Full text of "Protozoa in biological research"

See other formats

hiPiby

rte vias
tireless

dab

dee

tevin
eps:

olen
Me

Phe
Phries

bole}

ath;

WML D pe

e tities

thel
hehe

woolen ys
o— See:

eee eee Lt
ba eo vee ee Cee
=

title

Serer
Teste

de ites 2 poet ats

titiel

et ecrar
Si epeeeiyrece

Piebe
‘

- ~9~9- p=
rk

eis

ei
tH
teitiejertl

arate

aie

aero

= ce ll ae

—

e E525

TOO TOEO oO

HM/18WW

a nf f sayin
AR ee! i
yn i Wh
Mere eiys tis ti
TU

(h
Wi

PROMOZOA
IN BIOLOGICAL
IES EF ASh CH

PROTOZOA
DN BTORwOGIC AL
RESEARCH

Edited by
GARY IN. CALKINS
and
FRANCIS M. SUMMERS

I Ltrs BSA

New York: Morningside Heights
COLUMBIA UNIVERSITY PRESS
L941

Foreign agents; OxForD UNIVERSITY Press, Humphrey Milford, Amen
House, London, E.C. 4, England, AND B. I. Building, Nicol Road,
Bombay, India; MARUZEN COMPANY, LTD., 6 Nihonbashi,
Tori-Nichome, Tokyo, Japan

MANUFACTURED IN THE UNITED STATES OF AMERICA

PREFACE

NuMEROUS textbooks of varying degrees of excellence for the study
of Protozoa are now on the market and should be consulted for a
general treatment of these unicellular forms. This volume will not fill
such a need, but has been prepared as a result of a discussion by a
group of specialists assembled during the summer of 1937 at the
Marine Biological Laboratory at Woods Hole, Massachusetts, for the
purpose of ascertaining the best means to stimulate further research
on these unicellular animals.

As a result of these discussions it was decided that one good way to
attain our end would be to have a group of specialists in the field of
protozodlogy prepare a work on research in this field, each specialist
to provide a chapter on the subject in which he is best known, and
about which he can speak with authority.

Our first real difficulty was to select a limited number of topics from
a vast number of possibilities, and then to choose the biologists who, in
our opinion, were the best men to write these chapters. As can be
readily imagined, this opened up a long list of difficulties and led to
many vexatious troubles, but the present work, finally, is the outcome.

To our very great regret, one of the men chosen—Professor Noland,
of the University of Wisconsin—who was most enthusiastic over the
project, has been forced by reason of continued illness, to drop out.
The loss of others, on the plea of pressure of work, and so forth, has
also depleted our ranks. But the remainder have completed their labors,
have come through, and the results here speak for their continued in-
terest and assiduity.

While the arrangement of our material does not make a great
deal of difference, some order nevertheless is advantageous, and we
have therefore arranged the chapters, according to the general character
of their materials, into groups having a more or less common subject
matter.

Gary N. CALKINS
FRANCIS M. SUMMERS

New York City
January 2, 1941

ACKNOWLEDGMENTS

WE ARE INDEBTED to the American publishers of the following
books or periodicals for permission to use figures or other illustrations
which are reproduced in this book. In a few cases these have been
slightly modified by the contributors. Also we are under obligation to
numerous foreign publishers who, for obvious reasons, we have been
unable to ask for permission.

American Journal of Hygiene
Figures 188, 191, 192
Biological Bulletin
Figures 5, 10, 13, 74, 81, 82, 84, 96, 104, 181, 183, 185, 186, 187,
199A, 199B, 223
Biology of the Protozoa by G. N. Calkins, 1933. Lea and Febiger Co.
Bigures 12; 3A 3B, 9
Genetics
Figures 166, 168
Journal of Animal Behavior, Henry Holt and Co.
Figure 103
Journal of Cellular and Comparative Physiology, Wistar Institute
of Anatomy and Biology
Eigures 119.120, 121, 122) amd: Table 2
Journal of the Elisha Mitchell Scientific Society
Figures 202A, 202B
Journal of Experimental Medicine, Rockefeller Institute for Medical
Research
Figures 189, 190
Journal of Experimental Zoology, Wistar Institute of Anatomy and
Biology
Figures 14, 94, 97, 98, 102, 105, 106, 149, 156, 162, 164, 179, 182
Journal of Morphology, Wistar Institute of Anatomy and Biology
Figures 83, 91, 93, 154, 157, 160, 16%, 1163

Vill ACKNOWLEDGMENTS

Journal of Preventative Medicine, John McCormick Institute for In-
fectious Diseases
Figure 193
Light and Behavior of Organisms by S. O. Mast, 1911. John Wiley
and Sons, Inc.
Figures 99, 107
Physiological Zoology, University of Chicago Press
Figures 95, 113, 126, 180
Publications of the Carnegie Institute of Washington
Figure 214
Science
Figure 123
Transactions of the American Microscopical Society
Bigutes 36,537
University of California Publications in Zoology, University of Cali-
fornia Press
Figures 15, 16, 39, 49, 50, 53, 54, 55, 70) 85. 86, 87, esmao
146, 148, 150, 151, 196, 219, 225
United States Geological Survey of the Territories
Figure 221

Finally, it is a pleasure to recognize the care and patience on the part
of the staff of the Columbia University Press in the preparation and
distribution of this work. While all members have worked diligently
and harmoniously with us, we are particularly indebted to Miss Georgia
W. Read, Miss Ida M. Lynn, and Miss Eugenia Porter for the meticu-
lous care with which they have sought to unify and, in some cases, to
clarify the manuscripts on subjects unfamiliar to them.

THE Epirors

New York City
January 2, 1941

CONTENTS
Ieist: OL Ab eremiahionss ¢ Go on ke te es VO

I. General Considerations By Gary N. Calkins, Pro-
fessor Emeritus of Protozodlogy in Residence, Co-

lumbia University

Life and vitality—Fundamental and derived organiza-
tion—Some ecological consideraticns—Some historical
facts—The use of cultures—Factors influencing longev-
ity—Changes with metabolism—Reorganization of the
macronucleus and other derived structures in Ciliata—
Reorganization by endomixis and by conjugation—Lit-
erature cited

II. Some Physical Properties of the Protoplasm of the
Protozoa By H. W. Beams and R. L. King, State
Di A TAN TOOT oe eae ol NES A Loe Se ae 43

Introduction—Properties of protoplasm as exhibited in
Amoeba—Colloidal nature of protoplasm—Consist-
ency—Surface properties—Specific gravity or density—
Optical properties—Structural properties—Literature
cited

III. Cytoplasmic Inclusions By Ronald F., MacLennan,
OoeriniEal Can Pay ot TNIIS Bhs maa) ae, os LE

Mitochondria—The vacuome hypothesis—Digestive
granules—Segregation granules—Golgi bodies—Excre-
tory granules—Lipoid reserves—Carbohydrate reserves—
Protein reserves—External secretion—The granular com-
plex—The continuity of cytoplasmic granules—The
classification of cytoplasmic granules—Comparison with
cells of the Metazoa—Literature cited

IV. Fibrillar Systems in Ciliates By C. V. Taylor, Stan-
OCMUIZU CENTLY Aaya yeshi Ayia ee eel, wees: LDL

Introduction—Examples of fibrillar systems—Structural

53684

WAL,

VI.

VIII.

CONTENTS

analysis—Interpretation—Fibrillar systems of other
ciliates — Holotricha — Heterotricha — Oligotricha —
Hypotricha—Conclusions—Literature cited

Motor Response in Unicellular Animals By S. O.
Mast, Director, Zoological Laboratory, Johns Hop-
kins University .

Introduction—Responses to light—Rhizopods—Flagel-
lates—Ciliates—Colonial organisms—Responses to elec-
tricity —Rhizopods—Flagellates—Ciliates—Colonial or-
ganisms—Responses to Chemicals—Rhizopods—Ciliates
—Literature cited

Respiratory Metabolism By Theodore Louis Jahn,
State University of Iowa

Purposes of studying respiration-—Methods of measuring
aérobic respiration—Aé€érobic respiration—Investigations
which concern the source of energy—lInvestigations
which concern the mechanism of respiration—The
measurement of anaérobic metabolism and glycolysis—
Occurrence of anaérobiosis and glycolysis—Why are
anaérobes anaérobes, and aérobes aérobes ?—Oxida-
tion-Reduction potential versus respiration and growth—
Literature cited

The Contractile Vacuole By J. H. Weatherby, Medr-
cal College of Virginta

Introduction—The origin of contractile vacuoles—The
structure of contractile vacuoles—The function of con-
tractile vacuoles—Contractile vacuoles and the Golgi
apparatus—Conclusion—Literature cited

The Technique and Significance of Control in Proto-
zoan Culture By George W. Kidder, Brown University

Introduction—The problem of protozoan sterilization—
General material—General methods of sterilization—
Special methods and manipulations—The importance of
adequate sterility tests—Establishment of sterilized Pro-
tozoa in culture—Literature cited

Dik

352

404

448

CONTENTS xi

IX. Food Requirements and Other Factors Influencing
Growth of Protozoa in Pure Cultures By R. P. Hall,
University College, New York University . . . 475

Food requirements of Protozoa—Photoautotrophic nu-
trition—Photomesotrophic nutrition—Photometatrophic
nutrition — Heteroautotrophic nutrition — Heteromeso-
trophic nutrition—Heterometatrophic nutrition—Trophic
specialization—Specific growth factors, or vitamins—
Other growth factors—Growth stimulants—Growth in
cultures as a population problem—The initial popula-
tion—Growth in relation to waste products—Growth in
relation to food concentration—Growth in relation to
pH of the medium—Oxygen relationships—The redox
potential—Growth in relation to temperature—Growth
in relation to light and darkness—Acclimatization—
Literature cited

X. The Growth of the Protozoa By Oscar W. Richards,
Research Biologist, Spencer Lens Company . . . 517

Methods for the measurement of growth—The growth of
individual Protozoa—The growth of colonial Protozoa—
Pedigree isolation culture and life cycles—Protozoan suc-
cessions: nonlaboratory—Protozoan successions: labora-
tory—Autocatalysis and allelocatalysis—Nutrition and
growth—The growth of populations—The struggle for
existence—Literature cited

XI. The Life Cycle of the Protozoa By Charles Atwood
Kofoid, University of California, Berkeley . . . 565

Introduction—Asexual reproduction in alternating binary
and multiple fission (Type I)—Alternation of asexual
and sexual reproduction (Type I1)—The life cycle of
Eimeria schubergi—The life cycle of Plasmodium
vivax—The life cycle of Paramecium caudatum—Litera-
ture cited

XII. Fertilization in Protozoa By John P. Turner, Univer-
SO] MGHNESOldaeM <a eal, 2 Mien adie ~ 88?

Copulation—Gametic meiosis and fertilization—Autog-
amy—Zygotic meiosis—Significance of fertilization—

Xxil CONTENTS

Conjugation—The macronucleus during conjugation—
Conjugant meiosis—Literature cited

XIII. Endomixis By Lorande Loss Woodruff, Yale Uni-
UGESIL) tees: come EE. rapt S VE eels Mion. Spade? > EE

Macronuclear reorganization—Endomictic phenomena—
Autogamy—-Periodicity of endomixis—Genetical studies
on endomixis—Conclusions—Literature cited

XIV. Sexuality in Unicellular Organisms By T. M. Sonne-
born, Uniwersiy of Indiana = 2 VS) 3 G66

Sexuality in Chlamydomonas—The kinds of gametic
differences observed in Chlamydomonas—The nature of
the physiological differences between gametes in
Chlamydomonas—Interpretation of the Seal phe-
nomena in Chlamydomonas—Sexuality in Paramecium
and other ciliate Protozoa—Sexual differences between
conjugant individuals—Mating types in relation to the
Maupasian life cycle—The rdle of environmental con-
ditions in determining conjugation—Sex differences be-
tween gamete nuclei—Significance of the diversities be-
tween conjugants and between gamete nuclei—Literature
cited

XV. Inheritance in Protozoa By H. S. Jennings, Univer-
sity of (California LostAnccles ia. a vce eee

Types of reproduction and inheritance—Inheritance in
uniparental reproduction—Material processes—Inheri-
tance of characteristics—Changes in inherited characters
in uniparental reproduction—Age changes, sexual imma-
turity, and maturity—Inherited degenerative changes re-
sulting from unfavorable conditions—Inherited acclima-
tization and immunity—Inherited environmental modifi-
cations in form and structure—Variation and its inhert-
tance occurring without obvious action of diverse en-
vironments—Summary and interpretation—Inheritance
in biparental reproduction—Biparental inheritance in
haploids: Flagellata—Sex inheritance and sex-linked in-
heritance—Biparental inheritance in diploids: Ciliata—
Inheritance of mating type in Paramecium aurelia—In-
heritance of mating type in Paramecium bursaria—Effect
of the cytoplasm and its relation to nuclear constitution—
Literature cited

CONTENTS Xi

XVI. The Protozoa in Connection with Morphogenetic
Problems By Francis M. Summers, College of the
CMON Of hs fod wid gk Eizieyoh, (os es «2 772

Physiological regeneration—Some of the factors in tfe-
generation—External environment—Cyclical variations
—Racial variations—Degree of injury and reorganiza-
tion—The size factor—The nuclei in regeneration—
Behavior of fragments: grafting and reincorporation—
Regeneration and division—Polarity changes and proto-
plasmic streaming—Physiological gradients—Regenera-
tion in colonial forms—Literature cited

XVII. Certain Aspects of Pathogenicity of Protozoa By
Elery K. Becker, lowa State Gollege 5 29% *. 818

Problems of virulence and _pathogenicity—Amoebic
dysentery and bacterial complications—Malaria: Plas-
modium vivax—Variability in strains and in host re-
sponse—Coccidiosis in poultry—Nutrition and resistance
—Conclusions—Literature cited

XVIII. The Immunology of the Parasitic Protozoa By Wil-
liam H. Taliaferro, University of Chicago . . . 830

The physical bases of immunity—The cells involved in
immunity—Antibodies and antigens involved in immu-
nity—The cellular and humoral aspects of immunity—
Role of immune processes in the development of proto-
zoan infections—General methods—Malaria—Leishma-
niasis—Nonlethal infection with the Trypanosoma lewitsi
group of trypanosomes—Continuous fatal trypanosomi-
asis in the mouse and sometimes in the rat—Intermittent
fatal trypanosomiasis in various laboratory animals—
Practical applications of immune reactions—Artificial
immunization—Immunological reactions used in diagno-
sis—Immunological reactions in relation to classifica-
tion—Literature cited
XIX. Relationships between Certain Protozoa and Other
Animals By Harold Kirby, Jr., University of Cali-
fornia, Berkeley A At hE, Scien, Mae OO)

Accidental and facultative parasitism—Systematically
related free-living and symbiotic Protozoa—Mastigo-

X1V

CONTENTS

phora—Holotricha—Distributional host relationships
and host-specificity in representative symbiotic faunules—
General considerations—Ciliates of sea urchins—Proto-
zoa of termites and the roach Cryptocercus—aAdaptive
host relationships in morphology and life history—
General considerations—Thigmotricha—Ptychostomidae
—Astomata—Conidiophrys—aA postomea—Physiological
host relationships illustrative of mutualism and com-
mensalism—Flagellates of termites and Cryptocercus—
Ciliates of ruminants—Literature cited

XX. Organisms Living on and in Protozoa By Harold

Kirby, Jr., University of California, Berkeley .

Epibiotic schizomycetes—Schizomycetes on Mastigo-
phora—Endobiotic schizomycetes—Associations of a
constant character—Associations of an occasional char-
acter—S phaerita and Nucleophaga—Historical account
and distribution—Life history and structure—Effect on
host—Parasites of the nucleus of Trichonympha—
Phycomycetes other than Sphaerita and Nuacleophaga—
Protozoa—Phytomastigophora—Zobmastigophora—Sar-
codina—Sporozoa—Ciliophora—The genus <Amoebo-
phrya Koeppen—Metazoa—Literature cited

Index

1009

Pits

TABLES

1. Structural components of the fibrillar system of Paramecium

OST cs

10.

12:
13.
14.
ED:

16.

18.

. Rate of locomotion of Amoeba proteus in sodium and cal-

cium salt solutions

Sensitivity of respirometers .

Measurements of protozoan respiration .

Division rates of Protozoa with constant conditions .

The effect of hydrogen-ion concentration on the growth of
Protozoa .

Breeding relations in Chlamydomonas paradoxa and C.
pseudo paradoxa

. Breeding relations in Chlamydomonas sp. (coccifera?), C.

braunit, C. dresdensis, C. eugametos, and C. paupera .

. Grades of sex reaction in mixtures of sexes G to O from

the Chlamydomonas species C. braunii, C. dresdensis, and
C. eugametos

System of mating relations in Chlamydomonas braunii, C.
dresdensis, and C. eugametos. Observations and inter-
pretations of Moewus .

Results of mixing together animals from different cary-
onides of stock F, Paramecium aurelia .

The system of breeding relations in Paramecium aurelia

The system of breeding relations in Paramecium bursaria

Environmental modifications, Chlamydomonas debaryana

Early results of selection for low and high numbers of
spines, Difflugia corona . a es

Later results of selection for low and high numbers of
spines, Difflugia corona . ae eae

Inheritance of spine length, with regression toward the
mean, Difflugia corona .

Inheritance of mating types in Paramecium bursavia .

196
338
354
362
528
540

672

673

675

680

692
693
695
722

ai:

728
1D)

XV1 TABLES

19. Mating types of descendant clones, Paramecium bursaria

20. Lengths in microns of the two races crossed with the re-
sulting final lengths of the offspring, Paramecium cauda-
tum

21. Table of minimum volumes necessary for regeneration .

760

765
786

at CAL :
Kae
% (ye —_é e V<¢
= Lie R RY °
os 7
z Has
ILLUSTRATIONS \4 “s
~~ *
FIGURES
1. General morphology of Uronychia transfuga . 16
2. Uronychia transfuga; merotomy and regeneration 17
3A. Uroleptus mobilis; stages in the fusion of the Tearsanel
prior to cell division . Lg
3B. Uroleptus mobilis; the nuclei in ie ainisiont ead 20
4. Nuclear clefts in the macronuclei of Uroleptus halseyi ZA
5. Conchophthirus mytili; extrusion of chromatin during divi-
sion : sah 572
6. Aspidisca emeob: pen in aon fe emiton
during division 23
7. Lophomonas blattarum; avin ‘se feoroene atic a the
nucleus 26
8. Chilodonella uncinatus; replacement oF phage Beka:
and mouth . eT!
9. Uroleptus mobilis; old-age Speen sowing the eoetees:
tion of the macronucleus and the loss of micronuclei . 29
10. Uroleptus mobilis; graph representing the life history Zz
ten-day intervals 30
11. Glaucoma (Dallasia) emia sence meeeiologs a a
vegetative individual . a2
12. Gametogenesis in Glaucoma (diane) eae ; 28,
13. Uroleptus mobilis; conjugation and merotomy . oA
14. Uroleptus mobilis; formation of the new macronucleus nih
conjugation . aul
15-16. Mitochondria in EaE Pate ie sii
17. Ichthyophthirius multifiliis; series showing saiiealigack afd
the secretion of paraglycogen . 114
18-21. De novo origin of mitochondria in Riatoatiroe 114
22-25. Mitochondria in Amoeba proteus . 114
26-27. Mitochondria and protein reserves in A Porenia bienei 114
28-29. Mitochondria in conjugants of Bursaria truncatella . 114

XVili = ILLUSTRATIONS

30. The association of mitochondria with the gastriole in Amoeba
proteus

31. The association be the eeriolee aa ithe digestive pianales
in Ichthyophthirius multifiliis .

32-35. Morphological variations in the emma tion pomalee a
Opalina ranarum .

36-37. Segregation granules in ie ore ANE

38. Stages in the resorption of a segregation granule in Amoeba
proteus :

39. Dictyosomes from Heer el Spanien

40. Dividing dictyosome in Lecudina brasili . —

41. Stages in the secretion of neutral fat by Ie Beppe bids
multifiliis

42-45. Dictyosomes danine the We ae of Terwdina Bice

46-47. The effect of centrifuging upon the distribution of cyto-
plasmic granules .

48. Aggregation and Pstepenmies a Se Pons aa
ing pulsatory cycle of the contractile vacuole in is
ophthivius multipiliis .

49-50. Excretory granules and the roniactile acto in Poly:
plastron multivesiculatum

51. Excretory granules associated with the cone cele
in Dogielella sphaerii .

“Nephridialplasm” of CAraielll Agena ce.

es 55. Excretory granules in living ciliates .

56-64. Carbohydrate reserves in various Protozoa .

65. The association between glycogen and the parabasal pace
in Cryptobia helicis .

66. The formation and release of proren socanales fon the
macronucleus of Ichthyophthirius multifiliis .

67-69. External secretion in Evglypha and In Iyer

70. Accessory bodies being formed from the neuromotor ring in
Haptophrya michiganensis .

71. Pellicular pattern and longitudinal aa pontedine ell

granules in Paramecium .

72. Diagram of Gitter (lattice) with mtdelied ena and
of the neuronemes connecting bases of the cilia .

Wa)

130

133
134

136
141
141

141
141

142

146
146
146
146
149
156
158

164
167

‘79

195

TS:
74,
Yie¥
10:
Ta
78.
UP
80.
81.
82.

(S331
84.
3).
86.
S77.
88.
So.
LU
Ok.
92.
93:

94,

DD:

96.

The

98.

oo.

ILLUSTRATIONS

Connecting branch from neuroid to myoneme in Stentor

External fibrillar system of Explotes patella

Stalk of Zoothamnium arbuscula .

Spasmonem in Zoothamnium .

Three components of Spironem of abibane niin

Pellicular structure and myonemes in Zoothamnium .

Myonemes of stalk sheath of Vorticella .

Arrangement of second complex of body fibrils in Beer

Fibrillar system in Chlamydodon sp.

Fibrillar system of peristomal region in Gay see
magna

Neuromotor ears af Dile pie GIGAS .

Neuromotor system in Entodzscus pense

Pellicular fibrils of Entorhipidium echini .

Optical section of Expoterion pernix . sont

Neuromotor system of Haptophrya michiganensis .

Cross section of peripheral region of Balantidium coll .

Diagram of fibrillar system in Nyctotherus hylae .

Spivostomum ambiguum. Diagram of membranelles and
their intracytoplasmic structures ;

Fibrillar complex of cytostome in Oath

Section through anterior end of Uroleptus halsey: . ;

Camera sketch of horizontal optical section of Amoeba sie
teus SIG :

Curves aha eae paleion penned liaenineuie intensiigg reaction
time, stimulation period, and latent period in Amoeba
proteus TT

Camera drawings of Anas a lives He response to
localized illumination . :

Relation between adaptation to light af aideeea intensities
and rate of locomotion in Amoeba proteus . ee

Camera outlines representing different stages in the process
of orientation in Amoeba proteus .

Diagrams showing the position of the feelin a Englend
as seen in a viscid medium .

Euglena sp. in a crawling state, showiae. devi in tine anon
ess of orientation .

100. ,
. Graphs showing the relation between the direction of loco-

LTO:
HUT ke

ILItey
114.

apy.

LUG:

Ly.

ILLUSTRATIONS
Side view of anterior end of Euglena viridis .
motion of flagellates observed in a field of light produced

by two horizontal beams crossing at right angles and that
demanded by the ‘“‘Resultantengesetz’’

. Curves representing the distribution in the spectrum of stim-

ulating efficiency

. Camera drawings illustrating the eSnOnee of Peranema to

contact or to rapid increase in luminous intensity .

. Graph showing the effect of dark-adaptation on sensitivity

to light in Peranema trichophorum .

. Graphs showing rate of light-adaptation in Peranema .
. Graphs showing relation between luminous intensity and re-

action time, exposure period, latent ss and energy
(for Peranema)

. Stentor coeruleus in the process of orientation .
. Arrangement of zodids in a colony of Volvox .
. Sketches showing the structure of the eyespot in Volvox ane

its action on light entering the pigment cup at different
angles ; HANES :

Diagrammatic pe nao a he yas of orientation
in Volvox : soc! ;aaoapies

Sketches illustrating the effec Ap a Sieh. current on a
monopodal Amoeba moving toward the cathode .

Sketches illustrating the effect of a galvanic current on mono-
podal amoebae moving toward the anode in a weak cur-
rent :

Graphs showing comnnering ie: ee dierent Pee a
current on the rate of locomotion .

A series of camera sketches of an Amoeba, showin the ae
fect of an alternating current . :

Progressive cathodic reversal of the cilia and dunes a fot
in Paramecium as the constant electric current in made
stronger . : : At ena

Paramecium showing ee in the ditection ai he stroke
of the cilia in a galvanic current .

Sketch showing in a stationary photopositive Plone a Vol.

285

310

a2)

EO.

120.

PL,

123.

124.
125%
126.
L27-

128.
LA

130.
P30.
IS as

133:
134.

13De
136.

ILLUSTRATIONS

vox the effect of a galvanic current on the currents of
water produced by the flagella .

. Diagrams illustrating the effect of direct current on the dig.

tribution of ions in colonies of Volvox and their response
The relation between rate of locomotion, gel/sol ratio, and
hydrogen-ion concentration in a balanced salt solution
The relation between rate of locomotion, gel/sol ratio,
hydrogen-ion concentration, and sodium-ion concentra-
tion

. The relation eorcenet rate ot nucomonen eel /sol ratio, invate:

gen-ion concentration, and calctum-ion concentration .

The effect of adding calcium in different concentrations to
0.005 N sodium solutions, on the relation between hydro-
gen-ion concentration, rate of locomotion, and gel/sol
ratio

Capillary tube eed for the seechization ae Wr epariOne
hominis

Migration tube .

V migration tube for pemcold media be ih?

Migration-dilution apparatus drawn to show construction

Details of construction of the ioe Taco plunger for
the collection of cysts .

Growth phases in a hypothetical Sasamane

Hypothetical modifications of the normal growth Bee a popu-
lation, from the initial stationary to the maximal stationary
phase . Re

Growth in length alk in area af PE Paiin caudatum .

Growth in breadth and thickness of Paramecium caudatum

Growth in volume of Paramecium, Frontonia, and Hartman-
ella : wend:

Hypothetical curves to ieee piises Poptlibion eel

Population growth curves of Euglena, Paramecium, Stylo-
nychia, and Mayorella . ; seine

Diagram of the life cycle of Tnshontonias AUgUStA .

Diagram of the life cycle of Endamoeba coli (Councilmania
lafleur?)

XXI

328

330

334

313)

336

L5G,

13:
159:
160.
LGL:
162.
163.
164.

ILLUSTRATIONS

. Diagram of the alternating sexual and asexual reproduction

in the life cycle of Ezmeria schubergi .

. Diagram of the life cycle of Plasmodium vivax .

. Diagram of the life cycle of Paramecium caudatum .

. Copromonas subtilis in hologamous copulation .

. Autogamy in Sappinia diploidea .

. Actinophrys sol in autogamous fertilization .

. Progamic divisions in Monocystis rostrata .
. Isogamous gamete formation and fertilization in Ona

cystis mesnili

. Diagram of the life cycle of S$ eee te Pine

. A pair of Explotes patella in conjugation .

. Diagram of ciliate conjugation .

. Stages in the first maturation division aif Benores rae

. First maturation spindles of Uroleptus mobilis .

. Second meiotic division in Ezplotes patella . Swe
. Third maturation division and the fertilization nucleus in

Euplotes patella

. Migration of the pronuclei across othe prole closer rides

of conjugating Chilodonella uncinatus .

. Stages in the progress of the reorganization bands chroeen

the macronucleus of As pidisca

. Macronuclear dissolution in Blepharisma bet iis .
. General plan of the usual nuclear changes during endomixis

in Paramecium aurelia .
Possible methods of micronuclear ae cell ea at ne cli-
max of endomixis in Paramecium aurelia .

. Diagram of the normal process of endomixis in Tecbodte

sp. ike aE
Endomixis in Seo I say Pe :
Endomixis in Paraclevelandia simplex . pis
Nuclear changes during autogamy in Paramecium aurelia
Hemixis in Paramecium aurelia .
Climax of endomixis in Paramecium aurelia .
Autogamy in several races of Paramecium aurelia .
Graph of division rate of Paramecium aurelia .

165.

166.
167.

168.
169.
170.
ey

172.

173:

174.

£75.

L7G.
LET.
178.
19
180.
181.
SZ,
£33.

184.

185.

ILLUSTRATIONS

Group formation in Chlamydomonas, showing groups
formed in a mixture of cells differing in sex .

The mating reaction in Paramecium bursaria re

Conjugation in Cycloposthium bipalmatum showing Heer:
morphic pronuclet . ; ail

Difflugia corona; members of four aieerede nee showing
diversities in characteristics .

Polytoma uvella and P. pascheri; the unt races outed in oe
breeding experiments of Moewus . ;

Results of a cross between two species of Poly toma .
New combinations resulting from crossing over in Polytoma
Diagram of the sex chromosome of Polytoma Mere plus
and P. uwvella minus as
Diagram of the sex cheguipsomes produced By ensiie over
between the chromosomes of Polytoma pascheri plus and
P. uvella minus . PRE) = otk |

Diagram of pregamic divistodls ana proncelene ay in
ciliates tae ee

The four clones of Pueeie produced fron ie two ex-
conjugants of a pair, in the experiments of Sonneborn and
of Jennings .

Change of size resulting ha concen a intipieluals xi
large and small races of Paramecium caudatum . ,

Changes in mean size of the descendants of the two members
of an unequal pair of Paramecium caudatum

Different ultimate mean sizes reached by descendants of We
ferent pairs from crosses of the same two races of Para-
mecium caudatum . aT),

Regeneration in Uronychia ee Ga .

Regeneration in Uronychia uncinata .

Reincorporation in D/fflugia pyriformis . f !

Divisional and physiological reorganization in Gensehes

Diagram showing un regeneration in Paramecium cau-
datum ae

Successive stages in eke Paition ae the mouth in a nee
of Spirostomum with the mouth at the anterior end .

A relatively mature colony of Zoothamnium alternans .

XXiil

XXIV

186.

187.

188.

189:

£90:

po:

£92:

193.

194.

195.

196.

LOY.

Ss:

19:
200.
201.
202.
203:
204.
205.
206.
207:

ILLUSTRATIONS

A seventy-two-hour regenerate produced from a lateral cell
of the first branch generation in Zoothamnium .

Branch C of a Zoothamnium colony fifty-six hours after in-
jury to the neuromuscular cord .

The changes in number of Plasmodium ee ein al fe
percentage of segmenters during the acute rise and crisis
of the infection in a Central American monkey

The changes in number of Trypanosoma lewisi and the co-
efficient of variation and percentage of division forms dur-
ing the course of infection in the rat . iam

The demonstration of ablastin against Try Puede lewist
by passive transfer

The changes in number of Tey er osarae ideas wad
the coefficient of variation during the course of infection
in a mouse .

The changes in marae BF inp angiora phedeente dae
the coefficient of variation during the course of infection
in a guinea pig . ey: ee eta

The demonstration of a caeanaleia none a oe strain
of Trypanosoma equinum by passive transfer .

Posterior end of larva of Aédes (Stegomyia) scutellaris para-
sitized by ciliates .

One-day-old nymph of Rea er eee receiving Pine
todaeal food from the female termite . ae

Streblomastix strix attached to the lining of the piel a a
Zootermopsis angusticollis .

Fixation mechanisms in peritrichs at :

Fixation apparatus of Cyclochaeta (Urceolaria) ay eee

Thigmotricha

Ptychostomidae

Skeletal structures and praclenene qusarielleda in iglomae ;

Anoplophrya (Collinia) circulans in Asellus aquaticus .

Conidiophrys pilisuctor on Corophium acherusicum .

Synophrya hypertrophica; diagram of life cycle .

Ingestion of plant material by Ophryoscolecidae .

Ingestion and digestion of starch in Eudiplodinium medium

808

810

856

859

862

865

867

897

927,

930
Doh
9o2
939.
941
944
948
op) It
955
958
978
980

208.
209.
210;
Poll

WS
215.
214.

ZL:

206%
21:
218.
PANE
220:

220,
Posapee
22D:
224,
22).

226.

ILLUSTRATIONS

Fusiformis-like rods adherent to the surface of flagellates .

Adherent microdrganisms on flagellates of termites .

Adherent microorganisms on flagellates of termites .

Spirochetes adherent to Stephanonympha sp. from Neo-
termes insularis AAs AI:

Surface microOrganisms on various preeez0e

Bacteria adherent to ciliates .

Characteristic bacteria adherent to the mellecle a C ae
from the intestine of sea urchins . ;

Bacteria (Cladothrix pelomyxae Veley, and a anal ppecics)
in Pelomyxa palustris Greeff . :

Microérganisms in Stephanonympha and Guede ;

Developmental stages of Sphaerita in several Protozoa .

Nucleophaga . 4 ‘
Microorganisms in Tr chonsinpba ; ; : a
Nuclear parasites of Ty ae mpha sp. from Prey ptoter-

mes sp.
Filamentous fans paeiie on eee Pease
Small Protozoa ectoparasitic on Chilomonas and Col ee

Entamoeba in Zelleriella and Raphidiocystis on Paramecium

Cysts of two species of Amphiacantha, metchnikovellids
parasitic in the gregarine Ophiodina elongata .

O pisthonecta henneguyi, parasitized by Endosphaera en re
manni

Amoebophrya in San :

PLATES

1. Sluggish phagocytosis of Plasmodium brasilianum by macro-

phages in control of the American monkeys during the
acute rise of the malarial infection and the concentration of
P. brasilianum in Billroth cords of the spleen at the initi-
AGM Olp the. Etisisn:

2. Intense phagocytosis of Pladicdim Pete ain By macro-

phages in Central American monkeys at the height of the
crisis of the malarial infection and malarial pigment, the
residue of parasites after the intense phagocytosis, in the

macrophages shortly thereafter

XXV
1012

1014
1016

1017
1020
1023

1024

1026
1031
1049
1055
1060

1062
1066
1071
1077
1081

1088
1091

850

851

XXVi ILLUSTRATIONS

3. Portions of a venous sinus and Billroth cords in the spleen of
an uninfected rhesus monkey and comparable portions from
a rhesus monkey during the late acute rise of an infection
with Plasmodium cynomolgi . 852
4. A nodule in the white pulp of the plex nadine hated lie
perplasia associated with the late acute rise of Plasmodium
eyomolg? in a thesussmonkey, sae: 9. eee OnE

LIST OF ABBREVIATIONS

Abh. bayer. Akad. Wiss.: Abhandlungen der Kgl. Bayerischen Akademie der
Wissenschaften. Math.-Phys. KI. Munchen.

Abh. naturw. Ver. Bremen.: Abhandlungen hrsg. vom Naturwissenschaft-
lichen Verein zu Bremen.

Abh. sencken b. natur f. Ges.: Abhandlungen hrsg. von der Senckenbergischen
Naturforschenden Gesellschaft. Frankfurt a. M.

Acta Biol. exp.: Acta biologiae experimentalis. Warsaw.

Act. Zool. Stock: Acta Zoologica: Internationell Tidskrift for Zoologica.
Stockholm.

Act. Sci. Indust.: Actualités scientifique et industriel. Paris.

Allatt. Kézlem.: Allattani K6zlemények. Budapest.

American Academy of Arts and Sciences, see Mem. Amer, Acad. Arts Sci.;
Proc. Amer. Acad. Arts Sci.

Amer. J. Anat.: American Journal of Anatomy. Baltimore.

Amer. J. Bot.: American Journal of Botany. Lancaster.

Amer. J. Hyg.: American Journal of Hygiene. Baltimore.

Amer. J. Path.: American Journal of Pathology. Boston.

Amer. J. Physiol.: American Journal of Physiology. Boston.

Amer. J. Psychiat.: American Journal of Psychiatry. Baltimore.

Amer, J. publ. Hith.: American Journal of Public Health. New York.

Amer. J. Syph.: American Journal of Syphilis. St. Louis, Mo.

Amer. J. trop. Med.: American Journal of Tropical Medicine. Baltimore.

Amer. Midl. Nat.: American Midland Naturalist. Notre Dame, Ind.

Amer. mon. micr. J.: American Monthly Microscopical Journal. Washington.

Amer. Nat.: American Naturalist. Boston.

Anat. Anz.: Anatomische Anzeiger. Jena.

Anat. Rec.: Anatomical Record. Philadelphia.

An. Fac. Med. Porto.: Anais da Faculdade de Medicina do Porto.

An. Inst. Biol. Univ. Méx.: Anales del Instituto de biologia, Universidade de
México. México.

Ann. appl. Biol.: Annals of Applied Biology. Cambridge.

Ann. Bot., Lond.; Annals of Botany. London.

Ann. Fac. Med. S. Paulo.: Annaes da Faculdade de Medicina de Sao Paulo.

Ann. Igiene (sper.): Annali d’igiene (sperimentale). Torino.

Ann. Inst. océanogr. Monaco.: Annales de |’Institut océanographique de
Monaco. Monaco.

Ann. Inst. Pasteur.: Annales de |’Institut Pasteur. Paris.

Ann. Mag. Nat. Hist.: Annals and Magazine of Natural History. London.

i LIST OF ABBREVIATIONS

Ann. Microg.: Annales de Micrographie spécialement consacrée a la bac-
tériologie au protophytes et aux protozoaires. Paris.

Ann. Mus. Stor. nat. Genova.: Annali del Museo cirico di storia naturale.
Genova.

Ann. Mus. zool. polon.: Annales musei zoologici polonicit. Warszawa.

Ann, Natal Mus.: Annals of the Natal Museum. Pietermaritzburg.

Ann. N. Y. Acad. Sci.: Annals of the New York Academy of Sciences.
New York.

Annot. zool. jap.: Annotationes zoological japonenses. Tokyo.

Ann. Parasit. hum. comp.: Annales de parasitologie humaine et comparée.
Paris.

Ann, Physiol. Physicochim. biol.: Annales de physiologie et de physicochimie
biologique.

Ann. Protist.: Annales de protistologie. Paris.

Ann. Sci. nat.: Annales des sciences naturelles. (2) Botanique. (4) Zoologie.
Paris.

Ann. Soc. belge Micr.: Annales de la Société belge de microscopie. Bruxelles.

Ann. Soc. Sci. méd. nat. Brux.: Annales (et Bulletin). Société R. des sciences
médicales et naturelles de Bruxelles.

Ann. trop. Med. Parasit.: Annals of Tropical Medicine and Parasitology.
Liverpool.

Anz. Akad. Wiss., Wien.: Anzeiger der Kaiserlichen Akademie der Wissen-
schaften. Math. KI. Wien.

Arb. Gesundh. Amt. Berl.: Arbeiten aus dem Kais. Gesundheitsamte. Berlin.

Arb. Staatsinst. exp. Ther. Frankfurt.: Arbeiten aus dem Staatsinstitut fiir
experimentelle Therapie und dem Georg. Speyer-Hause zu Frankfurt a. M.
Jena.

Arb. zool. Inst. Univ. Wien.: Arbeiten aus den Zoologischen Institut der
Univ. Wien u. der Zoolog. Station in Triest. Wien.

Arch. Anat. micr.: Archives d’anatomie microscopique. Paris.

Arch, Anat. Physiol. Lpz.: Archiv fiir Anatomie und Physiologie. Leipzig.

Arch. Anat. Physiol. wiss. Med.: Archiv fiir Anatomie, Physiologie und
wissenschaftliche Medicin, Leipzig.

Arch. argent. Enferm. Apar. dig.: Archivos argentinos de enfernedades del
aparato digestivo de la nutricién. Buenos Aires.

Arch. Biol. Paris.: Archives de biologie. Paris.

Arch. exp. Zellforsch.: Archiv fiir experimentelle Zellforschung. Jena.

Arch. Hyg. Berl.: Archiv fiir Hygiene (und Bakteriologie). Miinchen u.
Berlin.

Arch. Inst. Pasteur Afr. N.: Archives des Instituts Pasteur de l'Afrique du
Nord. Tunis.

Arch. Inst. Pasteur Algér.: Archives de I’Institut Pasteur d’Algérie. Alger.

Arch. intern. Med.: Archives of Internal Medicine. Chicago.

Arch, int. Physiol.: Archives internationales de physiologie. Liége et Paris.

LIST OF ABBREVIATIONS XXIX

Arch, Méd. exp.: Archives de médecine expérimentale et d’anatomie patholo-
gique. Paris.

Arch. mikr. Anat.: Archiv fiir Anatomie (und Entwicklungsmechanik).
Bonn.

Arch. Mikrobiol.: Archiv fiir Mikrobiologie. Berlin.

Arch. Naturgesch.: Archiv fiir Naturgeschichte. Berlin.

Arch, parasit.: Archives de parasitologie. Paris.

Arch. Path. Lab. Med.: Archives of Pathology and Laboratory Medicine.
Chicago.

Arch. physiol. norm. path.: Archives de physiologie normale et pathologique.
Paris.

Arch. Protistenk.: Archiv fiir Protistenkunde. Jena.

Arch. russ. d’Anat.: Archives russes d’anatomie, d’histologie et d’embryologie.

Arch, russ. protist.: Archives russes de protistologie.

Arch, Schiffs- u. Tropenhyg.: Archiv fiir Schiffs- u. Tropenhygiene. . . .
Leipzig.

Arch. Sci. biol., St. Pétersb.: Archives des sciences biologiques. St. Péters-
bourg.

Arch. Sci. phys. nat.: Archives des sciences physiques et naturelles. Geneve,
Lausanne, Paris.

Arch. Tierernahr. Tierz.: Archiv fiir Tierernahrung und Tierzucht. Berlin.

Arch. wiss. prakt. Tierheilk.: Archiv fiir wissenschaftliche u. praktische Tier-
heilkunde. Berlin.

Arch. Zellforsch.: Archiv fiir Zellforschung. Leipzig.

Arch, zool. exp. gén.: Archives de zoologie expérimentale et générale. Paris.

Arch, zool. (ital.) : Archivio zoologico italiano. Napoli. Torino.

Arhiva vet.: Arhiva Veterinara. Bucuresti.

Arsskr. Lunds Univ.: Arsskrift-Lunds Universitet.

Atti Accad. gioenia.: Atti della R. Accademia gioenia di scienze natural.
Catania.

Atti della R. Accademia dei Lincei, see R. C. Acad. Lincei.

Atti Soc. Studi Malar.: Atti della Societa per gli studi della malaria. Roma.

Aust. J. exp. Biol. med. Sci.: Australian Journal of Experimental Biology and
Medical Science. Adelaide.

Bact. Rev.: Bacteriological Review. Baltimore, Md.

Beih. bot. Zbl.: Beihefte zum Botanischen Zentralblatt. Cassel.

Beitr. allg. Bot.: Beitrige zur allgemeinen Botanik. Berlin.

Beitr. Biol. Pfl.: Beitrage zur Biologie der Pflanzen. Breslau.

Ber. dtsch. bot. Ges.: Bericht der Deutschen Botanischen Gesellschaft. Berlin.

Ber. Ges. Wiss.: Berichte iiber die Verhandlungen der kgl. sachsischen Gesell-
schaft der Wissenschaften.

Berl. klin. Wschr.: Berliner klinische Wochenschrift. Berlin.

Ber. naturf. Ges. Freiburg i. B.: Berichte der Naturforschenden Gesellschaft
zu Freiburg i. Br.

XXX LIST OF ABBREVIATIONS

Ber. senckenb. Ges.: Bericht der Senckenbergischen Naturforschenden Gesell-
schaft in Frankfurt a. M. Frankfort a. M.

Ber. wiss. Biol.: Bericht tber die wissenschaftliche Biologie. Berlin.

Bibliogr, genet.: Bibliographia genetica. ’sGravenhage.

Bibliogr. zool.: Bibliographia zoologica. Lipsiae.

Biederm. Zbl.: Biedermanns Zentralblatt fiir Agrikultur-chemie und ratio-
nellen Landwirtschaftsbetrieb. Leipzig.

Bio-chem. J.: Bio-chemical Journal. Liverpool.

Biochem. Z.: Biochemische Zeitschrift. Berlin.

Biodyn.: Biodynamica. Normandy, Mo.

Biol. Bull. Wood’s Hole: Biological Bulletin of the Marine Biological Labora-
tory, Wood’s Hole, Mass.

Biol. Listy.: Biologické Listy. Prague.

Biol. Monogr.: Biological Monographs and Manuals. Edinburgh.

Biologe: Biologe; Monatsschrift zur Wahrung der Belange der deutschen
Biologen. Munich.

Biol. Rev.: Biological Reviews and Biological Proceedings of the Cambridge
Philosophical Society. Cambridge.

Biol. Zbl.: Biologisches Zentralblatt. Leipzig.

Biometrika.: Biometrika. Cambridge.

Bol. biol. Fac. Med. S. Paulo.: Boletim biologico, Laboratorio de parasitologia.
Faculdade de medicina de Sao Paulo.

Boll. Lab. Zool. agr. Bachic. Milano.: Bollettino del Laboratorio di zoologia
agraria e bachicoltura del R. Istituto superiore agrario di Milano.

Boll. Soc. eustach.: Bollettina della Societa Eustachiana. Camerino.

Botaniste.: Le Botaniste. Caen, Poitiers, Paris.

Bot. Gaz.: Botanical Gazette. Chicago.

Bot. Rev.: Botanical Review. Lancaster, Pa.

Bot. Ztg.: Botanische Zeitung. Berlin and Leipzig.

Brazil-med.: Brazil-medico. Rio de Janeiro.

Brit. J. exp. Biol.: British Journal of Experimental Biology. Edinburgh.

Brit. J. exp. Path.: British Journal of Experimental Pathology. London.

Bronn’s Klassen.: Bronn’s Klassen und Ordnungen des Tierreichs. Leipzig.

Bull. Acad. Med. Paris.: Académie de Médecine, Bulletin. Paris.

Bull. Acad. Sci. St.-Petersbourg.: Bulletin de l’Académie Impériale des sciences
de St.-Petérsbourg.

Bull. Acad. Sci. U.R.S.S.: Bulletin de l’Académie du sciences de I’U.R.S.S.

Bull. Amer. Mus. Nat. Hist.: Bulletin of the American Museum of Natural
History. New York.

Bull. Bingham oceanogr. Coll.: Bulletin of the Bingham Oceanographic Col-
lection, Yale University, New Haven.

Bull. biol. exp. med. U.R.S.S.: Bulletin de Biologique et de Medicine Ex-
perimentale de L’U.S.S.R. Moscow.

Bull. biol.: Bulletin biologique de la France et de la Belgique. Paris.

LIST OF ABBREVIATIONS XXxi

Bull. Inst. océanogr. Monaco: Bulletin de I|’Institut océanographique de
Monaco.

Bull. Inst. Pasteur.: Bulletin de I’Institut Pasteur. Paris.

Bull. int. Acad. Cracovie.: Bulletin international de I’Académie des sciences
de Cracovie (de ’l’Académie polonaise des sciences) .

Bull. int, Acad. Prag. Sci. Math. Nat.: Bulletin International. Ceska akademie
véd a uméni v Praze. Sciences, mathématiques et naturelles. Prague. (Ceska
akademie césare Frantiska Josefa pro védy, slovesnost a uméni v Praze.)

Bull. Mus. Hist. nat. Belg.: Bulletin du Musée Royal d’histoire naturelle de
Belgique. Bruxelles.

Bull. N. J. agric. Exp. Stas.: Bulletin of the New Jersey Agricultural Experi-
mental Stations. New Brunswick.

Bull. sci. Fr. Belg.: Bulletin scientifique de la France et de la Belgique.
Londres, Paris, Berlin.

Bull. Scripps Instn. Oceanogr. tech.: Bulletin Scripps Institution of Oceanog-
raphy. Technical Series. La Jolla, Calif.

Bull. Sleep. Sickn. Bur.: Bulletin. Royal Society Sleeping Sickness Bureau.
London.

Bull. Soc. bot. Fr.: Bulletin. Société botanique de France. Paris.

Bull. Soc. Hist. nat. Afr. N.: Bulletin de la Société d’histoire naturelle de
l'Afrique du Nord. Alger.

Bull. Soc. imp. Natur., Moscow: Société impériale des naturalistes, Bulletin.
Moscow.

Bull. Soc. méd.-chir. Indochine.: Bulletin de la Société medico-chiruricale de
Indochine. Hanoi.

Bull. Soc. Path. exot.: Bulletin de la Société de pathologie exotique. Paris.

Bull. Soc. zool. Fr.: Bulletin de la Société zoologique de France. Paris.

Bull. U. S. Bur. Fish.: Bulletin of the Bureau of Fisheries. Washington.

Bull. U. S. nat. Mus.: Bulletin. United States National Museum. Smith-
sonian Institution. Washington.

Carnegie Institution, see Pap. Tortugas Lab.; Publ. Carneg. Instn.; Yearb.
Carneg. Instn.

Camb. Phil. Soc. Proc.: Proceedings of the Cambridge Philosophical Society.
Cambridge.

Cellule.: La Cellule. Recueil de cytologie et d'histologie générale. Louvain.

Cellulose-Chem.: Cellulose-Chemie, (Papierfabrikant. Suppl.) Berlin.

Chem. Abstr.: Chemical Abstracts. New York.

Clin. vet. Milano.: La clinica veterinaria. Milano.

Cold Spr. Harb. Monogr.: Cold Spring Harbor Monographs. Brooklyn, N.Y.

Cold Spring Harbor Symp. Quant. Biol.: Cold Spring Harbor Symposium on
Quantitative Biology, 1933.

Coll. Net.: Collecting Net. Woods Hole, Mass.

Contr. zool. Lab. Univ. Pa.: Contributions from the Zoological Laboratory,
Univ. of Pennsylvania. Philadelphia.

XXXil LIST OF ABBREVIATIONS

Copeia.: Copeia. Published to advance the science of cold-blooded Verte-
brates. New York.

C. R. Acad. Sci. Paris.: Compte rendu hebdomadaire des séances de I’ Acad-
émie des sciences. Paris.

C. R. Acad. Sci. U.R.S.S.: Compte rendu de l’Académie des sciences de
rU.R.S:S.

C. R. Ass. Anat.: Compte rendu de I’Association des anatomistes. Paris and
Nancy.

C. R. Lab. Carlsberg.: Compte rendu des travaux du Laboratoire de Carls-
berg. Copenhague.

C. R. XII* Cong. Int. Zool.: Comptes rendues XII® Congrés international de
la Zoologie. Lisbon, 1935.

C. R. Soc. Biol. Paris.: Compte rendu hebdomadaire des séances et mémoires
de la société de biologie. Paris.

Cytologia, Tokyo.: Cytologia. Tokyo.

Denkschr. Akad. Wiss. Wien.: Denkschriften der Kaiserlichen Akademie der
Wissenschaften. Math.-nat. KI. Wien.

Denkschr. med.-naturw. Ges. Jena.: Denkschriften der Medizinisch-natur-
wissenschaftlichen Gesellschaft zu Jena.

Dtsch. med. Wschr.: Deutsche medizinische Wochenschrift. Leipzig.

Dtsch. tierarzlt. Wschr.: Deutsche tierarztliche Wochenschrift. Hannover.

Dutch East Indies Volkksgesondheid.: Dutch East Indies. Dienst der Volks-
gesondheid in Nederlandsch-Indie. Mededeelingen.

Ecol. Monogr.: Ecological Monographs. Durham, N.C.

Ecology.: Ecology. Brooklyn.

Edinb. New phil. J.: Edinburgh New Philosophical Journal, 1826, 1854,
1855-'64.

Ergebn. Biol.: Ergebnisse der Biologie. Berlin.

Ergebn. Hyg. Bakt.: Ergebnisse der Hygiene, Bakteriologie, Immunitats-
forschung u. experimentellen Therapie. Berlin.

Ergebn. inn. Med. Kinderheilk.: Ergebnisse der inneren Medizin u. Kinder-
heilkunde. Berlin.

Ergebn. Physiol.: Ergebnisse der Physiologie. Wiesbaden.

Ergeb. Zool.: Ergebnisse u. Fortschritte der Zoologie. Jena.

Fauna u. Flora Neapel.: Fauna u. Flora des Golfes von Neapel u. d. angrenz.
Meeresabschnitte. Berlin.

Flora, Jena.: Flora, oder allgemeine botanische Zeitung. Jena, Regensburg.

Folia haemat.: Folia haematologica. Leipzig.

ForschBer. biol. Sta. Plén.: Forschungsberichte aus der Biologischen Station
zu Plon. Stuttgart.

Gegenbaurs Jb.: Gegenbaurs morphologisches Jahrbuch. Leipzig.

Genetics.: Genetics: a Periodical Record of Investigations bearing on
Heredity and Variation. Menasha, Wis.

LIST OF ABBREVIATIONS XXXII

G6ottingen, mathem.-physical. KI.: Nachrichten von der K6nigl. Gesellschaft
der Wissenschaften zu Gé6ttingen. Mathematisch-physikalische Klasse.
Gottingen.

Gréce méd.: Gréce médicale. Syra.

Growth.: Growth. (A journal.) Series of numbered fasciculi at irregular
intervals.

Handb. Vererbungsw.: Handbuch der Vererbungswissenschaft. Berlin.

Illinois biol. Monogr.: Illinois Biological Monographs. Univ. of Illinois.
Urbana.

Indian J. med. Res.: Indian Journal of Medical Research. Calcutta.

Indian med. Gaz.: Indian Medical Gazette. Calcutta.

Indian med. Res. Mem.: Indian Medical Research Memoirs. Calcutta.

Indust. Engng. Chem.: Industrial and Engineering Chemistry. Easton, Pa.

Int. Rev. Hydrobiol.: Internationale Revue der gesamten Hydrobiologie u.
Hydrographie. Leipzig.

Iowa St. Coll. J. Sci.: Iowa State College Journal of Science. Ames.

. Acad. nat. Sci. Philad.: Journal of the Academy of Natural Sciences of

Philadelphia.

. agric. Res.: Journal of Agricultural Research. Washington.

Amer. med. Ass.: Journal of the American Medical Association. Chicago.

. Amer. statist. Ass.: Journal of the American Statistical Association. Boston.

. anat. Paris.: Journal de l’anatomie et de la physiologie normales et

pathologique de I’homme et des animaux. Paris. .

Anim. Behav.: Journal of Animal Behaviour. Boston.

. appl. Physics: Journal of Applied Physics. Menasha, Wis.

Bact.: Journal of Bacteriology. Baltimore.

. biol. Chem.: Journal of Biological Chemistry. Baltimore.

. cell. comp. Physiol.: Journal of Cellular and Comparative Physiology.

Philadelphia.

. Coll. Sci. Tokyo.: Journal of the College of Science, Imp. University of

Tokyo.

. comp. Neurol.: Journal of Comparative Neurology (and Psychology).

Philadelphia.

. comp. Path.: Journal of Comparative Pathology and Therapeutics. Edin-

burgh, London.

. Coun. sci. industr. Res. Aust.: Journal of the Council for Scientific and

Industrial Research, Australia. Melbourne.

. Dairy Sci.: Journal of Dairy Science. Baltimore.

. Dep. Sci. Calcutta Univ.: Journal of the Department of Science of Cal:

cutta University. Calcutta.

. Elisha Mitchell sci. Soc.: Journal of the Elisha Mitchell Scientific Society,

Chapel Hill, N.C.

. exp. Biol.: Journal of Experimental Biology. Cambridge.

. exp. Med.: Journal of Experimental Medicine. New York.

XXXIV LIST OF ABBREVIATIONS

. exp. Zool.: The Journal of Experimental Zoology. Philadelphia.

. Fac. Sci. Tokyo Univ.: Journal of the Faculty of Science, Tokyo Imperial

University. Tokyo. (4) Zool.

. Genet.: Journal of Genetics. Cambridge.

. gen. Physiol.: Journal of General Physiology. Baltimore.

. Helminth.: Journal of Helminthology. London.

. Immunol.: Journal of Immunology. Baltimore.

. industr. Engng. Chem.: Journal of Industrial and Engineering Chemistry.

Easton, Pa.

. infect. Dis.: Journal of Infectious Diseases. Chicago.

. Lab. clin. Med.: Journal of Laboratory and Clinical Medicine. St. Louis, Mo.

. linn. Soc. (Zool.): Journal of the Linnean Society. (Zoology.) London.

. Malar. Inst. India.: Malaria Institute of India, Journal.

. Mar. biol. Ass. U. K.: Journal of the Marine Biological Association

of the United Kingdom. Plymouth.

med. Res.: Journal of Medical Research. Boston.

. Microbiol.: Journal de microbiologie. Petrograd. Moscou.

Morph.: Journal of Morphology (and Physiology). Philadelphia, Boston.

nerv. ment. Dis.: Journal of Nervous and Mental Diseases. New York.

Parasit.: Journal of Parasitology. Urbana, Ill.

. Path. Bact.: Journal of Pathology and Bacteriology. London.

phys. Chem.: Journal of Physical Chemistry. Ithaca, N.Y.

. Physiol.: Journal of Physiology. London and Cambridge.

. Physiol. Path. gén.: Journal de physiologie et de pathologie générale. Paris.

. prev. Med. Lond.: Journal of Preventive Medicine. London.

prev. Med., Oshkosh, Wis.: Journal of Preventive Medicine. Oshkosh,

Wis.

R. Army med. Cps.: Journal of the Royal Army Medical Corps. London.

.R. micr. Soc.: Journal of the R. Microscopical Society. London.

. Sci. Hiroshima Univ. Journal of Science of the Hiroshima University.

Hiroshima, Japan.

J. trop. Med. (Hyg.) : Journal of Tropical Medicine (and Hygiene). London.

Jap. J. Zool.: Japanese Journal of Zoology. Tokyo.

Jber. Fortschr. Anat. EntwGesch.: Jahresberichte tiber die Fortschitte der Ana-
tomie und Entwicklungsgeschichte. Jena.

Jb. wiss. Bot.: Jahrbuch fiir wissenschaftliche Botanik. Berlin.

Jena. Z. Naturw.: Jenaische Zeitschrift fiir Naturwissenschaft. Jena.

Jena. Z. Naturgesch.: Jenaische Zeitschrift fiir Naturgeschichte. Jena.

Kiev. obshch, estest. Zap.: Kievskve obshchestvo estestvoispytatelei Zapiski.
Kief.

Kryptogamenfl. Mark Brandenb.: Kryptogamenflora der Mark Brandenburg
und angrenzender Gebiete. (Botanische Verein der Provinz Brandenburg).
Leipzig.

Matemat. termesz. estesit6.: Matematikai és természettudomanyi éstesito.

ey ey I | SS he | SS ee _ o_o | —a

LIST OF ABBREVIATIONS XXXV

Math. naturw. Ber. Ung.: Mathematische und naturwissenschaftliche Berichte
aus Ungarn. { Berlin: Budapest:} Leipzig.

Math.-nat. Kl., Heidelb. Akad. Wiss.: Mathematisch-naturwissenschaftliche
Klasse, B. Biologische wissenschaften. Heidelberger Akademie der Wissen-
schaften.

Medicine.: Medicine. Baltimore, Md.

Med. Paises calidos. Medicina de los paises calidos. Madrid.

Mém. Acad. roy. Belg.: Académie royale des sciences de Belgique, Mémoires.
Brussels.

Mém. Acad. Sci., Paris.: Mémoires de |’Académie des sciences de I|’Institut
de France. Paris.

Mem. Amer. Acad. Arts Sci.: Memoirs of the American Academy of Arts
and Sciences. Boston.

Mém. Cl. Sci. Acad. polon.: Mémoires de la classe des sciences mathématiques
et naturelles. Académie Polonaise des Sciences et des Lettres. Cracovie.

Mem. Coll. Sci. Kyoto.: Memoirs of the College of Science, Kyoto Imp. Uni-
versity. Kyoto.

Mém. Inst. nat. genev.: Mémoires de I'Institut national genevois. Geneve.

Mem. Inst. Osw. Cruz.: Memorias do Instituto Oswaldo Cruz. Rio de Janeiro.

Mém. Mus. Hist. nat. Belg.: Mémoires du Musée royal d’histoire naturelle
de Belgique. Bruxelles.

Mém. Soc. Phys. Genéve.: Mémoires de la Société de physique et d'histoire
naturelle de Geneve.

Mem. Soc, zool. tchec. Prague.: Ceskoslovenska spolecnost zoologicka (Soci-
etas Zoologica Cechoslovenica). Prague.

Microgr. prép.: Micrographe préparateur; journal de micrograprie générale,
de technique micrographique et revue des journaux francais et étrangers.
Paris.

Microscope.: Microscope. Ann Arbor; Detroit; Washington.

Midl. Nat.: Midland Naturalist (Midland union of natural history societies).
London; Birmingham.

Midl. Nat.: Midland Naturalist. Notre Dame, Ind.

Mikrokosmos.: Mikrokosmos (Deutsche mikrologische Gesellschaft, . . .)
Stuttgart.

Mitt. zool. Inst. Univ. Miinster.: Mitteilungen aus dem Zoologischen Institut
der Westfalischen Wilhelms-Universitat zu Miinster i. W.

Mitt. Zool. Sta. Neapel.: Mitteilungen aus der Zoologische Station zu
Neapel.

Monatsber. preuss. Akad. Wissensch.: Monatsberichte K. preussische Akad-
emie der Wissenschaften zu Berlin.

Monit. zool. ital.: Monitore zoologico italiano. Sienna; Firenze.

Monogr. Inst. Pasteur.: Monographies de I’Institut Pasteur. Paris.

Morph. Jb.: Morphologisches jahrbuch. Leipzig.

Nature.: Nature. London.

XXXVI LIST OF ABBREVIATIONS

Naturwissenschaften.: Naturwissenschaften. Berlin.

Nova Acta Leop. Carol.: Nova Acta Leopoldinisch-Carolinische deutsche
Akademie der Naturforscher. Jena.

Occ. Publs. Amer. Ass. Adv. Sci.: Occasional Publications, American Associ-
ation for the Advancement of Science.

Ohio J. Sci.: Ohio Journal of Science (Ohio State University . . . Ohio
Academy of Science). Columbus.

Ost. bot. Z.: Oesterreichische botanische Zeitschrift. Wien.

Pap. Tortugas Lab.: Papers from the Tortugas Laboratory (Department of
Marine Biology) of the Carnegie Institution of Washington. Washington.

Parasitology.: Parasitology, a supplement to the Journal of Hygiene. Cam-
bridge.

Pasteur Institute, see Ann. Inst. Pasteur; Arch. Inst. Pasteur Afr. N.; Arch.
Inst. Pasteur Algér.; Bull. Inst. Pasteur; Monogr. Inst. Pasteur.

Pflig. Arch. ges. Physiol.: Pfliigers Archiv fiir die gesamte Physiologie d.
Menschen u. d. Tiere. Bonn.

Philipp. J. Sci.: Philippine Journal of Science. Manila.

Philos. Trans.: Philosophical Transactions of the Royal Society. London.

Physics.: Physics. Minneapolis.

Physiol. Rev.: Physiological Reviews. Baltimore.

Physiol. russe.: Physiologiste russe. Moscow.

Physiol. Zo6l.: Physiological Zodlogy. Chicago.

Planta: Planta. (Archiv fiir wissenschaftliche Botanik. Berlin. Abt. E.)

Poult. Sci.: Poultry Science. Ithaca, N.Y.

Proc. Acad. nat. Sci., Philad.: Proceedings of the Academy of Natural
Sciences of Philadelphia.

Proc. Amer. Acad. Arts Sci.: Proceedings of the Academy of American Arts
and Sciences. Boston.

Proc. Amer. phil. Soc. Proceedings of the American Philosophical Society.
Philadelphia.

Proc. Boston Soc. nat. Hist.: Proceedings of the Boston Society of Natural
History. Boston.

Proc. Calif. Acad. Sci.: Proceedings of the California Academy of Sciences.
San Francisco.

Proc. Davenport Acad Sci.: Proceedings of the Davenport Academy of
(Natural) Sciences. Davenport, Iowa.

Proc. Acad. Sci. Amst.: Proceedings of the Royal Academy of Sciences,
Amsterdam.

Proc. Indian Acad. Sci.: Proceedings of the Indian Academy of Sciences.
Bangalore.

Proc, K. Akad. Wetensch.: K. Akademie van Wetenschappen, Amsterdam.
Proceedings of the Section of Sciences (translated).

Proc. Linn. Soc. N.S.W.: Proceedings of the Linnean Society of New South
Wales. Sydney.

LIST OF ABBREVIATIONS XXXVil

Proc. Minn. Acad. Sci.: Proceedings of the Minnesota Academy of Sciences.
Minneapolis.

Proc. nat. Acad. Sci., Wash.: Proceedings of the National Academy of Sci-
ences. Washington.

Proc. Pa. Acad. Sci.: Proceedings of the Pennsylvania Academy of Science.
Harrisburg, Pa.

Proc. R. Irish Acad.: Proceedings of the Royal Irish Academy. Dublin.
London.

Proc. roy. Soc.: Proceedings of the Royal Society. London.

Proc. roy. Soc. Edinb.: Proceedings of the Royal Society of Edinburgh.

Proc. R. Soc. Med.: Proceedings of the R. Society of Medicine. London.

Proc. Soc. exp. Biol. N.Y.: Proceedings of the Society for Experimental
Biology and Medicine. New York.

Proc. zool. Soc. Lond.: Proceedings of the General Meetings for Scientific
Business of the Zoological Society of London.

Protoplasma.: Protoplasma. Leipzig.

Protop.-Monog.: Protoplasma-Monographien. Berlin, 1928+-

Protozoology.: Protozoology, a Supplement to the Journal of Helminthology.
London.

Publ. Carneg. Instn.: Publications. Carnegie Institution of Washington.
Washington.

Publ. Fac. Sci. Univ. Charles.: Spisy Wydavané Prirodovédeckou Fakultou
Karlovy University. Praha.

Publ. Hlth. Rep. Wash.: Public Health Reports. Washington.

Quart. J. exp. Physiol.: Quarterly Journal of Experimental Physiology.
London.

Quart. J. micr. Sci.: Quarterly Journal of microscopical Science. London.

Quart. Rev. Biol.: Quarterly Review of Biology. Baltimore.

Radiology.: Radiology. St. Paul.

Rasseg. faunist. Roma.: Ressegna faunistica. Roma.

R. C. Accad. Lincei.: Atti della R. Accademia dei Lincei. Rendiconti. Cl. di
sci. fis. mat. e nat. Roma.

Rec. Malar. Surv. India.: Records of the Malaria Survey of India. Calcutta.

Rec, zool. suisse.: Recueil zool. suisse. Geneva.

Rep. Brit. Assoc. Adv. Sci.: Report of the British Association for the Ad-
vancement of Science. Bath.

Rep. N. Y. St. Conserv. Comm. Report of the New York State Conservation
Commission. Albany.

Rep. Sleep. Sickn. Comm. roy. Soc.: Report of the Sleeping Sickness Com-
mission. Royal Society. London.

Rep. vet. Res. S. Afr.: Report on (of Director of) Veterinary Research.
Department of Agriculture, Union of South Africa.

Rev. biol. Nord Fr.: Revue biologique du Nord de la France. Lille.

XXXVIIiI LIST OF ABBREVIATIONS

Rev. Microbiol. Saratov.: Revue de microbiologie (et) d’épidémiologie, (et
de parasitologie). Saratov.

Rev. sci. Instrum.: Review of Scientific Instruments. (Optical Society of
America) Rochester, N.Y.

Rev. Soc. argent. Biol.: Revista de la Sociedad argentina de biologia. Buenos
Aires.

Rev. suisse Zool.: Revue suisse de zoologie et Annales de la Société zoolo-
gique suisse et du Muséum d'histoire naturelle de Genéve. Geneva, 1893+

Rif. med.: Riforma medica. Napoll.

Riv. Fis. mat. Sci. nat.: Rivista di fisica, matematica e scienze naturali. Pavia.

Riv. Malariol.: Rivista di malariologia. Roma.

Roux Arch. EntwMech. Organ.: Wilhelm Roux Archiv fiir Entwicklungs-
mechanik der Organismen. Leipzig. (Abt. D, Zeitschrift fiir wissenschaft-
liche Biologie, Berlin.)

Russ. J. Zool.: Zoologicheskii viestnik. Journal Russe de zoologie. Petrograd.

Russk. zool. Zh.: Russkii zoologicheskii zhurnal. Revue zoologique russe.
Moscow.

S. Afr. J. Sci.: South African Journal of Science. Capetown.

S. B. Akad. Wiss. Wien.: Sitzungsberichte der Kais. Akademie der Wissen-
schaften in Wien.

S. B. béhm. Ges. Wiss.: Sitzungsberichte der Kgl. Bohmischen Gesellschaft
der Wissenschaften. Prag.-Math.-Nat. Klasse.

S. B. Ges. Morph. Physiol. : Sitzungsberichte der Gesellschaft fiir Morphologie
und Physiologie in Miinchen. Miinchen.

S. B. Ges. naturf. Fr. Berl.: Sitzungeberichte der Gesellschaft Naturforschen-
der Freunde zu Berlin.

S. B. preuss. Akad. Wiss.: Sitzungsberichte der Kgl. Preussischen Akademie
der Wissenschaften zu Berlin.

Science. Science. New York.

Sci. Prog.: Science Progress; a quarterly review of current scientific investiga-
tion. London, 1894-98; Sci. Prog. Twent. Cent. Science Progress in the
Twentieth Century, a quarterly journal of scientific thought. London,
1906.

Scientia, Bologna.: Scientia, rivista di scienza. Bologna.

Scientia, Sér. biol., Paris.: Scientia. Série biologique. Paris. 99-04

Sci. Mem. Offrs. med. san. Dept. Gov. India.: Scientific Memoirs by Officers
of the Medical and Sanitary Department of the Government of India.
Calcutta.

Sci. Mon., Lond.: Scientific Monthly. London.

Sci. Mon., N.Y.: Scientific Monthly. New York.

Sci. Proc. R. Dublin Soc.: Scientific Proceedings of the Royal Dublin Society.
Dublin.

Sci. Rep. Tohoku Univ.: Science Reports of the Toéhoku Imp. University.
Sendai. S.4, Biology. Sendai.

LIST OF ABBREVIATIONS XXXIX

Sci. Rep. Tokyo Bunrika Daig.: Science Reports of the Tokyo Bunrika
Daigaku. Tokyo.

Scripta bot. Petropol.: Scripta botanica. Leningrad. (Glavnyi botanicheskii
sad).

Smithsonian Institution, see Smithson. misc. Coll.; Bull. U. S. nat. Mus.

Smithson. misc. Coll.: Smithsonian Miscellaneous Collections. Washington.

Soc. geol. France.: Société géologique de France. Paris.

Special Rep. Ser. med. res. Counc.: Special report series, Medical research
council, London.

Stain Tech.: Stain Technology. Geneva, N.Y.

Stat. Rep. N.J.: New Jersey. Statistical Report. Trenton, N.J.

Stazione Zoologica. Naples.: Fauna und Flora des Golfes von Neapel. 1880--

Sth. med. J., Nashville.: Southern Medical Journal. Nashville, Tenn.

Studies Inst. Divi Thomae.: Studies of the Institutum Divi Thomae, Cin-
cinnati, Ohio.

Tabl. zool.: Tablettes zoologiques. Poitiers.

Tijdschr. ned.: Nederlandsch Tijdschrift voor Geneeskunde. Amsterdam.

Trab. Lab. Invest. biol. Univ. Madr.: Trabajos del Laboratorio de investi-
gaciones bioldgicas de la Universidad de Madrid.

Trans. Amer. Fish. Soc.: Transactions of the American Fisheries Society. New
York.

Trans. Amer. micr. Soc.: Transactions of the American Microscopical Society.
Menasha, Wis.

Trans. Amer. phil. Soc.: Transactions of the American Philosophical Society.
Philadelphia.

Trans. Faraday Soc.: Transactions of the Faraday Society. London.

Trans. N. Y. Acad. Sci.: Transactions of the New York Academy of Science.
New York.

Trans. roy. Soc. Edinb.: Transactions of the Royal Society of Edinburgh.
Edinburgh.

Trans. R. Soc. trop. Med. Hyg.: Transactions of the Royal Society of Tropical
Medicine and Hygiene. London.

Trav. Inst. biol. Peterhof.: Travaux de I’Institut des Sciences naturelles de
Peterhof (Biologische Institut zu Peterhof). Peterhof {Petergof}.

Trav. Lab. zool. Sebastopol.: Travaux lu Laboratoire zoologique et Station
biologique de Sébastopol. St-Pétersbourg.

Trav. Soc. Nat. St.-Pétersb. (Leningr.): Travaux de la Société Imp. des
naturalistes de St-Pétersbourg (Leningrad).

Trav. Sta. limnol. Lac Bajkal.: Travaux de la Station Limnologique du Lac
Bajkal.

Trop. Dis. Bull.: Tropical Diseases Bulletin. London.

Univ. Cal. Publ. Bot.: University of California Publications in Botany.
Berkeley.

Univ. Cal. Publ. Zool.: University of California Publications in Zoology.
Berkeley.

xl LIST OF ABBREVIATIONS

Univ. Mo. Stud.: University of Missouri Studies. Columbia.

Unters. Bot. Inst. Tubingen.: Untersuchungen, Botanische Institut. Tubingen.

U. S. Geol. Surv. Terr.: U. S. Geological and Geographical Survey of the
Territories. Washington.

U. S. Public Health Reports, see Publ. Hlth. Rep., Wash.

Verh. dtsch. zool. Ges.: Verhandlungen der Deutschen zoologischen Gesell-
schaft. Leipzig.

Verh. Ges. deutscher Naturf. Artzte: Verhandlung Gesellschaft deutscher
Naturforscher und Artzte. Berlin. (Amtlicher Bericht tber die zweiund-
zwanzigste Versammlung deutscher Naturforscher und Artzte in Bremen.
Bremen.)

Verh. naturh.-med. Ver. Heidelberg.: WVerhandlungen des Naturhistorisch-
medizinischen Vereins zu Heidelberg.

Verh. naturh. Ver. preuss. Rheinl.: Verhandlungen des Naturhistorischen
Vereins der Preussischen Rheinlande, Westfalens u. des Reg.-Bez. Osna-
briick. Bonn.

Wiss. Arch. Landw.: Wissenschaftliches archiv fiir landwirtschaft. Berlin;
Leipzig.

Yearb. Carneg. Instn.: Yearbook of the Carnegie Institution of Washington.

Z. allg. Physiol.: Zeitschrift fiir allgemeine Physiologie. Jena.

Z. Biol.: Zeitschrift fiir Biologie. Miinchen and Berlin.

Z. ges. Anat. 1. Z. Anat. EntwGesch, 2. Z. KonstLehre. 3. Ergebn. Anat.
EntwGesch.; Zeitschrift fiir die gesamte Anatomie. Abt. 1. Zeitschrift
fiir Anatomie u. Entwicklungsgeschichte. Abt. 2. Zeitschrift fiir Konsti-
tutionslehre. Abt. 3. Ergebnisse der Anatomie u. Entwicklungsgeschichte.
Berlin.

Z. ges. Neurol. Psychiat.: Zeitschrift fiir die gesamte Neurologie und Psychia-
trie. Berlin.

Z. Hyg. Infektkr.: Zeitschrift fiir Hygiene. Leipzig, 1886-91; became (Z.
Hyg. InfektKr.) Zeitschrift fiir Hygiene und Infektionskrankheiten. Leip-
zig.

Z. ImmunForsch.: Zeitschrift fiir Immunitatsforschung und experimentelle
Therapie. Jena.

Z. indukt. Abstamm.- u. VererbLehre.: Zeitschrift fiir induktive Abstam-
mungs- u. Vererbungslehre. Berlin.

Z. InfektKr. Haustiere.: Zeitschrift fiir Infektionskrankheiten, parasitire
Krankheiten u. Hygiene der Haustiere. Berlin.

Z. mikr.-anat. Forsch.: Zeitschrift fiir mikrospopische-anatomische Forschung.
Leipzig.

Z. Morph. Okol. Tiere.: Zeitschrift fiir Morphologie und Okologie der Tiere.
(Zeitschrift fiir wissenschaftliche Biologie. Berlin. Abt. A.)

Z. Naturw.: Zeitschrift fiir Naturwissenschaften. Leipzig.

Z. Parasitenk.: Zeitschrift fiir Parasitenkunde. (Zeitschrift fiir wissenschaft-
liche Biologie, Berlin. Abt. F.)

LIST OF ABBREVIATIONS xli

Z. phys. Chem.: Zeitschrift fiir physikalische Chemie, Stéchiometrie und
Verwandtschaftslehre. Leipzig.

Z. Tierz. ZiichtBiol.: Zeitschrift fir Tierziichtung und Ziichtungsbiologie.
Berlin.

Z. vergl. Physiol.: Zeitschrift fiir vergleichende Physiologie. (Zeitschrift fiir
wissenschaftliche Biologie. Berlin. Abt. C.) Berlin.

