the union of two cells with
fusion of their nuclei. The cells are known as gametes, while the single
uninucleated cell resulting from the union is called a zygote, and the nucleus
of the zygote, which is the product of the union of two gamete nuclei, is
the synkarion. The uniting gametes may be the ordinary individuals
which have ceased multiplying, or an ordinary individual, by a special
process of multiplication, may give rise to a number of smaller gametes

72

SYNGAMY IN PROTOZOA

which unite in pairs. In the latter case, the individual which gives rise
to the gametes is known as a gametocyfe, and the process by which it gives
rise to the gametes as gametogony.

The gametes which unite may be alike in size and shape, in which
case they are known as isogametes, and the process of union as isogamy.
On the other hand, they may be recognizably different from one another
in size or structure, and are known as anisogametes. The process is then
called anisogamy or heterogamy. If the gametes differ in size, the large

Fig. 41. — Syngamt of Cercomonas longicauda { x ca. 2,000).

1916.)

(After Woodcock,

1. Two individuals uniting by their posterior ends.

3. Still later stage after nuclei have fused.

4. Stage in which fiagella are lost and body rounded.

2. Later stage in the union.
5. Encysted zygote.

one is called the macrogamete and the small one the microgamete. It
usually happens that the small gamete or microgamete is actively motile,
on which account it is regarded as the male gamete, as it corresponds in
function with a spermatozoon of higher animals. The larger macrogamete,
which is usually a passive body heavily charged with food reserve material,
corresponds with the ovum. There is every transition between the process
of isogamy and anisogamy. Thus, in some cases the gametes are equal in
size, but differ from one another only in the size of their nuclei. In other

COPULATION

73

cases one gamete is only slightly larger than the other, and there is every
gradation towards forms like coccidia or malarial parasites, in which the
macrogamete is a comparatively large cell and the microgamete a very
minute one.

As an illustration of syngamy in which two ordinary individuals unite,
the case of Cercomonas longicauda, as described by Woodcock (1916), may
be considered (Fig. 41). Two flagellates of the ordinary type come
together and unite by their posterior ends, the union gradually extending
forwards. After the two flagellates are completely fused their nuclei
unite to form a synkarion. The zygote which is produced may commence
multiplying by binary fission in the usual manner, or it may encyst.
A similar process occurs in Polytoma uvella, but is modified as a result of
the protective covering of the body (Fig. 42). Two flagellates unite by
their anterior ends, and the cytoplasmic contents of one flow into the other,

Fig. 42. — Polytoma uvella: The Process of Isogamy as observed during thS;
Course of Three Hours ( x 1,500). (Original.)

The contents of one flagellate flow into the other, which gradually becomes spherical. Finally, a
cyst is formed after complete fusion has occurred. The nuclei were no longer visible in the
later stages. The dark rods are the stigmata.

giving rise to a spherical zygote which becomes encysted. In the case of
Copromonas suhtilis two individuals fuse completely, and Dobell (19086)
has stated that each nucleus before union gives off two reduction bodies
(Fig. 48). All these instances are cases of isogamy, in which the gametes
differ little, if at all, from the ordinary adult individuals. Isogamy has
been described by Woodcock (1916) for Sj)iromonas angtista and Co'pro7no7ias
fuminantium.

In most cases, however, certain individuals termed gametocytes, which
may differ from the ordinary reproducing forms, by a special type of
multiplication {(jametogony) give rise to a number of gametes, which then
unite. Syngamy of this type occurs amongst non-parasitic Protozoa, and
has been described, in the case of Foraminifera [TrichosphcEriii'm), Radio-
laria {Thalassicola), and other forms, but the best-known instances occur
amongst parasitic Sporozoa. In the reproduction of Monocystis magna,

74 SYNGAMY IX PROTOZOA

a gregarine of the earth-worm, Cuenot (1901) described the process of
syngamy. Two individuals (gametocytes) encyst together in a common
cyst (gametocyst), and each gives rise to a large number of gametes which
appear to be completely alike (isogamy). The gametes produced by one
individual unite with those produced by the other. The zygotes thus
formed become encysted in secondary cysts (oocysts). In his description
of syngamy in Monocystis rostrata, another gregarine of the earth-worm,
Muslow (1911) also found that there was complete isogamy.

From this condition of complete isogamy, various transition stages
leading to marked anisogamy are known amongst gregarines. Thus, in
the case of Lanlcesteria ascidice, Siedlecki (1899), and in the allied form
Lankesteria culicis of A'edes argenteus, studied by the writer (1911a), the
gametes produced by each gregarine are alike in size, but differ from one
another in that those produced by one gregarine have smaller nuclei than
those produced by the other (Fig. 465). A gamete with a small nucleus
unites with one which has a large one. In the case of Stylorhynchus longi-
collis, heger, L. (1904/^) noted that the gametes produced by one gregarine
were spherical bodies, while those produced by the other were spindle-
shaped structures, each provided with a flagellum. The spindle-shaped
motile gametes were actually larger than the spherical ones, so that if
the former are to be regarded as the male gametes, this instance affords
an exception to the general rule that the male gametes are smaller than
the female (Fig. 482). In the case of the gTeganne Pterocephalus nobiJis,
Leger, L. and Duboscq (1903a) describe the gametes which are formed from
one individual as sniall curved structures (microgametes), and those from
the other as large elongate bodies (macrogametes). In this instance there
is an approach to the condition which is characteristic of the coccidia.
Amongst the coccidia, female gametes or macrogametes are spherical or
ovoid bodies filled with food reserve material in the form of globules, while
the male gametes or microgametes are minute, elongate, sickle-shaped
bodies usually provided with two flagella. The microgametes, which are
provided with two flagella, are composed of chromatin covered by a thin
layer of cytoplasm, and in many respects resemble the spermatozoa of
higher animals (Fig. 337). A similar difference in size exists between
the gametes of the pigmented blood parasites of the genera Plasmodium
and Hwmoproteus and the non-pigmented Leucocytozoon (Figs. 383 and 391 ).
Where a special type of individual, the gametocyte, produces a number
of gametes, the actual number produced by each varies considerably in
different groups. Amongst the gregarines, where two gametocytes are
enclosed in a gametocyst, it is evident that the chance of gametes going
astray is reduced to a minimum, so that both gametocytes produce approx-
imately the same number of gametes. In the majority of gregarines there

COPULATION— C0NJUGATI(3N 75

are many gametes (Fig. 465); there may be not more than a dozen, as in
Schizocystis (Fig. 469), while in the case of Ophnjocystis each gametocyte
produces only a single gamete (Fig. 468).

Amongst the true coccidia or Eimeriidea, the male and female game-
tocytes are not associated, but develop in separate cells of the intestine or
other organ. The number of gametes produced by each individual may be
very unequal in number (Fig. 337). The gametocyte (macrogametocyte)
which gives rise to the macrogamete becomes directly transformed into
a single macrogamete, while the microgametocyte produces a large number
of microgametes. The latter are provided with flagella, and swim away
in search of a macrogamete, which is not itself, endowed with the powers
of movement. It seems evident that the production of large numbers of
microgametes is correlated with the greater uncertainty of the micro- and
macro-gametes coming together. In the case of the malarial parasites
and allied organisms (Hsemosporidiidea), in which fertilization takes place
in the stomach of a blood-sucking insect, the macrogametocyte produces
a single macrogamete, while the microgametocyte gives rise to from
six to ten microgametes (Fig. 391). In the coccidia belonging to the
Adeleidea, the macro- and micro-gametocytes develop in actual contact
with one another. The result of this close association is that, though the
macrogametocyte gives rise to a single macrogamete, the microgametocyte
produces only four microgametes (Fig. 338). In the hsemogregarines of
the genus Karyolysus, in which a similar association of the gametocytes
occurs, Eeichenow (1921) has shown that the microgametocyte produces
only two microgametes (Fig. 457). The marked difference in size betw^een
the microgametes and macrogametes in these cases is associated with the
conditions under which future development will take place. The macro-
gamete is provided with a large amount of cytoplasm heavily loaded with
food reserve material to enable it to survive and develop without nourish-
ment in the encysted condition. As a result of this provision, as in the case
of the ovum, its power of movement has been lost. The male gamete
merely functions as a fertilizing agent, for which its nucleus alone is required,
and for the fulfilment of which a high degree of motility is an advantage.

CONJUGATION. — In the type of syngamy which has just been
described, the two gametes unite completely and their nuclei fuse. This
process is know^n as copulation, to distinguish it from conjugation, which
occurs amongst the Euciliata. In typical conjugation two individuals
associate, and one of the two nuclei, which each then possesses, passes into
the other individual and unites w^ith the nucleus which has remained
stationary. As pointed out above, it is possible to regard the two ciliates
as each producing two gametes, the small gamete (migratory nucleus)
produced by one individual uniting with the large gamete produced by

SYNGAMY IN PROTOZOA

M N U P

Fig. 43. — Syngamy in Paramecium putrmum. (After Doilein, 1916, Slightly

Modified.)

[For description see opposite page-

CONJUGATION 77

the other. The process, which is an exceedingly complicated one, has
been studied in detail, especially in species of Parameciu7n. In the case
of Paramecium putrinum,, for instance, each individual possesses a macro-
nucleus and a micronucleus. When two individuals associate in con-
jugation, they become closely united by their peristomes and the side of
their bodies behind this (Fig. 43). The macronuclei become elongated
and undergo a series of divisions till a large number of fragments are
produced. All these ultimately degenerate and disappear. Meanwhile,
the micronuclei have divided by mitosis, and the two nuclei thus formed
in each ciliate again divide by mitosis. At this stage each ciliate or
conjugant, as it is called, contains four nuclei and a number of degenerating
bodies derived from the macronucleus. Three of the nuclei in each now
degenerate, so that each conjugant is left with only one. This one now
divides again, and of the two resulting nuclei in each conjugant, which as
far as can be seen are exactly alike, one is a stationary nucleus and the
other a migratory one. The migratory nucleus of each conjugant now
passes over and fuses with the stationary nucleus of the opposite conjugant.
The resulting nucleus, which is a zygote nucleus, now divides to give rise
to two, these two to give four, and the four to produce eight nuclei. At
this stage the ciliates, each of which has eight nuclei and still the remains
of the degenerating nuclei, separate from one another and swim away.
Of the eight nuclei, four increase in size and become macronuclei, three
degenerate, while the remaining one retains its character as a micronucleus.
The latter divides to form two micronuclei, and this is followed by division
of the ciliate itself in such a manner that two of the macronuclei and one
of the micronuclei pass to each daughter ciliate. At the next division of
these daughter ciliates the micronucleus divides, and each resulting ciliate
receives one of the two macronuclei and one of the tw^o micronuclei.
Thus, the nuclear condition of the original ciliate is regained. At all
subsequent divisions of the ciliate, both the macro- and micro-nuclei divide.

A. Two associated conjugants with intact macronuclei and commencing division of micronuclei.

B. Macronuclei and micronuclei dividing.

C. Bivided-up macronuclei and two dividing micronuclei in each conjugant.

D. Three of the four micronuclei in each conjugant have degenerated, while the remainmg one is

commencmg to divide.

E. The micronucleus of each conjugant is drawn out into a long spindle.

F. Four resulting micronuclei near the point of union of the two conjugants.

G. Union of the micronuclei in jDairs.

H-L. Progressive division of the micronuclei till each conjugant has eight. The conjugants
finally separate (L.)

M-N. Three micronuclei degenerate, four become macronuclei, while one remains and divides.
The ciliate divides.

0. One product of division of the form with four macronuclei and two micronuclei. It contains
two macronuclei and one dividing micronucleus. The ciliate divides.

P. One product i )f the division of the form with two macronuclei and two micronuclei. It contains
one macronucleus and one micronucleus, and thus resembles the ciliates before they com-
menced conjugation.

78

SYNGAMY IX PROTOZOA

Fig. 44. — Diagrammatic Representation of Nuclear Changes during
Syngamy in YortieeUa nelmlifem. (After Maupas, 1889.)

[For de-^criplion see opposite page.

CONJUGATION 79

This complicated process is best comprehended by reference to the diagram
(Fig. 43). Except for variations in detail, the conjugation of other ciliates
in which the process has been studied takes place in a similar manner. In
Paramecium 'putrinurn the two conjugants are equal in size. In other
ciliates a large individual conjugates with a smaller one, while the most
extreme condition is reached in Vorticella and its allies, in which a small
free-swimming ciliate budded of! from a large pedunculate individual
conjugates with one of the large forms (Fig. 44). The macronuclei in both
degenerate, and the micronuclei undergo a number of divisions, as in
Paramecium. All these degenerate except one which divides to give rise
to a stationary and a migratory nucleus. Each individual, one a large
and the other a small one, now contains two nuclei. Exchange of nuclei
then occurs, as in Paramecium, but the small individual, instead of pro-
ceeding to further development, shrinks and dies, while the large individual
alone survives. The single nucleus of the large surviving individual divides
repeatedly, and a number of macronuclei and one micronucleus are
produced. By successive divisions of the ciliate, similar to those occurring
in Paramecium 'putrinutn, the original condition is regained. In the case
of Paramecium caudatum, the process of syngamy is similar to that of
P. putrinutn (Fig. 45), but in the case of P. aurelia, owing to the fact that
the ciliate possesses two micronuclei instead of one, it is modified in certain
respects. When conjugation occurs, the two micronuclei of each conjugant
divide twice, so that eight are formed. Of these seven degenerate, leaving
in each conjugant one micronucleus and one degenerating macronucleus.
The single micronucleus divides and exchange of nuclei occurs, as in P. jnitri-
num and P. caudatum. After union of the two nuclei the single nucleus
divides twice till four are present, and of these two become macronuclei
and two remain as micronuclei. Each of the latter divides once, so that in
each ciliate there are now two macronuclei and four micronuclei. The ciliate

A. Union of the small free-swimming conjugant with the large attached one.

B. Fragmentation of the macronuclei and division of the micronuclei.

C. D. E. Further divisions of the micronuclei leading to four in the large conjugant and eight

in the small one.

F. All the daughter micronuclei have degenerated except one in each conjugant.

G. The two micronuclei are dividing with the axis of division across the plane of union of the two

conjugants.
H. The two micronuclei in the large conjugant are uniting, while those in the small one remain

separate.
I. The two micronuclei m the large conjugant have united, while those in the small one are

degenerating.
J. The micronucleus of the large conjugant is dividing, while the small conjugant is shrinkmg.
K. The small conjugant has disappeared, while the micronuclei of the large one are dividing.
L, 31. Further divisions of the micronuclei to give rise to eight.

X. Transformation of seven micronuclei into macronuclei and division of remaining micronucleus.
0, P. Division of the body has taken place, giving rise to an individual with four macronuclei

and one micronucleus (O), and one with three macronuclei and one micronucleus (P). By

further divisions the original condition is reached in which the micronucleus and one macro -

nucleus are present.

80

SYNGAMY IN PROTOZOA

Fig. 45. — Diagrammatic Representation of the Nuclear Changes during
Conjugation of Paramecium caudatum. (After Jennings, 1920.)

A. Two associated conjugants.

B. T)egeneration of macronucleus and first division of niicionucleus.

C. Second division of micronuclei to give rise to four, of which three degenerate.

D. Division of remaining micronuclei to produce the gamete nuclei.

E-F. Union of gamete nuclei. G. Separation of the conjugants.

H-J. Division of the nuclei to give rise to eight, of which four increase in size to become macro-
nuclei, while three degenerate.

K. After division of the single micronucleus the ciliate itself divides.

L. After a further division of the micronucleus the daughter ciliates again divide to give rise t(3 the
normal type.

CONJUGATION 81

whicli has separated from its partner divides into two daughter ciliates,
each of which has a single macronucleus and two micronuclei, as in the
original conjugants.

In the conjugation of Collinia branchiarum described below, the two
ciliates unite as inParameciu )ii , and exchange of nuclei takes place(Fig. 495).
The macronuclei, however, behave in a remarkable manner. Each becomes
much elongated, and when exchange of micronuclei is taking place, the
two long macronuclei arrange themselves side by side across the point of
union of the ciliates in such a manner that half of each macronucleus is in
each ciliate. When the ciliates separate the macronuclei divide, so that

Fig. 46. — Conjugation of the Ciliate Cycloposthium bipalmatum, showing
Differentiation of Conjugating Nuclei into Male (^J) and Female ( 2 )
( X ca. 300). (After Dogiel, 1923.)

M., Macronucleus ; Sk., skeletal plate ; An., anus; Ph., pharynx ; My., myonemes ; D., degenerating
micronuclei; Sp., remains of central part of spindle.

each ciliate receives half of each macronucleus. Though this occurs, the
macronuclei ultimately degenerate, and a new macronucleus is formed
from the micronucleus. It will thus be seen that in the Euciliata each of
the two conjugants ultimately contains two nuclei which are exactly alike,
except that one is a migratory or male nucleus, and the other a stationary
or female nucleus. This difference in behaviour is the only indication of sex
differentiation. In the case of Cycloposthium hipalmatum, a ciliate parasitic
in the intestine of the horse, Dogiel (1923, 1925) has noted that, though
conjugation between two individuals takes place in the usual manner, the
two nuclei which take part in the syngamic process differ in that the
migratory one assumes the characters of a male gamete in becoming a
I. ■ 6

82 SYNGAMY IN PROTOZOA

filament provided with a head, while the stationary one retains its original
form (Fig. 46). This observation is a confirmation of the view that the
migratory nuclei in other ciliates are actually male nuclei.

GONOMERY. — A remarkable process of syngamy was described by
Hartmann and Nagler (1908) for Sappinia diploidea, an amoeba isolated
from lizards' faeces. The amoeba is peculiar in being binucleate, the
two nuclei lying close together (Fig. 47). When encystment occurs, two
individuals enter a common cyst. The two nuclei of each individual now^
fuse and then undergo reduction divisions, the reduction bodies degenerat-
ing. After this the two amcebse unite, the nuclei approach one another,
but do not fuse. The amrrbn then leaves the cyst and commences to

d e f

Fig. 47. — Sapjyinia dij)loidea : Fkee and Encysted Stages ( x ca. 1.500). (After
Hartmann and Nagler, 1908.)

a. Usual form with two nuclei. I. Form with dividing nuclei.

c. Dividing form producing two binucleated daughter amcebse.

d. Two amcebse in common cyst. e. The two nuclei in each amoeba have united.

/. The bodies of the two amoebae have fused, giving rise to a binucleated amoeba which escapes
from the cyst and reproduces by binary fission, as at n, h, and c.

multiply by binary fission, the two nuclei dividing by mitosis side by side.
These nuclei are regarded as gamete nuclei, which, however, do not actually
unite, though dividing many times during asexual reproduction till
encystment again occurs. This condition is one of delayed union of gamete
nuclei, a process which is known to occur in higher animals, and which has
been teTmed gono7nery.

METHOD OF UNION OF GAMETES.— The actual union of gametes
during syngamy takes place in a variety of ways, which are dependent on
the structure of the gametes themselves. In the case oi Polytoma uveUa,
Copromonas subtilis, and other forms, the two flagellates approach one

GONOMENY— UNION OF GAMETE8

83

anotlier, and unite first by their anterior ends near the flagellar origin
(Figs. 42 and 48). During this process the flageUates are actively motile.
Their nuclei approach one another and come into contact, and the nuclear
membrane disappears at the line of contact till a common membrane is
formed. In the case of Cercomonas longicauda, Woodcock (1916) observed
union to take place first near the posterior end (Fig. 41).

Union of gametes within the gametocysts of gregarines takes place
in a similar manner. As already explained, sometimes the gametes are

Fig. 48. — Syngamy of Copromonas suhUlis ( x ea.

1. Individual flagellate as seen in living condition.

3. Nuclei dividing to form tirst reduction body.

4. Nuclei dividing t(i fmin sccdud ivdurtion body.

5. Union of nuclei and formation nfcNst

2.000). (After Dobell, 1908.)
2. Early stage in union of gametes.

6. Fully formed zygote in cyst.

alike, and are merely spherical bodies which, coming into contact with
one another, gradually fuse, while their nuclei unite. In other cases the
gametes produced by one gregarine are elongate and provided with
flagella, as in StylorhijncJms, while those produced by the other are spherical
bodies (Fig. 482). Union takes place by one of the elongate flagellated
gametes attaching itself to one of the spherical forms by its pointed
anterior extremity, after which fusion takes place, while the flagellum
disappears. Amongst the coccidia the minute flagellated microgamete
swims activelv and comes in contact with one of the larger immobile

84 SYNGAMY IX PKOTOZOA

macrogametes. Sometimes this occurs before the oocyst has formed; at
other times after its formation, in which case a pore, the micropyle, is
present at one end of the cyst, and through it the microgamete makes its
way. The microgamete enters the cytoplasm of the macrogamete, which
immediately commences to secrete a substance which closes the micropyle.
Though several microgametes may be attracted towards one macro-
gamete, immediately one has entered its cytoplasm this attraction ceases.
The nucleus of the. macrogamete has meanwhile been drawn out into a
long spindle, the fertilization spindle, on the fibres of which the chromatin
granules are distributed. The microgamete nucleus breaks up into
granules, which gradually become distributed upon the fertilization
spindle. The spindle now retracts, and a spherical nucleus containing
chromatin from both the macrogamete and microgamete is again formed
(Fig. 337).

Amongst the pigmented blood parasites of the genera Plasmodium
and Hcemojjroteus a similar type of union occurs. The nucleus of the
macrogamete moves towards the surface of the body, which is raised up
at this point into a small elevation. The elongate motile microgamete
enters this elevation, and its nucleus unites with that of the macrogamete
(Figs. 383 and 391).

Amongst the Euciliata, when conjugation occurs amongst free-swim-
ming forms, it is usually by the peristomes that they become attached
to one another. Actual continuity of cytoplasm appears to take place
just behind the peristomes, to allow of the interchange of nuclei, as
described above. In the attached forms, such as Vorticella, conjugation,
as already noted, takes place between a large attached individual and
a small free-swimming ciliated form which has been budded of! from
another individual. The small free-swimming form attaches itself to the
larger one at a point near the insertion of its stalk, and wdien exchange
of gamete nuclei has occurred it degenerates (Fig. 44).

METHOD OF FORMATION OF GAMETES.— The actual method by which
gametes are formed from gametocytes varies to some extent. Amongst
the gregarines, the nucleus of the gametocyte multiplies by a series
of divisions till the requisite number of nuclei are present (Fig. 465).
These are then arranged upon the surface of the gametocyte, and little
elevations of the cytoplasm are formed. Into each of these there passes
a nucleus. Each small cytoplasmic elevation or l)ud, which has acquired
the characteristic form of the gamete, is now separated by a constriction.
A large amount of the cytoplasm is usually left over as a residual body.
In the case of the coccidia and allied forms, where there is an extreme
condition of anisogamy, one gametocyte, the macrogametocyte, gives rise
to a single macrogamete. It is supposed that this transformation takes

FORMATION OF GAMETES— AUTOGAMY 85

place by the extrusion of one or more reduction bodies. In the case of
the microgametocyte, nuclear multiplication takes place till numbers of
nuclei are formed (Fig. 337). These nuclei at first appear as minute
aggregations of chromatin granules. They change their form on the
surface of the cytoplasm till they appear as dense comma-shaped struc-
tures. Each is then separated with a small amount of cytoplasm, which
contributes to the formation of flagella. In the blood parasites belonging
to the genera Plasmodium, Hcemojjroteiis, and Leucocytozoon, the macro-
gametocyte produces a single macrogamete, as in the coccidia, by the
rapid extrusion of reduction bodies. The microgametocyte gives rise
in the course of a few minutes to six or ten microgametes by a violent
process known as exflageUation, which occurs normally in the stomach of
the invertebrate host, but which may be observed in an ordinary moist
preparation of blood under the microscope (Fig. 381). The details of the
process will be described below in the section devoted to these parasites,
but it may be noted here that the function of the reduction bodies referred
to above is far from clear, and the assumption that the process is com-
parable with the formation of polar bodies during maturation of the
ovum of higher animals does not appear to be correct.

AUTOGAMY. — A process of syngamy which may be defined as self-
fertilization has been described for certain Protozoa under the name of
autogamy. In its most complete form the nucleus of a single individual
divides to form two daughter nuclei. Each of these undergoes reduc-
tion divisions, after which the two surviving nuclei unite. In the
case of Entamoeba coli, Schaudinn (1903) described autogamy in the
encysted stages. The single nucleus of the encysted form divides to give
rise to two nuclei. Each of these gives off two reduction bodies, after
which they divide to form four nuclei, wliich are arranged in pairs at
opposite sides of the cyst. One of each pair is a stationary nucleus and
one a migratory nucleus. The migratory nuclei move to opposite sides
of the cyst, where they unite with the stationary nuclei. The cyst again
has two nuclei, which proceed to divide till the characteristic eight nuclear
stage is reached. The writer (1907) saw certain stages in the development
of the cysts of Entamoeba miiris, which appeared to supply a confirmation '
of Schaudinn's account of E. coli, but there is little doubt that the appear-
ances were capable of another interpretation. All evidence goes to
show that no such process actually occurs in the cysts of E. coli or any
other anio-ba. A somewhat similar process was described by Prowazek
(1904f/) in the cysts of Prowazekella laceyta', while Schilling (1910) recorded
its occurrence in Trypanosofna lewisi. It seems perfectly clear that in
none of these cases was there sufficient evidence to justify the conclusions
whicli were made.

86 SYNGAMY IN PR(3T0Z0A

Hartmann (1909) gave a general account of autogamy amongst Pro-
tista, but a perusal of his paper shows that most, if not all, of the alleged
instances are based on very slender evidence.

PEDOGAMY.— There is another type of self-fertilization which differs
from true autogamy in that a single individual first divides into two
daughter forms after division of its nucleus. When the nucleus of each
has undergone maturation or reduction divisions, the two daughter cells
which are gametes unite. The process which is known as jjedogarny has

W

^^,

.-^^^..^.^

F

Fig. 49. — Pedogamy in Actinosphceriuni eicMomi ( x 80). (From Lang, 1901,
AFTER Richard Hertwig, 1898.)

A. A single multinucleated individual in a primary cyst.

B. Division into a number of uninucleated individuals which become enclosed in secondary cysts.
(!. The contents of each secondary cyst divide into two.

D. The division completed, after which each nucleus undergoes two reduction divisions.

E. The two gametes in each secondary cyst imite.

F. Secondary cysts containing zygotes resulting from the union of the gametes.

been studied by Richard Hertwig (1898) and others in the multinucleated
Heliozoon Actinosphcerium eichkorni (Fig. 49). An individual encysts
and divides into a number of uninucleate forms, which become enclosed
in secondary cysts. Within each secondary cyst a further division into
two individuals takes place. The nucleus of each of these undergoes
two reducing divisions, after which union takes place. In this case the.

PEDOGAMY

87

:-',1^.

w

3

'WW

// ^^

Fig. 50. — Pedogamy in Actiriophri/s sol, Ehrenberg ( x ca. 800). (After
Belak, 1923.)

1 . Loss of pseudi)])odia and their axial filaments.

2. First nuclear division and commencing formation of gelatinous cnvelii])e.

3. Two gamete nuclei in early division stage in the dividing cyti|iiasin within the cyst membrane.

4. Two sejiarate gametes each with a nucleus in process of first niatuiation division (reduction

division).

5 . Later stage of reduction division of the gamete nuclei: the chromosomes in conjugation.

. Two later stages in the reduction division of the gamete nuclei.

7. Completion of the reduction division: each gamete has a nucleus and a reduction body (de-
generate nucleus).

[Continued on j)- 88.

88 SYNGAMY IN PROTOZOA

two gametes are formed from a single individual in the secondary cyst.
In such an example there is an extreme instance of inbreeding. More
recently Belaf (19216, 1923) has described in detail a similar process of
pedogamy for another Heliozoon, Actinophrys sol (Fig. 50). A single
uninucleate individual encysts and divides into two daughter forms, which
become gametes. The nuclei undergo two divisions, one of which is
a reducing division in that the number of chromosomes is halved. One
of the products of each nuclear division degenerates. The two gametes
within the cyst then unite. The development is comparable with that
which occurs in the secondary cysts of Actinosphcerium. In the case of
Actinophrys sol, Schaudinn (1896a) stated that two individuals entered
the cyst, but doubt was thrown upon this by Distaso (1908) and Prowazek
(19136), who stated that the two gametes were derived from one individual.
Belaf has finally confirmed the statements of the latter observers. He
has also noted that occasionally two individuals encyst together, and that
each divides to form two gametes, so that four gametes occur within the
cyst. After the maturation divisions have taken place, the gametes
unite in such a way that those formed from one individual unite with
those from the other. In some cases, of the two gametes formed from one
individual, one is motile and the other not, so that a distinction between
male and female gametes can be drawn (Fig 50, lo).

PARTHENOGENESIS. — Amongst higher animals it sometimes happens
that the ovum, which usually develops only after fertilization, does so
without this having taken place. It is evident from what has already been
explained that in such a case the nucleus will only possess half the number
of chromosomes that it would have had if fertilization had occurred. It
has been found that during the parthenogenetic development of the
ovum the nucleus behaves in a variety of ways, by which the double
number of chromosomes is regained. Another feature of parthenogenesis
is that, though the ovum which develops without fertilization may give
rise to the same type of individual as it does when fertilized, this is not
necessarily the case. Thus, the ova of the honey-bee if fertilized develop
into females, if unfertilized into males. Amongst the Protozoa, several
observers have attempted to establish the occurrence of parthenogenesis.
The most notable instance is that described by Schaudinn (1902tt) for
the malarial parasite, Plasmodium vivax of man. This observer supposed
that the female macrogamete, which usually develops only after fertiliza-

8. Two stages in second maturation division of the gamete nuclei.

9. Completion of second maturation division and formation of second i eduction body: the two

reduction bodies are still present in later gamete.

10. Mature gametes, showing sexual dimorphism: the male gamete has pseudopodia.

11. Union of two gametes and commencing fusion of their nuclei.

12. Zj'gotc within its cyst.

PARTHENOGENESIS 89

tion in the mosquito's stomach, is sometimes able to do so in the human
blood-stream without fertilization. The nucleus is described as dividing
into two parts, one of which is cast oli with a portion of cytoplasm and
degenerates. The remaining nucleus multiplies, and reproduction by
schizogony occurs. In this manner it is supposed that the asexual or
schizogony cycle is started again, and it was claimed that this afforded
an explanation of the occurrence of relapses in malaria. The writer has
long held and taught that the parthenogenetic forms depicted by
Schaudinn were instances of red blood-corpuscles doubly infected with
a gametocyte and a schizont (Plate XII, 19, p. 926). Thomson, J. D. (1917),
also came to this conclusion, and showed conclusively that Schaud inn's
figures purporting to represent a parthenogenetic process were really
instances of doubly infected cells.

The cases of parthenogenesis recorded by Prowazek (1904) for Herpe-
tomonas muscarum and by Gonder (1910a, 19116) for Theileria jmrva have
even less evidence to support them than the instance described above.

The various methods by which syngamy is accomplished amongst the
Protozoa may be grouped as follows:

1. Copulation. — Complete union of two individuals.

(1) Two individuals having the characters of the ordinary repro-

ducing forms unite.
((/) The uniting forms are equal in size (isogamy).
(h) The uniting forms are unequal in size (anisogamy).

(2) Two individuals (gametocytes) give rise to a number of smaller

forms (gametes) which unite in pairs,
(a) The gametes produced by the gametocytes are equal in

size and characters (isogamy).
(6) The gametes produced by one individual are unlike
those produced by the other (anisogamy).

(i.) The number of gametes produced by the gameto-
cytes are equal, or approximately equal, in
number.

(ii.) One gametocyte (macrogametocyte) gives rise to
one large gamete (macrogamete), while the other
(microgametocyte) gives rise to a variable
number of small motile gametes (microgametes).

2. Conjugation. — Two individuals (conjugants) associate, their nuclei
divide, and exchange of daughter nuclei takes jjlace, after which the
conjugants separate.

(1) The conjugants are equal in size.

(2) The conjugants are unequal in size, one, a small one (micro-

conjugant), associating with a large one (macroconjugant).
In some cases, after interchange of nuclei the microconjugant
degenerates.

90 NUCLEAR DIVISION IN PROTOZOA

3. Autogamy.- — The nucleus of a single individual divides into two.
Each of these daughter nuclei undergoes reduction divisions, after which
they unite. It is extremely doubtful if this process ever occurs.

4. Pedogamy. — A single individual divides into two. Reduction
divisions of the nuclei of these two daughter individuals which are gametes
take place, after which the gametes unite and their nuclei fuse.

5. Parthenogenesis. — Part of the nucleus of a gamete, which normally
develops only after union wdth another gamete, is extruded, after which
multiplication occurs. There appear to be no convincing records of such
a process amongst the Protozoa.

NUCLEAR DIVISION AMONGST THE PROTOZOA.

The division of a nucleus which takes place by simple constriction
into two parts without formation of chromosomes is known as amitotic
division, to distinguish it from mitotic division, in which definite chromo-
somes and a spindle, associated with the presence of centrosomes, occur
as described above for the nuclear divisions of the cells of higher animals.
Between what appears to be true amitosis and mitosis there occur many
gradations. In some cases the appearances are in every way comparable
with what has been described above as typical mitosis in the cells of
higher animals. In other instances the nuclear membrane persists, and
the whole process of mitosis occurs within the nuclear membrane. In
other cases, again, there appear to be no centrosomes associated with
mitosis within the nuclear membrane, though many observers describe an
intranuclear structure, called the centriole, which is supposed to function
as a centrosome. As regards the nature of this body and its actual
existence there is much diiierence of opinion. That the formation of
a spindle may occur without definite centrosomes being identifiable has
long been recognized in higher plants, so there is no reason to suppose
that this may not happen amongst the Protozoa. When mitosis occurs
within the nuclear membrane, definite chromosomes may be formed at
the equator of the spindle, and these divide into daughter chromosomes
in the usual manner. In other cases, though a spindle is formed, the
chromatin granules become arranged irregularly upon the spindle fibres
without uniting into definite chromosomes. No equatorial plate is formed,
and the nucleus merely constricts into two parts. It is possible that in
some of these instances of irregularly arranged chromatin granules there
are produced a very large number of minute chromosomes which actually
divide. In order to distinguish these intermediate types of mitosis from
typical mytosis, the term promitosis has been introduced by Nagler (1909).

A good illustration of complete mitosis is afforded by the nuclear

MITOSIS

91

%

.v:'^^v.

I ^

'^Vf^n^V^

r7-/-r

S>^J.

^yf0^

i'.

'^-.

^-^^f

Fig. 51. — Stages in the Nuclear Division of Acanthocijstis avideata in which
THE Central Granule Functions as a Centrosome ( x ca. 690). (After
SCHAUDINN, 1896.)

1 . Ordinary individual, showing nucleus and central granule, from which radiate the axial fibres

of the pseudopodia (axopodia).

2, 3. Changes in nucleus nt ci mi men cement of division.

4. Division of central L;iauiile ami iiu •leu- in s|i;reine stage.

5, 6. Formation <>i eludmosoines in nucleus, a- it takes up a central position on the spindle

which forms between the two granules.

7. Disapijearance of nuclear membrane : formation of equatorial plate.

8. Sejiaration of daughter plates of chromosomes.

9. Cytoplasm dividing and two nuclei and central granules returning to the condition of individual

at 1.

92 NUCLEAR DIVISION IN PROTOZOA

division of Acanthocyslis aculeata, one of the Heliozoa, as described by
Scliaudinn (189G6), (Fig. 51). In the ordinary individual the centre of
the body is occupied by a granule, from which radiate the axial fibres
supporting the fine pseudopodia. The nucleus, which has a membrane
and large central karyosome, lies at one side of the central granule. When
division is to take place, the nucleus increases in size and the karyosome
becomes loculated, broken into a number of separate parts, and finally
disintegrated as minute granules which arrange themselves in the form
of a spireme or coiled thread. Meanwhile the supporting fibres of the
pseudopodia have disappeared, while radiating fibres develop in the
cytoplasm in connection w^ith the central granule, which, on account of
the part it plays in nuclear division, must be regarded as the centrosome.
The latter structure divides, and as the two daughter centrosomes separate
a spindle is formed between them, while radiating fibres form two asters.
The nucleus, within which the spireme has segmented into a number of
separate parts, now moves to the equator of the spindle. The nuclear
membrane disappears, and a number of small chromosomes take up a posi-
tion on the spindle as an equatorial plate. The individual chromosomes
divide, and there are formed two daughter plates which move towards
opposite poles of the spindle. At this stage the body of the Heliozoon,
which has become elongated, begins to show a constriction around its
centre. The spindle is finally divided at its centre, and the daughter
chromosomes of each plate become transformed into a karyosome, while
a new nuclear membrane is developed. The centrosome remains as the
central granule of the daughter individual which has been formed, and
new axial fibres are developed. In this division, practically all the stages
of mitosis as seen in the Metazoan cell occur.

Typical examples of mitosis occur also in the case of gregarines, the
nuclei of which divide repeatedly to form the gamete nuclei. Muslow
(1911) has described the process as it occurs in Monocystis rostrata, one
of the species of Monocystis which inhabit the vesicula seminalis of the
earth-worm. The resting nucleus consists of a nuclear membrane and
large central karyosome. When the first nuclear division is to take place
after two gregarines have become encysted together in the gametocyst,
the large karyosome breaks up, while a long twisted thread of chromatin
granules appears at one side of the nucleus (spireme stage). Meanwhile,
from two small areas on the surface of the nuclear membrane, radiations
appear in the cytoplasm to form the two asters. Between these, spindle
fibres develop, and with the disappearance of the nuclear membrane the
chromatin thread becomes segmented into eight looped chromosomes,
which arrange themselves at the equator of the spindle. Each chromo-
some becomes divided longitudinally, and the two groups of eight daughter

MITOSIS

93

4

*i?.^l^

.^1

Fig. 52. — Various Stages in the Late Mitotic Division of the Nucleus of

Monocystis rostrata (1-3x2,000; 4-8x1,700). (After Muslow, 1911.)
1. Resting nucleus. 2, 3. Formation of eight chromosomes.

4. Commencing splitting of the chromosomes.

5. Daughter chromosomes separating at equator of spindle, which is devoid of eentrosomes and

asters.

6. Daughter chromosomes moving towards the poles of the spindle.

7. Chromosomes breaking up into gametes.

8. Reconstitution of the nuclei

94 NUCLEAR DIVISION IN PROTOZOA

chromosomes move to opposite poles of the spindle. The central part
of the spindle disappears, the chromosomes break up into granules, and
with the formation of a nuclear membrane the nucleus is reconstructed.
In subsequent divisions the process is very similar, except that a spindle
is formed without definite centrosomes or asters (Fig. 52).

Very similar mitotic divisions of the nucleus were described by Brasil
(1905) also in the case of a species of Monocystis of the earth-worm (Fig. 53).
In both these instances the nuclear membrane disappears during division,
but in other cases the nuclear membrane persists during the whole mitotic
division of the nucleus.

In the case of Actinosjjhcprium eichhonii, the life-history of which has
been described in detail by Richard Hertwig (1898) in a classic memoir,

• -%

^/

\v

1 2

Fig. 53. — Nuclear Divisions in Associated Monocystid Gregaeines
(Monocystis sp.) of the Earth-Worm (x 900). (After Brasil. 1905.)

1. First nuclear division, showing centrosomes, spindles, and elongate daughter chromosomes.

2. Later nuclear divisions in various stages of mitosis.

very clear examples of mitosis occur. The multinucleate organism, as
mentioned above, becomes encysted in a large primary cyst, within which
it divides into a number of daughter individuals round which secondary
cysts are formed (Fig. 49). Within the secondary cyst a further division
into two individuals takes place. The nucleus of each of these divides
by mitosis to form two nuclei, one of which degenerates. A second
division of the surviving nucleus takes place, and again one of the
resulting nuclei degenerates. After this, the two individuals or gametes
in the secondary cyst unite and their nuclei fuse. The various nuclear

MITOSIS 95

divisions take place by mitosis. When the nucleus of a gamete in the
secondary cyst is about to divide for the first reduction division, there
appears at one side of the nucleus an area of clear cytoplasm towards
which the linin network of the nucleus with its chromatin granules is
drawn (Fig. 54). Into this clear cytoplasm some of the chromatin granules
of the nucleus are attracted, and by their aggregation give rise to the
centrosome. It is possible the centrosome was already present, either
in the nucleus or outside it, and that the commencement of its activities
results in the concentration of the nuclear elements at this pole of the
nucleus, and even the escape of some of the chromatin into the cytoplasm.
Whether Hert wig's account of the origin of the centrosome is correct or
not, when it becomes apparent it is situated at some distance from the
nuclear membrane, and is surrounded by radiations, the bulk of which
are directed towards the nuclear membrane. Division of the centrosome
takes place, and one of the resulting pair takes up a position at the oppo-
site pole of the nucleus. There are now two asters between which spindle
fibres appear. The nucleus occupies a position between the two centro-
somes, and the spindle fibres extend through the nuclear membrane and
the substance of the nucleus, so that there is both an extranuclear and
an intranuclear portion of the spindle. The chromatin granules of the
nucleus now form a series of chromosomes which become arranged in the
form of a plate across the equator of the spindle within the nuclear
membrane. Each chromosome divides, and there result two daughter
plates which, just behind the ends of the elongating nuclear membrane,
move towards the centrosomes. The nuclear membrane, which has
divided, now closes round the chromosomes, which gradually disintegrate,
so that daughter nuclei are formed. One of the nuclei now degenerates.
As already remarked, the nuclei of the two individuals in the secondary
cysts undergo two such divisions, the description just given applying to
the first of these. The second division is of a similar type, and again one
of the daughter nuclei degenerates. Richard Hertwig regards both these
divisions as reduction divisions, though he believed that the chromosomes
actually divide in each instance. It seems reasonable to suppose, from
wdiat is now known to occur in other Protozoa, that in one of the two
divisions splitting of the chromosomes does not take place, but that they
separate into two groups, so that the number of chromosomes in the
daughter or final gamete nucleus is halved. This is all the more probable
since, in Actinophnjs sol, an allied form which has a similar syngamic
process, Bela' (19216, 1923) has noted that in the first of the divisions
the chromosome number of forty-four is reduced to twenty-two (Fig. 50).
To return to AcfinnspluBnuni eichhorni, the many nuclei which an adult
contains become the nuclei of the daughter individuals which form the

96

NUCLEAR DIVISION IN PROTOZOA

■.-tj'^,1 .

^■7^

^Ov

^ J

^.^0'

c„

t->

0>*

,3u-^"*"7~7"1'i

;u>^

^OV'',!M.I J lil ||

S'»jr,i??i?'ri

^^HJ^infiV;

•^^o:,:;^^ '^

'^05^^" 6

v-'V^J'';:./- '//"^!-.>

■^ .it* iis!^^ii'l/4'

%vV/^ 7

••'/'/,

Fig. 54. — First Eeduction Division of the Nucleus of One of the Two
Oametes in the Secondary Cyst of Actinosjyhcerium eichhorni ( x ca. 1,200).
(After E. Hertwig, 1898.)

[For descriplion see opposite page.

MITOSIS 97

secondary cysts. These nuclei are the result of repeated mitotic divisions
of the nucleus of the parent. These divisions differ from those described
as taking place in the gametes in the secondary cysts, in that definite
centrosomes do not occur. Similarly, when the daughter individual in
the secondary cyst divides to form the two gametes, its nucleus divides
without the formation of centrosomes (Fig. 55). Indications of longi-
tudinally arranged fibres can, however, be detected within the nuclear
membrane, and also in a cone-shaped portion of cytoplasm which occupies

huh

^

Fig. 55. — First Nuclear Division in the Secondary Cyst of Aciinospluerium
ciclihorni ( x ca. 1,200). (After E. Hertwig, 1898.)

1. Chromosomes forming in the nucleus. 2. Chromosomes arrtanged as an equatorial plate.

.3. Daughter chromosomes separating as two j^lates.

No definite centrosomes appear at any stage.

the poles of the elongating nucleus. Chromosomes are formed, become
arranged as an equatorial plate, and divide into daughter chromosomes
in the usual manner.

As an illustration of another type of mitosis in which a definite spindle
and chromosomes are formed associated with disappearance of the nuclear
membrane and complete absence of centrosomes, Amoeba glebcB {Hart-
mannella glebce), a soil amoeba described by Dobell (1914c/), may be con-

1. Centrosome with radiations.

2. Two centrosomes at opposite ]iolcs of nucleus in which chromosomes are commencing to form.

3. The spindle has formed between the centrosomes. and chi'omosomes have taken u]) a position

as an equatorial plate. 4. Commencing division of the chromosomes.

5. The chromosomes have divided and two equatorial ]i!ates are foimcd.

6. Passage of the daughter chromosomes towards the ctntrdsomes.

7. Later stage, in whiiji the nuclear membrane is closinu round the chromatin granules.

8. Two daughter uiulci have formed, though the n'liiiiiiis of the spindle and the radiations from

the centrosome, which has itself disappeared, are still to be distinguished.

98

NUCLEAR DIVISION IN PROTOZOA

/5«v^"'

M

i'm)

;\v

#

•»,.—• ^.

/

'•Jt-..»..-,

,„:y

^:

/<9

^:;^

'#>'i
^^J

X'

//

■'' 9

,\

12

Fig. 5G. — Ilartmannella glebce : Binary Fission to show Various Phases of
Nuclear Division (x 2,000). (After Dobell, 1914.)

[ For dcscriplion see opposite pfuje]

MITOSIS 99

sidered (Fig. 56). The resting nucleus consists of a fairly thick membrane
and a large central karyosome round which are arranged a series of
granules. When nuclear division, preparatory to division of the amoeba,
commences, the nuclear membrane becomes thin and the karyosome
fragments into a number of fine granules, while those which surround
the karyosome disappear. Those originating from the karyosome run
together to form larger granules, which become arranged as a long-coiled
chain of beads which, decreasing in length, finally occupies the equator
of the nucleus as a ring. The linin network of the nucleus now shows
indications of spindle-fibre formation and the nuclear membrane dis-
appears. The spindle, which has rounded ends and no centrosomes or
asters, becomes slightly elongated, while the chromosomes, sixteen in
number, which are arranged as a ring round the equator of the spindle,
divide so that two rings of daughter chromosomes are formed. These
separate from one another as the spindle itself becomes greatly drawn
out. Finally, each ring of daughter chromosomes which has moved to
the end of the spindle is broken up and a nuclear membrane is formed.
The daughter nucleus is at first flattened, but gradually increases in size,
and, with reconstruction of the karyosome, assumes the characters of
the original parent nucleus. Before this stage is reached the amoeba,
which has become elongated, is divided by constriction into two parts.
In this division there are no granules which could be interpreted as
centrioles at the apices of the spindle, nor was it possible to discover any
indications of a centrodesmose, so that it would appear that centrosomes
and centrioles are completely absent.

The division of the nucleus of Entmnoeha histolytica, as seen in the
encysted forms, is of a similar type, but the nuclear membrane remains
throughout the process (Fig. 57). The earliest stage appears to be the
division of the minute central karyosome. The two daughter karyosomes
separate, while a spindle forms between them. On the equator of the
spindle, which is surrounded by the elongating nuclear membrane, appear
a ring of chromosomes in an equatorial plate. These divide to form
daughter chromosomes, which pass towards the poles of the elongating
spindle in an irregular manner. According to Kofoid and Swezy (1924o,
1 925) the chromosome number is six. As the spindle elongates the daughter

1. Usual type of amoeba : nucleus with large central karj'osome surrounded by granules.

2, 3. Karyosome breaking up into granules.
4. Chromatin arranged as irregular loop.

."). iJi.sappearance of nuclear membrane : spindle with equatorial plate of chromosomes.

()-9. Division of chromosomes to form daughter plates, which pa.ss to the poles of the elongating

spindle.
1(1. Ciiinmencing division of amoeba.
II. Disappearance of spindle, reconstruction of nuclear membrane, and commencing reconstrue-

tion of karyosome. 12. Encystr d amoeba.

100

NUCLEAR DIVISION IN PROTOZOA

/:

1

<y

§:

%

^^<^^

<'~\

^(y

Fig. 57. — Divisions of the Nuclei in the Cysts of Entamfeha histoli/tim ( x 2,000).

(Original.)

The deeply stained chromatoid bodies are shaded, while the limits of the cytoplasm and vacuoles
are shown in outline.

MITOSIS 101

karyosomes disappear, and there cannot be detected any structures like
centrosomes at the apices of the spindle. When the nuclear membrane
commences to divide, karyosomes of the daughter nuclei reappear.

A modification of the preceding type of division is seen in an amoeba
described by Dobell (1914a) under the name Amoeba lacertce. The amceba
is a common parasite of the intestine of Lacerta muralis and other lizards.
In the resting condition the nucleus consists of a nuclear membrane and
large central karyosome, in which all the chromatin of the nucleus is said
to be aggregated (Fig. 58). When nuclear division commences, coarse
granules of chromatin can be distinguished in the karyosome. These

•^- *:

.^

■•^^

Fig. 58. — Nuclear Division in Valilkampfia dobelli {Amceba lacertm, Dobell, 1914)
(x 2,000). (After Dobell, 1914.)

1. Ordinary form with nucleus containing large karyosome.

2. Karyosome breaking into granules.

3. Elongation of karyosome and arrangement of chromatin granules in meridional lines.
4 7. Elongation and constriction of karyosome.

8-9. Comijletion of nuclear division and commencing division of cytoplasm.
10. Encysted form.

become finer and arranged in meridional lines on the surface of the karyo-
some, which now becomes elongated, as does also the nuclear membrane.
On the surface of the elongated karyosome granules of chromatin are
arranged in longitudinal rows, and some indication of fibres can be de-
tected. The granules gradually collect at the two poles of the karyosome,
which itself becomes constricted at its centre and finally divided into
two parts. This is followed by constriction and division of the nuclear
membrane, which has persisted throughout the division process. The
daughter karyosomes contract to the spherical form, while the granules
of chromatin unite to form larger granules. During this division the

102 NUCLEAR DIVISION IN PROTOZOA

essential features are the appearance in the karyosome of granules which
become irregularly arranged in longitudinal rows on the fibres which
appear in the elongating karyosome. Spindle fibres are thus produced,
but the chromatin granules do not unite to form chromosomes, nor is
an equatorial plate developed. Nothing in the nature of a centrosome
is present.

The stages in the division of Amoeba hyalina (Hartmannella hyalina),
described by Hartmann and Chagas (19106), illustrate the type of division
in which a centriole is supposed to be present (Fig. 59). The resting
nucleus is described as having a centriole within the karyosome. When

0)- ■^- -# ■"^•

'^^M^

5

r*vsi.

)' iw^.

)V

Fig. 59. — Stages in Mitotic Division of Nucleus of Hartmannella hyalina, in

WHICH it is supposed THAT A CeNTRIOLE FUNCTIONING AS A CeNTROSOME IS

PRESENT (x 3,700). (After Hartmann and Chagas, 1910.)

division commences the centriole divides, and as the two halves separate
they are connected by a centrodesmose. The chromatin of the karyosome
breaks up into granules, which become arranged as chromosomes in an
equatorial plate. Each daughter centriole has now taken up a position
at the apex of the spindle-shaped nuclear membrane, within w^hich is
a system of exceedingly fine spindle fibres. The daughter plates of
chromosomes are formed, and these move towards the poles of the spindle.
Finally, the chromosomes at each end run together to form the karyosome,
in which the centriole is included, while the intermediate part of the
spindle disappears. It is by no means clear that the above is an accurate
description. Other observers who have investigated the nuclear division
of this or similar amoebse have failed to detect the centrioles (Fig. 89).
Arndt (1924), in describing the nuclear division of HartmanneUa

MITOSIS

103

klitzkei, which, according to him, takes place by typical mitosis with
extranuclear centrosomes, states that he has been able to demonstrate
simihxr centrosomes in four species of Hartmannella. The centrosome,
which does not originate from an intranuclear centriole, is easily overlooked,
and requires very special technique for its demonstration (Fig. 60). In
the resting nucleus it lies against the outer surface of the nuclear mem-
brane, and when division commences it divides into two daughter centro-
somes, which take up positions at the poles of the spindle. It is evident,
in the light of these observations, that the granule described as a centriole

a ^.

J I

I !

Fig. 60. — Stages in the Nuclear Division of Hartmannella Iditzlcei to snow
THE Presence of the Extranuclear Centrosome as revealed by Mann's
Stain ( x 2,500). (After Arndt, 1924.)

by Hartmann and Chagas cannot be a centrosome, and that the cases of
mitosis which have been recorded as taking place without centrosomes
require reinvestigation.

Another type of nuclear division which is distinct from those described
above occurs in amoebae belonging to the genera Vahlkampfia and Dima-
stigamoeha (Fig. 61). The resting nucleus has a large central karyosome
and peripheral chromatin in the form of fine granules within the nuclear
membrane. The nuclear membrane persists throughout nuclear division,
during which the karyosome becomes elongate and then dumb-bell-shaped,
and finally constricted into two daughter karyosomes. These may
remain connected by a fibre or centrodesmose, which in some cases can
be seen to unite two granules which are embedded in the dense daughter
karyosomes. Between the two karyosomes and surrounding the centro-

104 NUCLEAR DIVISION IN PROTOZOA

desmose are spindle fibres, at the equator of which chromosomes become
arranged. These are formed from the peripheral chromatin granules
of the nucleus, and possibly some which have separated from the karyo-
some. The chromosomes split to form two plates, which move towards
the daughter karyosomes. The centrodesmose and the spindle fibres
disappear, while the nuclear membrane is divided by constriction. Two
daughter nuclei, each with a larger central karyosome and peripheral
chromatin granules, are reconstructed.

In many cases, as, for instance, in trypanosomes and allied flagellates,
in which the nucleus consists of a nuclear membrane containing a large
central karyosome, all that can be detected in nuclear division is the
elongation of the nuclear membrane, within which the karyosome becomes
drawn out and finally dumb-bell-shaped. The narrow intermediate
portion may be quite short, or it may be very much drawn out. In
either case it finally disappears, leaving two daughter karyosomes. By
constriction and division of the nuclear membrane two daughter nuclei
are formed (Fig. 156). It is maintained by the advocates of the centriole
theory that the narrow intermediate portion in the dumb-bell stage
represents a centrodesmose connecting two daughter centrioles which are
lodged in the daughter karyosomes. Certain appearances which are some-
times seen might lend support to this view. Occasionally, a dividing
nucleus may be seen, in which a small granule is situated at each end of
the elongated nuclear membrane. These are connected by a fine fibre,
at the centre of which the still intact karyosome lies. Such an arrange-
ment might be interpreted on the supposition that the centriole within
the karyosome has divided prematurely, and that the two daughter
centrioles have passed out of the karyosome, which has not yet shown
any sign of division. At a later stage the karyosome divides, and the
two daughter karyosomes pass to the ends of the nucleus and again enclose
the centrioles. Such appearances, however, are unusual, and may be
merely accidental arrangements of chromatin granules. Hartmann and
Noller (1918) have given another account of the nuclear division in
Trypanosoma theileri (Fig. 156). They maintain that the apparently
elongated karyosome is really a spindle, at each apex of which is a centriole,
and that fine granules of peripheral chromatin form chromosomes which
become arranged as an equatorial plate. Mitotic divisions of trypanosome
nuclei have been described also by Chagas (1909) and Nieschulz (19226).

As already intimated, the Protozoan nucleus sometimes contains a body
which is entirely devoid of chromatin, and appears to consist of plastin
material alone. In nuclear division it may disintegrate and disappear,
to be re-formed again in the daughter nuclei. In some cases, however, it
divides into two parts, which pass to the poles of the spindle with the

MITOSIS 105

daughter chromosomes, and finally enter the daughter nuclei. Bodies
of this kind have been described by Reichenow (1921) in Karyolysus, and
the writer has seen them in Hepatozoovi baJfouri (Fig. 35). These plastin
bodies are not essentially different from karyosomes, which consist mainly

%

•!»

<^*.

'9

Fig. 61. — Amgeboid Phase of Dimastigamceha gruberi from Culture on Agar
Plate showing Method of Nuclear Division ( x ca. 1,350). (Original.)

1. Usual type of amoeba.

2. Commencing nuclear division. The karyosome has become elongate and granular.

3. The karyosome has become dumb-bell-shaped and the nucleus is filled with granules.

•4. There is an equatorial plate of dividing chromosomes, and the dividing karyosome has formed
the pole caps, which are still united by a fibre (centrodesmose).

5. The daughter chromosomes have become aggregated, and are passing towards the pole caps,

which have lost the connecting fibre. Each pole cajj has a central granule.

6. The nucleus has divided and each half is retracting.

7. Slightly later stage witli tlaughtcr nuclei still further retracted.

8. Form with two reconstituted nuclei.

9. Form with two nuclei in division: equatorial plate stage.

10. Form with two nuclei in different stages of division.

of achromatic material. In nuclear division the plastin substance,
whether it be regarded as a karyosome or not, may divide into two parts,
one of which goes to each daughter nucleus, as in Dimastigamoeba (Fig. 61),
or it may break up and disappear as a single body, to re-form in the
daughter nuclei, as in HartmanneUa (Fig. 56).

106 NUCLEAR DIVISION IN PROTOZOA

When reproduction by binary fission occurs, division of the nucleus
is followed by division of the body of the organism into two parts. When
multiplication by schizogony takes place, or when a number of gametes
are produced, the nucleus divides into two, these into four, and so on,
till the requisite number is reached. The multinucleated organism then
buds from its surface a number of daughter individuals. The repeated
divisions of the nuclei frequently take place by mitosis, especially when
they are multiplying to form gamete nuclei, as in the case of gregarines
or coccidia. It sometimes happens that before the spindle of one division

f/

^

if..

■A

Fig. 62. — First Nuclear Division in One of a Pair of Associated

MONOCTST'ID G-REGARINES {MonocysUs SP.) OF THE EaRTH-WORM. (AfTER

Brasil, 1905.)

1, 2. Two centrosomes are present, the nucleolus is breaking up, while the chromatin has collected
at the centre of the nucleus ( X 900).

3. The spindle has formed, the nucleus has been extruded, and chromosomes are found at the
equator of the spindle { X 9C0) .

•1. The chromosomes have divided and are passing to the poles of the spindle, where the centro-
somes have already divided for the succeeding division ( X 900).

5. Though the nuclei have not been definitely reconstituted, the spindles for the next division
have formed ( X 800).

has disappeared the two asters and the centrosomes, if these be present,
divide again, so that two asters are formed at each end of the spindle.
These may separate and form a new spindle between them, so that when
the daughter chromosomes reach the pole of the original spindle they are
already at the equator of a new one. In this manner very complicated
poly-aster figures may arise. Precocious formation of daughter asters
while the original spindle is still present has been shown to occur in a
gregarine (Monocystis) of the earth-worm by Brasil (1905) (Fig. 62).
Very complicated poly-aster figures similarly occur during nuclear

MITOSIS

107

multiplication in species of Aggregata, as described by Moroff (1908)
and other observers (Fig. 63).

The main types of nuclear divisions of Protozoa may be thus
classified :

1. Mitotic division with centrosomes, asters, achromatic spindle,
chromosomes, equatorial plates, and all the stages seen in the typical
nuclear division of higher animals. The nuclear membrane may or may
not persist during division. A nucleolus or plastin body, if present, may
be divided into two parts, one of which goes to each daughter nucleus,
or it may break up and disappear, the daughter nuclei re-forming their
nucleoli when division is approaching completion.

Fig. 63. — Poly-aster Figure resulting from Successive Nuclear Divisions
IN Male Gametocyte of Aggregata jacquemeti ( x 750). (From Minchin, 1912,

AFTER MOROFF.)

2. Mitotic division of the above type, except that centrosomes and
asters have not been detected.

3. Division in which there is formed within the nuclear membrane
a spindle upon which chromatin granules are irregularly arranged. There
are no asters or centrosomes. It is possible that the granules of chromatin,
though not arranged as an equatorial plate, are actually chromosomes,
which divide into daughter chromosomes as they do in the preceding
types of division. The karyosome may divide into daughter karyosomes,
or break up to be re-formed in the daughter nuclei,

4. Division in which the large central karyosome elongates and becomes
constricted. The two halves move to the ends of the elongating nuclear
membrane to form the pole caps between which a spindle is formed.
The peripheral chromatin becomes arranged as chromosomes in an

108 CHROMOSOMES DURING SYNGAMY IN PROTOZOA

equatorial plate. The pole caps become the karyosomes of the daughter
nuclei.

5. Division in which the karyosome becomes elongated and divided
within the nuclear membrane without development of spindle fibres
or chromosomes. This type of division is seen in the nuclei of small
organisms, and it is probable that it is actually similar to type 4, the
spindle fibres and chromosomes escaping detection owing to their
minuteness.

In those cases in which a centrosome is not present, some observers
claim that its place is taken by an intranuclear centriole.

BEHAVIOUR OF CHROMOSOMES DURING SYNGAMY.

Reference has already been made to the nuclear changes which occur
during the development of the ovum and the spermatozoon, and it has
been pointed out that the chromosome number of the zygote nucleus is
not doubled as a result of syngamy owing to the fact that after meiosis
the nuclei of the uniting gametes contain half the normal number of
chromosomes. Several instances of similar reduction divisions of Pro-
tozoan nuclei, whereby the number of chromosomes is halved, have been
recorded.

Muslow (1911) gives a clear account of a supposed reduction division
in Monocystis rostrata (Fig, 64). The nuclei of the two gregarines which
enter the gametocyst multiply by repeated mitotic divisions in which
eight chromosomes are present, as noted on p. 92. Eventually, after
nuclear division has ceased, a number of gametes are budded off from
each gregarine, and these unite in pairs and their nuclei fuse. During
the last nuclear division, whereby the gamete nuclei are formed, though
eight chromosomes appear on the equatorial plate, when the daughter
plates are formed, there is no splitting of the chromosomes, as has occurred
in previous divisions, but the eight chromosomes are separated into two
groups of four, which move towards the poles of the spindle. It was noted
also that the eight chromosomes composing the equatorial plate consisted
of four pairs of homologous chromosomes, the members of each pair
differing from those of other pairs, and that one of each pair entered each
daughter plate of chromosomes. This last division, which gives rise to
the nuclei of the gametes, is thus a true reduction division or meiosis, like
that which occurs in the production of gametes in higher animals. When
the gametes unite, the nucleus of the zygote, receiving four chromosomes
from each gamete nucleus, again has eight chromosomes or four pairs of
homologous chromosomes.

In the case of Diplocystis schneideri, a gregarine of the cockroach,

MEIOSIS 109

Dobell and Jameson (1915) have given a description of a reduction division
which differs from that of Muslow. According to these observers, during
all the division stages of the nuclei, including the last division which gives
rise to the gamete nuclei, there are three chromosomes which divide to
form the chromosomes of the daughter nuclei (Fig. 65). The nuclei of
the gametes thus have three chromosomes, as do the nuclei of the preceding
stages. When the gametes unite and their nuclei fuse, the zygote nucleus
has six, or double the number of chromosomes found at other stages.
The zygote nucleus now proceeds to division, and it is in this division that
the reduction occurs, three of the six chromosomes passing to each daughter
nucleus. At all subsequent division stages of the nuclei the three chromo-
somes are divided. In Muslow's account of Monocystis rostrata it was

? ^

1 2

Fig. 64. — Last Nuclear Division in One of Two Associated Gregarines,
Monocystis rostrata, to show the Reduction of the Chromosome Number
FROM Eight to Four in the Gamete Nuclei ( x 5,000). (After Muslow, 1911.)

1. Eight chromosomes in nucleus. 2. Eight chromosomes arranging themselves in pairs.

.•{. Separation of the individual chromosomes of each pair.

4. Four chromosomes moving to each pole of the spindle to form the gamete nuclei.

during the last nuclear division in the production of gamete nuclei that
the number of chromosomes was halved, whereas in Dobell and Jameson's
account of Diplocystis schneideri the reduction does not occur at this
stage, but at the first division after the zygote nucleus has been formed.
According to Muslow, the haploid number of chromosomes of Monocystis
rostrata is four, and occurs in the gametes, while all other nuclei have the
diploid number of eight chromosomes; on the other hand Dobell and
Jameson in Diplocystis schneideri find that the diploid number six
occurs only in the zygote, all other stages showing the haploid number
three. The latter observers have noted the same condition in the case
of the coccidium Aggregata eberthi (Fig. 66). In this parasite, during
schizogony nuclear divisions occur in which six chromosomes appear in
the equatorial plate, and they all divide so that the daughter nuclei have

no CHROMOSOMES DURING SYNGAMY IN PROTOZOA

1

iX

m^

r>

/2

'^

r of

/J

Fig. 65. — Nuclear Division in the Gregarine, Di])locysUs sclmeideri, to illus-
trate THE EeDUCTION IN THE CHROMOSOMES IN THE FiRST NuCLEAR DIVISION

IN THE Zygote ( x 2,500). (1-3 after Dobell and Jameson, 1916; 4-14 after
Jameson, 1920.)

1-3. First division in the cregarine, showinfr division and sojjaiation of the three chromosomes.
4-0. Third division in the gregarine, in a\ hich tlucc cliniiiKiscjnics auain divide.
7. Last nuclear division to form the gamete niu^lei: three chroniosumes again divide; there is
no reduction. 8-9. Zygote nucleus with six chromosomes.

10. The six chromosomes arranged in three pairs.

11, 12. Separation of the chromosomes in two groups of three (reduction).
13, 14. Rcconstitution of two nuclei, each with three chromosomes.

MEIOSIS 111

each six chromosomes. Finally, male and female gametocytes which give
rise to male and female gametes are formed. The nucleus of the male
or microgametocyte multiplies by repeated divisions in which the series
of six chromosomes are present (Fig. 66, A to D). They are filamentous
except when arranged as the equatorial plate, when they are contracted
and more or less spherical, though maintaining the same relations as
regards size. At the equator of the spindle the chromosomes divide by
constriction, and the two groups of six daughter chromosomes separate
and. become filamentous again. By repeated divisions of this kind, in
which the daughter asters divide before actual nuclei are formed, very
complicated poly-aster figures are produced. Eventually, as in the schizont,
nuclei which lie on the surface are constituted, and from them the micro-
gametes are formed. The latter are elongate bodies provided with two
flagella at the anterior end (Fig. 376). Meanwhile, certain merozoites of the
female line have become female- or macro-gametocytes. A complicated
series of changes takes place in the nucleus. The nucleolus or karyosome
is thrown out, the nuclear membrane disappears, and a series of six long
chromosomes appears (Fig. 66, E). Finally, a fertilization spindle is formed,
on w^hich the chromatin of the female nucleus is arranged in the form of
granules (see p. 873). The chromatin of the male nucleus, derived from
the microgamete, now enters the spindle, which retracts to form the zygote
nucleus (synkarion). This nucleus now proceeds to division by mitosis,
and the chromosomes are reconstituted (Fig. 66, F to K). It is found
that there are twelve of these — a series of six pairs, the two constituting
each pair being equal in size. Undoubtedly one chromosome of each
pair is derived from the microgamete nucleus and one from the macro-
gamete nucleus. The twelve chromosomes now pass to the equator of
the spindle and become globular in form, and the two constituents of
each pair now unite, giving a stage in which there are only six double
chromosomes (Fig. 66, G). The union, however, is not permanent, for
separation takes place, and one chromosome of each pair passes to one
pole of the spindle, while the other goes to the opposite pole (Fig. 66, H).
In this process there has been no division of the chromosomes, so that
in each daughter group there are only six chromosomes, whereas in the
zygote nucleus (synkarion) there were twelve. The first division of the
synkarion is thus a true reduction division, whereby the original number
of six is regained. It will thus be seen that in every stage of development
of this parasite the nuclei have six chromosomes, except in the synkarion
formed by union of the male and female nuclei, in which there are twelve.
The daughter groups of six chromosomes resulting from the division of
the synkarion now proceed to division again, but, as in the case of the
nuclear multiplication in the schizont and microgametocyte, at each

112 CHROMOSOMES DURING SYNGAMY IN PROTOZOA

M

(3

:<

bb'

cc G

Fig. 66. — Chromosomes of Aggregate eberthi ( x 2,000). (After Dobell
AND Jameson, 1915.)

A . Nucleus of male, showing six long chromosomes at prophase stage of first division .

B. Later stage of first division of nucleus of male: the chromosomes have become compact and

are arranged as an equatorial plate.

C. Later stage: each chromosome has divided to give rise to two groups of six daughter chromo-

somes.

D. One of the groups of six dumlitcr cliroinnsonics arisiim from first division of male nucleus

elongating to form the chroinnsdiiics of one of tlio dauuhtcr nuc-k'i.

E. Nucleus of female before fertilization, sliow ini: six limu chromosomes.

F. Chromosomes in zygote nucleus: early sta^c of first division, showing twelve chromosomes,

six (a-/) derived from the male, and six (■'-/') fnun the female.

G. Chromosomes in zygote nuolens: equatorial ])late stage of first division: the twelve chromo-

somes have contracted and Ixcome associated as six double chromosomes.
H. Chromosomes in dividinu zyt:ote nucleus: the individual chromo,somes of each pair have

separated, giving rise to two groups of six.
K. End of first division of the zygote nucleus : one of the groups of .six chromosomes, which have

elongated, entering the daughter nucleus.
L. Group of six daughter chromosomes on sjiindJe of a later nuclear division of the zygote.
M. Group of six chromosomes forming equatorial plate at second division of the spore

nucleus.

MEIOSIS 113

division the six chromosomes divide, so that each daughter nucleus has
six chromosomes (Fig. 66, L and M). Eventually, a large number of
nuclei are formed. These arrange themselves on the surface of the cyto-
plasm, which segments into a number of sporoblasts.

An exactly comparable process has been described by Reichenow
(192 1) in the case of haemogregarines of the genus Karyolysus (Fig. 457).
Here the haploid number of chromosomes is four, and these occur in nuclei
of all stages except those of the zygotes, which have the diploid number
of eight. When the zygote nucleus divides, four closely united pairs of
chromosomes occur at the equator of the spindle. One chromosome of
each pair then passes towards the pole of the spindle, so that the resulting
daughter nuclei have again only four (see p. 1098). These accounts agree
in that the reduction division occurs at the division of the zygote nucleus,
and not, as Muslow maintains, in the last division which gives rise to
the gamete nuclei. It seems highly improbable that Monocystis rostrata
would differ from other gregarines or coccidia in this respect, and Dobell
and Jameson have suggested that possibly Muslow was dealing with a
mixed infection of two gregarines, one of which has a chromosome number
of four and the other of eight, and that what he considered to be the
reduction division of the form with eight chromosomes was in reality the
ordinary division of the form with four chromosomes.