Z. wiss. Bot.: Zeitschrift fiir wissenschaftliche Botanik. Zurich.

Z. wiss. Mikr.: Zeitschrift fiir wissenschaftliche Mikroskopie und fiir mikros-
kopische Technik. Leipzig.

Z. wiss. Zool.: Zeitschrift fiir wissenschaftliche Zoologie. Leipzig.

Z. Zellforsch.: Zeitschrift fiir Zellforschung und Mikroskopische Anatomie.
Berlin. (Zeitschrift fiir wissenschaftliche Biologie. Berlin. Abt. B [2}.)

Z. Zell.- u. Gewebelehre.: Zeitschrift fiir Zellen- u. Gewebelehre. Berlin.
(Abt. B [1] Zeitschrift fiir wissenschaftliche Biologie, Berlin.)

Zbl. Bakt.: Zentralblatt fiir Bakteriologie, Parasitenkunde u. Infectionskrank-
heiten. Jena.

Zool. Anz.: Zoologischer Anzeiger. Leipzig.

Zool. Anz. Suppl.: Zoologischer Anzeiger Supplement (Bibliographia zoolo-
gica of the Concilium Bibliographicum). Leipzig.

Zool. Jb.: Zoologische Jahrbiicher. 1 Abt. fiir Anatomie. 2 Abt. fiir Sys-
tematik. 3 Abt. fiir allgemeine Zoologie und Physiologie der Tiere. Jena.

Zoologica.: Zoologica. Scientific Contributions of the New York Zoological
Society. New York.

Zool. Zbl.: Zoologisches Zentralblatt. Leipzig.

PROTOZOA
LN BLOL © GICAL
RESEARCH

CHAPTER I

GENERAL CONSIDERATIONS

Gary N. CALKINS

ALTHOUGH Protozoa are known in all parts of the world, and evidence
is at hand that they—in some cases even the same genera—have lived and
thrived in all periods of the earth’s history, little has been accomplished
to show how this remarkable phenomenon of longevity has been brought
about. Within the last century, however, the matter has been the subject
of many studies, both theoretical and experimental, although the latter,
it must be confessed, are nearly always combined with the former.

LIFE AND VITALITY

For many years it has seemed to the present writer that /7fe and
vitality are concepts which have so often been confused that at the pres-
ent time they are held by many biologists, and by most philosophers, to
be synonymous. There is reason, however, especially in connection with
the protoplasm of Protozoa, for distinguishing between them, as I have
maintained in my recent textbook, The Biology of the Protozoa.

It is generally recognized that life cannot be measured nor analyzed
as such, except through its manifestations of vitality. It has long been
known that each type of living thing has a specific organization which
is carried on, subject to adaptations through reactions to the environment,
or by inheritance, from generation to generation. At the present time we
do not know what this finer organization is, but it is assumed that specific
proteins form the basis of species differences, and that these, in combina-
tion with water, carbohydrates, fats, and salts of different kinds, pro-
vide the materials for metabolic activities. Thus we have the possibility
of arriving at at least two concepts; first, the concept of the physical
and chemical make-up, or, in general, the organization; and second, the
concept of that same organization in action. I would apply this second
concept to protoplasm during its activity and would limit the term
vitality to this activity. It follows that life may be defined as specific

4 GENERAL CONSIDERATIONS

organization having the possibility, or the potential of vitality. Such
activity of protoplasm involves the interaction of its component parts
with one another and with the environment. Life thus may be conceived
as static, or analogous to an automobile in its garage, and dynamic, as an
automobile in motion. A dried rotifer is almost, if not entirely static,
but retains its organization; place it in water and it becomes dynamic
within a few minutes. Similarly with an encysted protozodn, which,
within its cyst walls is not freely exposed to water and oxygen but
retains its specific organization and is apparently static and, like a dried
rotifer, it may remain in this desiccated condition for years without losing
its potential of vitality so that, when again placed in water, or culture
medium, it soon emerges from its cyst, develops motile organs and other
adult structures, and begins again its metabolic activities. The protoplasm
within the cyst is undifferentiated, but soon after active metabolism
begins, it becomes differentiated with structures peculiar to the species.
After this the organization changes with each act of a metabolic nature.

FUNDAMENTAL AND DERIVED ORGANIZATION

We have reason, therefore, to speak of a fundamental organization of
protoplasm, characterized by undifferentiated protoplasm of a cyst, or
of an egg, and of a derived organization which comes from the funda-
mental through metabolic activity and is characteristic of the adult and
all of its parts. It follows that death is not of necessity the absence of
vitality but is due to the derangement or breakdown of the organiza-
tion.

Thus all species of Protozoa have within themselves the potential
of an endless existence, subject, of course, to the vicissitudes of the
daily life of the adults, each of which is the custodian of a limited portion
of the fundamental organization.

SOME ECOLOGICAL CONSIDERATIONS

As well known, Protozoa may be found wherever there is moisture
without deleterious substances and the same species may be found in the
littoral waters of the sea and in inland fresh-water lakes, ponds, and
pools. Although many species are cosmopolitan, they tend to accumulate
in certain places where the environments best suit their needs; hence it
is possible to outline certain ecological limitations, although these must

GENERAL CONSIDERATIONS 5

not be too closely limited. We speak, for example, of the water-dwelling
forms, having reference to the great multitude of types which may be
found in exposed waters of the earth’s surface; or of an even greater
number of species which live in the sea at all depths, from the surface
down to 3,000 feet, while some Radiolaria are present in the most ex-
treme depths of the ocean. Where salt and fresh waters mix we find
special groups of brackish water fauna which are represented by thou-
sands of species.

The fresh-water species are so numerous and so varied that some help
is gained by grouping them in “habitat groups” which are adapted more
or less to similar environmental conditions. An attempt to classify such
fresh-water species on an ecological basis was made by Kolkwitz (1909),
although this classification has never been widely used in protozoan
taxonomy. It was based, in the main, on the amounts and conditions of
organic matter and oxygen present in the water. The habitat groups
were described by him under the terms katharobic, oligosaprobic, meso-
saprobic, and polysaprobic types. The first are relatively rare types, being
found in fresh-water springs, running rivers and streams, and wells
which have little organic matter but are rich in oxygen.

Oligosaprobic types are characterized by a small amount of organic
matter but are rich in minerals. Hence lakes and reservoirs become the
haunts of chlorophyll-bearing forms in particular, which often accumu-
late to incredible numbers and frequently cause disagreeable odors and
tastes in drinking waters and render them unpotable.

Mesosaprobic types are the most common of all fresh-water Protozoa,
for here active oxidation is going on and organic matter, in the presence
of sunlight, is in all stages of decomposition. Here the microscopist finds
his richest collecting place.

Polysaprobic types, finally, live in waters with little or no free oxygen,
but with an abundance of sulphureted hydrogen, carbon dioxide, and
other products of putrefaction which are advertised by their foul odors,
due to the gases which are formed. Here belong Lauterborn’s (1901)
“sapropelic fauna,’ which live, for the most part, as anaerobes, and
which are often characterized by fantastic shapes and inability to live
under aérobic conditions.

Another group have an almost terrestrial habitat and may be found in
damp moss, sphagnum, or similar environments. A few types of ciliates

6 GENERAL CONSIDERATIONS

and flagellates, and some testate rhizopods, may be found here, but one
must be an optimistic collector, and an opportunist, to get good results.

It is somewhat the same with soil-dwelling Protozoa, among which it
is to be expected that water-dwelling forms would occasionally be found
and interpreted as casual soil-dwelling types. Sandon (1927), however,
has shown that there is a characteristically well-defined group of forms
living in this environment, although, as would be expected, there is a
wide difference in soils, both as to depth of occurrence of Protozoa and
chemical make-up. In arable soils, Sandon finds that not only are they
most abundant, but that they live at a depth of four to five inches and,
for the most part, are bacteria eaters. He, with other observers, has
described some seventy-five species of flagellates as fairly common; and
about eighty-five species of rhizopods and ciliates which are less com-
mon. There is not much evidence that life in the soil leads to any particu-
lar type of morphological adaptations, but there is a possibility that species
adapted to life deep in the soil are already partly anaerobic, and such
forms may more easily become parasitic.

Lackey (1925) enumerates no less than nineteen common forms of
Protozoa which live in the sewage of Imhof tanks, while five common
species of rhizopods, four of ciliates, and about twenty less common
species of flagellates are occasionally found.

So-called coprozoic forms are rarely segregated, but may be found
more or less sporadically almost anywhere on the earth’s surface. These
are Protozoa which are taken into the digestive tract by all animals, and,
as encysted forms, become stored up in the intestine until they are passed
out with the feces. In water, such cysts accompanied by nutrient material
from the feces, develop into adult and active forms which may be mis-
taken by the unwary as entozoic parasites. So great and complete is the
specificity of internal parasites, however, that such cysts, while not repre-
senting parasites of the host providing the feces, may nevertheless be
cysts of active parasites of other hosts which now pass with disastrous
results, into the digestive tract of a new definitive host by which they
have been eaten with contaminated food and water.

In numbers of species there is little doubt that the free-living and
water-dwelling Protozoa stand first, but the parasitic forms make a
close second, for no type of animal is free from the possibility of in-
fection by one or more species of parasitic Protozoa.

GENERAL CONSIDERATIONS v

While parasitism will be dealt with by others in this volume (see
Becker, infra, Chapter XVII; and Kirby, zvfra, Chapters XIX, XX),
I will speak here merely of one or two types of adaptations which have
arisen as a result of this mode of life. Ectoparasites and endoparasites
have developed somewhat differently, as a result of their different modes
of life and different needs. In common, they all possess the first great
need of all parasites, viz. reproductive power, thus obeying a first law
of nature to the effect that the number of offspring produced should vary
according to the difficulties encountered in their youth and during de-
velopment to maturity.

Adaptations of ectoparasitic types are mainly morphological, and here
some striking structures may be developed, as a few illustrations will
show. All may have a special feeding advantage by being transported
from place to place; or when attached to gills or other structures of their
hosts, they are continuously bathed by food-bearing currents of water. At-
tached forms live on algae, exoskeletons and appendages of arthropods,
shells of molluscs, or gill filaments and gill bars of all kinds of water-
dwelling animals. Thus we find species of Zoothamnium or of Lagen-
ophrys on the legs of Gammarus, while species of Spzrochona or Den-
drocometes are on the gill filaments of the same hosts, or on Asellus.
The suctorian Trichophrya adheres like a saddle to the gill bars of Salpa,
while the vorticellid El/obiophrya encircles a gill filament of the lamelli-
branch Donax vittatus by the union of two branch outgrowths of a more
common adhesion disc (see Kirby, Fig. XXX). The latter is almost
always provided with hooks or suckers, or both, to form the “scopula,”
as in species of Trichodina or Cyclochaeta, parasites on Hydra. A special
thigmotactic reaction appears to keep the ciliate Kerona pediculus on the
surface of Hydra fusca. Such forms seem to have no ill effects on their
hosts and scarcely qualify as parasites. Schréder calls them “‘epibionts.”’
Real ectoparasites are rare, as a matter of fact, and they are little differ-
ent from free water-dwelling forms in structure. They do occur, how-
ever, especially on fish hosts. The flagellate Costa necathrix increases to
such numbers that normal functions are impeded, and young fish are
frequently killed. Ichthyophthirius multifiliis bores into fish skin and
brings about distributed ulcerations which may be fatal.

Endoparasites may be more destructive, and, while they are relatively
simple morphologically, they may be highly differentiated physiologically.

8 GENERAL CONSIDERATIONS

Knowledge of the life histories of endoparasites, particularly those of
man, has grown amazingly, and prophylaxis has grown with it (see
Becker, 7nfra, Chapter XVII; and Kirby, svfra, Chapter XIX-XX; and
for immunity, see Taliaferro, snfra, Chapter XVIII).

SOME HISTORICAL FACTS

Each of the myriads of species represented in this diversity of habitats
has its own specific fundamental organization which carries the possibility
of indefinite life in the future. We cannot visualize the conditions under
which life came into being in times past, but we can observe, study,
describe, and in part measure the manifestations—vitality—which have
kept it going and enabled it to withstand all of the vicissitudes of nature,
through upheavals, floods, droughts, and other symptoms of the might
of nature which have played so prominent a part in the history of the
earth’s activity since the dawn of life.

We have as a basis for such study the vast mass of knowledge which
has accumulated since Protozoa were introduced to science by the Dutch
naturalist Leeuwenhoek in the latter part of the seventeenth century.
Many different kinds were soon recognized, and this recognition led to
classifications and logical groupings in general which facilitated the dis-
covery and descriptions of new species, modes of life, adaptations to
changing conditions, and the like.

Almost from the time of their discovery, the Protozoa have played
an important part in problems of general biological interest. Spontaneous
generation, for example, or origin of living things from nonliving mat-
ter, which was popularly and generally believed up to the sixteenth
century, had received hard knocks from the experiments of Redi, Spal-
lanzani, Harvey, and other scientific men. All life from life, all living
things from eggs (omne vivum ex ovo) supplanting the common belief
regarding generation.

The use of the microscope, revealing a novel and marvelous world
of living things, gave a new lease of life to the theory of spontaneous
generation. Appearing in containers of pure water, it was asked ‘‘How
could such water become animated with living things if these had not
arisen there by spontaneous generation?” The problem, thus reopened
with the discovery of Leeuwenhoek’s animalcula, was not solved until
near the end of the nineteenth century by the careful work of Pasteur,

GENERAL CONSIDERATIONS

Lister, and a growing school of biologists who pushed back farther and
farther the organisms supposed to arise de novo. Lower invertebrates,
algae and Protozoa, and finally bacteria, one after the other forming the
fighting lines of the army of ignorance, now fell back, little by little,
before the slow but sure advance of science.

In connection with this theory of spontaneous generation, a novel
conception sprang up with the observations of the French naturalist
Buffon in 1749. This conception, expanded by Needham (1750), was
a suggestive forerunner of the cell theory outlined by Schleiden and
Schwann in 1839-40. The wealth of different forms of life and the
numbers of each type in natural waters in which decomposition was under
way, was interpreted by Buffon and Needham as the result of disintegra-
tion of animals and plants in water and the resulting liberation of myraids
of “organic particles’ (Buffon) of which such animals and plants were
composed.

Protozodlogy, like cytology, owes its birth and development to the
use of the microscope. There has been much discussion and much diver-
sity of opinion over the discovery of the microscope, meaning the com-
pound instrument, the significant principles of which have been adapted
and improved until the beautiful microscopes of today are the outcome.
Woodruff (1939) writes:

Galileo (1610) was the first to use the instrument,—but the first figures
ever made with the aid of a compound microscope to appear in a printed
book, were by Francesco Stelluti in 1625. But an Englishman, Robert Hooke,
was the first to realize to the full the importance of using instruments which
increase the powers of the senses in general and of vision in particular, and
to express it convincingly in 1665, in a remarkable book: the ‘‘Micrographica.”’
Here he described and emphasized for the first time, the “little boxes or

cells” of organic structure, and indelibly inscribed the word ‘‘cell’’ in bio-
logical literature. (Woodruff, Joc. cit., p. 2.)

As stated above, the real discoverer of the Protozoa was a Dutch mi-
croscopist, Anton von Leeuwenhoek (1632-1723) who, using crude
lenses of his own make, was one of the first to apply the microscope to
scientific investigation. His contributions to microscopic anatomy and to
physiology, inaugurating, as they did, the invaluable services of the
microscope in biological research, marked an epoch in the history of
science. In a letter to the Royal Society in 1675, Leeuwenhoek wrote that
he had discovered

10 GENERAL CONSIDERATIONS

living creatures in rain water which had stood but four days in a new
earthen pot, glased blew within. This invited me [he continues} to view
this water with great attention, especially those little animals appearing to
me ten thousand times less than those represented by Mons. Schwammerdam
and by him called Water fleas or Water-lice, which may be perceived in the
water with the naked-eye. The first sort by me discover’d in the said water,
I divers times observed to consist of 5, 6, 7, or 8 clear globuls, without
being able to discern any film that held them together, or contained them.
When these animalcula or living atoms did move, they put forth two little
horns, continually moving themselves. The place between these horns was
flat, though the rest of the body was roundish, sharp’ning a little towards
the end, where they had a tayl, near four times the length of the whole
body, of the thickness (by my microscope) of a spiders-web; at the end of
which a globul, of the bigness of one of those which made up the body;
which tayl I could not perceive, even in very clear water, to be mov’d by
them. These little creatures, if they chanced to light upon the least filament
or string, or other such particle, of which there are many in water, especially
if it hath stood some days, they stook entangled therein, extending their
body in a long round, and striving to dis-intangle their tayls whereby it
came to pass, that their whole body lept back towards the globul of the
tayl, which then rolled together Serpent-like, or after the manner of copper
or iron-wire that having been wound about a stick, and unwound again,
retains those windings or turnings. This motion of extension and contraction
continued awhile; and I have seen hundreds of these poor little creatures,
within the space of a grain of gross sand, lye cluster’d in a few filaments.
(From Calkins, 1901, p. 5.)

This is the first description of a protozoén; and though the descrip-
tion is incomplete, it undoubtedly refers to a species of Vorticella. Leeu-
wenhoek observed several other forms, but their identity is uncertain.

Leeuwenhoek allowed his imagination to see what his eyes could not.

When we see [said he} the spermatic animalcula [spermatozoa} moving by
vibrations of their tayls, we naturally conclude that these tayls are provided
with tendons, muscles, and articulations, no less than the tayls of a dormouse
or rat, and no one will doubt that these other animalcula which swim in
stagnant water [Protozoa} and which are no longer than the tayls of the
spermatic animalcula, are provided with organs similar to those of the
highest animals. How marvellous must be the visceral apparatus shut up
in such animalcula! (Quoted from Dujardin, 1841, pp. 21-22.)

In a letter a year later Leeuwenhoek further says: ‘“The fourth sort
of little animals . . . were incredibly small; nay, so small, in my sight
that I judged that even if one hundred of these very wee animals lay

GENERAL CONSIDERATIONS vi

stretched out one against another, they could not reach to the length of
a grain of coarse sand.” Later he discovered parasite Protozoa in man
and beast, and bacteria in the human mouth. Of course Leeuwenhoek
made many other discoveries during his long life—his studies were not
confined to animalcules—but it is enough that he is justly regarded as
the Father of Protozodlogy and Bacteriology (Woodruff, loc. cit., p. 3).

Obviously an immense field awaited intensive study, and this was be-
gun in a desultory way by many amateur and professional biologists dur-
ing the closing decades of the eighteenth century, the outstanding con-
tributions being made by O. F. Miiller in 1773 and 1786. And then
over half a century passed before Ehrenberg, in 1838, and Dujardin, in
1841, afforded a sufficiently broad view of the ‘‘simple’’ animals to justify
the establishment of the phylum Protozoa by von Siebold in 1845
(Woodruff, Joc. cit., p. 4).

Miiller, adopting the Linnzan nomenclature, described and named
some 378 species, of which about 150 ate retained today as Protozoa.
His classification was the first successful attempt to bring order out of
the heterogeneous collection of forms included under the name animal-
cula. He used Ledermiiller’s (1760-63) term Infusoria for the name of
the entire group, which he placed as a class of the worms (see Biitschli,
1883, p. 1129). While he eliminated many inaccuracies, he confirmed
the substantial observations of the earlier observers, extending many of
them to all groups of the Protozoa. He ascertained the presence of an
anus, showed that many Infusoria are carnivorous, and observed the
process of conjugation, his description of the latter being more accurate
than that of any of his followers until the time of Balbiani, in 1858-59.

Like his predecessors, Miiller included among the Protozoa many other
organisms, placing here diatoms, nematode worms, Distomum larvae,
and larval forms of coelenterates and molluscs. The majority of these
miscellaneous forms were, however, properly classified before 1840,
while finally spermatozoa (discovered by Ludwig Hamm, who is said
to have been a pupil of Leeuwenhoek) and which had been universally
regarded as animalcula inhabiting the seminal fluid, were withdrawn
during the last century.

Following John Hill (1752), Miller did not regard the Protozoa as
complicated animals, but considered them as the simplest of all living
beings, composed of a homogeneous gelatinous substance, a view in

12 GENERAL CONSIDERATIONS

which he was followed by a majority of the “‘nature-philosophers,” most
of whom gave little or no attention to the Protozoa, but, accepting Miul-
lers’ work as final, based many of their speculations upon it.

It is rather remarkable that fifty years or so later, when the biological
atmosphere was saturated with the idea of the cell theory, the justly
famous microscopist of Europe, C. G. Ehrenberg (1795-1876), using
much finer achromatic lenses, should have returned to the crude view
of Leeuwenhoek, assigning to the Protozoa a system of minute but com-
plete organs. His conclusions on Protozoa were brought together in one
great work, the title of which alone shows his point of view (Dve
Infusionsthierchen als vollkommene Organismen, 1838). From the sup-
posed possession of many stomachs he gave to one of his groups the
name Polygastrica or Magenthiere, making it a sharply defined class
of the animal kingdom. One of the sub-classes in which these supposed
stomachs were apparent he called the Enterodela, while all other forms he
included as Anentera. The red pigment spots of many forms were inter-
preted as true eyes, but as eyes could not be conceived without an ac-
companying nervous system, he sought for nerve ganglia in different
organisms and found what he was looking for in a species of Astasia.
He described the “‘eye”’ in this form as seated upon a “spherical granular
mass’ which he considered as equivalent to the suprapharyngeal ganglion
of the rotifers. The myonemes in the stalks of Vorticella, in Stentor, and
in many other ciliates he interpreted as muscles. Pigment spheres and
protoplasmic granules were described as ovaries, the nucleus as a testis,
while the contractile vacuole was at first regarded as a respiratory organ
(see Weatherby, infra, Chapter VII).

A formidable opponent of Ehrenberg soon appeared in France—Felix
Dujardin, who, influenced by long study of the Foraminifera, came to
the conclusion in 1835 that these rhizopods, which up to that time had
been classified with the cephalopod molluscs, were in reality the simplest
of animal organisms, composed of a simple homogeneous substance to
which he gave the name Sarcode.

Dujardin is best known by his systematic treatise on the Protozoa which
he published in 1841, and in which he laid the basis for the modern
classification of these unicellular forms. The first suggestion that Proto-
zoa might be single cells was made by Meyen (1839), who compared the
infusorian body with a single plant cell. But, according to Biitschli

GENERAL CONSIDERATIONS a

(1883), the cell theory was first applied directly to the Protozoa by Barry
(1843), who asserted that Momnas and its allies among the flagellates
were single cells, and that the nucleus found within them was the equiva-
lent of the cell nucleus of higher forms. At the same time Barry expressed
the view that cells increase only by division, and he compared the proc-
esses of multiplication in Volvox and Chlamydomonas with the cleavage
of eggs (cf. Buitschli, 1887, p. 1152).

Barry’s view was accepted, in part, by Owen, who thought, however,
that the Infusoria could not be included with the flagellates as single
cells because of their differentiation. It was von Siebold (1845) who
finally asserted the unicellular nature of all Protozoa.

Balbiani’s researches on the life history of Protozoa at first led him
into a curious error, a reminiscence, apparently, of Ehrenberg’s and the
older point of view. O. F. Miller had observed and had correctly in-
terpreted conjugation in different forms, but his successors down to Bal-
biani regarded this as incorrect, maintaining that Miiller had seen only
stages in simple division.

Balbiani (1861) returned to Miller’s view, and clearly stated that,
in addition to simple division, another and a sexual method of reproduc-
tion occurs. His interpretation of the sexual organs of the Protozoa was
given in 1858, when he maintained that the larger of the two kinds
of nuclei of Infusoria, the macronucleus, is the ovary, and that the smaller
one, the micronucleus, is the testis. He saw and pictured the striped ap-
pearance of the micronucleus prior to its division and interpreted the
stripes as spermatozoa. The eggs were said to be fertilized in the macro-
nucleus, and then deposited on the outside where they developed into
new ciliates. Stein at first (1859) opposed this assumption, but in the
second volume of his work on the Infusoria (1867), misled by his own
Acineta theory, in which certain Suctoria were thought to be stages in
the life cycle of certain ciliates, he practically adopted it, maintaining
however, that the young forms developed first in the nucleus and only
later left the mother organism. Biitschli (1873) was apparently the first
to point out Balbiani’s error, and in his epoch-making work of 1876,
after demonstrating the striped appearance of the nucleus in egg cells
(mitotic figure) during division, he concluded that the “‘stripings’” which
Balbiani held to be spermatozoa were no other than this striated con-
dition of the nucleus during division. Biitschli further showed that dur-

14 GENERAL CONSIDERATIONS

ing conjugation the macronucleus disintegrates, and that the parts which
Balbiani had considered eggs are resorbed in the protoplasm, the whole
nucleus being replaced by one of the subdivisions of the Nebenkern
(micronucleus). Biitschli’s interpretation of the process of conjugation
was equally happy. After observing that continued asexual division of
certain forms resulted in decreased size and a general “‘lowering of the
life energy,” he concluded that the function of conjugation is to bring
about a rejuvenescence (Verjungung) of the participants (see Turner,
infra, Chapter XII).

In the same year Engelmann (1876) obtained very similar results.
Quite independently of Biitschli he proved the error of Balbiani’s view,
and came to the conclusion not far different from Biitschli’s: ‘“The con-
jugation of the Infusoria,”’ he said, ‘‘does not lead to reproduction
through eggs, embryonic spheres, or any other kind of germ, but to a
peculiar developmental process of the conjugating individuals which
may be designated Reorganization (loc. cit., p. 628).

THE USE OF CULTURES

Toward the end of the last century new methods of studying Pro-
tozoa gradually evolved; at first structures, or morphological considera-
tions were predominant; then the uses of these structures led to a general
treatment of physiology, and a vast literature on the functions of differ-
ent parts of Protozoa grew up. All of this, in the main, was founded
upon observations and little was done in the experimental field of proto-
zoan research. Staining technique aided, and great strides were made
in knowledge of the microchemistry of all classes of Protozoa. Variations
in structures and in functional activities became apparent, and the con-
ception of the life cycle, first definitely outlined by Schaudinn (1900)
as a series of forms and activities consecutively produced and performed,
was recognized as characteristic of every species (see Kofoid, zmfra,
Chapter XI). The larger fields of study thus inaugurated have become
the starting point for many lines of research, and the single individual
has long since ceased to be the most important goal in study of Protozoa.

Any work on the life history, or portion of it, begins with observa-
tions on food and feeding habits of the protozoén in question. Experi-
ments must first be undertaken to find a suitable culture medium upon

GENERAL CONSIDERATIONS 15

which to grow the chosen form. The most universal of such media, in
all probability, is the “Shay infusion,” in which hay in water, preferably
after boiling, is allowed to stand for a certain time for bacteria to grow
before the protozoén is placed in it. If this is a satisfactory medium, the
organism will respond by dividing at a certain rate. After twenty-four
hours the culture medium is replaced by fresh medium, made in the
same way. In this manner the culture is inaugurated and carried on, and
mass cultures are provided and sustained from the reserve individuals that
are produced every day. In this manner material is produced for study
of individual structures and for various activities characteristic of the
different phases of the life cycle.

In many inaugural cultures made in this way, the outcome is different.
No culture is started, or at best a very poor one. The reasons for this
are manifold. Usually in such cases the medium turns out to be a suit-
able culture medium for some types of organisms, primarily bacteria,
which are noxious to the protozo6n under study. Hence a knowledge of
bacteriological technique is valuable in determining the proper bacterial
food to be used (see Kidder, zfra, Chapter VIII).

A successful culture of a ciliated protozoén, for example, provides
ample material for study of structures and functions; for encystment;
or for the minutiz of cell division, conjugation, sex phenomena, and the
like. The appearance of derived structures of all kinds may be followed
in sequence, from their origin in the fundamental organization as it ap-
pears in the recently encysted individual, to the active adult. Euplasmatic
and alloplasmatic materials and their functional purposes in the cell may
be determined, and the investigator proceeds with the confident expecta-
tion that plenty of material will be on hand for future study.

Very often the organism to be studied is carnivorous, and it is neces-
sary to provide suitable food material, which must be cultivated for the
purpose. Thus Actinobolina radians lives on Halteria grandinella,
Didinium nasutum on Paramecium, and Spathidium spathula on Col-
pidium, and practically pure cultures of these food organisms must be
kept on hand. For many purposes bacteria-free cultures must be pre-
pared, and for this a knowledge of bacteriological technique is not only
desirable but essential (see Hall, ivfra, Chapter IX; Kidder, ifra,
Chapter VIII).

16 GENERAL CONSIDERATIONS

FACTORS INFLUENCING LONGEVITY

The first factor in longevity is the set of functions which the isolated
individual must perform in its daily life. It must react to stimuli, it
must digest its food, it must excrete its waste matters (see Weatherby,
infra, Chapter VII), and it must experience the processes involving
katabolism. The euplasmatic structures having to do with these func-
tions gradually lose their vitality, and, perhaps as a consequence of this,
changes in activity are set up which lead to the second factor in lon-

Figure 1. Uronychia
transfuga, with giant cirri,
membranelles for swim-
ming, ten macronuclear seg-
ments, and single micro-
nucleus. (After Calkins,
1933.)

gevity—reorganization through cell division. These changes have only
within the last few decades been recognized and studied.

With every activity of the euplasmatic constituents of the protoplasmic
make-up, the derived organization is changed. These changes may be
studied day by day, and their significance in the life history of the organ-
ism under culture may be ascertained. Thus we learn that the macro-
nucleus is not a portion of the fundamental organization, but is one of
the derived organs of the ciliated Protozoa. These finer changes of the
organization are difficult to note in the living animal, on any morpho-
logical basis, but physiologically it is possible to show that the organiza-
tion is not equally responsive at all stages between divisions, and the
implication is that protoplasmic changes must have taken place.

A merotomy experiment indicates this. A marine ciliate—Uronychia

GENERAL CONSIDERATIONS icy

transfuga (Fig. 1)—is cut transversely through the center so that ap-
proximately half of the thirteen or fourteen beads which constitute the

\\ LW) 3

(ACAD

Figure 2. Uronychia transfuga, merotomy and regeneration. 1. Cell immediately after
division, cut as indicated; 2, Fragment A of 1, three days after the operation, no re-
generation; 3. cell cut five hours after division; 4, fragment A of 3, three days after
operation, no regeneration; 5, cell cut at beginning of division as indicated into prospec-
tive fragment A, B, and C; A’, B’, C’, fragments A, B, and C twenty-four hours after
the operation; fragment A regenerated into a normal but amicronucleate individual
(A’); B, C, divided in the original division plane forming a normal individual C’ and
a minute but normal individual B’. (After Calkins, 1933.)

macronucleus are left in each of the two halves, while the micronucleus
is left undisturbed in one half. If the operation is made on an individual
three to five hours, or less after the last previous division, the fragment

18 GENERAL CONSIDERATIONS

with the micronucleus regenerates perfectly, but the amicronucleate frag-
ment, while it may live for a few days, never regenerates the missing
structures. The same result is obtained if the organism cut is from ten
to fifteen hours old. If, however, individuals older than this are cut in
the same manner, an increasing percentage of complete regenerations,
varying with increasing age, results (Fig. 2). At the time of the experi-
ment the interdivisional period was twenty-six hours. If individuals
were cut after twenty-one hours of age, regeneration of the amicronu-
cleate individuals was invariable. The experiment indicates a progres-
sive differentiation in respect, at least to the power to regenerate and,
to that extent, a change in organization. Furthermore, if one individual
is similarly cut while the two daughter cells at division are still con-
nected, or shortly afterwards, there is no regeneration of the amicronu-
cleate fragment. This indicates that the condition which underlies the
power to regenerate is lost with processes of division and is not regained
until the young cell has undergone a considerable period of normal
metabolism (Calkins, 1911). This experiment was confirmed by Young
(1922) (see also Summers, infra, Chapter XVI).

CHANGES WITH METABOLISM

There is an accumulating amount of morphological evidence that a
derived structure, such as the macronucleus, is constantly changing with
continued metabolism. In Uroleptus halseyi reorganization after con-
jugation requires from four to five days for completion. At first there
is no chromatin in the young macronucleus, which stands out clearly
in the young organism, and attempts to stain it after fixation are futile.
By use of the Feulgen nucleal test, however, the chromatin reaction be-
comes increasingly intense and the chromatin granules more distinct
toward the end of the reorganization period; the nucleus disappears
from sight in the living organism, but now stains intensely with any
nuclear dye. The young macronucleus divides three times with the first
division of the ex-conjugant and each of the daughter cells receives
eight macronuclei, the chromatin staining deeply in all of them. This
chromatin is in the form of discrete granules of similar form and uni-
form size, but during the interdivisional period there appear a few
(three to five) larger granules, which are dissolved by the Feulgen

—

f
—_=
a
ee &
<L
ae

Figure 3A. Uroleptus mobilis Engelm. Stages in the fusion of the macronuclei prior to
cell division. (After Calkins, 1933.)

Figure 3B. Uroleptus mobilis Engelm. Further stages in preparation for division. (After
Calkins 1933.)

GENERAL CONSIDERATIONS 21

method, leaving vacuoles in the nuclear substance.
Toward the beginning of the following period of
division, these X-granules, as they were called, be-
come larger and more numerous, and collect, form-
ing a disc which extends across the nucleus nearer
one end than the other. This disc stains with acid
dyes and is entirely dissolved by the Feulgen hy-
drolysis. It forms the familiar Kernspalt of the
ciliate macronucleus and its substance appears to
act as a catalyst, for the smaller portion of the
nucleus between the disc and the nearer end of
the nucleus separates from the larger moiety (Figs.
3A, 3B; Fig. 4), and eight of these large frag-
ments now unite in the cytoplasm to form a single
division nucleus, while the eight smaller fragments,
with their contained X-bodies and chromatin, are
resorbed in the cell.

Here, then, is evidence of metabolic activity and
change leading to the phenomena of cell divi-
sion.

REORGANIZATION OF THE MACRONUCLEUS AND
OTHER DERIVED STRUCTURES IN CILIATA

Analogous processes of chromatin elimination,
which numerous observers have referred to as evi-
dence of “nuclear purification,’ occur in different
ways in other ciliates. Kidder (1933) described a
core of modified chromatin accumulating in the
center of the macronucleus of Concho phthirus ( Kid-

Figure 4. Urolep- ane
ie ee cae deria) mytili (Fig. 5). This core condenses into a

X-bodies, chromatin small deeply staining ball which, upon division of

climination, and nu- macronucleus, remains for a time in the connecting
clear cleft, in prep-

aration for division Strand of the daughter nuclei, but ultimately dis-
of the macronucle- appears in the cytoplasm. A similar protrusion,

. (After Calkins, Sis :
ee) eee veferted to only incidentally by Rossolimo and

22 GENERAL CONSIDERATIONS

Jakimowitch (1929), occurs in C. steenstru pi, where it is in the form
of a finely granular substance which comes from the macronucleus and
remains for a time between the nuclear halves after division, but ulti-
mately disappears in the cytoplasm.

There is reason to believe that this phenomenon has something

Figure 5. Conchophthirus (Kidderia) mytili, Extrusion of chromatin during division.
(From a preparation made by Dr. G. W. Kidder.)

to do with restoring full metabolic powers of the macronuclear chroma-
tin, possibly by the elimination of waste products of chromatin activity.

Another process of macronuclear reorganization during division which
does not involve the visible elimination of nuclear substance, occurs in
species of the families Aspidiscidae and Euplotidae of the Hypotrichida.
At the approach of division in Aspidisca (Fig. 6), according to Sum-
mers (1935), the area of reorganization first appears as a wedge-shaped
cleft at the approximate center of the convex side of the C-shaped

GENERAL CONSIDERATIONS 25

macronucleus. A small granule in the center of the cleft represents the
first of the reorganized nuclear material. As the cleft pushes across the
diameter of the nucleus, the central reorganized chromatin increases in
amount. Two separate “reorganization bands’’ result when the wedge-
shaped cleft reaches the opposite side of the macronucleus. These bands
then move in opposite directions, traversing the entire macronucleus and
disappearing at the two ends. The increasing zone of staining chromatin
granules, is quite different in appearance and in staining capacity from
the chromatin in parts of the nucleus which have not been traversed

Figure 6. Aspidisca lynceus. Origin and further history of the reorganization bands.
(After Summers, 1935).

by the bands. After disappearance of the bands at the ends, the nucleus
condenses to form the typical division macronucleus of Asp7disca. Here
again, therefore, there has been a physical change in the chromatin and a
change that is brought about through activity of substances which form
the nuclear cleft.

A similar process takes place in the genus Evp/otes, the reorganization
bands of which have been studied by numerous investigators. Griffin
(1910) was the first to describe them fully as ‘reconstruction bands,”
but by later writers, beginning with Yocom (1918), they have been
known as “reorganization bands.” In Ezplotes the bands begin one at
each end of the macronucleus and proceed toward the middle, where
they meet and disappear. There is no unanimity of opinion as to what
takes place during this passage of the bands, but all agree that some re-
organization of the chromatin occurs. Griffin (/oc. cit.) expressed the
opinion that all of the chromatin is disolved and later re-formed without
the erstwhile impurities. Yocom (Joc. cit.), on the other hand, holds that

24 GENERAL CONSIDERATIONS

the chromatin does not go into solution in that part of the band known
as the “solution plane,” but appears there as closely packed granules
and then passes to the part called the ‘reconstruction plane,” not as
precipitated granules, but as granules which have undergone some
physical or possibly chemical change. Turner (1930), working on Ez-
plotes patella, cites the absence of granules in the solution plane and re-
gards the substance of this plane “‘as in the state of a colloidal solution.”
The chromatin reticulum in the center of the nucleus he interprets as in
a continuous phase, while the karyolymph is dispersed. After action in
the reorganization bands this condition is reversed, the chromatin gran-
ules being in the dispersed phase and the karyolymph continuous. Phe-
nomena of an exactly similar nature have been observed in Diophrys,
Stylonychia, and a host of other forms so that little doubt can be enter-
tained as to the probability that this is a highly critical period in the daily
life of a ciliated protozoan.

At the time of division, not only in the macronucleus but throughout
the organism there is a wave of general house cleaning. Morphological
parts which have been active in the metabolic reactions receive attention
and all are built up afresh. This principle was recognized and applied
to all parts of the cell by Wallengren in 1900 and again in 1901 when
he wrote his illuminating paper in German.

In the Hypotrichida in particular, in which there is no covering of cilia
but instead motile organs called cirri, of a more complex type, Wallen-
gren was the first to show, in great detail, that the old cirri, just prior
to cell division, are gradually resorbed, while new ones arising from
the cortex at an adjacent spot, grow out slowly to take their places. In
like manner cilia are also replaced, and thus it comes to pass that, inside
and outside, the active organs of a ciliate are composed of new materials
derived from the fundamental organization contained in every protozo6n.

It is not only the ciliates that possess this apparent fountain of eternal
youth; other groups of Protozoa manifest similar, if not identical phe-
nomena.

With few exceptions, cell division in flagellates is longitudinal, be-
ginning as a rule at the anterior or flagellar end, the cleavage plane
passing down through the middle of the body. As the process continues,
the two daughter cells separate and usually come to lie in one plane, so
that final division appears to be transverse.

GENERAL CONSIDERATIONS 25

As there are few details in the structure of a simple flagellate on
which to focus attention, descriptions of division processes are practically
limited to the history of the nucleus, kinetic elements, and the more
conspicuous plastids. Here, in the main, are fairly prominent granules
of different kinds, which divide as granules, and, save for the chromatin
elements of the nucleus, without obvious mechanisms (see MacLennan,
infra, Chapter III).

In the simpler cases there is little evidence that can be interpreted to
indicate reorganization at the time of division, and that little is con-
fined to the motile organs. In the more complex forms, however, there
is marked evidence of deep-seated changes going on within the cell.

The earlier accounts of cell division in the simpler flagellates de-
scribed an equal division of all parts of the body, including longitudinal
division of the flagellum, if there were but one, or equal distribution
if there were more. One by one such accounts have been checked by
use of modern methods, until today there remains very little substantial
evidence of the division of the flagellum. The basal body and the
blepharoplast usually divide, but the flagellum either passes unchanged
to one of the daughter cells, as in Crithidia, Trypanosoma, and others,
or is resorbed into the cell. In some doubtful cases it may be thrown
off. If the old flagellum is retained in uniflagellate forms, the second
flagellum develops by outgrowth of the basal body or the blepharoplast.
If the old flagellum is resorbed, both halves of the divided kinetic body
give rise to flagella by outgrowths. Similarly if there are two or more
flagella, one or more may be retained by each daughter cell, while the
others, making up the full number, are regenerated.

Reorganization is indicated, to some extent, by these cases in which
the old flagellum is resorbed. It is still better indicated by a number
of flagellates in which the cytoplasmic kinetic elements, as well as the
flagella, are all resorbed and replaced by new combinations in each of
the daughter cells. Thus in Spongomonas splendida, according to Hart-
mann and Chagas (1910), the old blepharoplasts and two flagella
are resorbed and new ones are derived from centrioles of the nuclear
division figure. The phenomenon cannot be regarded as typical of the
simple flagellates, for in the great majority the kinetic elements are
self-perpetuating, even the axostyles, according to Kofoid and Swezy
(1915), dividing in Trichomonas. This, however, requires confirmation.

Figure 7. Lophomonas blattarum Janicki. Division of the nucleus and reorganization.
(After Bélar, 1926.)

GENERAL CONSIDERATIONS zy

An extreme case of reorganization in flagellates is apparent in the
two species of Lophomonas (L. blattae and L. striata) first described by
Janicki (1915). Here the parental calyx, basal bodies, blepharoplasts,
and rhizoplasts all degenerate and are resorbed during division (Fig. 7).
At the beginning of division, a cytoplasmic centriole divides with a con-
necting fibril, which is retained throughout as a paradesmose. The
nucleus emerges from the calyx in which it normally lies and moves
with the spindle to the posterior end of the cell. The spindle takes
a position at right angles to the long axis of the cell. Chromosomes,

Figure 8. Chilodonella uncinatus. New pharyngeal basket and mouth, replacing old
ones. (After MacDougall, 1925.)

probably eight in number, are formed and divided, and two daughter
nuclei result, each of which is enclosed in a new calyx, while new basal
bodies and new blepharoplasts apparently arise from the polar centrioles
Giigs'7 ):

This phenomenon in Lophomonas is strikingly similar to the re-
organization processes occurring in one of the Chlamydodontidae—
Chilodonella. Here, according to the observations of Enriques (1908),
Nagler (1911), and MacDougall (1925), the old mouth of the cell
and the oral basket of trichites are discarded and disappear in the cell,

28 GENERAL CONSIDERATIONS

while two new oral aggregates are provided for the daughter individ-
uals (Fig. 8).

In the majority of Sporozoa and Sarcodina simple division is not the
usual mode of reproduction. Where it does occur, as in Amoebida,
characteristic derived structures are absent and, if reorganization takes
place, it is confined to the cytoplasm, where we have little evidence of
change. A modified type of division, called “budding division,” is widely
spread in testacea. Here, water is absorbed by the old protoplasm, fol-
lowed by lively cyclosis and the protrusion of a protoplasmic bud from
the shell mouth. This bud grows, assumes the form of the parent
organism, and secretes its own chitinous membrane on which foreign
bodies (Arcellidae), or plates manufactured by the parent protoplasm
(Euglyphidae), are cemented. Apart from withdrawal of old pseudo-
podia, there is little evidence of reorganization. In Heliozoa, however,
pseudopodia, with their axial flilaments, are drawn in and new ones
are formed by the daughter cells.

In Radiolaria, Foraminifera, and Mycetozoa, indeed in the majority
of the Sarcodina and in most Sporozoa binary fission is replaced by
multiple division. The nuclei divide repeatedly, and a portion of the
cytoplasm is finally parceled out to each of the nuclei. The minute cells
thus formed leave the parent organism usually as swarm spores. Meta-
bolic products, waste matters, and certain structures of the derived
organization are left behind, and shells and skeletons alone mark the
previous existence of living cells. There is often a small amount of
protoplasm retained in these alloplasmatic products and it is not alto-
gether fantastic to see in these remains what Weismann (1880-82)
denied as occurring in Protozoa—viz., a corpse.

It might seem that these methods of restoring protoplasm to its
full vitality by processes of division are adequate to account for indefinite
longevity of Protozoa. This, however, is not the case, with the possible
exception of the animal flagellates, in which division in some form
is the only means of reproduction known. In ciliates the ability to divide,
if other methods of reorganization are prevented, gradually weakens;
the interdivisional periods are gradually lengthened, and the degenera-
tion of the derived structures finally results. The micronucleus may
hypertrophy (Calkins, 1904) or disappear (Maupas, 1888), and the

GENERAL CONSIDERATIONS 29

motile organs may be lost (Maupas, /bid.). In Uroleptus mobilis the
chromatin of the macronucleus ultimately disappears and only a few
X-granules remain. The protoplasm apparently dies from “‘old age”
(Figs 9).

The effects of continued division in ciliates, if other means of re-
organization are excluded, are clearly shown by the method of isolation

Figure 9. Uroleptus mobilis Engelm. Old-age specimens showing the degeneration
of the macronucleus (M) and loss of micronuclei. (After Calkins, 1919.)

cultures. A single individual, preferably an ex-conjugant, is isolated
in a suitable medium in a culture dish such as a Columbia isolation cul-
ture dish. The next day such an individual, for example a U. mobilis, is
represented by four or eight daughter individuals, the number depending
upon two or three divisions of the original one isolated. In our practice
five of these are isolated in separate dishes, and five independent
lines are started, all representing protoplasm which had been a part
of the protoplasm of the original individual isolated. A single individual
is isolated daily from each of these five lines and a daily record of

30 GENERAL CONSIDERATIONS

generations is kept for all lines. The total number of divisions 1s
recorded for all lines and these are averaged for ten-day periods. The
division rate is taken as a measure of vitality and the history of the
protoplasm is shown graphically by plotting the ten-day averages on
a graph in which the ordinates represent the number of divisions and
the abscissas the consecutive ten-day periods. Such a graph for U. mobilis
is shown in Figure 10, which is a composite graph of 23 different series,

ne eareT aT Tod J. PN Neo men ec a fT
Vir
pr==))

U 10 20 30 [2

Figure 10. Uroleptus mobilis Engelm. Graph representing the life history by ten-
day periods. (After Calkins, 1933.)

averaged for successive ten-day periods in isolation culture. It will be seen
that there is a high initial vitality which gradually wanes through 360
divisions during 300 days. In these isolation cultures the individuals
do not encyst nor conjugate; hence division is the only means of re-
organization.

That division is not effective in checking waning vitality is shown
by the fact that, in my experience, all such cultures of Uroleptus finally
die, in some cases in from fourteen to sixteen months, in other cases
in from three to ten months.

The macronucleus is probably the most important of all derived
organs of Uroleptus. With each division of the cell, as shown above, it
undergoes a process of reorganization whereby the chromatin is restored

GENERAL CONSIDERATIONS 31

to a virile condition. This may be repeated upwards of 300 times, until
reorganization, if it occurs, is ineffective and the protoplasm dies. Thus
the macronucleus, like all other derived structures of the cell which
come from the euplasmatic substances, apparently has a limited potential
of activity. But the macronucleus may overcome this difficulty through
the more deeply reaching phenomena in the life history, viz., endomixis
and conjugation.

REORGANIZATION BY ENDOMIXIS AND BY CONJUGATION

In isolation cultures of any ciliate, if the extra individuals which
remain over after the daily isolation is made on any day are put into
a larger container with abundant food, a so-called ‘‘encystment test’’
or “conjugation test’ is started. In my experiments these are begun
regularly every ten days. Here the individuals multiply by division until
there are thousands in the container. In the early stages of the life cycle
of U. mobilis all such individuals die of starvation, but in a month
or six weeks after the initial conjugation from which the series is
started, such tests result in an increasing number of encystments. A
type of breakdown which is not seen in the division phenomena is now
manifested. The macronucleus is fragmented, and the fragments are
distributed in the protoplasm where they ultimately disappear, while
a new macronucleus is formed from a product of micronuclear division.
Other structures of the derived organization are resorbed, waste matters
and water are voided to the outside, and a cyst membrane is formed
within which the organism may remain in a partially desiccated condi-
tion for months. When it is recovered from the cyst and reéstablished in
isolation culture, it has an optimum vitality and passes through a com-
plete life cycle exactly like that of an ex-conjugant. This phenomenon
of endomixis, except for encystment, is the equivalent of endomixis
in Paramecium aurelia as originally described by Woodruff and Erdmann
(1914). Endomixis thus brings about a more far-reaching and more
complete reorganization than does division; the new macronucleus aris-
ing from a micronucleus, is provided with a new potential of vitality.
For an up-to-date account of endomixis, see Woodruff, mfra, Chap-
ter XIII.

An interesting phenomenon which I interpreted as analogous to endo-
mixis occurs in the ciliate Glaucoma (Dallasia) frontata (Fig. 11). At

32 GENERAL CONSIDERATIONS

an early stage in its life history, a stage corresponding to the period
of encystment in Uroleptus, this organism loses its normal form which
is assumed under certain feeding conditions (see Kidder, imfra, Chap-
ter VIII), becomes elongate and cylindrical, and darts about in the
medium with unusual speed (Fig. 12). Soon its mouth and oral mem-

Figure 11. Glaucoma
(Dallasia) frontata Stokes.
Vegetative individual. A,
anus; BC buccal system;
CV, contractile vacuole;
LS ladder system; LU left
undulating membrane; M
mouth of buccal cavity;
MOT, region of motorium;
RU right undulating mem-
brane; T, ‘tongue’ in buc-
cal cavity. (After Calkins
and Bowling, 1929.)

branes are resorbed and it divides into two cylindrical individuals.
These divide into four, the four into eight, and the eight into sixteen,
all without intervening growth. The last-formed individuals have no
resemblance to the original parent form, but each has a macronucleus

GENERAL CONSIDERATIONS 33

and a micronucleus which has undergone maturation divisions and is
haploid as to chromosomes. The final sixteen are associated in pairs,
which become surrounded by a cyst membrane within which they fuse
(Calkins and Bowling, 1929). For other variations in the phenomena
of endomixis, see Diller, 1936; and especially Woodruff, znfra, Chap-
ter XIII.

This unusual phenomenon in Dallasia, which was not encountered by
Kidder in his studies on Glaucoma, finds its closest parallel in a group

Figure 12. Glaucoma (Dallasia) frontata Stokes. History of normal vegetative in-
dividual with successive divisions resulting in copulating gametes and encysted zygotes.
(After Calkins and Bowling, 1929.)

of ciliates in each of which the life history is unique. This group includes
the ectoparasite Ichthyophthirius multifiliis, according to Buschkiel
(1911) and Nerescheimer (1908), and Opalina ranarum, according to
Metcalf (1909) and Nerescheimer (1907). With these forms we have
good evidence, together with Dogiel’s ‘transformation of a male pro-
nucleus into a spermatozo6n” in the Ophryoscolecidae, that the intra-
cellular micronuclei forming pronuclei are a reminiscence of a brood
of gametes (Dogiel, 1923; see also Turner, zvfra, Chapter XII).

34 GENERAL CONSIDERATIONS

The longevity of a ciliate’s protoplasm in the past is shown by the
fact that it is before us today and it has a possibility of indefinite
longevity in the future. We know very little about the secrets of proto-
plasmic organization which underlie continued life, but we can analyse
some, at least, of the conditions under which it maintains its animation.
A proper environment and adequate food are essential factors. These
lead to growth and to reproduction by division. We have seen that these
latter bring about a reorganization and a return to full metabolic ac-

Figure 13. Uroleptus
mobilis Engelm. Conjuga-
tion and merotomy. (After
Calkins, 1921.)

tivity. The periodic restoration of these processes might well be enough
to ensure protoplasmic longevity, as seems to be the case with the
animal flagellates. At endomixis the ciliate macronucleus follows the
fate of other parts of the derived organization; a new one is formed
from the fundamental organization, and the result is, again, increased
vitality of the protoplasm. This phenomenon, recurring every thirty
days, ensured the longevity of Paramecium aurelia in Woodruft’s hands
for many years and through thousands of generations by division. As
Woodruff states, it may be adequate, indeed, to ensure indefinite life
in the future, as division alone is apparently adequate for animal
flagellates.

In a conjugation test made with Uroleptus, the results showed that
if the series is sixty or more days old, the individuals multiply by

GENERAL CONSIDERATIONS 35

division until there may be thousands in the container. If conditions are
appropriate, this massing may be followed by an epidemic of con-
jugations (see Sonneborn, snfra, Chapter XIV). In this process two
individuals fuse at the anterior ends and remain united for approxi-
mately twenty-four hours (Fig. 13). They then separate and each of
the ex-conjugants begins a process of reorganization which lasts for
four or five days. If such an ex-conjugant is isolated and fed, it will
give rise to a new series and its progeny will pass through all the stages
of vitality that the parent series passed through. At the outset its vitality
will be approximately the same as the original vitality of the parent
series, but it will be greater than that of the parent series at the time
of conjugation. It passes through the same history of waning vitality,
and its protoplasm finally dies, although this may be months after the
death of the parent protoplasm.

These experiments with Uroleptus, continued for more than ten
years and with 146 different series, always gave the same result. The
length of life of each series varied from three months to over a year
and all were descendants of a single bit of protoplasm making up the
body of one Urole ptus cell.

There is little reason to doubt that some change in the protoplasmic
make-up occurs with continued metabolic activity through many suc-
cessive generations by division. In isolation cultures it is manifested by
an increasing time interval and by a change in the physical properties
of the protoplasm whereby fusion of cells, total or partial, is possible.
A type of breakdown, not manifested early in the series, is set up. The
cortex is liquified, in most cases in the region of the peristome, and two
individuals fuse in conjugation. In some cases the entire cortex is thus
modified, and individuals will fuse at any point on the periphery. I
have often referred to one case where no less than nine Paramecium
caudatum were thus fused into one amorphous mass.

The stimulus of normal fusion results in activities not manifested
before. The micronuclei divide as they do in endomixis, and their
division involves reduction in number of chromosomes and in the
formation of pronuclei which meet and fuse. These copulating micro-
nuclei, as pronuclei, are usually interpreted as a reminiscence of an
ancestral gamete brood which is realized in Dallasia (Glaucoma) fron-
tata, O palina, and in gregarines. In the latter, as is well known, each indi-

36 GENERAL CONSIDERATIONS

vidual of a syzygy forms a brood of gametes which copulate with
similar gametes from the other individual of the pair (see Turner, znfra,
Chapter XII; and Kofoid, zvfra, Chapter XI).

Coming at the end of a vegetative period during which single or
multiple divisions may have occurred, such gamete broods indicate a
change in organization which, if copulation does not occur, results in
death of the gametes. In other words gametes are so specialized that
they require a complemental combination to ensure normal metabolism
and life. In some gametes differentiation has been in the direction of
greater constructive activity, resulting in food-stored macrogametes
equivalent to egg cells; in others in the direction of greater katabolic
activity, resulting in a brood of microgametes equivalent to spermatozoa,
as in Coccidiomorpha.

The happenings at conjugation need only be recapitulated, for the
phenomena are so well known that description here is unnecessary (see
Turner, 7nfra, Chapter XII, for detailed accounts). In Uroleptus, shortly
after fusion of the anterior ends, the micronuclei of both individuals
begin a series of maturation divisions, and the third division gives rise
to the gametic nuclei. One of a pair of these remains 77 stu, while the
other, or migrating pronucleus, passes through the protoplasmic bridge,
unites and forms a fertilization nucleus by fusing with the quiescent
pronucleus of the other conjugant. Thus a mutual fertilization occurs,
the two migrating pronuclei in Uroleptus passing each other at the
apex of the united pair of cells (Fig. 13). After this is accomplished,
the two individuals separate, the amphinucleus of each conjugant di-
vides twice, and one of the products forms a new macronucleus, which,
after four or five days, is ready to divide. Another product forms a
new micronucleus. In the meantime the old macronucleus begins to
degenerate and is ultimately resorbed in the cytoplasm. The other
structures of the derived organization of the old organism are resorbed
and replaced by new ones. The young organism, with this new set
up, starts a new life cycle with an optimum of vitality (Fig. 14).

Through these activities of conjugation, an old protoplasm with low
vitality is made over out of its own contained substances, into a new pro-
toplasm with high vitality. What explanation of this remarkable phenom-
enon can be given? The only apparent difference between the happen-
ings at division and at conjugation are: (1) fertilization, and (2)

GENERAL CONSIDERATIONS yi

loss and replacement of the old macronucleus, and so forth. Renewed
vitality may be due to one or to both of these phenomena. Conjugation
differs from endomixis mainly in gelatinization of the cortex and in

Figure 14. Uroleptus mobilis Engelm. Formation of new macronucleus after conjuga-
tion. 1. First metagamic or zygotic division of the amphinucleus; 2. One of the progeny
of this division dividing again; 3, 4, 5, telophase stages of this divison resulting in a
new macronucleus (above) and a degenerating nucleus (below) ; 6 to 10, stages in the
differentiation of the new macronucleus and disintegration and resorption of the old
macronucleus. In 10, two new micronuclei are in mitosis preparatory to the first division
of the ex-conjugant. (After Calkins, 1919.)

amphimixis. Fertilization, with its sequela of hereditary possibilities,
is a highly important result (see Jennings, imfra, Chapter XV; and
Sonneborn, infra, Chapter XIV). It is also assumed to be the raison

38 GENERAL CONSIDERATIONS

d’étre of all sorts of subsequent peculiarities, but for the phenomenon
of increased vitality under consideration, a very simple experiment with
Uroleptus mobilis shows that fertilization with amphimixis has little to
do with the problem. A conjugating pair is cut across the apex with a
scalpel. One individual of the two thus separated is fixed and stained, to
show the stage of conjugation when cut, while the other one is cultivated
in an isolation culture dish. The experiment is particularly striking when
the two wandering pronuclei are cut off with the apical piece (Fig. 13).
The cultivated individual goes through exactly the same processes as
though conjugation had been completed in the usual manner, and a
normal life cycle results. Amphimixis, however, had been prevented;
conjugation apparently had been transformed into endomixis (Calkins,
1921). Here, however, the possibility remains that one of the super-
numerary micronuclet of the amputated individual may divide and one
part may unite with the pronucleus already present.

The loss and replacement of the old macronucleus, together with
that of the old derived structures generally remains as a possible ex-
planation. This phenomenon is common to endomixis, conjugation, and
conjugation merotomy, and in all cases a renewed vitality results. This
does not happen with division, although, as shown above, characteristic
changes occur in the macronucleus. With its disintegration and re-
sorption in the cytoplasm, a new supply of nucleoproteins and other
chemical compounds useful in metabolic activities are distributed in the
cell, and these may be potent factors in the new vitality. Added to these
is the fact that an entirely new and powerful organ of the cell—the
macronucleus—has been supplied from the fundamental organization.

The majority of the derived structures of the cell have a relatively
short life, being resorbed and renewed at division (cirri and other
motile organs; others, such as membranelles and oral structures, appar-
ently have a longer life. Of all derived structures the macronucleus has
the longest life; it divides at cell division and may remain functional
through entire life cycles, including many hundreds of divisions. It 1s
probably the chief metabolic agent of the cell, yet it apparently lacks
the power of continued life which the micronucleus possesses. It 1s
essentially somatic in character, and, like the soma in metazoa, it ulti-
mately wears out; if not replaced, the rest of the cell, including the
micronucleus depending on it, dies with it. It is quite possible, indeed

GENERAL CONSIDERATIONS ao)

I regard it as probable, that the gradual deterioration of this important
organ of the ciliate cell, is the underlying cause of waning vitality and
ultimate death of protoplasm in isolation cultures (Fig. 10).

With parasitic forms there is probably the same underlying variation
in vitality, but there is not the same possibility of measuring it, at least
not in any direct way.

Remarkable as these phenomena are, they leave us cold so far as the
matter of protoplasmic vitality is concerned. The interpretations pre-
sented here are, after all, essentially mechanistic, and even with the
ciliates the phenomena described are by no means universal, while in
some groups of Protozoa they are not shown at all, or else only in a
vague and indefinite manner. In ciliates there is one organoid of the
cell, the micronucleus, which transcends all other structures of the cell,
and, although it is apparently not functional except in heredity and
activities connected therewith, such as regeneration, and so forth, it
does appear to be the most essential morphological element of the
fundamental organization. Its agent in metabolism is the macronucleus,
which is derived from it. For the secret of life and longevity in ciliates
we must turn to this inconspicuous and often overlooked structure of
the cell and of the cyst.

In other groups of the Protozoa, the homologue of this important
element of the cell lies in the usually single nucleus. Furthermore, in
the micronucleus it is probably the chromatin content that gives it its
power; and, in other groups than the ciliates, it is the chromatin con-
tent that makes the nucleus what it is. The value and importance of
chromatin is seen by the meticulous care with which it is distributed
to daughter cells and to progeny generally, while the maturation divi-
sions bespeak its significance in heredity. The secret of life and vitality
must thus be sought not in the daily activities of living things, but in
that enigmatical substance—chromatin—which is about us in all living
things, including ourselves. That secret may never be disclosed.

LITERATURE CITED
Balbiani, G. 1859. Du réle des organes généateurs dans la division spont. des
infusoires cilies. J. Physiol. Pathigén., 111: 71-87.
—— 1861. Recherches sur les phénoménes sexuelles des infusoires. J. Physiol.
Pathigén., : 102-30, 194-220, 431-48, 465-520.
Barry, M. 1843. On fissiparous generation. Edinb. New phil. Jour., 35: 205-
20. .

40 GENERAL CONSIDERATIONS

Belar, K. 1926. Der Formwechsel der Protistenkerne. Ergebn. Zool., 6: 235-
654.

Buffon, G. L. de. 1749. Histoire naturelle générale et particuliere.

Buschkiel, A. L. 1911. Ichthyophthirius multifiliis. Arch. Protistenk., 21: 61-
102.

Bitschli, O. 1873. Einiges ber Infusorien. Arch. mikr. Anat., 9: 657-78.

1876. Studien uber die ersten Entwicklungsvorgange der Eizelle, die

Zelltheilung, und die Konjugation der Infusorien. Abh. senckenb. naturf.

Ges., 10: 213-462.

—— 1887-89. Bronn’s Klassen, und Ordnungen des Thier reichs. Erster Band.
Protozoa. Leipzig. Pp. 1-2028.

Calkins, G. N. 1901. The Protozoa. New York, 1901.

1904. Studies on the life history of Protozoa. 1V. Death of the A-series.

J. exp. Zool., 13 423-59:

—— 1911. Regeneration and cell division in Uronychia. J. exp. Zool., 10:
95-116.

—— 1919. Uroleptus mobilis Engelm. 1. History of the nuclei during division
and conjugation. J. exp. Zool., 27: 293-357.

—— 1921. Uroleptus mobilis Engelm. Effect of cutting during conjugation.
J. exp. Zool., 34: 449-70.

—— 1930. Uroleptus halseyi. Il. The origin and fate of the macronuclear
chromatin. Arch. Protistenk., 69: 151-74.

—— 1933. The Biology of the Protozoa. 2d ed., Philadelphia.

— 1934. Factors controlling longevity in protozoan protoplasm. Biol. Bull.
Woods Hole, 67: 410-41.

Calkins, G. N., and R. Bowling. 1929. Studies on Dallasia frontata Stokes.
II. Cytology, gametogamy and conjugation. Arch. Protistenk., 66: 11-32.

Diller, Wm. F. 1936. Nuclear reorganization processes in Paramecium aurelia,
with descriptions of autogamy and “hemixis.” J. Morph., 59: 11-51.

Dogiel, V. 1923. The transformation of a male pronucleus into a sperma-
tozodn. Zool. Lab. Univ. Petrograd, 1923.

Dujardin, F. 1835. Observations sur les rhizopodes et les infusoires. C. R.

Acad. Sci. Paris, Nov. 1835, pp. 338-40.

1841. Histoire naturelle des zodphytes infusoires. Paris.

Ehrenberg, C. G. 1938-39. Die Infusionsthierchen als vollkommene Organis-
men. Leipzig.

Engelmann, Th. W. 1876. Uber Entwicklung u. Fortpflanzung der Infusorien.
Morph. Jb., 1: 573-635.

Enriques, P. 1908. Die Conjugation und sexuelle Differenzierung der In-
fusorien. Arch. Protistenk., 12: 213-74.

Griffin, L. E. 1910. Euplotes worcesteri, sp. nov. II. Division. Philipp. J. Sct.,
De Z Ola

Haas, G. 1933. Beitrage zur Kenntnis der Cytologie von Ichthyophthirius
multifiliis. Arch. Protistenk., 81: 88-137.

GENERAL CONSIDERATIONS 4]

Hartmann M., and C. Chagas. 1910. Flagellaten Studien. Mem. Inst. Osw.
Giz, 25 fase. I:

Hill, John. 1752. History of Animals, 3d vol. of a gen. Nat. Hist., 1748-52.

Hooke, Robt. 1665. The Micrographia. London.

Janicki, C. 1915. Parasiten Flagellaten, Teil 2. Z. wiss. Zool., 112: 573-691.

Kidder, G. W. 1933. Studies on Concophthirius mytili de Morgan. I.
Morphology and division, Arch. Protistenk., 79: 1-24.

Kofoid, C. A., and O. Swezy. 1915. Mitosis in Trichomonas, Prec. nat. Acad.
Scie ash. de 3i5-2 16

Kolkwitz, R., and M. Marsson. 1909. Oekologie der tierischen Saprobien.
Int. Rev. Hydrobiol., 2:

Lackey, J. B. 1925. Studies on the biology of sewage disposal. The fauna of
Imhof tanks. Bull. N. J. agric., Exp. Stas., No. 417.

Lauterborn, R. 1901. Die ‘‘Sapropelische’”” Lebewelt. Zool. Anz., 24: 50-55.

Ledermiiller, M. F. 1760-63. Mikroskopische Gemiiths. u. Augenerg6rtzungen.
Nurnberg, 1760: 88.

Leewenhoek, A. van. 1676. Observations concerning little animals by him
observed in rain, well, sea, and snow water wherein pepper had lain
infused. Philos. Trans., 12: 821-31.

MacDougall, M. S. 1925. Cytological observations on gymnostomatous ciliates,
etc. Quart. J. micr. Sci., 69: 361-84.

Maupas, E. 1888-89. Le Rejeunissement karyogamique chez les ciliés. Arch.
Zool. Exp; gent, (2) 7,8.

Metcalf, M. M. 1909 Opalina. Its anatomy, etc. Arch. Protistenk., 13: 195-
37).

Meyen, J. 1849. Einige Bemerk. tiber den Verdauungsapparat der Infusorien.
Arch. Aanat. Physiol. Lp. z., 1839: 74-79.

Moody, J. 1912. Observations on the life history of two rare ciliates—
Spathidium spathula and Actinobolus radians. J. Morph., 23: 349-99.

Miller, O. F. 1773. Vermium terrestr. et fluviatil. s. animal. infusor. etc. Haf-

niae u. Lipsiae, 1773.

1786. Animalc. infusoria, fluviat. et marina, etc. (posth.) Hafniae et

Lipsiae.

Nagler, K. 1911. Caryosom u. Centriol Beimteilungsvorgang von Chilodon
uncinatus. Arch. Protistenk., 24: 142-48.

Needham, T. 1750. A summary of some late observations upon the genera-
tion composition and decomposition of animal and vegetable substances.
Philos. Trans. n.s., 45: 615-66.

Nerescheimer, E. R. 1907. Der Zeugungkreis von O palina. S.B. Ges. Morph.
Physiol. Miinchen, 22:

—— 1907. Die Fortpflanzung der Opalinen. Arch. Protistenk., Festschr.
Hertwig.

Rossolimo, L. I., and Frau K. Jakimowitsch. 1929. Die Kernteilung bei Con-
cophthirius steenstrupii St. Zool. Anz., 84: 323-33.

42 GENERAL CONSIDERATIONS

Sandon, H. 1927. The composition and distribution of the protozoan fauna of
the soil. Edinburgh.

Schaudinn, F. 1900. Untersuchungen uber Generationswechsel bei Coccidien.
Zool. |b. Anat, Abt. Wy 1320177-292.

Siebold, Th. von. 1845. Lehrb. der Verg. Anatomie d. Wirbellosen Thiere.
Hf. 1, 1845.

Stein, F. von. 1859. Der Organismus der Infusionsthiere, Abth. I, Hypo-
trichida. Leipzig.

— 1867. Der Organismus der Infusionsthiere, Abth. II, Heterotrichida.
Leipzig.

Summers, F. M. 1935. The division and reorganization of the macronuclei
of Aspidisca lynceus Miller, Diophrys appendiculata St. and Stylonychia
pustulata Ehr. Arch. Protistenk., 85: 173-208.

Turner, J. P. 1930. Division and conjugation in Evplotes patella Ehr. Univ.
Cal. Publ. Zool., 33: 193-258.

Wallengren, H. 1901. Zur Kenntnis der vergl. Morphol. der hypotrichen
Infusorien. Zool. Jb., Anat. Abt. 1, 15: 1-58.

Weismann, August. 1880-83. Essays on life and death and heredity. London.

Woodruff, L. L. 1905. An experimental study on the life history of hypo-
trichus infusoria. J. exp. Zool., 2: 585-632.

—— 1939. Some pioneers in microscopy, with special reference to proto-
zoology. J. Trans. N. Y. Acad. Sci., Ser. 2, 1: 1-4.

Woodruff, L. L., and R. Erdmann. 1914. A normal periodic reorganization
process without cell fusion in Paramecium. J. exp. Zool., 17: 425.
Yocom, H. B. 1918. The neuromotor apparatus of Emplotes patella. Univ.

Cal. Publ. Zool., 18:337-96.

Young, D. B. 1922. A contribution to the morphology and physiology of
the genus Uronychia. J. exp. Zool., 36: 353-90.

Young, Dixie. 1939. Macronuclear reorganization in Blepharisma undulans.
J. Morph., 64: 297-347.

CHAPTER’ Il

SOME PHYSICAL PROPERTIES OF THE PROTOPLASM
OF THE PROTOZOA

H. W. BEAMS AND R. L. KING

INTRODUCTION

DUJARDIN in 1835, a little over a century ago, was the first to care-
fully describe the physical properties of protozoan protoplasm which
he termed “‘sarcode,’”’ although other earlier observers had seen and
drawn living amoebae. For instance, R6sel von Rosenhof drew an amoeba
in 1755, O. F. Miller described living amoeba in 1773 and Ehrenberg,
a pioneer protozodlogist, undoubtedly observed living protozoan proto-
plasm. However, none of these observers emphasized the protoplasm as
the living substance as did Dujardin. In addition, Dujardin’s description
of protoplasm was so accurate that his definition of it as a ‘‘living jelly,
glutinous and transparent, insoluble in water, and capable of contract-
ing into globular masses and of adhering to dissecting needles so that it
can be drawn out like mucus’ can be little improved upon today (see
Fauré-Fremiet, 1935).

Perhaps no group of animals has served as the basis for so many
and so extensive studies on the structure of protoplasm as the Protozoa.
This is, no doubt, in part due to the fact that many of the earlier
workers labored under the assumption that the “simplest type’’ of proto-
plasm should be looked for in the “lower forms’’ of animals. In addi-
tion many of the Protozoa are comparatively large and discrete cells,
thus offering little mechanical difficulties to direct microscopic observa-
tions upon their living structure.

In a modern discussion of the physical properties of protoplasm, one
must bear in mind the fact that the manifold chemical and physiological
properties of living matter are intimately connected with the structure of
protoplasm. That is, one must conceive of a system which is kept in
existence, in spite of the fact that the katabolic phases of energy ex-
change tend to destroy its integrity; this involves a continuous auto-

tt PROTOPLASM OF PROTOZOA

matic replacement of worn-out parts or the growth of new ones. Chemical
processes must often be definitely localized in a small region within the
framework of a single cell. However, visible differentiation usually does
not take place within a single cell in the Metazoa to such an extent as
in the Protozoa, where we have a whole series of special differentiated
organelles for performing particular functions.

A protozoan is usually regarded as both an organism and a cell; in
the Metazoa protoplasmic differentiation tends to be irreversible, and
in Protozoa to be reversible, so that the most highly differentiated cells
ever observed are found as individual Protozoa. This extreme differentia-
tion is, of course, surprising to one accustomed to the usual structural
simplicity of metazoan cells, and is probably the main reason for the
position taken by Dobell (1911) and others who deny the cellular
nature of the Protozoa. Several degrees of permanency of differentiation
may be distinguished among the Protozoa, although of course the groups
are not mutually exclusive. Thus we may distinguish: (1) temporary,
completely reversible structures or differentiations of the protoplasm,
such as pseudopodia and spindle elements; (2) differentiations usually
irreversible and lasting throughout the life of the organism, which may
be the seat of active chemical energy changes, such as cilia, flagella, and
myonemes, or, with little chemical changes, such as morphonemes and
pellicle; and (3) differentiations formed by protoplasm which are
chemically distinct from it, such as shells and secretions.

Every time a protozoan divides, conjugates, or encysts there is a
tendency toward dedifferentiation, followed by reorganization, so that
the individual emerging from these processes often has developed a
new set of structures derived from the fundamental structure. These
changes must be accompanied or caused by physical realignments of
protoplasmic elements, of which little is known. Perhaps in no other
phylum of animals can such a wide range of type of mitosis and
cytokinesis be found as exists in the Protozoa. The morphological de-
tails of the division process in these forms are fairly well known, and
evidence is continually accumulating which shows that mitotic and
cytokinetic mechanism does not differ fundamentally from that of
higher forms.

In the Protozoa, as in other types of cells, little is actually known
regarding the initial stimulus that starts the organism to divide, to

PROTOPLASM OF PROTOZOA 45

undergo certain sexual phenomena, or to form cysts. However, environ-
mental conditions, such as amount and kind of food present, tempera-
ture, and so forth, have been generally observed to affect the rate at
which these phenomena take place. Since Protozoa under normal condi-
tions seem to be limited to a more or less uniform maximum size for
a given species, various suggestions have been made to the effect that
the stimulus for division is in part controlled by definite ratio of volume
to body surface, or to a possible ratio of nuclear volume to cytoplasmic
volume. However, whatever may prove to be the explanation of this,
it is an observed fact that in most cases conditions suitable for most
rapid growth are the same as those best suited for most rapid division.
On the other hand, relatively unfavorable conditions, such as scarcity
of food or the concentration of metabolites, have been observed to in-
duce sexual activity in the various Protozoa (see Giese, 1935, for litera-
ture). Practically nothing can be said of the physical changes under-
gone by the protoplasm of the organisms just before they start division,
conjugation, or endomixis, except that before division they may shorten
and thicken (Uroleptus, Calkins, 1919), or become spheroidal in form
(Amoeba, Chalkley, 1935). Such observations possibly indicate a change
in viscosity, the nature of which awaits investigation.

In certain of the hypermastigote flagellates Cleveland (1934, 1935)
has found one of the best materials for the study of the cytology of
cell division, not only in the Protozoa but of cells generally. Here the
mitotic spindle, with its chromatic and achromatic parts, is clearly seen
in the living condition; it may also be easily preserved and stained by
ordinary techniques. In addition, experiments on pulling the centrosomes
demonstrate the elasticity and contractility of the extranuclear chromo-
some fibers and those of the central spindle. Accordingly, in this ma-
terial the various elements of the mitotic apparatus must be considered
real and therefore demand careful consideration in any discussion of the
mechanics of mitosis, not only in these forms but in astral types of
mitotic divisions generally. Furthermore, a study of the physical prob-
lems involved in the origin and nature of the centrosomes, spindle
fibers, asters, degeneration and reorganization of certain of the locomotor
organs, as well as the correlated mechanics of cytokinesis in these forms
would be most valuable. However, for the present we can only assume
that the physical-chemical changes giving rise to these various struc-

46 PROTOPLASM OF PROTOZOA

tures are similar to those which occur in the mitotic divisions of cells
of higher forms. For a discussion see Gray, 1931; Heilbrunn, 1928;
and Chambers, 1938.

PROPERTIES OF PROTOPLASM AS EXHIBITED IN AMOEBA

To obtain a concept of protoplasmic structure no better course can
be followed than to secure a microscope, some amoebae, and study them
under relatively high power. Efforts to obtain this information from the
literature alone often lead to confusion, because of the wide variety of
terminology used by the various workers on the structure of amoebae.
Careful microscopic examination of the protoplasm of Amoeba proteus
in locomotion will reveal that it is composed of an outer, thin, colorless,
hyaline layer which is for the most part optically structureless, and an
inner granular region which makes up the greater portion of the
animal. A detailed study of the granular portion will reveal that it is
composed of a colorless continuous phase (hyaloplasm), in which are
suspended crystals and granules of various sizes and shapes, nucleus,
food vacuoles, contractile vacuole, refractive bodies, oil globules, and
possibly other inclusions. Long intervals of observation on these struc-
tures will show that some of them seem to be permanent and self-
perpetuating while others are only transitory. The question that naturally
arises in this connection is, which are living and which are nonliving
bodies? A further discussion of this point will be given elsewhere.