The nuclear division during the vegetative reproduction by binary
fission, the formation of gametes, and their maturation in the Heliozoon
Actinophrys sol has been the subject of detailed study by Belaf (1923),
as mentioned above. The organism reproduces by simple division.
Finally, encystment occurs and the uninucleated individual within the
cyst divides to form two gametes (Fig. 50). The nucleus of each gamete
divides and one of these degenerates. The remaining nucleus then
divides, and one of the resulting nuclei degenerates. There have thus
been two maturation divisions of the gamete nuclei. Conjugation of
gametes then occurs.. During vegetative reproduction the nucleus
divides without centrosomes by mitosis, while retaining its nuclear mem-
brane. When the chromosomes, which number forty-four, first appear
during nuclear division they are thread-like, but as the equatorial plate
stage is reached they become much shortened, and finally roughly spheri-
cal, in which condition they divide to form daughter chromosomes.
When the encysted individual divides to form the two gametes, the nuclear
division is of the same type as that occurring during the ordinary vegeta-
tive reproduction. The forty-four long chromosomes become arranged
in twenty-two pairs, the members of each pair being closely applied to
one another. Finally, when the equatorial plate stage is reached, there
are present at the equator of the spindle twenty-two pairs of more or

114 CHROMOSOMES DURING SYNGAMY IN PROTOZOA

less rounded cliromosomes. Each chromosome splits into two, so that
the daughter plates and finally the daughter nuclei also contain twenty-
two pairs of chromosomes. Each resulting nucleus then undergoes two
maturation divisions. In the first of these at the equatorial plate stage
there are twenty-two pairs of rounded chromosomes, but when the
daughter plates form the chromosomes do not split, as in the preceding
nuclear division. One chromosome of each pair passes to each daughter
plate, which thus contains only twenty-two chromosomes instead of
twenty-two pairs. The process is similar to that shown at Fig. 4, except
that in the place of the four chromosomes there are forty-four. Of the
resulting nuclei, one degenerates and the survivor divides by mitosis as
before. During this division twenty-two chromosomes appear at the
equator of the spindle, and each divides, so that each resulting nucleus
has twenty-two chromosomes. After union of the gametes, the zygote
nucleus has forty-four chromosomes. During all these divisions the
chromosomes are long filaments at the commencement of nuclear division,
but they gradually retract and finally become roughly spherical, in which
form they are arranged as the equatorial plate.

In connection with the conjugation of ciliates, similar reduction
processes have been described. In these Protozoa, as explained above,
it is only the micronucleus which takes part in syngamy, the macro-
nucleus degenerating. The micronucleus in one individual divides to
form two nuclei, and these again to form four. Of these four, three
degenerate. The remaining one divides again, so that each of the two
associated ciliates contains two nuclei. One of the nuclei in each indi-
vidual now passes over to the other and unites with the stationary nucleus,
after which the ciliates separate. Here, again, if the number of chromo-
somes in the uniting nuclei has not been reduced, it is evident the zygote
nuclei will have double this number. Several observers have maintained
that the first of the three divisions of the micronucleus is really a reducing
division. Hertwig (1889) noted that in Paramecium aurelia, the nucleus
of which has a large number of chromosomes during division, the nuclei
which unite have approximately half the number of chromosomes seen
in the ordinary divisions of the micronucleus during reproduction by
fission. Calkins and Cull (1907), in the case of Paramecium caudatum,
noted that the number of chromosomes in the ordinary dividing nucleus
is about 165. During the first two divisions of the micronucleus during
conjugation there is a reduction in the number to about half this. On
account of their large number it is difficult to count the chromosomes
accurately. Prandtl (1906) found that in Didinium nasutum the first
division of the micronuclei during conjugation was associated with the
reduction of the chromosomes from sixteen to eight. In CoUinia

MEIOSIS 115

branchiarum, Collin (1909) described a reduction of from six to three
(Fig. 495, 5 and 6), while Enriques (1908a) in Chilodon uncinatus saw
a reduction of four to two, and (1907) in Opercularia coarcta a reduction
of sixteen to eight (p. 1174), In all these cases the conjugating or gamete
nuclei possess half or the haploid number of chromosomes, while the nuclei
resulting from the union of the gamete nuclei have the full or diploid
number, which is maintained at all subsequent divisions. This is the
reverse of what occurs in the gregarines and coccidia, as described by
Dobell and Jameson, and Reichenow.

In connection with the process of union of gametes many so-called
reduction or maturation processes have been described. In Eimeria
schubergi, Schaudinn (1900), for instance, described as a maturation pro-
cess the breaking up and extrusion from the nucleus of the macrogamete
of the large karyosome (Fig. 337, ii). From what has been said above of
the reduction division of the nuclei of coccidia, gregarines, and ciliates, it
seems highly improbable that such a process is a reduction at all. In
the case of Cyclosjpora caryohjtica, another coccidium, Schaudinn (1902)
described the macrogamete nucleus as dividing twice, one of the products
of each division degenerating (Fig. 341). This again is explained as a
maturation process for the macrogamete nucleus before it is fertilized by
the microgamete. A similar process is said to take place in the case of
the parasites of malaria. The macrogamete, before fertilization in the
mosquito's stomach, is supposed to extrude one or two polar bodies which
contain some of the chromatin of the nucleus (Fig. 391, i6). In the case
of the conjugation of the flagellate Copromonas subtilis described by Dobell
(19086), where two individuals fuse, before the union of the nuclei each
nucleus is said to divide twice to form two reduction bodies which de-
generate (Fig. 48). After this, the nuclei of the conjugating individuals
unite. From what has been discovered during the past few years regarding
the methods of reduction of the number of chromosomes in connection
with the union of gametes in the Protozoa, it is evident that many
of the processes previously interpreted as reduction or maturation
divisions of the nuclei need to be re-examined in the light of what
is now known. Till this has been done it is useless to speculate as to
their meaning. \

BLEPHAROPLASTS, PARABASALS, AND KINETOPLASTS.

It has been explained above that amongst the Mastigophora the axis
of the flagellum is a filament (axoneme) which arises from a granule called
the blepharoplast. When there are two or more flagella, there arc a
corresponding number of axonemes and blepharoplasts. The several

IIG BASAL GRANULE OF FLAGELLUM

blepliaroplasts, when more than one is present, are often so closely packed
together that it may be difficult to distinguish them individually.

The blepharopjast may be situated upon the nuclear membrane, as
in Cercomonas, or quite separate from it, as in the majority of other
flagellates. It has already been shown above that certain observations
tend to indicate that the blepharoplast is of nuclear origin. In certain
stages a flagellate m.ay lose its flagellum or flagella and become a rou.nded
body with a single nucleus. When the flagellum is about to be re-formed,
it is claimed that a granule separates from the karyosome of the nucleus
and passes out into the cytoplasm through the nuclear membrane (Fig. 31).

Fig. 67. — Trichomonas atufiistd, showing the Spiral Parabasal Body immediately

ANTERIOR TO THE NUCLEUS ( X CU. 2,5()0). (AfTER AlEXEIEFF, 1924.)

An axoneme is then formed from it as an outgrowth, and when the surface
of the body is reached it takes with it a sheath of cytoplasm and becomes
a flagellum.

In association with the blepharoplast, whether it is on the nuclear
membrane or separate from it, there may occur one or more masses of
a substance which stains deeply with many chromatin stains. To such
bodies Janicki (1911) has given the name parabasal (see p. 53). The
name kinetoplast is employed here to designate the compound structure
consisting of a united parabasal and blepharoplast. Kinetoplasts are
typically seen in trypanosomes and allied flagellates. Parabasal bodies
have been described as occurring in Trichomonas by Janicki (1915),
Wenrich (1921), and Alexeieff (1924), but they are only detected after
special fixation — e.g., osmic acid (Figs. 67 and 275).

BLEPHAROPLAST PARABASAL KINET0PLA8T 117

AVhen a flagellate is about to divide, the blepharoplast is usually the
first structure to show any indication of division. It becomes elongated
and constricted into two parts. Very often the two daughter blepharo-
plasts (or two groups of daughter blepharoplasts when several are present)
remain connected by a fibre which may be called the paradesmose, as
suggested by Kofoid and Swezy (1915), to distinguish it from the centro-
desmose which unites the daughter karyosomes, or centrioles which are
supposed by some observers to occur within the karyosome, during division
(Fig. 272). As the blepharoplast elongates and divides and the daughter
blepharoplasts separate, the parabasal also becomes elongated and divides.
If several parabasals are present, without dividing individually, they
separate into two approximately equal groups. The blepharoplast thus
leads the way in division of the parabasal. It sometimes happens that
the blepharoplast divides before the parabasal shows any signs of division.
A figure may be produced in w^hicli the two daughter blepharoplasts are
connected by a paradesmose, at the centre of which the still undivided
parabasal lies. The parabasal now divides, and the two halves move
towards the daughter blepharoplasts. There is some resemblance to
mitosis in this type of division, which has been employed as an argument
in support of the view that the blepharoplasts are centrosomes and
that the kinetoplast is actually a nucleus. The parabasal, however,
does not form chromosomes, nor are spindle fibres developed between
the blepharoplasts, though some claim to have observed these structures
during the division of the kinetoplast of trypanosomes. After the
blepharoplast and parabasal have commenced to divide, the nucleus
itself begins to show signs of division.

In flagellates like Heteromita uncinata and Cerco-monas longicavda, in
which the blepharoplast is on the nuclear membrane, a condition is
seen in which the blepharoplast appears to function as a centrosome
(Fig. 68). The blepharoplast upon the membrane divides, and the two
halves separate. They finally take up positions at opposite poles of the
nucleus, and a definite spindle is formed between them. The karyosome
breaks up, and chromosomes appear at the 'equator of the spindle. The
chromosome plate divides into two daughter plates which move towards
the blepharoplasts. Finally, the nuclear membrane is divided, the
chromosomes disappear, and with the formation of the karyosomes the
nuclei are reconstructed.

It seems difficult to resist the conviction that in such a division the
blepharoplast has fulfilled the function of a centrosome. Its behaviour,
however, may be merely due to its position on the nuclear membrane,
for in flagellates like Parajpolytoma satura, described by Jameson (1914),
in which the blepharoplast is separated from the nuclear membrane,

1].

BASAL GRANULE OF FLAGELLUM

8 9

Fig. 68. — Binary Fission in Ileteromita uncinata ( x 4,000). (Original.)

1 . Normal flcagcllate with blepharoplast on surface of nuclear membrane.

2. The flagellate has become rounded and its blepharoplast divided, while two new flagella have

foiiiKMl. The kaiyosonie has hioken \\\, into granules.

3. The lilc|ili;irnplastsnccu])y the poles of a s|iin(llc whitli has an ecpiatorial plate of chromosomes.

4. The elu-omusonics have divided to form two daughter plates.

5. End view of the equatorial plate. " G. The daughter plates are separating.

7. Formation of two nuclei and the reconstruction of karyosome.

8. Commencing division of the flagellate.

9. The karyosome has re-formed and the flagellate is about to divide.

BLEPHAROPLAST PARABASAL KINETOPLAST 119

mitotic division of the nucleus occurs without any centrosomes at the
poles of the spindle. Instances are known, however, in which the blepharo-
plasts which are separate from the nucleus occupy during nuclear division
positions upon the spindle which centrosomes would be expected to
occupy. Such an example is seen in the division of Oikomonas termo
described by Martin (1912) (Fig. 135).

In the case of Proivazekella lacertce, which has an axoneme originating
in a blepharoplast on the nuclear membrane, the nucleus has one or more
parabasals surrounding it. When division of the nucleus takes place,
the daughter blepharoplasts occupy the poles of the spindle and mitotic
division takes place, as in Heteromita and Cercomonas. The parabasal, if
there is a single one outside the nucleus, becomes elongated and divided
into two parts, one of which passes to each daughter nucleus. When there
are several parabasals they separate into two groups without dividing
individually, very much like the behaviour of mitochondria during division
of spermocytes in the process of spermatogenesis (Fig. 254, s-x).

The function of the centriole in nuclear division has been discussed
above. It will be seen that in Heteromita uncinata, Cercomonas longicauda,
and other forms in which the blepharoplast occurs on the nuclear mem-
brane, and in certain cases where it is separated from the membrane,
the daughter blepharoplasts occupy during nuclear division the same
positions that the daughter centrioles are said to occupy. It is claimed
that as the centriole is functionally a centrosome, the blepharoplasts of
flagellates must also be centrosomes. It is further assumed that, in
those cases in which the blepharoplast occupies a position in the cyto-
plasm apart from the nucleus, it represents a centriole or centrosome
which has left the nucleus or is the result of division of the centrosome
into two parts, one of which remains in the nucleus and still functions
as a centrosome during its division, while the other has left the nucleus
to become a blepharoplast.

The whole subject of the relation of blepharoplasts to centrosomes
is a very complex one, and depends largely on the exact definition of a
centrosome. Some observers definitely assert that the blepharoplast is
a centrosome. Minchin (1914), for instance, stated that in his opinion it
was a well-established fact that in a great many cases blepharoplast and
centrosome were one and the same body. It seems difficult to doubt
this in view of the fact that in the developing spermatozoon of higher
animals the axial filament of the tail which corresponds with an axoneme
is known to be formed as an outgrowth from the centrosome. In fact, the
tail with its axial filament arising from the centrosome is exactly com-
parable with the flagellum with its axoneme and blepharoplast.

The question of the nature of the numerous blepharoplasts possessed

120 BASAL GRANULE OF FLAGELLUM

by the Hypermastigida and the basal granules of the cilia of Ciliophora,
which are to all intents and purposes blepharoplasts, is still more difficult
to answer.

Another point in connection with the blej)haroplasts of flagellates
must be mentioned. Many observers have described fibres which connect
the blepharoplasts with the karyosome of the nucleus, and they suppose
that these fibres represent centrodesmoses which were formed when the
supposed intranuclear centriole divided off the blepharoplasts. As
already remarked, when several blepharoplasts are present, they are
usually packed so closely together that they cannot be distinguished
individually. It not infrequently happens, however, that in certain
individuals of any species of flagellate the blepharoplasts are more dis-
persed, so that it is possible to recognize the actual nvimber present.
Kofoid and Swezy (1920) have described a very complicated system of
fibrillar connections between the various blepharoplasts of Chilomastix,
and they introduce into their scheme a definite centrosome which they
state is present upon the nuclear membrane and is connected by a fibre
with one of the blepharoplasts (Fig. 69). If such a centrosome and
system of fibres is present in this flagellate, it has at any rate escaped
detection by most observers. The complicated system of fibres which
they describe as being present, together with the karyosome, centrosomes,
blepharoplasts, flagella, and other motor organs and marginal filaments
of the cytostomal groove, they name the neuromotor syste?n. This term
has been extended by them to include the fibrillar structures which occur
in other flagellates, such as the complex organisms parasitic in termites,
while Sharp (1914) employs it for the fibrillar apparatus of the ciliate
Diplodinium ecaudatum (Fig. 520). It is quite possible that some of the
fibres have a motor function, but others appear to be merely supporting
rods, while there is at present no direct evidence to prove that they are
comparable to nerve fibrils which the name neuromotor suggests. In
using the term " neuromotor system," groups of structures which are not
necessarily homologous in different organisms have been united under
one name. Kofoid and Swezy, for instance, homologized one of the
fibres which support the margin of the cytostome of Chilomastix, the
basal fibre of the undulating membrane in Trichomonas, and the two
structures of unknown function which commonly occur in the posterior
region of Giardia as parabasals. There seems to be no real evidence that
these are in any way homologous with the true parabasals of other flagel-
lates, and it is worthy of note that several observers have described what
are probably true parabasals in certain species of Trichomonas.

The growth and the formation of new flagella are intimately bound
up with the activities of the blepharoplast. When the blepharoplast of

BLEPHAROPLAST PARABASAL KIXET0PLA8T

121

a flagellate divides, the axoiieme which arose from it remains, as a rule,
attached to one daughter blepharoplast, while a new axoneme grows out

Fig. 69. — Chilomastix mesnili : Free and Encysted Forms, to illustrate the
Structures described by Kofoid and Swezy ( x 6,370). (After Kofoid
AND Swezy, 1920.)

A. Normal flagellate viewed from the ventral or oral side., and showing all the structures of the
body. B. Cyst viewed from the ventral or oral side.

Cent., Centro.3ome; cent.k.. central karyosome; r//s<, cyst wall; cy<.,cytostome; cyt.fl., cytostomal
flagellum or undulatinu; mcnildanc; i iil.iltiz., intranuclear rhizoi^last; l.a.fl., left anterior
flagella; nuc, nucleus; inir.rlii-... iiudrai' rhizoplast; par.b., parabasal body; parast., para-
style; per/s<./.. peristomal liluc; /iriiii.h/i j>li ., primary blejiharojDlast; r.a.fl., right anterior
flagellum; secMeph., secondary blepharoijlast; sp/r.jrr., spiral groove; tert.bleph., tertiary
blepharoplast; tr.rhiz., transverse rhizoplast.

from the other to form a new flagellum. When a group of blepharoplasts,
in flagellates with more than one flagellum, divides into two groups, some

122 BASAL GRANULE OF FLAGELLUM

of the axonemes and flagella remain with one group and some with the
other. There seems to be no regularity in their distribution. Those
blepharoplasts which have no axonemes then form new ones. Very
frequently, before the blepharoplast has actually divided, a new axoneme
grows out from the part of the elongating blepharoplast which will become
one of the daughter blepharoplasts. It may happen that the new axoneme
actually passes into the cytoplasmic sheath of the old flagellum, so that
finally longitudinal splitting of the flagellum occurs. In such a case
division of the sheath of the flagellum alone takes place. It seems highly
probable that in no case does an axoneme itself divide longitudinally. A
new axoneme is invariably formed as a result of the outgrowth from the
daughter blepharoplast. In cultures of Leishmania the flagellum is formed
by outgrowth of the axoneme, which can usually be detected in properly
stained specimens of the parasites as they occur in tissues (Fig. 192).

Certain structures other than axonemes take origin in granules, which
are usually regarded as blepharoplasts. Thus, the two fibres which border
the cytostomal groove in CJiiloynastix arise each from a granule or blepharo-
plast (Fig. 69). Similarly, the basal fibre of the undulating membrane in
Trichomonas originates in a blepharoplast, and when division occurs a
second basal fibre grows out from one of the daughter blepharoplasts into
which the original one has divided. The axostyle of Trichoynonas likewise
arises from the blepharoplasts (Fig. 26). The writer (1907), as well as
Kofoid and Swezy (1915, 1915a), describes the axostyle as splitting
longitudinally during division of Trichomonas muris and other species.
Dobell (1909) stated that the new axostyles in T. batrachorum are formed
from the two halves of the divided paradesmose, which connects the
daughter blepharoplasts during division. Kuczynski (1914, 1918) claims
that the old axostyle degenerates, and that new ones are formed as out-
growths from the daughter blepharoplasts, while the paradesmose dis-
appears. Wenrich (1921) has described a similar origin for the new
axostyles in Trichomonas 7nuris (Figs. 271 and 272).

A great variety of fibres directly or indirectly connected with the
blepharoplasts have been stated to occur in flagellates. Thus, Schaudinn
(1904) describes numerous structures of this kind in Trypanosotna noctucc,
Prowazek (1903, 1904) in Trypanosoma lewisi and in Herpetomonas
tnuscarum,, while McCulloch (1915) figures a very complicated system of
fibres in Crithidia leptocoridis (Fig. 154). It seems that the majority,
if not all, of these are accidental structures, which cannot be considered
as definite organs of the normal flagellates. Whether the marginal fibres
of the cytostomal groove of Chilomastix, the basal fibre of the undulating
membrane, and the axostyle of TricAomowas, and other similar structures
which are connected with blepharoplasts, are to be homologized with

NUTRITION 123

flagella cannot be considered as definitely established. It may also be
open to question if the granules in which they originate, and which are
generally styled blepharoplasts, are actually of this nature.

PHYSIOLOGY OF THE PROTOZOA.

Many of the physiological processes which regulate the life of Protozoa
have been referred to above. It will only be necessary to review these in
a general manner under the headings Nutrition, Movement, Reaction to
Stimuli, Influence of Environment, Influence of Syngamy.

NUTRITION.— The essential food requirements of Protozoa are those of
living matter in general. There is a constant expenditure of energy,
necessitating a continuous supply of nourishment, which includes oxygen,
simple chemical compounds, more complex organic substances, or highly
organized proteid materials. Oxygen is an essential requirement, as it is
of all living matter, but the method by which it is obtained varies, as it
does between the vegetable and animal kingdoms. There are no special
organs of respiration, so that absorption of oxygen and discharge of carbon
dioxide takes place by a process of diffusion through the surface of the
body. Certain Protozoa, like plants, possess chromatophores, and by
means of their pigments or chromophyll are able, in the presence of sun-
light, to obtain oxygen from the carbon dioxide which is in solution in
the liquids in which they live, or which is formed by the organism itself.
The chromatophores, which are green when they contain chlorophyll or
red when the pigment is haematochrome, multiply by binary fission, as do
also certain refringent granules called pyrenoids which they contain.
They behave in many respects as independent organisms, and this has
given rise to the view that they may be actually organisms living in a
condition of symbiosis with the cells in which they occur. This method of
nutrition is described as being holophytic, in contrast to the holozoic type,
which is characteristic of Protozoa, which are devoid of chromatophores,
and which must of necessity absorb oxygen directly from the liquid in
which they live. In either case the organisms require oxygen, so that the
two types of nutrition, the holophytic and holozoic, do not imply any
essential difference in the character of the protoplasm of which their bodies
are composed. This is well illustrated by certain species of Euglena, which
normally have chromatophores, and lead a holophytic mode of existence
(Fig. 6). Under certain conditions, as when cultivated in the dark with
consequent loss of the pigment, they behave as organisms devoid of
chromatophores. The holophytic forms nourish themselves like plants,
and, in addition to the power conferred on them by the coloured pigments
of being able to utilize carbon dioxide for the purpose of acquiring a supply

124 PHYSIOLOGY OF PROTOZOA

of oxygen, tliey are able to elaborate relatively simple chemical compounds
into the protein materials necessary for their existence. Such forms may
be cultivated in solutions of various salts, and, like plants, commonly
elaborate starch or other amyloid substances as one of the products of
assimilation, and not infrequently build for themselves capsules composed
of cellulose. Between these and the completely holozoic forms, which
require, in addition to oxygen, ready-formed proteid materials, either solid
or in solution, there exists a group of organisms known as saprophytes.
These do not possess chromatophores, but are able to live in fluids con-
taining oxygen and complex organic compounds, which nevertheless are
simpler than the proteid materials required by the truly holozoic types.

Amongst the holozoic Protozoa two methods of obtaining proteid
material occur. In the one the organism ingests solid proteid material,
mostly in the form of other living organisms, such as bacteria and other
Protozoa, or, as in the case of parasitic forms like Entamoeba histolytica,
the cells of the host's body (Fig. 95). This solid matter is ingested either
through a definite mouth opening or cytostome, or, when such is not present,
through any part of the body surface by means of pseudopodia which
surround it, or by a movement of the cytoplasm over the object, which
appears to sink into its substance. In the other method, the proteid which
is in solution is absorbed in liquid form. There is no mouth opening, the
material merely passing into the body by osmosis. The latter method is
characteristic of many parasitic Protozoa, such as trypanosomes, malarial
parasites, coccidia, and gregarines. Other parasitic forms, such as the
amoebse, Trichomonas and Balantidium, ingest solid matter either by means
of pseudopodia or definite cytostomes (Figs. 26 and 14).

In the case of Suctoria, which obtain their food by means of sucking
tentacles, these are applied to solid objects, from which the proteid is
extracted in a liquid form, probably as a result of ferments acting at the
points of contact (Fig. 15).

As regards the proteid material ingested, two conditions result. When
it is absorbed in solid form it is enclosed in food vacuoles, in which the par-
ticles are found in various stages of digestion (Fig. 70). When the proteid is
absorbed in a state of solution no such food vacuoles are formed. From
a study of the changes which occur during digestion in food vacuoles it
has been found that when a living organism is ingested it is at first killed
and then gradually digested, leaving finally a residuum of fsecal matter
which is got rid of by the vacuole approaching the surface of the body
and discharging its contents. In the ciliates there frequently exists
a definite anal opening or cytopyge, usually at the posterior end of the
organism, through which the residue is discharged (Fig. 512). The process
of digestion is evidently the result of ferments which are secreted by the

NUTRITION 125

cytoplasm, as various ferments have been extracted from Protozoa.
Generally speaking, the reaction of a food vacuole is at first acid when the
ingested organism is killed. The reaction then becomes alkaline. It is
probable that during the acid phase a peptic ferment is active, while a
tryptic ferment is present during the alkaline phase. Fats also are
capable of being digested. It sometimes happens that the contents of
a food vacuole are alkaline from the commencement, and it appears that
the cytoplasm has some power of varying its response to different types
of food.

The proteid material absorbed from the food vacuoles, or from the
medium in which the organism is living, enters the cytoplasm, and is
immediately elaborated into the constituents of the cell or leads to the
formation of various intermediate bodies. The latter may be regarded as
food-reserve materials which are merely accumulations resulting from the
intake of excess of nourishment, or definite reserves intended for a period
of excessive activity, such as occurs during the sporogony process of
coccidia and gregarines, or the continued development when access to
nourishment is prevented, as when a cyst wall is present.

In the organisms which have a holophytic method of nutrition the
food reserve is stored largely as starch or allied substances of an amyloid
nature. In gregarines preparing for sporogony in the gametocysts the
cytoplasm becomes charged with refractile globules of a substance called
paraglycogen. The macrogaraetocytes of coccidia, which are to continue
development in an oocyst, likewise become loaded with refractile globules
of an albuminous substance. Similarly in the encysted stages of Entaynoeha
histolytica, lodamoeha biltscMii, and other forms, a large amount of a
glycogenic substance is present. It is gradually used up during the period
passed by the encysted form in awaiting a suitable opportunity for emerging
from the cyst. Another substance which is often present is volutin, which
appears in the fresh condition as greenish refractile globules. It stains
deeply with many nuclear stains, and has been supposed to be a forerunner
of chromatin, but of this there is no direct evidence. Many of the granules
which have been described as chromidia are probably of this nature. It
commonly occurs in flagellates, and is often abundant in trypanosomes,
appearing as deep red granules in specimens stained with Romanowsky
stains. Fat globules also occur in Protozoa, and are commonly present
in Radiolaria. The identification of the various granules and reserve
substances is a very difficult matter, dependent on microchemical tests,
solubility in various fluids, and reaction to different stains.

The residue from food digestion, as pointed out above, is discharged
from the body. This may occur immediately after digestion is completed,
or it may be deferred. The substances may assume different forms.

126 PHYSIOLOGY OF PROTOZOA

They may become crystalline excretory crystals, or remain as amorphous
masses. Amongst the Sporozoa, when reproduction by schizogony takes
place, a certain amount of cytoplasm is usually left over as a residual body,
which takes no part in the formation of merozoites. In it is got rid of
a certain amount of excretory substance. Malarial parasites thus dis-
charge the pigment granules which accumulate as a result of digestion
of haemoglobin.

In addition to the substances which have been referred to, and which
may be regarded as steps in the formation of protoplasm or the waste
products from the food, there occur other substances which are elaborated
to fulfil some special function. The conspicuous so-called chromidial
body of shelled amoebae may have to do with the formation of the shell.
The various skeletal structures which occur in the cytoplasm of Radiolaria,
the supporting rods which form the axes of the pseudopodia of many
Heliozoa, and, indeed, the external coverings like the shells of Foraminifera
and the cyst walls themselves, are to be regarded as products of metabolism.
It is evident that the Protozoa which produce such structures must absorb
special substances for the purpose.

Quite apart from the excretion of substances no longer required by the
organism by the rupture of vacuoles containing them at the surface of
the body, there is another method of excretion, which is carried out by a
rhythmically contracting vacuole which is situated near the surface of the
body. Such a contractile vacuole, when fully formed, suddenly contracts,
so that the clear liquid contents are discharged through the surface of the
body. In a short time the vacuole re-forms, and, gradually increasing in
size, reaches its maximum, when it again contracts. In some cases definite
channels in the cytoplasm conduct fluid to the vacuole. The rate of
pulsation varies with temperature and the presence of substances which
affect the density of the medium. It is supposed that the vacuole is a
means of discharging carbon dioxide and other soluble excretory substances,
but the fact that contractile vacuoles are absent in marine Protozoa
and. many parasitic forms, and that fresh- water forms lose the contractile
vacuole when made to live in salt water, suggests that such a vacuole may
be a means of accommodating the organism to the medium in which it lives,
rather than an organ primarily excretory in function. It can hardly be
supposed that marine or parasitic forms are less dependent on excretion
for their existence than those which live in fresh water. It has been con-
jectured that the contractile vacuole may counteract the tendency of the
cytoplasm to become overcharged with water due to the greater absorption
in fresh than in saline water.

On the method of nutrition of any particular organism depends the
character of the medium in which it can be cultivated. Forms like

MOVEMENT 127

Euglena, whicli possess chromatopliores and behave like plants, can be
grown in distilled water in which certain inorganic salts are dissolved.
Saprophytic forms require more complex substances, while holozoic ones
will grow only in media in which proteid material is present. This is
usually in the form of bacteria, which form the staple food of amoebge,
flagellates, and ciliates, when grown on the surface of agar plates or in
liquid media. In other cases, as in the cultures of trypanosomes and
leishmania, bacteria are absent, the proteid materials being derived from
blood-serum.

MOVEMENT. — The power of movement is one of the properti<^s of
cytoplasm in general, and amongst the Protozoa it is seen in its simplest
form in organisms like amoebse, and is most highly developed when special
motile organs are present, such as flagella, cilia, the contractile filaments
in the stalks of the attached Protozoa, and the myonemes of gregarines
and other forms. The cytoplasm is in constant movement wathin the
organism. This streaming of the cytoplasm is undoubtedly the result of
chemico-physical changes which are taking place. In highly-organized
Protozoa, like the ciliates, the currents in the cytoplasm are constant in
their direction, and the various food vacuoles which move with them
perform a definite circuit. In the amoebae, which do not have definitely
orientated bodies, there is more irregularity. It is as a result of this
streaming of the cytoplasm that organisms like amcBbge are able to move
and form pseudopodia. When resting on a surface, the portion of cyto-
plasm in contact with the surface is prevented from movement, while the
streaming of the internal cytoplasm in one direction leads to a forward
movement, which is best illustrated by the roHing movement of a bag of
fluid on an inclined plane. In this manner the whole amoeba may progress
in one direction, or, when the streaming of the cytoplasm is limited, only
portions will move forwards, with the result that pseudopodia are formed.
By changes in the direction of the stream the pseudopodia are withdrawn
and others protruded. Certain pseudopodia, like those of Heliozoa, are
supported by axial fibres, which render them more permanent structures.
They are, nevertheless, capable of performing swinging or bending move-
ments. Whether these are the result of movements of the cytoplasmic
covering or of the axial fibre has not been satisfactorily determined.
As, however, fine pseudopodia devoid of axial fibres can perform such
movements, it would seem that the axial fibre may be purely elastic
in nature, with the function of bringing the pseudopodium back to its
original extended position when the movements of the cytoplasmic covering
cease. The more actively motile flagella and cilia of the Mastigophora and
Ciliophora have essentially the same structure as the axopodia of Heliozoa.
There is an axial fibre (axoneme) covered by a thin sheath of cytoplasm.

128 PHYSIOLOGY OF PROTOZOA

and it may be supposed that tlieir movement is brought about in a similar
manner by changes which occur in the thin cytoplasmic covering, the
axial fibre acting as an elastic support. Similarly, the myonemes which
occur in gregarines and other Protozoa may not in themselves be contractile,
though they may limit the contraction of the cytoplasm itself to definite
channels. It is generally supposed, however, that the filaments themselves
are contractile. In the case of attached forms like Vorticella the stalk
is composed of an axial fibre and a sheath of cytoplasm; when retraction
takes place, the axial fibre assumes the form of a compressed spiral. During
extension it appears that the elasticity of the axial fibre, which returns to
its original condition, is responsible for the extension of the stalk, and it is
possible that the sheath of cytoplasm is the sole cause of the retraction.
That cytoplasm itself, quite apart from the presence of myonemes or other
filaments, is able to perform sudden and rapid movements of contraction
is illustrated by the behaviour of contractile vacuoles.

Another series of internal movements which are common to all cells
provided with nuclei are those associated with nuclear division. The
complicated process of mitosis, with the formation of the spindle and
chromosomes, and the subsequent separation of daughter chromosomes,
is in many cases carried out under the influence of the centrosome. In
many Protozoa, however, no centrosome is visible, but in neither case
has a satisfactory explanation of the phenomenon been given. AVhen a
centrosome is present, it appears to be the centre of activity, for it is
towards it that the rays of the aster and the spindle fibres are directed.
For those who regard the blepharoplasts of flagella as centrosomic in nature,
the action of the flagella is supposed to be another illustration of the motor
activities of the centrosome.

The movements of the cytoplasm which have been considered are
distinct from the locomotion of the Protozoa themselves. An organism
which is in a resting condition and undergoing no changes in shape may
still show the streaming movement of the cytoplasm, but it is nevertheless
these movements of the cytoplasm which bring about the changes in
shape and actual locomotion when these occur. Progressive formation
of pseudopodia and changes in shape in amoebse are the result of continued
streaming movements in one direction, as explained above. In the case
of Mastigophora and Ciliophora it is the result of the continuous action
of the special organs of locomotion, which are so arranged that when
they are in activity the organism is propelled through the liquid medium.
The peculiar gliding or slug-like progression of gregarines has been sup-
posed to be due to the rapid secretion of a tenacious fluid from numerous
pores in the longitudinal grooves of that portion of the ectoplasm which
is in contact with the surface on which the organism is resting. It is

REACTION TO STIMULI— INFLUENCE OF ENVIRONMENT 129

possible that the gliding movements performed by the small gregarine-like
merozoites or sporozoites may be explained in a similar manner.

REACTION TO STIMULI. — The actual direction of progression is the
direct result of external stimuli acting on the organism. Practically all
Protozoa react to stimuli, whether mechanical, chemical, thermal, electric,
or photic. The response to such stimuli has been chiefly studied in the
case of ciliates, in which it has been frequently found that the region of
the cytostome is the most sensitive part of the body. It is evident that
for any Protozoon there is an optimum condition of the medium in which
it lives, and if, during progression, it reaches an environment which is
less favourable to its existence than that which it has just left, there will
be a stimulation of the sensitive area of the body. This stimulation will
result in an altered action of the organs of locomotion, with a consequent
withdrawal from the unfavourable stimulus. The movements of ciliates
when subject to adverse stimuli are very precise, and have been the
subject of extensive investigations. The attraction and repulsion are
known as positive and negative taxis respectively. Generally speaking,
positive taxis indicates a movement towards and a negative taxis a
movement from any particular environment.

INFLUENCE OF ENVIRONMENT.— The actual condition of the environ-
ment in which a Protozoon finds itself is a very important factor in its
development. As already remarked, for each there is an optimum
condition which suits it best. Departures from this are followed by
conditions of depression resulting in degeneration or even death. Lack
of food or excess of it, leading to starvation or overfeeding, also brings
about degenerative changes which are seen in alterations in the structure
of the nuclei, which frequently become enlarged. In certain cases the
nuclei break up entirely, leading to the final death of the organism. To
a certain extent Protozoa can be gradually adapted to changes in environ-
ment, provided these are not brought about too suddenly. It is possible
by gradually raising the temperature of cultures to obtain a race of
organisms which can live at a temperature which would have quickly
killed if applied suddenly. Provided that degeneration has not proceeded
too far, recovery is possible if the conditions are improved. Regeneration
of the degenerate parts takes place. Similarly, Protozoa which have been
mutilated or deprived of portions of their bodies are able to regenerate
themselves, provided the nucleus remains intact.

The majority of Protozoa are able to protect themselves against adverse
conditions by the process of encystment. The tough resistant capsule
which is secreted shuts them off from their environment, so that they are
able to survive unharmed till conditions favourable to a free-living

I. 9

130 PHYSIOLOGY OF PROTOZOA

existence recur. Within the cysts the organism either undergoes no
change or it may continue to multiply. In parasitic forms the cyst
protects the organism during its passage from one host to another, the
encysted form being known as the infective stage. Amongst the Sporozoa
it usually happens that a period of asexual reproduction is followed by
one in which sexual forms are developed. The appearance of these is
generally supposed to be an indication that unfavourable changes are
taking place in the environment, and that encystment, which occurs in
association with conjugation and the production of the zygote, is necessary.

Another feature characteristic of many parasitic forms is the difference
in environment associated with different stages of development. Thus,
in the case of malarial parasites the human blood supplies the conditions
necessary for asexual reproduction and the production of gametocytes.
In the body of the mosquito all asexual stages quickly perish, while the
gametocytes continue their development, which was arrested in the human
blood. The sporozoites ultimately produced will develop no further in
the mosquito, but with the change brought about by their injection into
man further progress occurs. Similarly in the case of trypanosomes the
forms taken up from the blood by the transmitting host quickly lose
their power of developing in the blood, though they do so in the body
of the invertebrate. The metacyclic trypanosomes which are eventually
produced have regained the power of development in the blood.

As a result of abundance of nourishment in the medium the cytoplasm
may become charged with globules of food-reserve material which appear
to be far in excess of that actually required. Thus, the ciVmie Balantidium
coli may be packed with such substances. In many cases this has
apparently little effect on the vitality of the organism, though it has
been shown that in certain forms degenerative changes result.

A feature of this over-nourishment may be seen in certain cases of
gigantism. Thus Tricliomonas vaginalis is often very much larger than
Trichomonas hominis of the intestine. If, however, both these organisms
are cultivated in the same medium, the forms which appear are exactly
alike, so that it would seem that the large size of T. vaginalis is merely
an indication of overgrowth. Similarly the giant forms of Herpetotnonas
mirabilis, which occur in the Malpighian tubes of certain flies, can probably
be accounted for in similar manner.

INFLUENCE OF SYNGAMY.— As already remarked. Protozoa which
become degenerate or pass into a state of depression may recover if
conditions of life become favourable. It is supposed that a similar
recovery may result from the process of syngamy. In the majority of
Protozoa, however, syngamy is not known to occur. In many cases
this is undoubtedly due to the fact that the complete life-history has not

INFLUENCE OF SYNGAMY 131

been elucidated. In some instances, however, unless it is assumed that
syngamy must of necessity take place from time to time, it appears that
reproduction by simple binary fission is continued indefinitely. Such
an organism divides into two daughter individuals, and when these have
become fully grown, division again takes place. A simple life-cycle of this
kind is characteristic of the amoebae, and it is only interrupted by the
amoebae becoming encysted under certain circumstances.

Within these cysts, which are purely protective in function, the
amoebae may or may not continue to multiply by fission. When condi-
tions again become favourable, the cyst is ruptured and the amoebae
escape to continue their multiplicative existence. Similarly, many
trypanosomes can be handed on indefinitely from one animal to another
by simple inoculation of infected blood. There appears to be a continuous
process of reproduction by binary fission without the intervention of
either syngamy or encystment. Under natural conditions, however,
direct transference from vertebrate to vertebrate, except in the case of
Trypanosoma equiperdu?n, does not occur, the life-history being varied
by alternate multiplication in a vertebrate and an invertebrate. As far
as is known at present, multiplication in both hosts is by continuous
binary fission, though some authorities assume that a syngamic process
will be found to occur in the invertebrate. When such a change of hosts
is obligatory, the parasite is said to require an alternation of hosts for the
continuance of its life-cycle. In the case of certain blood-inhabiting
Sporozoa (malarial parasites) the alternation of hosts is characterized
by the occurrence of asexual multiplication in the vertebrate and syngamy
followed by the production of sporozoites in the invertebrate.

Until recently it was considered that the periodic occurrence of
syngamy was essentia] for the continued existence of the race. This view
was the outcome of researches conducted on ciliates by Maupas and
Eichard Hertwig. Thus it was fdiown that Paramecium caudatum, after
a varying period of multiplication by fission, proceeded to conjugate.
Calkins (1904) found that, if conjugation was prevented, the ciliates,
though they continued to reproduce, gradually weakened and died.
Similar results had previously been obtained by Maupas (1888, 1889)
in the case of Stylonychia pustulata and other forms. It was believed
that these experiments proved that a race would invariably die out if
conjugation did not occur. Enriques (1903), working with Glaticoma
scintillans and G. pyriformis, and Woodruf? (1917) with Paramecium
aurelia, proved that this was not the case. The latter observer (1925),
having commenced with a single individual, has carried on the culture
by separating the daughter individuals produced at each division for a
period of fifteen years, during which over 10,000 divisions have taken

132 PHYSIOLOGY OF PROTOZOA

place. Great care was taken to keep the culture medium favourable,
and it was found that the ciliates were just as vigorous at the end
of this period as was the original parent. It is thus evident that,
even in the case of an organism which under natural conditions conjugates
from time to time, the race may survive and still remain in vigorous
condition when this is prevented. Woodruf! (1921) showed that during
this period of repeated binary fission the process of renewal of the macro-
nucleus from the micronucleus, known as endomixis, took place at intervals
(see p. 54). In the case of the ciliate Spathidium spathula, Woodrui?
and Moore (1924) have demonstrated that reproduction can be continued
indefinitely without recourse to endomixis or conjugation when suitable
environmental conditions are supplied.

From the work of Richard Hertwig and Maupas, who considered that
conjugation was essential to survival of the race, arose the theory of
rejuvenescence, which supposes that any race of ciliates dies out through
loss of vigour if conjugation does not take place. It has generally been
assumed that both these observers thought that the rejuvenating process
showed itself in an increase in the rate of multiplication. According to
Jennings (1920) this is a misrepresentation of their views, for it was
definitely stated that the rate of fission before and after conjugation was
not altered. Their view of the change which takes place in conjugation
is that the ciliates which would otherwise have died now continue to live,
and this continued existence itself is a sign of rejuvenescence. Calkins
(1919a), however, has definitely asserted that the failing energy and rate
of multiplication of the pre-conjugation period is abolished by conjugation,
and that in the post-conjugation period the rate of multiplication is
increased. Quite recently Woodruff and Spencer (1924), working with
Spathidium spathula, have clearly shoAvn that conjugation actually does
increase the rate of multiplication, and, furthermore, that on an average
cultures made from forms which have conjugated outlive those from forms
which have not, so that the chances of any particular line surviving are
increased. Careful experiments have not only shown that conjugation
is not necessary to continued existence, but appear to have demonstrated,
in many cases, that following it there is actual depression as regards rate
of division, likelihood of death, and in other respects. If conjugation does
not lead to some change of this kind, it is extremely difficult to account
for the process of syngamy at all. It appears to be unnecessary, yet it
takes place in nature. Minchin (1912) expressed the opinion that it
tends to level down individual variations and keeps the species true to
type. The true explanation may, however, be the reverse of this, as
Jennings (1920) has pointed out.

It has been explained above that there occurs a reduction in the number

INFLUENCE OF SYNGAMY 133

of chromosomes in the nuclei of gametes, or in the two nuclei into which
the zygote nucleus divides. In this reduction division the individuals
of each pair of homologous chromosomes are separated, one of each pair
going to each daughter nucleus. If, for instance, there are four pairs of
homologous chromosomes grouped as ka, B6, Cc, Dr/, at reduction division
one of each pair passes to a daughter nucleus, so that the daughter nucleus
may receive chromosomes in many possible combinations — -ABCD, ABCc?,
ABcrf, khcd, abed, A6CD, etc. In all, there may be sixteen different
combinations. When syngamy occurs, any one of these groups in one
gamete will unite with any one in the other gamete, so that the zygote
nucleus containing eight chromosomes will have a still larger number of
possible combinations, the actual number being eighty-one.

It has been abundantly demonstrated in the higher animals and plants
that the hereditary characters are intimately bound up with the various
chromosomes occurring in the nuclei of the gametes, so that it is clear
that union of gametes with four chromosomes will give rise to eighty-one
different combinations of hereditary characters. In ordinary division
without conjugation all the chromosomes split longitudinally, and half of
each chromosome passes to each daughter nucleus, so that the hereditary
characters are more equally distributed to the daughter nuclei. On this
account, Jennings (1920) sees that the progeny resulting from conjugation
show a greater diversity of hereditary combinations than do the progeny
arising from multiplication by fission. From the point of view of survival
of the race, the diverse individuals resulting from conjugation will be
more likely to provide at least some forms which will tolerate any new
condition of the environment than are the more uniform individuals
which result from continued asexual reproduction alone. The group of
organisms which result from conjugation will be at a distinct advantage
when compared with others when changes in environment take place.

LIFE-HISTORY OF PROTOZOA.

The life-history of a Protozoon is one of continued growth and repro-
duction, which may or may not be interrupted at intervals by a process
of syngamy. When syngamy occurs, two ordinary individuals which do
not appear to differ from those which have been dividing may copulate,
as in Copromonas, or conjugate, as in Paramecium, after which reproduction
is resumed. On the other hand, it may happen that certain young
individuals which arise in the usual manner, and which do not appear to
differ from others which are destined to develop into forms like the parent,
become transformed into individuals of a special type. They are known
as gametocytes, which, when fully grown, produce a number of gametes.

134 LIFE-HLSTORY OF PROTOZOA

The latter unite in pairs to form the zygotes, which give rise to typical
daughter individuals, known as sporozoites. These grow into adults,
which reproduce repeatedly in the manner characteristic of the repro-
ductive phase till certain of the progeny again become gametocytes.
The various forms which occur during the multiplicative phase, which is
known as agamogom/, belong to the asexual generation, while the indi-
viduals themselves are agamonts. In contrast to these, the gametocytes
and the gametes to which they give rise, the zygotes, and the sporozoites
which are ultimately formed, belong to the sexual generation. The
process of development from gametocyte to sporozoite is known as
sporogonij, while the gametocytes themselves are sporonfs. These two
phases of development alternate in that, after reproduction has been
repeated a number of times (agamogony), the sexual method of multiplica-
tion (sporogony) supervenes. The sequence of the two phases is known
as alternation of generations, which is a characteristic of the majority of
the Sporozoa. Amongst the typical gregarines, however, the asexual
generation and agamogony does not occur, the sporozoites into which the
zygotes divide growing directly into gametocytes, which again produce
gametes. The whole life-cycle of a typical gregarine is thus one of
sporogony.

As already indicated, the life-cycle of a Protozoon may at any time
be interrupted by the formation of protective cysts secreted from the
ectoplasm. Some organisms cease multiplying when they become en-
cysted, others continue to multiply within the cyst, while others again
never reproduce except in the encysted condition. Sometimes, as in the
case of parasitic amoebse, special individuals (precystic amoebse) alone
are capable of forming cysts. Amongst the Sporozoa, encystment only
occurs in association with sporogony. A cyst may be formed around
two gametocytes, as in the case of gregarines. It is then distinguished
as a gametocgst. After syngamy has taken place, the resulting zygote
may secrete a cyst known as an oocyst. The zygote may divide into a
number of sporoblasts, and these, either within the oocyst or after their
escape from it, become enclosed in secondary cysts called sporocysts.
Oocysts and sporocysts occur typically amongst the Sporozoa. The cysts
usually have very tough and resistant walls ; at other times they are little
more than thin membranes.

Protozoa may be free-living organisms which spend the whole of their
life in water or in moist situations, or they may be more or less intimately
associated with other animals. According to the degree of this dependence
three classes are usually recognized. There are commensals, which live
in or upon another organism, and, though deriving benefit from this
association, do not injure the host in any way. They deprive it of an

PARASITISM 135

inappreciable amount of material wliicli it might use itself, or feed upon
the waste products. Others are regarded as symbionts, which, living in
similar circumstances, not only derive benefit themselves, but contribute
to the well-being of the host. Thus, Cleveland (1923) has shown that
termites, which feed upon wood, do so by virtue of their intestinal Protozoal
fauna, which actually digest the wood to form substances on which the
life of the termites depends. Other forms are parasites, which deprive
their hosts of their own fluids or tissues, and damage them by destruction
of tissues either directly or indirectly through the formation of toxins.
The line of demarcation between these various types is very indefinite,
so that it is often impossible to decide to which group any particvdar
organism belongs. The numerous discussions which have arisen as to
the pathogenicity of the intestinal flagellates of man is a case in point.

When true parasitism is considered, it must be remembered that the
degree of harm inflicted on the host has a direct bearing on the continued
existence of the parasite. A parasite is an organism which has become
adapted to an existence in another, and has lost at the same time the
power of living outside this host. At some period of its existence it must
be transferred to a new host if it is to survive. This transference may
take place by the production of encysted forms which escape from the
body and are taken up casually by a new host, or an invertebrate
may take up the parasites from the blood and later introduce
them to new hosts. In the first case the parasite does not appear
to be able to produce the encysted stages till some time after infec-
tion of a new host has taken place, and in the second a period must
elapse before the appearance in the blood of the forms capable of infecting
the invertebrate. In any case, the chance of a parasite gaining access to
a new host is a precarious one, and it is evident that the longer a parasite
can survive in one host, the better is its chance of bringing about infection
of another. If, then, a parasite is so virulent that it very quickly destroys
its host, its chances of continued existence are definitely diminished. It
is found in nature that there is such an adaptation of parasite to host,
and vice versa that in all cases of parasitism the parasite damages its host
to the least extent compatible with its own continued existence. When-
ever a parasite is discovered which brings about the death of its host in
a short time, it may safely be assumed that the host is not the natural
one, or that it is a natural one which is in some unnatural condition. In
the case of the pathogenic trypanosomes of Africa, the natural hosts are
the antelopes, to which they do comparatively little harm, while human
beings and domestic animals are unnatural hosts, as they are much more
seriously affected. After a time adaptation may occur, and a host which
was at first an unnatural one may gradually become a natural host.

136 LIFE-HISTORY OF PROTOZOA

Man seems already to have become a natural host to Trypanosoina
gambiense, but to be only in process of becoming so for T. hrucei {T.
rhodesiense).

An important feature of parasitism is the specificity of any particular
parasite for its host. It is found in nature that some parasites are unable
to live in any other host than the one in which they naturally occur.
This undoubtedly depends upon the peculiar character of the body fluids
of these animals. Some parasites have become so specialized that they
cannot survive in any other fluid than the one to which they have become
accustomed. Very frequently, however, a particular parasite is able to
live in hosts which are nearly related, the fluids of which may be presumed
to differ only slightly from one another. Thus Plasmodiuin vivax, which
causes benign tertian malaria, cannot survive in any other vertebrate
host than man, though Mesnil and Roubaud (1920) have shown that it
may multiply for a short period in the chimpanzee. Other parasites are
much less specific, for many of the pathogenic trypanosomes can develop
in small rodents, which under natural conditions are never infected by
them. In such cases it seems probable that, quite apart from the suita-
bility of the fluid of a host, the rapidity with which a host can develop
antibodies is the determining factor as to whether a parasite can establish
itself or not. Instances are known in which it is only after many attempts
to introduce a parasite into a host that success is at last attained. An
instance of this is quoted below (p. 576), where Watson, attempting to isolate
a strain of Trypanosoma equiperdum from horses in laboratory animals,
only succeeded in one after inoculating over 600 animals. The infection,
once established, was then readily inoculated from one animal to another.
It is evident that here the fluids of the animal which gave a successful
result differed from those in which inoculation had failed, or that amongst
the organisms injected on the successful occasion there happened to be
a few which found the environment congenial and were able to resist
the antibodies developed. The fact that subsequent subinoculations were
easily carried out seems to suggest that the explanation is to be found
in the parasites themselves. Not infrequently an animal which has
acquired an infection will free itself, after which it is found to be immune
to further inoculations. On the other hand, it has been shown that in
some cases, when an infection has disappeared or has been much reduced,
further inoculations of the same organism may bring about a super-
imposed infection which may be more severe than that first produced.
Such an instance has been described by Ndller (1917) in the case of frogs
infected with Trypanosoma rotatorium.

It may be stated as a general rule that the specificity of parasitic
Protozoa for their particular hosts is much more marked than is the case

PARASITISM 137

with vegetable parasites, such as bacteria, yeasts, and allied organisms.
It often happens that a parasite in one host may be morphologically
indistinguishable from one in another, yet experimentally it is impossible
to produce cross-infections. Whether such biological races are to be
regarded as distinct species or not is a problem which still requires solu-
tion. From the strictly zoological point of view they should be regarded
as belonging to one. This highly developed specificity of Protozoan
parasites may be kept in mind when organisms of a doubtful nature are
being dealt with. The group of parasites known as Toxoplasma, which
most observers regard as Protozoa, may actually be vegetable organisms,
for it has been found that they are inoculable into a variety of different
hosts.

Another feature exhibited by parasites is one which is termed increase
in virulence. Here, again, illustrations occur amongst the trypanosomes.
T. gatnbiense can be inoculated from man to laboratory animals. In the
first passage the infection may be of slow development, but with successive
passages through these animals a strain wall develop which in its behaviour
differs from that originally introduced. Whereas at first it may have
taken a year to kill the animal in which the trypanosomes were always
scanty, finally it brings about a fatal issue in two or three weeks, the
trypanosomes reproducing rapidly till the blood of the animal is teeming
with them. It is evident that during successive passages the trypano-
somes have gradually adapted themselves to these animals. In the case
of naturally occurring infections, wdiich are characterized normally by
a balance between host and parasite, occasionally infections occur in
which such a balance does not exist. In naturally occurring malarial
infections amongst native children exposed to the bites of infected
mosquitoes there is a balance between the host and parasite, so that
the host appears to be little inconvenienced. Sometimes, however,
severe and fatal cases occur, either because the natural resistance of the
host is low or because the parasites have become peculiarly virulent.
These severe infections are of more frequent occurrence amongst human
beings who have come from non-malarial countries and are suddenly
exposed to infection. It is often claimed that these cases result from
a specially virulent strain of parasite, but it seems more probable that
the host is at fault, and that the fluids of the body differ from those of
the natural hosts. Another illustration is seen in the case of Entamoeba
histolytica. In the majority of cases of infection with this amcBba, the
organism produces a minimum of inconvenience to its host, which is
known as a carrier, but in a small percentage of cases the balance is
broken down and acute symptoms of amoebic dysentery reveal themselves.
It is found that the reaction of a host varies with the strain or race of

138 IMMUNITY IN PROTOZOAL INFECTIONS

any particular parasite emj^loyed. Two strains of the same sj^ecies of
trypanosome may produce very different results. An animal inoculated
with one strain may acquire an infection from which it will recover.
It may have developed an immunity and be no longer inoculable with
this particular strain, though it is still susceptible to inoculation with
another strain of the same species. On this account it is exceedingly
difficult to differentiate species of trypanosome by what have been termed
immunity experiments.

The mechanism of these various phenomena are far from being properly
understood, and it appears that a real explanation will never be obtained
till the biochemist has obtained more information regarding the chemistry
of the living cell and the fluids to which it gives rise.

IMMUNIIY IN PROTOZOAL INFECTIONS.

Immunity in connection with parasitism amongst the Protozoa will
be referred to below in connection with individual parasites, but it will
be necessary to discuss more fully some of the general features which
have just been mentioned above.

NATURAL IMMUNITY.— As remarked above, each parasite has its
own particular host or group of hosts in which it can live, and outside
these limits it is impossible for it to establish itself. This specificity, as
it is called, is well illustrated by the malarial parasites of man. Exactly
how infections are prevented in one host while they take place readily
in another is not properly understood, but, as a result of extensive re-
searches, it is evident that cells and fluids of the body of refractory
animals are of such a nature that parasites introduced cannot develop
and are finally killed. That the serum of the blood is largely responsible
for this natural resistance is proved by the experiments of Laveran (1904ft),
who showed that the blood-serum of baboons, which are usually refractory
to inoculation with Trypanosorna gam.biense, when injected into mice will
cause the disappearance of T. gambiense from their blood, or even prevent
infection if injected forty-eight hours before inoculation with tlie trypano-
some. Such an immunity against infection is a natural immunity. It
is possible, however, in some cases to overcome the natural resistance.
This may be effected either by lowering the resistance of the inoculated
animal, an illustration of the well-known fact that a person in good health
is less liable to disease than one who is in poor condition, or by increasing
the virulence of the parasite. As a rule mice and guinea-pigs are quite
refractory to inoculations with Trypanosoma hwisi of the rat, but Eoudsky
(1910ft, 1911), as will be mentioned below, was able to increase the viru-
lence of the trypanosome, so that mice and guinea-pigs were susceptible.

NATURAL IMMUNITY 139

It is thus evident that in a study of the interrehitions of a host and the
parasite both the condition of the host and that of the parasite have to
be taken into account. The increase in virulence of Tryjmnosoma lewisi
produced by Roudsky was artificial, and it is probable that under natural
methods of transmission such a change would rarely, if ever, take place.
Nevertheless, the observation is an important one, for it demonstrates
that a trypanosome may become modified to such an extent that it will
produce infections in animals in which normally it fails to develop. It
is a generally accepted fact that the animal trypanosome, Tnj'panosoma
hrucei, does not as a rule infect man who is constantly exposed to the
bites of infected tsetse flies, yet there occurs in man in Rhodesia a
trypanosome which has been given the name Tryjpanosoma rhodesiense,
which in all respects appears to be identical with T. hrucei. It is main-
tained by some that it is distinct from T. hrucei, and by others that it is
identical with it. It has, however, to be recognized that it is quite within
the bounds of possibility that the animal trypanosome T. hrucei may
occasionally change, for reasons not yet discovered, so that it becomes
capable of infecting man, or that man may occasionally be in a condition
which will permit infection with the unaltered trypanosome. Duke
(1923, 1923a) believes that an outbreak of trypanosomiasis amongst
human beings in the Mwanza district of Africa, in which the trypano-
some was of the T. rhodesiense type, was due to the inoculation of
the animal trypanosome T. hrucei as a result of the lowered resistance
of the population after a period of famine and heavy ankylostome
infection.

There are many examples of variation in virulence of parasitic Protozoa.
It is well known that if Trypanosoma gamhiense is inoculated from the
blood of man into a rat, the type of infection produced is a chronic one,
very few trypanosomes being present in the blood of the rat at any one
time, the inoculated animal often surviving for many months. In suc-
cessive passages in rats the virulence increases, till finally a strain is
produced which multiplies very rapidly, so that the blood is soon swarming
with parasites, which bring about the death of the host in about ten days.
By passage of the strain through a different host such as the guinea-pig
this virulence for rats may be largely lost. It is regained, however, by
further passage through the rat. Duke maintains that in the spread of
sleeping sickness the epidemic outbursts of this disease are due to direct
passage of the trypanosome from man to man by mechanical transmission
in which some biting insect merely conveys blood from an infected to
a healthy person, just as in laboratory experiments the syringe conveys
blood from an infected to a healthy animal. It is supposed that in this
way the virulence of the trypanosome, which is kept relatively avirulent

140 IMMUNITY IN PROTOZOAL INFECTIONS

under ordinary conditions by a definite cyclical development in the tsetse
fly, is greatly increased.

It is noteworthy that Blanchard and Blatin (1907) have shown that
the marmot during hibernation at a temperature of 6° C. becomes resistant
to trypanosomes, with which it can readily be inoculated when it is in
an active condition. Brumpt (1908a) found that the dormouse showed
a similar immunity during hibernation, though it was observed that the
trypanosome (T. blanchardi) with which it may be naturally infected per-
sists in its blood during the hibernation period. The natural susceptibility
or the resistance of animals to infection with parasites has been advocated
as a means of differentiating species. The method has been mostly
used in the case of trypanosomes, but it has been also applied to other
parasitic Protozoa. As an example may be quoted the effect of inoculating
into rats the two trypanosomes T. congolense and T. nanum, which in
their natural hosts are morphologically indistinguishable from one another.
When inoculated into rats T. congolense gives rise to an infection, while
T. nanum does not, and it is claimed by the advocates of the specific
value of this test that the dift'erence justifies the separation of the two
species. That the test is not as straightforward as at first it might appear
is illustrated by the fact that if T. congolense is inoculated into a goat,
it will be found to have lost its power of infecting rats. It follows, there-
fore, that distinction of species based solely on the ground of resistance
of certain animals is zoologically unsound. Another application of the
same test was made by Adler (1924), who discovered a coccidium in the
intestine of the civet cat in West Africa. Morphologically it resembled
Isospora rivolta, a parasite of dogs and cats. Attempts to infect dogs
and cats with the parasite of the civet cat having failed, it was thought
justifiable to establish a new species. Looking at the question from the
reverse point of view, the susceptibility of a number of different hosts
to a parasite derived from one host is strongly suggestive of the identity
of the parasites which may occur naturally in a variety of hosts. Thus,
birds are very liable to natural infection with a malarial parasite, Plas-
7nodium prcecox. The demonstration that the parasites from one bird
can be inoculated into birds belonging to other species is a valuable
indication that the one parasite may, under natural conditions, occur in
a variety of hosts. The converse is not necessarily true, for development
in one host may bring about such a change in the parasite that it is no
longer able to infect a host which was originally susceptible to it. The
example of passage of Trypanosofna congolense through the goat, referred
to above, is a case in point.

In connection with natural immunity it has to be remembered that
much depends upon the number of parasites — the dose of virus — intro-

RECOVERY FROM INFECTION 141

duced. Theoretically it would be expected that in the case of susceptible
hosts the introduction of a single parasite would bring about infection.
This has actually been demonstrated in the case of Trypanosoma brucei
and mice by Oehler (1913), who showed that the introduction into the
peritoneal cavity of a single trypanosome gave rise to infection. In
other cases, as, for instance, in the inoculation of Leishmania donovarii
to animals, no infection can be detected unless comparatively large doses
are employed. In animals with absolute immunity no infection occurs
even after the use of massive doses. Experiments such as these have been
conducted with animals which are not natural hosts of the parasites
concerned, but there is evidence that even in the case of natural hosts
infection does not always follow exposure, a result which may depend
on the dose of the virus.

Even when a host is a natural one there are always certain individuals
which resist infection. It is well known that, though human beings are
very susceptible to malaria, there are certain individuals who appear to
have a natural immunity, and never show any evidence of infection,
though constantly exposed to the bites of infective mosquitoes. Miihlens
and Kirschbaum (1924), during the inoculation of human beings with
malaria, observed one case which proved resistant to four inoculations,
but became infected after a fifth.

RECOVERY FROM INFECTIONS. — It is a general rule that when once
a parasite has established itself in a host it multiplies actively for some
time, so that the intensity of the infection rises to a maximum, after which
it gradually subsides till finally there may be every reason to suppose
that the infection has completely died out. This recovery may be due
to two causes. Firstly, the fluids of the host may gradually change with
the production of substances injurious to the parasite, or possibly by the
loss of substances which are necessary to the continued development of
the parasite; secondly, the parasite itself may become exhausted and
no longer capable of multiplication unless some change takes place. In
the case of coccidial infections of animals, during the early stages there
is active multiplication by schizogony in the intestinal epithelium.
Gradually this multiplication subsides, and there are produced an increasing
number of male and female gametocytes, which lead to syngamy and the
formation of oocysts, which leave the body. Eventually the sexually
differentiated forms alone can be found, and finally the infection ceases
when all these have been eliminated. In this case it is possible that the
host produces substances which act deleteriously on the parasite, and lead
to the production of the sexual stages, which are bound up, in the case
of the coccidia and other forms, with the distribution of the parasite to
other hosts. On the other hand, it may be that each sporozoite freshly

142 IMMUNITY IN PROTOZOAL INFECTIONS

introduced is only capable of reproducing asexually a certain number of
times, and that when this is completed sexual forms are produced. It
seems clear that the production of substances Vvdiich are generally termed
antibodies in the blood of the host plays some part, for when once a host
has passed through an acute infection it is rarely possible to produce as
intense infection again, while in many cases a complete immunity to
further infection is developed. But the second factor also comes into
play, for it has been shown that as one infection is subsiding it may be
possible to reinoculate the host with the same organism, so as to produce
a superimposed infection. Noller (1917) has shown that frogs which
have passed through the acute stage of an infection with Trypanosoma
rotatorium may be reinfected, though trypanosomes remaining from the
first infection are still present in the blood in small numbers. Such a
superimposed infection may become as intense as the first one, and even
bring about the death of the host. Similarly in the case of piroplasmosis
of cattle, Ed. Sergent and his co-workers (1924) have demonstrated that
superimposed infections are possible. They found that the appearance
of parasites in the blood after the second inoculation was not accompanied
by any of the symptoms which followed the first infection. The animals
had been rer^^ered partially immune, so that the injurious effects of the
parasite were resisted, though its development was not prevented. In
order to distinguish this partial immunity or tolerance immunity from
an absolute or true immunity they have introduced the term " premuni-
tion." It occurs in the infections with Babesia bigemina. The term is
not applicable to infections with Babesia nmtafis, which can also be super-
imposed on an already existing infection, for the first infection is not
accompanied by any recognizable symptoms. This parasite appears to
produce no immunity whatever. Hoare (1923) found that sheep, when
constantly infested with keds, always harbour Trijixinosoma melo'pliagium ,
but if the animals are freed from keds the infection in the sheep gradually
subsides, till after two or three months it can no longer be detected. It
is evident that the batch of parasites introduced by the keds on one
occasion have only a limited term of existence in the sheep, and it wovdd
appear that this is dependent rather on what may be termed an exhaustion
of the parasite than on changes in the sheep, for infection may at any
time be re-established by further introduction of trypanosomes from the
keds. This exhaustion, however, may be the result of continued action
of the antibodies producing a gradual weakening of the parasite.

It seems clear that in the case of many human Protozoal infections,
such as malaria, trypanosomiasis, and amoebiasis, in localities in which
these diseases are prevalent, individuals are constantly being infected
with fresh batches of parasites, and a condition resembling that in the

EECOVERY FROM INFECTION 143

sheep, just mentioned, occurs. In malarious countries, from their birth
children are constantly being bitten by infected mosquitoes, and it is
not unreasonable to suppose that the long duration of malarial infection
in cliildren in these countries is due to continuous reinfection. It has
been demonstrated by Miihlens and Kirschbaum (1924) that human
beings can be reinoculated with malaria when apparent recovery from
a first infection has taken place. They can even be inoculated a third
time, but the successive infections are of decreasing intensity. In view
of the difficulty in determining the complete elimination of parasites
from infected individuals, it is possible that some of these cases were
illustrations of superimposed infections. Recently Van Loon and
Kirschner (1924) in the Dutch East Indies have noted that the native is
relatively immune to inoculation of malarial parasites. In certain cases
it was found to be impossible to produce infection, though large doses of
blood heavily infected with Plasmodium, vivax were injected four or five
times. In other persons who had not experienced a lifelong exposure
infection was readily produced. Sergent, Et. and Ed. (1921c), have,
however, shown that birds in the chronic phase of a malarial infection
do not respond, or respond very slightly, to inoculations with a further
infective dose of parasites. A very striking illustration of the effect of
repeated doses of a virus was an observation made by Miller (1908) on
the haemogregarine Hepatozoon miiris of rats. As a rule these animals
which are infected by the ingestion of mites, acquire an infection which
does not appear to disturb the host in any way. Miller, however, found
that a batch of rats, which were so heavily infested with mites that con-
stant infection with large doses of virus was occurring, were very heavily
infected with the parasite, and that a definite pathological condition
resulted. When recovery from an infection is considered, a distinction
has to be drawn between the cases which have had a single dose of virus
and those which are repeatedly inoculated. Though recovery in a com-
paratively short time appears to be characteristic of many Protozoal
infections, this is not invariably the case. Animals such as cattle, horses
and dogs, which are liable to piroplasmosis, pass through an acute phase
when parasites are exceedingly numerous in the blood. Afterwards the
infection subsides, so that finally the organisms can no longer be detected
by microscopical examination of the blood. Nevertheless, it can be
demonstrated that they are still present and persist for years, bv the
inoculation of large quantities of blood into animals which have never
had the infection. In many cases of infection with Entamceba histolytica
the aniffibse persist in the intestine indefinitely. In these cases a balance
between the host and parasite has been reached, so that the former is
injured to a minimal extent, while the parasite can reproduce sufficiently

144 IMMUNITY IN PROTOZOAL INFECTIONS

to maintain itself. Hosts in this condition are usually termed carriers.
The practical difficulty associated with this type of infection is the im-
possibility of being absolutely certain that any infection has entirely
vanished. In the treatment of trypanosomiasis, leishmaniasis, malaria,
amoebic dysentery, and other infections, this difficulty is constantly being
encountered.

Another feature of recovery from infection has to be noted, and that
is that frequently during the period of abatement of the infection, when
the host may be said to be obtaining a mastery over the parasite, a relapse
occurs in which a fresh outburst of activity on the part of the parasite
leads again to an intense infection. It must be supposed that under
these conditions the control of the host over the parasite has broken down,
and anything which leads to this may bring about a relapse. It is well
known that in malarial infections of man a sudden exposure to cold,
shock resulting from accident, or the intercurrence of some other infection,
may lead to the appearance of large numbers of parasites in the blood.

Such periodic variations in the intensity of infections may, however,
be a feature of the development of the parasite. This periodicity is quite
distinct from the periodicity which results from the developmental cycle,
like that of parasites of malaria, which reproduce only at regular intervals.
In human trypanosomiasis, and also in animals experimentally infected,
it has been frequently noted that the number of parasites in the blood
is not constant. The trypanosomes may be comparatively numerous
on one day and absent on another. This is probably due to variations
in the rate of multiplication, but it is possible that it is also dependent
on variations in the rate of mortality of the trypanosomes resulting from
irregularities in the antibody content of the body fluids of the host. No
satisfactory explanation of this type of periodicity has been discovered.