The question may now be asked, how are the various microscopically
visible inclusions of Amoeba protoplasm kept in suspension? This may
be because their density is only slightly different from that of the sur-
rounding protoplasm and because of the viscosity of the protoplasm.
Mast and Doyle (1935a) have presented evidence that the refractive
bodies, which are the heaviest inclusions of the protoplasm, are found
near the lower surface in living amoebae in which the protoplasmic vis-
cosity is low; that they are distributed by protoplasmic flow has been
demonstrated by Mast. and Doyle (1935b). Upon centrifuging, the
cytoplasmic constituents of amoeba are layered out, in order of their
relative specific gravity, with the refractive granules at the centrifugal
pole. Upon recovery pseudopods form first at or near the lighter end,
and the heavy refractive bodies then flow forward followed by the other
layers of constituents until all are thoroughly mixed.

PROTOPLASM OF PROTOZOA 47

The granular region of an amoeba is made up of an outer stationary
layer, which forms a hollow cylinder through which an axial stream of
protoplasm flows. The outer stationary layer has been called the ecto-
plasm and, more recently, because of its firm consistency, plasmagel;
the axial flowing layer, endoplasm, and, more recently, because of its
fluid consistency, plasmasol. More difficult to observe, because of its
extreme thinness, is the surface layer, or plasmalemma, just external to
the clear hyaline layer.

Ordinary methods of fixation often result in an apparent loss of the
constituents which may be seen in the living cell; however, by the use
of suitable fixing methods Mast and Doyle (1935a) were able to show
that in A. proteus the cytoplasmic constituents may be preserved in a
form scarcely distinguishable from the living condition, except for the
nuclear granules and the water-soluble salts. Since protoplasm is an un-
stable intimate association of salts in solution, proteins, fats, and other
materials which may give it a fibrillar or an alveolar appearance, we
cannot hope to preserve it unchanged. Indeed, we may not even ob-
serve it in the living condition unchanged, as change is a universal
characteristic of protoplasm. It must be constantly kept in mind that
what is observed in a permanent fixed preparation is not living material,
but likewise it must also be recognized that while the fixed material 1s
not protoplasm, it is at least a significant artifact derived from proto-
plasm, since it has had its origin in the coagulation of proteins and
other significant elements of living material. Most of the established
facts of cytology have been first observed in fixed material, and later
corroborated by a study of the living. However, it should be pointed
out, from the works of Hardy and of Fisher, that probably many of
the older theories of protoplasmic structure, such as the granular, fibril-
lar, alveolar, and reticular, have been based in part, at least, on artifacts
induced by the methods employed.

Probably the most significant feature of the protoplasm of Amoeba
is its ability to change from a relatively fluid state to a more solid jelly-
like condition. Upon mechanical agitation, such as occurs in trans-
ferring an amoeba to a slide or by tapping on the slide, the organism
may assume a globular form which may have short projecting pseudo-
podia. If the temperature be raised to 30° C., the projections may dis-
appear (K. Gruber, 1912). This means that the protoplasm present

48 PROTOPLASM OF PROTOZOA

has become relatively fluid, because only a liquid immiscible with the
surrounding medium shows the globular form which is essential in the
principle of minimal surfaces. This change in consistency may be
brought about by mechanical agitation (Chambers, 1921) and has been
experimentally studied by Angerer (1936), who presents data to show
that mechanical agitation of amoebae causes a decrease in viscosity in the
plasmagel (A. proteus) and an initial decrease followed by an in-
crease in viscosity in the plasmasol (A. dubia). Extreme mechanical
agitation according to Angerer, leads to a minimum viscosity of the
plasmagel, which is eventually followed by the disintegration of the
organism, the substance of which mixes with the surrounding medium.

If enough pressure is applied to the cover glass, the amoeba will
burst, and it may be observed, providing the injury is not too extensive,
that an effort is made on the part of the protoplasm at the region of
rupture to form a water-insoluble membrane, thus inhibiting further
mixing of the protoplasm with the water. This process has been re-
cently termed “the surface precipitation reaction” by Heilbrunn (1928).
If, however, the injury has been sufficient so that the interphase be-
tween the ruptured protoplasm and the surrounding medium is too
great, the surface precipitation reaction is not sufficient to prevent a
complete dissolution of the amoeba into the water.

Small hemispheres of clear protoplasm (the incipient pseudopodia)
usually appear quickly upon the surface of a quiescent spheroidal
amoeba in the form of liquid extrusions from the main mass of proto-
plasm. Into these extrusions, which gelate equatorially, a central flow
of granular protoplasm may be seen streaming forward from the amoeba
in the direction of the advancing pseudopodium, which constantly moves
distally. As the pseudopodium advances, it may be seen to consist of an
outer cylinder of motionless protoplasm and an inner streaming fluid
protoplasm. That the cortical layer is gelated is indicated by the fact
that its granules and other inclusions are stationary. As the central
stream of flowing protoplasm (endoplasm or plasmasol) reaches the
hyaline cap, it moves peripherally in all directions and gelates (1.e.,
becomes ectoplasm or plasmagel). At the other end of the amoeba the
plasmagel becomes plasmasol and passes forward as a fluid core through
the cylinder of plasmagel (Mast, 1926). The cause of the forward
flow of protoplasm is obscure, but the suggestion has been made by

PROTOPLASM OF PROTOZOA 49

Hyman (1917) and many others that it is due to the tension exerted
upon the fluid plasmasol by the elastic plasmagel.

There has been much discussion concerning the physical characteristics
of the outermost layer, or plasmalemma, of different species of amoeba.
In certain forms, such as A. verrucosa, the pellicle may be lifted with
microdissection needles and stretched, apparently without injuring the
organism in any way (Howland, 1924c). Seifriz (1936) also found
a thin outer membrane on A. protews which was resistant, elastic, and
highly viscous except at the advancing tip of the pseudopodia. Likewise
Chambers (1924) was able to lift the plasmalemma in A. proteus by
injecting water beneath it, thus causing large blisters to form between
the plasmalemma and the underlying surface which burst upon punctur-
ing, leaving the pellicle collapsed. Mast (1926) caused blisters to
appear by local pressure; further pressure caused disruption of the
pellicle, the frayed ends of which were clearly observed.

Practically all investigators are agreed that the plasmalemma moves
forward, at least on the upper surface of a moving amoeba. In addition,
the method of formation of new food vacuoles requires a structure
of the plasmalemma of such nature as to form new surfaces immediately
by the replacement of large areas of the plasmalemma which have been
used in forming the boundary of the food vacuoles (Schaeffer, 1920).

Immediately beneath the outer layer, or plasmalemma, is the clear
hyaline layer. That this layer is fluid may be shown by the fact that it
contains scattered granules which are in Brownian movement (Mast,
1926) and by the injection of a suspension of lamp black into it, the
injected lamp-black particles spreading throughout this clear hyaline
layer (Chambers, 1924). However, Schaeffer (1920) holds that some-
times it is more rigid than endoplasm, and at other times not.

COLLOIDAL NATURE OF PROTOPLASM

Any attempt to understand the processes occurring in protoplasm
must depend for its success not only upon a knowledge of the chemical
constitution of protoplasm but also of its physical structure. For this
reason much ingenuity has been exercised in trying to discover the
physical structure of the protoplasm in such relatively simple forms as
Amoeba, in which permanently differentiated structures are at a mini-
mum. For example, in A. proteus apparently only the nucleus is perman-

50 PROTOPLASM OF PROTOZOA

ently differentiated; the contractile vacuole and other structures are
evanescent, at least in part. We have already mentioned the more ob-
vious physical characteristics of Amoeba protoplasm, which consists, as
does all protoplasm, of a complex heterogenous colloidal system, made
up of a suspension (granular, fibrillar, and alveolar) of many different
materials (fats, carbohydrates, and proteins) dispersed in a supporting
continuous liquid part. The greatest volume ingredient in protoplasm
is water; in solution in the water are various salts. Protoplasm may be
deprived of many of its visible inclusions without killing it, leaving
often a clear, colorless, optically homogenous hyaloplasm. Thus the
microscopic structure of protoplasm gives no direct evidence of the
finer submicroscopic structure. However, it is generally agreed that the
finer structure of protoplasm is dependent upon its colloidal nature,
which, because of the relatively large size of the particles present, results
in enormous intracellular surfaces.

Then too, the taking up of water by colloids is influenced both by the
solutes present and by the previous history of the colloid itself (hys-
teresis). Colloids often have the property of changing reversibly from
a relatively liquid (sol) to a relatively solid condition (gel). A gel
has many of the properties of a solid, among them elasticity, apparently
due to its structure; but differs from a solid in that diffusion in gels of
low concentration is often the same as in a simple solution, and in that
chemical reactions often can occur at velocities unaffected by the gel
condition. Every organism is dependent upon the temporal and spatial
coordination of its chemical reactions, and this depends largely on the
degree of dispersion and kinetic activity, because these regulate re-
action velocities. Thus the organization of chemical events is due in
some way to the nature and architecture of the colloidal system in which
they occur. Therefore, biologists attempt to explain the physiological
action of various factors as influences on the colloids of protoplasm.

CONSISTENCY

It is a phenomenon of general observation that protoplasm flows,
but that it resists pressure. The former is a property commonly attributed
to liquids, the latter to solids. In liquids there is great internal mobility
of the molecules; this is essential to many physical activities, such as

PROTOPLASM OF PROTOZOA 51

movement, and to chemical reactions in protoplasm. On the other hand,
solids have great internal cohesion of molecules,:i.e., they retain their
shape, show elasticity; this is essential to the maintenance of continuity
and form of protoplasm. Colloids quite generally show changes from a
relatively fluid to a firm jelly-like consistency, so that protoplasm as a
colloidal system may partake of the nature of a solid (in the gel con-
dition) or that of liquid (in the sol condition). Thus changes in vis-
cosity are one of the essential factors in ameboid movement. In addi-
tion, the viscosity of the protoplasm as a whole, and particularly the
changes in viscosity within a given portion of a protozoan, are very
important factors to be considered in connection with a study of mitosis,
cytokinesis, cyclosis, rate of diffusion of various substances, protoplasmic
reorganization, and functioning of such organs as the contractile vacuole.

Besides the qualitative methods of estimating the viscosity of proto-
plasm by observing the presence or absence of movement (Mast, 1926b),
presence or absence of Brownian movement (Bayliss, 1920; Pekarek,
1930; Brinley, 1928), microdissection studies (Kite, 1913; C. V. Tay-
lor, 1920; Chambers, 1924; Howland, 1924c; and others), certain
experimental methods, 1.e., centrifugation (Heilbrunn, 1928; 1929b;
Fetter, 1926), electromagnetic methods (Seifriz, 1936), and rate of
diffusion of certain dyestuffs (Chambers, 1924; Needham and Need-
ham, 1926) have been used to reveal data as to the relative and absolute
viscosity of the protoplasm of various Protozoa. For a detailed discus-
sion of these various methods the reader is referred to the works of
Chambers (1924), Heilbrunn (1928), and Seifriz (1936).

The Protozoa show a wide range of viscosity values. In fact a varia-
tion in protoplasmic viscosity from 2 times that of water in Amoeba
(Heilbrunn, 1929b) to over 8,000 times that of water in Paramecium
(Fetter, 1926) has been reported. Undoubtedly a much higher viscosity
exists in many protozoan cysts and other forms characteristic of re-
sistant stages. Seifriz (1936) has reported that during the winter the
plasmodium of a myxomycete becomes as hard and as brittle as a thin
sheet of dry gelatin.

In addition to the information obtained about the consistency of
various Protozoa from multilation experiments, for example those of
Calkins (1911) and Peebles (1912) on Paramecium caudatum, the

I2 PROTOPLASM OF PROTOZOA

microdissection apparatus has proved to be a useful instrument in a
study of the structure of Protozoa. By use of this method, Kite (1913)
determined the ectoplasm of A. proteus to have a moderately high
viscosity and cohesiveness. The substance forming the wall of the
contractile vacuole seemed to possess a much higher viscosity than the
surrounding endoplasm. The nucleus seemed to behave as a highly
rigid granular gel. Kite further observed that the protoplasm of Para-
mecium was a soft, elastic, and somewhat glutinous gel; the surface
seemed to be more viscous than the interior. More recent studies by
Chambers (1924); Howland (1924a, 1924b); Howland and Pollack
(1927); Seifriz (1936), and others on various species of Amoeba
have confirmed in a general way the observations of Kite. In addition,
C. V. Taylor (1920, 1923) has described the pellicle of Eplotes patella
as a firm, fairly tough, rigid substance. The micro- and macronuclei were
found to be highly gelatinous, rather rigid structures imbedded in a
viscous hyaline matrix. Needham and Needham (1926) in attempting
to inject Opalina ranarum made the observation that the outer mem-
brane is composed of a thick, tough substance; the inner cytoplasm they
found to be jelly-like, so that injected indicators would not spread
within it. The suggestion was tentatively made that the failure of the
injected indicators to spread within the Opalina might be due to its
consistency or to membranes surrounding the numerous nuclei. Follow-
ing injection, the organisms continued to swim about, and in a few
moments the injected portions suddenly dropped out, often leaving
the animals quite riddled with holes. See also Chambers and Reznikoff
(1926); Reznikoff (1926), and Morita and Chambers (1929), on in-
jection experiments in Amoeba with similar results.

Heilbrunn (1929b) has used the centrifuge method to study the
absolute viscosity of A. dubia, which he found to be approximately 2
times that of water at 18° C. However, the viscosity was found to vary,
with changes in temperature, from about 2 times water at 18° C. to
25 times water at 214° C. (1929a). Pekarek (1930), by studying the
Brownian movement in Amoeba, has estimated its viscosity to be about
6 times that of water. More recently Seifriz (1936), by observing
Brownian movement in Amoeba, estimated the viscosity in a quiet form
to be 700 to 800 times that of water; in an active form it was much
lower. Likewise Pantin (1924b) estimated the absolute viscosity of

PROTOPLASM OF PROTOZOA 53

certain marine amoebae to be comparable to that of vaseline, i.e., over
1,000 times that of water.

Fetter (1926), using the same method as Heilbrunn, found the
absolute viscosity of Paramecium to be 8,027 to 8,726 times that of
water. Hyman (1917) has found that the heliozo6n Actinos phaerium
eichhornii can be cut into pieces as if it were solid. In addition to the
viscosity and the changes in viscosity of the protoplasm which may take
place within organisms under “normal” conditions, certain experi-
mental conditions, such as abnormal salt concentration, acids, and
alkalies, abnormally high or low temperature, mechanical agitation,
changes in hydrogen-ion concentration, anesthetics and narcotics, radia-
tion, sound waves, and so forth, may cause marked changes in the
viscosity of the protoplasm.

The effect of various agents (chemical, mechanical, electrical, and
so forth) upon the viscosity of protozoan protoplasm has been deter-
mined by studying their effect upon locomotion, Brownian movement,
body form, pseudopod formation, rate of action of the contractile
vacuole, as well as by centrifuging and by the microscopic appearance
of the cytoplasm.

THE EFFECTS OF WATER

Since the principle solvent of protoplasm is water, any condition
which tends to increase or decrease the water content of the organism
also tends to change the viscosity or consistency of its protoplasm. Thus
hypertonic solutions and desiccation usually cause the cells to shrink,
this being accompanied by an increase in the viscosity of their proto-
plasm; a hypotonic solution or the injection of water directly into the
organism tends to induce a swelling, accompanied by a decrease in the
viscosity of the protoplasm (see Pantin, 1923).

DHE EFFECTS OF SALTS

The effects of salts on the consistency of certain Protozoa, particularly
Amoeba, have been rather extensively studied. Giersberg (1922), Ed-
wards (1923), Chambers and Reznikoff (1926), Reznikoff and Cham-
bers (1927), Pantin (1926a, 1926b), Brinley (1928), Heilbrunn and
Daugherty (1931, 1932, 1933, 1934), Thornton (1932, 1935), Pitts
and Mast (1934), Butts (1935), and others have studied the action

54 PROTOPLASM OF PROTOZOA

of various salts and salt antagonisms upon the consistency of Amoeba
protoplasm. In general it has been found that sodium and potassium
ions tend to increase the viscosity of the internal protoplasm, while
calcium and magnesium tend to lower it. However, Heilbrunn and
Daugherty (1932, 1934), have found that this does not hold for the
plasmagel (1.e., outer layer) of A. proteus. Here calcium produces a
pronounced stiffening of the cortical gel, and this effect tends to be
antagonized by Na, K, and Mg. Potassium has the strongest liquefying
effect, Mg next, and Na the least action. The degree of reaction, par-
ticularly the antagonism of salts, seems to vary somewhat, depending
upon the hydrogen-ion concentration (Pitts and Mast, 1934).

Greeley (1904) observed that KCl coagulates or increases the
viscosity, whereas NaCl liquefies or decreases the viscosity of the proto-
plasm of Paramecium. Heilbrunn (1928) reports that unpublished
work of Barth shows that lithium salts cause a coagulation or increase
in viscosity of the protoplasm of both Stentor and Paramecium. Heil-
brunn (1928) found that sodium, potassium, ammonium, and lithium
chlorides all cause coagulation of Stentor protoplasm and that weak
solutions of HgCl, produce a coagulation of the protoplasm of Ezglena.
The effect of salts upon reproduction in Amoeba has been studied by
Voegtlin and Chalkley (1935) and by Butts (1935). Oliphant (1938)
observed that potassium, lithium, sodium, and ammonium salts induce
reversal in the direction of the effective beat of the cilia of Paramecium,
whereas calcium and magnesium do not. The reversal in direction of the
beat of the cilia is thought to be associated with an increase in viscosity
of the cytoplasm. See also the work of Spek, 1921, 1923, and 1924,
on the action of various salts on Actinosphaerium, O palina, and other
Protozoa.

Chambers and Howland (1930) have cut or torn Spzrostomum
in CaCl, solutions; the exposed protoplasm coagulates into a dense mass
which the uninjured part of the organism pinches off. Injection of
CaCl, produces localized coagulated regions which are pinched off.
Potassium chloride and NaCl cause liquefaction. Ephrussi and Rap-
kin (1928), however, report that CaCl, facilitates ‘‘l’explosion” of this
ciliate; KCl and NaCl render explosion more difficult.

Chambers and Howland (1930) have further performed injection
and immersion experiments with A. eschhornii, a heliozoén with grossly

PROTOPLASM OF PROTOZOA =)

vacuolated protoplasm. Immersion in NaCl or KCI dissolves the vacuolar
membranes, with a dissolution of the intervacuolar protoplasm. Im-
mersion in strong concentrations of CaCl, causes coagulation of the
protoplasm; in weak solutions the coagulation may be local, and the
living remnant rids itself of the coagulated regions. After immersion
in strong concentrations of MgCl, the protoplasm coagulates into a
flabby mass; in weaker solutions localized regions rupture. Injections
result in similar but more localized effects, except in the case of MgCl.,.

THE EFFECTS OF ACIDS AND ALKALIES

There seem to be no general agreement on the effects of acids upon
protozoan protoplasm. Some authors report that they cause an increase
in viscosity, while others find that they produce a decrease in viscosity
of the protoplasm.

Jacobs (1922), by bubbling CO, through the culture medium con-
taining Paramecium and Colpidium, observed that short exposure of
these organisms to the CO, caused a decrease in the viscosity of the proto-
plasm, while longer exposures increased it. Brinley (1928) found that
CO, caused gelation of the ectoplasm and solation of the endoplasm of
A. proteus. Reznikoff and Chambers (1927), after injecting bubbles of
CO, into A. dubia, observed that it produced a decrease in the viscosity
of the protoplasm and that the animal was not irreversibly injured un-
less the CO, destroyed the cell membrane. Hydrochloric acid has been
observed to produce an increase in the viscosity of the protoplasm of
Amoeba (Chambers, 1921; Edwards, 1923; Brinley, 1928). Heilbrunn
(1937) finds that acids cause an increase in viscosity of both the plasma-
sol and plasmagel of Amoeba.

Chambers (1921) reports that basic dyes, which contain a relatively
strong acid radical, jelly the protoplasm of Amoeba, whereas acid dyes,
with a strong basic radical, liquefy it.

The action of alkalies has been reported to decrease the viscosity of
A. proteus (Chambers, 1921; Edwards, 1923; Brinley, 1928). How-
ever, Heilbrunn (1937) reports that alkalies increase the viscosity of
the plasmasol and decrease the viscosity of the plasmagel in Amoeba.

THE EFFECT OF TEMPERATURE

As pointed out by Brues (1927), the Protozoa are among the most
resistant of all animals to high temperatures; they have been found liv-

56 PROTOPLASM OF PROTOZOA

ing in hot springs at temperatures between 50° and 60° C. Motile forms
seem to be much more susceptible to high temperature than do en-
cysted forms (cysts of Co/poda are not killed at 100° C. dry heat for
three days), a condition probably associated with the low water con-
tent of the organisms (Bodine, 1923). That Protozoa show ability
gradually to acclimate themselves to increased temperatures has been
shown by Jacobs (1919).

Since temperature changes are known to affect the rate of action of
molecules in liquids, it is only reasonable to assume that their effect is
somewhat similar upon protoplasm. In general it may be stated that a
slight increase of temperature over that of the normal, causes a decrease in
protoplasmic viscosity; a slightly higher temperature, a reversible co-
agulation, and a still higher temperature, an irreversible coagulation
and death. Davenport (1897) has referred to these conditions as con-
traction, heat rigor, and death rigor respectively. See also the discus-
sion of the action of temperature on protoplasm by Heilbrunn, 1928.

Pantin (1924a), working on two species of marine Amoeba, found
that the viscosity was high near 0° C. and decreased with rise in tempera-
ture. The primary effect of temperature upon locomotion, he holds to be
a direct effect upon the change of state of the protoplasmic sol-gel trans-
formation.

Perhaps one of the most complete studies of the action of temperature
upon protozoan protoplasm is that of Heilbrunn (1929a) on A. dubia.
He found that at temperatures from about 3° to 10° C., the viscosity
value was about 22 to 23 times that of water. From 10° to 18° C. there
was a very rapid decrease in viscosity, approaching about 2 to 3 times
that of water, while at 20° to 25° C. there was an increase in viscosity to
about 8 to 9 times that of water, followed by a decrease to about 2 times
that of water at 30° to 35° C. Although the range of Heilbrunn’s expert-
ments was not extended beyond 35° C., he states that higher tempera-
tures cause a coagulation of the protoplasm. Thornton (1932) has
observed that the maximum viscosity of the plasmagel of A. proteus
occurs at 4.5° C. Between 4.5° and 30° C. the viscosity decreases pro-
gressively, with rise in temperature, until at 30° C. the decrease is more
rapid. Thornton (1935) has further found that the action of certain
salts does not alter the fundamental effect of temperature on the vis-
cosity of the plasmagel of A. proteus. Daniel and Chalkley (1932) ob-

PROTOPLASM OF PROTOZOA 2

served that the rate of mitosis, including nuclear and cytoplasmic divi-
sion, of A. proteus varies inversely with temperature from about 4° to
30° C. From about 30° to 40° C. these processes vary directly with the
temperature.

Greeley (1904) found that the consistency of the protoplasm of vari-
ous Protozoa, like organic colloids, varies directly with the temperature,
within certain limits. As the temperature is elevated above the normal,
the protoplasm absorbs water, so that its fluidity and motility are greatly
increased. These changes continue until a critical point is reached, at
which coagulation occurs. The resistance of P. caudatum to a tempera-
ture of 40° C. has been shown to vary with the hydrogen-ion concentra-
tion of the medium and to exhibit two maxima of resistance: one on
the alkaline and one on the acid side, with a region of minimal resist-
ance at neutrality (Chalkley, 1930). In saline solutions the mechanism
of death by heat seems to vary with different hydrogen-ion concentra-
tions. At PH 6 or less the cell coagulates, at pH 8 or more the organism
disintegrates; between these two extremes death occurs from rupture by
swelling. Furthermore, Chalkley also found an increase in thermal re-
sistance of Paramecium on the addition of Ca, and a decrease on addition
of K. Oliphant (1938) found that the rate at which the cilia of Para-
mecium beat in reverse varies directly with temperature, a condition he
implies is associated with changes in viscosity of the organisms. For a
further discussion of the effects of high temperatures on organisms, in-
cluding the Protozoa, see Bélehradek (1935).

It has been shown by Greeley (1901) that when Stentor coeruleus is
suddenly lowered to the freezing point of water, it is usually killed.
However, when the temperature is lowered slowly to 0° C., a remark-
able dedifferentiation of the animal takes place. The resorption of the
cilia and the gullet, and the throwing off of the ectosarc was observed,
and there was finally formed a spherical cyst-like undifferentiated cell,
which Greeley referred to as a “resting” cell. When returned to room
temperature, a reverse process takes place, and the cyst-like organism
becomes active. With the lowering of the temperature the organism was
observed to lose water. Greeley (1903) further found that the method
of reproduction in Monas could be controlled by temperature; at 20° C.
the organisms reproduced sexually and by fission; at from 1° to 4° C.
they reproduced by asexual spores.

58 PROTOPLASM OF PROTOZOA

According to Luyet and Gehenio (1938), Becquerel (1936) found
that certain Amoeba in dry soil were not killed when subjected to
—269° to —271° C. for 7.5 days, or when subjected to —190° C. for
480 hours. See the above-mentioned work of Heilbrunn (1929a) for
absolute viscosity values of the protoplasm of A. dubia at low tempera-
ture.

Chambers and Hale (1932) observed that Amoeba exposed to —5°
C. were not killed. However, by inserting an ice-tipped pipette into the
interior of the organism, fine feathery crystals of ice were observed to
appear immediately at —0.6° C.

In general it may be stated that low temperatures tend to increase the
viscosity and thus to decrease the rate of locomotion, and to favor cyst
formation in Protozoa. Motile forms are usually killed as the tempera-
ture of the water reaches 0° C. However, the Protozoa show some degree
of acclimatization to low temperatures.

MECHANICAL AGITATION

Mechanical agitation may cause a marked change in the consistency
of Amoeba protoplasm, apparently by causing a breakdown (thixotropic
collapse) of its internal structure. Chambers (1921) has shown that
churning Amoeba by microneedles caused a liquefaction of its proto-
plasm. Vigorous shaking (Angerer, 1936) caused at first a liquefaction
of the plasmasol of A. dubia, followed by an increase in viscosity; con-
tinued agitation caused the complete dissolution of the organism. How-
ever, in A. proteus agitation caused a decrease in viscosity in the plasma-
gel to a minimum, without the subsequent increase observed in the
plasmasol of A. dubia. High-frequency sound waves have been observed
to produce whirling of the inclusions in the small vacuoles of A. proteus
and A. dubia; higher intensities cause a mild whirling of the more liquid
regions, followed by rupture of the organisms (E. N. Harvey, E. B.
Harvey, and Loomis, 1928). A decrease in viscosity of the endoplasm
was also observed.

HYDROGEN-ION CONCENTRATION

That Protozoa can live in wide ranges of hydrogen-ion concentrations
is evident from the work of Alexander (1931) on Exglena which he

PROTOPLASM OF PROTOZOA 59

found lived in ranges from pH 2.3 to pH 11. Studies on the effects of
hydrogen-ion concentration on protoplasmic viscosity are complicated by
the rate of entrance of the ions and by the fact that they may be neutral-
ized by the buffers of the protoplasm (Pollack, 1928a).

Pantin (1926a) has found that the movement of certain marine
amoebae takes place between pH 6 and 10. It is reversibly inhibited at
the acid limit, but alkaline inhibition is reversible only after a brief im-
mersion. The rate of movement he holds to depend upon the rate of
change in sol—gel transformation. In addition, the water content of
A. proteus has been shown to vary with the hydrogen-ion concentration
of the medium (Chalkley, 1929). The effect of changes in hydrogen-ion
concentration upon the action of cilia has been studied by Chase and
Glaser (1930).

THE EFFECTS OF NARCOTICS

For a summary of the literature on the effects of narcotics in cells gen-
erally, the reader is referred to the work of Winterstein (1926) and of
Henderson (1930). In general it may be stated that the action of nar-
cotics upon cells is to change their permeability and viscosity, to inhibit
enzyme action, and to affect the electric potential.

Alcohol has been reported to produce a lowering of the viscosity of
the protoplasm of A. proteus (Edwards, 1923; Brinley, 1928). Ether
and chloroform likewise have similar effects (Brinley, 1928). Certain
paraffin oils have also been reported to have a narcotic effect on A. dubia
(Marsland, 1933). More recently Daugherty (1937) found that the
higher alcohols and ether in concentrations just below lethal cause lique-
faction of the plasmagel of Amoeba; the same concentrations of the
lower alcohols produce gelatin of the plasmasol; the higher alcohols and
ether, liquefaction of the plasmasol. More dilute solutions of the higher
alcohols and ether produce first liquefaction and then gelatin of the
plasmasol (see also Frederikse, 1933a). Potassium salts and fat solvents
liquefy the plasmagel in A. proteus (Heilbrunn, 1931).

Makarov (1935) has studied the effects of narcotics on various Infu-
soria, using vital stains in conjunction with the ultramicroscope. Narcotics
cause a change in the dispersion of the colloids, which is reversible.
Strong concentrations cause an irreversible coagulation.

60 PROTOPLASM OF PROTOZOA
THE EFFECTS OF RADIATION

The specific effects of radiation upon the cell are unknown. Further-
more, whether the nucleus or the cytoplasm is more susceptible to radia-
tion is a debated question. According to Heilbrunn and Mazia (1936),
Glocker and Reuss claim that isolated cells are less sensitive to Roentgen
rays than are cells in mass.

The permeability of Paramecium and Stylonychia to NH,OH has been
shown to increase with increased exposure of the animals to radiation
(Packard, 1923, 1924). After reviewing the literature on the biological
effects of radiation, including numerous studies on Protozoa, Heilbrunn
and Mazia (1936) reach the conclusion that ultra-violet rays, Roentgen
rays, and radium all cause liquefaction of the protoplasm and, with an
increase in exposure, coagulation. Coagulation of the protoplasm is fre-
quently preceded by extensive vacuolization. Furthermore, Heilbrunn
and Daugherty (1933), from their work on the effects of ultra-violet
rays on Amoeba, offer the theory that the effect of radiation is to release
the bound calcium from the cell cortex; it then enters the endoplasm,
causing first liquefaction and then gelation. Likewise, ultra-violet radia-
tion causes a release of fat in Amoeba°(Heilbrunn and Daugherty,
1938). For the recent ingenius method of ‘“‘microdissection”’ with ultra-
violet rays, see Tchakhotine (1937).

THE EFFECT OF HEAVY WATER

E. N. Harvey (1934) has studied the effects of heavy water on Para-
mecium, Amoeba, Euglena, and Epistylis (see also Taylor, Swingle,
Eyring, and Frost, 1933). Harvey found that paramecia were killed by
80 to 100-percent heavy water in from 6 to 10 hours. They first swim
slowly, appearing bloated; the contractile vacuole stops functioning; blis-
ters appear, followed by disintegration. However, they were not markedly
affected by 0.2-percent heavy water. Amoeba rounds up and is killed in
from 4 to 6 hours in 80 to 100-percent heavy water. Euglena, on the
other hand, is not irreversibly injured by 90 to 97-percent heavy water.
Gaw (1936b) found Blepharisma to become more spherical in 95-per-
cent heavy water, which indicates a change (decrease in viscosity) in the
physical nature of its cytoplasm.

PROTOPLASM OF PROTOZOA 61

HYDROSTATIC PRESSURE

High hydrostatic pressure (500 atmospheres) causes collapse of
pseudopodia, and a rounding up of A. proteus (Brown and Marsland,
1936). This effect is apparently the result of liquefaction of the plasma-
gel, an effect which inhibits the normal sol-gel transformation. However,
similar high hydrostatic pressures seemed to have little or no effect on the
beating of flagella or cilia of other Protozoa (Marsland, 1939).

THE EFFECTS OF ELECTRIC CURRENT

The effects of electric current on many Protozoa have been studied
(see Hahnert, 1932, for literature).

Bayliss (1920) has shown that on passage of an electric current of
the proper intensity through Amoeba, immediate gelation of the proto-
plasm takes place and all Brownian movement stops. As the Amoeba te-
covers, the protoplasm solates and the particles again take up their active
motion. Strong electric shocks caused irreversible coagulation. More re-
cently Luce (1926), Mast (1931b), and Hahnert (1932) have studied
the effects of electric current on Amoeba. In general, as stated by Hah-
nert, a constant electric current provokes responses in Amoeba by defi-
nite polar actions at the ends of the organism. Destruction or solation
of the plasmagel occurs at the cathode end immediately, then contractive,
and later, disintegrative processes occur at the anodal end. These re-
sponses are directly dependent upon the strength of the current.

IRREVERSIBLE COAGULATION

Acids and certain salts, such as mercuric chloride, in the proper con-
centrations, cause irreversible coagulation of protoplasm. Protozoa are
therefore often ‘‘fixed” in such solutions for morphological studies. The
causes of the irreversible coagulation which occurs at ‘“‘normal’’ death
are obscure.

SURFACE PROPERTIES

A discussion of the physical properties of the surface layer in Protozoa
involves its structure, which has been analyzed by a study of its gross
morphology, elasticity, contractility, extensibility, viscosity, and ultra-
microscopic architecture. It is obvious that any conception of the physi-
cal structure of the surface membrane must be in harmony with its

62 PROTOPLASM OF PROTOZOA

functions, such as preserving the integrity of the organism (by being
immiscible with water), controlling the diffusion of materials in and out
of the cell (permeability), acting as a seat of electromotive forces (mem-
brane or diffusion potentials), maintaining form (possessing tension),
forming secretions (for protection, adhesion, forming cyst walls, armor,
and so forth) and for the reception of stimuli, and so on.

STRUCTURE AND ORIGIN

The problem of surface structure in the Protozoa is often complicated
by a failure of many investigators to define clearly what they refer to as
the “cell membrane.”’ From a review of the literature it is quite obvious
that the vital membrane essential to the cell may be reduced to only
a very thin ultramicroscopic film, such as that which forms at the torn
surface of an amoeba. In other cases the “cell membrane” is of micro-
scopic dimensions and displays the physical properties of a thick, tough
pellicle. In this connection the work of Nadler (1929) showed that the
pellicle could be completely removed from Blepharisma without killing
or even affecting the shape of the organism; after a few days a new pel-
licle may be formed in these animals. Could it not be, therefore, that in
forms with thick surface layers, the physiologically active vital mem-
brane is mainly limited to a thin film, and that the remaining part of
the thick surface layer serves mainly for protection, support, giving an-
chorage to locomotor organs, secretion of slime for adhesion, forming
cyst walls, or in the formation of other surface structures which possibly
aid the animal in coping with its environment?

In addition to the vast literature describing definite morphological
membranes at the surface of various Protozoa, both in the living and
fixed condition, considerable experimental work has been done on the
physical nature of the surface layers. Kite (1913), Chambers (1924),
Howland (1924c), Howland and Pollack (1927), Taylor (1920),
Needham and Needham (1925, 1926), and many others have actually
established the presence of the surface membrane by puncturing it, tear-
ing it, and in some cases actually removing it. In general it has been
found to vary in thickness from that of a delicate film to that of a tough
pellicle. Microdissection methods have also shown it to possess measut-
able elasticity and contractility, and to vary considerably in consistency
from that of the underlying protoplasm.

PROTOPLASM OF PROTOZOA 63

The tension at the surface of Amoeba has been measured by E. N.
Harvey and Marsland (1932). They injected drops of paraffin oil or
olive oil into A. proteus and A. dubia and then subjected them to centri-
fugal force in the microscope-centrifuge. Because of the bouyancy of the
oil, the organisms became stretched. The amount of distortion was photo-
graphed and, under certain assumptions, a value for the order of mag-
nitude of the surface forces was calculated. In this way these authors
found the tension at the surface of A. dubia to give values of one to three
dynes per centimeter. In this form they concluded that “there can be no
appreciable turgidity due to resisting surface layers.” However, in A.
proteus it was impossible to pull even large oil droplets out of the or-
ganism by the highest centrifugal force available. For this reason no
tension at the surface of this organism could be calculated, but they esti-
mated it to be about thirty times that for A. dubia. The surface of A. pro-
teus was described as a firm, tough, external layer.

Since the surface membrane may be of ultramicroscopic dimensions,
its physical properties are not easily determined, and for this reason
much of our knowledge is of a theoretical nature. It has been held that
the appearance of a new membrane at the surface of a torn bit of proto-
plasm is due to the accumulation at the surface of substances, chiefly
lipoid, which tend to lower surface tension. However, Heilbrunn thinks
of this process of new surface membrane formation as a “surface precipi-
tation reaction,’ comparable in many ways to the clotting of blood. He
has produced evidence to show that the presence of calcium is a pre-
requisite for the formation of new surface membranes. In addition, he
holds that any factors which cause a release of calcium from its protein
binding cause the ‘‘surface precipitation reaction” to take place within
the cell interior, giving to it a froth-like appearance.

Whatever may be the exact mechanism of new membrane formation,
the fact that a time factor is involved in its production from the cyto-
plasm, that it assumes increased tension over that of the underlying
cytoplasm, that its consistency and durability depend both upon the en-
vironmental medium and the specific character of the protoplasm from
which it is formed, as well as its semipermeable properties, all point to
the surface membrane as being a definite, organized structure (Cham-
bers, 1924).

Recent researches upon the optical properties of cell membranes other

64 PROTOPLASM OF PROTOZOA

than those of Protozoa, by means of polarized light and X-ray diffrac-
tion methods, seem to indicate that they are constructed of lipoid and
protein molecules, with their long axes arranged perpendicular and
parallel, respectively, to the surface of the cell (Schmitt, 1938). E. N.
Harvey and Danielli (1936) also hold the cell membrane to be com-
posed of lipoid and protein substances. Furthermore, Langmuir has
shown that the structure and surface properties of certain nonliving
membranes are frequently determined by the orientation of the indi-
vidual molecular layers; these may, under certain conditions, undergo
almost instantaneous reversal or reorientation. This overturning may
markedly alter the chemicophysical properties of the membrane, a fact
which may prove to be of considerable interest in the study of the
permeability of cells generally. As pointed out by Harvey (1936), one
would seem justified in conceiving of the surface membrane in different
cells as composed of a monomolecular layer and a polymolecular film, or
as a polymolecular oil film with oriented adsorbed protein molecules
varying from a rather liquid to solid consistency.

The remarkable film-forming properties of the ciliate Sp/rostomum
have been studied by Fauré-Fremiet, Ephrussi, and Rapkine (1926).
This organism explodes when it comes into contact with the air-water
interface, and its solid constituents spread over the surface of the water.
By first dusting the water with talc, it was determined that the surface
film was 4.2 to 5.7 1 up thick, and probably monomolecular.

PERMEABILITY

1. Cell membrane.—In Protozoa as in all other types of cells one of
the most important properties of the protoplasm is its ability to form
new surface membranes which have a selective permeability. In addition
to preserving the integrity of the cell, the surface membrane regulates
to a large extent the passage of dissolved foodstuffs and oxygen into the
cell and the diffusion of waste materials from it. The rate of this exchange
depends in part upon the degree of the permeability of the surface mem-
brane and in part upon the osmotic concentration of the cell and of the
surrounding medium.

It is not our purpose to discuss generally here the physiological prob-
lems of permeability and diffusion. However, many fresh-water Proto-

PROTOPLASM OF PROTOZOA 65

z0a possess an interesting mechanism for controlling their osmotic state.
The protoplasm of these organisms possesses a much higher osmotic
concentration than that of the surrounding medium. For this reason
water tends continually to enter them, and, were it not for the controlling
mechanism of the surface membrane and the continuous bailing out
process of the contractile vacuole, the animals would swell up and burst.
Adolph (1931) has reviewed the literature dealing with the rate of water
exchange in Protozoa and has found that the fastest turnover is in
Cryptochilum, which excretes its body volume in 2 minutes; the slowest
water exchange was in Amoeba, which requires 31.5 hours, while P.
caudatum eliminates its own body volume of water in from 15 to
20 minutes.

Water, when injected in small amounts into an amoeba, readily dif-
fuses throughout the cytoplasm, causing a temporary cessation of ame-
boid movement (Chambers, 1924). However, if the injection be great
in amount i.e., equal to half the body volume, the water tends to collect
on one side in the form of a large blister, which is eventually pinched off,
and within a short time the amoeba resumes its normal activities (How-
land and Pollack, 1927). In addition, a marked increase in contraction
rate and water output of the contractile vacuole was noticed.

In contrast to the above experiment, Mast and Doyle (1934) caused
A. proteus, A. dubia, A. radiosa, and A. dofleini to lose water by plac-
ing them in 3-percent egg albumen, hypertonic salt solutions, or solutions
of calcium gluconate. The organisms decreased in size and their surfaces
became wrinkled and covered with protuberances, folds, and crevices.
The crests of some of the adjoining protuberances and folds fuse, giv-
ing rise to tubes open at one end. The region of the fused folds push
out, forming a pseudopodium, thus extending the tubule, the mouth of
which was observed to expand and contract, drawing the fluid in. Later
the tubule disintegrates, releasing the fluid to the inside of the organ-
ism. This “drinking” of water by means of tubules seems to be an im-
portant mechanism, to compensate for the rapid loss of water in the
organism. Further experiments by Mast and Fowler (1935) showed that
when A. proteus was placed in Ringer solution containing various con-
centrations of lactose, it would decrease as much as 88 percent in total
volume without injury. The rate at which the water leaves the cell was

66 PROTOPLASM OF PROTOZOA

found to be approximately 0.026 cubic micra per minute through each
square micron of surface. These authors concluded that the permeability
to water was regulated by the plasmalemma.

It is well known that many marine Protozoa, especially Sarcodina, do
not possess contractile vacuoles, but, when transferred to fresh water,
contractile vacuoles may appear (Schaeffer, 1926). The contractile vacu-
oles of fresh-water forms either work very slowly or disappear entirely
upon the organism being transferred to various concentrations of sea
water. Furthermore, some Protozoa of the same species, for example
Actinophrys, may be found in both fresh and salt water. The cytoplasm
of the fresh-water form is greatly vacuolated and possesses a contractile
vacuole, while the cytoplasm of the salt-water form is relatively free of
vacuoles, including the contractile vacuole, and the general appearance
of the cytoplasm has changed to a granular condition. Thus a gradual
acclimatization of fresh-water Protozoa to salt water seems to reduce the
difference between the external and internal osmotic pressures by a loss
of water from the cell. This is probably accompanied by changes in the
physical state of the protoplasm, particularly its consistency and specific
gravity. It is of interest to recall here the extraordinary case of Noctiluca,
in which the specific gravity of the organism is less than that of the sur-
rounding sea water, owing to lower concentration of salts. Here the
water, instead of entering the organism, tends to diffuse out of it, so
that osmotic work must be done to retain its constant state.

Experiments designed to test the selective permeability of the surface
membrane of the Protozoa have been carried out by utilizing various
dyestuffs (Chambers, 1922; Ball, 1927). For example, according to
Chambers (1922) an aqueous solution of eosin does not stain Amoeba
from the exterior. However, if injected into the interior of the cell, it
readily diffuses throughout the protoplasm.

Attempts to visualize the mechanism of permeability control in sur-
face membranes have been made by assuming a solubility of the permeat-
ing substance in the membrane inself or by assuming the membrane to
have a sieve-like structure similar to a filter, but much smaller. In any
case, whatever may prove to be the final answer to this problem, it will
undoubtedly involve a change in molecular aggregation, organization,
and polarity of the elements of the physiological membrane.

2. Nuclear membrane.—The interphase nucleus, like the cytoplasm of

PROTOPLASM OF PROTOZOA 67

the Protozoa, is surrounded by a limiting surface membrane which may
be demonstrated in many forms by the usual cytological techniques (see,
for instance, Chalkley, 1936). In addition, experimental studies on the
living nucleus by Kite (1913), Chambers (1924), C. V. Taylor (1920),
and many others have substantiated the view of the presence of a defi-
nite nuclear membrane, often extremely thin, but usually composed of a
moderately tough, solid substance, which, upon rupture of the surface
of the organism, may preserve the integrity of the nucleus for a consider-
able time. Seifriz (1936) reports that the nuclear membrane of Amoeba
may be readily removed by microneedles, following coagulation of the
nucleus as a whole.

Morita and Chambers (1929) have shown that the nuclear membrane
of Amoeba is permeable to acid, while the general body surface of the
animal is not.

King and Beams (1937) report that the macronucleus of P. caudatum
in the vegetative stage was greatly stretched by centrifugation. In some
specimens it was separated into a relatively heavy chromatic portion and
a relatively light achromatic portion. Animals with their macronucleus
separated in this way were able to live and carry on apparently normal
metabolic processes. Here it is evident that a mechanical disruption of
the macronucleus did not cause a marked physical change of the cyto-
plasm, as often happens when the nucleus is punctured or cut by a needle
(Kite, 1913; Peebles, 1912). King and Beams were unable to differenti-
ate a limiting membrane surrounding the two separated portions of the
macronucleus, and, if present, it must have been of ultramicroscopic di-
mensions.

Luyet and Gehenio (1935) were unable to demonstrate a definite
membrane surrounding the macronucleus of P. cawdatum by means of
ultra-violet absorption methods.

Whatever, in the final analysis, the physical structure of such ultra-
microscopic nuclear membranes may prove to be, it will probably in-
volve a special molecular behavior, characteristic of surfaces much like
that which is thought to occur between nonliving immiscible fluids.

3. Contractile vacuole —For the early literature dealing with the long-
disputed question of the presence or absence of a permanent membrane
surrounding the contractile vacuole in Protozoa, the reader is referred to
the works of C. V. Taylor (1923), Lloyd (1928), Howland (1924a),

68 PROTOPLASM OF PROTOZOA

King (1935), and Kitching (1938). It is well known that the contractile
vacuole often arises from the coalescence of smaller vacuoles.

C. V. Taylor (1923) studied the structure of the contractile vacuole
of Exzplotes and found that it appeared to be composed of a definite
“wall” of measurable thickness. However, he later concluded that the
apparent ‘wall’ was an optical illusion and that careful study revealed
only the internal surface of the “wall” to be sharply delimited; externally
it merges not abruptly, but gradually, into the surrounding medium. By
moving the needle point against and about the contractile vacuole, he
found it could be displaced and that its boundary was tolerably durable
and its viscosity distinctly higher than the surrounding endoplasm. With
completion of contraction, the vacuole wholly disappears.

In a similar study upon the contractile vacuole of A. vérrucosa and P.
caudatum, Howland (1924b) and Howland and Pollack (1927) have
been able to dissect the contractile vacuole out of the organisms and ob-
serve it floating freely in the water. Here it may be stained by alizarin
blue and manipulated by microneedles. Upon puncture, the surface of
the membrane was observed to wrinkle. In other experiments, when the
contractile vacuole was forced into contact with the plasmalemma, a
fusion took place and, because of this fact, Howland and Pollack were
led to suggest that the surface membranes of both the cell and the con-
tractile vacuole must possess similar physical properties. These authors
further found the contractile vacuole to lie in a region of gelated endo-
plasm, a condition they think necessary for its functioning.

Mast (1938) has described the membrane at the surface of the con-
tractile vacuole in A. proteus as a well-differentiated structure about
0.5 micron in thickness.

King (1935) has described the permanent components of the contrac-
tile vacuole system of P. multimicronucleata as including the pore with
its discharging tubule, and the feeding canals, each made up of a distal
excretory portion, an ampulla, and an injection tubule. The membrane
of the contractile vacuole itself is considered a temporary structure, dis-
appearing at systole and closing the pore, which ruptures at the next
systole. The new membrane of the contractile vacuole appears by the
coalescence of the membranes of vesicles which lie just under the pore,
and becomes continuous with that of the pore. In centrifuged P. cauda-
tum, King and Beams (1937) observed that in some cases the vacuole

PROTOPLASM OF PROTOZOA 69

seems to have been moved out of its position in contact with the mem-
brane closing the pore. In spite of this, the feeding canals continue to
form other main vacuoles, each of which expels its contents through
the pore in normal fashion. The displaced vacuole is free in the endo-
plasm, and, if not too large, may move about in the protoplasmic stream.
Such vacuoles have been observed to be present in the cytoplasm twenty-
four hours after centrifuging. These observations were interpreted in
further support of the view that the main contractile vacuole and its
membranes are purely temporary, forming anew before each systole by
the fusion of feeding vesicles formed at the vacuolar ends of the feeding
canals. The feeding canals are markedly osmiophilic, and for this reason
they have been described by Nassonov (1924) as homologous to the
Golgi apparatus. However, this view is not supported by Beams and
King (1932) and King (1935). Gelei (1928) thinks of the cytoplasm
surrounding the feeding canals as ‘“‘nephridial-plasma’” and suggests a
parallelism between the nephridial system in Paramecium and that of
higher organisms. Metcalf (1910) observed, in the cytoplasm surround-
ing the contractile vacuole of A. proteus, small round granules which
he termed “excretory granules.’’ These bodies, which are permanent
structures, are thought by Metcalf to be functionally connected with
secretion. However, Mast (1938a) has been unable to substantiate this
view.

It is interesting to note that the permeability of the cell membrane and
that of the contractile vacuole system are in some ways similar. For in-
stance, Morita and Chambers (1929) report that in A. dubia both the
surface membrane and the contractile vacuole membrane are imperme-
able to HCl. Kitching (1936) reports that the vacuolar surface, like
the cell surface, is relatively impermeable to salts in the peritrich ciliates.
He further presents arguments to show that the vacuolar system in these
forms actively secretes water.

4, Food vacuoles —In many free-living Protozoa the ingested food
particles are surrounded by a distinct vacuole. It is here that the digestive
enzymes collect and act upon the solid food particles, converting them
into a dissolved form suitable for use by the organism. The so-called food
vacuolar membranes, in forms such as Amoeba and Paramecium, must
perform much the same function as the cell membranes surrounding the
cells in the intestine of higher organisms. In other words, the semiperme-

70 PROTOPLASM OF PROTOZOA

able membrane delimiting the food particles from the surrounding cyto-
plasm prevents the diffusion of undigested substances, such as proteins
and starches, until they have been broken down into diffusable com-
pounds of much smaller dimensions.

The formation of food vacuoles by means of food cups (Kepner and
Taliaferro, 1913; Schaeffer, 1916) is of interest in connection with a
consideration of their surface structure. In Amoeba the membrane sur-
rounding the food particles has been observed to be derived directly
from the surface of the organism i.e., the plasmalemma. Schaeffer
(1916) reports that A. proteus may form several hundred such food
vacuoles a day, a fact which would necessitate considerable replacement
of the plasmalemma. If this observation be correct, one need only as-
sume a structure for the food vacuole membrane like that of the plasma-
lemma. Furthermore, it has been shown that the membrane of a food
vacuole which has completed its function in the cell may fuse upon eges-
tion with the cell membrane from which it has been derived (Howland,
1924c). In addition, some vital stains, such as neutral red, readily pene-
trate the surface membranes of many Protozoa and stain the food vacu-
oles, which suggests similar permeable properties for both the mem-
branes surrounding the surface and those surrounding the food vacuoles.
In contrast to its penetration of the surface and food-vacuole mem-
branes, neutral red does not readily diffuse through the membranes
of the nucleus or that of the contractile vacuole.

Dissection and multilation studies upon a number of Protozoa have
shown that the food vacuoles are capable of existing free of the cyto-
plasm for relatively long intervals of time. For instance, King and
Beams (1937) have observed food vacuoles in water to retain their
form for over one-half hour, after which time the vacuolar membrane
was observed to wrinkle, followed by a breakdown of the vacuole.

Dogiel and Issakowa-Keo (1927) immersed Paramecium in various
salt solutions and India ink. In solutions of MgSO,, MgCl,, and FeSO,
the food vacuoles are much elongated. These sausage-like food vacuoles
may swell up or may be extruded through the gullet. In BaCl, the food
vacuoles are small and spindle-shaped. Whether this effect is produced on
the membrane of the food vacuole or upon the cytoplasm is not clear.

Mast (1938b) has recorded for A. proteus that a food vacuole may

PROTOPLASM OF PROTOZOA 71

divide several times, forming a number of vacuoles. In other cases food
vacuoles have been observed to fuse (Mast and Hahnert, 1935).

From the comparatively few accounts of the physical structure of the
food vacuole membrane, it is not possible to give an analysis of its struc-
ture. However, it seems reasonable to conclude that it is usually of mo-
lecular dimensions, capable of resisting deformation, and that it is
permeable to enzymes, water, certain dyes, and digested food materials.
Its architecture is probably much the same as that of the surface mem-
brane, except perhaps for its thickness.

5. Other types of vacuoles —tIn addition to the contractile vacuolar
system and the food vacuoles, many other types of vacuoles may be found
in the cytoplasm of the Protozoa. Such vacuoles may be characteristic
of the cytoplasm, as the crystal vacuoles in Amoeba (Mast and Doyle,
1935a), transitory vacuoles (Hopkins, 1938), large acid-filled vacuoles
associated with changes in specific gravity of Noctiluca (E. B. Harvey,
1917; Lund and Logan, 1925); or they may be induced by certain ex-
perimental methods, such as change in the salt content of the environ-
ment (Schaeffer, 1926), exposure to dyestuffs, X-rays, poisons, and so
forth (Heilbrunn, 1928). These vacuoles, too, frequently have been
reported to fuse; normally as in the coalescence of vacuoles in Noctiluca
(Lund and Logan, 1925), and experimentally in Paramecium in which
King and Beams (1937) observed the crystal vacuoles to fuse when cen-
trifuged.

Little is known concerning the actual physical structure of the mem-
branes of such vacuoles, but there is no reason for believing that their
structure differs greatly from that of other surface membranes surround-
ing vacuoles, such, for instance, as the feeding vesicles of the contractile
vacuole of a form like Ezp/otes or Paramecium.

ADHESIVENESS OR STICKINESS

Among the physicochemical properties of the protoplasm-liquid
medium interface, adhesiveness is of importance in considering such
subjects as ameboid movement, tissue culture, leucocyte activity, im-
munity, and cell movements in embryology. Pfeiffer (1935) has listed
the literature on adhesiveness, which is scattered very widely.

It is well known that amoebae creep on vertical surfaces, even on the

72 PROTOPLASM OF PROTOZOA

under side of the surface film of water, creeping on this as though it
were a solid body, but Bles (1929) denies that Arce//la can move on a
clean surface film. Specimens of Amoeba on the under side of the cover
glass swell up when disturbed, but may still stay attached to the ‘‘ceil-
ing’ (Dellinger, 1906). Adhesion to the substratum, according to
Mast (1926b), is due either to the secretion of an adhesive substance or
to a state of the plasmalemma. According to Rhumbler (1898) and
Jennings (1904), an amoeba probably adheres to the substratum by a
mucus-like secretion. Many observers have reported that if the surface
of an amoeba is touched with a fine glass rod, it adheres to the glass
rod, so that sometimes a bit of the organism may be pulled off (Mast,
1926b) or the whole organism may be dragged about by a thread of
mucus from the amoeba which has become attached to the glass rod
(Rhumbler, 1898; Jennings, 1904). According to Chambers (1924), a
stationary amoeba accumulates a considerable amount of slime, by
which it is attached to the substratum; an amoeba dragged out of posi-
tion and then released will be pulled back toward its original position.
However, Schaeffer (1920) has not been able to convince himself that
amoebae secrete mucus; nevertheless, whatever the method, the tips of the
pseudopods often adhere so firmly to the substratum that strong squirts
from a pipette are necessary to dislodge them (Dellinger, 1906). Hy-
man (1917) applied a needle to the posterior end of an amoeba and
pulled the animal in two, in spite of the tensile strength of the ecto-
plasm.

It is commonly held that attachment is one of the important factors
in ameboid movement: when the organism is not attached there is no
locomotion, although protoplasmic flow and the gel-sol and the sol-gel
processes may be observed. Mast (1929) has studied the factors in-
volved in attachment of Amoeba to the substratum and finds that simple
agitation of the dish in which the amoeba are cultured may cause an 1n-
crease in the firmness of adhesion. There was slight attachment in pure
water (see also Parsons, 1926), but strong attachment over a great
range of hydrogen-ion concentration, pH 4.6 to 7.8). All the salts
tested by Mast caused a decrease in time for attachment, and an increase
in firmness of attachment; since non-electrolytes have no effect, Mast
believes that charges on the ions may change the surface charge of the
amoeba or may change unknown internal forces. Bles (1929) has shown

PROTOPLASM OF PROTOZOA 73

that under conditions of low oxygen tension, Arce/la releases its attach-
ment and floats to the surface.

According to Chalkley (1935), locomotor activity and attachment to
the substratum are important factors in cytoplasmic fission in Amoeba.
If one of the daughter cells is detached while the other remains attached,
fission is not completed, but the unattached daughter flows back into the
attached. However, if both amoebae are detached, as in distilled water,
fission may be completed by the pushing of the pseudopodia of one ani-
mal against those of the other, thus breaking the narrow cytoplasmic
bridge between the daughter cells.

In Testacea with lobose pseudopods, such as D/fflugia and Centro-
pyxis, the organism is pulled along by means of contraction of pseudo-
pods, the tips of which have become attached to the substratum (Dellin-
ger, 1906). That this attachment is of special nature and of considerable
strength has been shown by Mast (1931a), who prevented the shell from
being dragged along by the contraction of the pseudopods, under which
conditions the pseudopods were torn loose and snapped back toward the
shell. Testacea, such as Difflugia, Lesquereusia and Pontigulasia, attach to
Spirogyra and devour the cell contents (Stump, 1935). According to
Penard (1902), the filose pseudopods of such forms as Psewdodifflugia
and Cy phoderia have as their principle function the fastening of the ani-
mal to the substratum, to which they adhere with extraordinary tenacity.
Acanthocystis ludibunda (Helizoa) moves by adhesion of its axopods
to the substratum, after which they contract, rolling the animal along
over a distance twenty times its diameter in one minute (Penard, 1904).
Schaeffer (1920) has estimated that the axopods of this form must ad-
here, contract so as to pull the animal along, and relax their hold, all in
the short time of two seconds. The reticulose pseudopods of the Forami-
niferan, Astrorhiza limicola, attach and contract much as do the lobo-
podia of the Testacea. Here, however, the organism leaves a trail of
slime and bits of pseudopodia behind it, according to Schultz (1915).
In Gromia squamosa the reticulose pseudopods play a very slight part
in locomotion, but serve mainly as organs of attachment and food cap-
ture (Penard, 1902).

The whole outer layer of many rhizopods is sticky: it is well known
that certain shelled rhizopods collect foreign particles to be included in
their tests. According to Stump (1936), Pontigulasia will not reproduce

74 PROTOPLASM OF PROTOZOA

unless shell materials are present in the cultures in which the organisms
are grown; shell materials are collected just previous to division, but
before nuclear changes have begun. Verworn (1888) has found that
only after mechanical irritation of the pseudopodia of Difflugia do they
become sticky enough for glass particles to adhere to them, to be later
drawn into the shell. Foreign particles may adhere to the surface of a
moving amoeba and be carried forward by the outer layer, sometimes
making many complete revolutions. Parsons (1926) has observed that
carmine granules adhere to and move over the surface of an amoeba
floating in distilled water, although there has been a definite loss of ca-
pacity for adherence to substratum. Then, too, the outer layer of Fo-
raminifera and Heliozoa is quite fluid, serving to capture food organisms
which adhere to the surface. Certain of these organisms (i.e., Actz-
nophrys) have been reported to form temporary colonies and to capture
large objects of prey; such temporary colonies may be induced by me-
chanical means (Looper, 1928). However, it has been shown by Daw-
son and Belkin (1929) and Marsland (1933) that the adhesion between
A. dubia and an oil surface is distinct from the process of ingestion,
since a cap of oil may adhere to and spread over the tip of a pseudopod
without ingestion taking place. The relation of adhesion to phagocytosis
has been extensively studied in the amebocytes of the invertebrate Meta-
zoa (see Fauré-Fremiet, 1930; Loeb, 1927, 1928).

A remarkable example of the stickiness of protoplasm is shown by
the Choanoflagellata, in which the flagellum is surrounded by a proto-
plasmic collar; food particles, brought to the collar by the movements
of the flagellum, adhere and are carried to the point of ingestion by the
flowing protoplasm of the collar. Some flagellates (Heteromita and
Ozkomonas) have pseudopodia which are primarily adhesive in func-
tion. While flagella are primarily organs of locomotion, they frequently
are used as organs of attachment. This is commonly observed in Costa,
Chilomonas, Heteromita, Pleuromonas, Anisonema, and Petalomonas.
The nature of this adhesion is not known.

Certain of the developmental forms of trypanosomes become at-
tached by their flagella to the walls of the organ of the invertebrate host
in which they are found. Lwoff (1934) has mentioned that Strzgomonas
oncopelti and S$. fasciculata may swim free in cultures or may fasten to
the glass side of the culture dish; they release upon a few seconds heating

PROTOPLASM OF PROTOZOA 75

at 55° C. On the other hand, in Leptomonas ctenocephali the attached
forms are held in position by the slimy enlarged tip of the flagellum,
which is much shortened; they are killed at 55° C. before they can re-
lease their hold.

Adhesion phenomena have recently been introduced for diagnosing
trypanosomiasis. In adhesion tests a drop of Trypanosoma suspension
is added to one drop of equal parts of blood and 2-percent sodium cit-
rate. If the blood comes from an infected animal, the red blood cells,
and occasionally blood plates as well as bacteria, adhere to the trypano-
somes. The immune serum contains an antibody that apparently acts
upon the surface of trypanosomes to make it sticky. This test has been
recently used by Taliaferro and Taliaferro (1934) in connection with
other tests in equine trypanosomiasis; the adhesion phenomenon may
persist for more than two years in animals that have been infected.

Fauré-Fremiet (1910) has emphasized the fact that while motility
is the more general, it is not the only property of cilia. They may be-
come immobile and serve as rigid stalks, or may be reduced to short rods
and serve for fixation or for protection to the cell that bears them. It has
frequently been observed that the movements of ciliates often cease tem-
porarily when they come in contact with a firm surface. According to
Jennings (1906) “‘the cilia that come in contact with the solid cease
moving, and become stiff and set, seeming to hold the Paramecium
against the object.” Saunders (1925), however, holds that partially
extruded trichocysts with sticky tips serve as temporary attachments in
Paramecium. The hypotrichs generally can creep along vertical surfaces
and on the under surfaces of the cover glass. The hypotrich Ancystro-
podium mau pasi may attach itself by its posterior cirri (Fauré-Fremiet,
1908), and the holotrich Hemzspeira by a bundle of fixative cilia (Fauré-
Fremiet, 1905). Kahl (1935) thinks that such adhesion depends upon
the ability of cilia to become sticky, at least at the tips. Cilia are associated
with attaching organs in Trichodina and in Ellobiophrya (Chatton and
Lwoff, 1923a). Chatton and Lwoff (1923b) have also described organs
of attachment derived from the posterior ciliated region in the Thigmo-
tricha. Fauré-Fremiet (1932) has studied the fixatory apparatus of
Strombidium calkinsi which is constituted by two dorsal membranelles
nearly as long as the body and made up of coalesced cilia which separate
from each other at the adhesive distal extremity. At times the ciliate

76 PROTOPLASM OF PROTOZOA

seems to be walking on the solid surface, the two membranelles moving
one after the other, somewhat like the cirri of the hypotrichs. The mem-
branelles are not contractile but very elastic; the Strombidium may un-
fasten itself all at once and swim hastily away. Similar fixing organs have
been described for other oligotrichs (S. wrceolare and S. clavellinae).
Metacystis lagenula retracts by means of a large filament (a modified
ciltum glazed with sticky protoplasm), which adheres to the interior of
the test, while M. recurva and Vasicola gracilis seem to be kept in their
tests by a specialized cilium, a veritable bristle, which adheres to the
inside of the test (Penard, 1922). Mesodinium pulex may adhere by
means of tentacle-like structures, and various species of Stentor attach
themselves to the substrate by means of cilia (Fauré-Fremiet, 1910)
which form together with ectoplasmic extensions the so-called pseudo-
podia of the attaching disk of this form. The presence of slime-like or
mucus secretions for attachment have been described for Spirostomum
(Jennings, 1906), Urocentrum turbo and for Strombilidium gyrans. In
the latter the slime, which is secreted by an attaching organ, the scopula,
derived from modified cilia, congeals upon contact with the water into
a very resistant thread which is attached to some structure in the medium
(Fauré-Fremiet, 1910; Penard, 1922). The Strombilid?tum may then
swing back and forth like a pendulum, held in position by the thread
of slime which can be seen only because small particles of debris in the
culture medium adhere to it. Certain observations of Chambers and
Dawson (1925) on Blepharisma seem to show that the ability of cilia
to combine into composite organelles, such as undulating membranes,
membranelles, cirri, and so forth, may be dependent upon the presence
of a slime-like secretion which spreads over the cilia and joins them into
what looks like a homogeneous structure. At any rate these composite or-
ganelles are often seen to break down into their constituent cilia, after
which they may be recombined. This separation of composite motor
organelles into their components often takes place upon fixation.
According to Mast (1909), the prey of Didinium is held by means of
the seizing organ, which in some way adheres to the surface when con-
tact is made. Capture and ingestion of food depend upon the adherence
of the seizing organ of Didinium and the strength of the ectoplasm of
its prey, which is usually Paramecium. There are two different explana-
tions of how the tentacles of the Suctoria adhere to their prey: by the

PROTOPLASM OF PROTOZOA 7

presence of a viscid secretion, or by active suction of the hollow tentacles.
However, the character of the outer surface of the prey is an important
factor in its capture (Root, 1914).

SPECIFIC GRAVITY OR DENSITY

W hole organisms.—Since their specific gravity is slightly greater than
that of water, most unicellular organisms can remain suspended in fresh
or salt water only by the use of special locomotor organs such as cilia
or flagella. Among the advantages of this slightly greater specific gravity
is that the organisms are not caught in the surface film, nor in the con-
gealing water on the surface when their locomotor activities are depressed
because of low temperature; then, too, the simple methods of locomotion
of Protozoa would hardly suffice to move bodies of very great density or
to keep them suspended against the pull of gravity. In this connection the
work of Jensen (1893) should be mentioned. In spite of the inaccuracy
of his absolute measurements, it is clear that a Paramecium can lift 9
times its own weight in water. Probably a Paramecium with a specific
gtavity above 1.35 could not keep itself in suspension because a very in-
significant amount of energy 1 1 of the total is available for

> 100 1000
locomotion in this form (Ludwig, 1928a, 1930).

Certain Protozoa of floating habit frequently have hydrostatic devices
which aid in flotation, such as the gas bubbles secreted in the protoplasm
of Arcella and Difflugia; special layers of vacuolated protoplasm, such
as the calymma of Radiolaria; or very highly vacuolated protoplasm, as
in Noctiluca and in Heliozoa.

According to Bles (1929) the gas bubbles of Arce/la are formed in
the marginal protoplasm and are filled with oxygen. They are secreted
when the oxygen tension is reduced experimentally and are adaptive, in
that they reduce the specific gravity of the organism so that when oxy-
gen tension is low the organism may float to the surface, where the oxy-
gen tension is always somewhat higher. When Arcella is turned upside
down so that the external pseudopods cannot adhere to the substratum,
gas bubbles appear in from three to six minutes, before the animal be-
gins to right itself and aid in this process by lowering the specific gravity.
The bubbles disappear rapidly after the righting process is completed.
When there is more than one bubble present all grow at the same time

78 PROTOPLASM OF PROTOZOA

and decrease at the same time. In the Radiolaria periodic migrations take
place to and from the surface layers of the sea; these are brought about
by changes in the vacuolar contents of the hydrostatic layer, which, ac-
cording to Brandt (1885), is lighter than water. Schewiakoff (1927)
has described the presence of a clearly defined gelatinous hydrostatic
layer in the Acantharia (Radiolaria). Although the specific gravity of
Noctiluca (1.014) is less than that of sea water (1.026) in which it
floats, according to Massart (1893), E. B. Harvey (1917) has shown
that this form can lessen and increase its specific gravity in a regulatory
fashion. Lund and Logan (1925) have shown that the increase in specific
gravity, following strong mechanical shock or electrical stimulus, is
caused by the coalescence of large vacuoles and the liberation of their
contents, which diffuse through the pellicle. The density of the solution
in the vacuoles is less than that of sea water; according to Ludwig
(1928b) this is because of its lower salt content, its osmotic pressure
being about half that of sea water. Most marine animals possess body
fluids almost isotonic with the external fluid, but Noctéluca, together with
the marine teleosts, are hypotonic and, to a great extent, osmotically
independent. Such organisms must be impermeable to water, absorb
water in some way without salts, or take in sea water and excrete salts.
Marine teleosts apparently take in sea water and excrete the excessive
salts, and, since the membrane of Noctiluca is permeable to water, os-
motic work (negative osmotic force) must be done by the membrane
(E. B. Harvey, 1917) in maintaining this steady state instead of os-
motic equilibrium.

The protoplasm of marine Protozoa frequently becomes much vacu-
olated upon transfer to fresh water. The marine variety of Actinophrys
sol, according to Gruber (1889), has thick, granular protoplasm poor
in vacuoles and entirely lacking a contractile vacuole; during gradual
transfer to fresh water the protoplasm becomes foamy with bubbles and
a contractile vacuole appears, so that the organism is indistinguishable
from the fresh-water variety. The formation of vacuoles and the entrance
of water into them undoubtedly lowers the specific gravity in this form,
when transferred from salt to fresh water. The spine-like pseudopodia
of Heliozoa, Radiolaria, and other floating forms also serve as a protec-
tion against sinking.

The first estimation of the specific gravity of a protozoan apparently

PROTOPLASM OF PROTOZOA 7D

was that of Jensen (1893), who attempted to determine the energy rela-
tions of the movement of P. aurelia. He obtained the value of 1.25 by
suspending the organisms in solutions of potassium carbonate, a proce-
dure which gave too high values because of excessive shrinkage, due to
osmotic pressure. Later Platt (1899) suspended killed or anaesthetized
Paramecium and Spirostomum in solutions of gum arabic and found
their specific gravity to be 1.017. Lyon (1905) centrifuged living Para-
mecium in solutions of gum arabic and obtained 1.048 or 1.049. This
was repeated by Kanda (1914, 1918), who finally arrived at a value of
1.0382 to 1.0393 for Paramecium and 1.028 for Spirostomum. Fetter
(1926) utilized approximately the same value, 1.038, which she ob-
tained by centrifuging Paramecium in sugar solutions, in calculating
the protoplasmic viscosity of that form.

Leontjew (1927) has determined the density of various Protozoa
(Fuligo, Stemonitis, slime molds; Naegleria, an amoeba; and Dunaliella,
a flagellate) to be 1.020 to 1.065. Some of his readings on Fuligo varians,
obtained with a micropyknometer, are interesting enough to be men-
tioned in detail: in moist weather the density was 1.016; in dry, 1.040,
and 11 hours before spore formation, 1.065.

Heilbrunn (1929a, 1929b) used 1.03 as the specific gravity of the
protoplasm in his studies on viscosity of A. dubia. Motile amoebae
(Naegleria) have a density of 1.043, according to Leontjew (1926a),
and cysts 1.060 to 1.070 (Joschida, 1920, cited by Leontjew, 1927);
cysts of Hartmanella hyalina, a soil amoeba, have a specific gravity of
1.084 (Allison, 1924).

It is, of course, generally recognized that the protoplasm of encysted
Protozoa contains less water than that of active forms. Allison (1924)
determined the specific gravity of cysts of Colpoda by the time required
to fall through water. He finds that four-day cysts averaging 40.1 microns
in diameter, have a density of 1.042; while twenty-day cysts, averaging
25.1 microns, have a density of 1.061. Similar results were found for
cysts of Gonostomum. The decrease in size and increase in specific grav-
ity are apparently caused by water loss.

The specific gravity of protoplasm other than that of Protozoa has
been found to vary from about 1.02 to 1.08, with average values about
1.045. The publication of Pfeiffer (1934) gives a résumé of the meth-
ods and results of such studies.

80 PROTOPLASM OF PROTOZOA

Relative specific gravity of cell inclusions and components.—It has
long been known that the various inclusions of protozoan cells are of
different specific gravities. In centrifuging cultures to obtain large num-
bers of organisms for fixation previous to morphological studies, it is
often noticed that certain crystals have been displaced centrifugally. Mc-
Clendon (1909) was one of the earliest workers to fix and stain Parame-
cium after long-continued centrifuging; he found that the crystals and
nucleus were displaced centrifugally. Heilbrunn (1928) mentions that
a centrifuged Ezglena loses its spindle-shaped contour and becomes
spherical, with the granular inclusions packed at the centrifugal end.
The same author (Heilbrunn, 1929b) has used the speed of movements
of crystals centrifugally through the cytoplasm of A. dubia to estimate
the absolute viscosity of the protoplasm; the specific gravity of the
crystals was estimated to be approximately 1.10. E. N. Harvey (1931)
records that the crystals of A. dubia fall down so rapidly that their
velocity can hardly be determined in the microscope-centrifuge; and
that the crystals of Paramecium were rapidly thrown down, as was the
nucleus. He was also able to cleave living Stentor into two parts in the
microscope-centrifuge; the lighter, oral half contained none of the
Zodchlorellae which had been moved into the basal part. E. N. Harvey
and Marsland (1932) observed the movement of cytoplasmic particles
through the protoplasm of A. dzbia and found them to be layered out in
the following order: coarse granules and crystals, most centrifugal; nu-
cleus, a visibly empty zone, a zone of fine granules, and, most centripetal,
the contractile vacuole. Mast and Doyle (1935b) have recorded as
follows the relative specific gravities of the various cytoplasmic com-
ponents in A. proteus, from centrifugal to centripetal: refractive bodies,
beta granules (mitochondria) and food vacuoles containing little or no
fat, nucleus and food vacuoles containing much fat, hyaline protoplasm,
contractile vacuole, crystal vacuoles without crystals, and fat globules.
The position of the crystal vacuoles varies with the size of the included
crystals: those with large crystals are heavy and move centrifugally in
the centrifuge; those with small crystals are lighter. The small alpha
granules, which are about 0.25 micron in diameter, are not layered out.
All the refractive bodies, a large proportion of the crystals, and all the
fat may be removed, with no injurious effects, from a centrifuged
amoeba by cutting off the light and heavy ends. However, removal of

PROTOPLASM OF PROTOZOA 81

the beta granules (mitochondria) resulted in the death of the amoeba.
Singh (1939) has also centrifuged A. proteus ‘Y,’ and found the order of
layering to be: nutritive spheres, nucleus, crystals, neutral red bodies,
mitochondria, cytoplasm, contractile vacuole, and fat.

Patten and Beams (1936) centrifuged Evglena and found that the
chloroplasts form a middle belt, having on the centrifugal side paramy-
lum and neutral red bodies, while the clear cytoplasm containing the small
spherical mitochondria is at the centripetal pole. In Menodium the
heaviest inclusions are the paramylum and neutral red bodies; in Ch7-
lomonas the starch and neutral red bodies are heaviest. Johnson (1939)
has confirmed the results of Patten and Beams; in Evglena rubra, how-
ever, hematochrome is present and is displaced to the centripetal pole
with mitochondria.

King and Beams (1937) ultracentrifuged Paramecium; in this form
the various components and inclusions were layered in the following
order from centrifugal to centripetal: crystals in vacuoles, compact
chromatin of the macronucleus, food vacuoles and neutral red inclusions,
achromatic matrix of the macronucleus, endoplasm, large clear vacuoles,
and fat. Here the chromatin may be removed from the achromatic matrix
of the macronucleus; the chromatin regenerates a macronucleus; the
achromatic matrix persists for some time and apparently interferes with
subsequent divisions. Browne (1938) has ultracentrifuged Sprrostomum
and has found the contents of the cell to be layered as follows: centrifu-
gally located are the mitochondria, food vacuoles, and macronucleus; cyto-
plasm; Golgi bodies; and centripetally, vacuoles.

Daniels (1938) has used the ultracentrifuge in a study of gregarines;
here the paraglycogen and chromidial granules are heaviest; next, the
mitochondria and nucleus; cytoplasm; then the larger Golgi bodies; and
lightest the smaller Golgi bodies and fat globules. In the gregarines
studied the karyosome moved centrifugally in the nucleus, and the con-
tents of the deutomerite layered independently of those in the primite
because of the presence of the transverse septum.