ACQUIRED IMMUNITY.— Under this heading will be considered the
immunity to infection which a host acquires as a result of an infection.
It has already been shown that in some cases infection may persist for
many years in a latent form, and though there may be considerable
difficulty in determining the complete elimination of an infection, there
is reason to suppose that sometimes a host becomes completely free.
After recovery of this kind the host may be absolutely immune to further
infection, the type of immunity being known as active immunity. The
observations of Van Loon and Kirschner, who failed to produce malarial
infections in natives of the Dutch East Indies, have been referred to above.
In human infections with Leishmania tropica the disease oriental sore, if
allowed to run a natural course, will produce in most cases an absolute
immunity to further infection, so much so that artificial production of
oriental sore by inoculation on an unexposed part of the body has been

ACQUIRED IMMUNITY 145

employed as a means of avoiding the risk of the disfiguring natural
infection on an exposed part such as the face. Another illustration of
absolute immunity conferred by a single infection occurs in the case of
East Coast fever of cattle due to infection with Theileria parva. Animals
which have recovered from one attack are immune for the rest of life.
The same remark applies to rats which have recovered from an infection
with Trypanosoma lewisi. Again, in the case of many of the disease-
producing trypanosomes it has been found that certain animals, such as
the goat and sheep, though acquiring an infection, eventually recover to
such an extent that trypanosomes can no longer be detected. In this
condition they are immune to further inoculations with the same trypano-
some. As in the case of naturally immune animals, these actively im-
munized hosts have been employed as a means of differentiating species.
If it is desired to distinguish two trypanosomes which resemble one
another morphologically, one of them is inoculated into a goat. When
the animal has recovered and is no longer susceptible to inoculation with
this trypanosome, it is inoculated with the other. If infection occurs, it
is assumed that the trypanosomes are different. Though the experiment
undoubtedly indicates a physiological difference between the trypano-
somes, it is far from clear that they belong to distinct species. The test
has been applied by Laveran and Mesnil and others to a group of trypano-
somes which resemble Trypanosoyna evansi, with the result that a number
of species of very doubtful value has been created. Similarly, in the
case of piroplasmosis the test has again been applied. Animals which
recover from an acute attack pass into a chronic phase, during which the
parasites show a gradual diminution in their numbers, till finally they can
no longer be detected except by the inoculation of comparatively large
quantities of blood into a susceptible animal. It has been shown by
Ed. Sergent and his co-workers (1924) that in the case of Babesia bigemina
it is possible to produce a superimposed infection in wdiich parasites
appear in the blood, but this is unaccompanied by symptoms. The infec-
tion, moreover, is less intense than the original one, the parasites quickly
disappearing again. Stockmann and Wragg (1914) showed that cattle
which had recovered from an infection with B. bigetnina, and were
immune to further inoculations with this parasite, were nevertheless
susceptible to Babesia bovis, and behaved, as regards symptoms and
intensity of infection, as animals at their first infection. In this instance
there were morphological differences which justified the separation of
the two parasites as distinct species. On the other hand, a form of piro-
plasmosis in cattle in South America is due to a parasite resembling
B. bovis. Brumpt (1920) showed that cattle which had recovered from
the infection with this parasite were still susceptible to inoculation with
I. 10

146 IMMUNITY IN PROTOZOAL INFECTIONS

the one from South America. There appear to be slight morphological
differences between the two, but whether these are sufficiently distinct
to justify the recognition of the South American form as a distinct species,
Babesia argentina, apart from the cross-immunity test, is open to question.
In connection with piroplasmosis of horses, Nuttall and Strickland (1910)
and du Toit (1919) showed that animals recovered from infections with
Babesia caballi were still liable to infection with Babesia equi. Here again
morphological characters enable the species to be distinguished. The
difficulty of accepting the test as a means of distinguishing species is
illustrated by the experiments of Laveran and Nattan-Larrier (1913) on
canine piroplasmosis. The disease occurs in dogs both in France and
North Africa, and on morphological grounds appears to be due to the same
parasite, Babesia canis, in both places. Yet dogs which have recovered
from infection with the French virus and are completely immune to
further inoculations are susceptible to the North African virus. It would
appear imj)ossible on these grounds alone to recognize two species of
parasite.

As in the case of natural immunity, acquired immunity is dependent
on antibodies which appear in the blood, for the serum of the animals
which have recovered or have been infected for a length of time sufficient
to allow of the production of these substances can be employed as a
curative agent in the case of infected animals. Furthermore, the serum,
when injected into an animal before it is exposed to infection, may entirely
prevent an infection. In this case the immunity is known as passive
immunity, because the host itself has taken no part in the production
of the antibodies, which are merely introduced from another animal.
The extensive investigations of Rabinowitsch and Kempner (1899), and
of Laveran and Mesnil (1901a), on infections of rats due to TryjKinosoma
lewisi threw considerable light on this subject. Infected rats pass through
an acute phase followed by a chronic one, from which ultimate recovery
takes place. The animals are completely immune from reinfection. A small
quantity of the serum (0-5 c.c.) of a recovered animal, if inoculated into
the peritoneal cavity of a rat, will entirely prevent infection when trypano-
somes are inoculated twenty-four hours later. This property is possessed,
though to a less extent, by the serum of animals, such as goats and sheep,
which have recovered from infections with the pathogenic trypanosomes,
and animals, such as cattle, which are in a very chronic stage of infection.
Taliaferro has shown, in the case of T. leivisi, that this is due to the
appearance in the blood of the rat of a substance which inhibits the repro-
duction of the trypanosomes (see p. 467).

Many attempts to produce an active immunity by other means than
actual infection and natural recovery have been made. So-called attenu-

ACQUIRED IMMUNITY 147

ated strains, such as trypanosomes which, as a result of exposure to heat
or other adverse conditions, have lost their power of producing actual
infection, have been injected into animals. In a similar manner killed
trypanosomes, trypanosomes which have been broken up by immersion
in fluids which bring about cytolysis, dried trypanosomes, as well as
cultural forms of trypanosomes, which often have ceased to be infective
to animals, have been tried, but in none of these cases was satisfactory
evidence obtained that the animals inoculated with these altered trypano-
somes had acquired any immunity to inoculation with a virulent strain,
though the application of certain serological tests, such as that of the
complement fixation, has demonstrated that a specific change may have
taken place in the serum of the animals. The response as regards pro-
duction of immunity cannot be compared with that which occurs in the
case of bacteria. Ponselle (1923a) has found that by keeping the heart-
blood of a mouse containing Trypanosoma brucei for twenty-four hours in
a medium of dihydrogen potassium phosphate and hydrogen disodium
phosphate it loses its power of infecting mice, but if injected will render
mice immune to infection with unaltered Trypanosoma brucei (see p. 454).
The bulk of work in connection with the production of immunity in
Protozoal infections has been carried out with trypanosomes, but certain
investigations have been made with other Protozoa. Thus, the Sergents,
Et. and Ed. (19216), have produced a certain degree of immunity in the
case of the parasite of bird malaria, Plasmodium, prcecox. Normal canaries
were very easily infected with this parasite, only 0-72 per cent, resisting
infection out of 965 birds inoculated. If canaries are inoculated with
the sporozoites of the parasite which have been rendered non-infective by
keeping them for twelve to forty-eight hours after removal from the
mosquito, a certain degree of immunity results. It was found that
29*5 per cent, of twenty-four canaries thus treated resisted subsequent
inoculation with the parasite. Similarly, it was found that if the blood
of a canary was drawn off after it had been inoculated with the parasite,
and before the infection had established itself by the appearance of
parasites in the blood, this blood, if injected into healthy birds, produced
an immunity which protected from subsequent inoculation 21 "3 per cent,
of sixty-one canaries.

Many observers have attempted to produce immunity in cattle against
infection with Babesia bigemina and Theileria parva. From both these
infections animals may recover naturally, and possess an absolute immunity
to further infection, but the death-rate is ahvays high, especially in the
case of East Coast fever. No means of producing an immunity apart from
actual infection are known, though in the case of piroplasmosis it is
possible to inoculate the animals at a time when they are best able to

148 IMMUNITY IN PROTOZOAL INFECTIONS

withstand the disease. It is known that young animals recover more
easily than older ones, and that the disease is less severe at a certain
season. It has been shown by a number of observers that by inoculating
young animals with Babesia bigefnina at this particular season it is possible
to obtain a higher percentage of recoveries, and hence of permanently
immune animals, than if they had been exposed to natural infection.
In the case of East Coast fever also young animals are less seriously
affected than older ones, and it would be expected that a similar method
of protection could be applied. As will be shown below, it is not as.
a rule possible to transmit this disease by the inoculation of the blood
of an infected animal, but Meyer (1909) found that this could be effected
by inoculating the macerated spleen and lymphatic glands in which the
reproducing forms occur. By the inoculation of young animals with
emulsions of these organs Theiler (1911a, 19126) noted that though a
number acquired a severe and fatal disease, a much larger number survived
and recovered completely. As many as 50 per cent, of those which
survived proved resistant when exposed to infection by ticks under
natural conditions. Somewhat similar results were obtained by Wolfel
(1912) and Spreull (1914). In the production of immunity by these
methods it is important, as demonstrated by Theiler (1908) and Lignieres
(1903), to employ the particular strain of virus to which subsequent
exposure will occur. A previous infection with Babesia bigemina of
European origin w^ill not produce immunity against the parasite of South
Africa.

Mechanism of lynvnunity. — During the development of an immunity
the blood of the animal acquires certain properties which it did not pre-
viously have, but which are possessed by the blood of naturally immune
animals. It has already been pointed out that the serum of such an
animal will produce a degree of passive immunity when injected into a
healthy animal, which is thereby protected against inoculation with the
organism. Such passive immunity is usually of much shorter duration
than active immunity, which is due to the production of antibodies by the
host itself as a result of an actual infection, or the introduction of modified
or dead parasites, or the products of their dissolution, which stimulate the
host to produce the antibodies without actually giving rise to an infection.
Where active immunity is produced without infection, the substance
introduced is termed a vaccine. It is evident that the immunity produced
is dependent upon the presence of several distinct substances, each of
which has its special action. It was first shown by Laveran and Mesnil
(1901a) that during the course of an infection with Trypanosoma lewisi
the leucocytes of the rat's blood are constantly ingesting trypanosomes,
which are ultimately destroyed. It appears that the serum of an immune

MECHANISM OF IMMUNITY 149

animal actually stimulates this phagocytosis, for Laveran and Mesnil
found that if the serum of such an animal was mixed with trypanosomes
and injected into the peritoneal cavity of a rat, there appeared in the
peritoneal fluid numerous leucocytes which devoured the trypanosomes
with avidity. If the trypanosomes were injected alone, this phenomenon
was not observed to anything like the same extent. Levaditi and Mutter-
milch (1911) showed that the serum affected the trypanosomes in such a
way that they attached themselves to the leucocytes. This was inde-
pendent of the actual process of phagocytosis, for it was found that
attachment to killed leucocytes also occurred. It was shown by Mesnil
and Brimont (1909) that if immune serum were allowed to act upon
Trypanosoma lewisi a change took place, so that the trypanosomes were no
longer able to infect rats even if they were carefully washed free of serum.
It would thus appear that the protective action of the serum is a result
of its power of causing the trypanosomes to attach themselves to the leuco-
cytes which then engulf them. The serum of animals which are immune
to Trypanosoma lewisi also has the property of causing trypanosomes
to become agglutinated into clumps when blood containing them is
mixed with the serum (see p. 452). The presence of agglutinins in the
serum has been shown to occur in the case of other trypanosome
infections.

Another property which the serum may acquire is that of producing
cytolysis, or the gradual swelling up and dissolution of trypanosomes
exposed to its action. It was shown to occur in the case of infections
of animals with the pathogenic trypanosomes by Levaditi and Mutter-
milch (1909), amongst other observers. They also demonstrated that the
serum acquired the property of deviating the complement, a reaction
which has found a practical application in the diagnosis of trypanosome
infections (see p. 452). It seems evident that recovery from any infection
is dependent on the development of antibodies in the blood, which act
upon the particular parasites in various ways. This action of the serum
of an immune animal is specific for the parasite which stimulated its
production. On this account serological tests, which are similar to the
inoculation tests referred to above, have been employed as a means of
differentiating parasites. If the serum of an immunized animal behaves
towards an unidentified trypanosome as it does towards the one which
caused the immunity, then, provided that there is morphological similarity,
it is concluded that they are identical. On the other hand, it is main-
tained by some that, in spite of morphological identity, if the serum fails
to act it is proof of a specific distinction. It is possible that a natural
recovery would never take place unless antibodies were produced, and
that a parasite would continue to multiply continuously till the host

150 ACTION OF DRUGS IN PROTOZOAL INFECTIONS

was destroyed. Certain strains of pathogenic trypanosomes can be
handed on indefinitely from mouse to mouse by direct inoculation of
blood without there being any evidence that the rate of multiplication
by binary fission slackens in any way. In these cases the trypanosomes
multiply so rapidly that the host is overcome by the parasite before any
degree of immunity capable of checking the infection has been developed.
At each inoculation the trypanosomes are introduced into a new host
which has no immune bodies, and multiplication is continued with the
same result. For the development of immunity it is essential that the
rate of multiplication of a parasite shall not be so great as to bring about
destruction of the host before it has time to respond to the infection by
the production of sufficient antibodies to check the development of the
parasite. From the point of view of the parasite this is the condition
most favourable to its survival, and it appears to be the one which obtains
in most, if not all, natural infections.

A parasite may acquire the power of resisting the antibodies in the
serum. Jacoby (1909a) obtained a strain of Trypanosoma brucei which
was resistant to human serum, which normally will cause the disappear-
ance of the trypanosomes from the blood of mice. By repeatedly injecting
small quantities of normal human serum into an infected mouse and con-
tinuing the process in subinoculated mice, a strain of trypanosomes was
eventually secured which, as regards its development in mice, was un-
influenced by as large a dose (2 c.c.) of human serum as the mouse could
tolerate. Leboeuf (1911) in a similar manner obtained races of T. brucei
which were resistant to the serum of baboons.

ACTION OF DRUGS IN PROTOZOAL INFECTIONS.

It is possible that the disappearance of parasites as a result of the
administration of drugs is, in many cases at least, not the result of a
direct poisonous action of the drug upon the parasite. It would seem
natural to suppose that the good effects observed in amoebic infections
which result from the use of emetine and those following the ingestion
of quinine in malaria are due to the direct effect of the drugs upon the
parasites concerned. It appears that the action may be a much more
complicated one, and that drugs may act indirectly by stimulating the
tissues of the host to produce substances which may be regarded as anti-
bodies which are directly responsible for the suppression of the infection.
In support of this contention may be urged the fact that drugs such as
emetine, which are therapeutically active, are not more toxic to the
organisms when tested in vitro than other drugs which have no thera-
peutic properties. The investigations of Dale and Dobell (1917) on the

DRUG-FAST STRAINS 151

action of emetine are discussed below (p. 255). Morgenroth (1918)
believes that quinine combines with the red blood-corpuscles, and thus
prevents the entry into them of the merozoites of the malarial parasites.
Quite recently Yorke and Macfie (1924a) have suggested that in malaria
quinine acts by causing a destruction of a certain number of parasites,
the broken-down parasites then acting as a vaccine in stimulating the
host to produce antibodies, which finally rid the host of all remaining
parasites. So far the presence of the antibodies has not been demonstrated.
Another illustration of what may be the indirect action of a drug is seen
in " Bayer 205." This medicament is remarkably trypanocidal when in-
jected into animals infected with certain trypanosomes. Animals which
have recovered as a result of treatment or uninfected animals which have
received a dose of the drug remain immune from infection for compara-
tively long periods. It is possible that this resistance is due to the pro-
duction by the host of antibodies as a result of the action of the drug
upon its cells. On the other hand, it has to be remembered that when
a drug is administered to an animal it does not follow that the drug
remains unaltered. The fluids of the body act upon it chemically, and
may in this way produce other substances which are definitely toxic to
the parasites. It is known that arsenic compounds in which the arsenic
is in the trivalent form are toxic to trypanosomes in vitro, and are also
therapeutically active, whereas when the arsenic is in the pentavalent
form there is no action in vitro, though there is a therapeutic action which,
however, requires some time to develop. This difference has been
explained by the fact that in the body of an animal the pentavalent
arsenic radical is transformed into a trivalent one.

Another feature of the action of drugs on Protozoa is the development
of drug-fast strains. In the case of mice, for instance, infected with
pathogenic trypanosomes, the repeated treatment of the infection with
such a drug as atoxyl in doses which are insufficient to prevent a sub-
sequent relapse will finally result in a strain of trypanosome which is
quite unaffected by the drug administered to the animals. This strain
maintains its resistance when passed through a series of new mice, but
as Mesnil and Brimont (1908 b) discovered, it is susceptible to the drug
when inoculated into rats, and is still resistant when again passed into
mice. Such a fact appears to be explicable only on the assumption that
the trypanosomes have not become resistant to atoxyl itself, but to a
substance resulting from the action of the drug on the tissues of the
mouse, but not of the rat. Furthermore, it has been demonstrated that
trypanosomes can be rendered arsenic resistant by the inoculation of
infected mice with substances which contain no arsenic. Many writers
refer to quinine-fast strains of malarial parasites and emetine-fast strains

152 STATUS OF PROTOZOA

of Entamoeba histolytica, but at present there is no reliable evidence that
these actually exist. A drug which fails to act on a parasite may do so
because of some peculiarity on the part of the host. The whole subject
of the method of action of drugs in the treatment of Protozoal infections
is exceedingly complicated, and opens a field for extensive investigations.
A very instructive resume of the subject has been made by Dale (1924).

STATUS OF THE PROTOZOA IN THE ANIMAL KINGDOM.

It is usual to regard the Protozoa as constituting a Phylum wdiich
corresponds in status to one of the various Phyla, such as the Mollusca,
Arthropoda, Vertebrata, etc., into which the rest of the animal kingdom
is divided. This is the view adopted by most zoologists, but Dobell and
O'Connor (1921) have recently expressed the view that the Protozoa
constitute a group of organisms which has a status equal to the rest of
the animal kingdom. According to Dobell's contention, discussed earlier
in this work, the Protozoa are non-cellular animals, while the rest of the
animal kingdom includes all cellular animals. On this account he
divides the animal kingdom into two sub-kingdoms — the Protozoa and
the Metozoa. Such a distinction may still be admitted, though there
would be less reason for its recognition if the generally accepted view were
held that the Protozoa are unicellular, and not merely non-cellular animals.
Dobell, having raised the Protozoa to the rank of sub-kingdom, raises
to the status of Phyla the various classes in which they are divided. For
purposes of this work, however, it is unnecessary to discuss this very
intricate subject, and, following the more orthodox view, the Protozoa
will be still regarded as constituting a Phylum.

PART II

SYSTEMATIC DESCRIPTION OF THE PROTOZOA

WITH SPECIAL REFERENCE TO PARASITIC AND COPROZOIC FORMS

CLASSIFICATION OF THE PROTOZOA.

SUB PHYLUM :

PLASMODROMA

CLASS: RHIZOPODA

Order: AM(EBIDA
HELIOZOA
RADIO LARIA
FORAMINIFERA
MYCETOZOA

C'i.-4.s.s. MASTIGOPHORA
SUB-CLASS: Phytomastlgina

Order: GHRYSOMONADIDA
CRYPTOMONADIDA
DINOFLAGELLATA
EUGLENOIDIDA
PHYTOMONADIDA

SUB-CLASS: Zoomastigina

Monozoic Forms

Order: PROTOMONADIDA
HYPERMASTIGIDA
CYSTOFLAGELLATA

Diplozoic Forms

Order: DIPLOMONADIDA

Polyzoic Forms

Order: POLYMONADIDA

CLASS: CNIDOSPORIDIA

Order : MYXOSPORIDIIDA
Suh-Order : Eurysporea
,, Sphaerosporea

„ Platysporea

Order: MICROSPORIDIIDA

Suh-Order: Monocnidea

„ Dicnidea

Order: ACTINOMYXIDIIDA

UNDETERMINED

SARCOSPORIDIA

GLOBIDIUM

HAPLOSPORIDIA

155

PHYLUM: PROTOZOA

CLASS: SPOROZOA
SUB-CLASS: Coccidiomorpha

Order: COCCIDIIDA

Suh Order: Eimeriidea

,, Haemosporidiidea

„ Piroplasmidea

Order: ADELEIDA

Suh-Order: Adeleidea

„ Haemogregarinidea

SUB-CLASS: Gregarinina

Order: SCHIZOGREGARINIDA
EUGREGARINIDA

Sub Order: Acephalinidea
„ Cephalinidea

SUB-PHYLUM: GILIOPHORA

GROUP 1: PROTOCILIATA
CLASS: OPALINATA

GEO UP 2: EUCILIATA
CLASS: CI LI ATA

SUB-CLASS: Aspirigera

Order: HOLOTRICHIDA

Sub-Order .

Astomatea
Stomatea

Section 1 : Gymnostomata
Section 2 : Trichostomata

SUB-CLASS: Spirigera

Order: HETEROTRICHIDA
OLIGOTRICHIDA
HYPOTRICHIDA
PERITRICHIDA

CLASS: SUCTORIA

156 PHYLUM: PROTOZOA

PHYLUM: PROTOZOA GOLDFUSS, 1817.

The phylum Protozoa^ as defined above, is the subdivision of the animal
kingdom in which all unicellular animals are grouped. It may be divided
into two sub-phyla, as suggested by Doflein (1901). The first of these
is the PLASMODROMA, which includes the forms which have
pseudopodia or flagella, and in which syngamy, where it is known to
occur, consists in the complete fusion of two gametes. The second sub-
phylum is the GILIOPHORA, which comprises those Protozoa
which have numerous cilia as motile organs, a special type of binuclearity
(macronucleus and micronucleus), and a process of syngamy in which two
individuals temporarily associate, undergo exchange of nuclei, and then
separate. The class Opalinata, in which syngamy is of the type seen
amongst the Plasmodroma while the binuclearity characteristic of the
other classes of the Ciliophora is wanting, forms a connecting link between
the two sub-phyla.

A. SUB-PHYLUM: PLASMODROMA DOFLEIN, 1901.

This, the first of the sub-phyla into which Doflein divides the Protozoa,
includes forms which have either pseudopodia or flagella as organs of
locomotion, and the parasitic Sporozoa which, owing to their mode of life,
have been modified in various ways. There is either a single vesicular
nucleus or more than one are present. Syngamy takes place by the
complete fusion of gametes, which may be alike (isogamy) or different
(anisogamy). In many forms, after a period of asexual reproduction,
syngamy, followed by a different method of reproduction, occurs (alterna-
tion of generations).

The sub-phylum contains four classes of Protozoa, two of which include
mainly free-living forms, while two contain forms which are exclusively
parasitic. One class is characterized by the amoeboid form of the body
which produces pseudopodia as organs of locomotion, while in another,
though the body may be amoeboid, it possesses one or more flagella. The
Protozoa of the first type belong to the class RHIZOPODA, and those
of the second to the class M ASTIGOPHORA. The separation of these
two classes is rendered difficult by the fact that certain organisms which
are amoeboid and devoid of flagella for the greater part of their existence
may at certain stages develop flagella, while, conversely, forms which
usually possess flagella may have a purely amoeboid phase.

As regards the parasitic types, many observers have grouped them
together in the one class Sporozoa, which was divided by Schaudinn

PLASMODKOMA AND CILIOPHORA 157

(1900) into the Telosporidia and Neosporidia. It appears, however, that
these two groups are so fundamentally different that it is better to follow
Hartmann (1907) and place the Neosporidia in a separate class, for which
Doflein's name Cnidosporidia may be employed, and to reserve the
Sporozoa for the forms included in Schaudinn's group Telosporidia. The
class CNIDOSPORIDIA includes parasitic Protozoa, which are either
amoeboid or almost, if not entirely, motionless. They produce, by a com-
plicated process of development in which several cells take part, very
characteristic encysted stages or spores which are peculiar in possessing
special bodies called polar capsules, from which long filaments can be ex-
truded. The class SPOROZOA also comprises parasitic forms, which
reproduce characteristically by schizogony. After syngamy the zygote
gives rise to a number of sickle-shaped sporozoites. These are either free
within the oocyst which forms around the zygote, or they are enclosed in
a number of secondary cysts, the sporocysts, which are formed inside
the oocyst. Schaudinn included the Sarcosporidia in his group Neosporidia.
These parasites, however, have little in common with the true Cnidosporidia,
and though they produce bodies which are called spores, these are structur-
ally quite different from those of the Cnidosporidia. In fact, very
liUle is known about the true nature of the Sarcosporidia, which will be
considered with certain other parasitic forms (Haplosporidia, Globidium)
of undetermined affinities.

B. SUB-PHYLUM: CILIOPHORA DOFLEIN, 1901.

Ciliophora is the name given by Dofiein to the second of the two sub-
divisions into which he divides the Protozoa. The organisms included in
this group have a comparatively complex structure, and in this respect
may be considered to be the most highly specialized of the Protozoa
(Fig. 70). The body is not, as a rule, subject to changes of shape, unless
as a result of external pressure, there being a definite body form for each
individual. The most characteristic feature is the possession of numerous
hair-like processes, the cilia, which cover either the whole or only part
of the body surface. The cilia are used as organs of locomotion, or for
producing currents in the water for the intake of food. They may also
serve as organs of special sense, such as taste or touch.

The cytoplasm is differentiated into an endoplasm, which contains the
nuclei, contractile vacuoles, and food vacuoles, and a highly-organized
ectoplasm. The latter consists of a superficial membrane, the pellicle,
within which is a layer containing myonemes or contractile fibres, spaces
and canals of an excretory system, basal granules from which the cilia
arise, and sometimes trichocysts, which are small bodies from which

158

PHYLUM: PROTOZOA

Fig. 70. — Diagrammatic Figure of Parame-
cium caudatum ( x ca. 500). (From Minciiin,
1912, AFTER Lang.)

P., Peristome groove; o, mouth; ces., oesophagus with
undulating membrane; f.v.', food vacuole forming
at end of oesophagus; /.r., other food vacuoles; c.v.,
contractile vacuole with surrounding channels lead-
ing to it; ex., excretory crystals; N, macronucleus;
74, micronucleus ; tm, trichocj'sts ; al., alveolar layer;
p., pellicle; um, undulating membrare

threads are discharged. A de-
finite mouth opening or cyto-
stome may, or may not, be
present.

Though the Ciliophora agree
with one another in the posses-
sion of cilia, they differ funda-
mentally as regards their nuclei.
In what may be regarded as the
more primitive forms (Opali-
nata) there are present in each
individual two or more nuclei
which are all of one type, in
which respect an approach to
the Plasmodroma is made.
When syngamy occurs uninu-
cleated forms are produced, and
these, which are gametes, unite
in pairs, with complete fusion of
the bodies and nuclei. In other
forms there are typically two
morphologically distinct nuclei,
one of which is a macronucleus
and the other a micronucleus.
During syngamy the macro-
nucleus disintegrates and takes
no part in the process, while
the micronucleus divides. Two
individuals associate, and one
of the daughter micronuclei of
each individual migrates into
the other and unites with its
remaining daughter micronu-
cleus. When this has taken
place, the associated or con-
jugating individuals separate
and continue to lead an inde-
pendent existence. On the basis
of this distinction Metcalf (1918)
recognizes two groups, the
PROTOCILIATA and the
EUCILIATA. The members

PLASMODROMA AND CILIOPHORA 159

of the group Protociliata (OPALINATA) possess cilia during the whole of
their existence, whereas amongst the Euciliata certain forms (CI LI ATA)
constantly have cilia, while others (SUCTORIA) have them only in their
youngest free-swimming stages, which, however, soon attach themselves
to objects, lose their cilia, and develop suctorial tentacles.

Multiplication amongst the Ciliophora is by binary fission or bud
formation. Amongst the multinucleated Protociliata nuclear division
proceeds somewhat irregularly, and division of the body leads to the
production of two daughter multinucleated individuals, which may, or
may not, possess an equal number of nuclei. In the case of the Euciliata,
which typically possess one macronucleus and one micronucleus, both
these nuclei divide, so that each daughter individual possesses a pair of
nuclei similar to that of the parent.

From the foregoing remarks it will be seen that the phylum Protozoa
may be subdivided as follows:

A. SUB-PHYLUM: PLASMODROMA DOFLEIN, 1901.—
Movement is effected by pseudopodia or flagella, and syngamy, where it
is known, takes place by the complete fusion of gametes.

I. CLASS: RHIZOPODA VON SiEBOLD, 1845.— The predominating
phase is amoeboid, locomotion being effected by means of pseudopodia.

II. CLASS: MASTIGOPHORA Diesing, 1865.— The predominat-
ing phase is flagellate, locomotion being effected by means of flagella.

III. CLASS: CNIDOSPORIDIA Doflein, 1901.— Parasitic forms
which are frequently amoeboid, but which produce characteristic spores
provided with polar capsules from which long filaments can be extruded.

IV. CLASS: SPOROZOA Leuckart, 1879.— Parasitic forms which
reproduce typically by schizogony, and which give rise to sporozoites
enclosed in resistant oocysts after syngamy has occurred.

B. SUB-PHYLUM: CILIOPHORA DOFLEIN, 1901.— Move-
ment is effected by means of cilia.

GROUP 1: PROTOCILIATA Metcalf, 1918.— There are two or more
nuclei, which are all of one type. Syngamy is effected by the complete
fusion of uninucleated gametes.

I. CLASS: OPALINATA.— With the characters of the group.

GROUP "1: EUCILIATA Metcalf, 1918.— There is a definite nuclear
dimorphism, the nuclei being of two types (macronuclei and micro-
nuclei). When syngamy takes place the macronuclei disintegrate, the
micronuclei alone taking part in the process, which is characterized
by the exchange of the products of division of the micronuclei between
two temporarily associated individuals.

160

CLASS: RHIZOPODA

Cilia are present throughout

I. CLASS: CI LI ATA Perty, 1852
the life of the organism.

II. CLASS: SUCTORIA Claparede and Laghmann, 1858.— Cilia
are present only during the young stages, which usually attach themselves
to objects, lose their cilia, and develop suctorial tentacles,

A. SUB-PHYLUM: PLASMODROMA.

I. CLASS: RHIZOPODA V. SlEBOLD, 1845.

CLASSIFICATION.

CLASS: RHIZOPODA

Order: AMCEBIDA

Family: AMCEBID.^:.

Genibs : Amoeba.

,, Hartmannella.

,, Vahlkampfia.

„ Sappinia.

„ Pelomyxa.

„ Entamoeba.

„ Endamoeba.

„ Endolimax.

„ lodamoeba.

„ Dientamoeba.

Family: PARAMCEBID^.

Genus : Paramoeba.
Family: DIMASTIGAMCEBID^.

Genus : Dimastigamoeba.
Family : RHIZOMASTIGID^.
Genus : Mastigamoeba.
Mastigella.
,, Mastigina.
Order: HELIOZOA

RADIOLA.RIA
FORAMINIFERA
Genus : Chlamydophrys.
Order : MYCETOZOA

The Protozoa belonging to the class Hhizopoda (= Sarcodina Hertwig
and Lesser, 1874) are typically organisms which move, and ingest food
by means of pseudopodia. These are cytoplasmic processes of varying
form which are protruded from the surface of the body, and which,
after fulfilling their function, are withdrawn. They may be merely
short, stumpy elevations, or more elongate finger-like processes (Fig. 5).
Sometimes they are very fine, and give the organism a radial appear-
ance. Such radiating pseudopodia, seen typically amongst the Heliozoa,
may be supported by stiff axial fibres, which cause them to be more
permanent structures (Fig. 71). There may be but a single pseudo-
podium, another one being protruded only when the first has been
withdrawn; several may be developed at one time, or large numbers are
produced simultaneously from the whole surface of the body. In the
latter case, anastomoses may be formed between adjacent pseudopodia,
so that the organism has the appearance of being surrounded by a loose
network of cytoplasm. They may be shorter than the diameter of the body,
or many times this length. The cytoplasm may be differentiated into a

ORGANIZATION OF RHIZOPODA

161

superficial clear hyaline layer, the ectoplasm, and a more granular fluid,
endoplasm. A pseudopodium may be formed of ectoplasm alone, or it
may have a core of endoplasm. Within the endoplasm are to be found
the nuclei, food vacuoles, and various granules, while contractile vacuoles
are present in the forms which are not parasitic.

In some Rhizopoda (Foraminifera) the ectoplasm secretes a protective
shell known as a theca, which covers the
body almost entirely (Fig. 72). A pore is left,
through which pseudopodia are protruded,
to enable the organism to move about and
secure its food. In addition to the main open-
ing, the shell may be perforated by niimerous
minute pores. Shells of this kind may be
formed when the organism is only partially
grown, and with increase in size a new and
larger shell is made. With further growth
others still larger are produced, and these,
remaining attached to one another, give rise
to many chambered shells, the separate
sections of which are variously arranged
according to the particular species. The
Radiolaria have a perforated membranous
central capsule, which divides the cytoplasm
into a central mass in which the nucleus
lies, and an extracapsular portion or mantle.
In the latter siliceous skeletal structures
of various kinds are developed. These take
the form of shells or spicules, which are often
conspicuous for the beauty of their design.
Whatever may be the character of the organ-
ism, the predominating phase in develop-
ment is one which produces pseudopodia,
and in the majority no other phase is
known to exist. In some, however, a
transitory flagellate phase occurs, during
which the organism resembles in every
respect a member of the class Mastigophora. On this account it is
exceedingly difficult to define accurately the limits between the two
classes Rhizopoda and Mastigophora. In the latter the flagellate phase
is the predominating one, while in the former it is the pseudopodial or
amoeboid phase. It has been demonstrated in the case of certain organ-
isms (DiniasdtfdiHdha) that the ama'boid or flagellate phase can be pro-

I. 11

Fig. 71. — Actinosphceyiuni eich-
horni : An Entire Individual
( X 90) AND Portion of An-
other ( X 360). (From Lan-
kester's Treatise on Zoology,
after Leidy, 1879.)

c.y.i,Contractile vacuole ;c.«.o, position
of another contractile vacuole which
has just collapsed; cr.fndd vacuole;
r., rotifer just engulfed; y/.v , pscudd-
Tiodium ; psa.,axis of pscutlupodiuni ;
N., nucleus.

162

CLASS: RHIZOPODA

diiced by altering tlie character of the medium in which the organisms
are growing.

I '/w n\MX wv'■

Fig. 72. — Types of Shelled Riiizopoda. (From Lang, 1901, A and B, after
Max Schultze; C, after R. Hertwig.

A. Gromia oviformis with ingested Navicula and seven nuclei ( x ca. 50). The

pseudopodia round the shell should be three times as long as represented.

B. Rotalia freyeri, showing spirally arranged chambers (x ca. 90).

C. Spiroloculina sp., showing four chambers and nuclei (x ca. 30),

In some instances {Mastig amoeba, Mastigina, Mastigella) the body
of the organism resembles an amoeba, in that pseudopodia are formed for

ORGANIZATION OF RHIZOPODA

163

the purpose of locomotion and ingestion of food, while a flagellum is present
as a permanent structure (Fig. 73). Such organisms, though usually placed
amongst the Rhizopoda, might with equal justification be classed with
the Mastigophora.

The majority of the Rhizopoda possess a single nucleus, which divides
only when multiplication occurs. In some cases, however, two nuclei are
present, while in others the organisms are multinucleate. Some of the
multinucleate forms (Mycetozoa) are relatively large, each consisting of
a sheet of cytoplasm (plasmodium) easily visible to the naked eye. Re-

\

m

- o

I.

M

'P

m 9j

m

m^

Fig. 73. — Mastigina hylw : Free and Encysted Stages ( x 1,
(After Collin, 1913

1. Free amoeboid form with four nuclei, to one of which a flagelkim is attached.

2. Encysted form with two nuclei. 3. Encysted form with four nuclei.

production amongst Rhizopoda usually takes place by binary fission, or
simple division into two more or less equal parts. In association with
encystment, when a protective capsule is formed around the organism,
the single nucleus, by repeated divisions, may give rise to a number of
nuclei, and the multinucleate cytoplasmic body within the cyst then
segments into a corresponding number of daughter individuals. The
latter may be amoeboid organisms, like the adults from which they were
derived, or they may be flagellated bodies which swim about for some
time before losing their flagella and again becoming amoebae. In the case
of some of the Foraminifera, there is a complicated life-cycle involving

164 CLASS: RHIZOPODA

an alternation of generations (Fig. 74). Thus, in Polystomella crispa,
a many-chambered shelled form studied by Lister (1895) and Schaudinn

u ^

^

>1

Fig. 74. — Stages in the Life-Cycle of Polystomella crispa ( x ca. 70). (After

Lang, 190L)

A. Young megalospheric individual with three chambers.

B. Fully-grown megalospheric individual.

C. Megalospheric individual in process of formation of flagellated spores.

D. Flcigellated spore more highly magnified. E. Fully -grown microspheric individual.
F. Microspheric individual in jirocess of formation of daughter amoeboid forms which become

megalospheric forms.
1-3, nuclei of various sizes; 4, fragmenting nucleus; 5, chromatin granules.

(1895), the individual (microspheric form) becomes multinucleate, and
then gives rise to a number of daughter amoeboid forms which escape
from the shell (Fig. 74, E and F). Each of these forms a relatively large

ORDERS: AMCEBIDA AND HELIOZOA

165

shell, and grows into a many-chambered individual of another type
(megalospheric form), while the cytoplasm within the shell again gives
rise, by multiple segmentation, to daughter individuals (Fig. 74, A to C).
In this case, each daughter form which escapes from the shell is provided
with two flagella, by means of which it swims about till it meets another
similar form which has been produced by another individual. Conjuga-
tion takes place, and the zygote, losing the flagella, becomes an amoeba,
which forms a small shell and grows into a many-chambered individual
of the first type (microspheric form). In the great majority of the
Rhizopoda, however, no sexual process has been observed.

The class Rhizopoda may be sub-divided into the five orders:
AMCEBIDA, HELIOZOA, RADIOLARIA, FOEAMINIFERA, AND MYCETOZOA.

1. Order: AMCEBIDA Calkins, 1902.
The body consists of cytoplasm unprotected by any shell or skeletal
structure, while movement is effected by the formation of pseudopodia
from any part of the body sur-
face. There is usually a single
nucleus, but some forms have
two and others many nuclei.
The cytoplasm is generally dif-
ferentiated into a softer and
vacuolated inner portion, the
endoplasm, in which the nucleus
and food materials lie, and an
outer, more hyaline, and clearer
layer, the ectoplasm. This order
includes the organisms which
are generally known as amoebae,
and to it belong the various
parasitic forms which occur in
the intestinal canal of man and
animals.

2. Order: HELIOZOA Haeckel,
1866.

Fig. lo.^Actifiophrys sol ( x ca. 600). (From

MiNCHIN, 1912, AFTER GRENACHER.)

N., Nucleus from which radiate the axial fibres of
the pseudopodia; ps., pseudopodia; ax., axial
; C.V., contractile vacuole;/.?;., food vacuole.

The forms included in this
order have a characteristic radial

appearance, the result of fine spiky pseudopodia (axopodia), which are
stiffened and rendered permanent by axial fibres. The latter may radiate
from a granule, probably centrosomic in nature, situated at the centre of
the organism, while the nucleus lies to one side of this (Fig. 51). The

166

CLASS: RHIZOPODA

Heliozoa are popularly known as sun animalcules, and are mostly found
in fresh water. Two common forms are Actinosphcerhwi eichhorni (Fig. 71),
which is multinucleated, and Actinophrys sol (Fig. 75), which has a single
nucleus. Both have been much studied from the point of their nuclear
divisions and pedogamy, as described above (p. 86). Members of the genus

Fig. 76. — Vampyrella later itia : A Single Individual at Different Stages of its
Attack on an Alga ( x 300). (After Cash, 1905.)

1. The free individual. 2. The same applied to the surface of the filament.

3. The filament has been broken, and one segment evacuated.

4. Later stage with four segments detached, two of which are evacuated.

VampyreUa are parasitic forms which bore their way into the cells of alga),
in which they live and multiply (Fig. 76). Another genus, Nuclearia,
parasitizes not only algse, but also other Protozoa.

3. Order: RADIOLARIA Haeckel, 1861.

The members of this order, like those of the preceding one, show
a tendency towards a radial arrangement of the pseudopodia, but
morphologically they are more complicated than the Heliozoa. Various
skeletal structures are commonly produced, while a perforated mem-
branous structure, the capsule, divides the cytoplasm into a central
intracapsular portion, which contains the nucleus, and an extra-

ORDER: RADIOLARIA

CK

167

„, /^vV/iV|||fi|i,,||\.\^.^\vxv^ p

Fig. 77. — Thalassicola x>elagica : An Inhabitant of the Ocean Surface Waters
( X 25). (From Gamble's Article in Lankester's Treatise on Zoology, 1909.)

CK, Central caj^sule; EP, extracai^sular cytoplasm; al, carbonic acid filled vacuoles (alveoli);
2>s., ji.seudopodia.

^S,

f

I

I

Fu;. 78. — Aeanthometra elastica ( x ea. 150). (From Minchin's Protozoa, 1912.)

'<}) , Radiating spines; ps., pseudopodia; c, calymma; ex., central capsule; N., nuclei; x, yellow
cells; 7111/., myophrisks (rod-like bodies).

168

CLASS: RHIZOPODA

capsular portion (Fig. 77). The skeleton may be in the form of radiating
spines, tangentially arranged rods, or definite fenestrated shells (Fig. 78).
The latter may be spherical, with perforations, and several such shells
may be formed concentrically, one within the other, as the animal in-
creases in size, or they may have a definite axis, and be shaped like a cone
or bottle. In many forms the cytoplasm contains peculiar yellow cells
about 15 microns in diameter. These are known as zooxanthellse, and
each is an independent vegetable organism possessing a cellulose wall and
containing a nucleus and chloroplasts. It is probable that they live in
a condition of symbiosis with the host. The Radiolaria are marine
organisms which are found floating on the surface of the ocean. Their
shells are found in large numbers in the deposits of the ocean bed.

4. Order: FORAMINIFERA D'Orbigny, 1826.
These Rhizopoda ( = Testacea Schultze, 1854) may be regarded as amoebae
which have the body protected by an external shell or theca. In the simplest
forms the shell has a single

opening, through which the or- \ V\^ ^ tYxXv

ganism protrudes pseudopodia ^ ^\Wv

for locomotion purposes and
the capture of food, very much

Fig. 79. — Arcella vulgaris, show-
ing Outline of Shell, Side
View of the Circulak Chro-
MiDiAL Body, and Two
Nuclei ( x 1.000). (Original.)

Fig. 80. — Glohigerina buUoides from Ocean
Surface Waters ( x 70). The Shells form
the Main Constituent of the "Globiger-
iNA Ooze " of the Ocean Bed. (After
Ehumbler, from Lister's Article in Lan-
kester's Treatise on Zoology, 1903.)

as a snail emerges from its shell (Imperforata Carpenter, 1862). Such
forms {Arcella, Difflugia, etc.) are very commonly found in stagnant water
(Fig, 79). The shells may be strengthened by adherent sand grains or other
material (Fig. 8). When reproduction is to take place the nucleus divides,
a portion of cytoplasm with one of the daughter nuclei is protruded through
the opening, a new shell is formed around this, and another shelled individual

OKDER: FORAMINIFERA

169

separated by division of the cytoplasm. In other cases, with growth of the
organism, a new and larger shell, which remains adherent to the original one,
is formed to accommodate it. A succession of new shells may be produced,
and these remain attached to one another in such a way as to give rise to
complicated compound shells which are constant in arrangement for any
particular species. In addition to the main aperture the shells may have
numerous minute pores, through which filose pseudopodia may be pro-
truded (Perforata Carpenter, 1862) (Fig. 80). Reproduction of the simpler
forms is by binary fission, while the more complicated types may show an
alternation of generations with the production of flagellated gametes, as
described above (p. 164). The Foraminifera occur either in fresh water or
in the sea. The simpler ones occur in
the former situation, while the more
complicated types are marine forms.
Chalk deposits are composed largely
of shells of marine Foraminifera (Fig.
81). Those which occur in fresh water
are often placed in a separate order,
Thecamoebida (Delage and Herouard.
1896), but there is no sharp line of de-
marcation between these and the true
marine Foraminifera. Some forms,
such as Chlamydojphrys, may pass
through the intestine of an animal in
the encysted condition, and emerge
from the cyst and develop their
characteristic thecse in faeces after
they have left the body.

Chlamydophrys stercorea Cien-
kowsky, 1875.— This shelled amoeba

is of interest, as it is commonly present in fseces of such animals as horses
and pigs, as well as frogs and toads. In the freshly passed faeces, it occurs
in the encysted condition which has passed through the intestine. If
the faeces are kept moist for a few days or planted on agar plates, the
amoebae emerge from their cysts and secrete a thin, egg-shaped, trans-
parent shell, which has a pore at its narrower end, through which the
organism protrudes pseudopodia (Fig. 82). There is a single nucleus with
a large central karyosome. Dobell (1909) gave the measurements of an
average-sized individual as 20 by 14 microns. The writer, who has
obtained cultures from frogs' faeces as well as from dirty water, has
observed forms which are much smaller than this, some of them being
barely 15 microns in length. The organisms readily encyst. If they

Pig. 81. — Shell of Nummulites cum-
mingii ( x 20). Portion of Wall
removed to show the chambers.
(From Lang, 1901, after Brady.)

170

CLASS: RHIZOPODA

have no shell, they merely become spherical and form a cyst; if they are
shelled, they escape from the cyst first. The cysts vary from 6 to 17
microns in diameter. Multiplication takes place by division of the
nucleus, followed by the extrusion, through the pore, of half the cytoplasm
into which one of the nuclei passes. A new shell is secreted round this
portion with its pore directed towards that of the original shell. Finally,
division of the narrow neck of cytoplasm uniting the two shelled individuals
takes place.

Schaudinn (1903) stated that the cysts of Chlamydophrys stercorea
passed through the human intestine, and that sometimes the amwbse
escaped from their cysts and multiplied while still in the intestine. He

Fig. 82. — Chlamydophrys stercorea feom Pigs' F^ces ( x 1,000). (Original.)

A. Ordinary individual. Clear area round nucleus is the chromidial body.

B. Process of binary fission: daughter individual being formed as a bud.

also made the statement that a supposed amoeba, Leydenia gemmipara,
which Ley den and Schaudinn (1896) had found in human ascitic fluid,
was no other than the free amoeboid stage of Chlamydophrys stercorea
which had wandered from the intestine to the peritoneal cavity. There
seems to be no evidence of this whatever, and as Schaudinn was unaware
of the existence of such parasitic forms as Endolimax nana, it is highly
probable that the amoebae he saw in the human intestine and regarded
as C. stercorea were in reality E. nana. As to the nature of Leydenia
gemmipara, there is no reason to suppose that it was anything more than
body cells in a degenerate condition in the peritoneal exudate.

Belaf (1921) has reviewed the genius Chlamydophrys, and concludes
there are six distinct species, which differ from one another in size, method

CHLAMYDOPHRYS STERCOREA

171

of nuclear division, and other details. C. stercorea, according to liim,
measures from 30 to 40 microns in length.

Noller, Krosz, and Arndt (1921) have cultivated from horse and pig
dung a number of thecamoebee belonging to the genera Chlcmiydophrys,

X,

Fio. 83. — Trinema acinus: A Shelled Rhizopod from Pond AVater ( x 2,000).

(Original.)

Fig. 84. — Cochliopodium bilimbosuni ( x 1,000). (After Leidy, 1879.)

Plagiophrys, Trinema (Fig. 83), Groniia, and Cochliopodium (Fig. 84).
Many of these forms multiply readily on agar plates. If pigs' faeces are

172

CLASS: KHIZOPODA

kept moist in a Petri dish for some days, many of these forms appear along
with other coprozoic Protozoa.

Fig. 85. — A PortioiN ui- a LAitGii Plasmodium, possuily a Spkciks of Badhamia,

WHICH WAS GROWN ON AN AGAR PlATE. (ORIGINAL.)

A. General appearance under low magnification ( X 16).

B. Small portion more highly magnified, showing numerous nuclei and vacuoles with

inclusions ( x 1,000).

5. Order: MYCETOZOA De Bary, 1859.
The forms included in this order are characterized by a plasmodial
adult phase. The plasmodium is a large sheet of multinucleated cyto-
plasm which exhibits peculiar streaming movements associated with the

ORDERS: MYCETOZOA AND AMCEBIDA

173

production of branching and anastomosing pseudopodia (Fig. 85). . At
certain stages, portions of the cytoplasm become encysted in resistant
capsules (sporangia), which may be arranged on stalks (Fig. 86). In this
respect there is a striking resemblance to fungi, to which group the
Mycetozoa were originally thought to belong. The sporangia eventually
rupture, and may liberate flagellated organisms which, after a free-living
existence, assume the amoeboid form. By growth, accompanied by
nuclear multiplication, the large plasmodia are produced. The Mycetozoa
are terrestrial in habit, and are commonly found
on the moist surfaces of decaying wood and leaves,
or in similar situations. Some of them may be
grown on the surface of agar plates.

SYSTEMATIC DESCRIPTION OF THE ORDER
AMCEBIDA.

From the point of view of parasitology it is
chiefly members of the order Amoebida which
have to be considered. The vast majority of the
Rhizopoda are free-living organisms, and only a
comparatively small number are truly parasitic
and adapted to their hosts in such a way that
a free extra-corporeal existence does not occur.
The fact that many of the free-living non-para-
sitic forms are able to produce protective cysts
of a resistant nature to enable them to withstand
desiccation has led to some confusion
encysted forms are frequently eaten accidentally
by human beings or animals, and may pass un-
harmed through the intestinal canal. After
escape from the body in the dejecta, they may find themselves in an
environment which is favourable for further development. The amoebae
emerge from the cysts, and by active multiplication increase enormously
in numbers in a comparatively short time. In this way, erroneous
impressions as to their parasitic nature may be obtained. Care must
always be exercised to guard against the possibility of confusing
these coprozoic forms with true parasites. In the case of true
parasites, the only forms which survive outside the body are, as a rule,
the encysted forms, which remain quite passive and unchanged till they
are ingested by another host. The unencysted stages are present in the
freshly passed stool, and show a degeneration which becomes more marked
as the interval since their escape from the body increases. The non-

FiG. 86. — Badhaniia utri-
cularis. (After Lis-
ter, IN Lankester's
Treatise on Zoology,
1909.)

Such «• Group of sporangia ( X 12).

b. Cluster of spores (x 170).

c. Single sjjore.

d. Part of capillitium in in-
terior of sporangium (x 170).

174

FAMILIES OF THE AMCEBIDA

parasitic forms, which have passed through the alimentary canal in the
encysted state, are at the height of their free-living existence some time
after the escape of the cysts from the body.

In the order Amoebida are included a number of well-known free-
living amoebae, such as Amoeba proteus (Fig. 5) and Amoeba verrucosa
(Fig. 87). The majority are uninucleated, but some have tw^o nuclei
(A. binucleata), while others have many nuclei (Pelomyxa). In addition
to these larger forms, there are others which are smaller, and which are
of interest in that some of them are readily cultivated from the fseces of
man and animals, owing to the fact that their cysts are able to pass un-
harmed through the intestine. Such forms are known as coprozoic amoebae.

They have frequently been
referred to as Amoeba Umax, a
name given to an amoeba by
Dujardin (1841) which, accord-
ing to Dobell and O'Connor
(1921), is not now identifiable.
Some of the amoebae ascribed
to this species have been
shown to be the amoeboid
phase of the flagellated organ-
ism Dirnastigamoeba gruberi
mentioned below. Others
appear to be true amoeba?
which have no flagellate
stage. There are many species
which are difficult to identify
on account of their resemblance to one another. They differ in the character
of the cysts they produce, the method of nuclear division, and other details.
The order Amoebida may be considered as comprising the following
families:

1. Family: AMCEBIDA Bronn, 1859. — Amoebae which are not able to
form flagella.

2. Family: PARAMCEBiD^ Poche, 1913. — Amoebae which, in addition
to a nucleus of the usual type, possess an accessory body (Nebenkorper)
which, during division, divides with the nucleus.

3. Family: dimastigamcebidtE. — Amoebae which in the adult form are
able under certain conditions to form two or more flagella, by means of
which they progress as flagellates.

4. Family: RHIZOMASTIGID.E Calkins, 1902. — Amoebae which are pro-
vided with a single flagellum during the greater part of the free-living
existence.

Fig. 87. — Amoeba verrucosa ( x 300)
Cash, 1905.)

(After

GENERA: AMCEBA AND HARTMANNELLA 175

Family: AMCEBID^ Bronil, 1859.
In this family are included a number of free-living amoebao, and most
of the parasitic forms which occur in the intestine of man and animals.
Not many years ago all amoebae, including the parasitic forms, were
placed in the genus Amceha. It is now recognized that several distinct
genera are represented, but the group has not been sufficiently studied
to enable precise definitions to be given. Many of the smaller free-living
forms which were grouped under the name Amceha Umax have been placed
in the genera Hartmannella, Sappinia, Vahlkampfia, which can be identi-
fied by the type of nuclear division and other details, while the parasitic
amoebae have been separated into the genera Entamceha, Endamceha,
lodamceba, Endolimax, and Dientamceha. The exact limits of the genus
Amoeba are doubtful, but the majority of the large free-living uninucleated
forms, such as Amoeba proteus and Amoeba verrucosa, which may have
a diameter of 500 microns or more, are regarded as belonging to it. Much
more information regarding the complete life-histories, the methods of
reproduction and encystment, and the details of nuclear division, are
required before the group can be satisfactorily defined.

Oemis : Amoeba Bory, 1822.
In this genus are included the vast majority of free-living amoebae.
In most cases they are placed in the genus because detailed informa-
tion regarding their structure and development is wanting. It seems
probable that future investigators will show that the only ones which
actually belong to it are the large free-living forms like Amoeba proteus,
Amoeba verrucosa. Amoeba vespertilionis, and Amoeba hydroxena described
by Entz (1912) as parasitic on Hydra oligactis.

Genus: Hartmannella Alexeieff, 1912.

The amoebae belonging to this genus are recognized by the character
of their nuclei and method of nuclear division. The nucleus is spherical,
has a large central karyosome, and peripheral chromatin in the form of
granules either on the inner surface of the nuclear membrane or in the
space between the membrane and karyosome. During division the
karyosome disintegrates, and a spindle is formed upon which definite
chromosomes become arranged as an equatorial plate (Fig. 88). The
nuclear membrane usually disappears at some stage of the division. The
cysts are spherical structures. The numerous species belonging to this
genus are distinguished by the details of nuclear division and the character
of the cysts.

Hartmannella hyalina (Dangeard, 1900).^ — This amoeba, which is often
found in stale fa>ces or in agar plate cultures made from dirty water,

176

FAMILY: AMCEBID^

faeces, or other material, has been referred to by various observers as
Amoeba hyaUna, a name given to it by Dangeard (1900). The generic
name Hartmannella was created by AlexeiefE (1912cf). The amceba
which was cultivated from human faeces by Musgrave and Clegg (1904)
in the Philippines, the one described by Liston and Martin (1911) in

m

0- A

(S^

Fig.

-Stages in the Nuclear Division of a Species of Hartmnnnella isolated
FROM Pigs' F.^ces {xca. 3,400). (Original.)

India as occurring in culture media inoculated with liver abscess pus,
and water, and the form growing on plates after exposure to the air,
as noted by Wells (1911), are probably this species.

The amoeba, when spherical, has a diameter of 9 to 17 microns. It
has a contractile vacuole, while the nucleus consists of a nuclear membrane

■■*■■■ . ' ■ . .^ -

Fig. 89. — Hartmannella hyalina ( x 2,000). (After Dobell and O'Connor, 1921.)

L Ordinary amoeba.

2. Division stage, showing pointed spindle with equatorial plate of chromosome.s.

3. Cyst with crinkled wall.

and large central karyosome (Fig. 89). Peripheral chromatin granules
occur on the nuclear membrane, and in the clear zone between it and the
karyosome. At the time of division the karyosome disintegrates, and
a spindle is formed, at the equator of which the chromatin, in the form

GENUS: VAHLKAMPFIA 177

of a ring of spherical chromosomes, is arranged. The nuclear membrane
disappears during the process, leaving a sharp, pointed spindle in the
cytoplasm. The spherical cysts measure from 10 to 14 microns in dia-
meter. They have a smooth inner wall and a much wrinkled outer one.
The amoeba does not multiply within the cyst, nor does its nucleus undergo
division. When grown on the surface of agar, it not infrequently happens
that amoebae with two or more nuclei encyst, in which case a correspond-
ing number of nuclei occur within the cyst.

There are other amoebse of relatively small size belonging to the genus
Hartmannella, which differ from one another in the details of their nuclear
divisions. Thus H. glehce-, described by Dobell (1914 a), is very similar
to H. hyalina (Fig. 56). The spindle formed during nuclear division has,
however, rounded ends instead of pointed ones. The cyst, moreover, has
a smooth outer surface. This form, or one closely allied to it, often occurs
coprozoically in faeces, and can be cultivated on agar plates (Fig. 56).
At the present time it is impossible to identify many of these coprozoic
amoeba?, but it appears that two fairly well-defined types commonly
occur — the one corresponding to H. hyalina, and the other to H. glebcp.

Genus: Vahlkampfia Chatton and Lalung-Bonnaire, 1912.
Vahlkampf (1905) studied the development of an amoeba, which he
designated Amceba Umax. The nucleus possessed a large central karyo-
some, and during multiplication the nucleus divided with the formation of
pole caps, as in the case of Dimastig amoeba gruberi (Figs. 61 and 90). A.
similar form was named Amoeba 'punctata by Dangeard (1910). Chatton
and Lalung-Bonnaire (1912) created the genus Vahlkampfia for amoebae
showing this type of nuclear division and possessing pores in the cyst wall.
Flagellate forms of the amoebae were not observed, but the conditions
necessary for the production of the flagellate forms were not provided
by them. Several other observers have described amoebae which show
nuclear division of the same type, and which have not been noted to give
rise to flagellate forms. It does not seem improbable that most, if not all,
of these forms would produce flagellate stages if the necessary conditions
existed. Dimastig amoeba gruberi remains as an amceba on agar plates, or
in cultures in egg-albumen water and other media, and does not become
a flagellate unless a sudden change occurs in the medium, as, for instance,
that produced by the addition of tap water. If this is done, flagellates
appear in three or four hours, but they revert to the amoeboid form again
in about a day (Fig. 120). It is probable that many of the amoebae which
have been placed in the genus Vahlkampfia would be capable of transforma-
tion into flagellate forms if they were similarly treated. It is possible, how-
ever, that some of them would not. Hogue (1921), for example, obtained a
I. 12

178

FAMILY: AMCEBID^E

culture of an amcBba, which she named Vahlkamjpfia patuxent, from the
stomach of oysters. Though its method of nuclear division resembled
that of Dimastigamoeba gruberi, she failed entirely to obtain flagellate forms,
though all the methods which cause Dimastigamoeba gruberi to develop
flagella were tried.

Calkins (1913) separated the amcebse which have this particular type
of nuclear division into two genera — viz., the genus VahUamjjfia, to include
the forms which do not develop a flagellate stage, and the genus Ncegleria
(created by Alexeieff, 1912) for those which have such a stage. The
latter forms, as pointed out by Alexeieff (1912a) really belong to
the genus Dimastigamoeba of Blochmann (1894), and will be considered

mm

• 6
Fig.

90.

7 8

Vahlkamiyfia punctata. (After Vaiilkampf, 1905.)

1, 2. Appearance of living amoeba and encysted form ( x 1,500 ?). 3-8. Stages in nuclear
division ( x 3,000 V).

below (p. 260), and as Chatton and Lalung-Bonnaire actually observed
markings which were undoubtedly pores on the cyst wall, it is probable
they were dealing with an organism belonging to the same genus. In
this case, both the names Ncegleria and Vahlkampfia are really synonyms
of Dimastigamoeba.

As already remarked, it is still doubtful if any of the ama>bfe having
the type of nuclear division of Dimastigamoeba gruberi are really incapable
of developing the flagellate stage. The majority, at any rate, have not
been investigated from this point of view. Most of these forms are free-
living amoebae, occurring commonly in damp soil or decomposing vegetable
material, but some of them have been found in the intestines of cold-
blooded animals. Others are to be regarded as coprozoic amwba?, as they

GENUS: VAHLKAMPFIA 179

appear in stale faeces, and have been cultivated from stools on agar plates.
A large number have been named, but it is very doubtful if these are
all distinct species.

Dangeard (1910) described as Atnceba punctata a form of this type
which had cysts with punctate markings. It was studied by Chatton
and Lalung-Bonnaire (1912), who obtained it from human faeces. They
placed it in a new genus as Vahlkampfia 'punctata. The punctate markings
strongly suggest the pores in the cysts of Diynastig amoeba gruberi.

Hartmann (1907a) gave the name Amceba froschi to an amoeba
showing the same type of nuclear division which he had seen in the faeces
of frogs, and the name Amoeba lacertce to a similar form in the intestinal
contents of lizards of the genus Lacerta. Both these forms were studied
by Nagler (1909). The form described by Dobell (1914a) as Amoeba
lacertce, which also occurred in the intestinal contents of lizards, differed
as regards the details of its nuclear division from the form studied by
Hartmann and Nagler. Hartmann (1914) accordingly renamed the form
studied by Dobell Amoeba {V ahlkaynpjia) dobelli. Caullery (1906) gave
the name Amoeba padophthora to an amoeba which parasitized the eggs
of the marine crustacean Peltogaster curvatus, while Chatton (1909)
described, under the name Amoeba mucicola, an amoeba which was parasitic
on the gills of a marine fish. Epstein and Ilovaisky (1914) gave the name
Vahlkampfia ranarum to a large amoeba, reaching 50 microns in diameter,
which they found in the intestine of frogs. Mackinnon (1914) saw an
amoeba, which she referred to as Vahlkampfia sp., in the intestine of the
larva3 of the crane-fly, Tipula sp. An amoeba, which was cultivated by
Whitmore (1911a) from human faeces, liver-abscess pus, and tap water
in Manila, and referred to as Amoeba liynax, was placed in the genus
Vahlkampfia as V. whitmorei by Hartmann and Schilling (1917). The
amoeba described by Porter (1909a) as Amoeba chironomi, from chiro-
nomous larvae, is possibly of the same type, though the nuclear division
was not described. Hogue (1921) recorded 7. patuxent from the stomach
of oysters in America.

In addition to the above-mentioned forms, which have a certain
association with higher animals, a number of free-living species have been
named. Nagler (1909) described Amoeba spinifera, A. lacustris, and
A. albida ; Aragao (1909), A. diplomitotica ; Glaser (1912), A. tachypodia ;
Belaf (1915), A. diplogena ; Jollos (1917), Vahlkampfia magna, V. debelis,
and F. sp.; and Hogue (1914), Vahlkampfia calkensi. Glaser (1912)
described the nuclear division of Ehrenberg's Avnoeba verrucosa as being of
the Vahlkampfia type. An amoeba first seen by Molisch (1903), and later
by Zacharias (1909), is parasitic on Volvox, while another. Amoeba bloch-
manni (Doflein, 1901), first noted by Blochmann (1886), is parasitic on

180

FAMILY: AMCEBID^E

Hwmatococcus. It is possible that both these forms, as well as the others
named above, should be included in the genus Vahlkampfia.

These various amreba? all agree with one another in that the nuclear
division, where it has been studied, is of the type first described by
Vahlkampf (1905), and it is highly probable that further investigations
will demonstrate, in some of them at least, the presence of pores in the
cyst wall and the occurrence of flagellate stages, in which case they wall
have to be transferred to the genus Diynastigamoeha. Meanwhile, however,
till more accurate data are forthcoming, it seems advisable to group these
amoebae under the name Vahlkampfia, which, as pointed out above, may
be a synonym of Dimastigamoeba, rather than to establish a new genus,
which will be necessary if they are finally proved to have no flagellate
stage.

Oeniis : Sappinia Dangeard, 189(5.

The amo'ba? belonging to this genus are peculiar in possessing two
nuclei, which are closely applied to one another. During division, both

Fig. 91. — Sax^pinia diploidea ( x 2,000). (After Dobelt. and O'Connor, 1921.)

1. Ordinary individual with two nuclei in apposition.

2. Cyst containing two individuals.

nuclei divide. When encystment takes place, two amoebae, each with
two nuclei, are enclosed in a common cyst. In the form S. pedata, studied
by Dangeard (1896 a), the free amoebae have the characteristic two nuclei.
The cyst, however, is peculiar in having a pedicle or stalk attaching it to
objects.

Sappinia diploidea (Hartmann and Niigler, 1908). — This is an amoeba
which was isolated by Hartmann and Niigler from lizards' faeces. Accord-
ing to Dobell and O'Connor (1921), it occurs rarely in human faeces, but more

GENUS: SAPPINIA— AMCEBiE OF PLANTS 181

commonly in that of animals, such as the ox and lizard. Hartmann and
Nagler (1908) gave it the name Amoeba diploidea, while Alexeieff (1912a)
placed it in Dangeard's genus Sappinia. The amoeba varies in size from
10 to 30 microns, possesses a contractile vacuole, and has a characteristic
thick pellicle, which is sometimes wrinkled (Fig. 91). It possesses two
nuclei which lie side by side in a central position. They are spherical, and
have large central karyosomes. The amoeba multiplies by binary fission,
the two nuclei dividing and producing two parallel spindles. The daughter
individuals thus have two nuclei. When the amoebse encyst, two in-
dividuals form round themselves a common cyst. According to Hartmann
and Nagler, the two nuclei of each amoeba now fuse. Each nucleus is then
said to give off reduction bodies, which degenerate, after which the cyto-
plasms of the two uninucleate ama?ba? unite. Their nuclei, however,
come into contact with one another, but do not fuse (Fig. 47). The amoeba
emerges from its cyst, and commences to multiply by binary fission as
before (see p. 82).

AMCEB^E OF PLANTS.

Franchini (1922 g, h, j, k, I) in a series of papers stated that he had found amoebse
in the latex of various plants. They occurred either alone or in association with
flagellates of the leptomouas or tryj)anosome type. The plants found infected were
Euphorbias, figs, and allied forms, as well as the lettuce, and were as follows:
Eiqihorbia vertirillatd,, Eiiphorhia nereifolia, Chlorocodon Whitei, Cryptostegia grandi-
Jhra. Stroph(nitliiis I!i<itili ;;iid N. s<-<ni<h-)iti, Acolcantliera venenata, Thevetias-p., Cerbera
Odollam,Fici(s IU'Uja))i'ni(i, Ficiix J'ierrei, Ficus Tholloni, Ficus carica, Ficusparietalis,
Antiaris toxicaria, Lakoocha artocarpus, Chrisophyllon sp., Labramia Bojeri, Tregtdia
Africana, Mimusops schimperi, Sideroxylon inerme, Lactuca sativa, Plumeria alba.

Cultures of some of the amcebse wore obtained by inoculation of blood-agar
plates (Noller's medium) with the latex of the plants, and in this medium most of the
amoebae were found to ingest red blood-corpuscles. In this way cultures were made
from Ficus carica, Chlorocodon Whitei, Cryptostegia grandiflora, Acokanthera venenata,
Plumeria alba, and the lettuce, Lactuca sativa. Three of these amoebae were named
Amoeba chlorocodonis. Amoeba cryptostegice, and Amoeba lactuca;. The descriptions of
the amoebse and the figures are such that it is impossible to form an opinion as to
their nature. It seems not improbable that the cultures obtained may have been
derived from amoebse or their cysts on the cuticle of the plants.

Further remarkable assertions are made by the author (Franchini, 1922 n) in
connection with the inoculation of kittens with cultures of amoebae from the plants
Acokanthera venenata and Plumeria alba. Kittens injected per rectum with cultures
of these amoebae were said to remain well for six to ten days, when they suddenly
became ill with dysentery, which persisted for about ten days. During this period
amoebse, some of which included red blood-corpuscles, were constantly present.
The animals recovered. The figures of these amoebae, again, are unrecognizable,
and cannot be distinguished from cells. It is further claimed that mice are suscep-
tible to inoculation with the amoebae cultivated from latex of Euphorbias, and that,
when these cultures contain trypanosomes and leishmania, as well as the amoebae,
a general infection is produced, and that all these organisms can be recovered by
culture from the heart blood. In another paper Franchini (1923) asserts that

182 FAMILY: AM(EBID^

mice fed ou the latex of the plants or injected intrapeiitoneally with cultures of the
amoebae and flagellates acquired liver abscess in which amcebse with included red
corpuscles occurred. These amoebae are said to reproduce by schizogony, and give
rise to forms like anaplasma, leishmania, and trypanosomes. The figures purporting
to show these forms are quite unrecognizable, and it is impossible to understand the
ai;thor"s conception of these Protozoa.

Genus: Pelomyxa Greef, 1874.

This genus includes certain large free-living multinucleate amoebae,
which may reach a diameter of 2 millimetres. They occur commonly in
stagnant water, and are easily visible to the naked eye. Each individual,
which may contain several hundred nuclei, moves slowly as it throws out
blunt pseudopodia (Fig. 92). The character of the cytoplasm varies with
the medium in which the organism is growing. It is usually much vacuo-
lated, and may be packed with small globules of a refringent substance.

Fig. 92. — Pelomyxa lyalustris : Ordinary Condition during Active Movement
(x 1,000). (After Cash, 1905.)

in addition to sand grains, diatoms, bacteria, and other objects, which
cause it to be peculiarly opaque. At other times the globules are absent,
and the cytoplasm is much clearer. Reproduction takes place by fission,
while gamete formation has also been described by Bott (1906). It is
supposed that uninucleated individuals are divided off, and that these
conjugate in pairs to give rise to zygotes, which grow into the multinucleated
adults (see p. 30).

Genus : Entamoeba Casagrandi and Barbagallo, 1895.

The members of this genus, which inhabit the intestine of higher
animals, vary in diameter from 5 to 40 microns. The pseudopodia are
usually blunt processes, and a new one is rarely formed before the previously
existing one is withdrawn. A central endoplasm can be distinguished
from a peripheral ectoplasm, while a contractile vacuole is not present.

GENERA: PELOMYXA AND ENTAMCEBA 183

The nucleus is spherical, and consists of a definite nuclear membrane, on
the inner surface of which the bulk of the chromatin of the nucleus is
distributed in the form of granules. There is a linin network, upon which
fine granules of chromatin may or may not occur, while a comparatively
small karyosome is present. Reproduction in the vegetative phase is by
simple binary fission, while transmission from host to host is effected by
means of encysted forms. The cyst is a transparent and smooth structure,
and the nucleus of the enclosed parasite, by repeated divisions, gives rise
to a number of daughter nuclei, which vary from four to multiples of four.
The encysted forms are passed out of the body of the host, and undergo
no change till they enter the intestine of another host, where the cyst wall
ruptures, and there is liberated either the multinucleated cytoplasmic
body, which then divides into a number of amoebae, or a number of amoebse
which have been formed before rupture of the cyst. It is not clear which
of these processes actually occurs. Each species of the genus tends to
produce a cyst which, when fully developed, contains a definite number
of nuclei. Thus, E. coli, E. muris, and other forms have eight, while
E. histolytica and E. rananim have only four. Occasionally, the nuclei
are in excess of the usual number. There may be sixteen or more in
E. coli, and more rarely eight in E. histolytica. The cysts of E. ranarum
may have a still larger number. This tendency to nuclear excess may occur
less commonly in the unencysted stages. In the case of E. coli unencysted
forms with eight nuclei have been described, but it is probable that these
were really irregularly-shaped encysted forms, the cyst walls of which,
in stained preparations, were not actually visible. Multinucleate free
forms of E. ranarum were described by Collin (1913), and similar s^^ages
were seen by Keilin (1917) in the case of E. ynesnili, and by the writer
in E. histolytica. It has been supposed by Mathis and Mercier (1917)
that the cysts with an abnormally large number of nuclei represent a
special type of multiplication by schizogony, while those with the normal
number are destined to give rise to gametes. They produced no con-
vincing evidence in support of this view. It is more probable that for
some reason the nuclear multiplication has continued beyond the usual
limits, possibly owing to excess of nutriment, or sometimes to an amoeba
having encysted just as it was about to divide in the free state.