It is obvious that the centrifuge may serve as an important research
tool for the identification and study of the form, relative volume, and
other characteristics of the components and inclusions found in proto-
plasm. For example, Holter and Kopac (1937), by cutting amoebae in
half after centrifuging, were able to demonstrate that the enzyme dipepti-

82 PROTOPLASM OF PROTOZOA

dase is apparently associated with the cytoplasmic matrix, independent of
all cytoplasmic constituents which could be stratified by centrifugal ac-
celeration.

OPTICAL PROPERTIES

The ordinary optical characteristics of protoplasm, such as its trans-
parency, color, and refractive index, do not seem to be of very great im-
portance except that the observation of living protoplasm is conditioned
by these properties. Too many have assumed that because a structure
cannot be seen in living protoplasm, it is therefore nonexistent.

TRANSPARENCY

The: protoplasm of the protozoan cell is generally transparent or
translucent, but in the presence of granular or other inclusions it may
appear to be opaque or nearly so. Many of the differentiations are so
nearly of the same index of refraction that special fixing and staining
methods are necessary in order to study them. The state of aggregation
of the colloids of the general protoplasm seems to be dependent, at least
to some extent, on the salt content of the surrounding medium. Thus
Actino phrys sol in sea water is densely granular, while in fresh water it is
alveolar and translucent (Gruber, 1889). Spek (1921) has shown that
Actinos phaerium becomes relatively opaque in certain salt solutions, and
that P. bursaria, which is glass-clear, becomes dark brown in artificial salt
solutions, owing to the collection of albuminoid substances into large
aggregates. Certain observations of Schaeffer (1926) on marine Amoebae
are of interest here: Flabellula pellucida, a most transparent marine
amoeba, becomes densely granular in 25-percent sea water, while F.
citata, another marine amoeba, is unusually transparent in 364-percent sea
water.

COLOR

Protoplasm is usually observed to be colorless or grayish; many of the
shades of blue, green, or yellow described for Amoebae are merely dif-
fraction phenomena or subjective in nature. The color of the endoplasm
of various amoebae has been described by Schaeffer (1926) as pale
bluish-green, yellowish-green, bluish-gray; Hyalodiscus elegans has endo-
plasm which is arange-yellow centrally and ashen-gray peripherally. The

PROTOPLASM OF PROTOZOA 83

contents of the contractile vacuole are often slightly pink. This may be
regarded as an optical illusion, since the color observed is complementary
to the usual bluish-green of the endoplasm. Very frequently the color
is obscured by the presence of colored inclusions of various kinds, or
caused by colored inclusions of the same index of refraction as the proto-
plasm and therefore difficult to differentiate.

Ciliates may be colorless, gray, pink, blue, or violet. The blue color
of S. coeruleus is caused by a coloring matter, called stentorin, diffused
through the cytoplasm, but in Blepharisma the color, which may vary
from none through pink and violet to purple, and varies with the cul-
tural conditions and from individual to individual in the same culture,
is apparently concentrated in the pellicle (Nadler, 1929). According
to Jennings (1906), most colorless Infusoria do not react at all to a
light of ordinary intensity; this has not been tested with forms such as
Blepharisma, in which the color varies.

In the plant-like flagellates color is usually caused by chromatophores,
which may be green, blue (Lackey, 1936), brown, or yellow. The most
interesting colored inclusion is the hematochrome, found in such forms
as E. rubra as red granules from 0.3 to 0.5 microns in diameter. These
euglenae form a green scum in shaded places; the green chloroplasts
mask the hematochrome, which is centrally located; in direct sunlight the
scum is red, the hematochrome being peripherally located and masking
the chloroplasts. Control of the distribution of the hematochrome is so
delicately balanced that if the euglenae are shaded for fifteen minutes,
they change from red to green (Johnson, 1939). The mechanics of
this control needs to be investigated.

REFRACTIVE INDEX

Even the finest strands of protoplasm can be seen in water, in spite
of the fact that they may be transparent and colorless. This is because
of their relatively high index of refraction. It is surprising that so little
is known about the optical characters of protoplasm which may be seen
to change during cell division. Schaeffer (1926) has shown that the
nuclei of certain marine amoebae become much more prominent by dilu-
tion of the sea water with fresh water, and Chalkley (1935) has shown
that there is a change in the refractive index of the nuclei of A. proteus
during division: the interkinetic nuclei can easily be seen at a magnifica-

84 PROTOPLASM OF PROTOZOA

tion of 200 diameters; those in division cannot. Very few measurements
of the refractive index of protoplasm have been made; Frederikse
(1933b) has reported a value of 1.40 to 1.45 for A. verrucosa; Mackin-
non and Vlés (1908) 1.51 for the cilia of Stentor, and 1.56 for the
flagellum of Trypanosoma (Spirochaeta) balbiani. Mackinnon and Vlés
made their determinations by immersing the organisms in media of
different refractive indices; double refraction, due to depolarization,
disappears in media of the same refractive index as the cilia and flagella.
Fauré-Fremiet (1929) found the index of refraction for entire amoebo-
cytes of Lumbricus to be 1.400, for the hyaloplasm 1.364; of Asterzas
to be 1.446 and 1.385 respectively (for methods see Pfeiffer, 1931).

STRUCTURAL PROPERTIES

It has long been known that the polarity of a cell may persist after
the relative positions of its various visible constituents have been changed;
this has led to the idea that polarity has its basis somehow or other in
the structure of homogenous cytoplasm, which remains unchanged in
spite of exposure to high centrifugal forces (Conklin, 1924). Polarity
and symmetry are generally present in the Mastigophora and Infusoria,
in which an anterior-posterior axis is usually persistent throughout active
life, and has been described as present in cysts (Lund, 1917, in Bursaria).
However, in Sarcodina such as Amoeba, polarity may be thought of as
continually changing, being bound up with the gel-sol process at the
temporary posterior end, the flow of protoplasm forward, and the sol-
gel process at the temporary anterior end. Hyman (1917) has demon-
strated that the temporarily differentiated anterior end of Amoeba is the
region of highest susceptibility to cyanide. Mast (1931) and others
have reported that electrical currents have a solating action on the
plasmagel, on the side directed toward the cathode.

Recently Chalkley (1935) has studied the process of cytokinesis in
A. proteus. He observed, with the onset of prophase, a loss in sensitivity,
a swelling up of the organism, a decrease in activity of the contractile
vacuole, and an increase in movement of the granules in the region
of the nucleus. With the separation of the daughter chromosome plates,
he observed a flow of the cytoplasm from the equator, in the same direc-
tion as the separating daughter plates. As the daughter plates approach
the surface of the cell and the new nuclei begin to form, a solation of

PROTOPLASM OF PROTOZOA 85

the plasmagel takes place, resulting in the formation of numerous
pseudopodia which become attached and undergo active ameboid move-
ment. At the same time, because of the flow of the cytoplasm from the
equator toward the two poles, the region of the Amoeba at the equator
has become narrowed to a thin neck. Presumably a solation of the
plasmagel at the equator, together with a pull exerted by the two ac-
tively dividing ameboid daughter cells, produces the final separation of
the organisms. The temporary polarity becomes immediately lost after the
completion of division.

The physical-chemical factors involving the change in polarity of the
protoplasm at the equator and the forces responsible for the flow of the
protoplasm from this point are unknown. However, Chalkley thinks
the fundamental principles involved in these processes are the same as
those described by Mast for ameboid movement, namely sol-gel trans-
formation. Further, it has been shown by Chambers (1938) that if the
nucleus is moved toward the cell surface, pseudopodia are induced in
that region. Becker (1928) has demonstrated that the factor which de-
termines the direction of streaming and hence polarity is located in
Mastigina, a flagellated amoeba, in the region of the nucleus. If the
nucleus is moved posteriorly, streaming ceases and is then resumed to-
ward the nucleus.

It should be pointed out that in the Protozoa nuclear division and
cytoplasmic fission may be closely correlated or widely separated in tempo,
and that they often exhibit a considerable degree of independence. How-
ever, the plane of separation of the nucleus usually determines the plane
of cytokinesis, in that they usually take place at or nearly at right angles
to each other. In organisms like the Mastigophora, the plane of nu-
clear division is parallel to the anterior-posterior axis and coincides
with the plane of cytokinesis of the organism. In Ciliata the plane of
nuclear division is perpendicular to the anterior-posterior axis and coin-
cides with the plane of cytokinesis.

In some ciliates there appears to be a definite and permanent divi-
sion zone laid down early in the life of the organism, which 1s not
disturbed by diverse multilations of the body (Calkins, 1926). Further-
more, in Frontonia this zone differs so markedly from the surrounding
cytoplasm that it can be easily seen in the living condition (Popoff,
1908).

86 PROTOPLASM OF PROTOZOA

Multilation studies by Calkins (1911) and by Peebles (1912) on
Paramecium have resulted in the production of numerous monsters.
Peebles describes this condition as due to an upset in nuclear and cyto-
plasmic division tempo; thus when the nucleus is ready to divide, the
cytoplasm is not, and vice versa. If this be true, the mitotic and cyto-
kinetic phenomena in this form must be closely integrated, and the
division mechanism of the organism as a whole be dependent upon the
proper coordination of both the nuclear and the cytoplasmic division
processes.

Child and Deviney (1926) and Child (1934), have shown that in
ciliates generally there is an anteroposterior gradient, due to the existence
of a physiological gradient in the longitudinal axis. The anterior end is
more susceptible to many agents, and there is also an axial differential
in the rate of reduction of methylene blue. Child is of the opinion that
this metabolic gradient is the only basis of physiological polarity. Lund
(1917, 1921) has found that reversal of polarity often occurs in cut
halves of Bursarza undergoing regeneration; it may also occur in normal
animals. An indication of this change in polarity was a reversed beat of
the cilia. He further found that Paramecium showed a reversed beat of
cilia, in direct electrical currents of proper strength. Verworn (1899)
has shown that paramecia and other ciliates orient themselves with the
anterior end of their bodies toward the cathode to which they swim.
On the other hand, many flagellates show an opposite behavior.

Schaeffer (1931) has presented evidence that the protoplasm of the
amoebae, and presumably of other organisms, consists primarily of spe-
cific molecules which are organized into definite patterns, and that most
or all of the characteristics of the organisms are due to or correlated
with positional relationships of the molecules.

It is generally thought at the present time that adjacent protein mole-
cules, because of their multipolar character, have an orienting effect
upon one another and that the resulting configuration may be equivalent
to a net-like structure, extended in three dimensions. That this is true
may be inferred from the anomalous viscosity of solutions of proteins
and protoplasm: they show non-Newtonian flow, i.e., their viscosity
varies with the stress applied, although they may outwardly conform to
true fluids in being free from rigidity.

Bensley (1938) has recently isolated from the cytoplasm of liver

PROTOPLASM OF PROTOZOA 87

cells a material called plasmosin, which he thinks is constituted of linear
micelles.

The formation of a fiber results from the end-to-end orientation of these
linear micelles. In protoplasm in the liquid state these micelles are probably
independent and irregularly arranged but in flowing protoplasm they would
be oriented parallel to the axis of flow. . . . From this state by simple
end-to-end combination all changes in viscosity are possible up to the forma-
tion of a fibrous gel . . . or even discrete fibers.

Bresslau (1928) has also shown that ‘“‘tektin,’” a material extruded by
ciliates, has anisotropic properties somewhat similar to those of plasmo-
sin.

The fact that the chromosomes probably represent gene-strings has
been of enormous importance in determining our ideas of significant pro-
toplasmic structure. The chromosomes apparently reproduce themselves
at each cell division, so that their individuality is retained in all cell
generations. The demonstration of the presence of these linear aggregates
of visible size, which are self-perpetuating, cleared the way for the micel-
lar theory of protoplasm structure.

Much of the evidence for the presence of linear aggregates in proto-
plasm has been obtained by microdissection, by the use of dark-field and
polarization microscopes, by studies on cohesion and swelling, and by
X-ray diffraction methods. Some of these have already been considered
and others will be discussed below.

ELASTICITY

According to Seifriz (1936), “elasticity is the best indication we have
of the structure of living matter” and is evidence for the presence of
linear aggregates. A body is said to be elastic if after having been strained
it tends to return to its original form when the stress is removed. Volume
elasticity is characteristic of fluids and solids; shape elasticity (rigidity )
of solids and colloids in the gel condition generally. The form assumed
by the bodies of various flagellates and ciliates is characteristic and
offers means of identification in many instances. Relative rigidity is of
common occurrence among those Protozoa, such as Euplotes, which have
a differentiated pellicle and a firm ectoplasm. C. V. Taylor (1920)
demonstrated this elasticity by applying pressure with a microneedle; the
body bent conspicuously over the needle but returned to normal shape

88 PROTOPLASM OF PROTOZOA

upon release of the pressure. Animals cut two-thirds across may keep
their shape; this argues for a stiff consistency of the ectoplasm, as well
as a tough pellicle. Any apparent modification in the shape of E. patella
occurs only from outside pressure, since the animal is unable to vary its
shape. Other hypotrichs may be similarly armored, as is shown by the
fact that they are broken in the ultracentrifuge (King and Beams, un-
published work) so that fragments are found swimming around as
though the whole animal were brittle rather than plastic. In other forms,
such as Paramecium, the body may be constricted when the animal
forces its way through obstacles. Upon ultracentrifuging in gum solutions
paramecia become much elongated and thin, because of the presence of
materials of different specific gravities in the cell. Such elongated animals
may survive and return to their normal shape unless the pellicle and
ectoplasm have been strained beyond the limit of their elasticity, in
which case the structure responsible for return to normal shape has been
destroyed and they die, permanently deformed (King and Beams, 1937).
That the form of Paramecium is determined by the relatively firm outer
layers has been shown by some observations of Chambers (1924), who
tore the ectoplasm with a microneedle. The fluid interior pours out into
the surrounding water and the ectoplasm soon disintegrates; but oc-
casionally the fluid endoplasm forms a delicate surface film which main-
tains the integrity of the extruded mass. Merton (1928) has studied
these so-called autoplasmic paramecia which, deprived of pellicle, cilia,
trichocysts, and ectoplasm, take on the form of a fan-shaped amoeba,
which may live for some days, divide, and exhibit locomotor activities.
Under unfavorable conditions a rayed stage, with long pseudopodia-like
extensions reminiscent of a A. radiosa, may be assumed. That the pellicle
is not the only element involved in rigidity has been shown for Blephayr-
isma by Nadler (1929). The pellicle of this form may be shed after im-
mersion in weak solutions of strychnine sulphate. The “naked” animal
emerges from the old pellicle with the shape and elasticity characteristic
of the species. Eventually the pellicle is reformed, and the process may
then be repeated.

That amoebae have elasticity of form to a considerable degree has
been shown by Jennings (1904), who bent a pseudopod with a glass
rod; when released the pseudopod sprang back into its original position.
Whole amoebae were also bent, with subsequent return to original form

PROTOPLASM OF PROTOZOA 89

after release; Dellinger (1906) and Hyman (1917) have repeated these
experiments, as indeed may be done by anyone. Howland (1924c) has
stretched the outer layer of A. verrucosa with microneedles; upon re-
lease the animal recovers its normal shape, apparently unharmed. The
protoplasm of plasmodia of slime molds (Seifriz, 1928) is at times
poorly elastic, and at other times it may be stretched into very fine, long
threads which snap back a goodly distance when released.

Seifriz has also determined elastic values by inserting minute nickel
particles into the protoplasm of slime molds and attracting these particles
electromagnetically. On release of the current the metal particles return
to their original position; the distance traveled is measured and used as
an indicator of elasticity. A maximum stretching value of 4.4 microns
was obtained for liquid, previously streaming protoplasm of myxomy-
cetes, a maximum value of 292 microns for quiescent, highly viscous
exuded masses of protoplasm from plasmodia. This latter value is slightly
greater than that for gelatin solutions and slightly greater than that of
fresh egg albumen.

The long thread-like pseudopodia of Foraminifera, which usually pull
the organism along by adhering to the surface terminally and then con-
tracting, have been shown to be elastic by Schultz (1915), who cut
these and observed them to snap back like a rubber band.

The reticulose pseudopodia (myxopodia) of the Foraminifera are very
different from the lobose and filose forms in other Rhizopoda. The
former have a soft miscible outer protoplasm which leads to fusion on
contact with one another and a relatively rigid inner axial structure
which shortens without wrinkling when the pseudopod is withdrawn.
As this denser core is formed as an elongation “in the direction of
growth, strains will be set up during the process which will give rise
to ordered and preferential arrangement tending toward the crystalline
state” (Ewles and Speakman, 1930). Thus it will be seen that the axial
solid protoplasm of these myxopodia, although it is not in the form of
a fiber, serves the same function as the axial filament of the Heliozoa
and Radiolaria.

Up to this point elasticity has been considered principally in connec-
tion with the protoplasm itself, or in its temporary completely reversible
structures. There remain for consideration those differentiations which
last the whole life of the organism and are usually irreversible, such as

90 PROTOPLASM OF PROTOZOA

flagella, axial filaments, cilia, myonemes, and supporting fibers (mor-
phonemes) of various kinds. The fact that these are elastic is too well.
known to need more than mention here. Spindle fibers, on the other
hand, are often thought to be artifacts, but Cleveland (1935), work-
ing on the hypermastigote flagellates, has pulled the centriole out of
position; the chromosomes were also displaced, but both centriole and
chromosomes immediately sprang back into position when released. This
argues not only for the reality of the chromosomal and spindle fibers, but
also that they are structures of considerable elasticity. If one considers
the aphorism of Needham (1936) “that biology is largely the study of
fibers,” these fibrillar structures of the Protozoa are of great interest
because they consist of parallel aggregations of the submicroscopic elon-
gated particles (micelles) of protoplasm (see Taylor, 7vfra, Chapter IV).

CONTRACTILITY

We may distinguish between active contraction, as in muscle fibers,
and elastic shortening after having been stretched, as in elastic fibers in
the higher animals. Although there are many examples of contractility
and elasticity, in the Protozoa, associated with differentiated myonemes or
morphonemes respectively, there are also many instances of active con-
traction in the absence of any optically differentiated structure. Accord-
ing to Lewis (1926), theories of contractility must be based on the
presence of a contractile molecule, because the fibrillae seen in heart
muscle in tissue culture are not “true” cytological structures but are
due to reversible gelation. Fauré-Fremiet (1930) also holds that the
gelified condition is often bound up with the existence of internal
fibrillar structures, which disappear when solution occurs. That such
fibrillae appear and disappear may, of course, be caused by aggregation
and disaggregation of smaller invisible fibrillae, or may even be due to
changes’ of refractive index. It is well known that objects that are of the
same refractive index, transparency, and color cannot be seen, even in
the dark field (Schmidt, 1929).

It is generally assumed that contraction of the gelled ectoplasmic
cylinder in Amoeba forces the more fluid endoplasm forward (Schaeffer,
1920; Pantin, 1923; Mast, 1926b). The contraction is thought to be
caused by the fact that the gel-sol process at the posterior end of the

PROTOPLASM OF PROTOZOA 91

amoeba results in an increase of volume and so stretches the gelled cylin-
der of protoplasm; the resulting elasticity forces the sol forward where a
decrease in volume has occurred upon gelation of the plasmasol. How-
ever, in shelled forms, such as Difflugia (Dellinger, 1906; Mast, 1931a)
and Centropyxis, there is an active longitudinal contraction of the plas-
magel cylinder which results in locomotion by pulling the shell along.
That the circular elastic contraction in Amoeba and the longitudinal
active contraction in Difflvgia are different mechanisms may be doubted.
However, Mast (1931a) has shown that D/fflvgia deprived of their
shells move much as does Amoeba. It is to be noted that the source of
energy is in the ectoplasm, so that the streaming of the endoplasm in
these forms must be of a different nature from cyclosis in other forms
such as Paramecium ot Frontonia, where protoplasmic streaming is ex-
tremely difficult to explain in terms of contraction.

There are many examples of local contractility in the literature of
ameboid movement. Swinging and revolving movements of lobose
pseudopods when not in contact with the substrate have been described
by Penard (1902), Jennings (1906), Hyman (1917), Kepner and
Edwards (1917), and many others. These differential local contractions
of the ectoplasm often approximate muscular activity, according to Kep-
ner and Edwards. Schaeffer (1926) has described corkscrew-shaped pseu-
dopodia in Astramoeba flagellipoda, with from two to eight spirals which
wave about quite like flagella, often making a complete revolution in
three seconds.

The most spectacular instances of local contraction are those in which
an Amoeba pinches a large ciliate in half. Mast and Root (1916) de-
sctibe this process as taking ten seconds for Paramecium and show that
it cannot be explained in terms of the surface tension of the Amoeba.
Beers (1924) describes the constriction of Frontonia by Amoeba until
the former was dumbbell-shaped, and ascribes the pinching to centripetal
pressure exercised by an extending collar of protoplasm which pinched
the prey in half in eight minutes. Kepner and Whitlock (1921) saw
a partly ingested Paramecium constricted much as described by Beers for
Frontonia, except for the loss of the cilia from the ingested part. The
figures and descriptions of the two latter instances are very similar to
those of Grosse-Allermann (1909) for ingestion by invagination in

92 PROTOPLASM OF PROTOZOA

A. terricola, and of Mast and Doyle (1934) for the ingestion of water
by various species of Amoeba in albumin solutions. Penard (1902) has
described and figured the pinching off of an extensive injured portion
by A. terricola as has Jennings (1906) for A. /7max. The process of
egestion, as figured by Howland (1924c) for Amoeba verrucosa, seems
also to involve extensive local contraction.

The lobopodia and filopodia of the Amoebida and Testacea are solid
peripherally with a central fluid region, while the pseudopods of the
Radiolaria and Heliozoa (axopodia) and those of the Foraminifera
(myxopodia) are more fluid peripherally and more solid axially. In
axopods and myxopods there can be no flow of endoplasm caused by
the elastic contraction of a gelled ectoplasmic cylinder. Roskin (1925)
describes the origin of the axopods of Actinosphaerium by the flowing
together and alignment of fibrillae, which eventually fuse into a hollow
tube, filled with fluid which is associated with rigidity and contractility.
That these axopods do contract rapidly has been shown for Acanthocystis,
which, according to Penard (1904), may traverse twenty times its own
diameter by rolling along the tips of the axopods, which must adhere,
contract, and then release very rapidly. In the foraminiferan Astrorhiza,
according to Schultz (1915), pseudopods stretch out five to six times
the length of the body, make ‘‘feeling’” movements and may finally either
adhere to the substratum or contract and be withdrawn. The organism is
usually fastened by three bundles of pseudopods; if one of these be torn
loose, the others contract rapidly and the animal is pulled forward. The
axial, more solid stereoplasm of these pseudopodia is distinctly fibrillar.
Schmidt (1929) has also described the formation of contractile pseudopo-
dia in the Foraminiferan Rhuwmblerinella by the alignment and coales-
cence of fibrillae.

The minute size of cilia and flagella makes it extremely difficult to
determine whether their movements are due to active contraction or to
changes occurring in the cell which bears them.

In addition to their usual method of locomotion by means of flagella,
many Mastigophora such as the euglenoids show euglenoid movement, or
metaboly. In these forms there are present in the pellicle more or less
spiral striations, which apparently are elastic in nature and tend to pre-
serve the form of the organism during and after the contraction of the
superficial layers of the body. In some cryptomonads (e.g., Chrodmonas

PROTOPLASM OF PROTOZOA 93

pulex) springing movements are brought about by the strong contrac-
tion of the outer layer of the body, the resulting locomotion being inde-
pendent of flagellar movement. Certain flagellates have an extraordinary
superficial resemblance to medusae both in appearance and in method of
locomotion: (Cystoflagellata: Leptodiscus, Craspedotella; Phytomona-
dida: Medusochloris; Dinoflagellata: Clipeodinium). In these types ac-
cording to Pascher (1917), the movements represent a special form
of metaboly and two mechanical systems should be present, one radial
and dilating, the other peripheral and contracting. The former could
of course be elastic only and bring about passive return after the con-
traction of the latter.

In the Infusoria there are many examples of specialized retractile
organelles: the tentacles of Suctoria may be retracted and extended much
as may the axopods of the Heliozoa. The structure of these tentacles is
quite similar to that of the axopods( Roskin, 1925). The remarkable
tentacles of the ciliate Actinobolina may be extended to a length twice
the diameter of the body or may be completely retracted. They are as-
sociated internally with two groups of fibrils which seem to wind up to
retract and unwind to extend them (Wenrich, 1929). In many ciliates,
such as Stentor and Spirostomum, there are actively contractile myonemes
in the ectoplasmic layer. In other related forms, such as Climacostomum,
the corresponding structures are elastic only and have been referred to
as morphonemes. The myonemes may even appear to be striated (Dierks,
1926, and others) but this has been denied (Roskin, 1923).

A most unusual case of extension and retraction occurs in the ciliate
Lacrymaria olor. Here the ‘“‘neck’’ may be extended to fifteen times the
length of the body, the form of which remains unchanged. This “‘elas-
ticity” is associated with the presence of what appears to be a series of
spiral striations. However, upon complete extension of the neck, Penard
(1922) has observed that there is only a single continuous spiral. No
experimental work has been done upon this form, so that the nature of
the extension and retraction is not understood. However, Verworn
(1899) has cut the neck of L. olor free from the head and body. The
neck retains its extensile and contractile properties exactly as when in
connection with the body.

The finer structure of the contractile stalks of the Vorticellidae has
been studied by Koltzoff (1912), Fortner (1926), and many others.

94 PROTOPLASM OF PROTOZOA

The stalk consists of an external wall, an inner liquid, and a spirally
wound contractile cord. Within the contractile cord is an excentrically
placed myoneme often called a spasmoneme, the contraction of which
causes the stalk to become coiled like a spring. The distal end of the
stalk is usually attached to the substratum, but in Vorticella natans and
V. mayeri the organisms are never attached but swim, stalk first, through
the water. In the former the stalk rolls up into narrow spirals, but in the
latter the stalk swings in a wide loop on contraction somewhat like a
flagellum. Bélehradek and Paspa (1928) have reconstructed a myogram
from moving pictures of the stalk of a vorticellid and find that the
spasmoneme does not function like a true muscle but like a modified
flagellum. Various attempts have been made to explain the contraction
and extension of the stalk of vorticellids as caused by the complex action
of two opposing bundles of fibers, or in terms of internal pressure against
coiling brought about by elastic fibers.

Myonemes are also well developed in the gregarines, where longi-
tudinal and circular myonemes are apparently responsible for bending
and peristalsis-like movements. Myonemes called myophrisks are also
present in certain Radiolaria. Here they are associated with the spreading
out of the gelatinous cortical layer, previous to their decrease in specific
gravity and subsequent rise to the surface (Schewiakoff, 1927).

ROPINESS, OR THREAD FORMATION

Living material is said to be ropy if it can be drawn out into threads;
ropiness is thought to be due to the micellar structure of the material.
The ability of a drop of a pure liquid to resist distortion is due to its
surface tension only, and in heterogenous mixtures the surface film may
approximate a solid consistency. A column of fluid breaks up into a
number of smaller spherical bodies. The formation of pseudopodia,
especially of filopodia, axopodia, and myxopodia, demonstrates the pres-
ence of solid structures in protoplasm. Then, too, protoplasm may be
drawn out into fine strands of considerable elasticity and tensile strength:
the ectoplasm of Amoeba has been drawn out into fine strands by Hyman
(1917) and many others; Schultz (1915) was able to draw out the
protoplasm of the foraminiferan Astrorhiza into long threads; this was
more marked in the outer layers than in the inner mass of protoplasm.

PROTOPLASM OF PROTOZOA 95

Many other observers, using microdissection methods, have confirmed
this for a whole series of animal and plant cells. The spinning of threads
from protoplasm is generally assumed to be dependent upon the sub-
microscopic fibrillar structure of the protoplasm, the latter being respon-
sible for its elasticity and high degree of extensibility. The tensile strength
of a strand of myxomycete protoplasm has been found by Pfeffer (quoted
by Seifriz, 1936) to be 50 mgm. per square millimeter. The ropiness of
protoplasm is of course conditioned by temperature, viscosity, hydrogen-
ion concentration, and other factors which affect protoplasm (review
by Jochims, 1930).

Further evidence of the micellar nature of protoplasm may be deduced
from the experiments of Seifriz (1936), who has examined the proto-
plasm of slime molds with a Spierer lens. When the protoplasm is quiet
it presents a mosaic appearance, but when in active flow or when formed
into a thread is presents a striated appearance. Harvey and Marsland
(1932) noted that the crystals of Amoeba fall in jerks when moving
under centrifugal force; this may be due to the presence of structural
elements in the protoplasm. Moore (1935) has found that slime molds
in the plasmodial stage will flow through pores 1 y in diameter. He also
forced this living material through bolting cloth of various mesh sizes.
The plasmodia survived after being forced through pores 0.20 mm. in
diameter or larger, but died when forced through smaller pores. Moore
thinks that there are fibrillar elements in slime mold protoplasm which
are destroyed if forced unoriented through a pore through which they
could flow if the micelles were properly oriented.

DOUBLE REFRACTION

The great advantage of the use of the polarizing microscope is that
characteristic structure which is otherwise imperceptible may be revealed,
even in the living organism, without any alteration of the specimen.
Amorphous and pseudoamorphous materials (i.e., materials in which
the orientation of the particles is a random one) are dark when viewed
between crossed nicol prisms. True double refraction is always the re-
sult of an orientation of optically anisotropic elements; many substances,
such as glass, become doubly refractive if deformed by external forces.
Many sols when in movement or when placed in an electric or a magnetic

96 PROTOPLASM OF PROTOZOA

field show double refraction, as do many gels when under pressure or
when drawn out into threads. Mechanical stress may produce double
refraction in gelatin even of 0.01 percent.

Phenomena of double refraction in protoplasm often indicate the
presence of mechanically and optically anisotropic elements oriented in
some definite way, and are among the best evidences for the presence
of micelles in protoplasm. See Schmidt, 1937, for a complete discussion.

Valentin saw double refraction in the cilia of O palina ranarum in 1861
and Rouget in the stalk muscle of Carchesium in 1862. Engelmann
(1875) carried out very extensive pioneering work on double refraction
on the Protozoa and other forms. He saw anisotropy in the pellicle
of large ciliates, such as Opalina, but was unable to distinguish double
refraction in the myonemes of Stentor because the entire outer layer is
doubly refractive. Mackinnon (1909) mentioned that the protoplasm of
Actinosphaerium is quite generally anisotropic, and Schmidt (1937)
saw faint but unmistakable traces of double refraction at the edge of the
surface layers of Amoeba, which became distinctly greater as the amoeba
became rounded up before encysting, the cyst wall showing it very
distinctly.

Engelmann (1875) also observed that the axopods of Actinosphaerium
were doubly refractive in the living condition, the anisotropy being
coextensive with the axial filaments which extend deep into the proto-
plasm. With the withdrawal of the pseudopods the condition vanishes.
Mackinnon (1909) confirmed the findings of Engelmann. The structure
of the axial filaments of these pseudopods is known to be fibrillar (Ros-
kin, 1925; Rumjantzew and Suntzowa, 1925). Schultz (1915) found
the fibrillae in the rhizopods of the foraminiferan, Astrorhiza, to be
doubly refractive, and Schmidt (1929) found weak anisotropy in the
axopods of the radiolarian, Thalassicolla. Schultz (1915) and Schmidt
(1929) report that the stereoplasmic axis of the pseudopods of the
Foraminifera are formed by the parallel alignment of fibers.

Cilia have been found to be doubly refractive by Valentin, Engel-
mann (1875), and Mackinnon and Vlés (1908). The latter authors
consider this to be caused by the depolarization of the light by reflection
from surfaces of the cilia, because of the difference of refractive index.
If air penetrates into the axis of a cilium, upon drying the anisotropy is
greatly increased (Schmidt, 1937). The so-called rootlets of the cilia

PROTOPLASM OF PROTOZOA . 97

are doubly refractive, but the basal granules are not (Engelmann,
1880.) Myonemes have been quite generally shown to be anisotropic,
especially those in the stalk muscle of the vorticellids (Engelmann, 1875;
Wrzesniowski, 1877; Mackinnon and Vlés, 1908). The myoneme of
the stalk spreads out into fibrillae in the base of the animal. These, too,
are doubly refractive. Engelmann (1875) found that the extensile neck
of Trachelocerca (Lacrymaria) olor, when stretched out, was positively
anisotropic in relation to its longitudinal axis. It is to be recalled that
the neck may be extended as much as fifteen times the length of the
body. Associated with this is a single spiral thread which becomes
straight upon extension of the neck (Penard, 1922).

Brandt (1885) has shown that the isospore nuclei of the Radiolaria are
anisotropic, but not the vegetative nor the anisospore nuclei. This has
been confirmed by Schmidt (1932) on living and preserved material
of the same form. Schmidt (1929) had also observed double refraction
of the nuclear membrane in living nuclei of a foraminiferan and of
Amoeba; in the latter he also observed weak anisotropy of small visible
granules (chromatin?) in the living nucleus. Finally, Kalmus (1931)
has recorded that certain elements of the division figure of the nuclei
of Paramecium show slight traces of double refraction during fission and
conjugation.

X-RAY DIFFRACTION AND ULTRACENTRIFUGATION

Early studies on cohesion and swelling relations of organic fibrillar
structures indicated that the finer structures of which they are composed
are micellar in nature. Recently X-ray diffraction methods have sub-
stantiated this view and have made it possible actually to measure the
dimensions of these structural units. Much of this work has been done
on keratin, elastin, chitin, myosin, cellulose, and other nonliving sub-
stances. Some observations have been made on living nerve fibers
(Schmitt, Bear, and Clark, 1935).

The evidence from X-ray diffraction shows that animal fibers owe
their anisotropic properties to the fact that they are composed of
longitudinally oriented protein chains. However, in most protoplasm the
configuration of such chains must be such that it may be altered rapidly
and reversibly. For a review of X-ray diffraction, see Frey-Wyssling,
1933:

98 PROTOPLASM OF PROTOZOA

Beams and King (1937) and King and Beams (1938) have shown
that the complicated process of karyokinesis may occur in eggs of
Ascaris, although cytokinesis does not usually take place at very high
centrifugal forces. They believe that if stratification occurred, it would
involve a breakdown of the normal, submicroscopic spatial relations,
which are of importance for the maintenance of life.

LITERATURE CITED

Adolph, E. F. 1931. The regulation of size as illustrated in unicellular or-
ganisms. Springfield.

Alexander, G. 1931. The significance of hydrogen ion concentration in the
biology of Exglena gracilis Klebs. Biol. Bull., 61: 165-84.

Allison, R. V. 1924. The density of unicellular organisms. Ann. appl. Biol.,
1: 153-64.

Angerer, C. A. 1936. The effects of mechanical agitation on the relative vis-
cosity of Amoeba protoplasm. J. cell comp. Physiol., 8: 329-45.

Ball, G. H. 1927. Studies on Paramecium. Ill. The effects of vital dyes on
Paramecium caudatum. Biol. Bull., 52: 68-78.

Barnes, T. C. 1937. Textbook of general physiology. Philadelphia.

Bayliss, W. M. 1920. The properties of colloidal systems. IV. Reversible
gelation in living protoplasm. Proc. roy. Soc. B., 91: 196-201.

Beams, H. W., and R. L. King. 1932. The architecture of the parietal cells
of the salivary glands of the grasshopper. J. Morph., 53: 223-41.

— 1937. The suppression of cleavage in Ascaris eggs by ultracentrifuging.
Biol. Bull, 73: 99-111,

Becker, E. R. 1928. Streaming and polarity in Mastigina hylae (Frenzel).
Biol. Bull., 54: 109-16.

Beers, C. D. 1924. Observations on Amoeba feeding on the ciliate Frontonia.
Brit. J. exp. Bioly 335-41:

Bélehradek, J. 1935. Temperature and living matter. Protop. Monog., vol. 8.
Berlin.

Bélehradek, J., and K. M. Paspa. 1928. Courbe myographique de la Vorticelle.
Arch. Int. Physiol., 30: 70-72.

Bensley, R. R. 1938. Plasmosin. The gel- and fiber-forming constituent of the
protoplasm of the hepatic cell. Anat. Rec., 72: 351-69.

Bles, E. J. 1929. Arcella. A study in cell physiology. Quart. J. micr. Sci., 72:
527-648.

Bodine, J. H. 1923. Excystation of Colpoda cucullus, Some factors affecting
excystation of Colpoda cucullus from its resting cysts. J. exp. Zool., 37:
115-25:

PROTOPLASM OF PROTOZOA oD

Brandt, K. 1885. Die koloniebildenden Radiolarien (Spaerozoen) des Golfes
van Neapel. Fauna u. Flora Neapel, Monogr. 13.

Bresslau, E. 1926. Die Stabchenstruktur der Tektinhiillen. Arb. Staatsinst.
exp. Ther. Frankfurt, H. 21 (Festschr. f. W. Kolle): 26-31.

Brinley, F. J. 1928. The effect of chemicals on viscosity of protoplasm as
indicated by Brownian movement. Protoplasma, 4: 177-82.

Brown, D. E. S., and D. A. Marshland. 1936. The viscosity of Amoeba at
high hydrostatic pressure. J. cell. comp. Physiol., 8: 159-65.

Browne, K. M. R. 1938. The Golgi apparatus and other cytoplasmic bodies
in Sprrostomum ambiguum. J. R. micr. Soc., 58: 188-99.

Brues, C. T. 1927. Animal life in hot springs. Quart. Rev. Biol., 2: 181-203.

Butts, H. E. 1935. The effect of certain salts of sea water upon reproduction
in the marine Amoeba, Flabellula mira Schaeffer. Physiol. Zodl., 8:
273-89.

Calkins, G. N. 1911. Effects produced by cutting Paramecium cells. Biol. Bull.,
21: 36-72.

—— 1919 Uroleptus mobilis, Engelm. I. History of the nuclei during divi-
sion and conjugation. J. Exp. Zool., 27: 293-355.

1933. The biology of the Protozoa. 2d ed., Philadelphia.

Chalkley, H. W. 1929. Changes in water content in Amoeba in relation to
changes in its protoplasmic structure. Physiol. Zodl., 2: 535-74.

— 1930. On the relation between the resistance to heat and the mechanism
of death in Paramecium. Physiol. Zo6l., 3: 425-40.

—— 1935. The mechanism of cytoplasmic fission in Amoeba proteus. Proto-
plasma, 24: 607-21.

—— 1936. The behavior of the karosome and the “peripheral chromatin”
during mitosis and interkinesis in Amoeba proteus with particular refer-
ence to the morphologic distribution of nucleic acid as indicated by the
Feulgen reaction. J. Morph., 60: 13-29.

Chalkley, H. W., and G. E. Daniel. 1934. The effect of certain chemicals
upon the division of the cytoplasm in Amoeba proteus. Protoplasma, 21:
258-67.

Chambers, R. 1921. The effect of experimentally induced changes in con-
sistency on protoplasmic movement. Proc. Soc. exp. Biol. N. Y., 19: 87-
88.

—— 1922. A micro injection study on the permeability of the starfish egg.
J. gen. Physiol., 5: 189-93.

—— 1924, The physical structure of protoplasm as determined by micro-
dissection and injection. Sec. V, General cytology, 234-309 (edited by
E. V. Cowdry). Chicago.

—— 1938. Structural aspects of cell division. Arch. exp. Zellforsch. 22:
252-56.

100 PROTOPLASM OF PROTOZOA

Chambers, R., and J. A. Dawson. 1925. The structure of the undulating
membrane in the ciliate Blepharisma, Biol. Bull., 48: 240-42.

Chambers, R., and H. P. Hale. 1932. The formation of ice in protoplasm.
Proc, Loy SOGHB11102336-52.

Chambers, R., and P. Reznikoff. 1926. Micrurgical studies in cell physiology.
1. The action of the chlorides of Na, K, Ca, and Mg on the protoplasm
of Amoeba proteus. J. gen. Physiol., 8: 369-401.

Chase, A. M., and O. Glaser. 1930. Forward movement of Paramecium as a
function of the hydrogen ion concentration. J. gen. Physiol., 13: 627-36.

Chatton, E., and A. Lwoff. 1923a. Sur l’évolution des Infusoires des Lamelle-
branches, Les formes primitives du phylum des Thigmotriches: le genre
Thigmo., Phrya C. R. Acad. Sci. Paris, 177: 81-83.

—— 1923b. Un Cas remarquable d’adaptation: Ellobiophrya donacis. n. g. n.
sp., etc. C. R. Soc. Biol., Paris, 88: 749-52.

Child, C. M. 1934. The differential reduction of methylene blue by Para-
mecium and some other ciliates. Protoplasma, 22: 377-94.

Child, C. M., and E. Deviney. 1926. Contributions to the physiology of
Paramecium caudatum. J. exp. Zool., 43: 257-312.

Cleveland, L. R. 1935. The centriole and its role in mitosis as seen in living
cells. Science, 81: 598-600.

Cleveland, L. R., in collaboration with S. R. Hall, E. P. Sanders, and J. Col-
lier. 1934. The wood-feeding roach Cryptocercus, its Protozoa ,and the
symbiosis between Protozoa and Roach. Mem. Amer. Acad. Arts Sci.,
17, No. 2; 187-342.

Conklin, E. G. 1924. Cellular differentiation. Sec. IX, General cytology, 539-
607 (ed. by E. V. Cowdry). Chicago.

Daniel, G. E., and H. W. Chalkley. 1932. The influence of temperature upon
the process of division in Amoeba proteus (Leidy). J. cell, comp.
Physiol., 2: 311-27.

Daniels, M. L. 1938. A cytological study of the gregarine parasites of Tenebrio
molitor using the ultra-centrifuge. Quart. J. micr. Sci., 80: 293-320.

Daugherty, K. 1937. The action of anesthetics on Amoeba protoplasm. Physiol.
Zobl., 10: 473-83.

Davenport, C. B. 1897. Experimental morphology. New York.

Dawson, J. A., and M. Belkin. 1929. The digestion of oils by Amoeba proteus.
Biol. Bull., 56: 80-86.

Dellinger, A. P. 1906. Locomotion of amoebae and allied forms. J.. exp.
Zool., 3: 337-58.

— 1909. The cilium as a key to the structure of contractile protoplasm.
J. Morph., 20: 171-209.

Dierks, K. 1926. Untersuchungen tiber die Morphologie und Physiologie des
Stentor coeruleus, Arch. Protistenk., 54: 1-91.

Dobell, C. C. 1911. The principles of protistology. Arch. Protistenk., 23:
269-310.

PROTOPLASM OF PROTOZOA 101

Dogiel, V., and M. Issakowa-Keo. 1927. Physiologische Studien an Infusorien.
II. Der Einflusz der Salzlésungen auf die Ernahrung von Paramaecium.
Biol. Zbl., 47: 577-86.

Edwards, J. G. 1923. The effect of chemicals on locomotion in Amoeba. I. J.
exp. Zool:, 38: 1-43.

Engelmann, T. W. 1875. Contractilitét und Doppelbrechung. Pfliig. Arch.
ges. Physiol., 11: 432-64.

—— 1880. Zur Anatomie und Physiologie der Flimmerzellen. Pfliig. Arch.
ges. Physiol., 23:: 505-35.

Ephrussi, B., and R. Rapkin. 1928. Action des différents sels sur le Spirosto-
mum. Protoplasma, 5: 35-40.

Ewles, N. A., and J. B. Speakman. 1930. Examination of the fine structure
of wool by X-ray analysis. Proc. roy. Soc. B., 105: 600-7.

Fauré-Fremiet, E. 1905. L’appareil fixateur chez les Vorticellidae. Arch.
Protistenk., 6: 207-26.

—— 1908. L’Ancystropodium maupasi. Arch Protistenk., 13: 121-37.

— 1910. La fixation chez les Infusoires cilies. Bull. Sci. Fr. Belg., 44: 27-50.

— 1929. Caracteres physico-chimiques des choanoleucocytes de quelques
invertébrés. Protoplasm, 6: 521-609.

— 1930. The kinetics of living matter. Trans. Faraday Soc., 26: 779-93.

—— 1932. Strombidium calkinsi, a new thigmotactic species. Biol. Bull.
62: 201-4.

— 1935. L’Oeuvre de Félix Dupardin et la notion de protoplasma. Proto-
plasma, 23: 250-69.

Fauré-Fremiet, E., B. Ephrussi, and L. Rapkine. 1926. Lames minces formées
par la diffluence du cytoplasma cellulaire. C. R. Soc. Biol. Paris, 94:
442-43,

Fetter, D. 1926. Determination of the protoplasmic viscosity of Paramecium
by the centrifuge method. J. exp. Zool., 44: 279-83.

Fortner, H. 1926. Zur Morphologie und Physiologie des Vorticellenstieles.
Zirwiss. Zool, 128° 114-32;

Frederikse, A. M. 1933a. Viskositatsinderungen des protoplasmas wahrend

der Narkose. Protoplasma. 18: 194-207.

1933b. Der Brechungsindex des Protoplasmas. Protoplasma, 19: 473-48.

Frey-Wyssling, A. 1938. Submikroskopische Morprologie des Protoplasmas
und seiner Derivate. Protop.-Monog. 15, Berlin.

Gaw, H. Z. 1936a. Physiology of the contractile vacuole in ciliates. 2. The
effect of hydrogen ion concentration. Arch. Protistenk., 87:
194-200. .

—— 1936b. Physiology of the contractile vacuole in ciliates 4. The effect of
heavy water. Arch. Protistenk. 87: 213-24.

Gelei, J. V. 1928. Nochmals tiber den Nephridialapparat bei den Protozoen.
Arch Protistenk., 64: 479-94.

Ginsberg, H. 1922. Untersuchungen zum Plasmabau der Amében, im Hin-

102 PROTOPLASM OF PROTOZOA

blick auf die Wabentheorie. Roux Arch EntwMech. Organ., 51: 150-250.

Giese, A. C. 1935. The role of starvation in conjugation of Paramecium.
Physiol. Zool., 8: 116-25.

Gray, J. 1931. A textbook of experimental cytology. New York.

Greeley, A. W. 1901. On the analogy between the effects of loss of water

and lowering of temperature. Amer. J. Physiol., 6: 122-28.

1902. The artificial production of spores in Monas by a reduction of

the temperature. Biol. Bull., 3: 165-71.

—— 1903. Further studies on the effect of variations in temperature on ani-
mal tissues. Biol. Bull., 5: 42-54.

—— 1904, Experiments on the physical structure of the protoplasm of
Paramoecium, etc. Biol. Bull., 7: 3-32.

Grosse-Allermann, W. 1909. Studien tber Amoeba terricola Kreeff. Arch.
Protistenk., 17: 203-57.

Gruber, A. 1889. Biologische Studien an Protozoen. Biol. Zbl., 9: 14-23.

Gruber, K. 1912. Biologische und experimentelle Untersuchungen an Amoeba
proteus, Arch. Protestenk., 25: 316-76.

Hahnert, W. F. 1912. A quantitative study of reactions to electricity in Amoeba
proteus. Physiol. Zodl., 5: 491-526.

Harvey, E. B. 1917. A physiological study of specific gravity and of lumines-
cence in Noctiluca, with special reference to anesthesia. Publ. Carneg.
Tnstita; INo: 251: 235-53.

Harvey, E. N. 1931. Observations on living cells, made with the microscope-
centrifuge. J. exp. Biol., 8: 267-74.

— 1934. Biological effects of heavy water. Biol. Bull., 66: 91-96.

—— 1936. The properties of elastic membranes with special reference to the

cell surface. J. cell. comp. Physiol., 8: 251-60.

1938. Some physical properties of protoplasm. J. appl. Physics, 9: 68-80.

Harvey, E. N., and J. F. Danielli. 1936. The elasticity of thin films in rela-
tion to the cell surface. J. cell. comp. Physiol., 8: 31-36.

Harvey, E. N., E. B. Harvey, and A. L. Loomis. 1928. Further observations
on the effect of high frequency sound waves on living matter. Biol.
Bull., 55: 459-69.

Harvey, E. N., and D. A. Marsland. 1932. The tension at the surface of
Amoeba dubia with direct observations on the movement of cytoplasmic
particles at high centrifugal speeds. J. cell. comp. Physiol., 2:
UaeMe

Heilbrunn, L. V. 1928. The colloid chemistry of protoplasm. Berlin.

1929a. Protoplasmic viscosity of Amoeba at different temperatures.

Protoplasma, 8: 58-64.

1929b. The absolute viscosity of Amoeba protoplasm. Protoplasma, 8:

65-69.

— 1931. Anasthesia in Amoeba. Anat. Rec., 51: 27.

—— 1937. An outline of general physiology. Philadelphia.

PROTOPLASM OF PROTOZOA 103

Heilbrunn, L. V., and K. Daugherty. 1931. The action of the chlorides of
sodium, potassium, calcium, and magnesium on the protoplasm of
Amoeba dubia. Physiol. Zo6l., 4: 635-51.

— 1932. The action of sodium, potassium, calcium, and magnesium ions
on the plasmagel of Amoeba proteus. Physiol. Zodl., 5: 254-74.

— 1933. The action of ultraviolet rays on Amoeba protoplasm. Protoplasma,
18: 596-619.

— 1934. A further study of the action of potassium on Amoeba proto-
plasm. J. cell. comp. Physiol., 5: 207-18.

—— 1938. Fat release in Amoeba after irradiation. Physiol. Zodl., 11: 383-87.

—— 1939. The electric charge of protoplasmic colloids. Physiol. Zodl., 12:
ibe 4

Heilbrunn, L. V., and D. Mazia. 1936. The action of radiations on living
protoplasm. Biological effects of radiation, (edited by B. M. Dugger) :
625-76. New York.

Henderson, V. E. 1930. The present status of the theories of narcosis. Physiol.
Revs. LOe 17 1-220°

Holter, H., and M. J. Kopac. 1937. Studies on enzymatic histo-chemistry
XXIV. Localization of peptidase in Ameba. J. cell. comp. Physiol., 10:
Aa

Hopkins, D. L. 1938. The vacuoles and vacuolar activity in the Marine amoeba,
Flabellula mira Schaeffer and the nature of the natural red system in
Protozoa. Biodyn., No. 34: 1-22.

Howland, R. B. 1924a. On excretion of nitrogenous waste as a function of
the contractile vacuole. J. exp. Zool., 40: 231-50.

—— 1924b. Experiments on the contractile vacuole of Amoeba verrucosa

and Paramecium caudatum. J. exp. Zool., 40: 251-62.

1924c. Dissection of the pellicle of Amoeba verrucosa. J. exp. Zool.,

40: 263-70.

Howland, R. B., and H. Pollack. 1927. Micrurgical studies on the contractile
vacuole. I. Relation of the physical state of the internal protoplasm to
the behavior of the vacuole. II. Micro-injection of distilled water. J.
exp. Zool., 48: 441-58.

Hyman, L. 1917. Metabolic gradients in Amoeba and their relation to the
mechanism of amoeboid movement. J. exp. Zool., 24: 55-99.

Jacobs, M. H. 1919. Acclimatization as a factor affecting the upper thermal
death points of organisms. J. exp. Zool., 27: 427-42.

— 1922. The effects of carbon dioxide on the consistency of protoplasm.
Biol. Bull., 42: 14-30.

Jennings, H. S. 1904. Movements and reactions of Amoeba. Publ. Carneg.

Instn., No. 16: 129-234.

1906. Behavior of the lower organisms. New York.

Jensen, P. 1893. Die absolute Kraft einer Flimmerzelle. Pflig. Arch. ges.

Physiol., 54: 537-51.

104 PROTOPLASM OF PROTOZOA

Jochims, J. 1930. Das Fadenziehen biologischer Substanzen. Protoplasma, 9:
298-317.

Johnson, L. P. 1939. A study of Exglena rubra Hardy 1911. Trans. Amer.
micr. Soc., 58: 42-48.

Jordan-Lloyd, D. 1932. Colloidal structure and its biological significance.
Biol. Rev., 7: 254-73.

Joyet-Lavergne, Ph. 1931. La Physico-Chimie de la sexualité. Protop.- Monog.,
Vol. V. Berlin.

Kahl, A. 1935. Wimpertiere order Ciliata (Infusoria), in Dahl: Die Tierwelt
Deutschlands. 18 Teil, Jena.

Kalmus, . 1931. Paramecium. Jena.

Kanda, Sakyo. 1914. On the geotropism of Paramecium and Spirostomum
Biol. Bull., 24: 1-24.

—— 1918. Further studies on the geotropism of Paramecium caudatum. Biol.
Bull., 34: 108-19.

Kepner, W. A., and J. G. Edwards. 1917. Food reactions of Pelomyxa caro-
linesis Wilson. J. exp. Zool., 24: 381-407.

Kepner, W. A., and B. D. Reynolds. 1923. Reactions of cell-bodies and
pseudopodia fragments of D/fflugia. Biol. Bull., 22-47.

Kepner, W. A., and W. H. Taliaferro. 1913. Reactions of Amoeba proteus to
food. Biol. Bull., 24: 411-22.

Kepner, W. A., and C. Whitlock. 1921. Food reactions of Amoeba proteus.
J. exp. Zool., 32: 397-425.

King, R. L. 1928. The contractile vacuole in Paramecium trichium. Biol. Bull.,
55: 59-64.

— 1935. The contractile vacuole of Paramecium multimicronucleata. J.
Morph... 58:9 555-74..

King, R. L., and H. W. Beams, 1937. The effect of ultracentrifuging on
Paramecium, with special reference to recovery and macronuclear reor-
ganization. J. Morph., 61: 27-49.

— 1938. An experimental study of chromatin diminution in Ascarzs, J. exp.
Zool., 77: 425-43.

Kitching, J. A. 1936. The physiology of contractile vacuoles. II. The control

of body volume in marine Peritricha. J. exp. Biol., 13: 11-27.

1938. Contractile vacuoles. Biol. Rev., 13: 403-44.

Kite, G. L. 1913. Studies on the physical properties of protoplasm. I. The
physical properties of the protoplasm of certain animal and plant cells.
Amer. J. Physiol., 32: 146-64.

Koltzoff, N. K. 1912. Untersuchungen tiber die Kontraktilitat des Vorticellens-
tiels. Arch. Zellforsch, 7: 344-423.

Lackey, J. B. 1936. Some freshwater Protozoa with blue chromatophores Biol.
Bull., 71: 492-97.

PROTOPLASM OF PROTOZOA 105

Leontjew, H. 1926a. Zur Biophysik der niederen Organismem. II. Mitteilung.
Die Bestimmung des spezifischen Gewicht und der Masse von Naegleria.
Pflug. Arch. ges. Physiol., 213: 1-4.

—— 1926b. Uber das spezifische Gewicht des Protoplasmas Biochem. Z.
170: 326-29.

—— 1927. Uber das spezifische Gewicht des Protoplasmas. III. Protoplasma,
2: 59-64.

Lewis, W. H. 1926. Cultivation of embryonic heart muscle. Publ. Carneg.
Instn., 363: 1-21.

Lloyd, F. E. 1928. The contractile vacuole. Biol. Rev., 3: 329-58.

Loeb, L. 1927. Amoeboid movement and agglutination in amoebocytes of
Limulus and the relation of these processes to tissue formation and
thrombosis. Protoplasma, 2: 512-53.

—— 1928. Amoebocyte tissue and amoeboid movement. Protoplasma, 4:
596-625.

Looper, J. B. 1928. Cytoplasmic fusion in Actinophrys sol, with special ref-
erence to the karyoplasmic ratio. J. exp. Zool., 50: 31-49.

Luce, R. H. 1926. Orientation to the electric current and to light in Amoeba.
(Abss)\;Anats Rees 32155:

Ludwig, W. 1928a. Der Betriebsstoffwechsel von Paramecium caudatum.
Arch. Protistenk., 62: 12-40.

—— 1928b. Permeabilitat und Wasserwechsel bei Noctiluca miliaris Suriray.
Zool, Anz..76: 273-85.

—— 1930. Zur Theorie der Flimmerbewegung. Z. vergl. Physiol., 13: 397-
504.

Lund, E. J. 1917. Reversibility of morphogenetic processes in Bursaria. J. exp.
Zool., 24: 1-33.

—— 1921. Experimental control of organic polarity by the electric current.
is JrexpsZool.,.34:,471-93,.

— Lund, E. J., and G. A. Logan. 1925. The relation of the stability of
protoplasmic films in Noctiluca to the duration and intensity of an ap-
plied electric potential. J. gen. Physiol., 7: 461-72.

Luyet, B. J., and P. M. Gehenio. 1935. Comparative ultra-violet absorption
by the constituent parts of protozoan cells (Paramaecium). Biodyn., No.
7: 1-14.

—— 1938. The lower limit of vital temperatures, a review. Biodyn. No. 33:
1-92;

Lwoff, A. 1934. Die Bedeutung des Blutfarbstoffes fiir die parasitischen
Flagellaten. Zbl. Bakt., 130: 498-518.

Lyon, E. P. 1905. On the theory of geotropism in Paramaecium. Amet. J.
Physiol., 14: 421-32.

McClendon, J. F. 1909. Protozoan studies. J. exp. Zool., 6: 265-83.

Mackinnon, D. L. 1909. Optical properties of contractile organs in Heliozoa.
J. Physiol., 38: 254-58.

106 PROTOPLASM OF PROTOZOA

Mackinnon, D. L., and F. Vlés. 1908. On the optical properties of contractile
organs. J. R. micr. Soc., 553-58.

Makarov, P. 1935. Experimentelle Untersuchungen an Protozoen mit Bezug
auf das Narkose-Problem. Protoplasma, 24: 593-606.

Marsland, D. 1933. The site of narcosis in a cell; the action of a series of
paraffin oils on Amoeba dubia. J. cell. comp. Physiol., 4: 9-30.

Marsland, D. A. 1939. The mechanism of protoplasmic streaming, etc. J.
cell, comp. Physiol., 13: 23-30.

Massart, J. 1893. Sur l’irritabilité des Noctiluques. Bull. sci. Fr. Belg., 25:
59-76.

Mast, S. O. 1909. The reactions of Didinium nasutum. Biol. Bull., 16: 91-118.

1926a. The structure of protoplasm in Amoeba, Amer. Nat., 60: 133-42.

— 1926b. Structure, movement, locomotion and stimulation in Amoeba.

J. Morph., 41: 347-425.

1929. Mechanics of locomotion in Amoeba proteus with special refer-
ence to the factors involved in attachment to the substratum. Protoplasma,
8: 344-77.

—— 1931la. Movement and response in D7/flugia with special reference to the
nature of cytoplasmic contraction. Biol. Bull., 61: 223-41.

—— 1931b. The nature of the action of electricity in producing response and
injury in Amoeba proteus (Leidy) and effect of electricity on the vis-
cosity of protoplasm. Z. vergl. Physiol., 15: 309-28.

—— 1938a. The contractile vacuole in Amoeba proteus (Leidy). Biol. Bull.,

74: 306-13.

1938b. Digestion of fat in Amoeba proteus. Biol. Bull., 75: 389-94.
Mast, S. O., and W. L. Doyle. 1934. Ingestion of fluid by Amoeba. Proto-

plasma, 20: 555-60.

—— 1935a. Structure, origin and function of cytoplasmic constituents in
Amoeba proteus. 1. Structure. Arch. Protistenk., 86: 155-80.

—— 1935b. II. Origin and function based on experimental evidence; effect
of centrifuging on Amoeba proteus. Arch. Protistenk., 86: 278-306.
Mast, S. O., and C. Fowler. 1935. Permeability of Amoeba proteus to water.

J. cell. comp. Physiol., 6: 151-67.

Mast, S. O., and W. F. Hahnert. 1935. Feeding, digestion, and starvation in
Amoeba proteus (Leidy). Physiol. Zodl., 8: 255-72.

Mast, S. O., and F. M. Root. 1916. Observations on Ameba feeding on
rotifers, nematodes and ciliates, and their bearing on the surface-tension
theory. J. exp. Zool., 21: 33-49.

Merton, H. 1928. Untersuchungen tiber die Enstehung amébenahnlicher Zellen
an absterbenden Infusorien. Math.-nat. Kl. Heidelb. Akad. Wiss., 5 B.
1928, Nr. 3: 51-29.

Metcalf, M. M. 1910. Studies upon Amoeba. J. exp. Zool., 9: 301-31.

Moore, A. R. 1935. On the significance of cytoplasmic structure in Plasmo-
dium. J. cell. comp. Physiol., 7: 113-29.

PROTOPLASM OF PROTOZOA 107

Morita, Y., and R. Chambers. 1929. Permeability differences between nuclear
and cytoplasmic surfaces in Amoeba dubia. Biol. Bull., 56: 64-67.

Nadler, E. J. 1929. Notes on the loss and regeneration of the pellicle in
Blepharisma undulans. Biol. Bull., 56: 327-30.

Nassonov, D. 1924. Der Exkretionsapparat (kontraktile vakuole) der Protozoa
als Homologon des Golgischen Apparats der Metazoazellen. Arch. mikr.
Anat., 103: 437-82.

Needham, J. 1936. Order and life. New Haven.

Needham, J., and D. M. Needham. 1925. The hydrogen-ion concentration
and oxidation-reduction potential of the cell interior: a micro-injection
study. Proc. roy. Soc. B., 98: 259-86.

—— 1926. Further micro-injection studies on the oxidation-reduction poten-
tial of the cell-interior. Proc. roy. Soc. B., 99: 383-97.

Noland, L. E. 1931. Studies on the taxonomy of the genus Vorticella. Trans.
Amer, micr. Soc., 50: 81-123.

Oliphant, J. F. 1938. The effect of chemicals and temperature on reversal in
ciliary action in Paramecium. Physiol. Zo6l., 11: 19-30.

Packard, C. 1923. The susceptibility of cells to radium radiations. Proc. Soc.

exp, Biol. NUY., 20: 226-27.

1924. The susceptibility of cells to radium radiations. Biol. Bull., 46:

165-77.

—— 1925. The effect of light on the permeability of Paramecium. J. gen.
Physiol., 7: 363-72.

Pantin, C. F, A. 1923. On the physiology of amoeboid movement. I. J. Mar.
Biol. Assoc., 13: 24-69.

——— 1924a. Temperature and the viscosity of protoplasm. Jour. Mar. biol.
Ass. U.K., 13: 331-39.

—— 1924b. On the physiology of amoeboid movement. II. The effect of tem-
perature. Brit. J. exp. Biol., 1: 519-38.

-—— 1926a. On the physiology of amoeboid movement. III. The action of

calcium. Brit. J. exp. Biol., 3: 275-95.

1926b. On the physiology of amoeboid movement. IV. The action of
magnesium. Brit, J. exp. Biol., 3: 297-312.

—— 1930. On the physiology of amoeboid movmeent: a.V. Anaerobic move-
ment. Proc. roy. Soc. B. 105: 538-55. b. VI. The action of oxygen. Proc.
roy. Soc. B., 105: 555-64. c.VII. The action of anaesthetics. Proc. roy.
Soc. B.; 105: 565-79:

Park, O. 1929. The differential reduction of osmic acid in the cortex of
Paramecium, and its bearing upon the metabolic gradient conception.
Physiol. Zodl., 2: 449-58.

Parsons, C. W. 1926. Some observations on the behavior of Amoeba proteus.
Quart. J. micr. Sci. 70: 629-48.

Pascher, A. 1917. Von der merkwurdigen Bewegungsweise einiger Flagel-
laten. Biol. Zbl., 37: 241-429.

108 PROTOPLASM OF PROTOZOA

Patten, R., and H. W. Beams. 1936. Observations on the effect of the ultra-
centrifuge on some free-living flagellates. Quart. J. micr. Sci., 78: 615-35.

Peebles, F, 1912. Regeneration and regulation in Paramecium caudatum. Biol.
Bull., 23: 154-70.

Pekarek, J. 1930. Absolute Viskositatsmessung mit Hilfe der Brownschen
Molekularbewegung. II. Viskositaétsbestimmung des Zellsaftes der Epi-
dermiszellen von Allium cepa und des Amdben-Protoplasmas. Proto-
plasma, 11: 19-48.

Penard, E. 1902. Faune Rhizopodique due Basin du Leman. Geneve.

1904. Les Héliozoaires d’eau douce. Genéve.

1922. Etudes sur les Infusoires d’leau douce. Genéve.

Pfeiffer, H. 1931. Uber mikro-refraktometrische methoden im Dienste der
Protoplasma-Forschung. Z. wiss. Mikr., 48: 47-62.

—— 1933a. Beitrage zur quantitativen Bestimmung von molekularkraften
des Protoplasmas. I. Eine Methode zur messung der Adhisionsarbeit
plasmatischer Oberflachen. Protoplasma, 19: 177-92.

— 1933b. Beitrige zur quantitativen Bestimmung von molekularkraften
des Protoplasmas. II. Eine modifikation der methode zur bestimmung des
Reinbungswiederstandes nackter. Protoplasten. Protoplasma, 20: 73-78.

—— 1933c. III. Die Bestimmung des Randwinkels aus der Form nackter
Protoplasten. Protoplasma, 20: 79-84.

— 1934. Versuche zur Bestimmung des spezifischen Gewichts nackter
Protoplasten. Protoplasma, 21: 427-32.

—— 1935. Literature on adhesiveness (stickiness) of protoplasm and related
topics. Protoplasma, 23: 270-81.

— 1936. Further tests of the elasticity of protoplasm. Physics, 7: 302-5.

— 1937. Experimental researches on the non-Newtonian nature of Proto-
plasma. Cytologia, Tokyo. (Fujii Jubilaei vol.) 701-10.

Pitts, R. F., and S. O. Mast. 1934. The relation between inorganic salt con-
centration, hydrogen ion concentration and physiological processes in
Amoeba proteus. Il. J. cell. comp. Physiol., 4: 435-55.

Platt, J. B. 1899. On the specific gravity of Spirostomum, Paramaecium, and
the tadpole in relation to the problem of geotaxis. Amer. Nat., 33: 31-38.

Pollack, H. 1928a. Intracellular hydrion concentration studies. III. etc. Biol.
Bull., 55: 383-85.

—— 1928b. Micrurgical studies in cell physiology. VI. Calcium ions in liv-
ing protoplasm. J. gen. Physiol., 11: 539-45.

Popoff, M. 1908. Experimentelle Zellstudien 1. Arch. Zellforsch., 1: 245-379.

Piitter, A. 1903. Die Flimmerbewegung. Ergebn. Physiol., 2: 1-102.

Reznikoff, P. 1926. Micrurgical studies in cell physiology. II. The action of
the chlorides of lead, mercury, copper, iron, and aluminum on the proto-
plasm of Amoeba proteus. J. gen. Physiol., 10: 9-21.

PROTOPLASM OF PROTOZOA 109

Reznikoff, P., and R. Chambers. 1927. Micrurgical studies in cell physiology.
III. The action of CO, and some salts of Na, Ca, and K on the proto-
plasm of Amoeba dubia. J. gen. Physiol., 10: 731-55.

Reznikoff, P., and H. Pollock. 1928. Intracellular hydrion concentration
studies. II. The effect of injection of acids and salts on the cytoplasmic
pH of Amoeba dubia. Biol. Bull., 55: 377-82.

Rhumbler, L. 1898. Physikalische analyse von Lebenserscheinungen der Zelle.
Roux Arch. EntwMech. Organ., 7: 103-350.

Root, F. M. 1914. Reproduction and reactions to food in the suctorian,
Podophrya collini, n. sp. Arch, Protistenk., 35: 164-96.

Roskin, G. 1923. La Structure des myonemes des Infusoires. Bull. biol., 57:
143-51.

—— 1925. Uber die Axopodien der Heliozoa und die Greiftentakeln der
Ephelotiden. Arch Protistenk., 52: 207-16.

Rumjantzew, A., and E. Suntzowa. 1925. Untersuchungen tiber den Proto-
plasmabau von Actinosphaerium Eichhornit. Arch. Protistenk., 52: 217-
64.

Saunders, J. T. 1925. The trichocysts in Paramecium. Biol. Rev., 1: 249-69.

Schaeffer, A. A. 1916. On feeding habits of Ameba. J. exp. Zool., 20: 529-84.

1920. Amoeboid movement. Princeton.

—— 1926. Taxonomy of the amebas. Publ. Carneg. Instn., No. 345.

— 1931. On molecular organization in ameban protoplasm. Science, 74:
47-51.

Schewiakoff, W. 1927. Die Acantharia des Golfes von Neapel. Fauna u. Flora
neapel. Monogr., 37.

Schmidt, W. J. 1929. Rheoplasma und Steroplasma. Protoplasma, 7: 353-94.

1932. Der submikroskopische Bau des Chromatins. II. Uber die Dop-

pelbrechung der Isosporenkerne von Sphaerozoen. Arch. Protistenk., 77:

463-90.

—— 1937. Die Doppelbrechung von Karyoplasma, Zytoplasma und Meta-
plasm. Protop. Monog., 11. Berlin.

Schmitt, F. O. 1938. Optical studies of the molecular organization of living
systems. J. Appl. Physics, 9: 109-17.

Schmitt, F. O., R. S. Bear, and G. L. Clark. 1935. X-ray diffraction studies
on nerve. Radiology, 25: 131-51.

Schultz, E. 1915. Die Hyle des Lebens. I. Beobactungen und Experimente an
Astrorhiza limicola. Roux Arch. EntwMech. Organ., 41: 215-36.

Seifriz, W. 1928. The physical properties of protoplasm. Colloid Chemistry

(edited by J. Alexander), 11: 403-50.

1936. Protoplasm. New York.

Singh, B. N. 1939. The cytology of Amoeba proteus “Y’.and the effects of
large and small centrifugal forces. Quart. J. micr. Sci., 80: 601-35.
Spek, J. 1921. Der Einfluss der Salze auf die Plasmakolloide von Actinosphae-

rium eichhornit. Acta Zool. Stock., 2: 153-200.

110 PROTOPLASM OF PROTOZOA

—— 1923. Uber den physikalischen Zustand von Plasma und Zelle der
Opalina ranarum, (Purk. et Val.) Arch. Protistenk., 46: 166-00.

—— 1924. Neue Beitrage zum Problem der Plasmastrukturen. Z. Zell. u.
Gewebelehre, 1: 278-326.

Spek, J., and R. Chambers. 1934. Neue experimentelle Studien tiber das
Problem der Reaktion des Protoplasmas. Protoplasma, 20: 376-406.
Stump, A. B. 1935. Observations on the feeding of Difflugia, Pontigulasia

and Lesquereusia. Biol. Bull., 69: 136-42.

1936. The influence of test materials on reproduction in Pontigulasia

vas (Leidy) Schouteden. Biol. Bull., 70: 142-47.

Taliaferro, W. H., and L. G. Taliaferro. 1934. Complement fixation, pre-
cipitin, adhesion, mercuric chloride and Wassermann tests in equine
trypanosomiasis of Panama (murrina). J. Immunol., 26: 193-213.

Taylor, C. V. 1920. Demonstration of the function of the neuromotor ap-
paratus in Ewplotes by the method of microdissection. Univ. Cal. Publ.
Zool., 19: 403-70.

—— 1923. The contractile vacuole in Evplotes: an example of the sol-gel

reversibility of cytoplasm. J. exp. Zool., 37: 259-89.

1935. Protoplasmic reorganisation and animal life cycles. Biol. Rev.,

LOZ T11-22°

Taylor, H. S., W. W. Swingle, H. Eyring, and A. A. Frost. 1933. The effect
of water containing the isotope of hydrogen upon fresh water organisms.
J. cell. comp. Physiol., 4: 1-8.

Tchakhotine, S. 1937. Radiations, cell permeability and colloidal changes.
Trans. Faraday Soc., 33: 1068-72.

Thornton, F. E. 1932. The viscosity of the plasmagel of Amoeba proteus at
different temperatures. Physiol. Zodl., 5: 246-53.

—— 1935. The action of Sodium, Potassium, Calcium, and Magnesium ions
on the plasmagel of Amoeba proteus at different temperatures. Physiol.
Zool., 8: 246-54.

Tiegs, O. W. 1928. Surface tension and the theory of protoplasmic move-
ment. Protoplasma, 4: 88-139.

Verworn, M. 1888. Biologische Protisten-Studien. Z. wiss. Zool., 46: 455-70.

1899. General physiology. London.

Voegtlin, C., and H. W. Chalkley. 1935. The chemistry of cell division. IV.
The influence of H,S, HCN, CO, and some other chemicals on mitosis
in Amoeba proteus. Protoplasma, 24: 365-83.

Wenrich, D. H. 1929. The structure and behavior of Actinobolus vorax. Biol.
Bull., 56: 390-401.

Winterstein, H. 1926. Die Narkose. Berlin.

Wrzesniowski, A. 1877. Beitrige zur naturgeschichte der Infusorien. Z. wiss.
Zool., 29: 267-323.

CHAPTER III

CYTOPLASMIC INCLUSIONS

RONALD F. MACLENNAN

ALL ACTIVE CELLS possess a large number of cytoplasmic granules
which change in number, size, shape, and composition in accordance
with the changes in the activities of the cell of which they are a part.*
The fact that small granules are so constantly present in the living substance
is an indication that such a fine suspension of material represents a colloidal
condition favorable for the life process. It seems certain that as the physiology
of the cell becomes more clearly understood there will be shown to be a
definite dependence of vital phenomena on the granular nature of protoplasm,
on the properties which it possesses by virtue of the fact that it is a suspen-
sion (Heilbrunn, 1928, p. 20).

The cytoplasmic granules are a visible part of the fundamental organi-
zation of the cell, and the elucidation of their functions contributes not
merely to a specialized branch of cytology but contributes directly to a
solution of the fundamental problem of protoplasmic organization.

The richness of the granular complex early attracted the interest of
cytologists, and many studies were made on their chemical composition.
The report of Biitschli’s discovery that the carbohydrate granules of
gregarines differ from those in vertebrates is one of the classic papers
in the group. During the first thirty years of this century the emphasis
shifted from the earlier cytochemical methods to an interest in certain
of the granules as permanent, self-perpetyating cytoplasmic organelles,
which could be classified by certain empirical reactions such as osmic
reduction or the segregation of janus green and neutral red. Dissatisfac-
tion with the specificity of these methods has resulted recently in a re-
newed emphasis on methods which yield specific information on the
chemical and physical nature of the cytoplasmic granules and their
cyclic changes. Too often, however, there has been a tendency to carry

* This paper is a contribution from the departments of Zoélogy of the State College of
Washington and Oberlin College.

12 CYTOPLASMIC INCLUSIONS

on these two types of investigation separately, with the result that in
many cases the morphological and functional studies of the cytoplasmic
granules have become separated. The one group emphasizes the classifica-
tion of granules into hard and fast categories of Golgi bodies, mitochon-
dria, vacuome and so forth, with little specific consideration of function,
while those engaged in functional studies tend to group all the granules
together or to devise entirely new systems, which hinder the comparison
of granules in several species. This review is an attempt to codrdinate
these two angles of approach, so that both may contribute to our under-
standing of the rdle of the cytoplasmic granules in the cell.

Since summaries are available of the characteristics of single groups
of granules, this review is not intended to provide an exhaustive catalogue
of the facts of any one group. Particular emphasis is placed on those
granules which have been described with sufficient completeness to fur-
nish evidence as to reactions, classification, and function, as well as their
relationship to other granules in the same cell. Specific directions on
standard techniques for demonstrating the various granules are avail-
able in the various books on microtechnique and histochemistry and so
are not described in detail here. The more recent publications will be
emphasized, since the specificity of methods has improved greatly and
summaries of the earlier papers are available in the works of Calkins,
Doflein-Reichenow, and others. The Protophyta have been omitted in
most cases, since their inclusion would complicate the picture unneces-
sarily.

MITOCHONDRIA

Undoubtedly many granules described in early cytological studies of
Protoza were actually mitochondria, but their status as a separate group
of cytoplasmic constituents in the unicellular organisms was not recog-
nized until the publication of the monograph of Fauré-Fremiet (1910),
which emphasized the concept that mitochondria are universal, self-per-
petuating cytoplasmic constituents.

The identification of mitochondria is not yet entirely satisfactory,
since it depends upon stains and fixatives of the lipoid component, a
material not restricted to mitochondria alone, or upon vital dyes which
are not as effective in the Protozoa as in the Metazoa and which in certain
cases stain other organelles as well. Typical mitochondria are refractile

CYTOPLASMIC INCLUSIONS 113

in life, become grey brown, or black in osmic techniques, but usually
bleach faster than the Golgi bodies, reduce pyrogallol, take basic stains
after fixation in lipoid preservatives, stain weakly or not at all after fixa-
tives containing acetic acid, and are stained vitally with janus green B.
This last method is often considered to be the final criterion, but un-
fortunately in many cases the mitochondria of Protozoa stain only a pale
green (Subramaniam and Ganapati, 1938, and others), not the dark
green described in metazoan cells. In addition, the stain is not always
specific, since Lynch (1930) found that any concentration from 1:2000
to 1:500,000 tints the entire organism (Lechriopyla), although the
mitochondria can be distinguished by their darker color. Hayes (1938)
found that none of the granules in Dé/eptus stained electively with Janus
green B. An additional source of difficulty is the fact that in flagellates,
the parabasal bodies are often stained as darkly as the mitochondria.
Although the parabasal bodies and mitochondria show additional simi-
larities in staining reactions and composition, Volkonsky (1933) points
out that the former are derivatives of the neuromotor system and cannot
be considered as homologous with the mitochondria.

Mitochondria present a wide variety of shapes, but most commonly
they are spherules (Fig. 17), chains of spherules (Fig. 26), short rods
(Figs. 22, 23), or dumb-bells. The filamentous structures found so often
in metazoan cells are found rarely in the Protozoa, but a good example
has been described in the phytoflagellate Polykrikos by Chatton and
Grassé (1929). Lynch (1930), in his studies on the ciliate Lechriopyla,
found a compound structure (Figs. 15. 16), composed of several discs.
Each disc is composed of chromophobic material, with a rim of chromo-
philic material which stains with Janus green and the other mitochondrial
dyes. In cases of secretion (Fig. 17), the secretion granules often appear
as a chromophobic center in the mitochondria (MacLennan, 1936), but
the mitochondrial material itself usually appears to be homogeneous
either in the living unstained ciliate or after any of the mitochondrial
stains. It is probable that the chromophobic center of the discs of
Lechriopyla represents material secreted by the rim, which is the mito-
chondrial part of the complex discs. In some cases, however, mitochondria
may have a true duplex structure, since Mast and Doyle (1935b) showed
that the outer surface of the mitochondria of Amoeba stain more deeply
than the center. This differentiation may be explained either as a definite

a ee Wl 2

af
"G4

°
Coat

—"- Flite a
: i :

oR OY - earner
- 2 t arash wali youn pdt tha ap Ta
gaan yer 0 E

"¢-

ie
xf

Figure 15-16. From Lechriopyla mystax: Figure 15, end view after Hirschler’s
mitochondrial technique; Figure 16, lateral view, after Champy-iron haematoxylin. (After
Lynch, 1930.) Figure 17. From Ichthyophthirius multifiliis, series showing mitochondria
and the secretion of paraglycogen, vital stain with janus green. (After MacLennan,
1936.) Figures 18-21. From Monocystis showing de novo origin of mitochondria: Fig-
ure 18, sporozoite; Figure 19, trophozoite; Figure 20, conjugating gametes from a cyst;
Figure 21, spores, Champy or Flemmings—iron haematoxylin. (After Horning, 1929.)
Figures 22-25. From Amoeba proteus: Figures 22-23, free in cytoplasm; Figure 22, nor-
mal: Figure 23, fixed in modified Regaud’s fluid; Figures 24-25, on surface of contractile
vacuole. (After Mast and Doyle, 1935.) Figures 26-27, From Aggregata eberthi: Figure
26, mitochondria proper; Figure 27, mitochondria associated with protein reserves.
(After Joyet-Lavergne, 1926.) Figures 28-29. From Bursaria truncatella: Figure 28,
section of early conjugant; Figure 29, section of later conjugant. (After Poljansky,
1934.)

In all cases, material which responds to mitochondrial stains is drawn in solid black;
associated granules are stippled.

CYTOPLASMIC INCLUSIONS 115

localization of stainable materials or as a dense surface with less dense
centers, with the type of material the same in both places.

The mitochondria in most Protozoa are fairly evenly distributed
through the cytoplasm, sometimes alone and sometimes associated with
various types of storage granules (see the discussion of function).
Fauré-Fremiet (1910) found that if any localization occurs, the mito-
chondria tend to concentrate beneath the pellicle and occasionally around
the contractile vacuole. Horning (1927) extended these observations and
contends that mitochondria tend to concentrate near all membranes—
particularly around the food vacuoles during active digestion, beneath
the pellicle, and around the nucleus. Hall and Nigrelli (1930) criticized
Horning’s identification of mitochondria, which was based largely on
dark-field observations, and showed that in Vorticella sp. the mito-
chondria are not associated with the food vacuole. Volkonsky (1934)
likewise rejected Horning’s identification and showed that the granules
associated with the digestive vacuoles, in a large number of species, are
stainable only with neutral red. MacLennan (1936) found a similar
situation in Ichthyophthirius. In other Protozoa, however, the accumula-
tion of mitochondria near membranes has been confirmed. Mast and
Doyle (1935b) find that the mitochondria in Amoeba are occasionally
associated with the gastriole (food vacuole) and they were able to cor-
relate the association with the type and stage of digestion. They also
demonstrated an association with the contractile vacuole, confirming the
earlier work of Metcalf (1910). Volkonsky (1934) found a small ag-
gregation of mitochondria around the food vacuole of Campanella and
Paramecium during the alkaline phase of digestion. Chatton and Grassé
(1929) showed that the filamentous mitochondria of Polykrikos tend to
accumulate near the pellicle, but instead of being parallel to the surface,
as in the ciliates described by Horning, are perpendicular to the surface.
Poljansky (1934) studied the changes occurring in the life cycle of Bur-
saria and found that the mitochondria of neutral individuals are uniformly
dispersed throughout the cytoplasm, but during conjugation (Figs. 28,
29) the mitochondria migrate to the periphery and form a definite zone
under the ectoplasm, and also around the micronuclear derivatives. The
chondriosomes again scatter during the growth of the macronuclear pri-
mordium. There seems to be neither universal nor permanent localization
of mitochondria near membranes, as is to be expected according to Horn-

116 CYTOPLASMIC INCLUSIONS

ing’s theory that mitochondria accumulate at the intracellular surfaces in
accordance with the Gibbs-Thompson Law. Doyle (1935) suggests that
mitochondria tend to collect at regions of active interchange, since
he found that mitochondria are the only granules which flow out into the
pseudopodia of the foraminiferan Irzdza. While this theory is attractive in
certain cases, it is difficult to see how this would apply to the concentra-
tions which occur in conjugating Byrsaria.

Mitochondria are more widely accepted as universal, permanent, and
self-perpetuating granules than any other cytoplasmic component, and for
this reason it is worth while to consider in detail the proof upon which
such statements rest. Rigid proof of this theory requires a demonstration
of mitochondria only, with, of course, a lack of evidence of any de novo
origin. Furthermore, it is obvious that proof of the continuity of any
cytoplasmic component cannot be based on a study of only one stage
in the life cycle, but must rest upon adequate studies of the whole life
cycle.

Mitochondria have been identified in a multitude of Protozoa of all
groups by Fauré-Fremiet (1910) and later authors, and as a result these
components are usually considered to be present in all Protozoa. How-
ever, recent evidence shows that this assumption is unjustified. An ex-
treme example is Trypanosoma diemyctyli, in which Nigrelli (1929)
was not able to demonstrate any pre-formed mitochondria, although
granules which satisfy the general criteria of mitochondria are induced
by exposure of the organisms to Janus green B. These induced granules
are not permanent, but disappear after about two hours. The marine
amoeba Flabellula is another species in which mitochondria are normally
lacking. Hopkins (1938b) found that the normal amoeba possessed no
granules which can be classified as mitochondria, but that when the
amoeba is disturbed in a variety of ways, granules are precipitated in
small pre-formed cytoplasmic vacuoles, the contents of which are nor-
mally a homogeneous fluid. After the recovery of the amoeba from the
disturbing conditions, the granules are resorbed. These temporary
granules possess the staining reactions of mitochondria, including the
ability to segregate Janus green B, and are the only granules in this
organism which can be classed in that group. As a contrast to these cases
of induced mitochondria, Kirby (1936) reports the experimental destruc-
tion of the mitochondria which are a normal component in the cytoplasm

CYTOPLASMIC INCLUSIONS 17

of flagellates from termites. In normal Psewdodevescovina, large num-
bers of mitochondria can be demonstrated by the Flemming-Regaud
method but, after feeding for three days on filter paper soaked in one-
percent Janus green B, no mitochondria can be demonstrated. Addi-
tional examples of the lack of mitochondria are furnished by studies of the
whole life cycle of certain Protozoa. Horning (1929) was able to demon-
strate mitochondria in most stages of Monocystzs (Figs. 19, 20), but
found that these granules disappear in the sporozoite stage (Fig. 21).
Beers (1935) and MacLennan (1936) also found that mitochondria
disappear in the later encysted stages of ciliates, although the same
methods give positive results in other stages of the cycle. These observa-
tions under both normal and experimental conditions demonstrate that
mitochondria are neither universal nor permanent cytoplasmic constitu-
ents.

The crucial point in the classification of mitochondria as autonomous
organelles is whether they always arise from preéxisting mitochondria.
The occurrence of dumb-bell-shaped mitochondria and other possible
division stages have been found so often in fixed and stained preparations
that it is unnecessary to quote this evidence here. The observations on
living material are few and perhaps the clearest is that of Horning
(1926), who reported division stages of mitochondria in a living
heterotrich and was able to confirm the descriptions based on fixed
material. Thus division is a factor in the increase in numbers of mito-
chondria, but returning again to the studies of the whole life cycle, we
find that division is not the only method, since mitochondria must be
formed de novo (Figs. 18-21) in those species in which the mito-
chondria have disappeared during the quiescent phases of the life
cycle. Mitochondria are not self-perpetuating organelles, but are differ-
entiations which may endure for a longer or a shorter period during
the cycle of the cell.

In the cases considered above, mitochondria are cytoplasmic in their
origin, but this is by no means the only possibility. Joyet-Lavergne
(1926) states that the group of mitochondria attached to protein granules
(Fig. 27) are derived from the nucleus along with the protein reserves
(for discussion of this point, see p. 163), but Daniels (1938) in the
same or related species could find no mitochondria attached to the
protein granules. Calkins (1930) found in Uroleptus one set of cyto-

118 CYTOPLASMIC INCLUSIONS

plasmic granules, which he traced back to macronuclear fragments dur-
ing conjugation, interpreting these as mitochondria. Since the staining
methods were not very specific and these granules do not stain with
Janus green, the statement that these are mitochondria must be accepted
with caution. Miller (1937) has endeavored to prove that mitochondria,
Golgi bodies, and other cytoplasmic inclusions in A. proteus are “‘bac-
teria spores, fungi, or yeasts, together with indigestible material of
certain food organisms.” This idea is, of course, similar to that of Wallin
and has been so thoroughly criticized by Cowdry and others that it need
not be considered here in detail. Miller was not able to culture these
cytoplasmic “bacteria’’ and his main argument seems to be based on the
observation that mitochondrial stains and Golgi type impregnations will
demonstrate granules in the culture medium. This merely shows that
the stains used are not always specific under all conditions, a fact which
has been pointed out many times. Miller does not present any evidence
which can stand up against the observations and experiments of Mast
and Doyle (1935a, 1935b), Holter and Kopac (1937), and Holter and
Doyle (1938) on the same species. The mitochondria are specializations
of the cell itself, probably in all cases from the cytoplasm, and are neither
artifacts nor invaders.

The composition of mitochondria is still incompletely known in any
exact sense, in spite of the large amount of work done on these com-
ponents. They have long been thought to be composed of both lipoids?
and proteins, because of their staining reactions and solubilities (Fauré-
Fremiet, 1910; Hirschler, 1924, 1927). Unfortunately no one has
yet repeated in Protozoa the work of Bensley (1937), who isolated
mitochondria from liver and was able to make both qualitative and
quantitative analyses. These analyses confirmed the cytochemical analysis
of lipoid and protein, but instead of the large amounts of phosphatids
and so forth, which were predicted from the cytological reactions, he
showed that the lipoid is largely neutral fat. Bensley also found that
many reactions (for example the osmic-acid reaction) are much weaker

* Lipoid is used here, as in most cytological works, in a very general sense. It includes
all materials which are soluble in ether, absolute alcohol, and so forth, and which stain
with Nile blue sulfate, the sudans, and other fat soluble dyes. Neutral fat, fatty acid,
phosphatids, and the like respond to these tests. Lison (1936) suggests the rather
awkward term sudanophil material, in order to emphasize the cytological side and to
avoid false implications as to chemical nature.

CYTOPLASMIC INCLUSIONS 119

in mitochondria in the cytoplasm than in mitochondria isolated from the
cytoplasm. These observations confirm the validity of the interpretation
of mitochondrial reactions in the Protozoa as indicative of a lipoid-
protein mixture, but emphasize the need of caution in more specific
interpretations before methods as specific as Bensley’s are applied to the
Protozoa. It is clear that the evidence now available as to the nature of
the lipoids and of the proteins in the mitochondria of Protozoa is sig-
nificant largely as a lead for further work.

Horning (1927) adheres to the view that the lipoid component of
mitochondria is a phosphatid, but presents no conclusive evidence for
this statement. Wermel (1925) found that the mitochondria (or lipo-
somes) of Actinosphaerium react with Ciaccio’s method, but according
to Lison the only valid interpretation is that unsaturated lipoids are
present. MacLennan (1936) stated that the lipoid material in the mito-
chondria of Ichthyophthirius ts a fatty acid, on the basis of a blue stain
with Nile blue sulphate, used according to Lorrain Smith’s method.
However, since Lison (1936) presents evidence against the specificity
of this method, the above interpretation is perhaps too strict, but it is
interesting to note that the original interpretation is in accord with
Bensley’s analysis of the mitochondria of liver. There is likewise a
lack of information on the nature of the proteins present. Hayes (1938)
demonstrated a positive reaction to fuchsin-sulfurous acid reagent and
claimed that nucleic acid is present. However, since lipoids may react
in this manner in the “‘plasmal reaction,” it is possible that this test was
concerned with the lipoid component rather than the protein portion.

The metallic impregnation of mitochondria, or the depth of stain taken
after the use of lipoid solvents, varies between the species of Protozoa
and has usually been considered to be a rough indication of the pro-
portion of lipoids present. Thus Scott and Horning (1932) find in
O palina a large amount of lipoid; Lynch (1930) in Lechriopyla, Patten
(1932) in Nyctotherus, MacLennan and Murer (1934) in Paramecium,
find some lipoid; while Beers (1935) in Dédinium finds little if any
evidence of lipoids in the mitochondria. Although this data is very
crude, foundation is provided for the working hypothesis that there
is a wide variation in the probable composition of mitochondria, rang-
ing from practically pure lipoid to almost pure protein. Much of this
difference represents constant differences between species and could be

120 CYTOPLASMIC INCLUSIONS

detected by Bensley’s mass technique. However, Fauré-Fremiet (1910)
found staining differences within the mitochondria of a single indi-
vidual, and Peshkowskaya (1928) reports that the ectoplasmic chondrio-
somes of Climacostomum are resistent to fixatives which usually dis-
solve mitochondria, although the endoplasmic mitochondria are much
more typical in their reactions. Pellissier (1936) found similar dif-
ferences, not within the cell but between various individuals, and was
able to show that all the mitochondria impregnate more deeply in the
vegetative stages than in the stages just before reproduction. By the
selection of species in which all granules are in the same stage at the
same time, Bensley’s mass technique could be used very profitably
in exploring the changes in mitochondrial composition.

MacLennan and Murer (1934) found heavy deposits of ash in the
typical mitochondrial rods, as well as in the other cytoplasmic granules
of Paramecium.

The presence of enzymes in mitochondria have been indicated in-
directly in many cases by the morphological association of these bodies
with structures in which digestive or synthetic activity is going on.
The only direct demonstration of the localization of cytoplasmic
enzymes is due to Holter and Kopac (1937) and Holter and Doyle
(1938), who showed that dipeptidase is not present in mitochondria,
but that amylase is. The method used was a combination of centrifugal
localization of granules and micro-methods for the measurement of
enzymatic activity. The mitochondria were concentrated in one end
of an Amoeba, which was then cut and the enzymatic activity of the
mitochondria-rich and the mitochondria-poor portions of the cytoplasm
compared. Since both the centripetal and the centrifugal portions had
the same amount of dipeptidase (measured by the ability to split
alanylglycine) per unit volume of cytoplasm, Holter and Kopac con-
cluded that the enzyme is in the matrix. They point out that this proves
nothing as to the origin of the enzyme, which might diffuse out from
a granule as fast as it is formed. Holter and Doyle found that the
middle region of the centrifuged Amoeba had the most amylase
(measured by the digestion of starch). The nucleus, crystals, digestive
vacuoles, mitochondria, and matrix are found in this zone of the centri-
fuged Amoeba. The enzyme could not be localized in the nucleus, since
non-nucleated fragments show no significant diminution in amylase,

CYTOPLASMIC INCLUSIONS 121

nor in the crystals since most of these are in the centrifugal end which
would thus have the highest enzymatic activity. They also ruled out a
localization in the digestive vacuoles by a demonstration that the enzyme
content of hungry and feeding Amoeba, with a resultant difference in
the number of vacuoles, is the same. The only structures the distribu-
tion of which after centrifuging corresponds with the distribution of
amylase are the mitochondria. The study of the centrifuged Amoeba
presents many difficulties, since the stratification is never complete and
there is always some mixing between the finishing of centrifuging and
cutting the amoeba in parts, but the further use and development of these
methods and their use in species in which stratification is complete
will undoubtedly aid in the complete analysis of mitochondria and other
cytoplasmic granules.

The theory that mitochondria are concerned with cellular respiration
has led to attempts to identify in the mitochondria the materials known
to be active in this respect. One of these is glutathione, in which the
physiologically active group is sulfhydril, demonstrable cytochemically
by the sodium nitroprusside reaction. Joyet-Lavergne (1927-29) found
that the mitochondria of Sporozoa give a positive reaction with sodium
nitroprusside, and this was confirmed by Cowdry and Scott (1928) in
Plasmodium. Chalkley (1937), however, found that the strongest re-
action in vegetative Amoeba is in the nucleus and that at the metaphase
this material is poured into the cytoplasm. Some granules in the nucleus
give a particularly strong reaction, but in the cytoplasm the coloration
is diffuse. These results of Chalkley’s extensive work on glutathione in
Amoeba suggest the desirability of a reinvestigation of the Sporozoa,
and certainly indicate that the materials containing the sulfhydryl group
are not always localized in the mitochondria. Joyet-Lavergne (1934) has
also shown that the mitochondria give a strong reaction with the anti-
mony trichloride test for vitamin A, and concludes that this is a part of
the respiratory mechanism along with glutathione. Although respiration
cannot be discussed in detail here, it should be pointed out that the
glutathione-vitamin A theory presents many difficulties and the system
more usually accepted is glutathione-ascorbic acid (Holmes, 1937).
Bourne and Allen (1935) and Bourne (1936) have demonstrated the
concentration of ascorbic acid in cytoplasmic granules by the acetic-
silver-nitrate method, but unfortunately have not correlated these with

122 CYTOPLASMIC INCLUSIONS

any particular type of granules. Daniels (1938) used this method in
gregarines, but although she was able to demonstrate granular accumula-
tions in the cells of the gut, the gregarines remained clear.

The functions ascribed to mitochondria in the Protozoa may be
grouped under two main headings: synthesis (or segregation), and
respiration. The first group includes a number of activities, all of which
involve the accumulation of materials and in some cases the synthesis
of new products from these raw materials. Examples of this type of
function for which there is definite evidence are the secretion of reserve
bodies, a digestive function in connection with the gastrioles, excretory
function in connection with the contractile vacuoles, and the transport of
materials from one organelle to another. Respiration also might be
considered to fall in this category, since it would depend upon the ac-
cumulation in the mitochondria of the substances responsible for the
oxidation-reduction processes of the cell.

The secretion of reserve bodies is cytologically the easiest phase of
accumulation to demonstrate. Fauré-Fremiet described the formation
of deutoplasmic granules by direct transformation of mitochondria, as
well as by the more common method of segregation adjacent to or
within the mitochondria which retain their identity. Joyet-Lavergne
(1926a) occasionally found a relationship of this type between the
mitochondria and paraglycogen and always found that protein granules
possessed a mitochondrial cap (Fig. 27), but denied that this indicated
anything more than a casual relationship. Horning (1925) described
mitochondrial rims around the protein granules in O palina (this identi-
fication of mitochondria is denied by Kedrowsky, 1931b) and accepts
it as evidence of secretory activity. MacLennan (1936) described the
origin of paraglycogen in the center of spherical mitochondria (Fig. 17).
The identification of these granules was made not only with the usual
permanent stains for mitochondria, but with specific microchemical
stains (Sudan III, Nile blue sulfate, iodine, chlor-zinc-iodide), and
their growth observed in live specimens stained with Janus green. The
fact that the paraglycogen first appears as a center in a solid mito-
chondrial sphere refutes the usual suggestions that the secretion merely
happens to be in contact with the mitochondria and that there is no
real connection between the two. There is no evidence as yet to show
whether these visible secretions are actually synthesized by the mito-

CYTOPLASMIC INCLUSIONS 3

chondria or are the result of the segregation by the mitochondria of
materials synthesized at other points. Wermel (1925) found that
certain mitochondria of Actinosphaerium have a high lipoid content,
as shown by Ciaccio’s lipoid methods, 1.¢., a so-called liposome rather
than an ordinary type of mitochondria, and concludes that they secrete
the lipoid reserves. Except for the fact that these granules stain weakly

Figure 30. The association of mitochondria with the gastriole in Amoeba proteus.
(From Mast and Doyle, 1935b.) A, 2-6 hours; B, 6-8 hours; C, 8-16 hours; D and E,
16-30 hours; b and bi, mitochondria; f, fat; s, starch; v, vacuole refractive bodies; c,
crystals; p, pellicle of Chilomonas.

with Janus green, they are like the intermediate lipoid bodies and could
perhaps be classed more conveniently with them. Zinger (1928)
counted the “‘spherical inclusions” and the mitochondria in O phryoglena
and found that they are roughly proportional in number. He concluded
that the spherical inclusions are derived from mitochondria. Since many
granules of entirely different origin will increase in number under
favorable conditions, this conclusion cannot be considered as proved.

The digestive function of mitochondria rests upon two types of
evidence: the actual demonstration of enzymes in these granules (see
p. 125), and the correlation between the periodic aggregation of mito-
chondria around the gastriole and the type of digestion taking place
within. Both types of evidence are available for A. proteus, so that the
results of observations may be checked against a direct knowledge of the

124 CYTOPLASMIC INCLUSIONS

enzymes actually present. In A. proteus, Mast and Doyle (1935b) find
that the mitochondria accumulate around the food vacuole six to eight
hours after its origin and then again at sixteen to thirty hours (Fig. 30).
During the first contact, digestion begins, starch is changed to erythro-
dextrine, and fat leaves the vacuole. In the second contact, vacuole re-
fractive bodies and crystals disappear. These authors never found the
mitochondria actually entering the vacuole. The relationship between
mitochondria and starch digestion was directly demonstrated by Holter
and Doyle, who showed that these granules contain amylase. Their
function is restricted with respect to digestion, since they lack dipeptidase,
according to similar studies by Holter and Kopac (1937). This situa-
tion is not universal, since Hopkins (1938b) also working with Mast,
showed that no formed granules are associated with digestion in the
marine amoeba, Flabellula, although a material is dissolved in the
vacuoles which when precipitated by disturbance, and so forth, forms
granules which stain with Janus green B and other mitochondrial stains.
Again, in many other Protozoa the mitochondria have no direct con-
nection with digestion. Thus mitochondria may contain enzymes in some
cases, but this is not a necessary association.

Excretory granules associated with contractile vacuoles have been
described many times, but only recently have mitochondria been proved
to be associated with the excretory process. Mast and Doyle (1935a)
have shown that the excretory granules of A. proteus (Figs. 24, 25, 47)
correspond in their staining reactions to mitochondria. By centrifuging
the majority of these bodies into one end of the amoeba and removing
this part, these authors showed that the formation of the vacuole 1s
dependent upon these granules and that if most of the mitochondria
are removed, death follows. In one experiment, most of the mito-
chondria were left, with the result that the average interval between
pulsations was 3.46 minutes. If few mitochondria were left, the time
was correspondingly longer, and when very few granules were left,
the average time between pulsations increased to twenty-five minutes.
Mast and Doyle interpreted these experiments as indicating that some
excretory material, toxic to Amoeba, is eliminated by the vacuole, and
that the mitochondria function as the means of transport to the vacuole.

Doyle (1935) harmonized the apparently discrepant functions of
digestion and excretion by pointing out that they may be united under

CYTOPLASMIC INCLUSIONS 125

the general heading of transport, and pointed out further that mito-
chondria tend to accumulate wherever exchanges are taking place, both
within the individual and at the outer surface. Mast and Doyle (1935b)
observed that the mitochondria accumulate around the crystal vacuoles
while the crystals decrease in size, and with the surface of the refrac-
tive bodies while the latter are increasing in size. The mitochondria of
Amoeba seem to be a mechanism for intracellular transport and for
carrying amylase to the food vacuoles, digested material from the food
vacuoles and crystal vacuoles to the refractive bodies, and metabolic
wastes to the contractile vacuoles. However, since Holter and Kopac
(1937) have shown that dipeptidase is not associated with mito-
chondria, and Volkonsky (1933) and MacLennan (1936) have shown
cases in which vacuome alone touches the food vacuole, and Hopkins
(1938b) cases in which no preformed granules are associated with the
vacuole, it is clear that the mitochondria are not necessary in all cases
either for transport function or a support for enzymes.

The supposed universality and permanence of mitochondria have led
to many suggestions that they are concerned with some vital part of
cellular activity, some function more universal than secretion and
storage. Some evidence to this effect has been presented by Joyet-
Lavergne (1927-35), both from the standpoint of the presence of
materials active in respiration (see p. 121) and from the standpoint
of a direct demonstration of respiratory activity. He finds that vital
dyes are reduced most strongly near mitochondria and that individuals
which have large amounts of mitochondria reduce the dyes faster than
individuals with less mitochondria. He was able to demonstrate gluta-
thione and vitamin A in the mitochondria and attempted to show that
these two form an oxidation-reduction system. Rey (1931a, 1931b)
repeated Joyet-Lavergne’s staining experiments and obtained the same
results, but criticized the latter’s interpretation of his findings. Rey
also repeated the experiments using an electrometric method for deter-
mining rH and found no significant differences. Wurmser (1932) like-
wise criticized Joyet-Lavergne’s interpretations of the stain reactions as
indications of oxidation-reduction differences. Bles (1929) found that
the oxidation-reduction reactions which he studied in Arce/la were asso-
ciated with the hyaloplasm, rather than with any granules. Since Joyet-
Lavergne found morphological continuity between the mitochondria and

126 CYTOPLASMIC INCLUSIONS

two types of reserve granules, the existence of a secretory function is
possible, and it is not necessary to invoke respiration in order to find
a function for these mitochondria. The respiratory function must be
regarded as unproved, either from the standpoint of the proof of the
presence of materials which could act as an oxidation-reduction system,
or from the standpoint of the direct measurement of localized oxidation-
reduction potentials. At best mitochondria as morphological entities
cannot be necessary for respiration, since many species lack them at one
time or another in the life cycle, and since in other cases they can be
eliminated experimentally without fatal results (Kirby, 1936).

All the various types of evidence—staining reactions, composition,
function, and tracing through the life history—show that mitochondria
in the Protozoa do not form a homogeneous group, but are actually a
heterogeneous assortment which are associated merely by their ability
to segregate Janus green B or by even less specific staining reactions. No
one type is found in all Protozoa, and in all cases which have been
carefully studied mitochondria are not self-perpetuating but arise de
novo at some time during the life cycle.

THE VACUOME HYPOTHESIS

According to the vacuome hypothesis as applied in the Protozoa, there
are only two fundamental cytoplasmic components in the Protozoa—the
chondriome and the vacuome, since the Golgi bodies and vacuome are
merely different aspects of the same thing. The term vacuome was
substituted for the earlier term segregation granule as an indication of the
supposed homology between the neutral red bodies in animal cells and
the vacuoles of plant cells. Volkonsky (1929 on), Kedrowsky (1931-
33), Hall and his associates (1929 on), Lynch (1930), and others have
upheld the general conclusion that the granules stainable with neutral
red are identical with the Golgi bodies; but Kirby (1931), MacLennan
(1933, 1940), Bush (1934), Kofoid and Bush (1936), Daniels
(1938), and others have demonstrated many cases of neutral red
granules which are not osmiophilic (for a more detailed discussion of
this point, see p. 140). It should be pointed out that the acceptance
of the vacuome hypothesis is by no means universal in the Metazoa or
Metaphyta, according to Weier (1933) and Kirkman and Severinghaus
(1938).

CYTOPLASMIC INCLUSIONS 127

The ability to segregate neutral red has been ascribed to a specific
reaction of the dye with a single material in the granule, the process
being called the “neutral red reaction” by Koehring (1930) and in-
cluded as a part of the “ferment theory of the vacuome”’ by Kedrowsky
(1932b). The substance involved is supposed to be a proteolytic enzyme,
a conclusion based on the work of Marston (1923), who showed that
these enzymes are precipitated by combination with neutral red, Janus
green, and other azine dyes. Le Breton (1931) has reviewed the rather
voluminous literature resulting from this suggestion and concludes that
the reaction is not specific, since ordinary proteins (an important con-
stituent of most segregation bodies) also are precipitated. From
the standpoint of cytology, the theory fails to explain how neutral red
and Janus green have such different staining reactions in the granules
of living cells. Hopkins’s (1938a) experiments with F/abellula show that
precipitates are formed in vacuoles after either Janus green or neutral
red. If Janus green was first used, and then neutral red added, a red
precipitate formed around the original green one. “The small neutral red
vacuoles are, then, the same as the vacuoles in which the condensation
granules are formed, but it appears that Janus green B stains a different
component of these vacuoles than does the neutral red.”

Not one factor, but many are responsible for the segregation of neutral
red by cytoplasmic granules. The rdle of pH in neutral red staining has
been demonstrated by Chambers and Pollack (1927) and Chambers and
Kempton (1937), who showed that neutral red tends to go from an
alkaline region to an acid region so that ‘‘segregation granules’ would
be those which are acid relative to the hyaloplasm. Kedrowsky (1931)
demonstrated in Opalina that normally the segregation granules have
this relationship with the hyaloplasm and that the staining reactions
can be changed by altering the pH of the cytoplasm. He also showed
that the granules will take up acid dyes in the presence of albumoses,
which was confirmed by Volkonsky (1933) and included under the
term “‘chromopexie.’’ Since neutral red has long been known as a
lipoid stain (Fauré-Fremiet, Mayer, and Schaeffer, 1910) it is possible
that this may play a rdle in the staining of bodies which contain lipoids,
such as the dictyosomes of gregarinida and the digestive granules of
Ichthyophthirius. From these brief examples it is clear that the segrega-
tion of neutral red and other vital dyes is influenced by many internal

128 CYTOPLASMIC INCLUSIONS

and external factors and that it cannot be considered specific in the
sense that it combines with a single definite substance.

Since many factors are involved in the segregation of neutral red,
it is to be expected that more than one type of granule will be revealed
by the use of this and similar dyes. Conclusive evidence of this lack
of specificity is furnished by several Protozoa in which two or more
types of granules are able to segregate neutral red at the same time, i.e.,
under identical conditions. Dangéard (1928) stained two types of
granules in Evglena with neutral red—the vacuome and the mucous ap-
paratus. The latter group may be extruded to form a mucous envelope
or mucous hairs—an interesting example of true external secretion, and
comparable to the staining of secretion granules in Metazoa by neutral
red. However, Dangéard rejected the mucous apparatus as vacuome
because it retains the neutral red after the death of the cell, while the
other granules—the true vacuome—do not. Finley (1934) found four
different groups of granules which segregate neutral red in Vorticella:
pellicular secretions, pellicular tubercles, thecoplasmic granules, and re-
fractile granules. He was careful to control the staining to avoid over-
staining, so that his results cannot be questioned on that ground. Bush
(1934) found in Haptophyra two sets of granules which are discon-
tinuous in size and distribution. Mast and Doyle (1935b) complete the
picture by showing that three groups of granules stain in Amoeba:
vacuole refractive bodies, refractive bodies, and blebs on crystals, and
they proved experimentally that these bodies are different in origin and
in fate. The experiments of Kedrowsky (1931) on Opalina show the
reverse picture—under certain feeding conditions, the growing segre-
gation vacuoles lose their ability to take up neutral red, just as they do
in vertebrate eggs. Hall and Loefer (1930) showed that the granules
in Exglypha may vary in the same specimen from pink to bluish red
or red violet.

Since neutral red stains several different groups of granules and also
does not stain all stages of the same granule, it does not of itself reveal
fundamental homologies, and it seems to me to be unjustifiable to group
all of these bodies as vacuome or under any other catchall term. As a
preliminary step toward an accurate classification of this group, the
digestive granules—i.e., those neutral red bodies associated with the
gastrioles—are separated from the segregation granules which cor-

CYTOPLASMIC INCLUSIONS 129

respond to the ‘‘vacuome de reserve” of Volkonsky and the segregation
apparatus of Kedrowsky. The segregation granules of necessity still
include a heterogeneous assortment and probably include granules other
than those associated with synthesis and storage, but at present they
cannot be classified properly because too many of them are known only
by their ability to segregate neutral red. Kedrowsky and Volkonsky
regard both types as carriers of enzymes, in one case acting to digest
proteins and in the other case to synthesize them. However, each group
appears de novo when the necessity arises, and the two groups show
no direct continuity with each other. Further evidence of the independ-
ence of these two groups of granules is furnished by Opalina, in which
only the segregation bodies are present, and by Ichthyophthirius, in which
only the digestive granules are present, these latter having no connection
with the storage of the numerous protein granules.

DIGESTIVE GRANULES

The digestive granules may be briefly defined as cytoplasmic granules
stainable with neutral red, which become associated with the newly
formed vacuoles containing food. This does not include all neutral
red granules in the food vacuole, since such granules as the vacuole
refractive bodies of Amoeba are derived from the food (Mast and
Doyle, 1935b) and thus are not cytoplasmic components. Volkonsky
(1934) points out that the term vacuole has been applied to so many
structures that it is a source of confusion, and he has substituted the term
gastriole. The fluid vacuole which contains ingested food is a pro-
gastriole, and with the addition of the digestive granules (vacuome in
Volkonsky’s terminology) becomes a gastriole. In addition to these
terms it is convenient to use postgastriole for the structures containing
undigested remnants.

There is no single pattern of the gastriole in the Protozoa. In
A. proteus, mitochondria alone aggregate periodically around the gas-
triole (Mast and Doyle, 1935b); in Paramecium and Campanella, the
digestive granules enter the gastriole, and mitochondria cluster around
the membrane in the alkaline phase; in Flabellula, the materials stain-
able with Janus green or neutral red are normally dissolved in the fluid
vacuole, which later forms the gastriole (Hopkins, 1938), and a some-
what similar situation is found in hypermastigote flagellates (Duboscq

130 CYTOPLASMIC INCLUSIONS

and Grassé, 1933); in Ichthyophthirius (MacLennan, 1936), the diges-
tive granules (Fig. 31) enter the gastriole, but the mitochondria are
never associated in any visible manner with the digestive mass. A more
detailed description of digestion will not be given in this section, since
attention is here centered on the granules.

All careful descriptions agree that the digestive granules vary in
size and shape during the gastriolar cycle, ranging from minute spherules
to relatively large rods. Both vital stains and metallic impregnation show
a homogeneous structure, and the deformation of these granules either

GS) Gas 8

Figure 31. The association of the gastrioles and the digestive granules in Ichthyoph-
thirius multifiliis. (From MacLennan, 1936.)

from other cytoplasmic granules or from outside pressure show that
they have a soft, semifluid consistency. Volkonsky (1934) has found
that the morphology of the gastriole varies in the same species with the
food used. In Acanthamoeba he was able to induce the formation of
large granules, small granules, or a homogeneous rim stainable with
neutral red, by varying the food used. No digestive granules were formed
around vacuoles which contained only starch.

The penetration of the digestive granules into the vacuole was not
observed by Koehring (1930), Hall and Dunihue (1931), Dunihue
(1931), or Hall and Nigrelli (1930), and the suggestion was made
that the granules in the food vacuole are derived from food particles

CYTOPLASMIC INCLUSIONS 131

rather than from the cytoplasm, a suggestion which later was proved to be
true, in the case of vacuole refractive bodies of A. proteus, by Mast
and Doyle (1935b). Volkonsky, however, checked his observations on
many ciliates and ruled out exogenous granules in the gastrioles of
Glaucoma by the use of bacterial free medium; he still observed diges-
tive granules in the vacuoles and was able to trace them from the cyto-
plasm. The migration of the digestive granules into the gastriole of
Ichthyophthirius was found by MacLennan (1936), but it was shown
that the granules do not penetrate any membrane. Instead, a new mem-
brane is formed (Fig. 31) around the whole gastriole and the inner
membrane, which is the original one, then disappears. The end result
is exactly the same as in the cases described by Volkonsky, but the
mechanism is somewhat different.

Volkonsky’s interpretation of the digestive granules as vacuome 1s
based upon their impregnation by the various Golgi-type methods. The
question as to the identification of the granules as shown by entirely
different methods is not present in this case, as it was in the case of the
scattered cytoplasmic granules (segregation granules), since the digestive
granules can be recognized independently of their staining reactions by
their relationship to the gastriole. Hall and Nigrelli (1937) claim that
the digestive granules are less consistent in impregnation than the
scattered cytoplasmic granules and dispute Volkonsky’s claim that they
can be considered as vacuome. MacLennan (1940) showed that the
various types of osmiophilic granules could not be distinguished on the
basis of impregnation alone; in particular the digestive granules of
Ichthyophthirius show 100-percent impregnation.

Few of the materials which occur in the digestive granules are known
from direct evidence. The digestive granules of Paramecium are high
in ash (MacLennan and Murer, 1934) and those in Ichthyophthirius
contain lipoids (MacLennan, 1936). These latter bits of information
are not as yet particularly useful and emphasize the need for more
specific knowledge. The presence of enzymes is suggested by the morpho-
logical evidence and this would be a fertile field for the use of the
microenzymatic methods.

The digestive granules are not permanent self-perpetuating structures,
but appear to rise in the cytoplasm in response to the stimulus of feeding.
Volkonsky (1934) found that when the preéxisting granules are utilized

132 CYTOPLASMIC INCLUSIONS

in the formation of gastrioles, new granules are formed in the cyto-
plasm. The digestive granules in Ichthyophthirius were observed by
MacLennan (1936) to arise de novo in direct contact with the ingested
particles of food. Final evidence of their de novo origin was furnished
by the fact that no digestive granules are present in the encysted stage,
after the gastrioles which were formed during the feeding stage have
disappeared. Volkonsky suggests that the materials of the digestive
granule exist in the cytoplasm in a diffuse state. In the hypermastigote
flagellates this material is concentrated in dissolved form in the gastrioles;
in Flabellula the materials are first concentrated in vacuoles, which con-
tribute the fluid part of the gastrioles; in the more common cases, the
materials are condensed to form digestive granules. We must not for-
get, however, that not all the digestive reactions result in granules, since
dipeptidase in Amoeba is independent of any granules. Whatever the
specific morphology of the reaction, whenever the cytoplasmic equilib-
rium is disturbed by the ingestion of food there is an effective mobiliza-
tion of this material to cope with the ingested food. Volkonsky calls
these varied changes the “vacuolar reaction,” an extremely useful term,
but since we reject the term vacuome, “‘gastriolar reaction’ would be
more appropriate.

SEGREGATION GRANULES

The segregation granules are bodies which are able to concentrate,
accumulate, and store within themselves vital dyes, proteins, and other
materials. Unlike the definitions of most cytoplasmic granules, this
definition is based upon function rather than upon morphology or
staining reactions. The evidence for this definition is due largely to the
work of Kedrowsky (1931-33) on Opalina, Spirostomum, and other
ciliates. Since the functions of most of the granules stainable with
neutral red have not been demonstrated, the work on Opalina will be
discussed first and granules in other Protozoa considered in the light of
this work. The accumulation of vital dyes in higher concentration than
they occur in the medium is, of course, one piece of evidence of the
segregating ability, even if this has’ not been demonstrated with the
materials which enter into the metabolism of the cell.

The segregation bodies of Opalina (Figs. 32-35) are the external
layer of granules or ectosomes and have been identified as Golgi bodies

CYTOPLASMIC INCLUSIONS Ei)

or as mitochondria by various authors, since they respond to some of
the Golgi and mitochondrial techniques, although they are not stained
specifically with Janus green B. Kedrowsky described four main morpho-
logical types—fine dispersed granules, large granules, alveoli, and
heteromorphic granules (Figs. 32-35). In some cases the dispersion of
granules is so accentuated that they lose their identity as granules. These
changes in morphology are associated under natural conditions with the
seasons of the year; for instance, the heteromorphic types are common
in the spring and early summer, and the large granular type is found
in the early spring. There is some variation between populations of
different frogs in the same season, but all members of the same popula-

Figures 32-35. Basic morphological variations in the segregation granules of Opalina
ranarum. Semischematic. (After Kedrowsky, 1931le): Figure 32. Dispersed type; Figure
33, homomorphic granular type; Figure 34, alveolar type; Figure 35, heteromorphic
granular type.

tion have essentially the same type of granules. These different forms
can be produced experimentally by changing the culture medium. The
colloidally dispersed type is typical of amino-acid cultures. In distilled
water, each ectosome swells and becomes a watery vacuole. The hetero-
morphic type is found in cultures which contain defibrinated and hemo-
lysed blood. These changes, which occur both naturally and in artificial
media, may well account for the disagreement among cytologists both
as to the morphology and the identification of these granules.

The segregation bodies of Opalina are not permanent organelles. As
indicated above, they may disperse homogeneously through the ecto-
plasm, which then takes a general pale stain with neutral red, and no
method applied at this stage shows any indication of a remnant of the
originally discrete granules. If the bodies are loaded with protein com-
pounds of metals, such as silver, they may finally be extruded from the
surface and be replaced by granules which arise de novo.

134 CYTOPLASMIC INCLUSIONS

The composition of the segregation bodies is as varied as their
morphology and results from the same causes—changes in the environ-
ment. Under natural conditions they may contain proteins (Millon’s
reaction), glycoproteins (Fischer's reaction), cholesterin (Schultz te-
action, digitonin reaction), or be stained with bile pigments. The
alveolar type of course contains mostly water. The segregation granules
will store basic dyes in salt solution, acid dyes in the presence of pro-
teins, silver in the form of kollargol or other similar compounds, iron
albuminates, cholesterin, and so forth. According to the ‘ferment theory
of the vacuome” of Kedrowsky (1932b), enzymes are present in the

Figures 36-37. Segregation granules in Trypanosoma diemyctyli, neutral red stain.
(After Nigrelli, 1929.) Figure 36, Preformed granules around the blepharoplast; Figure
37, preformed granules and granules induced by prolonged exposure to neutral red.

segregation bodies, but, as is the case with the digestive granules, no
direct proof of this is available.

The segregation bodies obviously function in the concentration and
storage of various materials, particularly proteins. This may be accounted
for on a purely physical basis, as, for instance, the tendency of basic
dyes to migrate to a more acid region, or the tendency of molecules to
migrate toward bodies of opposite charge. The exact mechanism is, of
course, a complex problem and cannot be taken up here, but the es-
sential point at present is that the granules may play a purely passive
role—the accumulation and storage of materials which originate else-
where.

Accumulation is merely one part of the function, according to the
enzyme hypothesis of the segregation granules, advanced at nearly the
same time in slightly varying forms by Koehring (1930), Kedrowsky
(1931 on), and Volkonsky (1929 on). Koehring based her conclu-
sions on the supposed specificity of the neutral red reaction for proteo-
lytic enzymes (see p. 178). Kedrowsky considers that both the synthesis

CYTOPLASMIC INCLUSIONS 155

of proteins and deaminization may occur in the segregation bodies. The
evidence of synthesis may be summarized simply as the increase in size
or number of segregation bodies in Opalima when immersed in various
nutrient solutions, and the subsequent identification in the granules of
materials from this culture medium. This certainly proves the segregat-
ing ability, but the actual synthesis might take place almost anywhere
and the increase in size be due simply to the segregation of these pre-
formed materials. The evidence of deaminization is based on the appear-
ance of glycoprotein in the late stages of the segregation bodies and
the fact that the segregation granules are able to oxidize Rongalit white
vitally. This evidence could hardly be called more than suggestive, but
the processes which are indicated by these tests seem to be localized in
the segregation granules.

The neutral red granules of Protozoa other than Opalina, to be con-
sidered in the rest of this section, excludes only the group which were
discussed above as digestive granules. It is thus essentially the group
called segregation apparatus by Kedrowsky (1931), or the vacuome of
Hall (1929 on). Since neither the history nor the function of most of
these granules is known, this is doubtless a heterogeneous group, but
I believe that further splitting at this time would merely add names
without increasing understanding.

The segregation bodies are normal cytoplasmic constituents and are
not induced by vital dyes, since they have been observed by many in-
vestigators in normal, unstained specimens (Hall, 1929 on; Finley,
1934; MacLennan, 1933, 1936; Volkonsky, 1929 on; Kedrowsky, 1931
on, and others). Prolonged staining (Fig. 23), it is true, may induce
the formation of new granules (Kedrowsky, 1931; Cowdry and Scott,
1928; Nigrelli, 1929), but this is not a universal phenomenon, since
many species, in my own observations, show the general diffuse stain-
ing of the cytoplasm and nucleus characteristic of severe overstaining,
without the appearance of new granules. The normal origin of all types
of segregation granules seems to be de novo. Mast and Doyle (1935b)
removed most of the refractive bodies from A. proteus and observed the
formation of new bodies several hours after feeding. The “blebs’’ on
the crystals likewise clearly originate de novo. These cases are too few
to justify any certain statements for all the many segregation granules,
but it is indicative that in all the many descriptions of these granules,

136 CYTOPLASMIC INCLUSIONS

there has been no clear case of division described—all the evidence indi-
cates a de novo origin.

Small homogeneous spherules are the commonest type of segregation
body and they are found in all classes of Protozoa. There are often more
than one group in a single individual, as shown by differences in size
(Bush, 1934) or differences in localization (Finley, 1934). Complex
bodies similar to those found by Kedrowsky in O palina have been found
in flagellates from termites (Kirby, 1932). In some cases such complex

; y o
=® f- SH ey
a |
A B Cc 02 E F

Figure 38. Segregation granule (refractive body) of Amoeba proteus in successive
stages of resorption. (From Mast and Doyle, 1935.) A, normal body; B-F, stages in
resorption, optical sections; D, surface view. Outer layer cross-hatched, shell black,
vacuole in outline.

bodies may be the result of precipitation of dyes or other materials in
an aqueous vacuole, but in the cases of the denser granules entirely,
different methods show the duplex structure. In the refractive bodies
of Amoeba (Fig. 38), microchemical tests show that the duplex struc-
ture represents an actual difference in composition (Mast and Doyle,
1935a).

The segregation granules are most often scattered at random through-
out the endoplasm (Hall and his associates, 1929 on), but occasionally
are more definitely localized. In Trypanosoma (Figs. 36, 37) they are
concentrated around the blepharoplast (Nigrelli, 1929), in the Ophry-
oscolecidae they are most common in the operculum and spines (Mac-
Lennan, 1933), and in Lechriopyla a marked concentration is found just
under the pellicle (Lynch, 1930). In Vorticella a group of discrete
globular inclusions is found scattered in the cytoplasm, and another group
is found in the stalk (Finley, 1934). The tubercles of the pellicle also
stain, as do the secretions of the pellicle, but the latter have been consid-
ered in the section on secretion granules and the former do not seem to
me to fulfill the definition of cytoplasmic granules, or at least are clearly
not in the same class as the ectosomes of O palina. As a contrast to these
Protozoa with several types of segregation granules, the ciliate Ichthy-

CYTOPLASMIC INCLUSIONS 137

ophthirius has none at all at any time in its life cycle (MacLennan,
1936), and the function of synthesis and storage of the protein re-
serves is taken over by the macronucleus.

The composition of the segregation granules of Protozoa other than
Opalina is known only in the case of the refractive bodies and blebs
of Amoeba. The osmiophilic shell of the refractive bodies of Amoeba
has a protein stroma impregnated with a lipid substance (Mast and
Doyle, 1935a). This portion stains with Sudan II only after Ciaccio’s
method for “unmasking” the lipoids, and is intensely blue in Nile blue.
These granules are not dissolved in alcohol in twelve hours, but they
do lose their positive reaction to fat soluble dyes. They respond to the
methylene-blue-sulphuric test for metachromatin and give a faint re-
action with Millon’s reagent. Within this layer a brittle carbohydrate
shell and an unknown fluid are found. The stainable blebs on crystals
in the same protozoan, when first formed, contain only lipoids, but as
they grow larger, protein is added so that their final composition is
similar to the shell of the refractive bodies of the cytoplasm. These
granules are therefore quite different from the segregation granules of
O palina, since Kedrowsky showed that whatever else might be stored
in these bodies, they do not segregate lipoids. However, since the re-
fractive bodies do segregate proteins and stain with neutral red, they
are included as segregation bodies. This is, of course, arbitrary, since
they overlap on the Golgi granules and on the reserve granules. Sufh-
cient mineral ash is present to mark the vacuome in incinerated speci-
mens of Opalina (Horning and Scott, 1933) and Paramecium (Mac-
Lennan and Murer, 1934). The only striking fact about the segregation
bodies in most Protozoa is that with the exception of those in O palina and
Amoeba, not even a sketchy outline of their composition is available.

The function, in so far as it is known, agrees closely with the storage
function described for the granules in Opalina. Variations of the num-
bers of the segregation granules have been found in Paramecium (Duni-
hue, 1931) and in Vorticella (Finley, 1934). Dunihue was able to
correlate the decrease in numbers with starvation, thus indicating a
storage function. Mast and Doyle (1935a) showed that both protein and
lipoid materials are found in the blebs on the crystals and on the re-
fractive bodies. Since the blebs appear shortly after feeding and since
they in turn disappear as the refractive bodies are increasing, the blebs

138 CYTOPLASMIC INCLUSIONS

seem to be temporary reserves, while the refractive bodies are the final
reserves. In the blebs, the protein portion appears later than the lipoid
portion. There is no evidence in these cases of anything more than the
accumulation of materials.

GOLGI BopDIEs

The term Golgi body has been applied in Protozoa to organelles
which differ fundamentally in both composition and function. These
structures include contractile vacuoles (Ramon y Cajal, 1904-5), gran-
ules (Hirschler, 1914), specialized regions of cytoplasm around the
contractile vacuoles (Nassonov, 1924), segregation granules (Cowdry
and Scott, 1928), osmiophilic nets (Brown, 1930), and the parabasal
apparatus (Duboscq and Grassé, 1925). The controversies which have
arisen between advocates of one or another of these structures have
been due not so much to disagreement upon the actual facts involved as
to disputes concerning criteria for the identification of Golgi material.
The selection of criteria is thus a crucial point in codrdinating the in-
vestigations of Golgi bodies; yet even after years of work on representa-
tives of all the major groups of animals and plants, and notwithstand-
ing periodic reviews of the field, few criteria seem to have unanimous
approval—few, indeed, have majority approval. The most recent re-
view (Kirkman and Severinghaus, 1938) after failing to demonstrate
any universal and objective basis of identification, quotes as follows
from Gatenby (1930): “modern workers in general have experienced
no difficulty in identifying Golgi bodies.” This is very satisfactory as
long as only one school of cytologists is considered, but such state-
ments lose their attractive ring of authority when one tries to correlate
the results presented by such experienced cytologists as Bowen, Parat,
Gatenby, Canti, or Ludford, to mention only a few.

This is particularly true when seeking criteria on which to base a
reasonable identification of Golgi bodies in the highly specialized cells
of the Protozoa. In the following paragraphs the major objective cri-
teria which have been used are discussed with particular reference to
their applicability to the Protozoa. In general these criteria involve two
points—consistent impregnation, and occurrence in all types of cells.

The first Golgi structures were discovered by the use of metallic im-
pregnation methods, and ever since these methods have remained as

CYTOPLASMIC INCLUSIONS Sy

the primary criteria (Gatenby, 1930), particularly since the discovery
that the supposedly typical reticular network is a relatively rare type
and in many cases is due to a temporary aggregation of granules (Hirsch-
ler, 1927). Both the osmic and the silver methods involve the same prin-
ciple—the adsorption of the reduced metal on particular structures,
although the actual reduction of the metallic compound may take place
either where the deposits are located or in other parts of the cell (Owens
and Bensley, 1929). Since the osmic acid methods have been most
thoroughly investigated, this discussion will be restricted to these tech-
niques, but the fact should be emphasized that the same general principles
are involved in the silver techniques as well.

The preliminary treatment of the cell strongly affects the subsequent
impregnation (Lison, 1936). Thus direct exposure of the protozoan
to osmic fumes (Hall, 1929) is not equivalent to the full technique
which involves preliminary fixation in such mixtures as Champy’s fluid.
Likewise, the blackening of granules by exposure to osmic fumes after
they have been stained with neutral red is not the equivalent of the
standard Golgi methods, since it has been shown in the case of Flabel-
lula (Hopkins, 1938b) that the blackening is produced only in the
presence of neutral red.

The Golgi methods are not specific for any single type of material,
since a series of different lipoids extracted from echinoderm eggs give
a typical Golgi reaction (Tennent, Gardiner, and Smith, 1931). Further-
more both lipoid and non-lipoid bodies of various types in the Protozoa
react typically and identically to the Golgi techniques (MacLennan,
1940). Although the method is not specific in the sense that it reveals
a single known substance, or some unknown Golgi material, the method
is consistent in the sense that under proper conditions the results can
be reproduced. In general, the reputation of osmic acid methods as
carpricious and erratic is due to the slow penetration of tissues by
osmium tetroxide, with the result that the cells in a block vary in ex-
posure to the unreduced reagent because of their varying distances from
the free surfaces. Another source of difficulty is the fact that the com-
position of cytoplasmic granules changes during the growth of that
granule (Kedrowsky, 193le; Mast and Doyle, 1935a, 1935b; Mac-
Lennan, 1936), and it is to be expected that the reducing power will
also vary. The Protozoa are admirably adapted to solve both of these

140 CYTOPLASMIC INCLUSIONS

difficulties. The individual cells are either separate or in such small
aggregates that all cells and all parts within the cell are exposed to
essentially the same concentration of reagents. The variations in the
stage of the Golgi granules can be observed directly in living Protozoa,
so that the impregnation of the granules can be compared at equivalent
stages, which can be identified with or without impregnation. In meta-
zoan cells the Golgi nets are extremely difficult to demonstrate in the
normal living cell, so that it is at present impossible in these cells to
check the structures independently of impregnation. When standard
conditions are achieved both around the cell and within the cell, abso-
lutely consistent results are attained, even with osmic acid (MacLennan,
1940). Under these conditions, 100 percent of the digestive granules,
fatty acid granules, excretory granules, and so forth, impregnate, with
the result that these types cannot be separated on the basis of the Golgi-
type reactions. The results of Hall and Nigrelli (1937), which seem
to show that excretory granules and digestive granules are erratic in
impregnation, is due to failure to allow for changes in composition and
aggregation or for the occasional lack of these granules at certain times
in the life cycle. All the stages have been lumped together, rather than
equivalent stages compared.

In view of the fact that osmic acid is reduced by many different sub-
stances, it is highly important to prove that the impregnated bodies
are not some other component (Hirschler, 1927; Bowen, 1928). Re-
sistance to bleaching by turpentine or hydrogen peroxide is more pro-
nounced in Golgi bodies than in most mitochondria, but unfortunately,
even with this method, there are too many border-line cases in which
the individual judgment of the observer is the determining factor.
However, this individual judgment can be eliminated in comparing
Golgi bodies and mitochondria by the use of such methods as the Alt-
mann aniline acid fuchsin stain after osmic impregnation. Such a com-
parison of Golgi bodies and segregation granules is not possible, since
the major criteria of the one is a fixation method and of the other
is a vital staining method. If the two components have a different
distribution, a comparison of different cells is sufficient to establish the
difference (MacLennan, 1933), but too often there is in both cases
merely a random distribution. The most famous case of this sort is that
of the gregarines, in which Joyet-Lavergne (1926b) described the Golgi

CYTOPLASMIC INCLUSIONS 141

bodies as being identical with the neutral red bodies because of simt-
larity in form and distribution, and this has been widely accepted in
spite of objections presented by Tuzet (1931) and Subramaniam and
Ganapati (1938). The question was not settled conclusively until
Daniels (1938) applied the centrifuge to these species. She found
that Golgi bodies always moved to the centripetal pole of the cell, but

Figures 39-45. Dictyosomes: Figure 39, Dictyosomes from Haptophrya michiganensis,
Champy-osmic (after Bush, 1934); Figure 40, diagram of a dividing dictyosome of
Lecudina brasili (after Subramanian and Ganapati, 1938); Figure 41, stages in the
secretion of neutral fat in Ichthyophthirius multifiliis, Lorrain Smith Nile blue-sulphate
method, black represents blue stain, stippling represents pink (after MacLennan, 1934) ;
Figures 42-45, dictyosomes during the life cycle of Lecudina brasili; Figures 42 and 43,
intracellular stages; Figure 44, growing trophozoite; Figure 45, ‘‘association’”’ stage,
either daFano or Nassonow impregnations (after Subramanian and Ganapati, 1938).

that the bodies stainable with neutral red were never displaced. Thus
in this case the Golgi bodies and the neutral red granules are not
identical. This does not mean that no osmiophilic granules segregate
neutral red, since, for example, the digestive granules react to both
impregnation and vital staining, but it does mean that the ability to
segregate neutral red is not a characteristic of all the Golgi bodies of
the Protozoa.

142 CYTOPLASMIC INCLUSIONS

The theory that the Golgi apparatus is a universal organoid of the
cell, as constant in its characteristics as is the nucleus, has given rise
to a series of criteria requiring permanence during the whole cycle of
the cell, as well as similarity in form and intracellular distribution. The
presence of Golgi bodies in all stages of the life cycle has been demon-
strated in Sporozoa (Joyet-Lavergne, 1926a) as well as their origin
by the division of preéxisting Golgi bodies (Subramaniam and Gana-
pati, 1938, Figs. 42-45). However, this is not universal, since neither

Figures 46-47. The effect of centrifuging upon the distribution of cytoplasmic
granules. Figure 46, diagram of a centrifuged gregarine, F, fat, GAa granular Golgi
material, GAb larger Goli elements, N nucleus, K karyosome, M mitochondria, P
paraglycogen, C ‘“‘chromidia,” neutral red bodies not shown; the paraglycogen mass
marks the centrifugal pole. (From Daniels, 1938.) Figure 47, contractile vacuole of
a centrifuged amoeba, v vacuole, b mitochondria; the mitochondria are thrown to the
centrifugal surface of the vacuole. (From Mast and Doyle, 1935b.)

of the types of Golgi bodies in Ichthyophthirius (MacLennan, 1936)
nor Amoeba (Mast and Doyle, 1935a) are self-perpetuating or even
present in all stages of the cycle, but arise de novo. Thus the Golgi
bodies are not universally self-perpetuating and permanent.

The criterion of similarity in form has received considerable support,
but the evidence as to what this form is has been discordant. Nassonov
(1924) described a net-like structure around contractile vacuoles and
homologized this with the Golgi net. Hirschler (1927) finds that the
typical Golgi bodies have an osmiophil cortex and an osmiophobe
center, this duplex structure being called a dictyosome. The complex
nets around the contractile vacuoles are, according to Hirschler, aggre-

CYTOPLASMIC INCLUSIONS 143

gations of the dictyosomes. Subramaniam and Ganapati (1938) insist
on this dictyosome structure, although they describe a homogeneous
spherule in one stage of the Golgi cycle:

a Golgi granule when it enlarges becomes differentiated into a vesicle having
chromophile and chromophobic regions. Rupture of the vesicle gives rise to
batonettes in which the chromophobic part is in relation with the cytoplasm.

Since all of these structures—spherules, dictyosomes, aggregations—may
be included in the cycle of a single granule (Kedrowsky, 1931; MacLen-
nan, 1936; Mast and Doyle, 1935a) or as the result of the periodic ag-
gregration of granules (Hirschler, 1927; MacLennan, OZ 33) nites
impossible to insist on one type to the exclusion of the others, and we
must conclude that morphology is not a criterion for the identification
of various types of granules.

Similarity in distribution within the cell has also been urged as a
criterion. Hall (1931) holds that a random distribution throughout the
cytoplasm is the typical configuration, while Subramaniam and Gopala-
Aiyar (1937) consider that an excentric juxtanuclear position, similar
to that found in spermatids or gland cells, is typical. In metazoan cells
both types have been found, and both may be typical of the same cell if
all stages of the life cycle are considered. Similar periodic aggregations
and dispersal of Golgi granules have been described in connection with
the pulsatory cycle of the contractile vacuoles of ciliates (MacLennan,
1933, 1936). These movements are comparable to the migration of
mitochondria and of digestive granules, which have likewise been as-
sociated with functional changes in the cell.

The chief characteristic either of form or of distribution of Golgi
bodies is a variability which is associated with functional changes, and
even this variability is not a criterion since it is characteristic of all gran-
ules which are actively concerned with the metabolism of the cell.

The criteria based upon impregnation and upon separation from other
granules which can be identified by more specific methods are the only
truly objective criteria, while the criteria of universality in form, dis-
tribution, and permanence are indefinite and are based upon various
theories of the function, form, or derivation of Golgi bodies. The term
Golgi body, being based upon nonspecific criteria (nor would retention
of specific form or distribution make the definition more exact), in-

144 CYTOPLASMIC INCLUSIONS

cludes a heterogeneous group of structures, including scattered endo-
plasmic granules and granules associated with contractile vacuoles or
fused to form heavy and permanent vacuolar membranes. A summaty
of the known functions of these granules includes the excretion of ma-
terials through the contractile vacuoles, the formation of granules of
neutral fat, the storage of lipoids other than neutral fat, a secretory cycle
which does not involve lipoids, as well as many functions as yet unknown.
These functions are different, but in a broad sense they are all varieties of
secretion and in this respect conform to the Nassonov-Bowen theory of
the relationship between Golgi bodies and secretion. The restriction of
criteria for Golgi bodies to that of impregnation alone thus does not do
violence to the concept of Golgi bodies as originally developed in verte-
brate tissues.

The Golgi bodies are simply those secretory bodies (exclusive of
mitochondria and segregation granules) which synthesize or store ma-
terials which can be preserved by Golgi-type fixatives, and after this
treatment are able to reduce (or adsorb the reduced metal) OsO, or
silver nitrate. This is not a natural grouping, since on the one hand it
includes several specific types of secretion, and on the other hand it does
not include all types. In several cases the Golgi bodies can be classified
according to function or composition, and in this discussion they are
referred to as excretory granules, intermediate lipoid bodies (see p.
151), and, when the bodies aré secretory in nature but the type of
secretion body unknown, secretory Golgi bodies. This leaves a miscel-
laneous group of Golgi bodies which are known only by their ability
to reduce osmium or silver and for which there is no evidence as to
either composition or function. Since the impregnation reactions them-
selves do not reveal homologies which must be based on composition
and function, the retention of the term Golgi body is merely a con-
venience to bridge the change from reliance on the nonspecific osmic
techniques alone to reliance upon specific cytochemical and physiological
criteria.

EXCRETORY GRANULES

Ramon y Cajal (1903-4) was the first to suggest that the contractile
vacuole is equivalent to the Golgi reticulum of the cells of the Metazoa.
The first confirmation of this view was the demonstration in several
ciliates by Nassonov (1924) that this vacuolar region is osmiophilic.

CYTOPLASMIC INCLUSIONS 145

Further work has extended the number of such cases in ciliates and
flagellates, but at the same time it has been definitely proved that the
vacuoles in several species of both classes are never osmiophilic. An
examination of these cases shows that they form a closely graded series
ranging from no impregnation at any time to complete impregnation at
all times. Fabrea has no ectoplasmic Golgi bodies and no osmiophilic
contractile vacuoles (Ellis, 1937). Lechriopyla has many ectoplasmic
Golgi bodies, but they never form an aggregation around the contractile
vacuole (Lynch, 1930). Epidinium, Eudiplodinium (Fig. 54), and oth-
ets show an accumulation of granules only during diastole (Kra-
scheninnikow, 1929; MacLennan, 1933). This same type is found in
Ichthyophthirius in the parasitic stages (Fig. 48), but neither free osmio-
philic ectoplasmic granules nor accumulations around the contractile
vacuoles during encystment (MacLennan, 1936) are present. Me/a-
dinium (Fig. 55) has a permanent granular nephridioplasm which
waxes and wanes during the pulsatory cycle (MacLennan, 1933). Para-
mecium caudatum and P. nephridiatum have a permanent osmiophilic
shell around the radiating canals, but not around the contractile vacuole
itself (Nassonov, 1924; von Gelei, 1928). Haptophrya possesses a vacuo-
lar apparatus which consists of a permanent, homogeneous osmiophilic
tube (Bush, 1934). This nicely graded series shows that the impregna-
tion of parts of the vacuolar apparatus is due to aggregations of osmio-
philic granules around the fluid vacuoles and their membranes. The
only cases in which the membranes themselves impregnate are those ex-
tremely specialized cases in which the osmiophilic material forms a
permanent shell around the fluid vacuole. The highly complex osmio-
philic apparatus in either Paramecium or Haptophrya is fundamentally
no different from those vacuoles with a granular layer, and the homo-
geneous osmiophilic shells are merely the result of the aggregation and
specialization of the ordinary undifferentiated ectoplasmic Golgi bodies.
This view is similar to that of Nassonoy (1924) and Hirschler (1927),
except that these authors view the osmiophilic portion as the outer por-
tion of the dictyosomes, and the fluid vacuole as the inner portion of the
dictyosome structure. Both views are, of course, the same, if the osmio-
phobic portion of the dictyosome be accepted as a secretion droplet and
not as an essential part of the dictyosome itself, as held by Gatenby and
Subramaniam and Ganapati.

146 CYTOPLASMIC INCLUSIONS

The demonstration that the osmiophilic granules are an addition to
the simple contractile vacuoles which may exist independently of the
granules, even in the same species, is a close parallel to the union of
the progastriole and digestive granules. The only difference is that no
gastriole yet discovered presents a permanent, highly developed mem-

50 Sy,

eye

Figures 48-52. Excretory granules and contractile vacuoles. Figure 48, aggregation
and disappearance of excretory granules during the pulsatory cycle, from Ichthyophthirius
multifiliis, Champyosmic impregnation (after MacLennan, 1934); Figures 49-50, from
Polyplastron multivesiculatum; Figure 49, cold impregnation; Figure 50, warm im-
pregnation (after MacLennan, 1933) ; Figure 51, from Dogielella sphaerii, Champyosmic
(after Nassonov, 1925); Figure 52, “nephridialplasm of Campanella umbellaria,
Flemming-glychémalum (after Fauré-Fremiét, 1925).

brane or a permanent granular region, as in the contractile vacuoles of
Metadinium, Paramecium, and Haptophrya.

The origin of a new vacuolar apparatus from the original structure of
the parent has been described and compared with dictyokinesis in germ
cells (Nassonov, 1924; von Gelei, 1928). The clearest case is that of
Haptophrya, in which the vacuolar apparatus is a tube extending the
full length of the ciliate. Studies of both live and fixed animals show
that this tube is permanent and that the transverse fission of the cells

CYTOPLASMIC INCLUSIONS 147

divides the tube in two parts, each of which continues to function in the
daughter cells (Bush, 1934). The only possible exception would be the
stages in which the parasite is transferred from one host to another. The
new vacuolar apparatus of Paramecium is said to arise by the multipli-
cation of canals and by the division of the whole vacuolar apparatus just
prior to fission (Nassonov, 1924). The formation of extra vacuoles has
been noted many times in living ciliates, but there is no recorded observa-
tion of the actual division in a living Paramecium, and the interpretation
of Nassanov’s figures of fixed material is susceptible to the difficulties
inherent in building any cycle from fixed material alone.

The majority of vacuolar systems, however, do not possess the thick,
permanent wall similar to that in Haptophrya, but a temporary aqueous
vacuole which certainly arises de novo (Taylor, 1923; Day, 1927; Mac-
Lennan, 1933, 1936). These vacuoles and their membranes are not
osmiophilic, the impregnation of the vacuolar system being due to the
aggregation of the osmiophilic excretory granules. The fundamental
question with respect to the origin of Golgi bodies is in these cases not
the origin of the vacuoles, but the origin of the individual excretory
granules. These granular aggregations in the Ophryoscolescidae have
been observed in living specimens (Figs. 53-55) to be ectoplasmic Golgi
bodies which migrate into the region of the vacuole during systole and
the earliest stages of diastole (MacLennan, 1933). In specimens fixed
during division of the ciliate, the newly arising vacuolar regions some-
times overlap the old ones and give the appearance of a division of the
old one as described in Didinium (von Gelei, 1938), but a study of
similar stages in living ciliates shows that they originate independently.
These granules are continually migrating toward the vacuoles and dis-
solving there, and no granules migrate outward, so the question of origin
is shifted to the granules at the time they are scattered in the ectoplasm.
No cases of division were observed, either in fixed or in living material,
at any place nor at any stage of the life cycle. This negative proof is not
entirely satisfactory, since these granules are small (0.25—0.50 ) and
a very rapid division might escape notice. This problem does not occur
in Ichthyophthirius, since all ectoplasmic granules (whether scattered or
around the vacuoles) are absent in the encysted stage (MacLennan,
1936), so that in this ciliate they must originate de novo in the young
parasites, whether or not they continue from them by division as the

148 CYTOPLASMIC INCLUSIONS

ciliate grows during the feeding stages. Persistence and genetic continu-
ity is restricted to a few very highly specialized types of vacuoles, and in
most cases there is no continuity either in whole or in part.

The osmiophilic reaction of the differentiated cytoplasm around the
contractile vacuoles has given rise to statements that this is a lipoid
structure (Nassonov, 1924; Volkonsky, 1933; Haye, 1930), and to the
interpretation of the vacuolar system as due to the accumulation of
lipoids at the vacuole-cytoplasm interface, which is similar to Parat’s
theory of the Golgi region around the vacuome. It is true that these
structures are partially destroyed by lipoid solvents, but such evidence
is not to be completely trusted and before acceptance must be corrobo-
rated by more specific methods. In Ichthyophthirius these granules are
negative to Sudan III and Nile blue sulphate. In Paramecium (unpub-
lished work) I have used Ciaccio’s long unmasking process and find only
a very faint reaction—only slightly more than in the hyaloplasm—and
negative results with Nile blue sulphate. These experiments show that
there is no concentration of lipoids, either in the excretory granules or
in the cytoplasm around the vacuoles.

The variations in impregnation during the pulsatory cycle and during
the life cycle suggest that the osmiophilic reaction is due to some reduc-
ing agent (not a lipoid) which is poured into the vacuole during di-
astole. The excretory theory of the contractile vacuole is indicated by the
name nephridialplasm (Fauré-Fremiet, 1925). The similarity between
the cytological changes during the pulsatory cycle and in the glandular
epithelium or renal tubules has been used in support of this theory (Nas-
sanov, 1924, 1925). Further support was given by the demonstration
that the osmiophilic granules dissolve in the vacuolar fluid which 1s then
discharged (MacLennan, 1933). In Amoeba the activity of the contrac-
tile vacuole is roughly proportional to the number of befa granules
around it (Mast and Doyle, 1935b). It is certain in these cases that
water plus some other material is being excreted.

The question, then, is not whether excretion in a broad sense takes
place, since water plus dissolved materials is certainly being excreted,
but what are the dissolved substances which are carried to the vacuole in
granular form? Nitrogenous excretion has often been assumed, but
Weatherby (1927, 1929) found that the vacuolar fluid extracted by an
application of the microdissection technique showed upon analysis too

Figures 53-55. Excretory granules in living ciliates. Figure 53, from Polyplastron
multivesiculatum ; Figure 54, from Eudiplodinium maggii; Figure 55, from Metadininm
medium (from MacLennan, 1933).

150 CYTOPLASMIC INCLUSIONS

little nitrogenous materials—probably not more than one percent—to
account for any significant part of the total excretion of Paramecium. The
application of these results to all Protozoa is by no means certain, since
I have shown previously (1933) that the pellicle of the Ophryoscolecidae
is highly impermeable and the only pathway for the excretion of kata-
bolic wastes is through the contractile vacuoles. Frisch (1938) has
demonstrated a similar impermeability of the pellicle in Paramecium
itself, and suggests that Weatherby’s experiments be repeated, with the
use of more delicate tests which have been devised recently.

The materials concentrated in the osmiophilic structures of the con-
tractile vacuole are not salts, since no significant accumulation is demon-
strated in the nephridialplasm by microincineration (MacLennan and
Murer, 1934). These materials may possibly be incidental in some
species, but Amoeba dies if this elimination is prevented (Mast and
Doyle, 1935b), demonstrating that these wastes are toxic and that in
this respect they have the known properties of the nitrogenous wastes of
metabolism. Evidence that these materials are the result of metabolism
is found in Ichthyophthirius (MacLennan, 1936). The vacuoles are
osmiophilic only during the active feeding phase, when large amounts of
food are being used and converted into storage bodies; but during en-
cystment, when these activities cease, the vacuoles are not osmiophilic.
The food of this protozoan consists entirely of epithelial cells, which are
largely proteins, but not more than a third of the reserves of the ciliate
are proteins, the rest being carbohydrate and fat. During the feeding
stage a large amount of the ingested protein would be deaminized to
form other reserves, with the result that much larger amounts of nitroge-
nous wastes would be formed during the feeding stage than in the en-
cysted stage.

Frisch (1938) suggests that the contractile vacuoles also function in
respiration. This function cannot be correlated with the variations in
the nephridialplasm in various Protozoa and thus, if this is a function of
the contractile vacuoles, it is probably independent of the granules which
are being considered here.

LIPOID RESERVES

The lipoid materials considered in this section include all lipoids
which are visible as granules and which are laid down during active

CYTOPLASMIC INCLUSIONS 151

feeding stages and are used during hunger or encystment. Zinger (1933)
included all sudanophil particles as lipoid reserves, but digestive gran-
ules, mitochondria, as well as other bodies respond to Sudan III because
of their lipoid content. Zinger pointed this out, for in his conclusion he
states that the sudanophil bodies are more than reserve materials. How-
ever, until more is known of the functions of the intracellular lipoids,
it is impossible to indicate accurately the boundary between reserve lipoids
and those active directly in the metabolism of the cell.

Lipoid reserves have been found in a large number of Protozoa (for
a detailed list see von Brand, 1935). Usually, if not always, these gran-
ules are in the endoplasm, either distributed at random, as in Ich-
thyophthirius (MacLennan, 1936), or concentrated at one end, as in
Anoplophrya (Eksemplarskaja, 1931). Although these visible lipoid
granules occur in many Protozoa, they are not universal. Trypanosoma
evansi lacks all lipoid reserves, a fact which is correlated with a lack of
lipase (Krijgsman, 1936). The Ophryoscolecidae and Cycloposthiidae,
noted for their tremendous glycogen reserves, have no important lipoid
reserves. Mesnilella multispiculata has no lipoid reserves, although five
other species of the same genus have many fat globules (Cheissin, 1930).

The formation of droplets of neutral fat inside a granule of fatty
acid has been demonstrated in O palina '(Kedrowsky, 1931) and Ich-
thyophthivius (MacLennan, 1936) by the Nile blue sulphate method
(Fig. 41). Since, after staining with Nile blue sulphate, very small
quantities of fatty acid dissolved in neutral fat result in an intense blue
color rather than the pink which is characteristic of pure neutral fats,
the pink stain observed in the cases above indicates that there are no free
fatty acids in the neutral fat granules, as would be expected if the fats
were synthesized on the surface of these granules. The fatty acids and
glycerine dissolved in the endoplasm are first segregated into granules,
and in these granules the neutral fat is synthesized. Then this fat is segre-
gated into the visible droplets of pure neutral fat inside the active gran-
ules. These latter granules are typical Golgi bodies (MacLennan, 1936,
1940), as indicated by the name endoplasmic Golgi bodies. However,
since these are functionally an intermediate stage in the development of
the fat reserves, the descriptive term “intermediate lipoid body’’ is more
appropriate in a functional classification.

In A. proteus the fat droplets grow in the cytoplasm without any in-

152 CYTOPLASMIC INCLUSIONS

termediate granules being visible. These cytoplasmic fat droplets are not
derived directly from fat in the food vacuoles, but the ingested fat is
absorbed as free fatty acid and glycerine, synthesized into neutral fat in
the cytoplasm and then stored as granules (Mast, 1938). Free fatty acids
were demonstrated in the food vacuoles when fat was being digested,
but none were demonstrated in the cytoplasmic fat droplets or on their
surface when they were being formed. In the cases in which this process
is not visible, either the synthesis is carried on elsewhere and the fat trans-
ported to the granules as such, or the fatty acids are never allowed to
accumulate sufficiently to show under the microscope.