This genus includes Entamoeba coli, the harmless amoeba of the human
intestine; E. histolytica, the pathogenic form producing amoebic dysentery
and liver abscess in man; E. gingivalis, an inhabitant of the human mouth;
and various species which occur as intestinal parasites of animals, such as
E. muris of rats and mice, E. pitheci and E. nuttalli of monkeys, E. bonis
of cattle, E. ovis of sheep, E. testudinis of the tortoise, E. ranarmn of frogs,
E. minchini of the larvae of Tipulid flies, and many other species. It is

184 FAMILY: AMCEBIDiE

probably safe to assume that practically every vertebrate animal, as well
as many invertebrates, will be found to harbour amoebae belonging to this
genus. In the great majority of cases they are of the non-pathogenic
variety, and in this respect resemble E. coli. In a few instances, forms
associated with dysenteric symptoms have been described from animals.
The various species resemble one another very closely, so much so that
in many cases they could not possibly have been regarded as distinct
species, apart from the fact that they occurred in different hosts.

There is some doubt as to the correct spelling of the name Entamceba.
The generic title was created in this form by Casagrandi and Barbagallo
(1895) for E. coli of the human intestine. Leidy (1879), however, had
given the name Endantceba blattcB to the amoeba of the cockroach. If this
form should prove to belong to the same genus as the human amoeba,
then Leidy 's name will have priority. As one of the cockroach amoebae
presents some peculiar features, it is better to regard it at present as
belonging to a distinct genus, Endamoeba (see p. 235).

entam(eb;e of man.

(a) Pathogenic Form.

Entamoeba histolytica Schaudinn, 1903. — Chief synonyms : "Amoeba coli"
Losch, 1875; '"Amoeba dysenteriae" Councilman and Lafleur, 1891; Amoeba coli
(Loscli) Kovacs, 1892; Amceba dysenterice (Councilman and Lafleur) Kovacs, 1892;
Entamceba dysenterice (Councilman and Lafleur) Craig, 1905; Entamoeba coli var. tetra-
gena Viereck, 1 907 ; Entamoeba africana Hartmann, 1907 ; Entamoeba tetragena ( Viereck)
Hartmann, 1908; Poneramoeba histolytica hiihe 1909; Eiitamoeba minuta JL\n\assia,n,
1909; Entamceba nipponica Koidznmi, 1909; Entamceba hartmanni Prowazck, 1912;
Loschia (Viereckia) tetrajsna Cliatton and Laluno-Bonnaire, 1912; Entamceba
brasiliensis AragXo, 1912; Liischia histolytica (Schaudinn) Mathis, 1913; Entamoeba
venaticum Darling, 1915 ; Entamoeba minuta Woodcock and Penfold, 1916 ; Enda-
moeba coli (Losch) Aragao, 1917 ; Endamoeba dysenterice (Councilman and Lafleur)
Pestana, 1917; Entamoeba tenuis Kuenen and Swellengrebel, 1917; Entamoeba
minutissima Bvng, 1917; Endamoeba histolytica (Schaudinn) Craig, 1917; Entamceba
coli communis Knowles and Cole, 1917; Entamceba paradysenterica Chaterjee, 1920;
Caudamceba sinensis Faust, 1923; Karyamoebina falcata Kofoid (and Swezy, 1924);
Entamceba disj^ar Brumpt, 1925.

Everyone who has studied the question is agreed that E. Jii.sfolytiea
was first seen and described by Losch (1875), and named by him " Amoeba
coli.'" Though this name was not correctly written in the original descrip-
tion given by Losch, it was employed for a long time for the amoebae of
the human intestine before it was fully realized that more than one species
existed. Similarly, Councilman and Lafleur (1891) proposed to call the
amoeba "Amoeba dysenteriae," another name which was not correctly
presented. Quincke and Roos (1893) and Roos (1894) were the first to
conclude that two types of amoeba occurred in man, the one an active

ENTAMCEBA HISTOLYTICA 185

form which produced cysts 10 to 12 microns in diameter and was pathogenic
to cats, and the other a less active form which produced cysts 16 to 17
microns in diameter and which did not give rise to infection in cats.
Kruse and Pasquale (1894) similarly described two forms, the one patho-
genic to cats and the other not.

Though Schaudinn (1903), in his account of the amoebse of the human
intestine, made many erroneous statements, he was the first observer to
appreciate clearly the fact that two distinct species exist, the one patho-
genic and the other harmless. Before this, the descriptions referred
sometimes to the one form and sometimes to the other, and often to
a mixture of both. In many cases it is only the association of the amoebae
with pathological conditions, and their occurrence in lesions of the intestine
and abscess of the liver, which are now known to be due only to invasion
of tissues by E. histolytica, that make it almost certain that some of the
earlier writers were actually dealing with this form. If recent investi-
gations had shown that both E. coli and E. histolytica were liable to invade
the tissues, then there would be practically no data whatever to enable
a decision to be made as to which of the forms the earlier writers were
referring. The experiences of the past few years have demonstrated
clearly that E. histolytica alone is responsible for the production of patho-
logical conditions, so that it is perfectly clear that the amoebae described
in the lesions of the intestine, liver, and brain by the earlier writers were
actually E. histolytica, though the descriptions of the amoebse themselves
were in most cases so imperfect that it would be impossible to identify
them. Though Losch, in his original description, expressed a doubt as
to the part played by the amoebae in the production of dysentery, his
really excellent figure depicts an organism which can hardly be any other
than that now known as Entamoeba histolytica.

If Schaudinn had recognized the fact that the amoeba, which Losch
called "Amoeba coli,'' was the pathogenic amoeba, and had given it the
name Entamoeba coli, endless confusion would have been avoided, but as the
matter stands at present there seems to be no alternative, unless further
confusion is to be caused, but to retain Schaudinn's name E. histolytica for
the pathogenic form and E. coli for the non-pathogenic one. The .whole
question of the nomenclature of the intestinal amoebae of man has been
reviewed very thoroughly by Dobell (1919), and readers are referred to his
book for more detailed information on this very intricate subject.

LIFE-HISTORY. — E. histolytica is to be regarded as a tissue parasite of
man, as first demonstrated by Koch and Gaffky (1887), and more clearly
by Kartulis (1885 and 1886). Infection is brought about by the ingestion
of encysted forms, first seen by Quincke and Roos (1893), which have been
passed in the faeces of some other infected person. Under the action of

186 FAMILY: AMCEBID^

the digestive fluids the cyst ruptures. From the work of Chatton (1917a),
who fed cats with material containing cysts, and that of Penfold, Woodcock,
and Drew (1916), who treated cysts with liquor pancreaticus, it appears
that it is the secretions in the small intestine which cause the cyst wall
to dissolve.

Chatton stated that the cyst liberated a four-nucleated amoeba, while
the other observers merely noted that a single amoeba escaped from the
cyst. Whether this happens in the human intestine or not cannot be
stated. Dobell and Stevenson (1918) and the writer have failed to bring
about any escape of amoebae from cysts by means of liquor ]Kmcreaticiis.

From experiments on cats, there can be no doubt that human beings
are infected by the ingestion of cysts. Whatever may be the exact method
of escape of the encysted amoebae and their development after this, it is
a fact that invasion of the intestinal wall by the amoebae quickly takes
place. In the earliest condition the amoebae make their way into the
glands of the large intestine, and crawl to the bottom of these. Here they
multiply, and partly by pressure, and possibly by the secretion of a toxin,
the gland cells degenerate and separate from one another. By this time
the tubule of the gland has probably become blocked, and if the adjacent
glands over a small area of surface are all similarly involved, as is usually
the case, a slightly raised yellowish nodule is produced. Meanwhile, the
amoebae have made their way into the interglandular connective tissue,
and a certain amount of necrotic material from broken-down cells has
collected. In this condition the yellow nodule is in reality a small
amoebic abscess of the mucosa. Very soon this abscess bursts into the
lumen of the intestine, and the contents are discharged, with the result
that a small undermined ulcer is formed (Figs. 93, 94). The amoebae which
thus escape invade other glands, causing the condition to spread, or they
are passed in the faeces with a certain amount of blood and mucus, which
represents the discharge from the abscess. The infected portions of the
intestine may be very limited, so that only a few scattered nodules are
formed, or there may be a more or less continuous infection of all the
glands. After rupture of the primary abscess the ulcer so formed becomes
gradually larger, the amoebae multiplying in the base of the ulcer and
extending over a wider area. They break through the muscularis mucosae,
and extend into the submucous tissues, producing eventually ulcers which
may reach an inch or more in diameter. These ulcers, like the small ones
originally formed, have undermined edges, and become filled with mucoid
material, debris of cells, and amoebae. It is probable that the plugs of
mucus admixed with blood, which occur in the stools of amoebic cases,
represent the evacuations from these ulcers. These masses of mucus
may contain enormous numbers of amoebae.

ENTAMCEBA HISTOLYTICA

187

At any time the discharged amoebae may infect fresh areas, so that
the large intestines of these cases show every stage in the formation of the
ulcers, from the smallest yellow nodules to large, undermined ulcers.

Ks,^

M

G

■uc~-

^

U

A

Fig. 93. — Amcebic Ulceration: Section of a Small Ulcer in a Human Large
Intestine ( x 30). (Original.)
The area marked by a square is shown under higher magnification at Fig. 94.

The best pictures from a histological point of view are obtained by
cutting sections of the small nodules which have not yet become subject
to secondary bacterial invasion, while at post-mortem examinations the
contents of one of these small nodules will often show still live and active

188 FAMILY: AM(EBID.E

amoebae which cannot be obtained so readily from the larger ulcers.
After the amoebae have multiplied and caused an extension of the ulcer
for some varying period a healing process sets in, the amoebae disappear,
and the site of the ulcer is finally represented by a puckered scar of fibrous
tissue, while the peritoneal surface opposite it may be considerably
thickened. In other situations, however, the process of invasion and
ulceration is still continuing, and this affords an explanation of the
persistence of infections with E. histoJytica, which are known to last for

^

3

o ♦

O

''1

Fig. 94. — Eniamceba histolytica in Tissue of Human Large Intestine ( x 500)

(Original.)
The area is shown in the square at Fig. 93.

many years, if not a lifetime, unless eradicated by suitable treatment.
In their extension through the tissues of the intestinal wall the amoebae
not infrequently make their way into blood-vessels, and are carried as
emboli to the liver, spleen, brain, or other organ, where they continue to
multiply and give rise to the well-known amoebic abscesses.

In an infected individual, if the amoebae are multiplying rapidly and
invading one portion of the large intestine after another in quick succes-
sion, the discharge from the ulcers is considerable, and much blood and
mucus will appear in the stool, which becomes of the characteristic

ENTAMCEBA HISTOLYTICA 189

dysenteric type. If the extension is not rapid, then only occasional plugs
of mucus, which may or may not be contaminated with blood, are passed,
and the individual may be quite unaware of his condition. The cases
of rapid extension are regarded as the acute ones, and the amoebae are
all of the large tissue-invading form, many of which contain red blood-
corpuscles. In other cases, where there is not rapid extension, though
a considerable area of the wall must be involved owing to the enormous
number of amoebae or their cysts which are passed in the faeces, a state
known as the " carrier condition " occurs. Exactly what happens in this
condition is not properly understood, for it is difficult to obtain perfectly
fresh post-mortem material from these cases. It is not possible to repro-
duce the carrier condition in animals, which always acquire an acute
infection which either terminates fatally or disappears. From what can
be observed in the stool, it is found that a smaller type of amceba occurs
in the faeces of carrier cases. These are in reality encysting forms (pre-
cystic amoebae), for in association with them are to be found cysts showing
one, two, or four nuclei. In some cases the precystic amoebae and the
encysted forms are passed together in the stool, while in others only the
amoebae or only the cysts are passed. This is probably dependent upon
the varying rate at which the large intestine evacuates itself. It seems
probable that the small amoebae arise in the ulcers by division from the
larger tissue-invading forms under certain conditions which may be
supposed to hinder their free and easy development. As a general state-
ment it can be accepted that Protozoa encyst when the conditions of life
are becoming unfavourable. The small precystic amoebae are formed
from the large ones which have become more superficial in position, and
it might be surmised that if the large amoebae which have escaped from
the tissues into the debris which fills the ulcer remain there for some time,
as they may be supposed to do in the slowly extending cases, no increase
in size occurs through lack of proper food, though they multiply and give
rise at each division to increasingly small forms. These amoebae, deprived
of their proper food, which is to be found only in the tissues in the deeper
parts of the ulcer, become encysted, and escape into the lumen of the
intestine when the ulcer discharges its contents. This discharge may
take place before actual encystment is complete, in which case the small
precystic amoebae will be found in the stool. In certain cases enormous
numbers of cysts are passed in the stool, and it must be supposed that
the process described occurs simultaneously at many parts of the intestine,
not necessarily in large evident ulcers, but in the very small superficial
ones which are not readily detected by the naked eye. The lesions in
these cases may be merely superficial, and, not being of an acute nature,
it is not surprising that certain individuals may be passing extraordinarily

190 FAMILY: AMCEBIDiE

large numbers of cysts without showing any symptoms whatever. In
view of the recent successful culture of E. histolytica by Boeck and
Drbohlav (1925) in egg media, it appears possible that the amoebse may
actually live and multiply on the surface of the intestine without giving
rise to any lesions. It may be that the infection of many symptomless
carrier cases is of this type, and that the precystic amoebae and cysts are
produced by amoebae living on the surface of the mucosa.

As regards the fate of encysted amoebae, there are two views. The
one which maintains that an amoeba which has once encysted in the gut
is unable to leave its cyst in the large intestine of the same host appears
to be in accord with the behaviour of parasitic Protozoa generally.
According to this view an encysted amoeba, in order to develop further,
must pass out of the intestine and be ingested by another or the same
host, so that the cyst may come under the influence of the digestive fluids
of the small intestine. A corollary to this is that if all the amoebae in
an individual could be induced to encyst, an automatic cure would result,
for all the cysts would have to be passed from the body. It is evident,
therefore, that it is just as incorrect to suppose that any case is resisting
treatment because of the impermeable cysts in the intestine as it would
be to conclude that a case of ankylostomiasis was not cured because the
eggs of the worm were too resistant. In the one case cure is effected
by killing the amoebae which produce the cysts, and in the other by killing
the worms which produce the eggs. In either case the presence of cysts
or eggs in the stool is an indication that the organisms producing them are
still present in the intestine, and that treatment has so far failed to kill
the organisms, and not that treatment has failed to kill the cysts or eggs.
According to the second view, though the majority of cysts must neces-
sarily escape from the intestine, some hatch in the large intestine before
they escape, so that the encysted stage can be regarded as a resistant one.
There is no evidence that this actually takes place in the large intestine
of man, though Sellards and Theiler (1924) have succeeded in infecting
kittens by injecting them per rectutn with material which they claim
contained only encysted forms of E. histolytica. Dr. Drbohlav informs
the writer that he has been able to confirm this observation, which has
been repeated by Hoare (1926). The writer has observed in stained
preparations cysts of E. histolytica which appeared to have ruptured and
to have developed hernia-like protrusions. It is just possible that this may
be a natural process, and represents the escape of amoebae from the cyst.

An individual who is in the carrier condition may at any time revert
to one of acute amoebic dysentery. An infected person frequently suffers
from periodic attacks of acute amoebic dysentery when only the large
tissue-invading forms are present in the stool. Between the attacks,

ENTAMCEBA HISTOLYTICA 191

when the acute symptoms have abated, the carrier condition maintains,
when precystic amoebae and cysts are passed. Certain individuals become
infected without suffering from acute dysentery, the infection being
detected only as a result of microscopic examination of the faeces. Those
who become carriers after acute attacks have been termed convalescent
carriers by Walker and Sellards (1913), and the others contact carriers.
Such carriers may remain infected for many years, probably for the rest
of their lives, without at any time being seriously troubled by their
infection. In this respect the infections with E. histolytica are very
similar to those produced by pathogenic bacteria.

As would be expected from the above account, the signs of an infection
with E. histolytica vary considerably. In the acute condition, if there
is extensive ulceration, the quantity of mucus and blood and the number
of amoebse passed in the stool may be considerable. The mucus is
generally of a brownish colour and the blood of a dark red tint. If
ordinary food has been continued, as is often the case, and the large
intestine has not been emptied of faecal matter, this will be present, and
mixed with the blood and mucus to a varying extent. It is not surprising
that the amoebae are found in largest numbers in the mucus which has
been discharged from the ulcers or from the irritated surface in their
immediate neighbourhood. In some cases where active multiplication
of amoebae is in progress over a large surface of the bowel, and food is
continually taken, the stool may be of a soft brown consistency, which
on first inspection appears to differ little from the normal. It will be
found, however, that there is an intimate mixture of faecal matter and
mucus in which large numbers of amoebae occur. Sometimes the stool
is more liquid and of diarrhoeic nature (amoebic diarrhoea), when careful
inspection will reveal small flakes of mucus in which numerous amoebae
may be found. Such cases may be due to superficial invasion of extensive
areas. In many cases it is impossible to decide whether the symptoms
noted are due entirely to the amoebae, or whether they are partly the
result of secondary bacterial infection of the already damaged tissues.
It would be expected that an ulcerated intestine, though producing no
symptoms, would be more liable than a healthy one to be irritated by
food or bacteria, and if diarrhoea results from such irritation it is difficult
to affirm that it is due to the amoebae, though many may appear in the
stool. It not infrequently happens that individuals who are undoubtedly
infected with E. histolytica rarely pass amoebae in the stools, so that many
examinations have to be undertaken before an absolutely certain diagnosis
can be made. In these cases inspection of the mucosa of the lower bowel
by means of the sigmoidoscope has yielded valuable information. Not
only can the ulcerated areas be seen, but scrapings from them will imme-

192 FAMILY: AMCEBIDiE

diately reveal amoebae even in cases which have proved negative after
many examinations of the faeces.

Some observers have attempted to discover a means of diagnosis in
the microscopic appearance of the cells in the stools of amoebic dysentery
cases, but apart from the amoebse there is nothing characteristic of the
condition. As a rule there occur a certain number of cells, including
degenerating epithelial cells, macrophages which have been discharged
from the ulcers, and some pus cells. They are usually present in com-
paratively small numbers, and it is only rarely that the stool contains
the enormous number of cells usually seen in acute bacillary dysentery.

Thomson, J. G. (1918), and Acton (1918) drew attention to the frequent
presence of Charcot- Ley den crystals in the stools of amoebic dysentery
cases, and the latter observer concluded that their presence was pathog-
nomonic of an infection with E. histolytica. Thomson, J. G., and Kobert-
son (1921 and 1921a) have published an account of observations which
tend to confirm the earlier conclusions. It is possible that Charcot-
Leyden crystals appear in any chronic ulcerative condition of the large
intestine, and that their association with E. histolytica is a result of the
amoeba being the most frequent cause of such a condition.

It has been noted above that E. histolytica may find its way to the
liver, spleen, or even the brain, and there give rise to abscesses. In these
situations the process of development is like that in the deeper tissues
of the intestinal lesions. Only the large amoebae are found, and there
seems to be no tendency to the production of the small, precystic amoebse
or cysts, which have never been demonstrated in these situations.

Wherever E. histolytica occurs in the tissues there is no tendency for
the area of invasion to be limited by the formation of fibrous tissue. On
this account the abscesses of the liver are not limited by a fibrotic wall,
as occurs in the case of chronic bacillary abscesses. If a section of the
wall of an amoebic abscess is examined, it will be seen that there is a
gradual transition from normal tissue to the completely necrotic area on
the surface of the abscess wall. The amoebse are found to be most
numerous in what may be called the intermediate zone. On this account
the examination of the pus which first discharges from an amoebic abscess
of the liver may reveal no amoebse. After a day or two, when apparently
the surface of the abscess is breaking away and being discharged, amoebse
may appear in the discharge in large numbers. These amoebse have the
same character as the larger forms found in the intestinal ulcers.

A number of records of the presence of amoebse in the urine have been
published. In the majority of cases these are more than doubtful, but
in one or two instances, as in the cases recorded by Walton (1915) and
Petzetakis (1923), it seems safe to suppose that the observers were actually

PLATE I.

Entamoeba histolytica (x looo) as seen in living condition in a portion of mucus from the
stool of a case of amcEbic dysentery. The mucus, in addition to the amoebae, contains
leucocytes and red blood corpuscles. Many of the amoebae show ingested red blood
corpuscles, from some of which the haemoglobin is diffusing into the cytoplasm, giving
a browinish tinge to the amcebae.

(Original.

[To fa

ENTAMCEBA HISTOLYTICA 193

dealing with amoebae which were of the E. histolytica type, and not with
tissue cells, which frequently lead observers astray. How the amoebse
gain access to the urine is not known, but it may be surmised that a
secondary infection of the urinary tract has taken place, and that amoebse
are discharged from the lesions into the urine, where, however, they
undergo degeneration more rapidly than after their discharge into the
lumen of the bowel from the intestinal ulcers. There is no reason for
regarding the urinary form as a species distinct from E. histolytica, though
Baelz (1883), who was the first observer to see amoebae in the urine,
proposed the name Amoeba urogenitalis.

Warthin (1922) observed E. histolytica in the vas deferens and the
lumen of the dilated tubules of the epididymis and testis. The patient,
a typical case of amoebic dysentery, died in spite of treatment which had
cleared the intestine of its infection. The amoebae were seen in section
of the tissues. They were mostly in clots of blood and fibrin in the lumen
of the dilated tubes, but in some places were invading the walls. They
were remarkable in that they had phagocyted not only red blood-corpuscles,
but also spermatozoa. Hines (1923) noted that a case of amoebic dysen-
tery suffered from enlarged and extremely tender seminal vesicles.
Seminal fluid expressed from the vesicles revealed typical active amoebae
with included red blood-corpuscles.

Petzetakis (1923 and 19236) in Alexandria describes amoebic bronchitis
in which, without actual abscess formation, the lungs appear to be in a
broncho-pneumonic state. There was no evidence of liver abscess, and
only certain cases gave a history of dysentery. The expectoration was
said to contain active amoebae, which in their movements, size, structure,
and included red blood-corpuscles resembled E. histolytica. Those cases
which were free from intestinal infection responded very readily to emetin
treatment. It is evident that these claims require confirmation. Libert
(1924) states that he obtained active forms of E. histolytica in a case of
hepatitis by means of the duodenal tube, an observation confirmed by
Boyers, Kofoid and Swezy (1925).

Several observers have recorded amoebic infections of the skin, but
in most cases there is little evidence that the structures described were
amoebae at all. Maxwell (1912) observed amoebae in fistulae about the
buttocks of cases in Formosa. In this instance it is not improbable that
amoebae had passed into the fistulae from the intestine. Engnian and
Heithaus (1919) gave a description and figures of what they regarded
as E. histolytica from ulcers on the skin of a case which was said
to have an intestinal infection. Judging from the figures and description
it is impossible to recognize the bodies as amoeba?, and it is evident the
authors have had little experience of these organisms. Kofoid and Swezy
I. 13

194 FAMILY: AMCEBIDiE

(1924 a) state, however, that they have examined the material from this
case, and can confirm the occurrence of E. histolytica in the skin lesions.
Furthermore, they claim to have seen another case showing the same
infection.

Smith, S. (1924) states that he has seen amoebse in pus from a knee-
joint, while Sharp and Morrison (1925) claim to have found them in pus
from abscesses in muscles.

MORPHOLOGY. — The morphology of E. histohjtica may be considered
under three headings corresponding with the three phases of. development
— namely, the tissue-invading form, the precystic form, and the cyst.

1. Tissue-Invading Forms. — These may be regarded as representing
the most active phase of development (Plate I., p. 192). They occur
normally in the walls of the intestinal ulcers and of the secondary lesions
produced in other parts of the body. They are to be found in the faeces
after discharge from the ulcers, in the pus draining from abscesses of the
liver and other organs, or in material coughed up after rupture of an
abscess into the lung. As has been pointed out by Dobell (1919), the
amoebae begin to degenerate soon after they have left the intestine, an
explanation of the many discrepancies which characterize the accounts
of the morphology of E. histolytica and the attempts at the establishment
of new species. Even when the amoebae are seen in perfectly fresh stools
within a few minutes of their escape from the body, changes may already
have occurred during their passage down the large intestine. It thus
happens that in most cases in actual medical practice a diagnosis has
to be made from forms which are abnormal, and which do not show the
true structure of the nucleus and cytoplasm of the amoebae as they appear
in the living tissues. Such alterations in character, however, do not
necessarily lead to the death of the amoebae, for kittens may be infected
by injection of material which was passed many hours before.

The tissue-invading form of E. histolytica as a rule varies in diameter
from 20 to 30 microns, but larger or smaller forms may occur (Fig. 95, 1-4).
A very characteristic feature of the amoeba is its activity, large,
blunt pseudopodia being formed and withdrawn in rapid succession.
Progression in one direction is effected by the formation of a pseudopodium
and the flowing of the entire cytoplasmic body into it. The pseudopodia
are often formed quite suddenly with almost explosive violence. The
remarkable activity of a group of these amoebae when seen in a freshly
passed and still warm portion of mucus can only be appreciated when
seen; no description can give a satisfactory picture of this really extra-
ordinary phenomenon. Not infrequently the amoebae become elongated
and glide in a slug-like manner over the surface of the slide without
noticeable change in shape. In so doing the posterior end may have

ENTAMCEBA HISTOLYTICA

195

/

\

s

'»

■■©;

Oi

Fig. 95. — Eniamceha histoJijtiea : Vegetative Forms (x 2,000). (Original.)

1. Large form with single nucleus, three included red blood-eorpiisclcs and two other bodies of
doubtful nature. 2. Large form with dividing nucleus.

3. Large form with two nuclei and food inclusions, possibly altered and swollen red blood-
corpuscles. 4. Large form with clear cytoplasm.

5. Form with spicular chromatoid bodies. 6. Four-nuclear forms from liver abscess pus.

7. Precystic form with large chromatoid body. 8. Precystic form with included bacteria.

9. Precystic form with clear cytoplasm. 10-11. Precy.stic forms of small race.

196 FAMILY: AMCEBIDiE

a ragged appearance, and to it bacteria and other debris may adhere.
This type of movement is common in certain free-living amoebas, and
from it the name " Umax " is derived. Though occasionally E. coli will
be seen to move with an activity almost, if not quite, equal to that of
E. histolytica, this is rarely the case, and the energetic movements of
E. histolytica serve as one of its most important distinguishing features.
When degeneration is advanced the movements become less evident, and
finally cease altogether, though very often evidently degenerate amoebae
will commence moving with remarkable activity when warmed on the
warm stage. In the formation of the pseudopodia the first indication is
a slight elevation of the ectoplasm, but as this increases in size the endo-
plasm quickly flows into it. A characteristic appearance of E. histolytica,
as seen in the stool, is that of an amoeba with a clear, broad, hyaline
ectoplasm sharply marked off from a granular endoplasm. Such forms
may be producing pseudopodia with great activity, and these frequently
consist entirely of ectoplasm. This extreme condition is probably the
result of degeneration. As E. coli does not produce appearances of this
kind, the marked ectoplasm of these altered E. histolytica serves as a dis-
tinguishing feature. In the perfectly fresh and normal individuals the
distinction between ectoplasm and endoplasm is much less marked. The
writer has examined portions of infected mucosa removed through the
sigmoidoscope. Though not more than one minute had elapsed after
removal, the amoebae could be seen actively motile within the pieces of
mucosa and forming ectoplasmic pseudopodia, as in the freshly passed
dysenteric stool. It hardly seems possible to regard the amoebae under
these conditions as being in any way degenerate. On the other hand,
the appearance of an amoeba with a relatively thick ectoplasm surrounding
a globular mass of granular endoplasm is undoubtedly due to a degenera-
tive change. The endoplasm has a ground-glass appearance, and, apart
from the nucleus and food vacuoles, contains, according to Dobell (1919),
numerous small granules which stain intra vita^n with neutral red. The
food vacuoles include red blood-corpuscles (Fig. 95, i), and sometimes
leucocytes or other cells in various stages of degeneration (Fig. 95, i).
Sometimes the whole endoplasm appears packed with red cells, and in
many cases, as these become dehsemoglobinized, the cytoplasm assumes
a yellowish tint. It is only rarely that other objects are ingested by the
amoebae. The number of amoebae in any particular specimen containing
red blood-corpuscles varies considerably. Sometimes as many as 25 per
cent, will show them, while in other cases a long search will reveal only
a single one, or none at all. Amoebae containing red cells may be found
in stools which do not show any blood or other abnormality on naked-eye
inspection.

ENTAMCEBA HISTOLYTICA 197

The writer and O'Connor (1917) noted the occasional inclusion of the
spores of a large bacillus, and on one occasion a large yeast-like organism.
E. histolytica is very fastidious about the kind of food it takes up, and in
the cases just mentioned, though many other structures were present in
the surrounding medium apart from the spores or yeasts, all the amcebse
had selected these particular objects for ingestion. The question arises
as to whether these were taken up by the amoebae before they were
discharged from the ulcers, as probably happens in the case of the included
red blood-corpuscles and other cells, or whether they were ingested during
the passage of the amoebae down the large intestine. E. coli, on the other
hand, ingests indiscriminately all kinds of objects in the intestine, but
apparently not red blood-cells, so that the presence of the latter in an
amoeba is strong presumptive evidence of its being E. histolytica. Occa-
sionally, however, undoubted E. histolytica, as seen in the stool, possess
vacuoles with included bacteria. These organisms are sometimes seen
in amoebae in sections of ulcers from human beings and cats when the
ulcer is invaded by intestinal organisms. This condition is only seen in
the superficial layers. Amoebae, both in faeces and in sections of intestinal
ulcers and liver abscess, especially in cats, may contain numerous irre-
gularly-shaped bodies which appear to be chromatoid in nature. Some-
times they bear some resemblance to Charcot-Leyden crystals (Fig. 95, 5).
In the cultures of E. histolytica, as described by Boeck and Drbohlav
(1925), the amoebae, when grown on egg media, feed largely on bacteria.
In blood media they ingest red cells also.

The nucleus of E. histolytica is a spherical structure 4 to 7 microns in
diameter. It consists of a fine membrane enclosing an alveolar substance
which in fixed material assumes the form of a network of linin threads,
some of which may be radial. At the centre of the nucleus is a small
karyosome surrounded by a clear area, the outer limits of which represent
the inner limits of the alveolar material. In fixed specimens, again, the
clear area appears in optical section to be limited by a ring of fine granules.
The small karyosome is homogeneous, and is said to consist entirely of
chromatin. Hartmann and others claim to have detected a centriole in
the karyosome, but it is very doubtful if such a structure exists. Chromatin
granules are arranged uniformly over the inner surface of the nuclear
membrane. In amoebae which have partially degenerated the nuclei
may have a very different appearance. The karyosome may appear larger
and be definitely excentric in position, while the chromatin on the
membrane may be distributed more irregularly in the form of several larger
masses. Amoebae with nuclei which do not conform to the type are
frequently encountered in perfectly fresh stools. The position of the
nucleus in the endoplasm varies considerably, and is subject to constant

198 FAMILY: AMCEBIDiE

change, as can easily be noted by observing living amoebse in which the
nucleus can be seen. Owing to the density of the cytoplasm and its high
refractive index, the delicate nucleus of E. histolytica is often difficult to
detect in the living amoebae. The nucleus of E. coli, on account of the less
dense cytoplasm, is more readily seen, while structurally it is very different
from that of E. histolytica.

E. histolytica mulitiplies in the tissues by binary fission. There is
first a division of the nucleus, the details of which have been described by
Dobell (1919), and this is followed by division of the cytoplasm into two
more or less equal parts. Kofoid and Swezy (1924a, 1925) state that there
are six chromosomes which appear during nuclear division (Fig. 57).
Reproduction by bud formation, as described by Schaudinn (1903), and
by a process of schizogony, as recorded by Job and Hirtzmann (1918),
are undoubtedly the result of observations on degenerate amoebse, or even
tissue cells. On one occasion the writer has observed amoebae with two
and four nuclei in liver-abscess pus (Fig. 95, 6).

2. Precystic Forms. — As already explained above, under certain con-
ditions E. histolytica becomes encysted, and as the cysts are smaller than
the tissue-invading amoebae, it is evident that before encystment smaller
amoebae are produced. These are probably developed from the large
amoebae by division, while the daughter amoebae, instead of increasing in
size as they do in the tissues, divide again, so that increasingly small forms
are produced. It is possible that the large amoebae, which have become
more superficial in position in the intestinal lesions, suffer from a lack of
fresh tissue or fluid nutriment on which to feed, so that after division
growth does not take place. This shortage of food may be the stimulus
which leads to encystment. The size of the amoebae which actually
encyst varies considerably, and evidence has been brought forward by
the writer and O'Connor (1917), and by Dobell and Jepps (1917, 1918),
that there exist definite races of E. histolytica which can be distinguished
from one another by the average size of the cysts. In the races with small
cysts these may have an average diameter of 7 microns only, while in
those with larger cysts it may be as much as 18 microns. It follows,
therefore, that the precystic amoebae may vary in diameter from 7 microns
upwards (Fig. 95, 7-11). There does not appear to be any evidence to
support the view that in those races with small precystic amoebae the
corresponding tissue-invading forms are smaller than in those producing
larger precystic amoebae.

The precystic amoebae have the same general structure as the tissue-
invading forms, but the cytoplasm is devoid of food vacuoles, the amoebae
having ceased to ingest red blood-corpuscles or other cells, and having
got rid of the remains of those taken in previously.

ENTAMCEBA HISTOLYTICA 199

The precystic forms of E. histolytica were first seen by Elmassian
(1909). He did not realize their nature, and, thinking he was dealing with
a new amoeba, gave it the name E. minuta. The name was employed
subsequently by Woodcock and Penfold (1916) for the smallest races of
E. histolytica, but Elmassian did not use it for the small race, of the
existence of which he was not aware, but for the one of average size which
everyone now admits is undoubtedly E. histolytica. Walker (1911), and
Walker and Sellards (1913), appear to have been the first to realize that
the small amoebae with clear cytoplasm were the precystic forms of the
large tissue-invading amoebae. This has been amply confirmed by many
observers. Shortly before encystment takes place the amoeba often
develops a vacuole containing glycogen, which colours brown with iodine,
as well as one or more refractile bodies. The latter, which often have the
form of rods with rounded ends, were named chroynatoid bodies by Dobell.
They show no marked affinity for iodine, but stain black with iron
hsematoxylin. It is very improbable that they are chromatic in nature.
They have well-defined edges, and are readily seen as greenish refractile
bars in the living amoebae or cysts. The margin of the glycogenic vacuole,
as stained with iodine, is not sharply defined, for it gradually shades off
into the surrounding cytoplasm. In the case of the cysts of lodatnoeba
butschlii, the substance in the vacuole is much denser than that in the
vacuole of the cysts of E. histolytica, and in iodine-stained specimens the
limits of the vacuole, or more correctly those of the glycogenic body
within it, are very sharply defined, the brown colour of the included
substance ceasing abruptly at the margin of the vacuole (Plate II., 5, 6, 9,
and 11-14, p. 250).

The nuclei of the precystic amoebae resemble those of the tissue-
invading forms, except that the chromatin on the membrane often occurs
in larger masses. In some cases the nuclei possess a single large crescentic
mass in addition to smaller ones. Dobell (1919) states that chromatin
granules occur also on the linin network, a condition which he does not
find in the normal nuclei of the tissue-invading forms.

The precystic amoebae are not so active as the tissue-invading forms,
and on account of the larger chromatin gratiules of the nuclei they may
be difficult to distinguish from the corresponding stages of E. coli. In these
cases it will be necessary to discover the characteristic cysts. The smaller
races are still more difficult to distinguish, as they may be confused with
Endolimax nana. The structure of the nucleus, as seen in stained pre-
parations, is important, and a final diagnosis may not be possible till
cysts have been found, it may be after repeated examinations on different
days.