The visible lipoids, i.e., those which are found in definite globules
and demonstrable by the ordinary fat-staining technique, include only
a part of the total lipoids of the cell. “The pathologists have known for
many years that the fats and fat-like substances of protoplasm are so
bound or united to proteins as to be for the most part non-recognizable
in the living or stained cell” (Heilbrunn, 1936). Besides the factors of
food and the formation of fat from other substances such as carbohy-
drate or protein, changes from bound lipoids to free globules must be
considered in any estimation of the reserve lipoids. Heilbrunn demon-
strated an increase in lipoid globules in specimens of A. proteus kept in
a dilute solution of ammoniumsalts. Three types of amoebae were found
—those which show lipoids in culture, those in which lipoid globules
appear after treatment with NH,Cl, and those in which no free lipoid
appears even after treatment. In similar experiments with Arbacia eggs,
Heilbrunn showed that the total lipoids of the protoplasm remained con-
stant; therefore the newly visible bodies are derived from bound lipoids,
not from new fat formation. Since ammonium salts in the culture medium
raise the pH of the immersed cells, the results were attributed to alka-
linization of the protoplasm. The fact that CO, bubbled in the medium
(which would tend to lower the protoplasmic pH), inhibits the forma-
tion of visible lipoids, confirms this hypothesis. Old cultures of Para-
mecium show larger amounts of fat than new cultures, although the
paramecia divide and show no ill effects (Zinger, 1933); and since such
cultures contain ammonia (Weatherby, 1927), the presence of abnormal
amounts of visible fats may be due to the resulting alkalinization of
the protoplasm.

Ultra-violet radiation causes a release of lipoid in Amoeba (Heil-

CYTOPLASMIC INCLUSIONS iS

brunn and Daugherty, 1938). The release of lipoids was greatly in-
creased by a preliminary immersion in ammonium chloride solutions.
“Further study is necessary in order to determine whether this fat re-
lease is due to a direct action of the radiation on the protein-lipoid bind-
ing or whether it may not be due indirectly to an alkalinization of the
protoplasm” (Heilbrunn and Daugherty, 1938). In the same publica-
tion it is stated that any stimulus in which localized increases in tempera-
ture occur is efficient in the release of fat. This is also shown by the
experiments of Sassuchin (1924), who compared the protoplasm of
O palina kept at room temperature with the protoplasm of those kept at
35-38° C. In the first group he found elongate mitochondria in the endo-
plasm (Kedrowsky’s endosomes), but in the heated group only fat
spherules and protein spherules, and these results were interpreted as due
to the separation of mitochondria into their two components. These latter
experiments should be repeated in individuals with little or no fat, and
in species as to which there is more agreement on the identification
of mitochondria.

In Paramecium (Zweibaum, 1921) and Stentor (Zhinkin, 1930) fat
is stored under conditions of low oxygen tension and lost when the oxy-
gen tension is restored. The rate of loss in this case is dependent upon
the temperature.

Pathological conditions are often marked by fatty degeneration in the
Protozoa. Degenerating coccidial oocysts show an increase in fat globules
(Thélohan, 1894), and in Awlacantha fatty vesicles are formed and the
nucleus is finally replaced by fatty bodies (Borgert, 1909). Individuals
of Actinophrys which show depression by a lowered division rate and
otherwise, have an abnormal number of lipoid bodies, and’ in extreme
cases show typical fatty degeneration. In the macronucleus of Paramecium
parasitism by bacteria also results in tremendous quantities of visible
lipoids in the cytoplasm and also of crystals (Fiveiskaja, 1929).

CARBOHYDRATE RESERVES

Granules containing carbohydrates are found in most Protozoa, al-
though in a few species this reserve is in a diffuse form which is pre-
cipitated as granules or irregular masses by fixation. The lack of any
carbohydrate reserve at all has been proved in only a few species, such as
Trypanosoma evansi.

154 CYTOPLASMIC INCLUSIONS

The carbohydrate reserves in Paramecium (Rammelmeyer, 1925) and
in the cysts of Bursaria (Poljansky, 1934) are probably dissolved in the
protoplasm, since they are visible in fixed specimens only as cloudy
masses, not in regular granules. Homogeneous vacuoles, granules, or
platelets visible in the living normal Protozoa are very common. They
are well known in lodamoeba and other intestinal amoebae. Large num-
bers of these granules are found in the flagellates from termites and
wood-eating roaches (Cutler, 1921; Kirby, 1932; Cleveland, 1934;
Yamasaki, 1937a). The carbohydrate granules of Stentor tend to be
localized in a peripheral sheath of the endoplasm (Zhinkin, 1930) and
just beneath the pellicle. In Arce//a these granules are embedded in the
chromidial net. In Ichthyophthireus these smaller granules are always
associated with mitochondria (MacLennan, 1936). Glycogen granules
are often associated with the parabasal bodies in flagellates (Duboscq
and Grassé, 1933).

Carbohydrate granules with definite internal structure are by no means
uncommon. The granules of Sporozoa (Fig. 60) have a cross or star-
shaped center (Joyet-Lavergne, 1926a; Daniels, 1938), the general ap-
pearance of which and ability to accumulate iodine suggest vacuoles.
Identification of a lipoid center (Erdmann, 1917) is based on insufficient
evidence and, in view of the later work quoted above, seems unlikely.
Vacuolated bodies are also found in Balantidium (Fig. 57) with the
added feature of crystals floating in some of the vacuoles (Jirovec,
1926). Two types of granules are found in D7fflzgza, small homogeneous
spherules and larger elliptical bodies with a center granule which stains
a pale blue after hematoxylin and a rim which is rose-colored after Best’s
stain (Rumjantzew, 1922).

In the Ophryoscolecidae, the granules possess a spherical center (Fig.
56) denser than the rest of the granule (MacLennan, 1934). The most
spectacular of the carbohydrate reserves are the skeletal plates of the
Cycloposthiidae, Ophryoscolecidae, and related families. The plates
themselves are probably supporting structures, but in their meshes are
platelets of the same type as the scattered cytoplasmic granules. The plate-
lets in the Cycloposthiidae (Fig. 61) are roughly spool-shaped with slen-
der strands connecting the flanges of adjacent granules (Strelkow,
1931), but in the Ophryoscolecidae (Fig. 62) the polygonal plates are
unconnected (MacLennan, 1934).

CYTOPLASMIC INCLUSIONS 153

The formation of paraglycogen bodies has been followed in only a
few cases. The bodies of Pelomyxa behave like permanent bodies with a
protein stroma and with the paraglycogen being built up or released as
the case demands (Leiner, 1924). The paraglycogen granules of Poly-
plastron are likewise independent of other formed components and are
apparently self-perpetuating (Fig. 56), since they show regular division
(MacLennan, 1934). The dense centers may be naked or, more often,
surrounded by an envelope of varying thickness. In the largest of these
compound granules, the centers are dumb-bell-shaped or double, and
in the latter the envelope also is constricted. These stages probably repre-
sent growth or utilization stages and division stages, although this was
not confirmed by following a single granule in live ciliates. The com-
plex granules of Amoeba hydroxena (Fig. 58, 59), in which a varying
number of glycogen granules are imbedded in glycoproteid (Wermel,
1925), suggests a conversion of glycogen into glycoprotein for storage
and the reversal of this process in utilization.

Some paraglycogen granules are formed in association with mito-
chondria instead of being independent bodies. The paraglycogen in
Ichthyophthirius first appears as a minute vacuole (Fig. 17) in the center
of a sphere of mitochondrial material (MacLennan, 1936). As the gran-
ules grow, this mitochondrial shell breaks into short rods fused to the
surface of the paraglycogen. The mitochondria disappear after the gran-
ule has attained full size. Joyet-Lavergne (1926b) also noticed a morpho-
logical relationship between mitochondria and paraglycogen of greg-
arines, but says “il y a la un simple rapport de contact et nous n’avons
aucune raison de suppose une intervention dans da génése du para-
glycogéne.” However, in the case of a granule in the center of an un-
broken sphere, as in Ichthyophthirius, it is difficult to list the relationship
as merely an incidental contact.

Duboscq and Grassé (1933) show that the glycogen granules of
Cryptobia helicis are not found scattered in the cytoplasm, but are formed
in close contact with or in the strands of the parabasal bodies (Fig. 65).
The glycogen is not laid down in the summer, but only in the winter, a
fact which, they point out, would explain the negative results of other
authors. This formation of glycogen by the parabasal body parallels the
secretion of protein granules by the macronucleus of ciliates—a part of
the segregation function which, in most species, is performed by isolated

Figures 56-64. Carbohydrate reserves, Figure 56, stages in the paraglycogen granules
of Polyplastron multivesiculatum, Champy-osmic impregnation followed by Sudan III in
hot paraffin (after MacLennan, 1934); Figure 57, growth of crystals in paraglycogen
granules of Balantidium elongatum, Zenkers-dahlia (after Jirovec, 1926); Figures 58-
59, glycogen droplets in a glycoproteid granule, from Amoeba hydroxena, Carnoy-Best
(after Wermel, 1925) ; Figure 60, vacuolated paraglycogen body from Sporozoa (after
Joyet-Lavergne, 1926) ; Figure 61, skeletal platelets of Cycloposthium edentatum, Lugol
(after Strelkow, 1929); Figure 62, skeletal platelets of Polyplastron multivesiculatum,
Champy-osmic-Sudan JII in hot paraffin (after MacLennan, 1934); Figures 63-64,
glycogen reserves in Trichonympha agilis, Best’s stain, 63 normal, 64 showing loss of
glycogen just before death under conditions of lowered temperature and raised oxygen
pressure (after Yamasaki, 1937).

CYTOPLASMIC INCLUSIONS 157

cytoplasmic granules, is performed in the one case by a nuclear structure
and in the other case by a neuromotor structure.

The differentiation between the various carbohydrates found in the
Protozoa is based on their staining reactions and solubility, since the
exact nature of the sugars involved in the formation of protozoan poly-
saccharides is unknown. Zhinkin (1930) and von Brand (1935) pointed
out that this is unsatisfactory and contend that no separation should be
made from glycogen until this is known. However, the differences are so
pronounced that it is convenient to retain the name paraglycogen.

Soluble glycogen as found in vertebrate liver cells is relatively rare.
The diffuse materials found in Paramecium and Bursaria are probably
of this type. The commonest carbohydrate is paraglycogen, distinguished
by Buitschli (1885) from glycogen on the basis of its relative insolubility
in water as compared with true glycogen. It is digested by ptyalin and
diastase and the sugar produced reduces Fehling’s solution. It stains a
light brown in todine and brown or brown purple in todine-sulphuric
acid or chlor-zinc-iodide. Probably all of the granular reserves of carbo-
hydrate in Protozoa are paraglycogen or some similar relatively insoluble
compound. The reserve granules of the flagellates of termites have been
identified as glycogen (Yamasaki, 1937a; Kirby, 1932); but in the re-
lated flagellates of the wood roach, since the Protozoa contain no enzyme
capable of breaking down glycogen, it has been suggested that the gran-
ules which stain with iodine consist of some other product which results
from the breakdown of cellulose (Cleveland, 1934). The material in
the platelets of the Ophryoscolecidae has been named ophryoscolecin on
the ground that it is unique in this family and is more like cellulose than
paraglycogen (Dogiel and Fedorowa, 1925). It was later identified as a
hemicellulose (Strelkow, 1929). This interpretation is based on slight
variations in solubility and color reactions, but other authors, using some
of the same methods and some different methods, were not able to find
any difference between the reactions of paraglycogen and the platelets
(Schulze, 1922, 1924, 1927; Weineck, 1931, 1934; MacLennan, 1934).
However, such arguments cannot be settled, as von Brand suggests, until
the exact structure of these polysaccharides is known, and the term para-
glycogen in this discussion is used in a rather general sense for carbo-
hydrates more insoluble in water than glycogen and differing in color
reactions from starch and cellulose.

158 CYTOPLASMIC INCLUSIONS

The presence of more than one type of material in the same granule
has been demonstrated in several cases, in spite of the relative crudity
of the cytochemical methods for the demonstration of carbohydrates. The
oval carbohydrate bodies of D7fflugza are not completely dissolved in
ptyalin, and their staining reactions suggest the presence of a glyco-
proteid (Rumjantzew, 1922). Two types of carbohydrate reserves have
been reported from Sporozoa by Dobell (1925). Chakravarty (1936)

Figure 65. The associa-
tion between glycogen and
the parabasal body in
Cryptobia helicis, winter
forms stained with iodine.
(After Duboscq and
Grassé, 1933.)

also differentiated two sets of granules by differences in the speed of
destaining after treatment in iodine. These authors refer to one set as
glycogen, the other as paraglycogen. Two types of carbohydrate have
been found in Actinosphaerium (Rumjantzew and Wermel, 1925), and
were identified as glycogen and a glycoprotein on the basis of their re-
action to Best’s and Fischer’s stains. Pelomyxa loses its paraglycogen dur-
ing prolonged starvation, but since the remnants of these granules may
be stained with haematoxylin (Leiner, 1924), it is probable that the
carbohydrate is here associated with a protein. The carbohydrate bodies

CYTOPLASMIC INCLUSIONS 159

of Amoeba hydroxena contain two different materials (Figs. 58, 59)
and on the basis of Best’s and Fischer's methods have been interpreted
as granules of glycogen embedded in glycoprotein.

The decrease in glycogen or paraglycogen during hunger or encyst-
ment, and its storage during the feeding stages, has been noted so often
that detailed descriptions of observations under controlled conditions
are rare. The glycogen in Stentor is deposited during low temperatures
and utilized at higher temperatures, and this process is accelerated by
starvation (Zhinkin, 1930). Fat, rather than glycogen, is deposited, if
the oxygen tension is lowered. Under such conditions some of the car-
bohydrate is probably converted into fat. In Trichonympha also, avail-
able food, oxygen tension, and temperature affects the amount of glyco-
gen present (Yamasaki, 1937b). The cytoplasm of this species is divided
into two parts by a fibrillar basket, which suspends the nucleus from the
anterior cone of the body and separates this portion from the rounded
posterior part in which the food vacuoles are formed. Both regions
normally contain glycogen (Fig. 63), but during starvation the glycogen
in the posterior part disappears first, the glycogen in the anterior part
then diminishes and disappears, and the death of the organism follows
shortly. Similar results are observed at high temperatures, or with oxy-
genation at room temperature. However, when the termites are oxygen-
ated at low temperatures (Fig. 64), the glycogen in the posterior por-
tion often shows little change, but the portion anterior to the nucleus
disappears rapidly. As soon as the glycogen in the corbula disappears the
protozoan dies, even though glycogen remains in the body region.
Yamasaki states that the posterior region is simply one of synthesis and
storage, while the anterior region is the region of consumption. He
concludes that defaunation by oxygen is due not only to toxicity but also
to a depletion of the glycogen available for the nucleus and motor or-
ganelles.

Trypanosoma evansi possesses no glycogen and, since it possesses no
amylase, is not able to synthesize it (Krijgsman, 1936). Other trypano-
somes do deposit glycogen, but at best it forms an insignificant reserve,
since trypanosomes may use three times their body weight in sugar in
twenty-four hours (von Brand, 1938). In this case the glycogen re-
serves of the trypanosomes are the liver glycogen of the host.

160 CYTOPLASMIC INCLUSIONS

PROTEIN RESERVES

This term is one of convenience and, as in the case of the term lipoid,
cannot be taken in a strict sense, but is used here to include, besides
true proteins, bodies which contain lipoids or carbohydrates, as well as
proteins, amino acids, nucleic acid, and so forth. Since most of the fixing
agents precipitate at least the protein portions of such granules, many
have been described, although relatively few have been identified by
acceptable microchemical methods. For this reason they have been de-
scribed under a variety of names, many of which mention incidental
staining properties. Some of the names which are most securely em-
bedded in the literature are chromidia, volutin, metachromatic granules,
basophilic granules, chromatoidal bodies, and albuminoid reserves. The
confusion in these terms is best illustrated by chromidia. This was orig-
inally used to designate chromatin bodies which are extruded into the
cytoplasm from the nucleus (Hertwig, 1902) and which have the abil-
ity to reaggregate to form new nuclei. Although this interpretation has
been disproved, the name may be retained to designate these granules
(Meyers, 1935). In other cases it is used to designate nonchromatin ma-
terial which is supposed to be extruded from the nucleus (Daniels,
1938). Other authors use it even more loosely to designate basophilic
and metachromatic cytoplasmic bodies which are secretory in nature
(Campbell, 1926). The elimination of the original meaning was due to
the improvement of both cytoplasmic and nuclear methods, accompanied
by detailed studies of life cycles. The last stronghold of this theory—
the Foraminifera—was eliminated by the tracing of the nuclear history
in live Patellina throughout the vegetative and sexual stages, with a
complete demonstration of the cycle with moving pictures (Meyers,
1935). The exclusion of chromidia in the original sense, with respect
to the cells of the Metazoa, has already been accepted (Wilson, 1928).

Many chromidia are actually mitochondria (Fauré-Fremiet, 1910)
which contain a high percentage of protein and are therefore resistant
to routine fixatives. This probably led to one revival of the chromidial
theory, according to which all cytoplasmic structures are formed from
mitochondria, which in turn originate from the nucleus as chromidia.
Alexeieff in a series of works on the Flagellata strives to prove that all cell

structures are formed at the expense of mitochondria. The latter, according to
Alexeieff, in their turn are not autonomic, as the majority of investigators

CYTOPLASMIC INCLUSIONS 161

suppose, but originate from the nucleus as chromidia. . . . In cases where
the autonomy of the mitochondria and of the blepharoplast is indisputable,
this author always attributes to them a nuclear origin though phylogenetic.

After this summary of Alexeieff’s theory, Milovidov (1932) rejects it.
Certain cytoplasmic granules of Uroleptus are derived from the nuclei
during reorganization (Calkins, 1930), and these granules were de-
scribed as mitochondria, but since they do not stain with janus green
they do not seem to be typical mitochondria.

The term chromidia, as now accepted, includes cytoplasmic granules
supposed to be derived from the nucleus (but not necessarily chromatin),
particularly in the Sporozoa. It also includes granules in the rhizopods,
at one time supposed to be examples of the chromidial theory, but now
retained without any such implication.

The chromidial net, characteristic of many of the rhizopods with shells,
is a definite morphological entity which may be recognized independently
of particular staining methods. The net itself is negative to Feulgen’s
stain in Arcella and Chlamydophrys, either with or without hydrolysis,
and is digested more rapidly than the nucleus by pepsin or trypsin
(Reichenow, 1928). Since the net in D/fflugia gives a positive reaction
with Ciaccio’s lipoid method (Rumjantzew, 1922), it probably has a
lipoid component in addition to the protein component in this species.
Although basophilic, it is not directly related to the nuclear material.
On the other hand, the net of Patellina is positive to Feulgen’s method,
but complete studies show that it is independent of the nuclei (Meyers,
1935). In both cases the net is a specialized mass of reserve protein, and
within it may be found two other types of reserve, volutin and glycogen
granules. This is not true in all species, since no glycogen is found in the
net of Difflugia (Rumjantzew, 1922). The chromidia of gregarines are
similar in ordinary staining reactions to the karyosome and to the pro-
tein reserves (Daniels, 1938).

The chromidia of several Sporozoa (Joyet-Lavergne, 1926a) are posi-
tive to Millon’s reagent and are therefore certainly protein and they
appear to be associated with mitochondria. In gregarines from meal-
worms, on the other hand, these granules are negative to both Millon’s
reagent and Feulgen’s reagent, and show no morphological relationship
with mitochondria (Daniels, 1938). Daniels found chromidia and volu-
tin similar in shape, distribution, and so forth, but found fewer black

162 CYTOPLASMIC INCLUSIONS

granules in haematoxylin preparations than blue granules after the
methylene blue method. She concludes that they are separate types of
granules, although there is a close relationship. Joyet-Lavergne was not
able to decide whether chromidia and volutin are really separate.

Daniels observed buds on the surface of the karyosome, then bodies
in the nuclear sap, and finally in the cytoplasm near the nucleus, but she
found no direct evidence as to how they penetrate the nuclear mem-
brane. On the basis of these suggestive observations, she concludes that
these bodies are derived from the karyosome. With regard to the validity
of this conclusion, the comment of Wilson (1928, p. 96) with regard
to a similar case in oogenesis is highly pertinent: ‘“To the writer none of
these cases yet seems to be satisfactorily demonstrated, and the question
is a most difficult one to be settled by studies on fixed material alone.”
Joyet-Lavergne (1926a) calls these protein granules albuminoid reserves,
a name far more appropriate than chromidia, which at least implies a
nuclear origin.

Volutin granules are basophilic granules which are also metachromat-
ic. Because of their pronounced basophilia, volutin granules have often
been linked with chromatin. However, they are negative to Feulgen’s
stain after hydrolysis, but give a positive reaction when the preliminary
hydrolysis is omitted (Reichenow, 1928), a characteristic of free nu-
cleic acid. The full Feulgen reaction apparently dissolves this type of
volutin granule, so that in Arcella there results a diffuse Feulgen reac-
tion in the chromidial net. This is a possible explanation of the positive
Feulgen test by the chromidial net of Patellina. The volutin granules of
Trypanosoma melophagium contain no nucleic acid (van Thiel, 1925),
while those of T. equinum do (Reichenow, 1928). The volutin bodies
of T. evansi were not tested in this respect (Krijgsman, 1936), although
they are listed as containing nucleic acid. Since reserve bodies are not
the same in all species of the genus (some trypanosomes are able to
store glycogen while others are not, according to von Brand, 1938),
both analyses of the basophilic granules may be correct.

Volutin granules increase and nuclear granules decrease in trypano-
somes which have been treated with atoxyl (Swellengrebel, 1908). This
fact in conjunction with the staining reactions of volutin, were inter-
preted as indicating a direct nuclear origin—in other words, a type of
chromidia. In these experiments the results are probably a degeneration

CYTOPLASMIC INCLUSIONS 163

phenomenon, since in Pelomyxa the expulsion of chromatin into the
cytoplasm is found just prior to death (Schirch, 1914). Hindle (1910)
thought of this as a degeneration phenomenon in T. gambiense. In
various phytoflagellates division stops when volutin is lost, and the volu-
tin was interpreted as a nuclear reserve (Reichenow, 1928), although no
direct connection between the two was demonstrated. A dehydrase has
been demonstrated in Trypanosoma by the leucomethylene blue method
and localized in the volutin granules (Krijgsman, 1936). Krijgsman,
however, holds to Reichenow’s views of volutin as a nuclear reserve.

Protein bodies in Oxymonas dimorpha, which are negative to Feul-
gen’s stain and stain with either basic or acid dyes (i.e., not metachromat-
ic), have been called volutin granules (Connell, 1930), although they
are not volutin in the sense used by Reichenow. However, in Oxymzonas,
as in the phytoflagellates of Reichenow’s experiments, division ceases
when these granules are exhausted. Since the division stages of Oxymonas
are also the flagellated stage, the protein granules could be explained
as reserve bodies for the expenditure of energy by these organelles.
Neither explanation has adequate proof, since each merely correlates
obvious phenomena.

Volutin is thus a term which has no standard usage, but wherever
microchemical tests have been made volutin has been found to contain
proteins, nucleic acid, or other similar materials. Since the available evi-
dence shows that it behaves as a reserve material, it seems to me to be
convenient to include it as one of the various types of protein reserves
and to eliminate the terms volutin and metachromatin, neither of which
seems to have been used consistently by protozodlogists.

The macronuclei of many ciliates contain one or more large, intensely
basophilic bodies lodged in vacuoles among the closely packed granules of
chromatin (Chakravarty, 1936; MacLennan, 1936). Since their num-
ber and size vary, it has been suggested that these are reserve materials
(Kazancev, 1928). In Ichthyophthirius these granules have been traced
in living ciliates from the macronucleus through temporary breaks in the
macronuclear membrane into the cytoplasm (Fig. 66) where they are
stored until resorption and utilization occurs in the encysted stages (Mac-
Lennan, 1936). These bodies take both acid and basic dyes even more
strongly than chromatin and, unlike the chromatin, are negative to
Feulgen’s reaction and Macallum’s tests for iron. These granules first

164 CYTOPLASMIC INCLUSIONS

appear as minute bodies at the lower limits of visibility, embedded in
the chromatin net during the feeding stages of the ciliate but not during
encystment. The macronucleus is positive to Feulgen’s reagent without
preliminary hydrolysis during the formation of these granules, but at
no other time. It seems probable that food materials are built up in the
macronucleus into chromatin, which is then split into a group contain-
ing iron and nucleic acid and another protein group which lacks these
substances. The first group is used to rebuild more chromatin and the
latter group is segregated into the granules which are ejected into the

Figure 66. The forma-
tion and release of protein
granules from the macro-
nucleus of Ichthyophthi-
rius multifiliis, Feulgen-
light green. (After Mac-
Lennan, 1936.)

cytoplasm. Since granules of this type are found in both the macto-
nucleus and the cytoplasm of so many ciliates, this is probably quite a
general phenomenon. The Protociliata lack macronuclei, but perform
this same function by the segregation apparatus.

The balls of chromatin and other macronuclear fragments which are
extruded during the various types of macronuclear reorganization are
only incidentally reserve material, if at all, and will be considered in
detail in the chapters on nuclear phenomena.

The crystals which are common in various Protozoa are often con-
sidered to be excretory products, and in some cases have been identified
as uric acid (fora discussion of this work, see Reichenow, 1929). Recent
work (Mast and Doyle, 1935b) shows, however, that some of the crystals
must be regarded as reserve material. A. proteus contains two types of
crystals, a bipyramidal type and a plate-like type, which are suspended
in vacuoles containing an alkaline fluid. A careful study of spectroscopic

CYTOPLASMIC INCLUSIONS 165

analysis, solubility, and form shows that the bipyramidal type probably
consists of a magnesium salt of a substituted glycine. The plate-like
crystals are insoluble in a saturated solution of leucine, and in structure
resemble leucine crystals. If the crystals are removed by centrifuging
and the Amoeba is then put in a solution which contains amino acids
and egg albumin, the platelets are formed in the vacuoles which con-
tain leucine, while the bipyramidal crystals are formed in all solutions.
“Crystals are normally formed from amino acids derived from food dur-
ing digestion” (Mast and Doyle, 1935b). These crystals decrease in
number just before the refractive bodies increase in number, indicating
that the crystals are an intermediate stage in the transfer of food from
the food vacuoles to the lipoprotein refractive bodies.

The chromatoidal bodies of various parasitic amoebae, are intensely
basophilic structures, the fixing and staining reactions of which suggest
a protein composition. They possess neither chromatin nor free nucleic
acid (Reichenow, 1928), so are not volutin; but they are similar in
their reactions to the protein bodies of ciliates. Since they disappear dur-
ing encystment they are reserve bodies.

The reserve proteins are often found in combination with other ma-
terials. In Amoeba, lipoids and proteins are bound together in the re-
fractive bodies (Mast and Doyle, 1935a). In Actinosphaerium, granules
of glycoproteid are present (Rumjantzew and Wermel, 1925). Similar
inclusions are found in Ophryoglena (Zinger, 1928). In the Foet-
tingeriidae the protein reserves have the characteristics of the vitellin of
the hen’s egg and in Polyspira there is a single central mass consisting of
protein associated with a carotenoid (Chatton, Parat, and Lwoff, 1927).
The protein portion alone is used, the carotenoid remaining in the old
cyst, and finally disappearing during encystment.

The protein bodies which are found throughout the Protozoa vary
greatly in their specific structure and composition. This variation, with
the resulting variation in staining reactions, has resulted in a complicated
nomenclature with the usage of terms proposed by each author. The
term chromidia is so definitely bound up with disproved theories that
it should be dropped. Volutin should either be dropped or definitely
restricted to metachromatic granules which respond to Feulgen’s stain
when used without hydrolysis. Whenever the function is that of a re-
serve, as in the majority of known cases, I believe these granules should

166 CYTOPLASMIC INCLUSIONS

be called simply protein reserve bodies, a usage found convenient by
Joyet-Lavergne (1926a). At the same time, it should be recognized that
a reserve function is the one most easily identified by morphological
methods, and that other functions must be investigated. The presence
of dehydrogenase (Krijgsman, 1936) in protein aie of Try pano-
soma evansi is one definite lead.

The protein reserve bodies are as catholic in origin as in structure and
may result from the activities of the segregation granules, macronucleus,
mitochondria, or food vacuoles, or may be independent of other formed
bodies. The crudity of our knowledge of cytoplasmic granules is illus-
trated by the fact that no suggestion of the significance of these differ-
ences can be made.

EXTERNAL SECRETION

This important cytological subject has been greatly neglected in the
Protozoa and is generally ignored in a discussion of the protozoan cyto-
plasm, except as the vacuolar apparatus is considered to be a secretory or-
ganelle. It is an important subject in itself and is most nearly comparable
to secretion studied in the Metazoa.

The attaching organs, or at least the cementing portion, of sessile
Protozoa are secreted structures. Just preceding the formation of the
peduncle of Campanella, granules are found in the basal region (Fauré-
Fremiet, 1905). T7ntinnopsis nucula is cemented to the lorica by a mucus
secretion which is derived from basophilic granules in the stalk (Camp-
bell, 1926).

The lorica of Favella is likewise derived from cytoplasmic granules
(Campbell, 1927). Granules which are to form the new lorica accumu-
late near the mouth, in dividing animals. After division these are forced
out through the cytostome, expand, fuse, and harden. At the same time
fecal pellets are molded into this secreted material and the whole lorica
is shaped by the activities of the motor organelles. There is in this form
a local zone of secretion, as in gland cells, not a general secretion over
the whole surface.

The shell of Exg/ypha is formed from separate shell plates, which are
secreted within the cytoplasm (Hall and Loefer, 1930). They appear
first as small refractive spheres in vacuoles, then enlarge and elongate to
become typical shell plates. (Fig. 67). The finished plates lie free in the

CYTOPLASMIC INCLUSIONS 167

cytoplasm. It was demonstrated that the reserve plates have no connection
with either the mitochondria or the neutral red globules.

The cyst of Ichthyophthirius is secreted in two parts: first a homo-
geneous clear membrane is formed (Fig. 68) and then individual
fibrils are extruded, apparently between the bases of the cilia (Mac-
Lennan, 1937). These sticky fibrils are stroked into rope-like fibers,
which adhere to the under side of the outer membrane (Fig. 69) by
the activities of the cilia. Although seven types of granules were demon-

50 eo gh
i

Figures 67-69. External secretion. Figure 67, inclusions in Evglypha alveolata, prob-
ably representing the formation of reserve shell plates (after Hall and Loefer, 1930) ;
Figures 68-69, secretion of the cyst wall in Ichthyophthirius multifiliis; Figure 68,
section of early stage showing only the homogeneous layer; Figure 69, section of
later stage showing the addition of the fibrillar layer (from MacLennan, 1936).

strated, no granules could be associated with the secretion of this cyst.
In Nyctotherus, variations in the thickness of the secreted cyst may be
correlated with the distribution of ectoplasmic structures (Rosenberg,
1937), but no granules responsible for the secretion were noted in this
form either. The lorica of Folliculina ampulla is likewise secreted in the
form of a clear fluid, which hardens to form a membrane just beyond
the tips of the somatic cilia (Fauré-Fremiet, 1932).

The secretion of vacuoles of oxygen in Arcel/a is not associated with
granules, but with nongranular regions of hyaloplasm, and is probably
a result of oxidative and reductive processes of the cell (Bles, 1929).

The most striking aspect of these examples of protozoan secretion is

168 CYTOPLASMIC INCLUSIONS

that none of them has been traced to Golgi bodies, mitochondria, or
segregation bodies—1.e., they do not react with osmic acid, Janus green
B, nor neutral red. It is evident that not all important segregation nor
synthesis is revealed by these stains.

The mucus granules of Evglena (Dangéard, 1928) and the pellicular
secretions of Vorticella (Finley, 1934) stain with neutral red and thus
might be classed the segregation granules of Opalina and others, the
only difference being that in the former is segregated mucus, which is
not used within the cell but is extruded in the normal functioning of
the protozodn, while in the latter are segregated proteins, which are
normally used within the cell. It is interesting, however, that Kedrowsky
found that when the segregation granules were filled with foreign ma-
terials, such as the organic silver compounds, the granules are extruded.
The expulsion of droplets containing neutral red may be induced in
Paramecium (Frisch, 1938) and other ciliates. These examples indicate
that the formation of the segregation granules and the secretion granules
is comparable, the only difference being that in one the material is used
internally and in the other externally.

THE GRANULAR COMPLEX

The detailed consideration of each of the types of cytoplasmic gran-
ules has resulted in the conclusion that there are no universal cyto-
plasmic components and that each of the terms mitochondria, Golgi
bodies, neutral red granules, and so forth has been applied to a hetero-
geneous assortment of granules of widely different functions. This con-
clusion, derived from a consideration of the types of granules separately,
becomes inescapable if we consider the whole granular complex. The
problem is on the surface one of classification, but fundamentally it 1s
one of function—what functions are performed by cytoplasmic gran-
ules, and is the same function always performed by the same type of
granule in different Protozoa? Since these granules are not independent
units but are part of a granular complex which in turn is a part of
the whole cell, this whole complex must be considered in seeking an
answer to these problems of function. The investigations which seem to
be suitable for this comparison are those of Mast and Doyle (1935a,
1935b), Holter and Kopac (1937), Holter and Doyle (1938), all on
Amoeba proteus; Hopkins (1938a, 1938b) on Flabellula mira; Ked-

CYTOPLASMIC INCLUSIONS 169

rowsky (1931-33) on Opalina ranarum; MacLennan (1936, 1937) on
Ichthyophthirius multifiliis; and Joyet-Lavergne (1926 on) on several
Sporozoa, supplemented by the observations and experiments of Daniels
(1938) on similar species. Since a summary of the individual granules
has been given in the previous sections, only a general account of each
granule will be given in this comparison.

The number of types of granules ranges from two in the marine
amoeba Flabellula to at least six in Ichthyophthirius and some of the
Sporozoa. In Flabellula there are only digestive granules and small gran-
ules of unknown composition and function. This small number contrasts
sharply with A. protews, which has four types of granules of cytoplasmic
origin: refractive bodies (dictyosomes), alpha granules unknown in
composition and function, mitochondria (beta granules), and neutral
fat granules. In addition to these granules there are two types of crystals,
blebs on these crystals, and vacuole refractive bodies, all of which arise
in connection with the food vacuole. The two ciliates Opalina and Ich-
thyo phthirius also show marked differences in number of granules—the
former with only four types and the latter with seven. O palina has segre-
gation bodies, endosomes (mitochondria?), intermediate lipoid bodies
(endoplasmic Golgi), and neutral fat, while Ichthyophthirius has inter-
mediate lipoid bodies (endoplasmic Golgi), neutral fat, excretory gran-
ules (ectoplasmic Golgi), mitochondria, paraglycogen, and protein
bodies. Gregarines and Coccidia have at least one type of Golgi body,
neutral fat, one or two types of mitochondria, paraglycogen, one or two
types of protein reserves, and neutral red bodies, a total of six to eight
types of granules, allowing for differences in the accounts of Daniels and
Joyet-Lavergne. Not all the Sporozoa present such a complicated picture,
since there are probably not more than three types of granules in Plasmo-
dium: mitochondria, segregation granules, and pigment. These marked
differences, which appear even with a crude comparison based only on
number of types of granules, show clearly that at best only very few gran-
ules could be universal. Furthermore, the number of granules varies in-
dependently of the relationships of the Protozoa involved, since both large
and small numbers of granules are found in Protozoa of the same class.

The immediate facts which stand out with respect to the staining re-
actions of these five species of Protozoa are that in each species are
granules or vacuoles which are stained specifically by Janus green B

170 CYTOPLASMIC INCLUSIONS

and other mitochondrial stains, in each are granules which segregate
neutral red, and in each, with the exception of Flabellula, are granules
which may be specifically impregnated by the Golgi methods. If we do
not press the comparison any further, we can say that chondriome and
vacuome are universal cell constituents but, even with the very crude
definition based on impregnation alone, Golgi bodies are lacking in one
of the five Protozoa discussed. However, this apparent uniformity
is reduced if these granules are compared with respect to other character-
istics than the so-called specific staining methods, which are in reality
quite crude in spite of brilliant contrasts. With respect to mitochondria,
the bodies range from one extreme of temporary induced granules in
Flabellula to two separate types in some of the Sporozoa. The neutral
red bodies are even less comparable: in Ichthyophthirius they are lipoid-
containing bodies, which are found only in association with gastrioles.
In Opalina they range from watery vacuoles to dense bodies of proteins
and are obviously not connected with any gastrioles. In Flabellula they
are vacuoles in which is also dissolved the material stainable with Janus
green. In Amoeba there are two types of neutral red bodies which are
of cytoplasmic origin, and both contain large amounts of lipoids and at
some stages proteins as well. One of these types, the refractive bodies,
are apparently built up from material derived from the blebs and the
crystals. In a structural sense also the refractive bodies are unique and
are much more complex than any of the other neutral red bodies. In
the Sporozoa, lipoid dictyosomes are weakly stainable with neutral red,
but in addition there are non-lipoid bodies which stain much more
specifically with neutral red. With respect to Golgi bodies, even if we
ignore Flabellula, harmony is not achieved. In Amoeba the two types
of neutral red bodies, as well as the mitochondria, respond to impregna-
tion and bleach with difficulty. A comparison between these lipoid bodies
and the endoplasmic Golgi bodies (intermediate lipoid bodies) of
Ichthyophthirius, O palina, and the dictyosomes of Sporozoa seems quite
logical, until we consider that in these latter three species, these fatty
acid bodies are an intermediate step in the formation of neutral fat,
while the refractive bodies of Amoeba are finished bodies, the granules
of neutral fat being morphologically independent of them. None of these
lipoid bodies is comparable with the non-lipoidal ectoplasmic Golgi

CYTOPLASMIC INCLUSIONS nA

bodies (excretory Golgi) around the contractile vacuoles of Ichthy-
ophthirius.

These comparisons demonstrate that with respect to actual composition
not even one single type of granule is found throughout these five species
of Protozoa, and the apparent universal presence of certain types of
granules is due to the lack of specificity in Janus green, neutral red,
osmic acid, and other stains. This is demonstrated in spite of the fact
that the known composition of these bodies can be stated only in
qualitative terms which are actually very broad—t.e., lipoid, non-lipoid,
protein, and so forth.

A comparison on the basis of composition alone is open to criticism
if it is not checked from other angles. The segregation apparatus of
O palina may be aqueous vacuoles, dense protein bodies, or it may contain
bile pigments, depending on the medium and the temperature. Using
composition alone as a criterion (or the neutral red reaction, for that
matter), these granules would be separately classified, actually Ke-
drowsky showed they are the same granules with the same function—
segregation and synthesis. In this case the stain reactions and the compo-
sition are incidental, and they are important in the classification of the
segregation apparatus only if they can be used to reveal the function.
Can groups of granules be demonstrated if the granules are compared,
not on the basis of structure nor of composition, but on function? If so,
are any of these groups represented in all of these five species which
we are considering? This comparison cannot be in any way as complete as
the comparison based on staining reactions and composition, since for
the most part this knowledge is restricted to those functions which have
a definite morphological expression—digestion, storage, external secre-
tion, and so forth. The apparent emphasis on these functions should not
be considered as an implication that these are the only functions in which
the cytoplasmic granules may play a rdle, but as an inverse expression
of the difficulties of localizing functions which do not produce visible
structures.

The segregation and storage of protein reserves is obvious morpho-
logically and the materials which are stored can be identified by adequate
cytochemical methods. All the five Protozoa, again with the exception
of Flabellula, have visible stores of proteins or derivative substances.

i/2 CYTOPLASMIC INCLUSIONS

A. proteus forms leucine and glycine crystals in the gastrioles (they are
therefore not strictly speaking cytoplasmic bodies), and these are then
separated from the gastriole and the materials are transported from the
crystal vacuoles to the growing refractive bodies and are there stored
in the form of the protein stroma of these lipoid-protein bodies. In
Opalina proteins are stored in the ectoplasmic segregation bodies. In
both these cases, the final structures are of cytoplasmic origin and al-
though they are so different they could perhaps be harmonized on a
functional basis. In Ichthyophthirius, on the other hand, the protein
spherules are stored and utilized in the cytoplasm, but originate in the
macronucleus by splitting from the chromatin a portion which contains
nucleic acid and iron, leaving a reserve protein in the form of large gran-
ules which are then discharged as completed bodies into the cytoplasm.
There is some evidence in the Sporozoa also of a nuclear origin of some
of the protein reserves, although it is entirely possible that they are
connected with mitochondria, since Joyet-Lavergne noted a morpho-
logical relationship between the two in the Sporozoa he studied. How-
ever, even if we disregard the somewhat questionable case of the Sporo-
zoa, we find that an identical function—the storage in the cytoplasm of
proteins—is accomplished in two cases by cytoplasmic structures, but in
a third case by the macronucleus.

Digestion, except in the astomatous species, is accomplished by the
gastriole, a structure formed by the union of a vacuole which contains
the food particles with granules or vacuoles of cytoplasmic origin. In
Ichthyophthirius the granules involved are cytoplasmic in origin, but
become enclosed within the vacuole; the cytoplasmic vacuoles of Fla-
bellula apparently furnish the fluid in the gastriole; while in A. proteus
the granules merely aggregate around the gastriole. From morphological
evidence, the granules in Ichthyophthirius and Flabellula are concerned
with all types of digestion, but in A. proteus both morphological and mi-
croenzymatic studies show that the mitochondria are concerned with the
digestion of carbohydrates and with the transport of digested materials
from the gastriole to such bodies as the refractive granules. There 1s
thus some variation in the digestive function, but clearer evidence of
differences is the fact that in both Ichthyophthirius and Flabellula the
diffusion of materials outward from the vacuole is accomplished without
the intervention of any visible granules. The granules of Amoeba, there-

CYTOPLASMIC INCLUSIONS 173

fore, have a transport function, but none of the granules of either
Ichthyophthirius or Flabellula give evidence of a relationship with the
gastriole which would permit such a function.

The storage of lipoids is accomplished in almost identical fashion in
O palina, Ichthyophthirius, and Gregarina by the formation of inter-
mediate lipoid bodies which are converted into neutral fats. A. proteus
also stores neutral fats, but in this case no visible intermediate bodies are
formed. This is not necessarily in conflict with the facts observed in the
other species, since it is very possible that the intermediate bodies of
fatty acid might be present but never get as large as the lower limits of
microscopic visibility. However, not all the visible lipoid reserves of
Amoeba are in the form of neutral fat; some are in the form of masked
lipoids in the refractive bodies, and none of the other Protozoa in this
group have granules which are strictly comparable with these complex
structures.

Carbohydrate reserves are found in the form of paraglycogen gran-
ules in Ichthyophthirius and the Sporozoa, and in both cases are secreted
by mitochondria. In O palina, glycoprotein is found in certain cases in the
segregation apparatus, but according to Kedrowsky, there are no im-
portant stores of carbohydrates in this species. For Amoeba likewise this
statement holds, the only carbohydrate being the shell between the fluid
and the lipoid-protein rim of the refractive bodies. No carbohydrate
reserves are found in Flabellula. From these cases it would appear that
the function of carbohydrate storage is largely accomplished by mito-
chondria, but it must be remembered that this is not a general rule, since
these reserves may be formed by independent bodies, as in the Ophryo-
scolecidae, or by the parabasal body, as in certain flagellates.

The vacuolar apparatus performs at least two functions—the excretion
of water to maintain the proper water balance, and the excretion of
other materials which are probably metabolic wastes. The first function
may be performed without the intervention of granules, as in the encysted
stages of Ichthyophthirius, but the excretion of other materials is accom-
panied by the periodic aggregation of granules around the vacuole in
Amoeba and Ichthyophthirius. In the former function the granules con-
form to the definitions of mitochondria, and in the latter to the Golgi
bodies. These bodies are certainly a group having to do, in Ichthyoph-
thirius, only with the contractile vacuoles; but in Amoeba they are appar-

174 CYTOPLASMIC INCLUSIONS

ently also associated with the food vacuoles, the refractive bodies, and so
forth, in a general transport function. The two groups are comparable,
but the granules of Ichthyophthirius perform only a part of the functions
assigned to them in Amoeba. It may be possible that the mitochondria
or beta granules of Amoeba include more than one type, but in view of
the detailed experiments which have been made on this form, this explan-
ation is hardly more than a possibility.

This survey of functions which have a granular basis fails to reveal
any general uniformity, even in this restricted group of five Protozoa.
The most clear-cut case of two different mechanisms having the same
function is that of the segregation of reserve protein in Ichthyophthirius
and Opalina. No matter how broad the definitions are made, the fact
remains that the identical result is attained through the mediation of two
different cellular mechanisms—in the one case the mechanism is the
macronucleus, in the other it is strictly cytoplasmic, the segregation
bodies. The general concept of transport introduced by Doyle helps in
several cases to group apparently diverse functions within a single func-
tional concept; but here again none of Kedrowsky’s published obser-
vations in the case of Opalina, nor my own observations in Ichthyoph-
thirius, would support this. The stored fat, paraglycogen, and proteins
merely decreased in size during encystment, and no intermediate bodies
aid in the redistribution of this material.

The granules which are produced in a particular species are typical of
that species, but in other species the same function may be accomplished
by granules different in composition and relationships from those of the
first species, or the same function may be accomplished without the form-
ation of visible granules. The cell is not restricted in the accomplishment

of its functions by any system of universal and invariable cytoplasmic
components.

THE CONTINUITY OF CYTOPLASMIC GRANULES

The failure to find evidence of universal cytoplasmic components by
the use of either composition or function as criteria, brings the discus-
sion to a much more general concept—the distinction between granules
as permanent organelles and as temporary reserve granules. This dis-
tinction can be traced back to Altmann’s bioblast theory, but it has been
applied more recently in a modified and refined form to the mitochondria,

CYTOPLASMIC INCLUSIONS 175

Golgi bodies, and the vacuome. The rejection of the universality of any
one of these components still leaves the possibility that in each cell there
are two sets of granules—the group which is permanent in organization,
and a group of temporary granules, usually “passive’’ reserve bodies,
which may be derived from the activities of the first. This is not a
restatement of Altmann’s theory, in the sense that it implies that the
first group are living units as such, nor even that these granules are
regarded in any strict sense as independent, since their maintenance
obviously depends upon their interaction with the other parts of the
cell. The question is whether any set of granules are present during the
whole life cycle, and further whether new granules of the same group
arise directly from the old granules and never arise de novo.

The morphological studies of Fauré-Fremiet (1910), Joyet-Lavergne
(1926a), MacLennan (1934), Subramaniam and Ganapati (1938), and
others have shown that mitochondria, Golgi bodies, glycogen granules,
and so forth, in various Protozoa, undergo division in such a manner as
to retain the original organization of the granules, and that these bodies
are found in all stages of the life cycle. On the other hand, Horning
(1929), Volkonsky (1929 on), MacLennan (1936), and Kedrowsky
(1931 on) find that one or more of the supposedly fundamental com-
ponents arise de novo either continuously or at some stage of the life
cycle. A de novo origin has been proved by Mast and Doyle (1935b) not
only for granules which are simple morphologically, but even for the
complex tripartite refractive granules of A. proteus. In Ichthyophthirius
apparently none of the granules are retained through the life cycle, thus
clearly eliminating in this protozoan any distinction based on continuity.
These morphological studies show that there may be a genetic continuity
with respect to some granules in some of the Protozoa, but that it is not
a general thing.

The observation that in some Protozoa all of the granules arise de
novo at some time or other, raises the question whether the observed divi-
sions are significant or are merely incidental. Kedrowsky was able to in-
duce typical division figures in the endosomes of O palina by altering the
culture medium. Horning (1929) showed that dividing mitochondria
are found in the trophozoite of Monocystis but that these granules dis-
appear completely during the spore stages and form de novo in the
newly liberated sporozoite. The digestive granules of Ichthyophthirius

176 CYTOPLASMIC INCLUSIONS

divide inside the gastriole to form a larger number of small granules,
and both the mitochondria and the intermediate lipoid bodies (endoplas-
mic Golgi) fracture and split into rods as the granule within the sphere
grows; yet in none of these cases is the division more than an incident
in the cycle of the original granule, which arises in all cases de novo. The
division of cytoplasmic granules is merely an indication that the granule
is unstable under the particular conditions of size, surface tension, and
so forth.

The term “‘vacuolar reaction’? was introduced by Volkonsky to de-
scribe the relationship between the formation of new digestive granules
(his vacuome) and the presence of food. The pattern of the reaction
depended both upon the species of cell and upon the type of food present.
This formation of granules as a response to a specific stimulus is by no
means restricted to the single case of the digestive granules. The forma-
tion of secretion granules in the Tintinnidae is a specific response to the
factors which require a new lorica, and these granules are present at no
other time. The formation of the complex refractive bodies in Amoeba
is a specific response of that particular protozodn to excess food; when
this condition no longer holds, the granules are resorbed. The excre-
tory granules of Ichthyophthirius are the response of this protozodn to
the presence of metabolic wastes, which result from active feeding and
growth and which disappear in encystment, when the original condition
no longer holds good. The segregation bodies of O palina likewise exhibit
changes which are specific responses to the particular food which is
available. Horning (1929) points out that the disappearance and re-
appearance of mitochondria in Monocysizs 1s correlated with the decrease
and increase of metabolism resulting from encystment and excystment.
Volkonsky’s vacuolar reaction is one case of general response, or “granu-
lar reaction,” of the cell to a host of stimuli. If the stimulus is always
present, the particular granules which are characteristic for the stimulus
and for the particular cell under consideration are always present, but if
the stimulus is intermittent, the particular granules involved are present
only for the corresponding period.

Continuity is of no significance in the evaluation of the granules, but
is rather a criterion of the continuity of the stimulus which induces the
formation of the granule. This, together with the demonstration that
the division of the granules is purely incidental, shows that it ts not possi-

CYTOPLASMIC INCLUSIONS U7

ble to distinguish between permanent organelles and temporary com-
ponents, nor between active and passive granules.

THE CLASSIFICATION OF CYTOPLASMIC GRANULES

The cytoplasmic granules are a visible reaction of the cell to various
stimuli, with the result that they show as great a variety as do the func-
tions of which they are the visible expression and as the cells which form
these granules. Any final classification must be based, then, on function,
composition, and origin, rather than on a few nonspecific stains which
give the impression of universal components, or on a distinction between
permanent organelles and temporary reserves. Since function, composi-
tion, and relationship vary widely from one cell to another, the cyto-
plasmic granules, even of the Protozoa alone, cannot be divided into
three or four sharply defined types, but must be separated into more
types, with a classification sufficiently flexible to allow for the combina-
tion of several functions in the same granule. Such an ideal classification
may be defined briefly as functional.

A functional classification is impossible at the present time, since the
usual cytological or cytochemical methods reveal only those functions
which result in the accumulation of visible masses of material—the
segregating functions. The general type of material which is segregated
has been identified in many cases, but usually there is insufficient evidence
to determine whether this is a simple segregation process or whether
there is actual synthesis involved. A functional classification on such a
narrow base would lack permanent value, but it is necessary to readjust
the present classification, in order to separate granules which are obvi-
ously unlike, even on the relatively scanty evidence now available. This
separation has been outlined in the previous sections with the detailed
evidence, but it is worthwhile to assemble these suggested changes here
in one place.

The mitochondria are those granules which respond to mitochondrial
methods, such as those employed by Regaud, Benda, and so forth, and
which usually segregate Janus green. This is admittedly a heterogeneous
group, but there is insufficient information at present to separate any
groups on a logical basis.

The term Golgi body is used to designate granules or structures which
impregnate specifically with the classic reduction methods, but excepts

178 CYTOPLASMIC INCLUSIONS

those bodies which are mitochondria, or which segregate neutral red
vitally. The term as used here is thus merely a convenient way to indicate
briefly certain techniques. Included in this group of granules are the
fatty acid bodies, which are simply a stage in the formation of neutral
fat granules and which have been called sntermediate lipoid bodies. The
granules or membranes which are associated with the contractile vacuoles
are a separate group in composition and function, and are called excre-
tory granules. There is a third group which display a characteristic secre-
tory cycle but which are neither lipoidal in composition nor excretory
in nature (Ellis, 1937). For these bodies and other unknown granules,
the term Golgi body is appropriate, since it merely designates them ac-
cording to the techniques used and implies nothing as to their composi-
tion or functions.

The term neutral red granule refers to any body which segregates
neutral red or similar basic dyes in the living normal protozo6n. Again,
it is a term which indicates only the technique used and is a convenience
when there is no evidence as to function. In this group are the segrega-
tion granules, which accumulate and perhaps synthesize proteins and
similar materials, as in Opalina. The refractive bodies of Amoeba may
be included here because of the neutral red reaction and because of the
protein stroma, or they could be listed in the lipoid reserves because of
their high lipoid content. The d/gestive granules are a separate group
and are associated with the gastriole. One of the problems in this con-
nection is how these digestive granules differ in function and composi-
tion from the mitochondria, which may also be associated with digestion.

Many different types of granules, some of them with the power to
segregate neutral red, are expelled from the cell in the formation of
shells, cysts, cement, and so forth, and are named secretion granules.
These granules should be given more attention, since they indicate a
situation similar to the secretion granules of gland cells.

The reserve bodies have been separated on the basis of the material
stored—protein, lipoid, and carbohydrate—which also allows for the
various combinations which do occur, and which will permit further
subdivision when justified by an increase in the precision of cytochemical
methods. This is convenient in summarizing the reserves, but for final
classification it is unsatisfactory, since it ignores the differences in origin—
whether they are independent bodies, as the segregation granules of
O palina and the paraglycogen granules of Polyplastron, or whether they

CYTOPLASMIC INCLUSIONS 179

are products of the macronucleus, mitochondria, and so forth. This is a
difficulty which cannot be overcome until the fundamental processes
which are involved have been worked out.

In addition to these granules are a heterogeneous group of wnknown
granules such as the alpha granules of Amoeba and the accessory bodies
formed by the neuromotor system of Haptophrya (Fig. 70); various
pigment granules, which may in some cases be part of the lipoid reserves,
or in some cases residues of food, as in Plasmodium and Ichthyophthirius,
this latter type of course not being true cytoplasmic granules. Crystals
also are often present, the ones in Amoeba being classed as a part of
the protein reserves (although here there is a question, since they orig-

Figure 70. Accessory
bodies being formed from
the neuromotor ring in
Haptophrya michiganensis,
Zenker’s haematoxylin.
(After Bush, 1934.)

inate in the food vacuoles) on the evidence of Mast and Doyle. Other
crystals, according to Reichenow, may be excretory granules.

COMPARISON WITH CELLS OF THE METAZOA

Cytological investigations in the Protozoa have always been influ-
enced by the transfer of concepts originally developed from a study of
the cells of the Metazoa, particularly the vertebrates, with the result that
the division of granules into mitochondria, Golgi bodies, vacuome, and
passive reserve bodies are as common in the literature of the Protozoa
as in that of the Metazoa. In spite of Dobell’s denial of the cellular
nature of the Protozoa, any consideration of the granules of the Protozoa
necessitates a comparison with the cytoplasmic granules in other animal
cells.

The lack of a “‘typical’ reticular net of Golgi in the Protozoa, the
infrequency of filamentous mitochondria, and other striking morpho-
logical differences between the protozoan and metazoan cells have been
stressed so often that it is well to present several cases of equally striking
similarities in both structure and function. Volkonsky (1934) found

180 CYTOPLASMIC INCLUSIONS

that the digestive granules and gastrioles of Protozoa, choanocytes, and
leucocytes are entirely comparable, and included all of these cells in his
vacuolar reaction. Fauré-Fremiet (1909) and later Kedrowsky (1932,
1933) compared the segregation granules of Protozoa and vertebrate
tissues and found they were similar in appearance, staining reactions, and
function. Chatton, Parat, and Lwow (1927), on the basis of specific
microchemical reactions, compared the protein reserves in certain of
the Foettingeriidae with the vitellin of hen’s eggs. Kedrowsky (1931)
and MacLennan (1936) have given figures of the formation of neutral
fat bodies in the Protozoa which are almost identical with the descrip-
tion and figures of Bowen (1929) in the relationship between Golgi
bodies and lipoid secretion in cells of the mouse. Examples could be
multiplied, but these are sufficient to emphasize the fact that there are
similarities as well as differences between protozoan and metazoan cells,
with respect to their cytoplasmic granules.

The same difficulties with respect to the so-called specific staining
reactions arise in both Protozoa and Metazoa. In a study of echinoderm
eggs, Tennent, Gardiner, and Smith (1931) showed that not one ma-
terial, but many reduce osmium in the Golgi techniques. The presence
of more than one type of osmiophilic granules has been proved by
Mast and Doyle (1935) and MacLennan (1936) in Protozoa. Although
Kirkman and Severinghaus (1938) hold to the idea of a particular
Golgi substance, they bear witness to the occurrence of additional osmio-
philic materials: “One often finds small osmiophilic granules of uncet-
tain significance in Kolatchev sections, but they are present in addition
to the Golgi apparatus and appear to bear no relation to the latter struc-
ture.” In neither group is there any evidence that there is a particular
Golgi substance, any more than a particular Golgi structure.

There is also at times an embarrassing overlapping in the results ob-
tained from the use of Janus green and neutral red. These dyes were
found by Hopkins (1938) to stain the same vacuoles in the marine
amoeba, Flabellula, and Uhlenhuth (1938) reports similar results in
the thyroid cells of amphibia. The mitochondria turn out to be not a
simple group but a complex group, as indicated by the distinction
between mitochondria and active mitochondria, or mitochondria proper,
by Parat (1927) and by Joyet-Lavergne (1926).

Evidence is accumulating in both Protozoa and Metazoa that no type
of cytoplasmic granule (with the the exception of the centriole) can be

CYTOPLASMIC INCLUSIONS 181

considered as permanent, self-perpetuating structures. Wilson and Pol-
lister (1937), in connection with an investigation on sperm formation
in scorpions, review the division and distribution of mitochondra, Golgi
bodies, and vacuome, and show that the supposed accurate division 1s
actually an incidental fragmentation of large masses and that the distri-
bution is random during the division of the cell. They state: ‘There 1s,
however, little ground for the contention that either Golgi bodies or
chondriosomes can be regarded as permanent individuals having the
power of self perpetuation by growth and regular division.” Exactly
the same situation has been shown in this discussion with respect to the
cytoplasmic granules of the Protozoa, from studies of both fixed material
and of living normal cells. The permanence of mitochondria in metazoan
cells has been summed up recently by Bensley (1937): “The disappear-
ance and reappearance of mitochondria in living cells under observation,
as described by Chambers, however repugnant the idea may be to those
who would elevate mitochondria to the dignity of living, self-reproduc-
ing units, must be definitely entertained as probable.” With respect to
the de novo origin of Golgi apparatus, Kirkman and Severinghaus
(1938) assert that “there is little to favor such a view,” although they
quote at least a dozen authors who have advanced evidence of a de novo
origin in one form or another, admitting in several cases that the evidence
presented has not been refuted. For a detailed discussion, the reader is
referred to the review of the subject by these authors and to the original
publications, but it is clear that not all cytologists agree with these authors
on the permanence of the Golgi bodies.

The concept that the cytoplasmic granules arise or are resorbed as a
result of specific conditions of metabolism in the cell—in other words
that there is a granular reaction—is a logical result of the evidence that
the granules are neither permanent nor self-perpetuating, and it 1s
therefore no surprise to find that this interpretation has been made with
respect to the cytoplasmic constituents of the Metazoa as well as of the
Protozoa. One of the clearest statements of this concept has been made
by Tennent, Gardiner, and Smith (1931): ‘‘The results of this research
have been the conviction that neither Golgi bodies nor Chondriosomes
are structural elements in the cellular architecture, but that both are
the chemical products of physiological processes.” Nahm (1933) like-
wise states that “they are the visible products of chemical reactions that
occur in the cell.”

182 CYTOPLASMIC INCLUSIONS

LITERATURE CITED

Beers, C. D. 1935. Structural changes during encystment and excystment in
the ciliate Didininm nasutum. Arch. Protistenk., 84: 133-55.

Bensley, R. R. 1937. On fat distribution in mitochondria of the guinea pig
liver. Anat. Rec., 69: 341-54.

Bles, E. J. 1929. Arcella, a study in cell physiology. Quart. J. micr. Sci. n. s.,
72: 527-648.

Borgert, A. 1909. Ueber erscheinungen fettiger Degeneration bei tripyleen
Radiolarien. Arch, Protistenk., 16: 1-25.

Bourne, G. 1936. The role of vitamin C in the organism as suggested by its
cytology. Physiol. Rev., 16: 442-49.

Bourne, G., and R. Allen. 1935. The distribution of vitamin C in lower or-
ganisms. Aust. J. exp. Biol. med. Sci., 13: 165-74.

Bowen, R. H. 1928. The methods for the demonstration of the Golgi ap-

paratus. VI. Protozoa. The vacuome. Plant tissues. Aust. J. exp. Biol.

med. Sci., 40: 225-76.

1929. The cytology of glandular secretion. Quart. Rev. Biol., 4: 484-519.

Brand, T. von. 1935. Der Stoffwechsel der Protozoen. Ergebn. Biol., 12: 161-
220.

— 1938. The metabolism of pathogenic trypanosomes and the carbohydrate
metabolism of their hosts. Quart. Rev. Biol., 13: 41-50.

Brown, V. E. 1930. The Golgi apparatus of Pyrsonympha and Dinenympha.
Arch, Protistenk., 71: 453-62.

Bush, M. 1934. The morphology of Haptophrya michiganensis Woodhead.
Univ. Cal. Publ. Zool., 39: 251-76.

Bitschli, O. 1885. Bemerkungen ueber einen dem glikogen verwandten
Koerper in Gregarinen. Z. Biol., 21: 603-12.

Cajal, Ramon y, Santiago. 1904. Variaciones morfologicas del reticulo nervioso
de invertebrados y vertebrados sometidos 4 la accién de condiciones
naturales. Trab. Lab. Invest. biol. Univ. Madr., 1: 3.

Calkins, G. N. 1930. Uroleptus halseyi n. sp. I. The origin and fate of the
macronuclear chromatin. Arch. Protistenk., 69: 151-74.

Campbell, A. S. 1926. The cytology of Tintinnopsis nucula (Fol) Laakman.

Univ. Cal. Publ. Zool., 29: 179-236.

1927. Studies on the marine ciliate Favella, with. special regard to the
neuromotor apparatus and its rdle in the formation of the lorica. Univ.
Cal. Publ. Zool., 29: 429-52.

Chakravarty, M. 1936. On the morphology of Balantidium depressum Ghosh
from a mollusc Pila globosa, with a note on its nucleal’ reaction and
cytoplasmic inclusions. Arch. Protistenk., 87: 1-9.

Chalkley, H. W. 1937. The chemistry of cell division VII. Protoplasma, 28:
489-97.

Chambers, R., and R. T. Kempton. 1937. The elimination of neutral red by
the frog’s kidney. J. cell. comp. Physiol., 10: 199-221.

CYTOPLASMIC INCLUSIONS 183

Chambers, R., and H. Pollack. 1927. Micrurgical studies in cell physiology.
IV. Colorimetric determination of the nuclear and cytoplasmic pH in
the starfish egg. J. gen. Physiol., 10: 739-55.

Chatton, E., M. Parat, and A. Lwow. 1927. La formation, la nature et |’evolu-
tion des réserves chez les Spirophrya, les Polyspira, et les Gymnodinioides
(Infusoires Foettingeriidae). C. R. Soc. Biol. Paris, 96: 6-8.

Chatton, E., and P. P. Grassé. 1929. Le chondriome, le vacuome, les vésicules
osmiophiles, le parabasal, les trichocystes, et les chidocystes du
Dinoflagellé Polykrikos Schwartzi Biitschli. C. R. Soc. Biol. Paris, 100:
281-85. ;

Cheissin, E. 1930. Morphologische und systematische Studien tiber Astomata
aus dem Baikalsee. Arch. Protistenk., 70: 531-618.

Cleveland, L. R. 1934. The wood-feeding roach Cryptocercus, its Protozoa,
and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts
Sci., 17: 185-331.

Connell, F. H. 1930. The morphology and life cycle of Oxymonas dimor pha
sp. nov. from Neotermes sim plicicornis (Banks) . Univ. Cal. Publ. Zool.,
36: 51-66.

Cowdry, E. V. 1924. General cytology. Chicago.

Cowdry, E. V., and G. H. Scott. 1928. Etudes cytologiques sur le paludisme.
III. Mitochondries, granules colorable au rouge neutre et appareil de
Golgi. Arch. Inst. Pasteur Afr. N., 17: 233-52.

Cutler, M. 1921. Observation on the Protozoa parasitic in Archotermopsis
wroughtoni Desn. Part III. Pseudotrichonympha pristina. Quart. J. micr.
Sci., 65: 248-64.

Dangéard, P. 1928. L’Appareil mucifére et le vacuome chez les Eugléniens.
Ann. Protist., 1: 69-74.

Daniels, M. L. 1938. A cytological study of the gregarine parasites of Tenebrio
molitor, using the ultra-centrifuge. Quart. J. micr. Sci., 80: 293-320.
Day, H. C. 1927. The formation of contractile vacuoles in Amoeba proteus.

J. Morph. 44: 363-72, 6 figs.

Dobell, C. C. 1925. The life history and chromosome cycle of Aggregata
eberthi, Parasitology, 17: 1-136.

Doflein, F., and E. Reichenow. 1929. Doflein, Lehrbuch der Protozoenkunde.
Sth ed., Jena.

Dogiel, V., and T. Fedorowa. 1925. Uber den Bau und die Funktion des
inneren Skeletts der Ophryoscoleciden. Zool. Anz., 62: 97-107.

Doyle, W. L. 1935. Distribution of mitochondria in the foraminiferan, Iridia
diaphana, Science, n.s., 81: 387.

Duboscq, O., and P. Grassé. 1925. Notes sur les protistes parasites des
Termites de France. Appareil de Golgi, mitochondries, et vésicules sous-
flagellaires de Pyrsonympha vertens Leidy. C. R. Soc. Biol. Paris, 93:
345-48.

—— 1933. L’Appareil parabasal des flagellés, avec remarques sur le tropho-

184 CYTOPLASMIC INCLUSIONS

sponge, l'appareil Golgi, les mitochondries, et le vacuome. Arch. zool.
exp. gén., 732 381-6217.

Dunihue, F. W. 1931. The vacuome and the neutral red reaction in Para-
moecium caudatum, Arch. Protistenk., 75: 476-97.

Eksemplarskaja, E. V. 1931. Morphologie und Cytologie von Anoplophrya
sp. aus dem Regenwurmdarm. Arch. Protistenk., 73: 147-63.
Ellis, J. M. 1937. The morphology, division and conjugation of the salt marsh
ciliate Fabrea salina Henneguy. Univ. Cal. Publ. Zool., 41: 343-88.
Erdmann, R. 1917. Chloromyxum leydigi und seine Bezeichnung zu anderen
myxosporidien. Arch. Protistenk., 37: 276-326.

Fauré-Fremiet, E. 1905. Sur l’organisation de la Campanella umbellaria. C. R.
Sec. Biol. Patiss 582 205-1.

—— 1909. Vacuoles colorables par le rouge neutre chez un infusoire. C. R.
Ass. Anat., 11: 286-88.

—— 1910. Etude sur les mitochondries des protozoaires et des cellules
sexuelles. Arch. Anat. micr., 11: 457-648.

— 1925. La Structure permanente de l'appareil excréteur chez quelques
vorticellides. C. R. Soc. Biol. Paris, 93: 500-3.

—— 1932. Division et morphogenése chez Folliculina ampulla OFM Bull.
biol., 66: 79-109.

Fauré-Fremiet, E., A. Mayer, and G. Schaeffer. 1910. Sur la microchimie des
corps gras. Arch. Anat. micr., 12: 19-102.

Finley, H. E. 1934. On the vacuome in three species of Vorticella. Trans.
Amer. micr. Soc., 53: 57-64.

Fiveiskaja, A. 1929. Einfluss der Kernparasiten der Infusorien auf den Stoff-
wechsel. Arch. Protistenk., 65: 275-98.

Frisch, A. 1938. The rate of pulsation of the contractile vacuole in Paramecium
multimicronucleatum. Arch. Protistenk., 90: 123-61.

Gatenby, J. B. 1930. Cell nomenclature. J. R. micr. Soc., 50: 20-29.

Gelei, J. von. 1928. Nochmals ber den Nephridialapparat bei den Protozoen.
Arch. Protistenk., 64: 479-94.

— 1938. Das Exkretionsplasma von Déidinium nasutum in Ruhe und
Teilung. Arch. Protistenk., 90: 368-82.
Hall, R. P. 1929. Modifications of technique for demonstration of Golgi ap-
paratus in free-living Protozoa. Trans. Amer. micr. Soc., 48: 443-44.
—— 1931. Vacuome and Golgi apparatus in the ciliate, Stylonychia. Z.
Zellfotsch., 13: 770-82.

Hall, R. P., and F. W. Dunthue. 1931. On the vacuome and food vacuoles in
V orticella. Trans. Amer. micr. Soc., 50: 196-205.

Hall, R. P., and J. B. Loefer. 1930. Studies on Exglypha. I. Cytoplasmic
inclusions of Exglypha alveolata. Arch. Protistenk., 72: 365-76.

Hall, R. P., and R. F. Nigrelli. 1930. Relation between mitochondria and
food vacuoles in the ciliate, Vorticella, Trans. Amer. micr. Soc., 49: 54-
aya

CYTOPLASMIC INCLUSIONS 185

— 1937. A note on the vacuome of Paramecium bursaria and the contractile
vacuole of certain ciliates. Trans. Amer. micr. Soc., 56: 185-90.

Haye, Ans. 1930. Uber den Exkretionsapparat bei den Protozoen, nebst
Bemerkungen uber einige andere feinere Strukturverhaltnisse der unter-
suchten Arten. Arch. Protistenk., 70: 1-87.

Hayes, M. L. 1938. Cytological studies on Dileptus anser. Trans. Amer. micr.
S0G315 75 11225:

Heilbrunn, L. V. 1928. The colloid chemistry of protoplasm. Berlin.

—— 1936. Protein lipid binding in protoplasm. Biol. Bull., 71: 299-305.

Heilbrunn, L. V., and K. Daugherty. 1938. Fat release in Amoeba after itra-
diation. Physiol. Zodl., 11: 383-87.

Hertwig, R. 1902. Die Protozoen und die Zelltheorie. Arch. Protistenk., 1:
1-40.

Hindle, E. 1910. Degeneration phenomena of Trypanosoma gambiense.
Parasitology, 3: 423.

Hirschler, J. 1914. Uber Plasmastrukturen (Golgi’scher Apparat, Mito-
chondrien, u.a.) in den Tunicaten-, Spongien, und Protozoen-zellen.
Anat. Anz., 47: 289-311.

—— 1924. Sur les composants lipoidiféres du plasma des Protozoaires. C. R.
Soc. Biol. Paris, 10: 891-93.

— 1927. Studien uber die sich mit Osmium schwarzenden Plasmakompo-
nenten (Golgi-apparat, Mitochondrien) einiger protozoenarten, nebst
Bemerkungen uber die Morphologie der ersten von ihnen im Tierreiche.
Z. Zellforsch., 5: 704-86.

Holmes, E. 1937. The metabolism of living tissues. Cambridge.

Holter, H., and W. L. Doyle. 1938. Uber die lokalisation der Amylase in der
Amében. C. R. Lab. Carlsberg (Sér. Chim.) , 22: 219-25.

Holter, H., and M. J. Kopac. 1937. Studies in enzymatic histochemistry.
XXIV. Localization of peptidase in the Amoeba. J. cell. comp. Physiol.,
10: 423-37.

Hopkins, D. L. 1938a. The mechanism for the control of the intake and out-
put of water by the vacuoles in the marine amoeba, Flabellula mira
Schaeffer. Biol. Bull., 75: 353.

—— 1938b. The vacuoles and vacuolar activity in the marine amoeba
Flabellula mira Schaeffer and the nature of the neutral red system in
Protozoa. Biodyn., No. 34, 22 pp.

Horning, E. S. 1925. The mitochondria of a protozo6n (Opalina) and their
behavior during the life cycle. Aust. J. exp. Biol. med. Sci., 2:

—— 1926. Studies on the mitochondria of Paramoecium. Aust. J. exp. Biol.
med. Sci., 3: 91-94.

—— 1927. On the orientation of mitochondria in the surface cytoplasm of
infusorians. Aust. J. exp. Biol. med. Sci., 4: 187-90.

—— 1929. Mitochondrial behavior during the life cycle of a sporozoén
(Monocystis) Quart. J. micr. Sci., 73: 135-43.

186 CYTOPLASMIC INCLUSIONS

Horning, E. S., and G. H. Scott. 1933. Comparative cytochemical studies by
micro-incineration of a saprozoic and an holozoic infusorian. J. Morph.,
54: 389-94.

Jirovec, O. 1926. Protozoenstudien I. Arch. Protistenk., 56: 280-90.

Joyet-Lavergne, P. 1926a. Recherches sur les cytoplasmes des Sporozoaires.
Arch. Anat. micr., 22: 1-128.

— 1926b. Sur la coloration vitale des elements de Golgi des gregarines.
C. R. Soc. Biol. Paris, 94: 830-32.

—— 1927. Sur les rapports entre le glutathion et le chondriome. C. R. Acad.
SciParis; 1s4ato srs

—— 1928. Le Pouvoir oxydo-reducteur du chondriome des Gregarines et
les procédés de recherches du chondriome. C. R. Soc. Biol. Paris, 98: 501.

—— 1929. Glutathion et chondriome. Protoplasma, 6: 84-112.

—— 1931. Le Potentiel d’oxydo-reduction et la sexualisation cytoplasmique
des Gregarines. C. R. Soc. Biol. Paris, 107: 951-52.

—— 1932a. Sur le pouvoir oxydant du chondriome dans la cellule vivante.
GOR Soe Biol’ Paris; 11107552255:

— 1932b. Sur la mise en evidence des zones d’oxydation dans la cellule
animale. C. R. Soc. Biol. Paris, 110: 663-64.

— 1934. Une Théorie nouvelle sur le mécanisme des oxydo-réductions

intracellulaires. C. R. Acad. Sci. Paris, 199: 1159.

1935. Réchérches sur la catalyse des oxydo-reductions dans la cellule

vivante. Protoplasma, 23: 50-69.

Kazancev, V. 1928. Beitrag zur Kenntnis der Grosskerne der Ciltaten. Trav.
Lab. zool. Sebastopol, Ser. II, 13: 1-30.

Kedrowsky, B. 1931a. Die Stoffaufnahme bei Opalina ranarum. I. Methodik
der Kultur in kiinstlichen medien, pH regulieren und Ionen Gleich-
gewichte im Kulturmedien. Protoplasma, 12: 356-79.

—— 1931b. II. Struktur. Wasseraufnahme und Wasserzustand in Proto-
plasma von Opalina. Protoplasma, 14: 192-255.

— 1931c, HI. Aufnahme und Speicherung von Farbstoffen. Z. Zellforsch.,
12: 600-65.

—— 193ld. IV. Die synthetische Fettspeicherung. Z. Zellforsch., 12: 666-
714.

—— 193l1e. V. Der Segregationsapparat. Z. Zellforsch., 13: 1-81.

—— 1932a. Vitalfarbungsstudien an Infusorien, Z. Zellforsch., 15: 93-113.

— 1932b. Uber die Natur des Vakuoms. Z. Zellforsch., 15: 731-60.

—— 1933. Neue Probleme im Studium des Etweissstoffwechsels der Zelle.
Arch. exp. Zellforsch., 14: 533-54.

Kirby, H. 1931. Trichomonad flagellates from termites. II. Eutrichomastix
and the subfamily Trichomonadinae. Univ. Cal. Publ. Zool., 36: 171-262.

— 1932. Flagellates of the genus Trichonympha in termites. Univ. Cal.
Publ. Zool., 37: 349-76.

CYTOPLASMIC INCLUSIONS 187

—— 1936. Two polymastigote flagellates of the genera Pseudodevescovina
and Caduceia. Quart. J. micr. Sci., n.s., 79: 309-35.

Kirkman, H., and A. E. Severinghaus. 1938. A review of the Golgi apparatus.
Anat. Rec., 70: 413-32, 557-74; 71: 79-104.

Koehring, V. 1930. The neutral red reaction. J. Morph., 49: 45-130.

Kofoid, C. A., and M. Bush. 1936. The life cycle of Parachaenia myae, gen.
nov., sp. nov., a ciliate parasitic in Mya arenaria Linn. from San Fran-
cisco Bay, Calif. Bull. Mus. Hist. nat. Belg., 12: 1-15.

Kraschenninikow, S. 1929. Uber den Exkretionsapparat einiger Infusorien-
atten der Familie Ophryoscolecidae. Z. Zellforsch., 8: 470-83.

Krijgsman, B. J. 1936. Verleichend physiologische Untersuchungen tiber den
stoffwechsel von Trypanosoma evansi in Zusammenhang mit der Anpas-
sung an das Wirtstier. Z. vergl. Physiol., 23: 663-711.

LeBreton, E. 1931. Mitochondries et ferments protéolytiques. Examen de
’hypothése de Robertson-Marston. Arch. Biol. Paris, 42: 349-63.
Leiner, M. 1924. Das Glycogen in Pelomyxa palustris Greef mit Beitragen zur

kenntnis des Tieres. Arch. Protistenk., 47: 253-307.

Lison, L. 1936. Histochimie Animale. Paris.

Lynch, J. E. 1930. Studies on the ciliates from the intestine of Strongylocen-
trotus. Il. Lechriopyla mystax, gen. nov., sp. nov. Univ. Cal. Publ. Zool.,
J. 907-50:

MacLennan, R. F. 1933. The pulsatory cycle of the contractile vacuoles in the
Ophryoscolecidae, ciliates from the stomach of cattle. Univ. Cal. Publ.
Zool., 39: 205-50.

— 1934. The morphology of the glycogen reserves in Polyplastron. Arch.
Protistenk., 81: 412-19.

— 1936. Dedifferentiation and redifferentiation in Ichthyophthirius. Il.
The origin and function of cytoplasmic granules. Arch. Protistenk., 86:
404-26,

—— 1937. Growth in the ciliate Ichthyophthirius. 1. Maturity and encystment.
J. exp. Zool., 76: 423-40.

—— 1940. A quantitative study of the osmic acid reaction in Protozoa.
Trans. Amer. micr. Soc., 59: 149-59.

MacLennan, R. F., and H. K. Murer. 1934. Localization of mineral ash in
the organelles and cytoplasmic components of Paramecium. J. Morph.,
55: 421-33.

Marston, H. R. 1923. The azine and azonium compounds of the proteolytic
enzymes. Bio-chem. J., 17: 850-59.

Mast, S. O. 1938. Digestion of fat in Amoeba proteus. Biol. Bull., 75: 389-94.

Mast, S. O., and W. L. Doyle. 1935a. Structure, origin, and function of
cytoplasmic constituents in Amoeba proteus. I. Structure. Arch. Pro-
.tistenk, 86: 155-80.

—— 1945b. Structure, origin, and function of cytoplasmic constituents in

188 CYTOPLASMIC INCLUSIONS

Amoeba proteus with special reference to mitochondria and Golgi sub-
stance. II. Origin and function based on experimental evidence; effect of
centrifuging on Amoeba proteus. Arch. Protistenk., 86: 278-306.

Metcalf, M. M. 1910. Studies upon Amoeba, J. Exp. Zool., 9: 301-32.

Meyers, E. H. 1935. The life history of Patellina corrugata Williamson, a
foraminifer. Bull. Scripps Instn. Oceanogr. tech., Ser. 3: 355-92.
Miller, E. D. 1937. A comparative study of the contents of the gelatinous
accumulations of the culture media and the contents of the cytoplasm of

Amoeba proteus and Arcella vulgaris. J. Morph., 60: 325-54.

Milovidov, P. F. 1932. Independence of chondriosomes from nuclear matter.
Cytologia, 4: 158-73.

Nahm, L. J. 1933. A study of the Golgi elements. J. Morph., 54: 259-301.

Nassonov, D. 1924. Der Exkretionsapparat (kontraktile Vakuole) der Pro-
tozoa als Homologen des Golgischen Apparats der Metazonzellen. Arch.
Mikr. Anat., 103: 437-82.

—— 1925. Zur Frage tber den Bau und die Bedeutung des lipoiden Excre-
tionsapparates bei Protozoa. (Chilodon, Dogielella). Z. Zellforsch., 2:
87-97.

Nigrelli, R. F. 1929. On the morphology and life history of Trypanosoma
diemyctyli and the relation of trypanosomiasis to the polynuclear count.
Trans. Amer. micr. Soc., 48: 366-87.

Owens, H. B., and R. R. Bensley. 1929. On osmic acid as a microchemical
reagent, with special reference to the reticular cipipeleans of Golgi. Amer.
J. Anat., 44: 79-110.

Parat, M. 1927. A review of recent developments in histochemistry. Biol. Rev.,
Za 3 5 -OY

Patten, R. 1932. Observations on the cytology of O palina ranarum and Nycto-
therus cordiformis. Proc. R. Irish Acad., 41: 73-94.

Pellissier, M. 1936. Sur certains constituants cytoplasmiques de I’infusoire cili€,
Trichodinopsis paradoxa Clap. et Lach. Arch. zool. exp. gén, 78: 32-36.

Peshkowskaya, L. 1928. On the biology and the morphology of Climacosto-
mum virens. Arch, russ. protist., 7: 205-35.

Poljansky, G. 1934. Geschlechtsprozesse bei Bursaria truncatella OFM. Arch.
Protistenk., 81: 420-546.

Rammelmeyer, H. 1925. Zur Frage ueber die Glykogendifferenzierung bei
Paramecium caudatum. Arch. Protistenk., 51: 184-88.

Reichenow, E. 1928. Ergebnisse mit der nuclealfaerbung bei Protozoen. Arch.

Protistenk., 61: 144-66.

1929. Doflein, Lehrbuch der Protozoenkunde. Sth ed., Jena.

Rey, P. 1931a. Potentiel d’oxydo-reduction et sexualité chez les Gregarines.
GARY Soe BioleParis: M07 Glee

—— 1931b. Coloration vitale et potentiel d’oxydo-reduction chez les gre-
garines. C. R. Soc. Biol. Paris, 107: 1508-11.

CYTOPLASMIC INCLUSIONS 189

Rosenberg, L. E. 1937. The neuromotor system of Nyctotherus hylae. Univ.
Cal. Publ. Zool., 41: 249-76.

Rumjantzew, A. 1922. Uber den Bau der Chromidial substanz bei Dzfflugia
pyriformis. Arch. russ. protist., 1: 87-105.

Rumjantzew, A., and E. Wermel. 1925. Untersuchungen ueber den Proto-
plasmabau von Actinosphaerium Eichhorni. Arch. Protistenk., 52: 217-
64.

Sassuchin, D. 1924. Zur Kenntnis der Plasmaeinschliisse bei den Opalinen.
Arch. russ. protist. 3: 147-54.

Schrich, P. 1914. Beitrige zur Kenntnis des Lebenscyclus.von Arcella vulgaris
und Pelomyxa palustris. Arch. Protistenk., 33: 247-71.

Schulze, P. 1922. Uber Beziehungen zwischen tierischen und pflanzlichen
Skelettsubstanzen und tiber Chitinreaktionen. Biol. Zbl., 42: 389-94.

—— 1924. Der Nachweis und die Verbreitung des Chitins mit einem Anhang
iiber das komplizierte Verdauungssystem der Ophryoscoleciden. Z.
Morph. Okol. Tiere, 2: 643-66.

— 1927. Noch einmal die ‘‘Skelettplatten’”’ der Ophryoscoleciden. Z. Morph.
Okol. Tiere, 7: 670-89.

Scott, G. H., and E. S. Horning. 1932. The structure of Opalinids as revealed
by the technique of microincineration. J. Morph., 53: 381-88.

Strelkow, A. 1929. Morphologische Studien uber Oligotriche Infusorien aus
dem Darme des Pferdes. Arch. Protistenk., 68: 503-54.

— 1931. II. Cytologische Untersuchungen der Gattung Cycloposthium
Bundle. Arch. Protistenk., 75: 191-220.

Subramaniam, M. K., and P. N. Ganapati. 1938. Studies on the structure of
the Golgi apparatus. I. Cytoplasmic inclusions in the gregarine Lecudina
brasili n.sp. parasitic in the gut of Lwmbriconereis. Cytologia, 9: 1-16.

Subramaniam, M. K., and R. Gopala Aiyar. 1937. The Golgi apparatus and
the vacuome in protozoa—some misconceptions and the question of
terminology. Proc. Indian Acad. Sci., Sec. B., 6: 1-18.

Swellengrebel, N. H. 1908. La Volutine chez les trypanosomes. C. R. Soc.
Biol. Paris, 64: 38-40.

Taylor, C. V. 1923. The contractile vacuole in Evplotes an example of the
sol-gel reversibility of cytoplasm. J. exp. Zool., 37: 259-82.

Tennent, D. H., M. S. Gardiner, and D. E. Smith, 1931. A cytological and
biochemical study of the ovaries of the sea-urchin Echinometra lucunter.
Pap. Tortugas Lab., 27: 1-46.

Thélohan, P. 1894. Nouvelles réchérches sur les coccidies. Arch. zool. exp.
gén., 3: 541-73.

Tuzet, O. 1931. Une Grégarine parasite de Bythinia tentaculata. Gonospora
duboscqui nov. sp. Arch. zool. exp. gén., 71: 16-21.

Uhlenhuth, E. 1938. A quantitative approach to the secretion process of the
thyroid. Coll. Net, 13: 76-87.

190 CYTOPLASMIC INCLUSIONS

Van Thiel, P. H. 1925. Was ist Rickettsia melophagi? Arch. Protistenk., 52:
394-403,

Volkonsky, M. 1929. Les phénoménes cytologiques au cours de la digestion
intracellulaire de quelques ciliés. C. R. Soc. Biol. Paris, 101: 133-35.
—— 1933. Digestion intracelluaire et accumulation des colorants acides. Etude
cytologique des cellules sanguines des Sipunculides. Bull. Biol., 67:

135-286.

—— 1934, L’Aspect cytologique de la digestion intracellulaire. Arch. exp.
Zellforsch., 15: 355-72.

Weatherby, J. H. 1927. The function of the contractile vacuole in Paramecium
caudatum; with special reference to the excretion of nitrogenous com-
pounds. Biol. Bull., 52: 208-22.

—— 1929. Excretion of nitrogenous substances in the Protozoa. Physiol.
Zobl., 2: 375-94.

Weier, T. E. 1933. A critique of the vacuome hypothesis. Protoplasma, 19:
589-601.

Weineck, I. 1931. Die chemische Natur der Skelettsubstanzen bei den Oph-
ryoscolec iden. Jena. Z. Natur., 65: 739-51.

—— 1934. Die Celluloseverdauung bei den Ciliaten des Wiederkduermagens.
Arch. Protistenk., 82: 169-202.

Wermel, E. 1925. Beitrage zur Cytologie der Amoeba hydroxena Entz. Arch.
russ. protist., 4: 95-120.

Wilson, E. B. 1928. The Cell. 3d ed., New York.

Wilson, E. B., and A. W. Pollister. 1937. Observations on sperm formation
in the centrurid scorpions with especial reference to the Golgi material.
J. Morph., 60: 407-44.

Wurmser, M. 1932. Sur l'emploi de certain colorants pour l’évaluation des
proprietés oxydantes du cytoplasme. C. R. Soc. Biol. Paris, 111: 690.

Yamasaki, M. 1937a. Studies on the intestinal Protozoa of termites. III. The
distribution of glycogen in the bodies of intestinal flagellates of Termites
Leucotermes and Coptotermes. Mem. Coll. Sci. Kyoto, B 12: 212-24.

—— 1937b. IV. Glycogen in the body of Trichonympha agilis v. japonica
under experimental conditions. Mem. Coll. Sct. Kyoto, 12: 225-35.

Zhinkin, L. 1930. Zur frage der Reservestoffe bei Infusorien (Fett und Gly-
kogen bei Stentor polymorphus). Z. Morph. Okol. Tiere, 18: 217-48.

Zinger, J. A. 1928. Morphologische Beobachtungen an Ophryoglena flava
Ehr. Arch. russ. protist., 7: 179-204.

—— 1929. Beitriige zur Morphologie und Cytologie der Siisswasserinfusorien.
Arch, russ. protist., 8: 51-90.

——— 1933. Beobachtungen an Fetteinschliissen bei einigen Protozoen. Arch.
Protistenk., 81: 57-87.

Zweibaum, I. 1921. Ricerche sperimentali sulla conjugazione degli Infusori.
II. Influenza della coningazione sulla produzione dei materiali di riserva
nel Paramecium caudatum. Arch. Protistenk., 44: 99-114.

CHAPTER IV

FIBRILLAR SYSTEMS IN CILIATES
Gir TAMIOR

INTRODUCTION

THE ESSENTIAL NATURE of Leeuwenhoek’s “‘little animals’ remained
obscure for more than 150 years, evidently because the methods of ob-
servation which characterized that ingenious microscopist of Delft were
replaced largely by fruitless speculation. Otherwise, man’s epochal dis-
covery of the cellular nature of living things might have been realized
sooner.

Meantime, it is true, a prodigious diversity of macroscopic forms had
been examined and classified. But the disclosure of cellularity, which
eventually unified all of this diversity in organic form, had to depend
upon the detailed analysis of organic structure.

During the hundred years that have now intervened, that common
denominator of organic form and function has come to be regarded, for
multicellular plants and animals, as a sort of master key to the solution
of their fundamental problems. And for the major advances in biology
during that memorable century, we are surely indebted primarily to this
cellular concept of the organization of living things.

For the microdrganisms, however, the concept of cellularity, although
generally conceded, has encountered not infrequently some confusing
difficulties. With von Siebold’s pronouncement in 1845 of the unicellu-
larity of the Protozoa, the way at first seemed clear toward simplifying
and unifying all forms of life, in terms of the cell as the universal
unit. Eventually, however, it was evident that, for the Protozoa, this
concept did not simplify matters so satisfactorily. The chief difficulty
here arose in trying to equate the protozoon cell with a tissue cell of
the Metazoa. And even in recent times this comparison has again been
challenged by Dobell (1911) and others, who would maintain that
Protozoa are not cells at all and so should be regarded as non-celluar
organisms.

192 CILIATE FIBRILLAR SYSTEMS

But similar difficulties in comparing microscopic with macroscopic
forms of life had confronted investigators several years before von Sie-
bold’s pronouncement and, in fact, before the concept of cellularity had
been definitely formulated. As is well known, this all culminated in
the Ehrenberg-Dujardin controversy, beginning in 1835. Obviously for
these investigations, the issue was not one of cellularity, but it had to
do with complexity versus simplicity in the organization of the Infusoria.
It seems probable that Ehrenberg defended his thesis of “complete or-
ganisms’’ partly in refutation of the theory of spontaneous generation,
then vigorously championed for microorganisms. At any rate, he sought
to identify in the Infusoria all the organs common to other animals.
Much of his adduced evidence, it will be recalled, was successfully re-
futed by Dujardin, who described among other things, his newly dis-
covered “‘sarcode”’ in support of his contentions for uniqueness and sim-
plicity in the organization of the Infusoria.

The essentials of these contrasting views of Ehrenberg and Dujardin
on the nature of infusorian organization have recurred, in varied guise,
many times in the literature since their day. These opposing viewpoints
have, of course, become translated into terms of the concept of cellu-
larity, so that now the nature of unicellular organization, or “‘proto-
plasmic differentiation,” is commonly contrasted with ‘“‘cellular differ-
entiation” of multicellular organisms.

Accordingly, in the following review of literature on fibrillar systems
in ciliates, it will become evident that some discrepancies in both the
analysis of structure and the interpretation of functions may owe their
origin largely to contrasting points of view on the essential nature of
“protoplasmic differentiation” in the Protozoa and ‘‘cellular differentia-
tion” in the Metazoa.

Before beginning that review, however, the fact should be emphasized
that, as Maupas (1883) has pointed out, the Ehrenberg-Dujardin con-.
troversy marks a turning point in protistological investigations. Not only
did it enlist a wider interest in these microdrganisms, but it made clear
the necessity of a critical structural analysis of their greatly diversified
types of organization and of a comparative study of such types before
any satisfactory interpretations were possible.

The literature resuiting from those analyses is so voluminous that
when one undertakes to review the accounts of a given system of organ-

CILIATE FIBRILLAR SYSTEMS 193

elles, such as the fibrillar system, and to condense that review within
reasonable bounds, the difficulties soon become evident. For this reason
it has seemed advisable in the review that follows, in the interests of
students and laity as well as of specialists, to present a fairly detailed
account of the structural analysis, together with interpretations of the
fibrillar differentiations of a well-known representative of each of four
major groups of ciliates. This is followed by a brief review of other
published work, mostly since 1920, on fibrillar systems in other ciliates,
with some suitable illustrations; and finally, a few paragraphs of general
discussion are added under the caption “Conclusions.”

The discussion of the structural analysis of the fibrillar systems of the
four representative ciliates, Paramecium, Stentor, Euplotes and Vorti-
cella, is offered first and separate from the interpretations for these four
ciliates, whose order is then, for convenience, reversed. This separate
treatment was decided upon primarily for the sake of accuracy and clarity.
Often in the literature the author’s interpretations are so intermixed with
his factual descriptions that it is sometimes very difficult to make certain
just what he observed and undertook to describe.

EXAMPLES OF FIBRILLAR SYSTEMS

A. STRUCTURAL ANALYSIS

1. Paramecium.—vThis familiar representative of the holotrichs has
doubtless been more generally used in both teaching and research labora-
tories than has any other of the numerous kinds of ciliates. Probably
its apparent simplicity, more than its smaller size, tended to discourage
a search for a fibrillar system, such as had been found in Stentor and
other forms.

In 1905, however, Schuberg described for both Paramecium and
Frontonia fibrillar differentiations which, running close under the pel-
licle, united the basal granules in the longitudinal rows of cilia. By
means of a bichromate-osmic fixative and Loefflet’s stain, not only was
this relationship of fibril and basal granules clearly defined, but also,
because of their staining properties, they could be well differentiated
from the hexagonal, or rhomboidal, pattern of the pellicle, as was well
illustrated in Schuberg’s several figures.

In 1925, J. von Gelei described in Paramecium nephridiatum a periph-
eral network of fibrils which was not connected with the familar po-

194 CILIATE FIBRILLAR SYSTEMS

lygonal pattern observable in the living organism. For fixation, he used
Apathy’s sublimate-osmium and stained with toluidin blue.

In the following year Klein (1926a) reported the results of his
studies on a peripheral fibrillar complex in certain ciliates by means
of a new silver-nitrate technique which involved no previous fixation.
The method, thus employed, is now well known as the “dry method,”
in contrast to von Gelei’s (1932a) “‘wet method,” and the resulting
silver-impregnated fibrillar complex is quite commonly referred to as the
“silverline system.”

The several subsequent publications of these two authors on the fibril-
lar system of Paramecium, using especially the silver-nitrate technique
but also other methods, admit of useful comparisons for this brief re-
view, so that their results will now be considered together.

In most of these various articles, the author’s account of the structures
that were clearly observed is at times so involved with his avowed inter-
pretations of their functions that it has been found difficult to sift out
the essential data for which this review is intended.

Their structural analyses of the fibrillar system of Paramecium have,
nevertheless, several important points in common which may now be
fairly, and as simply as possible, presented. On the basis of these com-
mon points, certain discrepancies will then be indicated.

To this end, it will be convenient to recall the findings of Schuberg
(1905). He observed (1) a differentially stainable pellicular pattern,
which was hexagonal over the body and rhomboidal on each side of
the mouth, and below this (2) a longitudinal fibril connecting (3) the
basal granules in each row of cilia—each such granule appearing below
the center of each pellicular polygon (Fig. 71).

In outline, the descriptions of both von Gelei and Klein present this
same general picture, which may now serve to simplify a brief com-
parison of their essential findings. In their later papers, both of these
authors agree that the fibrillar system of Paramecium is entirely sub pel-
licular. Bearing this in mind, we may note that:

1. Schuberg’s pellicular pattern corresponds im general outline to
von Gelei’s “‘Stiitzgitter System’ and to Klein’s ‘“Indirekt verbindung
System” (““Meridiaan II. Ordnung’’).

2. Both the Stiitzgitter System and the Indirekt verbindung System,
lying under the pellicle, comprise each: (1) a longitudinal fibril, between

CILIATE FIBRILLAR SYSTEMS 195

the rows of cilia; (2) cross fibrils, connecting adjacent longitudinal
fibrils; (3) opening for a trichocyst midway on the cross fibril; and (4)
a suture line at the anterior and the posterior poles. The resulting lattice,
therefore, has its counterpart in Schuberg’s pattern.

3. Beneath von Gelei’s Stiitzgitter System lies his “Neuronem Sys-
tem.”

4, Beneath Klein’s Indirekt verbindung System lies his ‘“Direkt ver-
bindung System.”

5. Both Neuronem System and Direkt verbindung System comprise

Lfio: ms i
bgr. -—--4 Nye
ges AL q ie 4 za

Figure 71. Pellicular Pattern and Longitudinal Fibrils Connecting Basal Granules in
Paramecium. (Modified from Schuberg, 1905.)
b. gr.—basal granule 1. fib—tlongitudinal fibril pell. p.—pellicular pattern

each: (1) an /nterciliary fibril, connecting the ‘“Basalapparaten” in each
longitudinal row of cilia; (2) cross fibrils, connecting the interciliary
fibrils; and (3) Relationskérner, which include the Basalap parat and the
“Trichocystenkorn.”

The interciliary fibril and the Basalapparaten, therefore, find their
counterpart essentially in Schuberg’s longitudinal fibril and its connected
basal granules.

For both von Gelei and Klein, accordingly, it is evident that the
fibrillar system of Paramecium includes two subsystems, or fibrillar com-
plexes. For present convenience and especially for later discussion, I
shall refer to these as von Gelei’s outer fibrillar complex and inner fibril-
lar complex and Klein’s outer fibrillar complex and inner fibrillar com-
plex respectively.

DO Geet h

(‘dioy ‘n 344g UsyIsIMZ) (aAoqe) Z ‘T uI—
AIOYIITT, JO pus Jayno ye a[nuvss v—usoyuaysA0yIIIT, “q

‘JrepnIIIZ = {JauloyuaqaNN OMY, (Z)
Ug usoxeseg (1)
(uoddnisiaiaiq ) yereddeyeseg ‘ev
(ua}yyUndSsso}¢ )
TJOUIOYSUOTILIAY “¢
a] qyorejuswe]q

s]iiqy Jo uorjounf }e duTpIoATIs WoIfZ astie

(sunyyds Aq "[ wosz paatiap) aypiqyrepnoirz *z

YUM “TSeJIVIIIIOJUT “7
‘PIO TIL FO YA AWNUTUOD UF s[Isqry

(wo}shg “pulgioA JYxPIP) SunupsiO "[ ueiprioy_ ‘TT

(e870 spiuqy yySIIpUT “Nn yyAIIP) s19}UT_Y “gq

atury JvjodumIny—y]og aapioA ‘ve
SHOTRMENE

UJoyUe}sADOYIII}OId JIvJUIWIpPNY “q

ysAD0YIII} JOF Furuado YW “ve

puesys | UPY} STOUT S spIIqY *jSUOT UIdMJagG—uOYTeqIaNEd °*Z
puvyjs T Wey} STOUT :¥ITID JO SMOI UaaMJoq—JosLJSULT *T
SUNUPIOP'T ut

“PHO Wo1Z paarsop ‘apoijod ay} Japun yIOMjOU INL}suOU YW

(Wia}shg “pUIqIoA }YPIIpUr) SunupsiO “[] uempusyy ‘T

ul21N

(7Z61 ‘seay) woysds IeT[IIqy d1}ewWse[desjuy

uaTIqIy apeuosviq “¢

VaTTqgIINO °Z

Ud] [IIqyssueyT *]

(é) UorUNF UF d9]1}9vI}UOD ‘ sSuIssoID ye AjIMUT}UOD UT ‘ssaUydIY}
jO Sopris JUaIayIp Jo somqy ‘aynuvis [eseq YUM oad] dures
ay} 3 pue Y}eaUaq pa}ed0] s[lIqy jo xatdurod dtwsedowo uy

(TaJ2H) UOA *D Jo) wayshsr9}IH orerpoeIjuy

Jasejierfiosjoyuy puv jeseddeyeseg surpunosins vusejdoman °*9
IWIVOINIUILI[III9JUI UO UIOYUa}sADOYIIIT, “q

uloyuaqeaN, (¢)
osejUY UIOX[eseg WN SulI jeseq (Z)
uloyyeseg (T)
yereddeyeseg ev
awauOINeU
Uo MoU asIIe JO UOIssy SuIINp aplAIp—souIOYsuOT}LAY *¢
‘Iqy € JO Z Jo—PIBeH PN Jodsayuryy ‘p
UIWIUOINIUSSULPT UddM}
-2q S[IIqy-sso1d jo woajsks J[vInsstwuwo0I—jYeNT [OdIapI0OA *¢
UasUNpUIqIaAIANG) *Z
noysnosy} eee “q
snonunuod nq SorelIovig “e
‘uvIs ysAIOYINI} Y}IM—JOSvJIEIIDIOWU] *T
I YIM payauUO0) jou Ss]IIqy JO YIOM}JIN
wajsk§ aWaUOININ

Jola}sog “q

JOMa}Uy ‘eB
arurpyeN *¢

pus Yove je UIOYIIVI YU “q

ysAI0YIIIT, JOJ Surusdo yA “ev

pueijs [—uor}I9s
“ur Area [| Suneredas ‘spriqy ‘ysuoy U39M}9G—Uoy[eqIand) *Z
pues}sS [—PI]ID JO SMOI UIIMJOG—UIasv]JssuLT “T
UOTLIVUIIOYIP Jtuwsvpdoyea {aypotyJad
ayy Jopun ApaIIp s[qy Jo YIOMJoU |. YsTURYOW-YDsTWV}s,, W

wayshg 19}}13Z)NIS

12/25) uo A

WIMDAWNVAVd -T ATAV YE

‘AI

‘Ill

CILIATE FIBRILLAR SYSTEMS 197

The several components of these fibrillar complexes, which were listed
above, constitute the main structural features shared by Klein and von
Gelei in their various accounts of the fibrillar system of Paramecium.
But certain discrepancies appear in their descriptions of these and espe-
cially of some other components.

Space does not permit a discussion of all of these discrepancies, but
most of them will be found listed in Table 1, which comparatively sum-
marizes all the structural components ascribed to Paramecium’s fibrillar
system by von Gelei and by Klein in their various publications. Some of

Figure 72. Diagram of G/tter (lattice) with attached trichocysts and of the neuro-
nemes connecting bases of the cilia. (Von Gelei, 1925.)
Git—Gitter neu.—neuroneme tri.trichocyst

the discrepancies may very possibly be attributable to species differences,
since von Gelei studied P. nephridiatum and P. caudatum, while Klein’s
descriptions are of P. awrelia.

A few discrepancies should, however, here be pointed out. The first
and most important of these has to do with any structural integration
between the two fibrillar complexes. Klein especially emphasizes the fact
that his outer fibrillar complex and inner fibrillar complex are a con-
tinuum through interfibrillar connectives. Von Gelei, on the contrary,
denies that any connection exists and so states that his outer and inner
fibrillar complexes are only contiguous (Fig. 72).

A further discrepancy concerns the basal apparatus of the cilium.
According to von Gelei (1932a), this apparatus consists of (1) a basal

198 CILIATE FIBRILLAR SYSTEMS

ring surrounding (2) the basal granule of the cilium, and (3) the
“Nebenkorn” occurring at the right of the junction of the basal ring
and the interciliary fibril (p. 158). According to Klein (1931), the
elements of the basal apparatus include (1) a ring (Zirkularfibrille)
within which are usually three granules (Drierkérner). The central
granule is the basal granule, and the other two are Nebenkérner. The
discrepancy here is not only in the relative number of granules in the
basal apparatus but in their relationship, since von Gelei regards his
‘“Nebenkorn”’ as identical with Klein’s basal granule, and accounts for
Klein’s third granule as being only a thickening of the basal ring.

Finally, mention may be made in this connection of an additional
system of fibrils described by Gabor von Gelei (1937) in three species
of Paramecium,—P. caudatum, P. multimicronucleata, and P. trichinum.
This third fibrillar complex, in addition to the two noted above, was
found at the level of and below the basal granules. Its fibrils spread
throughout the entire body surface, including the vestibule, where it
sends a thickened fibril into the cytopharynx between the membranelles.

The general pattern of this complex, made up of longitudinal and
cross fibrils, resembled that of the outer fibrillar complex, the meshes
of the former being smaller and more numerous, however, than those
of the latter. Also, the course of the fibrils of this third complex were
more irregular. Its longitudinal fibrils become fewer in the middle region
of the body and at times are directed diagonally, even spirally. A splitting
of fibrils was occasionally observed, as well as variations in their thick-
ness. They may alsc anastomose and form a ‘‘Schaumgiter.”

The author could discover no connection between this fibrillar complex
and either of the other two.

Apparently the most recent detailed account of the fibrillar system
of P. caudatum, including that of its cytostome, was made by Lund
(1933), working in Kofoid’s laboratory. After comparing the descrip-
tions of earlier workers (Engelmann, 1880; Maupas, 1883; Schuberg,
1905; Rees, 1922; von Gelei, 1925-32; Klein, 1926-31; Jacobsen, 1931)
with his own findings, Lund concluded that previous investigators had
confused “parts of at least two and possibly three quite different aggre-
gations of structures, namely, the pellicle, the trichocysts and the periph-
eral portion of the neuromotor system. In addition” they had failed

CILIATE FIBRILLAR SYSTEMS 199

“completely to demonstrate the great pharyngeal complex,” which is
an integral part of this system.

Lund was able to differentiate between these “‘different aggregations
of structures” by means of the silverline technique (Klein’s and von
Gelei and Horvath’s) on the one hand and, on the other, by the use of
iron-haematoxylin and Mallory’s stain.

The former method demonstrated von Gelei’s ‘‘Stiitzgitter System”
and Klein’s ‘‘Indirekt verbindung System,” i.e., their “outer fibrillar
complex’’ noted above. It also revealed essentially their “inner fibrillar
complex,’’ also as noted above.

But, according to Lund, these are separate and distinct “aggregates.”
The “outer fibrillar complex’ is not subpellicular, as both von Gelei
and Klein maintain, but represents rather the sculptural polygonal pat-
tern of the pellicle itself. A similar interpretation was made by Brown
(1930).

The “inner fibrillar complex’ of von Gelei and of Klein comprises the
basal granules, their connecting longitudinal body fibrils, transverse
fibrils connecting the longitudinal fibrils, and others which include the
“radial fibrils.” These last mentioned “originate as longitudinal fibrils
in the cytopharynx and oesophagus, spread radially out from the oral
opening over the body surface and terminate a short way from the cyto-
stome.”’

This inner fibrillar complex may be clearly demonstrated by the silver-
line techniques, especially by the wet method. There is, however, a
portion of the fibrillar complex within the cytopharynx and cytoesopha-
gus which is not wholly demonstrable by these techniques. This was well
differentiated by the iron-haematoxylin and Mallory’s methods, and
described as “seven major parts, namely: (1) the pharyngo-esophageal
network, (2) the neuromotorium, (3) the penniculus, (4) the oesopha-
geal process, (5) the paraesophageal fibrils, (6) the posterior neuromotor
chain, and (7) postesophageal fibrils.’

For the descriptive details of this very elaborate complex of fibrils
and associated parts, obviously the original account must be read. It is
evident, however, from this brief review of the results of these several
workers on the fibrillar system of Paramecium that a number of discrep-
ancies need to be cleared up and perhaps further structural analysis of

200 CILIATE FIBRILLAR SYSTEMS

this system made before we can hope to have a complete understanding
of the parts that are, or are not, structurally integrated.

In recent publications Chatton and Lwoff (1935, 1936) have de-
scribed a fibrillar complex in several ciliates, which has long been known
(Chatton and Lwoff, 1936) but has not been clearly distinguished from
Klein’s silverline system. The fibrils are visible 72 vivo and may be clearly
differentiated in preparations fixed in Bouin’s or Champy’s solution and
stained in iron-haematoxylin.

Each fibril (c/nétodesme) has connected, always along its left side,
the basal granules (c/nétosomes) of a longitudinal row of cilia. The
fibrils, together with their adjoined basal granules (the so-called infra-
ciliature), are each essentially an independent entity. They are never
united by anastomosis or otherwise at either body pole, and so include
no transverse or other fibrillar connectives throughout their course.

The fibrils of this 7fraciliature are entirely superficial and adhere
to the pellicle as rectilinear (never sinuous) threads. Other granules, as
well as the ciliary basal granules, appear likewise attached, and these
represent successive stages of the multiplication of the basal granules.
Fibrils and granules stain alike, but in some species the fibrils cannot
be impregnated with silver by the usual techniques. After fixation (osmic
acid, Da Fano, Champy) and covering with gelatin or gelose, the fibrils
may show, upon silver-nitrate treatment, the basal granules connected
to a sinuous thread which, with its various connectives, represents Klein’s
silverline ‘‘plexus.” This plexus, according to Chatton and Lwoff
(1935), is acid labile and cannot be stained.

The selective staining properties and relations of the infraciliature
show that it is quite distinct from the silverline fibrils, and is comparable
with the flagellar ridges of the Hypermastigidae, marking the place of
formation and of the insertion of the cilia.

It would appear that Chatton and Lwoff’s infraciliature may be identt-
fied with the longitudinal fibrils and basal granules of the inner fibrillar
complex reviewed above. The left lateral attachments of the czmétosomes
to the cinétodesmes is evidently a new finding.

2. Stentor—In his search for organs that would account for the
well-known contractile behavior of Stentor, Ehrenberg (1838) saw in
its conspicuous longitudinal bands the seat of that contractility. This
interpretation of its contractile mechanism was accepted by several later

CILIATE FIBRILLAR SYSTEMS 201

investigators including K6lliker (1864) and, according to Neresheimer
(1903), Haeckel’s (1873) “‘Myophanen”’ should be so construed.

It was between these longitudinal stripes, within the clearer non-
pigmented meridians (Biitschli’s ““Zwischenstreifen”), that Lieberkthn,
in 1857, found a distinct contractile fibril coursing from the basal disc
forward to the adoral zone. Greeff (1870) confirmed these findings
and Engelmann (1875) made detailed studies on the refractive and
contractile properties of the fibers which have come to be commonly
referred to as myonemes.

Four authors may be cited, among many others, for the descriptive
details of the fibrillar system of Stentor: Schuberg (1890), Johnson
(1893), Neresheimer (1903), and Dierks (1926). These have been
the main sources for the following brief review of this system.

Schuberg (1890) made several important observations on the arrange-
ment of the myonemes of Stentor coeruleus and an analysis of the basal
apparatus of the membranelles. He found that the course of the body
myonemes, from the basal disc to the peristome border, was not con-
stant. Instead, some showed bifurcations, with occasional re-branching.
This branching of myonemes followed consistently a corresponding
branching of the longitudinal rows of cilia and their adjacent, non-
pigmented bands. Similar relations of bands, ciliary rows, and myonemes
obtained also for the peristome field.

Schuberg further observed that the double row of cilia, comprising
a membranelle, was seated in an ectoplasmic basal platelet (‘“Basal-
saum’’), itself bipartite, below which appeared a triangular Jamella
(“‘Basallamelle”). The inwardly directed apex of this triangle was
continued as a fibril (““Endfadchen’’) which was, in turn, united to all
other such end fibrils by a basal fibril. The latter then ran rather deep
below and parallel to the entire series of membranelles.

Schuberg’s account of Stentor’s myonemes and his analysis of its
membranelles have been generally confirmed, with the exception of the
basal fibril. The latter was identified by Johnson (1893), Maier (1903),
and Schréder (1906). But Neresheimer (1903) and Dierks (1926)
are certain that, as such, it does not exist. It is worth noting that Schu-
berg’s ‘‘Basalfibrille’”’ has been widely cited in the literature.

Johnson’s (1893) work is not concerned primarily with a structural
analysis of Stentor’s fibrillar system, but his observations were thorough

202 CILIATE FIBRILLAR SYSTEMS

and critical and, for the most part, they have remained valid. Reference
now may be made to his search for the so-called myoneme canal, de-
scribed by Biitschli and Schewiakoff (1889, p. 1297).

Beneath the “Zwischenstreifen” they found a fairly spacious fluid-
filled canal which surrounded the myoneme throughout its course.
Johnson looked in vain for this canal, finally deciding that he was
“unable to find the least evidence of such a structure, either in optical
or actual sections.’’ The majority of authors—including Delage and
Herouard (1896), Maier (1903), Neresheimer (1903), and especially
Dierks (1926)—agree with Johnson that the canal does not exist except
as an artefact. Schroder (1907), on the contrary, affirms its form to be
oval or circular in cross-section, its shape and position varying with the
degree of body constriction. Roskin (1918) and von Gelei (1929)
claim also to have definitely identified it. The latter regards it as an
“organic part’’ of the myoneme, “‘solid and elastic.”

Neresheimer’s chief contribution to the microanatomy of Stentor was
his discovery of another complex of fibrils to which he gave the name
‘‘Neurophanen.” These were associated contiguously with the myonemes,
but coursed usually peripheral to them. In suitable preparations which
had been differentiated with Mallory’s triple stain, the myonemes were
distinctly red, whereas the neurophanes were colored a dark violet. The
Zwischenstreifen remained unstained. Schréder (1906) maintained that
these fibrils were rather only a structural feature of the “Zwischen-
streifen,” which, according to his results, also with Mallory’s stain, did
show an intense purple color. More recently, however, von Gelei (1925)
and Dierks (1926) have identified similar fibrils, as will be noted
further on.

Neresheimer traced these neurophanes as coursing, each fibril directly
over a myoneme, from the aboral plate to about halfway up the body.
Here some ended in a knob and all others disappeared before reaching
the peristome border. While the myonemes became shorter and thicker
in fully contracted Stentors, the neurophanes appeared sinuous but
otherwise remained unchanged. It is not clear, however, how Neres-
heimer could make sure of the changed or unchanged appearance of
these fibrils, since he stated that he was not able to fix Stentor in an
uncontracted state. Evidently the myonemes may be visible in the living
organism (Biitschli, 1889; Johnson, 1893), which may have been

CILIATE FIBRILLAR SYSTEMS 203

Neresheimer’s means of observing a ‘‘three-fold increase” in the thick-
ness of the contracted myonemes.

Perhaps the most complete structural analysis of the fibrillar differ-
entiations of S. coeruleus is that by Dierks (1926). His work, which
was carried out in Korschelt’s laboratory, considerably revised and
extended earlier accounts of the fibrillar system of this heterotrich.

He noted a gradual thickening of the myonemes from the peristome
border down to the aboral pole, where the fibers do not end abruptly,

Figure 73. Connecting
branch from neuroid to
myoneme in Stentor.
(Dierks, 1926.)
myo. str.—striation of myo-

neme
neu.—neuroid
neu. br.—neuroid branch to

myoneme

as Johnson (1893) thought, but bend sharply inward and revert an-
teriorly to form a pencil-like bundle (see also Schréder, 1906a). This
bundle soon becomes fimbriated, its component fibrils branch, and their
tapering ends disappear in the cytoplasm ‘“‘near the center of the con-
tracted animal.”

Dierks confirmed Neresheimer’s (1903) findings of a second fibril
coursing parallel and usually peripheral to the myoneme, both of which
also stained differentially by Mallory’s method. But the relationship of
these two sorts of fibrils was found by Dierks to be evidently more
intimate than Neresheimer had observed. In various sectioned and
stained preparations, the smaller fibril, or “‘neuroid,” gave off one or

204 CILIATE FIBRILLAR SYSTEMS

more branches (Fig. 73) to its adjacent myoneme, with which it
apparently united (cf. von Gelei, 1929b).

He observed also the knob-like endings of these neuroids, as described
by Neresheimer for his neurophanes, but Dierks apparently could ac-
count for such knobs as being merely the cross sections of fibrils that
happened to be bent near the plane of section.

The cross striations of the myonemes, described by Butschli and
Schewiakoff (1889), were observed by Dierks in the living organism
as well as in his preparations. Johnson (1893) had regarded these as
artifacts due possibly to wrinkling of the myonemes, but the regularity
of their recurrence and spatial relations seemed to preclude this. The
myonemes were usually elliptical in cross section, with the longer axis
of the ellipse directed toward the center of the body. This cross section
revealed definitely an outer cortex (Plasmahiille) and a medulla (Plas-
mamark) (cf. Roskin, 1918).

Dierks’ analysis of Stentor’s membranelle apparatus differs in several
points from most earlier descriptions. The membranelle platelet, sup-
porting each membranelle, represented essentially the aggregate of basal
granules of the component cilia. Continuintg from these granules into
the cytoplasm was a basal lamella, the outline of which was clearly
rectangular and not triangular as Schuberg (1890) had claimed for
his “‘Basallamelle.’’ Dierks’ rectangular lamella could appear as a tri-
angle, whose apex might be directed either toward or away from the
basal granules, depending upon their position when viewed. For these
lamellae were as ribbons, each about three times as long as broad, and
each alike was slightly twisted on its long axis. This, according to Dierks,
accounted for the erroneous interpretation of Schuberg (1890), Schroder
(1906), and others. Not only might the lamellae appear as triangles,
but also their inwardly directed “apexes’’ might then seem to be con-
tinued as a fibril (‘‘Endfadchen’’). To account for the basal fibril, which
Schuberg thought united all of the end fibrils (“Endfadchen’”’), Dierks
observed that his basal lamellae overlapped in such a way that their
ends could give the impression of a continuous fiber, comparable in
appearance, direction, and extent to Schuberg’s described “Basalfibrille.”

3. Euplotes——As a major group of ciliates, the hypotrichs probably
mark the acme of highly differentiated motor organelles (undulating
membranes, membranelles, and cirri) the related fibrillar system of

CILIATE FIBRILLAR SYSTEMS 205

which may appear correspondingly specialized. Another structural dif-
ferentiation, the pellicle, assumes in this connection a significant im-
portance in maintaining the flattened bodily form so characteristic of
the hypotrichs. The often remarkable rigidity of this pellicle has long
been recognized.

Euplotes, the representative whose fibrillar system will now be
reviewed, suitably illustrates these well-known characteristics of the
hypotrichous ciliates.

Maupas (1883) was apparently the first to identify fibrillar differ-
entiations in this genus. He described in Ewplotes patella var. a fibril
extending anteriorly from the basal plate of each of the five anal cirri.
These five fibrils united into a single fiber, which continued anteriorly
and disappeared near the bases of the adoral membranelles.

Maupas’s findings were essentially confirmed by Prowazek (1903)
in his brief account of protoplasmic reorganization in E. harpa. He
further observed the ‘‘solide und fest’’ nature of fixed anal cirri fibrils,
as indicated upon sectioning, when they might be pulled and bent
thread-like by the microtome knife. Similar fibrils were seen to extend
radially from the bases of the other cirri. Prowazek also described and
figured still finer fibrillar lines (‘‘Fibrillenziige’’) going in parallel to
the adoral membranelles. These finer fibrils have apparently not been
identified as such by later workers.

Some years later Griffin (1910) gave a fairly detailed description
of the fibrillar system which he discovered in E. worcesteri. From the
base of each of its five anal cirri, he observed a fiber extending ante-
riorly. All five fibers converged toward the adoral membranelles, near
which they disappeared ‘“‘close to each other.” Unlike similar fibers
described by Maupas (1883) and by Prowazek (1903) for other species
of Exzplotes, these of E. worcesteri apparently did not unite to form
a common strand and were not traceable to the membranelles. Several
finer fibers were found associated with the bases of some of the other
cirri. Their number and direction varied, however, and they had no
connection with those of the anal cirri. Griffin suggested that all of
these fibers might be comparable to myonemes, the number of which
had become reduced with a reduction in rows of cilia, as postulated for
the hypotrichs generally; but he noted also that some of the fibers may
be directed even transverse to the hypothetical original ciliary rows.

206 CILIATE FIBRILLAR SYSTEMS

Yocom (1918), working in Kofoid’s laboratory, found and described
in E. patella a fibrillar system much more extensive than that delineated
in other Explotes by Maupas, Prowazek, and Griffin, as noted above.
In addition to the anal cirri fibers, such as they had found, Yocom
discovered in E. patella ‘“‘a fiber connecting the inner ends of the
cytostomal membranelles’’ (“anterior cytostomal fiber’), and a “‘mo-
torium’’ (after Sharp, 1914) which united the membranelle fiber with
those from the anal cirri. A structural integration was traced, therefore,
between the cytostomal membranelles and the anal cirri. Similar fibers,
radiating from the bases of the other cirri, were also described, but no
connection was found between them and the others above mentioned.
In the “anterior lip” of this species, Yocom depicted a fibrillar lattice-
work which was united by “‘short rodlike projections” to the mem-
branelle fiber.

The intimate contiguity between this fibrillar system of E. patella
and its motor organelles was clearly detailed by Yocom. Certain minor
modifications and additions to his account were made by Taylor (1920),
from studies especially of dissected and slowly disintegrating organisms.
Following Sharp’s (1914) terminology for a comparable fibrillar system
which he had found and elaborately described in Diplodinium ecauda-
tum, Yocom designated this system in E. patella a “neuromotor ap-
paratus.”’

The neuromotor apparatus discovered by Yocom is to be distinguished
from an additional fibrillar system in this same species, which was
carefully worked out by Turner (1933) by means of his modification
of Klein’s (1926) and von Gelei and Horvath’s (1931) methods. His
technical procedure is here worthy of note. After fixing the organisms
in osmic acid vapor for about three seconds, and before the material
was quite dry, Turner added two or three drops of 2-percent silver
nitrate. Within four to eight minutes the nitrate was poured off and the
slide placed in distilled water, barely covering the preparation. Over a
white background, the slide was then exposed to the sun until the
reduction of the nitrate had progressed as desired, according to occa-
sional microscopic examinations. The preparation was then thoroughly
washed in distilled water, dehydrated, and mounted. “The method gives
strikingly clear-cut results.’’ For this study, various other techniques
were also employed, on both whole mounts and sections.

CILIATE FIBRILLAR SYSTEMS 207

By these several methods Turner was able to disclose an ‘‘external
fibrillar network,” which included:

1. The “dorsal network,’ comprising (1) seven to nine longitudinal rows
of granular rosettes, from each of which protruded a central bristle; (2) seven
to nine longitudinal fibrils, uniting the bristles of the rosettes and designated
primary fibrils; (3) longitudinal secondary fibrils, running midway between
the primary fibrils; (4) commissural fibrils connecting, midway between the
rosettes, the primary fibril and on either side its secondary fibril, “pulling the
latter slightly out of line.” The square meshes of this dorsal network, which
appeared remarkably constant, averaged about four microns across.

2. The “collar,” anterior to this dorsal network, comprising, (1) the
inclined row of parallel basal plates of the adoral membranelles, the lower
ends of which “‘rest’’ on the oral lip; (2) a posterior membranelle fibril,
connecting the “upper” ends of the membranelle basal plates; (3) an anterior

POSE SiON TID) pie Sau lene,

ie Se AE AY
a
VEO TINVIO. ea sey VANARAAD i. SPY __. sen. Bf.
von een plate vay los.
rer eyy TAs fa COUP TID
BOK ARG
ee ae ao
Figure 74. Euplotes patella: dorso-lateral view of external fibrillar system. (Turner,
1933.)
ant. m. fib—anterior membranelle fibril com. fib.—commissural fibril
med. m. f.—median membranelle fibril mem. pl.—membranelle plate
post. m. fib.—posterior membranelle fibril pri. fib.—primary fib.
ros.—rosette sec. fib—secondary fibril sen. br.—sensory bristle

membranelle fibril, connecting short commissures at the “lower” ends of
these basal plates; (4) a median membranelle fibril, attached on the basal
plates between the anterior and posterior membranelle fibrils.

These anterior, median, and posterior membranelle fibrils continue this
same relationship with the membranelle basal plates throughout the course of
the membranelles along the ventral ‘‘lapel’’ down to their ending in the
cytopharynx (Fig. 74).

Of primary importance is Turner’s observation that his anterior mem-
branelle fibril is identical with Yocom’s anterior cytostomal fiber, because this
obviously integrates structurally the nmeuromotor apparatus described by
Yocom and the external fibrillar system described by Turner.

3. The “ventral network,’ which is entirely comparable, in its general
features, with the dorsal network, and includes also primary and secondary
longitudinal fibrils and rosettes, with their central bristles.

208 CILIATE FIBRILLAR SYSTEMS

Its pattern, although “constant and characteristic,” is less regular and
“reminds one of badly treated chicken wire.”

Sectioned material showed the fibrils of both dorsal and ventral networks
“to be immediately wzder the pellicle and in contact with it.”

The fibrils stained zntra vitam were distinctly more delicate than those
impregnated with the silver.

Turner confirmed Yocom’s observations on the neuromotor apparatus,
excepting the motorium. In the E. patella which he studied, he was
unable to detect this cited organelle. Instead, the single fiber, formed
by fusion of the five anal cirri fibrils, was traceable to the “‘collar,”
without a break, where it continued as the anterior membranelle fibril
(Turner’s designation) noted above.

4, Vorticella—The vorticellids, by their size and quick reactions,
caught the eye of the earliest microscopists, including, of course,
Leeuwenhoek. The sudden contraction of the spiraling stalk along with
the inversion and closure of the adoral membranelles naturally invited
speculations on the kinds of mechanisms that might account for such
reactions. Geza Entz (1893) cites Wrisberg (1765) as among the first
to describe “mit recht treffenden Worden” this surprising behavior
and to point out its elastic nature.

To Ehrenberg (1838), however, apparently should go the credit for
the earliest detailed studies of the fibrous nature of the contractile stalk
and the detection of longitudinal and circular fibers in the body of
several vorticellids. He attributed to all of these fibers a contractile
function and described in the stalk “muscle’’ cross striations comparable
to those of the striated muscles of other animals.

This fibrillar complex in these peritrichs came to be a favorite object
of investigation by many able workers, especially during the latter half
of the past century: Dujardin (1841), Czermak (1853), Lachmann
(1856), Lieberkithn (1857), Kithne (1859), Rouget (1861), Cohn
(1862), Haeckel (1863), Metschnikoff (1863), Kollicker (1864),
Greeff (1871), Everts (1873), Engelmann (1875), Wrzesniowskt
(1877), Forrest (1879), Maupas (1883), Brauer (1885), Biitschli
(1889), Schewiakoff (1889), and Entz (1893). The literature for this
period has been reviewed by Greeff (1871), Wrzesniowski (1877), and
Biitschli (1889). Similar investigations on the vorticellids have been
relatively meager during the present century, and the most detailed and

CILIATE FIBRILLAR SYSTEMS 209

careful analysis of the finer structure of their fibrillar system, so far as
I have found, is that of G. Entz (1893) on “Die elastischen und con-
tractilen Elemente der Vorticellen.” In the brief review here presented
of this system, I have followed chiefly this excellent account.

In this review of the fibrillar system of Vorticella and its relatives,

Figure 75. Stalk of Zoothamnium arbuscula. (Entz, 1893.)
axo.—Axonem cyt.—Cytophane spr.—Spironem sps.—Spasmonem

it will be more convenient to consider first the fibrils of its contractile
stalk, then those of its body and its peristome.

Previous to the critical investigations of Entz (1893), the “Svzel-
strang”’ in the contractile stalk of the vorticellids, which had been
identified by Ehrenberg (1838) as the ‘“‘Stielmuskel,’ was found by
later analysts to be composed of two parts: (1) the “Stielmuskel,” a
cylindrical, or band-like, strongly refractive fiber, and (2) an adjacent,
granular “Proto plasmastrang,” which accompanied the former through-

210 CILIATE FIBRILLAR SYSTEMS

out its course. Both were surrounded by a delicate membrane, the
“Strangscheide,”” in the same manner that the whole Stielstrang is en-
closed within the outside membrane, ‘‘Stielscheide,”’ of the stalk.

The Stelmuskel, so most authors (e.g., Engelmann, 1875; Wrzesniow-
ski, 1877) agreed, was made up of distinct fibrils running variously
transverse or parallel to its long axis. For Biitschli (1889), however,
this composition of the Stielmuskel represented rather (and probably
more in line with his alveolar hypothesis) an attenuated meshwork. And
Ehrenberg (1838), Leydig (1883), ef al. could apparently see cross
striations in the Size/muskel of some vorticellids, comparable with those
of metazoan muscle.

Entz (1893) described for the giant stalk of Zoothamnium arbuscula,

Figure 76. Spasmonem
(cross section) in Zoo-
thamnium. (Entz, 1893.)

and as typical for all the Contractilia, a Stielstrang comprising three
well-defined fibers: a “Spasmonem,” a “Spironem,” and an “Axonem”’
(Fig. 75), all enclosed within the Strangscheide. He identified the
Spasmonem with the Stielmuskel (noted above), and the Spironem and
Axonem with the Proto plasmastrang.

In the smaller branches of Z. arbuscula, the Spasmonem (Stielmuskel)
is a round fiber which, in the main stalk, becomes compressed by the
adjacent Spironem so that a cross section of the former appears crescent-
shaped or, since one edge of the crescent is swollen, rather like the form
of a comma (Fig. 76). As shown by Engelmann (1875), it is bire-
fringent and may frequently be tinted a steel-gray or appear faintly
greenish or yellowish. It is stainable in carmine and disintegrates in
alkali and in mineral salts—teactions which are more rapid in the
younger stems, and which, according to Entz, would indicate that the
Spasmonem is not cellulose; neither is it chitin nor keratin, although
least unlike chitin.

This age difference shows itself also in the finer structure of the

CILIATE FIBRILLAR SYSTEMS pAligt

Spasmonem. In the younger branches it is fairly homogeneous, but in
the older stems and in the main trunk one may distinctly observe, in
its hyaline ground substance, parallel longitudinal fibrils that gradually
disappear toward the distal end. Nodal interruptions in these parallel
fibrils apparently account for the cross striations cited by earlier workers.
Outside these longitudinal fibrils is a single, or composite, spiraling
fibril, and centrally placed along the Spasmonem are ovoid discs, each
containing a central granule or “nucleus.”

Entz’s Spironem and Axonem constitute, as noted above, the Proto-

Figure 77. Three components of Spironem of Zoothamnium, (Entz, 1893.)
cir. filb.—circular fibril cyto.—cytophanes 1. filb—longitudinal fibrils

plasmastrang. This structural duality had been previously overlooked,
perhaps because the Spironem is wound closely around the Axonem.
Also, the spirals of the former, which are contiguous when the stalk
is fully contracted, separate increasingly as the stalk is extended, so that
the two fibers may easily appear as one in the completely extended stalk.

In its finer details, the Spironem (Fig. 77) shows beneath its investing
membrane a spirally wound fibril, under which are several longitudinal
fibrils. All these fibrils are birefringent and, as noted for those in the
Spasmonem, nodes of less refringence give here also the effect of cross
striations. Along the axis of the Spironem are oval bodies (Cytophanes)
containing each a central granule (Caryophane). These Cytophanes are
connected by a longitudinal fibril (Fig. 75) similar to a string of pearls.

In the Axonem (Fig. 75) longitudinal fibrils comparable to those in
the Spironem also occur, but their course is apparently completely inter-

212 CILIATE FIBRILLAR SYSTEMS

rupted at regular intervals by Cytophanes which are relatively much
larger than those found in the Spzronem.

It seems to be generally agreed that the stalk of all the vorticellids,
both the Contractilia and the Acontractilia, is a direct continuation of the
body. Bearing this in mind, we may now briefly review the fibrillar

Figure 78. Pellicular structure, and branching of longitudinal myonemes in Zootham-
nium. (Entz, 1893.)
lon. myo.—longitudinal myoneme pell.—pellicle

system of the body proper and later note how the parts of this system
are related to those in the stalk.

For convenience in description and with special reference to the genus
Vorticella, we may regard the body as divisible into three fairly well-
defined regions: (1) the funnel, lying between the stalk and the ciliary
ring; (2) the bell, that part of the body above the ciliary ring; and
(3) the disk, which includes the peristomal border, adoral zone, and
cytostome.

The entire body, like the stalk, is covered by a pellicle which, accord-

CILIATE FIBRILLAR SYSTEMS 213

ing to Entz (1893), is not homogeneous but distinctly sculptured (Fig.
78) as if composed of “Stabchen” that overlap, somewhat as tile on
a roof.

The fibrillar system of the body lies immediately beneath the pellicle
and comprises an outer and an inner complex of fibrils, or myonemes.
Each such complex is in turn composed of (1) an outer circular layer,
and (2) an inner longitudinal layer, making in all, then, four fibrillar
layers.

Lachmann (1856) was first to describe the outermost circular layer.

Figure 79. Myonemes of stalk sheath of Vorticella. (Entz, 1893.)
lon. my.—longitudinal myoneme 0. sp. my.—outer spiral myoneme

It was later recognized also by Stein (1867), but apparently overlooked
by other investigators previous to Entz (1893). As a single fibril, it
spitals directly beneath the pellicle and may be followed from the
attachment of the stalk uninterruptedly to the center of the disc. Its
spiral course accounts for the annular appearance of the pellicle. Entz
(1893) thinks also that the birefringence of the pellicle may be due
to this underlying fibril, since, as shown by Engelmann (1875), all the
fibrils are refractive in polarized light.

The fibrils of the next layer, the longitudinal fibrils, lie immediately
below layer (1), noted above, and likewise pass from the style’s attach-
ment to the center of the peristomal disc. On this disc they of course
are radially arranged. This layer was found by Greeff (1871), but
Butschli (1889) questioned its existence.

It should further be noted also that this outer fibrillar complex is
continued uninterruptedly into the protoplasmic lining of the style sheath
(Fig. 79). This will be referred to again in later discussion.

214 CILIATE FIBRILLAR SYSTEMS

i mer. go.

Figure 80. Arrangement of second complex of body fibrils in Epistylis. (Entz, 1893.)

fnl. msc.—funnel muscle mer. myo.—meridional myoneme
per. bdr.—peristome border

Of the second complex of fibrils, its outer component, unlike the outer
single fibril of the first complex, behaves rather as a large fiber that may
become split into several fibrils. This set of fibrils courses spirally around
only the lower half of the fannel portion of the body (Fig. 80). Toward
the ciliary ring it disappears and, according to Entz (1893), reappears
actually to form this ciliary ring “out of a number of fine fibrils (Myone-

CILIATE FIBRILLAR SYSTEMS 215

men).” This would account for the interlaced appearance of the ciliary
ring. Above the ring, in the be// part of the body, this set of fibrils could
not be detected except in the peristome border, where it forms a fairly
strong spiral, narrowly wound. Its occurrence there was described by
earlier authors as a ‘‘sphincter ring’ of the peristome border (Fig. 80).

On the disk, this fiber appears as a fairly thick strand, one end of
which originates in the center of the disk and, after following at their
base the several turns of the adoral membranelles along the outer margin
of the disk and through the mouth into the gullet, it continues down the
inner surface of the gullet the whole length. Along the gullet wall Entz
thought that, in some preparations, he could observe this fiber branching
into many increasingly fine fibrils.

The inner, longitudinal layer of the second fibrillar complex is a
direct continuation of the Spasmonem (Stielmuskel) of the contractile
stalk. Upon entering the body this Spasmonem breaks up into its com-
ponent fibrils, which diverge and so form the ‘funnel muscle” (Fig. 80)
of earlier authors. Higher in the funnel, these fibrils may rebranch, then
anastomose into a network. At the ciliary ring, the fibrils curve round
it as crescent-like spans, which are somewhat larger and apparently more
dense, and continue directly up the wall of the be// to the peristome
border. These meridional myonemes (Fig. 80) may end singly on this
border or, as Engelmann (1875) noted, they may bifurcate and unite,
each of a pair with its adjacent neighbor, to form “‘arcades”’ just below
the peristome border. The fibrils then span this projected border, much
as they curved over the ciliary ring, and thereupon proceed as radii to
the center of the disc. Toward this center, these fibrils may sometimes
branch and anastomose to form varied and striking patterns.

Finally, it may be mentioned that, in addition to the four layers of
fibrils noted above, Entz (1893) describes a fairly thick strand of other
fibrils which originate from the middle of the disc and project down-
ward through the endoplasm and finally disappear in the region of the
cytostome.

B. INTERPRETATION

The essential structural components of the fibrillar systems of several
representative ciliates have been briefly reviewed in the foregoing para-
graphs. It remains now to consider the several functions that have been

216 CILIATE FIBRILLAR SYSTEMS

ascribed in the literature to each of these fibrillar mechanisms, as illustra-
tive for similar systems that have been studied thus far in a large number
and variety of other ciliates.

It will be recalled that Ehrenberg (1838) saw in the “‘stalk muscle”’
of Vorticella cross striations which he regarded as comparable to those
of the striated muscles of other animals. Also, he detected both longi-
tudinal and circular fibers in the body of several other vorticellids. Quite
in line with his search for such comparisons, he attributed to all of these
fibrils a contractile function. For him they were literally miniature
muscles which had their structural and functional counterparts in the
muscles of macroscopic organisms (Maupas, 1883). Evidently, Ehren-
berg’s comparison between microérganisms and macroérganisms led him
to look for a one-to-one correspondence between microérgans and
macroorgans.

It will be recalled that what Ehrenberg regarded as the stalk muscle
(Stielmuskel) was analyzed into several components by later investi-
gators. These, according to Entz (1893), included: a cross-striated, rod
or band-like Spasmonem, and another rod-like strand, the Axonem,
around which was coiled the Sprronem. Each of these was, in turn,
analyzable into longitudinal fibrils, whose properties apparently ac-
counted for their birefringence and cross striations. The Spasmonem and
Spironem showed also, spiraling within their own spiral strand just
outside its longitudinal fibrils, a fine fibrillar coil coursing throughout
their length. Also, both Spironem and Axonem had, centrally located,
the ‘“‘cytophanes” which, with their longitudinal interconnections, ap-
peared like a string of pearls. Thus Ehrenberg’s “Stielmuskel” turns out
to be a highly complex mechanism which is structurally quite different
from the “‘muscles of other animals’ to which he had compared it.

Finally, mention was made also of the circular and longitudinal
myonemes of the protoplasmic lining of the style sheath, which are
continuations of the owter fibrillar complex of the body myonemes.

Taking into account this assemblage of highly differentiated com-
ponents within the stalk of the Contractilia, together with a few rather
inconclusive experimental results, various interpretations were advanced
to account for both the contraction and the extension of this stalk. These
may be summarized as follows:

1. The majority of investigators agree that the quick spiraling con-

CILIATE FIBRILLAR SYSTEMS JAF

traction of the stalk is referable to contractile properties of some one
or more of its components. Dujardin (1841) was apparently the only
one to regard the stalk sheath as the seat of that contractility. Ehren-
berg’s Stzelmuskel was for him the contractile component, but also the
only component that he had recognized within the stalk sheath. Of the
three fibers into which this Stzelmuskel was later analyzed, viz., the
Spasmonem and Spironem and Axonem, Kiuhne (1859) considered
the latter two, or Protoplasmastrang, as the contractile organelle. This
he likened to the sarcoglia of metazoan muscle. Other proponents of
this contractile theory (Czermak, 1853; Engelmann, 1875; Wrzesniow-
ski, 1877; and others) regarded the Spasmonem as the contractile fiber
which, for later authors, represented a bundle of fibrils or myonemes
that continued without interruption into the body of the vorticellid.

2. Apparently there is general agreement among all the investigators
that the mechanism of sfalk extension inheres in the elastic properties
of the pellicle (Bitschli, 1889; Entz, 1893). Only Kithne (1859), who
alone regarded the Spironem and Axonem as the contractile organelles,
attributed elasticity to both the pellicle and the Spasmonem, thus to
account for the stalk’s extension.

3. While the majority, as noted, support the contractile theory of
stalk contraction, there is a minority group who maintained that the
vorticellid stalk, in its quick spiraling retraction, actually does not con-
tract as such but instead recoils somewhat as a coiled spring.

A chief proponent of this elastic theory is Entz (1893), who sees the
Spasmonem not as a contractile fiber but, like Kiihne, as an elastic fiber.
This organelle is, according to Entz, primarily responsible for the sudden
recoil of an extended stalk. He likens this elastic fiber to a curly hair
which when stretched and then released will resume its spiral form.

The opposing force, tending to “stretch” this normally spiral Spas-
monenz, 1s inherent in the elastic pellicle. Thus pellicle and Spasmonem
constitute a pair of “antagonistic elements.”

Associated with this pair is another pair of antagonistic elements,
viz., (1) the longitudinal myonemes in (a) the stalk sheath and (b)
the Spironem; and (2) the circular myoneme in (a) the stalk sheath
and (b) the Sp/ronem. These opposing pairs of fibrils are relatively
weak, but of a strength sufficient to determine by their antagonistic
contractions whether the stalk ‘‘contracts’”’ with the recoil of the elastic

218 CILIATE FIBRILLAR SYSTEMS

Spasmonem or extends by means of the elastic pellicle. Accordingly the
longitudinal fibrils of the stalk sheath and the Spironem tend to reinforce
the elasticity of the Spasmonem, and the spiral myoneme of both sheath
and Spironem, upon relaxation of the longitudinal pair of myonemes,
tend to reinforce the elasticity of the pellicle.

Entz’s rather elaborate thesis at least makes evident the elaborate
differentiation of the vorticellid’s stalk, and so provides one working
hypothesis which might be experimentally tested. With modern tech-
niques, this should not prove very difficult. And, in the writer’s opinion,
the vorticellids offer extraordinary possibilities for some clear-cut and
fruitful experiments which should help toward a better understanding
of their fibrillar mechanisms.

It seems to be more generally agreed that the fibrillar complexes of the
funnel, bell, and disc are primarily contractile. If so, and if the
Spasmonem of the stalk is, as Entz and others claim, continued without
interruption into the funnel and bell as the longitudinal fibrils of the
“inner complex,” then it should follow that if these fibrils of the inner
complex are contractile, one should expect the Spasmonem also to be
contractile. Entz (1893) apparently does not discuss this point.

Finally, it may be noted that Entz’s thesis attributes no specific function
to the Axonem. Both he (1893) and Stein (1854) suggest that it might
function as a neuroneme.

Observations on the fibrillar system of Stentor have provided a basis
for some interpretations which appear more plausible than those cited
above for the vorticellids. These interpretations have to do exclusively
with the myonemes. The other fibrils are poorly understood.

Before discussing these, however, the essential components of this
system should be recalled to mind. As was pointed out, and as will
doubtless be familiar to the reader, the myonemes of Stentor are band-
like fibers which course beneath and slightly lateral to the longitudinal
rows of body cilia and the curved ciliary rows on the oral disc.

Associated with these band-like myonemes, but lying peripheral to
and mostly contiguous to them, are other fibrils which Neresheimer
(1903) described as “neurophanes” and which Dierks (1926) recently
further described and named “‘neuroids,” since he could not fully identify
these with Neresheimer’s neuronemes.

Dierks added one very significant observation, which, if verifiable,

CILIATE, FIBRILLAR SYSTEMS 219

would definitely suggest a conductive function for his neuroids. He saw,
in several preparations, that the smaller fibril gave off one or more
branches to its adjacent myoneme. As will be noted further on, this claim
has recently been challenged by von Gelei (1929b).

Another fibril, found and described by Schuberg (1890), connected
the basal lamellae of the entire series of membranelles. This basal fibril
was noted also by Johnson (1893), Maier (1903), and Schréder
(1906), but Neresheimer and Dierks maintain that such does not exist.
Dierks (1926) does describe a fibril coursing along the platelets of the
membranelles, which he thinks both Schuberg (1890) and Meyer
(1920) may have seen but misinterpreted.

The evidence supporting the interpretation that the myonemes are
contractile organelles has been supplied from many sources, beginning
with their discoverer, Lieberkithn (1857). Ehrenberg (1838) claimed
to have seen in a living Stentor that in the extended state the myonemes
were serpentine, while with the contraction of the body they became
shortened and straight. But since he misidentified the pigmented meridi-
ans as myonemes, the significance of his observation is uncertain.

Probably the most significant evidence for the contractile nature of
Stentor’s myonemes was provided by Johnson (1893) and verified by
Dierks (1926). The former investigator compressed the living organism
beneath the cover slip and could then observe the myonemes “alter-
nately to extend and contract,’ concluding that ‘“‘no one who has once
observed them under these conditions can doubt that they are responsible
for the contractions of the animals.” Biitschli (1889) apparently made
similar observations on living Stentor upon applying an electric stimulus.
Merton (1932) has confirmed these former evidences of the contractile
properties of Stentor’s myonemes.

Dierks (1926) states further that “these fibrils are definitely shorter
and thicker in the contracted animal and longer and shorter’? when the
organism is extended. He also noted, however, that this behavior was
lacking in the myonemes toward the posterior end of the body, and
supposed that here the body protoplasm contracted more or less inde-
pendently, suggesting a progressive differentiation in the myonemes
anteriorly along their course.

The assigned contractile function of the myonemes of Stentor appears
to be agreed upon without exception. Schroder (1906) added to the

220 CILIATE FIBRILLAR SYSTEMS

knowledge of their contractile function probably also that of conducting.
But for the other fibrils of this ciliate, evidence regarding their actual
function or functions is mostly wanting. Interpretations based on mor-
phological evidence concern chiefly the relations of these fibrils (neuro-
phanes or neuroids) to other organelles.

Neresheimer’s observation on the parallel course of the neurophanes
with the myonemes and the differential stainability of these two kinds of
fibrils, suggested for the neurophanes a conductive function. By means
of a drop-weight apparatus (Fallmachine) he tested the responses of
Stentor to a variety of narcotics: morphine, strychnine, atropine, caffein,
and so forth, and to other chemicals which are known to affect the nerves
of higher animals. On the basis of an assumed selective action of these
reagents, he derived evidence which seemed to favor assigning a nervous
function to the neurophanes. The technique for these tests was ingeni-
ously devised and might prove useful also for others. But owing to lack
of adequate controls and because of other possible interpretations, his
results do not seem very convincing.

Schroder (1906) considered Neresheimer’s interpretation of the
neurophanes invalid. He believed that Neresheimer had mistaken a struc-
tural feature of the “mid-stripes’”’ for his neurophanes, as indicated upon
comparing the latter’s Figures 7 and 8 with Schréder’s Figures 1-5.

From Dierks’ more extensive and detailed studies, however, there can
be no doubt about the identity of another fibril coursing with each of
the myonemes. The occurrence of these was consistent and their relation-
ship with the myonemes apparently significant. As formerly noted,
Dierks found that in good preparations branches were given off from
the neuroids to the myonemes which they evidently joined. He considered
several possible functions which this connection between neuroids and
myonemes might indicate, and finally, as a plausible hypothesis, sug-
gested for them a conductive function.

Von Gelei (1926c) rejects this interpretation. From his own previous
studies (1925) on Stentor, he concludes that Neresheimer’s neurophanes
and Dierks’s neuroids are real and are identical structures. But, according
to von Gelei, these neuroids are not fibers but bands composed of fibrils,
which are quite wide, especially in the aboral region. They are not a
structural feature of the pellicle (Schroder, 1906), but are swbpellicular
and fused to the pellicle. This was indicated in contracted Stentors by

CILIATE FIBRILLAR SYSTEMS 221

the body groove overlying the neuroid, as produced by the contraction of
the myoneme under the neuroid. The attachment of the pellicle to the
neuroid and the latter, in turn, to the myoneme would, upon contraction
of the myoneme, cause an ingrooving of the pellicle.

Studies on the hypotrichous ciliates yielded apparently the first re-
corded example of fibrils directly associated with ciliary locomotor
organelles. Engelmann (1880) traced such fibrils from the bases of the
marginal cirri of Stylonychia ‘‘nach der Mittellinie des Leibes.” He
postulated for these fibrils a conductive function in transmitting im-
pulses, as in nerves of higher animals, from the ventral region of this
hypotrich to the cirri, the movements of which might thereby be
regulated.

Engelmann’s interpretation was formulated entirely by analogy and
he offered no substantial evidence to support it. It seems to have been
generally rejected by his contemporaries. Maupas, who three years later
(1883) described similar fibrils in E. patella var., said, in a brief foot-
note (p. 622) concerning his own findings: ‘Quant a la signification
physiologique de ces racines ciliaires, j’avoue ne pas la connaitre.’”’ And
with reference to Engelmann’s discovery of ‘“‘fibrilles nerveuses’’ in
Stylonychia mytilus, Maupas adds: “‘Je ne sais comment concilier des
interpretations aussi divergents.’’ Biitschli regarded Engelmann’s inter-
pretation as untenable and proposed, on equally meager evidence, a
contractile function for these fibrils. Maier (1903), who attributed a
contractile function to the basal fibril associated with the basal lamellae
of membranelles in Stentor, concluded that the lateral cirri fibers in
Stylonychia were “required” for the support of these cirri.

Prowazek (1903), on the other hand, suggested that the anal cirri
fibers which he found in E. arpa might perform the dual functions of
contractility and conductivity. He noted, however, that E. arpa could
move each anal cirrus independently and could flex the tip of these cirri
quite at will. He observed, moreover, that a detached anal cirrus might
continue its contractions for a time. These important observations have
apparently been overlooked by later investigators.

It will be recalled that Griffin (1910) compared the anal cirri fibrils
of E. worcesteri, which he found and described, with myonemes whose
number might have become reduced phylogenetically during reduction
in the hypotrichs’s rows of cilia. He pointed out a difficulty in this

222 CILIATE FIBRILLAR SYSTEMS

interpretation, viz., that some of the few fibrils of other cirri in this
species were not aligned longitudinally, but might be even transverse to
the longitudinal axis.

Griffin based his concept of contractility for these fibrils in E. worces-
ter? only partly, however, on this comparison with myonemes in other
ciliates. He accepted Biutschli’s interpretation of a contractile function
for the marginal and anal cirri fibrils in Stylonychia as a much more
reasonable view, and also noted that ‘‘Every detail of arrangement and
structure indicates that the fibrils are, principally at least, contractile in
function.”” He observed, also, that the fibrils were developed around the
bases of the cirri in such a way as to assist in producing the ordinary
motions. ‘As the anal cirri have only a single strong motion, a vigorous
kick directed backward, each needs but a single strong fibril.”

Evidently Griffin had not seen any reversal in the effective stroke of
these anal cirri. In several other species of Ezp/otes this reversal is not
uncommon in both swimming and creeping movements. Should that be
the case in E. worcesteri, then a contractile function for the anal cirri
fibers is scarcely conceivable. The effective function of a contractile fibril
is obviously a pulling but not a pushing function. Moreover, Prowazek’s
account of the contractile behavior of the anal cirri in E. harpa, cited
above, largely vitiates any claims for contractility in the cirri fibrils of
this species, and similar behavior has been observed by the writer in the
anal cirri of E. patella. It was also noted (Taylor, 1920) that cutting
these fibrils apparently did not impair the effective stroke of the anal
cirri, whether that stroke was directed backward or forward.

The latter evidences were thus cited against the assumption that these
fibrils in E. patella were contractile. It was further shown by those
experiments that cutting the anal cirri fibrils interrupted the codrdinated
movements between anal cirri and adoral membranelles. Also, severing
the membranelle fiber likewise interrupted the codrdinated movements
of the membranelles on opposite sides of the incision. Incisions in other
parts of the body did not impair the codrdination of these organelles.
It should be pointed out, however, that those incisions which did inter-
rupt codrdinated movement of organelles cut not only the anal cirri
fibrils or the membranelle fiber, but also the peripheral fibrils which
have since been described especially by Turner (1933). Whatever rdle,
if any, these peripheral fibrils may have in E. patella’s co6rdinated

CILIATE FIBRILLAR SYSTEMS 223

behavior was not demonstrated by the writer's (1920) experiments.
Reinvestigation of this problem, especially on a more favorable form
such as Lichnophora (Stevens, 1891), ought, therefore, to be undertaken
in order to determine what relative rdles the so-called introplasmic fibrils
and the peripheral fibrils each perform in the codrdinated movements
of the organelles with which such fibrils are demonstrably associated.

Several investigators (Bélaz, 1921; Jacobson, 1931; Peschowsky,
1927) have maintained that all such cirri fibrils in the hypotrichous
ciliates and the fibrillar systems in various other ciliates are primarily
or exclusively supporting in function. Jacobson (1931), for example,
studied by means of various techniques, including the silver-nitrate
methods, the fibrillar systems of some twenty-seven ciliates. These com-
prised representatives of all the major groups and included among the
hypotrichs E. patella and E. charon. She concluded from the results that
no evidence was found in support of a conductive function for any of
the fibrillar systems studied. It was pointed out that in the hypotrichs
whose motor organelles are localized, fibrils are nevertheless present,
as on the dorsal side where cilia are wanting. Reference on this point
should be made to Turner’s (1933) studies on E. patella, which showed,
as previously mentioned, that longitudinal fibers connect the bases of
the dorsal and ventral rows of bristles, whose function is not known.
But they may have a function and if that function is, as has been
suggested, sensory in nature, it is surely not inconceivable that the
associated fibrils may facilitate its performance.