The precystic amoebae and the cysts of E. histolytica were first

200 FAMILY: AMCEBID^E

accurately studied by Huber (1903), though the cysts had previously been
seen and figured by Quincke and Roos (1893), and Rods (1894), They
were again seen by Viereck (1907), and by Hartmann and Prowazek (1907),
who regarded them as belonging to distinct species of amoebae, which
were named E. africana and E. tetragena respectively. This supposed
difference, however, was the outcome of Schaudinn's erroneous account
of the development of E. histolytica, which was almost entirely based on
the appearances seen in degenerating amoebse. There can be no doubt that
E. africana, E. tetrageyia, and E. ttmiuta are merely forms of E. histolytica.
The small amceba described by Prowazek (1912a) as E. hartmanni, and
by Kuenen and Swellengrebel (1917) as E. tenuis, is undoubtedly a small
race of E. histolytica, producing cysts 6 to 8 microns in diameter.

3. Cyst. — The cyst which is formed round a precystic amoeba seems
to be composed at first of a soft material which quickly shrinks and
hardens to a resistant, colourless, smooth, transparent capsule. It is
completely filled by the cytoplasm of the amoeba (Fig. 96). The cyst
wall is about 0*5 micron in thickness, its inner and outer margin being
visible in optical section. When first formed, it encloses the amoeba
and the structures it contains. Thus, the newly-formed cyst contains
the cytoplasm and nucleus, and also the vacuole and chromatoid bodies
if these happened to be present in the amoeba. The cysts are generally
spherical, but they may be elongated or even dumb-bell-shaped. Within
the cyst the single nucleus divides to form two nuclei, and these divide
again, so that in the mature cyst four nuclei are present (Fig. 57). Very
frequently the four nuclei are arranged in pairs at opposite sides of the cyst.
On Very rare occasions eight nuclei may be found. According to Dobell
(1919) the vacuole, if present, gradually disappears as the cyst develops.
It appears as if the glycogen of the vacuole is used up during the nuclear
divisions. The chromatoid bodies are similarly absorbed while the cyst
is waiting outside the body to be ingested by a new host. The chromatoid
bodies usually have the form of rods with rounded ends, and very com-
monly one, two, or three are present in the cyst. They vary in length
from 5 to 10 microns, but longer or shorter forms may occur. Sometimes
they ^e of a different shape, and may be more rounded or irregular in
outline. On other occasions they are filamentous structures, or a large
number of small, irregularly-shaped bodies may be present. When seen
in the living cyst they appear as homogeneous structures which have
a refractive index higher than that of the rest of the cyst. On this
account they are readily distinguished. The chromatoid bodies are of
great diagnostic value, for they occur much more rarely in the cysts of
E. coli, in which case the eight nuclei characteristic of the mature cysts
of this amoeba will be noted.

ENTAMCEBA HISTOLYTICA

201

^><8A

■7

iTk

^

/4

i5

fZ

/J

'#

Fig. 96. — Entamoeba histolytica: Encysted Forms ( x 2,000). (Original.)

1. Form with one nucleus and vacuole. 2. Form with one nucleus and chromatoid bodies.

.3. Form with one nucleus, large vacuole, and chromatoid bodies.

4. Irregularly shaped form with single nucleus, large vacuole, and chromatoid bodies.

5. Form with dividing nucleus.

6. Binucleated form without vacuole or chromatoid bodies.

7. Binucleated form with chromatoid bodies.

8. Binucleated form with numerous chromatoid bodies.

9. Form with four nuclei and chromatoid bodies.

10. Form with four nuclei, chromatoid bodies, and vacuoles with inclusions.

11. Form with four nuclei and two chromatoid bodies. 12. Form with four nuclei alone.
13. Form with six nuclei, two of the original four having divided. Similarly, forms with eight

nuclei occasionally occur. 14-16. Forms belonging to a small race.

202 FAMILY: AM(EBID.E

The cyst of E. histolytica, when seen in fresh material, has a greenish
refractile appearance. Owing to its refractiveness, which is much more
marked than that of the cysts of E. coli, it is sometimes very difficult to
distinguish the nuclei, though the chromatoid bodies may be easily seen.
In iodine solution, however, all the contents can be clearly distinguished
(Plate II., 5-IO, p. 250).

The cysts of E. histolytica vary in diameter from 5 to 20 microns
according to the particular race, but all the cysts of any one race are not
of the same size. Thus, in six cases studied by the writer and O'Connor
(1917) the diameter of the cysts varied as follows: 7 to 9 microns, 7 to 11
microns, 10 to 13 microns, 10 to 14 microns, 11 to 15 microns, 12 to 18
microns. The cysts remained constant in their average size during the
observation, which in some cases extended over several months, so that
it would appear that true races are represented (Fig. 10). Some observers,
however, believe that the small cysts belong to a distinct species of
amoeba. Prowazek (1912 a) gave the name E. hartmanni to these forms,
Kuenen and Swellengrebel (1917) the name E. tenuis, and Brug (1917)
the name E. minutissima. Though the writer has repeatedly observed
the appearance of cysts in the stools of cases in which the acute symptoms
of amoebic dysentery were subsiding, these have always been of the average
size, or larger than this. In no case has he seen the small cysts appear
under these circumstances. It cannot be regarded as finally established
that the races of E. histolytica which produce the small cysts are able
to give rise to amoebic dysentery. Drbohlav (19256) has cultivated a
small race. In the cultures the amoebae resembled the typical
E. histolytica. They did not, however, ingest red blood-corpuscles, and
though producing infection in kittens, failed to give rise to dysentery and
ulceration of the large intestine. The precystic amoebae as seen in faeces
correspond in size with the cysts, so that they are smallest in those
races which produce the smallest cysts. There are no data, however,
to show whether a corresponding variation in average size of the
tissue-invading forms occurs. Shimura (1918) described a race of
E. histolytica with small cysts as a non-pathogenic race, but it has to be
remembered that the majority of carriers who show no symptoms are
passing cysts of the average size. If carriers alone were examined, the
average-sized cysts might with equal justification be regarded as belonging
to non-pathogenic races. In the case of the smaller-sized cysts, diagnosis
from living specimens may be very difficult unless the characteristic
rod-like chromatoid bodies are present. In iodine the details are much
clearer, but it is often necessary to prepare stained films before making
a final diagnosis.

The cysts passed from the body may contain one, two, or four nuclei.

ENTAMCEBA HISTOLYTICA 203

It sometimes happens that the majority of the cysts seen in a specimen
of fseces are in the uninucleate condition, while two and four nuclear
specimens may be very difficult to find. In other cases the majority of cysts
have four nuclei. There seems to be no regularity regarding the stage
of development in which cysts are passed, and this is not surprising when
it is remembered that the evacuation of the large intestine depends upon
the host, and may occur either before or after encystment has commenced.

The cysts of E. histolytica will remain alive for a considerable time after
leaving the body in faecal matter, but if brought into clean water they
will survive a much longer period. During this time the chromatoid
bodies gradually disappear. When the cysts die, various degenerative
appearances become evident, and it is found that the cysts will stain
immediately if brought into eosin solution. It appears that eosin solution
may be used as a test of the life of a cyst, as also of the free amoebae them-
selves. The live cysts or amoebae will not stain immediately, whereas
the dead ones will become red at once. This can readily be demonstrated
by watching the effect of eosin on cysts before and after heating to a
temperature sufficient to kill them.

In his description of the development of E. histolytica, Schaudinn
(1903) described a method of reproduction by bud formation. The
nucleus was supposed to give off chromatin material into the cytoplasm
in the form of granules, which collected in groups on the surface of the
amoebae. Small cytoplasmic buds, each containing a group of chromatin
granules, were formed. These buds were described as becoming enclosed
in very resistant capsules, forming spores, which were much smaller than
the cysts of E. histolytica, as they are now known. Schaudinn claimed
to have produced infection in cats by means of these spores after complete
drying, a procedure which is known to kill immediately the cysts of
E. histolytica. Eecent investigations have failed entirely to confirm
Schaudinn's statements, so that it is safe to conclude that the budding
process and spore formation as described by him do not take place.

PATHOGENICITY.— It has usually been assumed that any individual
who is harbouring E. histolytica must have definite lesions of the intestinal
wall, in the tissue of which the amoebae are living, but though this may
be true to a very large extent, the successful culture of the amoebae in
tissue-free media suggests that they may sometimes live on the surface
of the intestine without giving rise to actual lesions. In some cases the
lesions give rise to large quantities of blood and mucus, so that the acute
condition of amoebic dysentery results. In the great majority of cases,
however, very few, if any, symptoms are noted, so that the infection can
only be detected by microscopic examination of the faeces. The fact
that in some individuals the symptoms of acute dysentery occur, while

204 FAMILY: AMCEBID^

in others the infection is of a mild nature, may be comparable with what
is known to occur in bacterial infections. Many individuals harbour
pathogenic bacilli in their throats without having symptoms of the
disease which may be caused by these organisms. Invasion of the tissues
may occur because the resistance of the host is lowered or because the
virulence of the organism is increased. The former seems to be the most
rational explanation, and in the case of E. histolytica infections it would
seem that the resistance of the intestine is lowered from time to time,
with the result that active multiplication of the amoebae with their exten-
sion into the tissues takes place, so that acute dysentery supervenes. On
the other hand, it has to be remembered that the virulence of protozoa
may vary considerably. In the case of trypanosomes it is well known
that passage of a strain from one animal to another may so change it
that it will bring about death in a few days instead of a few months.
It is possible that the virulence of E. histolytica may vary in a similar
manner, but there is no evidence that this occurs. By quick passage
of a strain from one man to another the virulence might be so increased
that eventually every individual infected would acquire an acute and
fatal amoebic dysentery. Whether this actually happens in nature is not
known, but judging from the results of experiments on kittens the writer
can find no reason to suppose that the amoebae from carrier cases with few
or no symptoms are less virulent than those from acute cases. Brumpt
(1925), however, suggests that there exist two types of amoeba included
under the name E. histolytica, the one infective to kittens and the other
not. To the latter he gives the name Entamoeba dispar, and suggests that it
accounts for many of the carrier cases in countries where amoebic dysentery
is uncommon. The writer does not believe that physiological data of this
kind aiiord a means of distinguishing species.

SUSCEPTIBILITY OF ANIMALS.— Losch (1875) succeeded in infecting
a dog with E. histolytica, and this experiment was repeated by Hlava
(1887), Kruse and Pasquale (1894), Harris (1901), and Dale and Dobell
(1917). Young cats, however, are more easily infected, and it is with
them that most experimental work has been done. Hlava (1887) was
the first to produce infection in these animals, and he was followed by
Kartulis (1891), Kovacs (1892), Quincke and Roos (1893), Kruse and
Pasquale (1894), Marchoux (1899), the writer (1912(?), and many others.
Kruse and Pasquale also infected cats with the amoebae obtained from
liver abscess. The infection has generally been produced by injections
of dysenteric stools per anum, and this is the most reliable method for
infecting these animals. Unless cysts are present the animals cannot
be infected by feeding, a fact first demonstrated by Quincke and Roos
(1893), the first observers to describe the cysts of E. histolytica. Huber

ENTAMCEBA HISTOLYTICA 205

(1903), who rediscovered the cysts, confirmed this observation, which was
repeated by Kuenen and Swellengrebel (1913), the writer and O'Connor
(1917), and Dobell (1917), and others. The infection in kittens is, as a
rule, of a very severe type, the whole of the surface of the large intestine
being infected with amoebae. The infection usually commences at the
lower part of the large intestine, and it is here that the changes in the
mucosa are most marked. Sellards and Leiva (1923a) have shown that
this is probably due to the natural stasis which occurs at this point. By
ligaturing the large intestine of cats at various levels and inoculating
infective material directly into the csecum they have demonstrated that
the infection commences and is most marked just above the ligature.
They see in this an explanation of the fact that in human beings amoebic
ulceration is most marked at the point where stasis occurs. If the animals
live long enough, definite ulcers occur as in human beings, but frequently
all the glands are infected over the whole gut wall and death results from
the general necrosis of the mucosa which is set up. Sellards and Leiva
(1923a) have shown that bacterial invasion of the blood also plays a
part, for they have cultivated various intestinal organisms from the
blood of infected cats. As a method of diagnosis of amoebic infection
in the cat they have employed a daily saline enema. The fluid is quickly
returned, and the flakes of blood-stained mucus which it carries with it
can be examined for amoebae. Infected cats frequently pass -per anum
a whitish fluid containing many broken-down cells and enormous numbers
of amoebae. In less acute cases the stools resemble those of amoebic
dysentery in man, there being faecal material containing masses of mucus
stained with dark red blood. Eecoyery rarely takes place in cats, and
when it does the infection dies out, there being no carrier condition
corresponding to that in human beings. The cysts of E. histolytica are
never formed in cats. As in man, secondary infection of the liver may
take place, leading to the formation of liver abscess, an observation
Avhich was first made by Marchoux (1899), and subsequently by Craig
(1905), Werner (1908), "^Huber (1909), the writer {I9l2d), Dale and
Dobell (1917), Mayer (1919), and Sellards and Leiva (1923ff). Harris
(1901) noted a similar condition in an experimentally infected dog.

The infection in cats may be maintained indefinitely by injecting the
intestinal contents per rectum from one animal to another. The cats
must be only a few weeks old, as large, full-grown animals are more
resistant to infection. As the cysts of E. histolytica never occur in cats,
the infection cannot be handed on from cat to cat by feeding with intestinal
contents. The writer has never succeeded in infecting kittens by means
of material from liver abscess, in spite of the presence of active amoebae.

Guinea-pigs have been infected by Baetjer and Sellards (1914), and

206 FAMILY: AMCEBIDtE

by Chatton (1917, 1918rf). This may be accomplished by injections
per anum or by feeding with cysts per os. In these animals dysenteric
symptoms do not appear, but large tumours develop about the caecum,
and these are found to consist of overgrowths of the tissues due to the
amoebae, which grow and multiply within them. Huber (1909) claims to
have produced a chronic ulceration of the caecum in rabbits by feeding
them with cysts, while Lynch (1915) and Brug (1919a) claim to have
infected rats. Kessel (1923a) states that he has infected rats and mice
with E. histolytica. He finds (1923) that natural amoebic infections of
these animals can be excluded by the examination on two successive days
of faeces obtained after the administration of a purge in the form of stale
bread soaked in magnesium sulphate solution. To such animals cysts
of E. histolytica were given. The infections produced are of a chronic
nature, and persist for months. Free forms, as well as characteristic
cysts, could be obtained in the faeces of the animals after giving them
magnesium sulphate. The infection was handed on from rat to rat.
Chiang (1925) has also infected rats. The amoebae from the experimental
rats, as well as a naturally occurring rat strain {E. histolytica var. murina),
gave rise to typical infections when inoculated to kittens. Clean rats
kept with infected ones contracted an E. histolytica infection.

Attempts which have been made to infect monkeys have been incon-
clusive, owing to the fact that these animals are liable to natural amoebic
infections due to two species of amoebae which are very similar to E. histo-
lytica and E. coli. These animals suffer from amoebic dysentery, and
even amoebic abscess of the liver, as pointed out by Eichhorn and Gallagher
(1916) and others (see p. 226).

CULTIVATION. — Many attempts have been made to cultivate E. histo-
lytica in artificial media, but the only successful results are those of Cutler
(1918) and Boeck and Drbohlav (1925). Other observers have cultivated
only coprozoic amoebae. Cutler used two media.

The first was made as follows: The entire contents of an egg were
broken up by shaking in a glass bottle with beads. To the broken-up
egg 300 c.c. of distilled water were added, and mixture was effected by
shaking. The fluid was then brought gradually to the boiling-point in
a water bath, and kept at this temperature for half an hour. During the
heating the mixture was shaken, so that a fluid was obtained in which
minute egg particles were suspended. It was then distributed in quanti-
ties of 5 c.c. in test-tubes and autoclaved. Before use a few drops of
blood were added to each tube.

The second medium was prepared by boiling 500 c.c. of human blood-
clot for an hour in a litre of water. To the filtrate was added 0-5 per
cent, sodium chloride and 1 per cent, peptone. The fluid was then tubed

ENTAMCEBA HISTOLYTICA 207

and sterilized by steaming for twenty minutes on three successive days.
As in the case of the egg medium, a few drops of blood are added before
inoculation.

Attempts were made to cultivate amoebae from forty-five samples of
fseces containing E. histolytica, and amoebas were grown from six which
contained blood and mucus. Bacteria grew in the media as well as the
amoebae, and it was necessary to subculture every twenty-four to seventy-
two hours on account of the quantity of acid produced by the bacteria.
A temperature of 28° to 30° C. was better than a higher one, as bacterial
growth was reduced. Subculture was effected by transfer of 0-5 to 1 c.c.
of the culture. By this means cultures were maintained for over three
months, and not only did multiplication of the amoebae take place, but
encystment also occurred. Cats were infected by inoculation per rectum
with cultures of more than two and a half months' standing, and typical
dysenteric symptoms with amoebae resulted, while post-mortem examina-
tion showed the characteristic amoebic lesions, from which fresh culture
was obtained. Other animals were infected by feeding them on cultures
containing cysts. Dobell (1919) stated that he attempted without
success to cultivate E. histolytica by this method, and concluded that there
must have been some fallacy in Cutler's work. The writer also failed to
repeat Cutler's experiments. Barret and Smith (1923, 1924), however,
obtained cultures of another amoeba, Entamoeba barreti of the turtle,
Chelydra serpentina. The medium used was a mixture of human blood-
serum 1 part and 0-5 per cent, sodium chloride solution 9 parts. In
each tube 5 c.c. of the mixture was used. A small quantity of mucus
obtained from the intestinal wall was inoculated at the bottom of the
tubes, which were kept at 10° to 15° C, or at room temperature. At first
it was necessary to subculture every twenty-four or forty-eight hours,
but when a culture was established a weekly transfer was sufficient. Two
strains were kept for nine months, during which thirty subcultures were
made. The amoeba? multiplied actively, and corresponded in every way
with those seen in the intestine of the turtles. No cysts were found,
however. Cultures of E. ranarum of the frog have also been obtained.
These results, which were obtained with amoebae of cold-blooded hosts,
led Barret and Smith to suggest that Cutler may have been more successful
with E. histolytica than some had supposed.

Quite recently Boeck and Drbohlav (1925) have cultivated E. histolytica
on solid egg and blood agar slopes covered with Locke's solution containing
serum or egg albumin. From two human cases E. histolytica was isolated
and maintained in subculture for many generations, in one case for more
than eight months, during which 150 subcultures were made. Sub-
culture was made every two or three days, and the tubes were kept at

208

FAMILY: AMCEBID^

30° to 37° C. Bacteria were constantly present in the amoebse. In the
blood medium red blood-corpuscles were frequently ingested by the
amoebae, which structurally corresponded with E. histolytica. Even after
as many as ninety-three subcultures kittens could be infected with the
cultural forms, and a condition exactly like that arising from the injection
of material from cases of amoebic dysentery resulted. In a few instances
the animals developed amoebic abscess of the liver. Cultures were also
obtained from the infected kittens. On one occasion cysts were observed
in the culture tubes. Drbohlav (1925rt) has repeated these experiments,
which have also been confirmed by Thomson, J. G. and Robertson (1925).
ABERRANT FORMS OF E. HISTOLYTICA.— Working in North China,
Faust (1923) has observed in four cases of dysentery a peculiar type of
amoeba which ingests not only red blood-corpuscles, but also bacteria

(Fig. 97). The characteristic
feature of the organism,
which has a diameter of 16 to
17 microns when quiescent
and globular, is its posterior
end. When active it is
definitely elongated, with a
rounded anterior end and a
tapering posterior end which
terminates in a pointed pro-
toplasmic structure (caudo-
style), surrounding which are
sometimes several smaller

..,.r.. ,A -r, ,^-.o X K----K— — proiections.

000). (After Faust, 192.3.) ^ ^ ^ ^

Debris tends to become

adherent to the region of the caudostyle. The nucleus, measuring 3 to 4*5

microns in diameter, is always situated in the rounded anterior end of

the organism. On the inner surface of the nuclear membrane are minute

chromatin granules. The karyosome is a star-shaped structure which may

have a central vacuole. The rays consist of chromatin granules. In two

of the cases examined the infection was a pure one, while in the other two

cases E. histolytica occurred in one and E. coll in the other. Faust states

that there was no difficulty in distinguishing these amoeba? from other

species. Though the cases were followed for some time, no encysted stages

of the organism were seen. The amoeba appears to fix and stain badly,

as compared with E. histolytica or E. coli, which sometimes occurred in

the same sample of faeces. Owing to the fea'.ures described above, Faust

places the amoeba in a new genus as Caudamaeba sinensis. He believes

that it is a cause of amoebic dysentery. As regards the validity of this

Fig. 97. — " Candamceba sinensis " from the Human , ,

•2.000^. fAFTER Faust. 192.3.^ protoplasmic

Debris tends

Intestine (

ENTAMOEBA HISTOLYTICA 209

species it is difficult to form an opinion, as the encysted stages were not
seen. In any case there seems to be little ground for the creation of a
new genus. It has to be remembered, however, that undoubted E. histo-
lytica often move in a slug-like manner, as noted by Dobell and O'Connor
(1921), and that many free-living amoebae, as well as E. histolytica, may
develop the slug-like form with tapering posterior end to which debris
adheres, while other amoebae in the same pure culture move in the more
normal amoeboid manner. Whether Candamoeha sinensis is actually
distinct from E. histolytica future investigations alone will show, but it
seems to the writer that sufficient evidence to justify the distinction has not
yet been produced. Eecently the writer has had an opportunity of observing
E. histolytica in cultures. The assumption of a slug-like form with tapering
posterior end to which debris adheres is quite common. The fact that bacteria
as well as red blood-corpuscles occurred in vacuoles is a feature which may
be met with in undoubted E. histolytica. Schubotz (1905) has figured an
elongated form of E. hlattcB of the cockroach which bears some resemblance
to C. sinensis, while Jepps (1923) has described a somewhat similar form
of E. gingivalis, and Keilin (1917) one in E. mesnili (Fig. 109).

Chaterjee (1920) gave the name Entamoeba jmradysenterica to amoebae
which he found post-mortem in dysenteric lesions, and which he regarded
as a distinct species on account of certain peculiarities of nuclear structure.
The writer has seen preparations of this amoeba, which is unquestionably
a degenerate E. histolytica.

Kofoid and Swezy (19246) gave the name Karyamceha falcata to
an amoeba of the human intestine. As the generic name was preoccupied,
they (1925a) changed it to Karyamoebina (Fig. 98). The amoeba was
first described from three cases. The first harboured, in addition, E.
histolytica, E. coli, Endolimax nana, Dientamceba fragilis, as well as the
form described as CounciUnania lafieuri ; the second E. histolytica ; and
the third E. histolytica, E. coli, and C. lafieuri. Three further cases were
reported in their second paper. The chief distinguishing feature is the
nucleus and the method of nuclear division. The nucleus has a definite
membrane, upon which the chromatin is massed in one or two, rarely more,
crescentic clumps. There is an excentric karyosome round which is a halo.
In division the nucleus elongates, and there is formed at each end a deeply
staining pole cap. On this account the amoeba is supposed to be allied to
members of the genus Vahlkampfia (see p. 177). In Vahlkampfia, however,
the pole caps are formed from the divided karyosome, and it is definitely
stated that in K. falcata the karyosome does not divide. In this form the
pole caps are merely terminal aggregations of the large chromatin masses on
the nuclear membrane. On this account the amoeba cannot be allied with
Vahlkampfia. It is said that in K. falcata about twenty chromosomes occur
at the equator of the elongating nucleus. Cysts have not been observed.
I. ' 14

210

FAMILY: AMCEBID^

As pointed out by the writer (1925), from the fact that the cases from
which K. falcata was first recorded harboured E. histolytica also, w^hile two
of them had other amoebae as well, it seems that definite proof that the
so-called K. falcata is a distinct entity has not been produced. It is
known that in E. histolytica the nucleus not infrequently shows chromatin
arranged in crescentic masses, and it has yet to be demonstrated that
in nuclear division such nuclei never assume the form supposed to be
characteristic of K. falcata.

Of quite another nature are the supposed amoebae which Kofoid and
Swezy (1922) and Kofoid, Boyers and Swezy (1922) have described from the

r

'-■^f
.^--

-- - ■:-■:/

\

Fig.

98. — Free Forms of " Kanjamcebina falcata '" ( x 2,000).
Swezy, 1924, Slightly Reduced.)

(After Kofoid and

1. Form with clear pseudopodium and single crescentic body on nuclear membrane.

2. Nucleus with two crescentic bodies united by a fibre.

3. Dividing form: nucleus with pole caps, centrioles united by centrodesmose, and equatorial

plate of about twenty dividing chromosomes.

bone marrow of cases of arthritis deformans, and from the hypertrophied
lymphatic glands in Hodgkin's disease. Because of a particular type of
division exhibited by the nuclei of certain cells, it is concluded tliat they
are not only amoebae, but actually E. histolytica. It must be apparent to
most protozoologists that far more convincing evidence is required before
this view can be accepted.

(b) Non-Pathogenic Forms.

Entamoeba coli (Grassi, 1879) Casagrandi and Barbagallo, 1895. —
Chief synonyms: "Amcebse" Lewis, 1870; "Anioebse" Cunningham, 1871; Amoeba
coli Crassi, 1879; "Amoeba coli mitis" Quincke and Eoos, 1893; "Amoeba intestini
vulgaris" Quincke and Roos, 1893; Entamceba coli, Casagrandi and Barbagallo, 1895;

ENTAMCEBA COLI

211

Entamceba hominis Casagrandi and Barbagallo, 1897; Entamoeba coli Scliaudinn,
1903; Amoeba coli Brumpt, 1910; Entamoeba williamsi Prowazek, 1911; Entamceba
hartmanni Prowazek, 1912 (pro parte); Entamoeba brasiliensis Aragao, 1912 (pro
parte); Loschia coli Chatton and Lalung-Bonnaire, 1912 ; Entamoeba coli communis
Knowles and Cole, 1917 (^^ro parte); Endameba intestinivulgaris Aragao, 1917 ;
Endameba coli Craig, 1917; Endameba hominis Pestana, 1917 ; Councilmania lafleuri
Kofoid and Swezy, 1921.

This amoeba is a harmless commensal of the digestive tract of man,
and is in no sense a tissue-invading amoeba like E. histolytica. According
to Dobell, it was first seen by Lewis (1870) in India, and was described
more accurately by Cunningham (1871). Grassi (1879-1888) gave various
descriptions of the organism, and erroneously believing it to be identical
with the form originally studied in dysenteric cases by Losch (1875),
gave it the name Amoeba coli, a name which shovdd have been employed
for the pathogenic form only. As has been explained above, Schaudinn
again committed this error, and though,
according to the strict laws of nomen-
clature, E. coli should be the name of
the pathogenic amoeba, its employment
in this sense would lead to endless
confusion, so that it is better to retain
the name E. coli for the harmless amoeba.
Grassi realized that the amoeba was a
harmless inhabitant of the human diges-
tive tract, for he found it not only
in sick, but also in healthy people.
Quincke and Eoos (1893) gave a good
description of E. coli, which they dis-
tinguished from E. histolytica, while
Casagrandi and Barbagallo (1895, 1897)
studied the same organism, which they

named Efitamoeba coli. They took a retrograde step in assuming that
this was the only form which occurred in healthy as well as in dysenteric
subjects. Schaudinn (1903) clearly stated that there were two amoebse,
the one a tissue-invading form and the other a harmless commensal, and
his reputation as a protozoologist resulted in a universal acceptance of
this view, which had been previously put forward by Quincke and Roos.
Since Schaudinn's time numerous names have been given to amoebse which
are undoubtedly merely forms of E. coli. These have been fully discussed
by Dobell (1919), and it is unnecessary to enter into the matter here.

Entamoeba coli is a very common parasite of the human intestine. In
tropical lands, or in other countries where sanitary arrangements are not
satisfactory, it is probable that no person escapes infection. Like E. histo-

FiG. 99. — Entamoeba coli with In-
gested Cyst of E. histolytica
(x ca. 2,000). (After Wenyon
AND O'Connor, 1917.)

212 FAMILY: AMCEBID^

lytica it lives in the large intestine, but it does not invade the tissues.
It develops in the intestinal contents, especially on the surface of the
mucosa, where it feeds on bacteria, yeasts, and other material. It will
ingest cysts of other Protozoa, such as those of Giardia and Isosiwra,
and even the cysts of E. histolytica (Fig. 99). It does not appear to ingest
red blood-corpuscles in its natural habitat. In cases of bacillary dysen-
tery, when enormous numbers of red cells occur in the stool, E. coli may
sometimes be seen moving about amongst them, and showing no inclina-
tion to take them in. The writer has seen red blood-corpuscles adhering
to the surface of motile E. coli, which, however, showed no tendency to
engulf them. Lynch (1924) has, however, been able to induce E. coli
to ingest red cells by incubating them with blood in a test-tube. In the
writer's experience this never occurs in the intestine, and, if it does, it
must be such a rare phenomenon that the general rule given above, that
an amoeba with included red cells is almost certainly E. histolytica, still
holds for all practical purposes. Like E. histolytica, E. coli becomes
encysted in transparent resistant cysts, and it is these forms which
spread infection from one individual to another.

MORPHOLOGY. — E. coli may be considered in three stages: the adult
form, the precystic form, and the cyst.

1. Adult Form. — The fully-groAvn E. coli (Fig. 100) is on an average
larger than E. histolytica, and as usually seen it has a diameter of 15 to
30 microns. Occasionally very much smaller forms, under 10 microns
in diameter, occur. Generally, the amoebae are much less active than
E. histolytica, the movements being very sluggish. Occasionally, however,
the writer has seen undoubted forms of E. coli moving with a rapidity
comparable with that of E. histolytica. The ectoplasm is not so clearly
defined as in E. histolytica, and in the normal individual there is merely
a superficial layer which is clearer than the endoplasm into which it
merges. The degenerating forms of E. coli do not show the exaggerated
extension of ectoplasm which is such a characteristic feature of the
abnormal forms of E. histolytica. The endoplasm of E. coli is often
extensively vacuolated, and the vacuoles contain a great variety of
objects which are chiefly bacteria. The general appearance of the amoeba
is that of a slightly greyish object, which contrasts with the greenish tint
resulting from the high refractive index of the denser E. histolytica.
E. coli is much more fluid in consistency than E. histolytica. Sometimes
the amoebae show various fissures or rectangular vacuoles, which are
probably the result of degenerative changes.

1-3. Forms with vacuolated cytoplasm, including bacteria. 4. Binucleated form.

5-6. Forms with irregularly shaped nuclei and very coarse chromatin masses.

7. Form which has ingested a small binucleated cyst.

8. Form showing excentric position of karyosome.

9-10. Small individuals. 11. Large precystic form with clear cytoplasm.

ENTAMCEBA COLI

213

10 ^1

Fig. 100. — Entamceha coli : Vegetative Forms ( x 2,000). (Original.)

[For di' ■■script inn see opposite page.

214 FAMILY: AMCEBID^

The nucleus of E. coli is a larger and coarser structure than that of
E. histolytica, and is readily distinguished in the living amoeba on account
of the low refractive index of the cytoplasm. In stained specimens it is
seen to have a thicker membrane than the nucleus of E. histolytica. The
chromatin granules are coarser and the karyosome, when it is a single
compact granule, is larger, as also is the clear area around the karyosome.
Dobell (1919) states that the karyosome is nearly always excentric, and that
chromatin granules occur on the linin network between the clear area and
the nuclear membrane. The nucleus of E. coli thus differs from that of
E. histolytica chiefly in its coarseness, and as the nucleus of E. histolytica
quickly changes in character as a result of degeneration, it is very frequently
impossible to distinguish the two amoebae as they occur in the stool from
the appearance of their nuclei alone. The presence of a larger number of
food vacuoles containing bacteria and other objects is a more reliable means
of recognizing E. coli. It must be admitted, however, that it is very often
impossible to distinguish between E. coli and E. histolytica in the free con-
dition. In such cases search must be made for the characteristic cysts.
E. coli reproduces by binary fission, like E. histolytica. The details of
nuclear division have not been followed completely in the free forms; they
are very similar to those of E. histolytica. During the division of nuclei in
the cysts Swezy (1922) states that there are probably six chromosomes.
Several observers, including Schaudinn (1903), Casagrandi and Barbagallo
(1897), and Mathis and Mercier (1917), have described a process of schizo-
gony of E. coli. In stained films it is often very difficult to detect the
wall of a cyst, which becomes highly transparent in cleared preparations.
If such a cyst has an irregular shape, as is not infrequent in prepara-
tions, the appearance of an amoeba with eight nuclei is produced.
The writer has seen and marked such forms as possible schizogony or
multinucleate stages, but in all cases it has appeared more probable that
they were distorted or irregularly shaped forms which were really encysted.
There seems no reason to suppose that E. coli in the free condition repro-
duces in any other way than by binary fission.

2. Precystic Forms. — As in the case of E. histolytica, prior to encyst-
ment there are produced amoebae which are smaller than the adult forms
and have a cytoplasm cleared of all food materials (Fig. 100, ii). The
precystic forms of E. coli are very similar to those of E. histolytica, but
as the average size of the cyst of E. coli is greater than that of E. histo-
lytica, so the precystic amoebae are correspondingly larger. These pre-
cystic forms are probably formed by division of the larger individuals.

3. Cyst. — A cyst wall is secreted round a precystic amoeba which has
become spherical. The nucleus divides to form two nuclei, these divide
to form four, and the four divide again to give the eight nuclei charac-

ENTAMCEBA COLI 215

teristic of the mature cyst (Fig. 101). Occasionally, a further division
will take place, giving rise to sixteen nuclei. The cysts with sixteen
nuclei, though uncommon, are much more frequently encountered than
the eight-nuclear cysts of E. histolytica. Very rarely, cysts with a larger
number of nuclei occur. During the process of nuclear multiplication some
of the nuclei may cease to divide, so that an irregular number of nuclei
of unequal size may result. According to Dobell (1919), soon after encyst-
ment, a glycogen vacuole forms in the cytoplasm, and this reaches its
maximum development at the two-nuclear stage. After this it gradually
shrinks till at the eight-nuclear stage it has disappeared. In the writer's
experience the precystic amoebse themselves may possess a large vacuole
or a series of vacuoles which run together after encystment has occurred.
This vacuole, however, is not always present.

The cysts of E. coli vary in diameter from 10 to 30 microns. They
usually measure from 15 to 20 microns, but larger ones may occur, as
recorded by the writer and O'Connor (1917), who saw one measuring
38 by 34 microns. The commonest type of cyst met with in the stool is
one containing a clear cytoplasm in which are embedded the eight nuclei
(Fig. 101, 4 and 8).

In most cases there occur also a smaller number of cysts of a different
type. These are usually larger than the ones just mentioned, and have
a large central glycogen vacuole which reduces the cytoplasm to a thin
layer lining the cyst wall (Fig. 101, 10-12). There are usually two
nuclei, which generally lie at opposite poles of the cyst. They often
appear as if flattened against the cyst wall by pressure of the vacuole.
In other cases the vacuole is smaller, and there is a thicker layer of cyto-
plasm. The vacuole contains glycogen, which stains brown with iodine
(Plate II., 2, p. 250). More rarely cysts of this type may be seen with
four nuclei, and still more rarely with eight nuclei. A modification is
occasionally seen in which a series of vacuoles occurs round the periphery
of the cyst (Fig. 101, 14), while the cytoplasm with the two, four, or eight
nuclei may occupy its centre. In optical section such cysts have a cart-
wheel appearance. Dobell (1919) considers that the vacuole occurs in the
normal course of development, and reaches its maximum size at the two-
nuclear stage, and that it t