Another and more significant observation was made by Jacobson, viz.,
that in Sciadostoma difficile, where three ciliary rows surround the
anterior body pole, no silverline connection exists between the basal
granules. An impulse, therefore, originating at the anterior ciliary ring
would need to pass nearly to the posterior body pole and back again in
order to affect adjacent cilia. Since our assumptions should be, first of
all, plausible, one would justifiably regard this morphological evidence
contradictory to the thesis that these silverlines in S. difficile are con-
ductive in function. It may be pointed out, however, that Chatton and
Lwoff (1935) apparently demonstrated that the fibrils (cinétodesme )
described for several holotrichous ciliates, which alone were connected
with the basal granules, could not be stained by silver impregnation,
whereas other adjacent fibrils (interpreted by them as silverline fibrils )

224 CILIATE FIBRILLAR SYSTEMS

could be silver impregnated but apparently were not in contact with the
basal granules.

The thesis that all fibrils of all ciliates are only supportive in function
is, of course, not tenable. One would at once except myonemes. But why
limit the exceptions to myonemes? If, in the eons of time, protoplasmic
fibrils have become differentiated so as to facilitate contractility in
protistan organisms, who can deny them the capacity to have become
differentiated also to facilitate conductivity or some other function in
these unicellular forms of life? All our assumptions should be both
plausible and reasonable assumptions, the validity of which may, in the
last analysis, be demonstrated only by experiment.

In conformity with the less specialized differentiation of its motor
organelles, the fibrillar system of Paramecium is also relatively less
specialized, as compared with those of the other three representative
ciliates reviewed in foregoing paragraphs.

From the accounts especially of Schuberg (1905), von Gelei (1925-
31), and Lund (1933), it appears that two separate and distinct com-
plexes have been described in the literature, which may be represented
by the outer fibrillar complex of von Gelei and of Klein (1926-32),
and by their inner fibrillar complex. For these latter authors, both com-
plexes are swbpellicular.

According to Schuberg (1905) and Lund (1933), howeyer, the
above-mentioned outer fibrillar complex is not subpellicular, but actually
represents the polygonal pattern of the pellicle itself. Lund emphasizes
the fact, therefore, that the essential fibrillar system of Paramecium 1s
exclusively the complex which is associated with the basal granules of
the entire motor mechanism, including the cilia of the mouth, cyto-
pharynx, and cytoesophagus, as well as the body cilia. Lund’s fibrillar
system would essentially include, therefore, the inner fibrillar complex
of von Gelei and of Klein.

The discrepancies just noted in the structural interpretations of
Paramecium’s fibrillar system are obviously crucial, since they go hand
in hand with discrepancies in the functional interpretations of that
system. This holds, of course, not only for the investigators cited above,
but for various others also.

Referring now to these diverse functional interpretations, Schuberg
(1905) suggested that the fibrils connecting the basal granules, such as

CILIATE FIBRILLAR SYSTEMS 225

he had described for Paramecium and Frontonia, might function in the
well known metachronism of ciliary movement. He was not inclined,
however, to compare these fibrils with Neresheimer’s neurophanes nor
with the neurofibrils of the Metazoa. Obviously, since it had not yet
been proved that the neurofibrils were the conductive elements of nerves,
then an analogy between these neurofibrils and the fibrils found in
Paramecium would not add to our understanding of either the one or
the other.

Mention may here be made of the system of fibrils described for
Paramecium by Rees (1922). These have an arrangement and relation-
ship quite different from any of the fibrils referred to above. According
to Rees, all of the fibrils he observed connected the basal granules of
the cilia of the body and cytopharynx with the motorium, located just
anterior to the cytostome, by coursing through the cytoplasm in several
graceful whorls.

From the results of his few experiments, Rees concluded that these
fibrils were conductive in function. By severing with a microneedle the
fibrils connecting the cytopharyngeal membranelles with the motorium,
the codrdinated movements of these membranelles was interrupted. Like-
wise, the codrdinated movement of body cilia was interrupted when the
neuromotor center was destroyed.

In view of the later descriptions of the more peripheral system of
fibrils in Paramecium, Rees’s experiments should be repeated. It may be
noted also that Jacobson (1931) reinvestigated this fibrillar complex
described by Rees, and concluded that he had observed not fibrils but
internally discharged trichocysts, as effected by the killing agents used.
Her figures of fixed Paramecium illustrating such trichocysts are com-
parable to some of Rees’s figures, except for certain regularities in the
“fibrillar whorls” depicted by Rees. One could suppose that the in-
ternally discharging trichocysts might tend to follow the course of these
whorls of fibrils and so reproduce that course in fixed material, which,
through differential staining, revealed the trichocysts but not the fibrils.
Only further careful investigation can clarify this discrepancy.

The interpretations of von Gelei (1925-31) and of Klein (1926-32)
agree in ascribing a conductive function to the inner fibrillar complex
of Paramecium, but their views on the relations and functions of its
outer fibrillar complex are not in accord. The basis for their interpreta-

226 CILIATE FIBRILLAR SYSTEMS

tions is confined primarily to morphological evidence. This evidence is
derived, however, not only from their studies on the fibrillar system of
Paramecium, but also from comparative studies of a considerable number
and variety of other ciliates.

According to von Gelei’s interpretation, Klein’s strongly birefringent
“indirekt System” (Meridian II. Ordnung) is nothing more than the
well known “‘pelliculare Gittersystem” of the ciliates, which functions
as a supporting network to maintain body form. As such, it has no
differentiated structural connections with the less refractive inner fibrillar
complex (von Gelei’s neuroneme system), which alone, therefore, 1s
the peripheral conductive mechanism. Von Gelei sees in the longitudinal
and transverse fibrils of his neuroneme network a mechanism which
structurally integrates the ciliary basal granules, the trichocysts, and even
the contractile vacuole pore into a codrdinated whole (“einer koordi-
nierten Einheit’’).

This integrated mechanism codrdinates automatically the metachronous
effective strokes of successive cilia. The direction of the stroke of one
cilium activates conditionally that of the next by way of the basal
apparatus (basal granule, basal ring, and ‘‘Nebenkorn’”’), which repre-
sents a sensory organelle. For the codrdinated activity of the organism
as a whole, however, von Gelei recognizes in his peripheral neuroneme
system the absence of a neuromotor center. Accordingly, he regards the
“intraplasmatic’’ fibrillar complex described by Rees as a centralized
mechanism which may complement the peripheral neuroneme complex,
thus to provide reasonably a unified neuromotor system. Von Gelet
(1929b) observed by means of his silver-osmium-formol method a
“platte’”” beneath the basal apparatus, which, he thought, might serve
as a sort of end plate connecting Rees’s intraplasmic fibrils with the
peripheral neuroneme complex.

As has been previously indicated, Klein’s (1926-32) interpretation
of his inner fibrillar complex (Meridian I. Ordnung) agrees essentially
with von Gelei’s concept of a conductive function for his peripheral
neuroneme network. Among their minor differences, Klein would ac-
count for the commonly observed reversal in the effective stroke of cilia,
not by means of Rees’s intraplasmic complex as von Gelei supposed, but
by way of a “‘primitive reflex arc.” This reflex arc includes the axial
filament of the cilium as the receptor, the basal apparatus as the “‘relator,”’

CILIATE FIBRILLAR SYSTEMS 227

and the protoplasmic sheath as the effector. The basal apparatus com-
prises the basal granule and two “Nebenkorner.” The latter serve to
spread the proximal part of the reflex arc and, with the basal granule,
may function as a kind of commutator that regulates the direction and
change in direction of the effective stroke of the cilium.

We may note that while such a mechanism might conceivably account
for a reversal in the effective stroke of a given cilium, obviously it does
not, as such, provide for the synchronous reversal of the many other
cilia, which is an essential part of the problem of codrdinated movement.

In this connection, reference may be made to a fairly recent paper
by J. C. Hammond (1935), who would refer the phenomenon of
synchronous and metachronous ciliary behavior to an anterior-posterior
physiological dominance in the organization of the cell, as opposed to
the concept of a neuromotor mechanism. This thesis might help to
account for this more general codrdinated behavior (Rees, 1921), but
it would presuppose a reversal in anterior-posterior dominance of the
physiological axis in order to explain the reversal of ciliary stroke so
common in the swimming or creeping behavior of ciliates. Moreover,
the well known localized reversal of a few or many of the cytostomal
membranelles in the intake or ejection of solids would obviously require
a similar presupposition.

The chief discrepancy between Klein’s and von Gelei’s interpretations
of the fibrillar system of Paramecium concerns the structural and func-
tional relations between their outer fibrillar complex and their inner
fibrillar complex. Klein (1928, p. 203) regards these two fibrillar com-
plexes as a continuum. His ‘‘Meridian II. Ordnung’” is a derivative of
his ““Meridian I. Ordnung,” as are also all cilia, basal granules, ‘‘Neben-
kérner,”” trichocyst granules, and protrichocyst granules (secretory, or
“Tektin” granules). In its re-genesis, observed during reorganization,
many more fibrils (Profibrille) are formed than are retained, varying
with the species and genus, and of those that persist some may unite to
form composite fibrils (Biindelfibrillen), as occurs in Paramecium
(Klein, 1932).

Klein’s silverline system incorporates as a unit, therefore, both struc-
turally and functionally, the outer and inner fibrillar complexes. In its
re-genesis, it functions as a ‘‘form-building system,’’ and some of its
fibrils become more resistant and rigid by the addition of a secondary

228 CILIATE FIBRILLAR SYSTEMS

substance, the “‘fibrillare Komponente”’ (1928, p. 255). By virtue of its
integral relation with the basal apparatus of the cilia and with the tricho-
cysts, he attributed to his silverline system also a specifically conductive,
coordinative function.

Chatton and Lwoff’s (1935-36) criticisms of Klein’s interpretations
bear mainly upon the structural relations of the fibril to which are ad-
joined, always on its left s7de, the basal granules. The former authors
think Klein’s silverline system is quite separate and distinct from their
infraciliature. It appears to the writer, however, that this discrepancy
might be completely resolved by identifying Chatton and Lwoff’s infra-
ciliature with the longitudinal fibrils and basal granules of Klein and
von Gelei’s inner fibrillar complex. Further critical investigation would
be needed, of course, to establish such identity.

FIBRILLAR SYSTEMS OF OTHER CILIATES

A. HOLOTRICHA

Ancistruma (Kidder, 1933).—The fibrils in Ancistruma mytili and
A. isseli are of three types—the Jongitudinal fibrils of the ciliary rows,
the irregularly distributed transverse fibrils, and the net complex of fine
fibrils in the peristomal region. The latter fibrils seem to connect di-
rectly with the basal bodies of the peristomal cilia, and they are coarser
and less numerous in A. /ssel7 than in A. mytilz. The longitudinal fibrils
in A, mytil? are continuous around the posterior end, but in A. ssselz
they center about a posterior suture.

A number of fine fibrils connect the inner dorsal row of peristomal
cilia to the outer dorsal row. These seem to be distinct from the net
complex and probably are in the nature of concentrated transverse
fibrils. In A. zsseli such fibrils are absent, and instead the fibril of the
outer ciliary row joins that of the inner dorsal row.

Fibrils resembling the interstrial fibrils of Boverza (Pickard, 1927)
are sometimes seen in A. mzytili, but these may represent a deep-lying
network of the same type regularly seen in the peristomal region.

METHODS

Fixatives: Schaudinn, sublimate-acetic, Bouin’s, Zenker’s, Champy’s.
Stains: Heidenhain’s and Delafield’s haematoxylin, crystal violet-sulph-
alizarinate (Benda’s).

CILIATE FIBRILLAR SYSTEMS 229

Klein’s silver method, modified to include fixation with osmic acid fumes
and impregnation period of one to three hours in 2-percent solution of
silver nitrate.

Boveria teredinidi Nelson (Pickard, 1927).—Surrounding the cyto-
stome is an oral rng which begins and ends in the motorium. From the
motorvium arise the anterior and the posterior adoral fibrils, which bound
the adoral zone. The posterior fibril joins the anterior fibril distally,
and the latter continues as the pharyngeal fibril. This enters the endo-
plasm in the region of the pharynx and spirals around the potential gul-
let. A fibril from a point on the ring opposite the motorium enters the
pharyngeal fibril near the margin of the peristome. Within the peristomal
field delicate fibrils connect the anterior adoral fibril with the ring.

The longitudinal lines of the body surface consist of contractile mzyo-
nemes and basal granules of the ciliary rows. Myonemes arise at the
anterior end directly from the posterior adoral fiber, or indirectly from
fibrils of the posterior granular line. The myonemes pass posteriorly and
in somewhat oblique parallel lines. They gradually converge in the pos-
terior field. Basal granules of cilia rest on myonemes, oral ring, and the
anterior and posterior adoral fibrils (except the free end of anterior
adoral fibril). A deep nerve net, consisting of longitudinal interstrial and
transverse fibrils, interconnect the area “between the myonemes and
their basal granules.”

METHODS
Fixatives: Schaudinn’s (60° C.), Bouin, Zenker, formalin, osmic acid,
Da Fano.
Stains: Delafield’s iron haematoxylin, Mallory’s triple, alum carmine, and
Yabroff’s silver-gold method.
Clearing: Xylol, oil of cedar; equal parts of bergamot, oil cedar, and
phenol, and sometimes before imbedding, in synthetic oil of wintergreen.

Chlamydodon sp. nov. (MacDougall, 1928) .—The structural analysis
of this new species of Chlamydodon disclosed “a complex neuromotor
apparatus, including a codrdinating center, and systems of fibers con-
nected with cilia, the mouth opening, the pharyngeal basket, and the
‘railroad track.’ ”’

The motorium was identified as a bilobed mass located just below the
anterior end of the large pharyngeal basket. This mass and all the

230 CILIATE FIBRILLAR SYSTEMS

fibrils of the neuromotor system stained bright red by Mallory’s method.
When dislodged along with the basket by means of microdissection, the
motorium was ‘a refringent body.” In stained sections, however, it ap-
peared granular.

‘Fans of fibrils” join the motorivm at both its anterior and posterior
ends. Those at the anterior end mark the convergence of longitudinal
frbrils (Fig. 81A) of the body cilia, of ventral fibrils from the posterior
region of the body and of fibrils from the mouth. The circular myonemes
of the mouth are traversed by many fine fibrils that continue on toward
the “‘railroad track.” A fibrillar fan from the posterior end of the moto-
rium continues as the dorsal fibers to the “railroad track,” which, in turn,
show a very complex system of fibrils associated with its trichites.

The basal bodies of the cilia are connected not only by the longitudinal
fibrils but also by cross fibrils (Fig. 81B).

By methods of microdissection it was shown that (1) after destruc-
tion of the motorium “there is a marked disturbance in the action of the
cilia, in no way comparable to the disturbance of the cilia if other parts
of the body are injured”; (2) the cilia may still exhibit a wave-like
motion, but they do not reverse after the moforium is destroyed. This
seemed to suggest a codrdinating function for the fibrillar system of this
Chlamydodon, whose inconspicuous motor organelles are not favorable
for an experimental study of modifications of their codrdinated activity.

METHODS

Fixatives: Schaudinn’s, Bouin’s, and strong Flemming’s.

Stas: Iron-haematoxylin, Mallory’s (after Zenker’s or picromercuric
fixation) .

Microdissection.

Concho phthirus mytili De Morgan (Kidder, 1933).—The peripheral
longitudinal fibrils linking the ciliary basal bodies originate from a
transverse fibril in the anterior ventral region. These almost parallel rows
of fibrils pass around the posterior end of the organism uninterrupted,
continue over the anterior end, and again return to the transverse fibril
of the ventral surface. Each basal body is furnished with a ciliary rootlet
extending toward the endoplasm.

An elongate mass of homogeneous material, the motorium, follows the
posterior line of the cytopharynx and continues into the endoplasm as a

CILIATE FIBRILLAR SYSTEMS 231

fib. f.

ees ab
1B:

Figure 81. Chlamydodon sp. (MacDougall, 1928.)
A. c. myon.—circular myoneme and traversing fibrils b. gr.—basal granule
B. fib. f—fibrillar fan c. fib—cross fibril

strand which appears to be made up of fibrils. This strand follows the
posterior margin of the gullet and frays out in the endoplasm near the
left side of the organism. From the outer end of the otorium originate
two fibrils: one joins the basal elements of the posterior brush, while
the other is the fiber of the dorsal peristomal row of cilia. The latter pro-
ceeds anteriorly to join the anterior fibril and thus to the transverse fibril
mentioned above.

232 CILIATE: FIBRILLAR] SYSTEMS

The fused basal elements of the large oral brush of cilia form a deeply
staining band or plate at right angles to the long axis of the motorium.
From its anterior end two fibrils are given off which join the anterior
and middle oral brush plates. Fine fibrils arise from the basal elements
of the large oral brush line of the cytopharynx and connect with the
motorium along its anterior side. Finally, a ventral peristomal fiber runs
under the basal plate of the long ventral peristomal cilia, curves slightly
to the outside, and ends just anterior to the oral brush.

The brushes of cilia about the mouth seem to connect directly with the
motorium but not with the peristomal fibers. This lack of connection is noted
in the movements of the cilia, the peripheral and peristomal cilia beating
regularly, continuously and metachronously while the beating of the oral
brushes is non-continuous and synchronous.

METHODS

Fixatives: Flemming’s, Zenker’s (for whole mounts), Bouin’s, Zenker’s,
strong Flemming’s (for sections).
Stains: Heidenhain’s haematoxylin, Mallory’s triple.

Concho phthirus (Kidder, 1934).—The “well integrated and closely
interconnected neuromotor systems” of three species of Concho phthirus
—C. anodontae Stein, C. curtus Engl., and C. magna sp. nov.—are quite
comparable. The description is given of the external, internal, and peri-
stomal fibrillar complexes, with reference especially to C. magna.

Most of the numerous, closely set ciliary rows originate in an antero-
ventral suture and terminate in a dorsal suture near the posterior end.
The ventral suture comprises two fibrils which are united at their ends
and are connected irregularly by cross fibrils. This ventral suture is con-
tinued posteriorly as the pre-oral connecting fiber, ftom which arise two
fibrils: (1) connecting the rows of basal granules of the dorsal lip, and
(2) connecting the basal granules of the pharyngeal ciliary row.

The pre-oral connecting fiber itself becomes the peristomal net fiber.
The latter gives off secondary fibrils to the peristomal field. On the left,
these secondary fibrils are bounded by a longitudinal znner net fiber
(Fig. 82) from which arise numerous fine fibrils that line the ventral
side of the peristomal basket. On the floor of this basket, these fine fibrils
join the nner basket fiber, which, in turn, gives off many branches that
line the dorsal surface of the basket. These branches then unite with the

CILIATE FIBRILLAR SYSTEMS 233

fiber (noted above) that connects the basal granules of the dorsal lip.

At the anterior, inner end of the pharynx is a large pharyngeal ring
fiber that fuses with the snner basket fiber, previously noted, to form
the fibrillar bundle. This point of fusion is regarded as comparable to the
motorium of C. mytil7, From the fibrillar bundle a gullet fiber extends
inward and courses throughout the floor of the gullet, finally fraying out
at the posterior end.

The inner basket fiber is united posteriorly with the posterior basket
connecting fiber. The latter, bending dorsally and to the right, comes to
join the post-oral connecting fiber. Thus a direct connection is made
between the peristomal region and the dorsal suture. From this post-oral
fiber, numerous cross fibrils connect with adjacent ciliary rows.

“This neuromotor system is thought to be mainly conductive but some
parts of it may possibly be contractile or even supportive.”

METHODS

Fixatives: Klein’s (1926), von Gelei-Horvath’s (1931), strong Flem-
ming’s for four hours.
Stains: Heidenhain’s haematoxylin, destained with hydrogen peroxide.

Dallasia frontata Stokes (Calkins and Bowling, 1929).—The most
conspicuous part of the neuromotor system of Dallasza frontata was found
to be the complicated apparatus of the mouth. This is composed of a
tongue running through the buccal cavity, supported by bars which are
anchored in long strands of dense material lying on the floor of the
buccal cavity. There are two of these /ongitudinal strands and two series
of bars from the tongue, one on each side. On the right and left sides
are undulating membranes. A Jadder-like organ originates anteriorly just
below the membrane of the buccal cavity and at the right side of the
mouth, and runs into the gullet.

A discoidal mass on the left side of the gullet is interpreted as the
motorium. It is connected by fibers directly to the proximal end of the
tongue and strands. Similar fibrils connect the outer and the inner margins
of the /adder-like structure with the motorium, and these fibers appear to
form the outer and inner margins of this organ. Minute granules are
present at the ends of each bar, at the points where the bars join with
the longitudinal fibers. Posteriorly two fine fibers run from the motorium

234 CILIATE FIBRILLAR SYSTEMS

a
<>)
el

ph. r. fib. er ee LY)
Fi DOB: ay TESTS Se a

Ary
CLS

4
SS

ANNA

Gf prom se

Figure 82. Conchophthirus magna. (Kidder, 1934.) Diagram of the fibrillar system
of peristomal region.

d. f. p. b.—dorsal fibers of peristomal basket d. I. c. r—dorsal lip ciliary row
fib. b—fibrillar bundle gul. fib—gullet fibril i. n. fib.—inner net fibril
in. b. filb—inner basket fibril 1, 2, 3, 4—rows of body cilia
p. b. c. f.—posterior basket connecting fibril p. o. c. f—pre-oral connecting fibril
p. o. c. f'.—post-oral connecting fibril ph. c. r—pharyngeal ciliary row
ph. r. fib.—pharyngeal ring fibril v. f. p. b—ventral fibril of peristomal basket

deep into the endoplasm, where they are lost, one in the vicinity of the
macronucleus, the other near the contractile vacuole.
Basal granules are described, but no connecting fibrils mentioned.
The function of this enigmatical organ is purely conjectural; possibly it
has something to do with the opening and closing of the mouth. . . . What-

ever the specific function may be there is little reason to doubt that it is
intimately connected with the irritability of the mouth region.

CILIATE FIBRILLAR SYSTEMS 235

METHOD (whole mounts and sections)
Fixatives: Schaudinn’s, made up in 95-percent ethyl alcohol, followed by
prolonged treatment with turpentine.
Stains: Iron haematoxylin; solution of acid fuchsin and methyl green.

Dileptus gigas (Visscher, 1927).—From a rod-like motorium found
near the base of the gullet, several sets of fibrils extend to different
parts of the body, as follows: (1) one set supplies the wall of the fun-
nel-shaped gullet, (2) two more distinct fibrils course anteriorly along
the proboscis, one on either side of a row of trichocysts; and (3) a set
of branching finer fibrils spreads posteriorly over the body, where they

Figure 83. Dileptus gigas. Neuromotor apparatus (camera drawing). (Visscher,
1927.)

a. fib.—anterior fibril f. fib—fine fibrils i. fib—inner fibrils

mot.—motorium o. fib.—outer fibrils t.—trichocysts

236 CILIATE FIBRILLAR SYSTEMS

are probably associated with the longitudinal rows of cilia running paral-
lel to the contractile fibrils (Fig. 83).

METHODS
Fixative: Schaudinn’s.
Stains: Iron-haematoxylin, acid borax carmine.

Entodiscus borealis (Powers, 1933) .—The following distinct groups
of fibrils and associated structures characterize the fibrillar system of
Entodiscus borealis: (1) the stomatostyle, with its dorsal and ventral
anterior horns and their adoral fibrils; the labial fibrils; the pharyngeal
fibrils; and the circum pharyngeal rods; (2) the anterior fibrillar center,
or motorium, with its anterior, posterior, and marginal strands; the pos-
terior auxiliary fibrillar center with its associated strands; and (3) the
peripheral transverse commissural fibrils interconnecting the basal gran-
ules. Closely associated with the commussural fibrils are the distal branch-
ing ends of most of the internal fibrils. The ventral peripheral layer
is further complicated by the long (6-12 y) célzary rootlets, which ex-
tend into the endoplasm from the basal granules of the cilia.

The pellicle of the ventral surface is thicker than elsewhere and is
highly differentiated owing to pellicular fibrils accompanying each
longitudinal ciliary row. These fibrils are conspicuous only at the anterior
end. They are interpreted as either supporting or contractile in nature
(Fig. 84).

METHODS

Fixatives: Schaudinn’s, Da Fano’s, 25-percent osmic acid, Flemming’s
without acetic acid (the two latter fixatives for sections).
Stains: Iron alum haematoxylin, Yabroff’s silver method.

Entorhipidium echini (Lynch, 1929).—One fibrillar system of Ento-
rhipidium echini consists of a motorium which is connected to a network
of peripheral fibrils linking all the basal granules. The other set of fibrils
is developed in the pellicle, chiefly on the ventral surface.

The motorium is located to the left of the buccal cavity. It is com-
posed of five heavy strands united into a rod-like body by /ongitudinal
fibrils which appear to be continuous with the newrofibrils of the basal
granules. These delicate Jongitudinal fibrils unite the basal granules of
each peripheral row of cilia, and other fibrils encircle the body, uniting

od. ad. #6.
Ger DNC.
eBwela@
Zon Tio:

Figure 84. Entodiscus borealis. (Powers, 1933.) A. Reconstruction of neuromotor
system, dorsal surface (modified from author). B and C. Relations of body cilia and their
basal granules with fibrils of neuromotor system.

a. fib. c-—anterior fibrillar center b. gr.—basal granule
cil. r—ciliary rootlet d. ad. fib.—dorsal adoral fibril
1. m. fib.—left marginal fibril 1. pell—tlongitudinal pellicular thickening
li. cyt—lips of cytostome p. fib.—posterior fibril
p. fib. c.—posterior fibrillar center ph. fib—pharyngeal fibril
r. m. fib.—tright marginal fibril stom.—stomatostyle

t. f—transverse fibril v. ad. fib.—ventral adoral fibril

238 CILIATE FIBRILLAR SYSTEMS

the basal granules with perfectly regular transverse commissures. In the
region posterior to the frontal lobe, the commissural fibrils branch, form-
ing numerous collaterals extending in various directions. These unite
with transverse and longitudinal fibrils.

The pellicular thickenings of the frontal lobe are present in the form
of heavy, deeply staining fibers which alternate with the ciliary rows. Be-
tween the anterior ends of these and the anterior ends of the dorsal
rows of cilia is a delicate fretwork, or polygonal area. The boundaries of
polygons correspond with the boundaries of a double row of large
vacuoles. Posterior to the frontal lobe, the eavy fibrils become fine and
lie so close to the xewrofibrils that they are almost indistinguishable from
them. Similar fibrils are evident on the dorsal surface of the organism
only at the anterior end (Fig. 85).

METHODS

Fixatives: Schaudinn’s, Da Fano’s, 2-percent osmic acid, Flemming’s with-
out acetic acid (the two latter for sections).
Stains: Iron-haematoxylin, Yabroff’s silver method.

Eupoterion pernix (MacLennan and Connell, 1931).—Most of the
longitudinal ciliary fibrils of the body take their origin from a heavy bar,
the anterior connective fibril, that lies dorsoventrally across the anterior
tip, whence they extend to the posterior end, where they fuse. Additional
fibrils arise in pairs along the pre-oral suture line and these fuse mostly
also at the posterior end. Each basal granule gives off one or two com-
missural fibrils to adjacent ciliary rows, resulting in a fairly regular lat-
ticework (Fig. 86).

The neuromotorium lies beneath the wall of the cytostome. The fibrils
of the four pairs of oral ciliary rows along the oral groove are fused in
a V-shaped figure at the apex of the suture line. All of these end directly
in the motorium or are closely connected to it by the transverse fibril or by
the longitudinal ciliary fibrils. The transverse fibral lies actoss the end
of the neuromotorium, its right end joins the pharyngeal fibrils, thus
forming a pharyngeal strand; and its left end fuses with the two outer-
most peristomal ciliary fibrils and ends farther left in a connective fibril
of adjacent outer rows of cilia. The two rows of cilia arising from the
anterior ends of the outer peristomial rows (of the ordinary peripheral

pp Te 4... br fib

ERre
aN

Figure 85. A. Entorhipidium echini. (Lynch, 1929.) Cross section, showing periplast
of the anterior dorsal fibril. B. Extorphidinm echini, tangential section of anterior ventral
surface.

A. b. gr.—basal granule B. comm.—commissural neuro-fibril
1. p. fib—longitudinal pellicular fibril |. fib—longitudinal fibril

t.—trichocyst p. fib.—pellicular fibril
tr. fib—transverse fibril

240 CILIATE FIBRILLAR SYSTEMS

type) are connected to the rest of the body rows by commissural fibrils,
thus uniting the peristomial cilia with the rest of the body cilia.

METHODS
Fixative: Schaudinn’s (60° C.).
Stain: Heidenhain’s iron haematoxylin.

Haptophrya michiganensis “Woodhead (Bush 1934).—Haptophrya
michiganensis has an integrated system of fibrils that center in a moto-

Figure 86. Expoterion pernix, optical section. (MacLennan and Connell, 1931.)

a. c. fib.—anterior connective fibril n. mot.—neuromotorium
b. gr.—basal granule ph. fib—pharyngeal fibril
cyto.—cytostome ph. str—pharyngeal strand
ectop].—ectoplasm tr. fib.—transverse fibril

rium. Within the sucker at the anterior end is a fibrillar ring, homologous
with the esophageal ring of stomatous ciliates. The motorium is located
in the center of this ring and is connected with it by radzal connectives.
Accessory bodies of the motorium are suspended from various points on
the inner edge of the ring. (These are not evident during fission.) Nu-
merous myonemes radiate from the inner edge of the fibrillar rng and
extend to the opposite walls, posteriorly and laterally, dividing into
fine fibrils at their outer ends. Equally spaced perspheral myonemes arise
from the external edge of the fibrillar ring, adhere to the inner layer of

CILIATE FIBRILLAR SYSTEMS 241

the ectoplasm, and extend to the posterior end of the animal. These
myonemes are “‘closely associated with the basal granules.” Commissures
connecting the basal granules form a close network over the entire body.
Supporting fibrils from the nuclear membrane, the endoplasmic cone, and
the contractile canal extend to the peripheral ectoplasm (Fig. 87).

The deeply staining mass was interpreted as a motorium because *' (1)
it is connected, directly or indirectly, with all parts of the fibrillar system;
(2) it is near the anterior end of the ciliate; (3) if, with this mass, the
sucker is removed, the animal loses its power of worm-like forward
movement even though the cilia continue to beat; and (4) a toxic sub-
stance acts first upon the anterior part, particularly the sucker, where-
upon the animal ceases its forward movement.”

METHODS
Fixatives: Schaudinn’s and Zenket’s.
Stains: Delafield’s and Heidenhain’s haematoxylin; also Kolatschev’s osmic
impregnation, as outlined by Bowen; Yabroff method.

Ichthyophthirius (MacLennan, 1935).—The longitudinal fibrils con-
necting the basal granules beneath the ciliary rows of the body surface
ate linked together anteriorly and to some extent posteriorly by small
centers, the anterior and the posterior fields. The centers are connected
by a “‘suture fibril” which marks the ventral side. The suture fibril is
interrupted by the oral region, thus dividing it into pre- and post-oral
segments. Concentric accessory suture fibrils lie on the sides of the oral
region and terminate anteriorly and posteriorly in the suture fibrils. The
lip of the oral opening is bounded by (1) the outer peristomal fibrils,
which are also linked to the suture fibril. (2) Circular fibrils line the
walls of the oral cavity and radial fibrils intersect the two sets of fibrils
at right angles. These transverse connections between the ciliary fibrils
are present only in the oral region.

Two heavy basophilic rods, each attached to a heavy esophageal fibril
are located near the esophageal plug. An inner peristomal fibril runs from
this bilobed newromotorium to the basal granules.

Ciliary rootlets are developed in the region of the inner peristomal
fibrils; they are less well developed in the region of the outer peristomal
fibrils, and not found in the region of ordinary body cilia. About 50-100
individual ciliary rootlets combine to form numerous esophageal strands,

Pell. 2-==n= NUC. Me

: i --- -- endopl. 116.
cont.can. f16. ia. a 3 = Se. Biel &

CONG. Cal.

eG fre
eR aiho:

io) ola a
comm. -

Figure 87. Haptophrya michiganensis. (Bush, 1934.) A. Diagrammatic section show-
ing one-fourth of the animal body with part of the macronucleus. B. Diagram of anterior
part of neuromotor system.

acc. mot.—accessory motorium mot.—motorium

b. gr.—basal granule macro.—macronucleus
comm.—commissures nuc. fib—nuclear fibrils

c. fib.—coarse fibrils nuc. m.—nuclear membrane
cont. can.—contractile canal pell.—pellicle

cont. can. fib.—contractile canal fibrils per. my.—peripheral myoneme
endopl. c.—endoplasmic cone rad. my.—tradial myonemes
endopl. fib—endoplasmic fibrils rad. conn.—radial connectives

fibr. r.—fibrillar ring ret. fib.—reticulate fibrils

CILIATE FIBRILLAR SYSTEMS 243

each of which turns sharply and continues to the endoplasm, parallel to
the main axis of the oral pit.

METHOD
Fixatives: Schaudinn’s, Zenket’s.
Stains: Heidenhain’s iron-haematoxylin, Delafield’s (for fibrils), Klein’s
(1926) (for ciliospores), Lund’s wet silver method (for adults).

Lechriopyla mastax (Lynch, 1930).—The cilia of Lechriopyla mastax
can be divided into three areas: (1) the cilia of the general surface of
the body, (2) the cilia of the peristome, and (3) a transverse band
of cilia known as the supraoral band. The basal granules of all these
cilia are connected by delicate longitudinal fibrils without transverse con-
nectives. Below the ciliary lines of the peristome and vestibule are verti-
cal pellicular lamellae, which are fused to the furcula described below.

A crescent-shaped neuromotorium lies beneath the pellicle at the
left end of the peristome. Its ends are continued as long fibers, the
anterior and posterior adoral fibers, which form a complete (or nearly
complete) ring about the peristome. From the ring arise the ciliary lines
which extend over the surface of the body, or pass into the peristome
and the pharyngeal involution. A variable number of fibrils arising from
the outer border of the motorium extend through the cytoplasm for vary-
ing distances, to fuse with the pellicle just beneath a ciliary line. They
may branch or anastomose, and they become more delicate distally.

The furcula, shaped much like a heavy tuning fork, partly surrounds
the vestibule. The ends fuse with the walls of the pharynx, and delicate
fibrils from the pharyngeal wall extend to the furcula. This organelle
may be an additional element of the neuromotor system.

A long pellicular fiber extends from the left end of the internal open-
ing of the cytopharynx to the middle of the posterior end of the organism
and occasionally curves along the right side for varying distances. This

fibril is not included in the author’s description of the neuromotor sys-
tem.

METHODS

Fixatives: Bouin’s, Schaudinn’s, Da Fano’s cobalt nitrate-formalin.

Stains: Iron alum haematoxylin, carmines, cochineals, Yabroff’s silver-
nitrate method (no success), Klein’s silver method.

Sections prepared in a variety of ways.

244 CILIATE FIBRILLAR SYSTEMS

Ptychostomum chattoni Rossolimo (Studitsky, 1932) .—The mouth of
this parasitic ciliate is at its posterior end. At the anterior end is a horse-
shoe-shaped sucker with a projecting rim for attachment.

The sucker (Frxationsapparat) is provided with a system of fibrils.
The largest fibril, the perspheral cord, borders the sucker and gives off
at its ends fine fibrils that extend into the cytoplasm. The sucker’s disc
has four sets of fibrils: (1) the deeper set comprises two groups of paral-
lel fibrils that cross each other at a sharp angle as they traverse the disc,
both groups coming to adhere to the per/pheral cord; (2) the uppermost
set, visible in the living organism, courses from right to left and from
anterior to posterior; (3) a third set of fine fibrils are attached to the
peripheral cord by their anterior ends; and (4) the fourth set, com-
posed of sixteen or seventeen strands that run from right to left and
from anterior to posterior, frays into four or five fine fibrils at the anterior
end of each strand, to become attached to the peripheral cord and to
other adjacent fibrils; the posterior ends of these strands become fimbri-
ated also into fine fibrils. All apparently serve for support.

METHODS

Fixatives: Schaudinn’s, Carnoy’s, Bouin’s, Champy’s and Benda’s (for
whole mounts), Altmann-Kull’s (for sections).

Stains: Heidenhain’s haematoxylin, safranin, von Gelei’s toluidin blue,
Mallory’s triple.

B. HETEROTRICHA

Balantidium coli Malmsten and B. sus sp. nov. (McDonald, 1922) .—
The motorium of Balantidium suis lies within the ectoplasm of the
apical cone, close to the right ventral wall of the esophagus. A fibril
encircling the esophagus arises and ends at the anterior end of the moto-
rium, where the ddoral ciliary fiber also arises. The circumesophageal
frbril has irregular enlargements, from which fibers pass both posteriorly
and anteriorly into the ectoplasmic mass of the anterior end. These fibrils
appear to fade out in the ectoplasm. The adoral fiber connects the basal
granules of the adoral cilia. The remainder of the fibrils are not directly
connected with the motorium.

The basal granules of the peripheral longitudinal spiral rows of cilia
are so Closely set that it has been impossible to see a fibrillar connection.
No transverse fibrils connecting the rows were observed. A ciliary rootlet

CILIATE FIBRILLAR SYSTEMS 245

extends from each basal granule, and a second small granule is found
at the junction of the ectoplasmic and endoplasmic layers. The ectoplasmic
layer is quite thin, except at the anterior end of the organism, where it
is deep and the distance between the granules of each ciltum correspond-
ingly long. The rootlets of the row of adoral cilia around the margin of
the peristome, the “radial fibrils,” are exceedingly long, ending in about
the posterior third of the body without connection or attachment. The
cilia immediately posterior also have long rootlets, but they become
shorter as they approach the base of the apical cone (Fig. 88).

METHODS
Vital stain: Neutral red (differentiates the neuromotor apparatus).
Fixatives: Schaudinn’s, Zenker’s, Formalin, osmic acid, picrocuric,
60-80° C.
Whole mounts and sections
Stains: Iron haematoxylin, Mallory’s triple stain (particularly for sections).

Figure 88. Balantidium coli, cross section of peripheral region. (McDonald, 1922.

b. gr.—basal granule gr. b.—granular band
cil. r.—ciliary rootlet hy. b.—hyaline band
endo.—endoplasm pell.—pellicle

Balantidium sushilii (Ray, 1932).—Fibrils associated with the con-
tractile vacuoles have been described in Balantidinm sushili, Each of the
two lateral vacuoles has a fibril running from the wall of its outer half
to the neighboring pellicle. The neck of the terminal vacuole 1s sur-
rounded by a diaphragm of fibrils running from the wall of the neck
to the surrounding pellicle. These unusual fibrils are described and figured
in the extended as well as the contracted condition.

An axial and a peripheral system of fibrils can be seen also in the
living organism. Stained preparations show that the former consists of

246 CILIATE FIBRILLAR SYSTEMS

three or four fibrils, parallel or twisted after the manner of a rope, which
originate below the pellicle at the anterior end and extend posteriorly
a short distance beyond the mouth. The individual fibrils gradually be-
come thinner toward the posterior end, where they come in contact with
the limiting membrane formed by some of the fibrils of the peripheral
system. At the anterior termination of these fibrils is found a knob-like
structure which may be imbedded in the epithelium of the host.

The peripheral system of fibrils is arranged in two conspicuous arches
along the left anterior border of the organism. Anteriorly, fibrils extend
both to the right and to the left peristomal lips. Posteriorly, the two
arches converge. Some of the fibers continue mesially and come to form
a kind of limiting membrane beyond which no fibrils are traceable.

The group of fibers forming the axial system, together with the borer
attached at its anterior end, is called the boring apparatus, as the first
of its kind to be noted. It may be compared with the axostyle of some
flagellates.

The peripheral system is believed to represent morphonemes.

METHODS

Fixatives: Brasil’s modification of Bouin-Duboscq’s (for whole mounts),
Bouin’s alcoholic, twenty-four hours (for sections 5 wp).
Stam: Heidenhain’s haematoxylin (whole mounts and sections).

Fabrea salina Henneguy (Ellis, 1937).—In Fabrea there are numer-
ous longitudinal rows of closely set body cilia (in pairs). These are
interrupted on the ventral side by a coiling adoral zone. The basal gran-
ules are connected by fine longitudinal fibrils. No transverse connections
or ciliary rootlets were observed. Each membranelle consists of two rows
of basal granules whose ciliary rootlets fuse into a single plate, the basal
lamella. Each longitudinal fibril of the dorsal and ventral surfaces, with
the exception of those that merge with one another, is connected at the
adoral zone with the basal lamella of a membranelle. The basal lamella
is connected with the adoral fibril by fibril running across the peristomal
groove. The adoral fibril starts at the anterior tip and follows the course
of the inner border of the adoral zone. The peristomal fibril is continued
beyond the end of the adoral zone on the wall of the funnel and ends
in a ganglion-like body on the left wall of the ventral lobe. From this
motorvium arise several fibrils—the adoral fibril, which follows the course

CILIATE FIBRILLAR SYSTEMS 247

of the adoral zone, and other fibrils which appear to end blindly in the
endoplasm of the ventral lobe. Anteriorly the fibrils of the frontal field
tend to converge and end very obliquely on the adoral fibril.

No pellicular pattern was demonstrated by any of the silver methods.
but they did show the longitudinal fibrils connecting the cilia.

METHODS

Fixatives: Schaudinn’s, Bouin’s and Flemming’s.
Stains; Iron-haematoxylin, Mallory’s triple.
Silver method: Yabroft’s modification of Da Fano’s.

Metopus circumlabens (Lucas, 1934).—The motorium of Metopus
circumlabens lies posterior to the cytostome. From its left side it gives rise
to a pair of ventral adoral fibrils which follow the peristomal curvature
outward to the oral margin and there end in a sort of arborization. Each
row of peristomal membranelles arises immediately in contact with a con-
nective between these two fibrils. A dorsal adoral fibril extends from the
right side of the motorium. At slightly irregular intervals is gives rise
to from ten to twenty heavy connectives, which may partially fuse in
pairs as they curve beneath the dorsal wall of the peristome to its left
side. There they turn ventrad and unite with the ventral adoral fibers.
A fibrillar pharyngeal strand arises from the posterior end of the moto-
rium. Its course varies in different organisms, but it usually lies along the
right lateral wall of the organism to the right of the cytopharynx. Near
the posterior end of the latter structure it forms a large spiral coil.

The entire body surface, except the right lateral margin, is covered
with rows of cilia. Longitudinal ciliary fibrils are present, but no com-
missural fibrils were observed. Each basal body in the most dorsal of the
five rows of the crest cilia gives rise to rootlets which end freely in the
cytoplasm. A relation between these peripheral fibrils and the fibrils of
the motorium seems to be suggested by the numerous fine branches which
arise from the ventral adoral fibrils. These extend indefinitely into the
cytoplasm toward the longitudinal ciliary rows of the ventral surface.

In view of the contrastingly striking and obvious specialization in the
fibrillar structure of the neuromotor system about the peristomal, pharyngeal,
and central endoplasmic regions of the cell, one is inclined to believe that
the neuromotor system of this ciliate is vitally, though not exclusively, con-

cerned in the conductile functions related to the metabolic activities of the
organism. It is possible that, because they are located within the mobile

248 CILIATE FIBRILLAR SYSTEMS

cytoplasm of the protozoan, the stouter of these various fibres may serve in
addition some function in the nature of support.

METHODS
Fixatives: Schaudinn’s, Bouin’s, Jorgensen’s, Van Rath’s.
Stains: Heidenhain’s iron haematoxylin, Regaud’s haematoxylin, Mallory’s
triple (whole mounts and sections).

Nyctotherus hylae (Rosenberg, 1937).—A detailed description of the
neuromotor system of Nyctotherus hylae is given, including two centers
and a group of special fibrils believed to control the reversal of ciliary
action. An incomplete account of some of these structures was given by
Kirby (1932) for N. sylvestrianus. The movements of N. Aylae were
studied by cinematographic methods.

From the main moforium located at the distal end of the cytopharynx
arise two sets of unbranching fibrils. One set extends along the anterior
border of the pharynx, eventually terminating at the end of the peristome.
The other set follows the posterior border and arm of the ectoplasmic
thickening. Some of the latter fibrils unite at the cytostomal border with
the peripheral per7stomal fibril. The so-called “reversal fibrils’ originate
from the posterior part of the moforium, radiate through the endoplasm,
and at their distal ends unite with the ciliary lines at several points, not
including the presutural ciliary lines.

The membranelles have each a basal plate and two rows of basal gran-
ules, from which fine fibrils connect with the circum pharyngeal fibrils.
The latter become the transverse peristomal fibrils, of which there are at
least two for each membranelle of the series.

A number of fibrils from the motorium directly connect with the
anterior neuromotor center. From this structure arise many ciliary lines
that connect rows of basal granules. Commissural fibrils between these
lines are present only in the apical post-sutural region. The lateral and
sagittal sutures which divide the ciliation into definite regions were
interpreted as probable conductors between ciliary lines.

The pharyngeal terminus, a deeply staining structure, gives rise to a
post-pharyngeal bundle of fibrils that have no apparent distal attach-
ment. Kirby (1932) described a similar “‘band formed structure” in
N. silvestrianus which may extend beyond the cytopharynx, its course
in the endoplasm varying in different individuals. He considers it “ho-

CILIATE FIBRILLAR SYSTEMS

249

mologous with the ‘continuation tube’ of the ‘subpharyngeal canal’ de-
scribed by Higgins (1929) in N. cordiformis” (Kirby, 1932, p. 298).
Whether or not this strand is a part of the neuromotor system is an

open question.

Fibrils interpreted as mor phonemes are as follows: (1) those extend-

comm. fib.

tr. perist. 116.

per. perist fib. _1, q

CieiOu) os
ana

moet.

sh, B fib. Af

4 0s,
a aoe oa

Figure 89. Nyctotherus hylae, fibrillar system, diagrammed. (Rosenberg, 1937.)

ant. neur. cen.—anterior neuromotor cen-
ter

c. b. b.—ciliary basal body

c. 1—ciliary line

ca. fib—caryophore fibril

cir. fib—circumpharyngeal fibril

comm. fib——commissural fibril

1. ph. fib—longitudinal pharyngeal fibril

lat. sut.—lateral suture

mot.—motorium

n. env.—nuclear envelope

p. |. ph. fib—posterior
pharyngeal fibril

p. ph. b.—post-pharyngeal bundle

ph. term.—pharyngeal terminal

per. perist. fib—peripheral peristomal fi-
bril

re. fib.—reversal fibrils

sh. s. fib—shelf supporting fibrils

tr. perist. fib.—transverse peristomal fibril

longitudinal

250 CILIATE FIBRILE ARTS SiEMS

ing from the right to the left of the body, (2) the caryophore fibrils,
and (3) the shelf-supporting fibrils (Fig. 89).

METHODS

Fixatives: Schaudinn’s (5-percent acetic), Flemming’s.
Stain: Heidenhain’s iron haematoxylin, aqueous and alcoholic.
Silver techniques: Klein, Gelei-Horvath, Yabroff (negative).

Spirostomum ambiguum Ehrbg. (Bishop, 1927).—The ridges and
furrows in the ectoplasm of Sprrostomum ambiguum follow a sinistral
spiral course from the anterior to the posterior end of the body. Be-
neath the furrows lie thread-like myonemes, somewhat beaded in ap-
pearance, but without light and dark alternating bands. The myonemes
taper gradually as they approach either end of the body and finally disap-
pear from view. They are not attached to any structure. Longitudinal
myonemes were found on either side of and running parallel to the band
of peristomal membranelles, except along those membranelles nearest
the cytostome. No evidence was obtained confirming the presence of
other fibrils such as neurophanes.

On the anterior side of each myoneme lie the basal granules of the
body cilia. The rows of granules are parallel to and slightly above the
level of the mzyonemes. No ciliary rootlets nor connections between basal
granules or myonemes were discovered.

The system of fibrils underlying the membranelles (Fig. 90) of S.
ambiguum includes:
an anterior basal fibril extending from the anterior end of the body to the
beginning of the peristomial depression; a middle fibrillar system which
varies in its course in different individuals, but which collects the end-threads
of the membranelles lying on the left side of the peristomial depression; and
a posterior basal fibril into which the end-threads of the membranelles at
the posterior end of the peristomial depression and in the cytopharynx join.
A connection between the posterior basal fibril and the middle fibrillar

system is seldom found, and there is always a break between the middle
fibrillar system and the anterior basal fibril.

A central body to which the fibrils join was found in no case.

METHODS
Fixatives: Schaudinn’s, picro-mercuric (hot).
Stains: ron-haematoxylin (alcoholic and aqueous), Mallory’s triple
(Sharp’s modification), Fuchsin S.

GIEVATE FIBRILLAR SYSTEMS 25 1

Figure 90. Spirostomum
ambiguum. Diagram of
membranelles and __ their
intracytoplasmic structures.
(Bishop, 1927.)

b. fib.—basal fibril
b. 1.—hbasal lamella
b. pl.—basal plate
e. thr.—end thread
memb.—membranelle

52 AA

b. fb.
C. OLIGOTRICHA

Diplodinium ecaudatum (Sharp, 1914).—The motorium is located
in the ectoplasm above the base of the left skeletal area and between the
left extremities of the dorsal and the adoral membranelle zones. Dorsally
it is connected with the bases of the dorsal membranelles by a dorsal
motor strand, which also sends a branch along the base of the inner
dorsal lip. A ventral motor strand connects the motorium with the bases
of the adoral membranelles and its branch and passes along the base
of the inner adoral lip. Numerous fibers from the motorium follow the
contour of the operculum and disappear near the base of the right skeletal
structure. A circumesophageal ring surrounds the esophagus at the level
of the outer adoral furrow, from which a fibril connects with the 70-
torium, Certain fibrils in the wall of the esophagus appear to unite with
the circumeso phageal ring; others are attached to skeletal structures and
are considered contractile fibrils. Rootlets from the oral cilia end in, or
close to, the ring. A codrdinating (conductive) function was ascribed
to this fibrillar system of D. ecaudatum, because of its initimate relation-
ships with the motor organelles and its complete structural integration
through the motorium.

METHODS
Fixatives: Schaudinn’s (alcoholic, hot), Zenker’s, Flemming’s, Worcestet’s,
Bouin’s, formalin 4 percent, osmic acid one percent (formalin 36° C.)
Stains: Heidenhain’s haematoxylin and Mallory’s triple.

22 CILIATE FIBRILLAR SYSTEMS

Diplodinium medium (Rees, 1931).—The structure of D. medium is
compared with D. ecaudatum as described by Sharp. The disagreement
between Rees’s interpretation and the interpretation of Sharp has to do
primarily with the ectoplasmic layer directly under the pellicle, except
in the region of the adoral lip and the inner boundary layer of the ecto-
plasm. In D. medium this layer, according to Rees, is more prominent
than in D. ecawdatum and consists, instead of fine alveoli, of an znter-
woven network, or complex system of fibrils. Serial cross and longitudi-
nal sections, 3 p. in thickness, made it possible to trace the ectoplasmic
layers.

Cross sections of D. medium show a fold of this middle layer of the
ectoplasm which corresponds in its position to Sharp’s motorium. Further-
more, the esophageal ring described by Sharp is interpreted as section
of the inner boundary layer of ectoplasm. The fibers connecting the
mortorium with the membranelles and esophagus could not be differ-
entiated.

The ciliary rootlets are attached to membranes composed of sheets of
the fused middle and inner layers of ectoplasm, which in turn are at-
tached to the fibrillar system of the ectoplasmic layers The structures are
membranes, according to Rees, because “‘one or the other of them occurs
in all longitudinal and oblique sections, whether cut with reference to
the parasagittal plane or to a plane at right angle or at any other angle
to it.” The esophageal tractor strands are considered to be a part of the
ectoplasmic network of fibrils. The membranes, instead of strands, are
believed by Rees to function in the retraction of the adoral and the dorsal
cilia.

The occurrence of fine fibrillae in the non-ciliated ectoplasm of Diplodinium
is of interest in connection with other papers on the neuromotor system. It is

obvious that in the latter ciliate the fibrillae of the ectoplasmic layers have no
relationship to a neuromotor system.

METHODS

Fixatives: Not listed.
Stains Iton-haematoxylin, Zirkle’s N-butyl alcohol method of dehydration.

Diplodinium Schbg. (Kofoid and MacLennan, 1932).—In addition
to the neuromotor apparatus as described by Sharp (1914) for D. ecauda-
tum and incidentally confirmed in this systematic investigation, a fibrillar

CILIATE FIBRILLAR SYSTEMS 253

complex also was found to occur in the caudal spines of D. dentatum,
in contrast to the apparently structureless spines in Entodinium. Along
the bases of the caudal spines appeared a heavy marginal fibril from
which finer anchoring fibrils extended into each spine, terminating under
the cuticle of its outer margin. A very heavy main anchoring fibril
bordered the inner edge of each spine. From the anchoring fibril of the
ventral spine smaller fibrils branched toward the anus, where they ended,
one on each side. Small branches from these coursed in the wall of the
rectum, parallel to its main axis.

No connection was evident between this fibrillar complex of the caudal
spines of D. dentatum and its neuromotor system. The location and re-
lationships of the former suggested a supporting function similar to that
of the longitudinal surface fibrils. Also, since these spines undergo a
change in their curvature such as might obviously be facilitated especially
by the main anchoring fibril together with the marginal fibrils, these
caudal fibrils were considered to be myonemes.

A similar fibrillar system had been described by Bélaf (1925) in
Epidinium caudatum, on the basis of which Reichenow (1929) denied a
neuromotor function for all fibrils of the Ophryoscolecidae. The clear
difference in the morphological relationships of the two fibrillar systems
in D. dentatum, however, indicated that these systems have quite differ-
ent functions:

The caudal fibrils are admirably situated to serve as supporting and contractile
structures. The motor fibrils are so situated as to be of little or no use
either as supporting or as contractile fibrils. The caudal fibrils show no con-
nection to the motor organelles. The motor fibrils link together (through the
neuromotorium) all the motor organelles of the individual.

METHODS
Fixative: Schaudinn’s.
Stain: Iron-haematoxylin.

Favella jorgensen (Campbell, 1927).—The neuromotorium 1s a
spindle-shaped body in the ventral ectoplasmic wall in the mid-region
of the gullet. This organelle gives rise to five intracytoplasmic fibrils as
follows: (1) the adoral fibril, extending to and interconnecting the mem-
branelles; (2) the circumesophageal fibril, with branches surrounding
the gullet; (3) a dorsal fibril which appears to connect with the striations

254 CILIATE. FIBRILLAR SYSTEMS

of the oral plug; and (4, 5) two ventral fibrils extending downward
and ending freely in the endoplasm. It was observed that the mem-
branelles not only serve the organism in feeding and in locomotion, but,
during periods of binary fission, function in the building of the lorica.
In addition to their fibrillar connection with the motorium, each mem-
branelle is supplied with three large basal bodies.

METHOD

Fixative: Schaudinn’s (aqueous and alcoholic), 90° C.
Stain: Iron-haematoxylin (whole mounts and sections).

Tintinno psis nucula (Campbell, 1926).—The somatic ciliation is con-
fined to the column and forms in longitudinal rows along the myonemes.
These showed basal granules, but without fibril connection. The myo-
nemes are ectoplasmic structures, arranged longitudinally and un-
branched. Anteriorly, they extend to the reflexed margin of the collar
and possibly connect with basal granules of the adoral membranelles;
posteriorly, they fade out.

At the base of each adoral membranelle are three basal granules con-
nected by fibrils. Through this triangular base passes the adoral motor
fibril. Three oral membranelles (flat plates of fine cilia) follow the
spiral of the gullet. Each oral membranelle ends in a distinct basal body.
The ciliary membrane (undulating membrane of unusual construction),
which functions in house-building and repair, is connected through its
basal granules to the adoral fiber. A retractile tentaculoid is found be-
tween each adoral membranelle. Tentaculoids, accessory combs, and
trichocysts have no known fibrillar connection.

The motorium is located in the ectoplasm of the ventral wall of the
column. From it arise directly (1) the adoral fiber (granular), which
connects with the adoral membranelles, the oral membranelles, and the
ciliary membrane; (2) two dorsal fibers, extending into the ectoplasm
adorally, where they end freely; and (3) the ventral fiber, which extends
downward and also ends freely in ectoplasm. The circumesophageal ring
is connected to the motorium indirectly by a single fiber. Short fibers
from the r7vg surround the gullet.

METHODS
Fixatives: Schaudinn’s, 90° C.
Stain: Heidenhain’s iron-alum haematoxylin, aqueous and alcoholic.

GILTATE FIBRIELAR SYSTEMS 255

D. HYPOTRICHA

Oxytricha (Lund, 1935).—The parts of the neuromotor system of
Oxytricha apparently are confined to the more specialized organelles. No
fibrils were found in connection with most of the ventral cirri.

A long membranelle fibril connects the inner ends of the membranelle
plate. The frontal membranelles, in addition, have célzary rootlets which
arise only from the most proximal basal granules of each of the three
rows. These combine into a stouter fibril for each membranelle, which
ends free in the endoplasm.

Along the ventral margin of the peristome are numerous fibrils (Fig.
91). One of these connects the basal granules of the undulating mem-

4 EE CEOS \ \ R

S S>
SSS >

Figure 91. Oxytricha. Diagram of fibrillar complex of cytostome, ventral view.
(Lund, 1935.)
b. fib. u. memb.—basal fibril of undulating membrane
b. pl. memb.—basal plate of membranelle d. cy. fib.—dorsal cytostomal fibril
ect. f.—ectoplasmic fold memb. r.—membranelle rootlet
mg. fib. memb.—marginal fibril of membranelles
mg. u. memb.—position of marginal undulating membrane
p. e. fib—post-esophageal fibril term, fil—terminal filament
v. cy. fib—ventral cytostomal fibril

256 CILIATE FIBRILLAR SYSTEMS

brane. Twenty-two originate near the anterior end of this fibril. Their
posterior destinations are as follows: ten terminate in ten small granules
attached to the marginal fibril in the region of the posterior seven or
eight membranelles; six pass along the dorsal wall of the gullet and
extend into the endoplasm to a point near the right side of the body;
and the other six continue into the gullet to form the ventral post-esopha-
geal fibrils. These fibrils along the right side of the peristome were ob-
served to be lax, apparently nonelastic, and capable of individual move-
ment.

A delicate fibril extends anteriorly from each of the five anal cirri.
In the region at the left of the posterior macronucleus they disappear
from view. Their position suggests, however, that they may join the
other parts of the neuromotor system, in the region of the posterior wn-
dulating membrane fibril.

METHODS

Fixatives: Schaudinn’s (with 5-percent acetic), Zenker'’s.

Figure 92. Uroleptus halseyi, section through anterior end. (Calkins, 1930.)

b. gr.—basal granule c. fib—codrdinating fibrils
b. pl.—basal plate mot.—motorium

CILIATE FIBRILLAR SYSTEMS Zoy,

Stains: Iron-haematoxylin, Mallory’s triple (Sharp’s modification) .
Microdissection

Uroleptus halseyi Calkins (Calkins, 1930).—The conspicuous parts
of this kinetic system are the motorium near the right side of the gullet
and, leading from it, the longitudinal anterior fibril which links a row of
endoplasmic granules. Each basal plate of the membranelle series is con-
nected by a short fibril to one of these basal granules, in regular order,
and additional connectives unite the basal granules of each frontal cirrus
with the chain. One short anterior fibril from the motorium extends to
the margin of the peristome; the other leads to the undulating membrane,
and two posterior fibrils are soon lost in the endoplasm. (Fig. 92).

METHODS

Fixatives: HgCl, saturated in 95-percent alcohol.
Stain: Iron-haematoxylin.

CONCLUSIONS

It is evident from the review presented in foregoing paragraphs that
the differentiation of fibrils in ciliates has been established for various
representatives of their major groups beyond any doubt. Such differ-
entiations, as revealed in fixed and stained material, are not artifacts,
for many may be seen in living or in slowly disintegrating organisms
(Worley, 1933).

It is not certain, however, that all of the structures thus identified are
actually fibrillar. Some may represent rather a sculptural, fibrillar-like
pattern in the pellicle.

It is also clear that the fibrils are not all alike, either structurally or as
related to other protoplasmic differentiations of the cell. This was well
illustrated in the several complexes of fibrils in the contractile stalk
of the vorticellids. Here the structural elements composing the Spzronem,
for example, differed partly in kind, but especially in arrangement,
from those of the Axonem. Also, their relations to the fibrillar com-
plexes of the be// were found to be different.

Again, it is known that many fibrillar complexes of various ciliates
are intimately associated with the basal apparatus of the motor organ-
elles. This was shown for the inner fibrillar complex, or the ifracilza-

258 CILIATE FIBRILLAR SYSTEMS

ture, of holotrichs such as Paramecium; for the membranelle fibril of
Stentor and of Euplotes; and for most of the fibrillar systems of the other
ciliates, the accounts of which were more briefly reviewed.

Also, the literature contains numerous records of fibrils, in a variety
of ciliates, which are structurally integrated into a so-called fibrillar
system. It is with such fibrillar systems that this review has been chiefly
concerned.

Having established the identity of these fibrils and fibrillar systems
and, for many, their structural continuity or contiguity with other organ-
elles of the cells, especially the motor organelles, the investigators’ further
interest has, of course, been concerned with the function or functions
which may be performed by such definitely related and integrated fibrillar
systems.

It was previously pointed out that the interpretation of the function
or functions of these fibrillar systems has been based largely on the evi-
dences of their structural integration and their relationship to other
organelles. Relatively few of the interpretations have been made from
experimental evidence. From both kinds of evidence, it was noted that
at last four elementary functions have been ascribed by the many in-
vestigators to these various fibrils or fibrillar systems: (1) elasticity, (2)
mechanical support, (3) contractility, and (4) conductivity.

Some examples of these included (1) Elasticity, the Spasmonem and
pellicle in the contractile stalk of the vorticellids (Entz, 1893), the axial
filament of cilia (Koltzoff, 1912); (2) Mechanical support, Stitzgitter
system of Paramecium (von Gelei, 1929); fibrils generally (Jacobsen,
1931); (3) Contractility, myonemes of Stentor (Johnson, 1893; and
other authors), and of Boveria (Pickard, 1927); (4) Conductivity,
neuromotor system of many ciliates (Sharp, 1914; Yocom, 1918; and
other authors).

A fifth function, ‘‘metabolic influence,” not previously noted, has re-
cently been proposed by Parker (1929) for the fibrillar complex in
Paramecium (Rees, 1922) and in other ciliates, comparable to the func-
tion of fibrils in nerve cells. The neurofibrillar hypothesis for conduc-
tivity in nerves was regarded as untenable by Parker (1929). After an
extensive review of the evidences for and against this thesis, including
Bethe’s (1897) experiment on the brain neurones in the crab Carcinus,
which showed that the nerve impulses did not have to traverse the fibrils

CILIATE FIBRILLAR SYSTEMS 259

of the cell body, Parker suggested that neurofibrils generally, and possi-
bly the fibrils described for certain ciliates, may serve to transmit, from
the metabolic center or nucleus, metabolic influences “‘essential for the
continued life of the whole neurone.’’ How these transmissions might
be made was not clear. He thought they might involve “chains of ionic
readjustment such as have been proposed as an explanation of the nerve
impulse.”’ Aside from whether or not such might apply to the function
of the fibrils in ciliates, however, he rightly observed that these fibrils
may not be intimately associated with the nucleus, as seems to be the
case 1n neurones.

Entz’s (1893) interpretation of an elastic function for the Spasmonem
in the recoiling stalk of the vorticellids was discussed under the caption
“Interpretations.” In addition to this, reference may be made briefly
to Koltzoff’s (1903, 1906, 1912) similar interpretations for elastic
fibrils in cilia and in cells generally. He would ascribe elasticity to all
fibrils in maintaining all organic form other than spherical. Since proto-
plasm is liquid, as shown by the sphericity of its enclosed vacuoles, then
elastic elements must be postulated to counteract the physical forces of
inner and outer osmotic pressure and surface tension, which tend always
to effect spherical form. Such elements are fibrillar, as observed in
the many kinds of cells investigated. The amount of evidence adduced
by Koltzoff is impressive, but his interpretation obviously cannot apply
exclusively to all fibrils.

Similar claims for a supporting function for fibrils are rather wide-
spread in the literature. Thus Jacobson (1931), as already stated, is
disposed to attribute a supporting function to all noncontractile fibrils.

These few citations, together with many others previously noted, may
serve to indicate the diversity of functions that have been variously
attributed to fibrillar differentiations in ciliates. In so far as they suggest
that these fibrils and fibrillar systems may differ in their structure, func-
tions, and relationships among the manifold kinds of ciliated Protista,
certainly no one could present conclusive evidence to the contrary. But
when, in the absence of proof, an investigator seriously contends that
in these unicellular organisms any and all fibrillar differentiations per-
form only one elementary function, whether it be that of elasticity,
mechanical support, contractility, conductivity, or “metabolic transmis-
sion,’ or when he assigns to these fibrils or fibrillar systems one or two

260 CILIATE FIBRILLAR SYSTEMS

functions to the exclusion of another possible function or functions, then
surely that investigator thereby adopts a point of view which is incon-
sistent and indefensible as well.

In such instances we begin to sense a recrudescence of the opposing
claims advanced in the Ehrenberg-Dujardin controversy and of the non-
cellular theory of protistan organization proposed by Dobell and others.
Once having denied the validity of Ehrenberg’s extreme contention that
the organs of the Infusoria are essentially miniature counterparts of
those of macroscopic organisms, a comparably extreme viewpoint is sub-
stituted, which would maintain that the Protista represent a complete de-
parture in the organization of living things and so belong in the wholly
exclusive category of non-cellular organisms. Thus the claims of these
counter extremists would have us search for identities in organization, on
the one hand, or only for differences in protistan and multicellular or-
ganization on the other hand.

In Ehrenberg’s day similar extreme points of view were quite irrec-
oncilable, but in our day they can scarcely represent anything less than
rash inconsistencies. Obviously the thesis of non-cellular organization
tends to place exclusive emphasis on d/fferences between protozoan and
metazoan organization and, if one is still inclined to accept that thesis,
one might well refer to Bélat’s (1926) excellent monograph on the
protistan nucleus. Variable as are the nuclei, in form and behavior, of
the many kinds there described and illustrated—where they appear to
differ from one another more than some differ from metazoan nuclei—
surely one cannot fail to recognize that their numerous modifications do
not represent discrete differences, but clearly betray the indelible marks
of a common origin. They are like the musical variations of some great
motif. They demonstrate irrefutably that living nature has been both
labile and stable in its evolutionary history, so that we are amply justified
in searching out and emphasizing not merely differences but also, and
more fundamentally, similarities, in both the structural and functional
processes of protoplasmic differentiation.

And since all cellular differentiation is referable in its last analysis to
protoplasmic differentiation, then certainly the fibrils and fibrillar sys-
tems of multicellular tissues, such as those described by Grave and
Schmitt (1925), may belong in the same category, both structurally and
functionally, as some fibrillar differentiations that have been described
and some that we may afford further to search for, in unicellular organ-

CILIATE FIBRILLAR SYSTEMS 261

isms. Knowing today the general properties and behavior of the long-
chain protein molecules, if such fibrils are proteinaceous, as evidently
they are, then fibrillar differentiation is one of the most likely kinds of
protoplasmic differentiation that might be expected.

But by the same token, we would not expect all such proteinaceous
fibrils to be alike, either structurally or functionally. Both by virtue of
their intrinsic properties and their relations to other organelles, some
fibrils of protistan cells, or of metazoan cells, may serve for support,
others for contraction, and still others for conduction of impulses to and
from motor or other organelles. Or any one fibrillar complex may per-
form more than one of these, or of other yet unkown, functions. And
this duality or plurality of fibrillar functions may obtain for protistan
cells and for tissue cells of multicellular organisms. Certainly we know
of no evidences contradicting this posszbil7ty.

The actual function or functions of most fibrils or fibrillar systems
are not as yet finally known. There can be no doubt about the contractile
properties of the myonemes of Stentor, and some experimental evidences
indicate a codrdinating (conductive) function for some fibrils in several
ciliates and in epithelial tissue. The outer fibrillar systems of Paramecium
and other ciliates may be fibrillar, or only apparently fibrillar, as an
integral part and pattern of the pellicle. If of the pellicle, then at least
one of its functions would evidently be that of support.

Much mote study and more critical analysis of these fibrillar systems
are greatly needed by both improved old and newly devised observational
methods, perhaps such as that of the recently developed electron micro-
scope. Then complementing these observational and comparative studies
will be required indispensably, as the crucial test of all of our hypotheses,
exceedingly refined and precise tools and methods of experimentation.
Even today there are many devices suitable for this purpose, if properly
adapted and fully utilized by the ingenious well-trained hands and eyes
of thoroughly informed, exceptionally endowed minds. It is a mistake
to suppose that such microtechniques are peculiarly difficult and that such
problems are really unapproachable. It is rather that these techniques are
different and that their use requires special training. With such training,
it may be easier to transect a ciliate or a marine ovum, with much more
accuracy, than to perform ‘‘free hand” some of the disections on macro-
scopic organisms.

By micromanipulative methods and with the aid of other modern

262 CILIATE: FIBRILLAR SYSTEMS

devices, such as the ultracentrifuge, and by micromethods of irradiation,
and the like, we may expect for the future, once the world has recovered
its sanity, notable advances in protistological investigations such as may
not have been dreamed of, even by the most sanguine of our predecessors.
No other group of organisms may offer more than the unicellular forms
toward the solution of some of our most fundamental problems. The re-
sults, therefore, will provide a better understanding not only of these
unicellular organisms, but of all forms of life. In their last analysis, how-
ever, all such problems must surely depend for any final solution upon
unique exacting methods of biological experimentation. ‘Belief uncon-
firmed by experiment is vain’’ (Francesco Redi, of Florence, 1668).

LITERATURE CITED

Alverdes, F. 1922. Untersuchungen tber Flimmerbewegung. Pfliig. Arch.
ges. Physiol., 195: 243-49,

Bélat, K. 1921. Protozoenstudien III. Arch. Protistenk., 43: 431-62.

— 1925. Hartmann, Allgemeine Biologie. Jena.

—— 1926. Die Formwechsel der Protistenkerne. Ergbn. Zool., 4: 235-664.

Belehradek, Jan., 1921. Rozbor pohybu Vorticell. Biol. Listy, 8: 49-53.

Beltran, Enrique. 1933. Gruberia calkinsi sp. nov., a brackish-water ciliate
(Protozoa, Heterotrichida) from Woods Hole, Mass. Biol. Bull., 64:
De

Bethe, A. 1897. Das Nervensystem von Carcinus maenas. 1. Arch. mikr. Anat.,
50: 589-639.

Bishop, Ann. 1927. The cytoplasmic structures of Spirostomum ambiguum
(CEhrbe)sOuatts | mict. Sci 7-47-72"

Brauer, A. 1885. Bursaria truncatella unter Beriicksichtigung anderer Hetero-
trichen und der Vorticellinen. Jena. Z. Naturw., 19: 489-519.

Bresslau, E. 1921. Die Gelatinierbarkeit des Protoplasmas als Grundlage
eines Verfahrens zur Schellanfertigung gefarbter Dauerpraparate von
Infusorien. Arch. Protistenk., 43: 469-80.

Bretschneider, L. H. 1931. Beitrige zur Strukturlehre der Ophryoscoleciden.
I. Ekto- und Entoplasma. Fibrillen. Zool. Ans. Suppl., 5: 324-30.

—— 1934. Beitrage zur Strukturlehre der Ophryoscoleciden. II. Arch, Pro-
tistenk., 82: 298-330.

Brown, V. E. 1930. The neuromotor apparatus of Paramecium. Arch. zool.
exp. gén., 70: 469-81.

Bush, Mildred. 1934. The morphology of Haptophrya michiganensis Wood-
head, an astomatous ciliate from the intestinal tract of Hemidactylium
scutatum (Schlegel). Univ. Cal. Publ. Zool., 39: 251-76.

Butschli, O. 1887-89. Protozoa. Bronn’s Klassen, Bd. 1-3.

CILIATE FIBRILLAR SYSTEMS 263

Calkins, Gary N. 1930. Uroleptus halseyi Calkins, HI. The kinetic elements
and the micronucleus. Arch. Protistenk., 72: 49-70.

Calkins, Gary N., and R. Bowling. 1929. Studies on Dallasia frontata. II.
Cytology, gametogamy, and conjugation. Arch. Protistenk., 66: 11-32.

Campbell, A. S. 1926. The cytology of Tntinnopsis nucula (Fol) Laackmann,
with an account of its neuromotor apparatus, division, and a new intra-
nuclear parasite. Univ. Cal. Publ. Zool., 29: 179-236.

—— 1927. Studies on the marine ciliate Favella (Jorgensen), with special
regard to the neuromotor apparatus and its rdle in the formation of the
lorica. Univ. Cal. Publ. Zool., 29: 429-52.

Chakravarty, M. 1936. On the morphology of Balantidium depressum
(Ghosh) from a mollusc, Pla globosa, with a note on its nuclear reac-
tions and cytoplasmic inclusions. Arch. Protistenk., 87: 1-9.

Chatton, E., and S. Brachon. 1935. Discrimination, chez deux Infusoires du
genre Glaucoma, entre systeme argentophile et infraciliature. C. R., Soc.
Biol. Paris, 118: 399-402.

Chatton, E., and A. Lwoff. 1930. Impregnation, par diffusion argentique, de
l'infraciliature des ciliés marins et d’eau douce, apres fixation cytologique
et sans desiccation. C. R., Soc. Biol. Paris, 104: 834-36.

1935. I La Constitution primitive de la strie ciliare des Infusoires. La

desmodexie. C. R. Soc. Biol. Paris, 118: 1068-71.

—— 1936. Les Remaniements et la continuité du cinétome au cours de la
scission chez les Thigmotriches Ancistrumidés. Arch. zool. exp. gén.,
78: 84-91.

Cohn, F., and C. V. Siebold. 1862. Ueber die contractilen Staubfaden der Dis-
teln. Z. wiss. Zool., 12: 366-71.

Czermak, J. 1853. Uber den Stiel der Vorticellen. Z. wiss. Zool., 4: 438-50.

Delage, Y., and Ed. Herouard. 1896. Traité de zoologie concrete. 1. La
Cellule et les protozoaires. Paris.

Dierks, K. 1926. Untersuchungen tiber die Morphologie und Physiologie des
Stentor coereuleus mit besonderer Beriicksichtigung seiner kontraktilen
und konduktilen Elemente. Arch. Protistenk., 54: 1-91.

D'Udekem, M. J. 1864. Description des Infusoires de la Belgique. I. Ser. Les
Vorticelliens. Mém. Acad. roy. Belg., 35: 1-52.

Dujardin, F. 1835. Sur les prétendus estomacs des animalcules infusoires et
sur une substance appelée sarcode. Ann. Sci. nat., 2: 4.

—— 1841. Histoire naturelle des (Zoophytes) Infusoires. Suites a Buffon.
Paris.

Ehrenberg, C. G. 1838. Die Infusionsthierchen als Vollkommene Organismen.
Leipzig. Atlas, 64 pls.

Ellis, John. 1937. The morphology, division, and conjugation of the salt-marsh
ciliate Fabrea salina Henneguy. Univ. Cal. Publ. Zool., 41: 343-88.

Dobell, C. C. 1911. Principles of protistology. Arch. Protistenk., 23: 269-310.

264 CILIATE FIBRILLAR SYSTEMS

Doflein, F., and E. Reichenow, 1929. Doflein, Lehrbuch der Protozoenkunde.
5th ed., Jena.

Engelmann, T. W. 1875. Contractilitét und Doppeltbrechung. Pflug. Arch.
ges. Physiol., 11: 432-64.

—— 1879. Physiologie der Protoplasma und Flimmerbewegung. Hermann’s
Handbuch d. Physiol., 1: 343-408.

—— 1880. Zur Anatomie und Physiologie der Flimmerzellen. Pflig. Arch.
ges. Physiol., 23: 505-35.

Entz, G. 1893. Die elastischen und contractilen Elemente der Vorticellen.
Math. naturw. Ber. Ung., 10: 1-48.

Everts, E. 1873. Untersuchungen tiber Vorticella nebulifera. Z. wiss. Zool.,
23:5592-622.

Fabre-Doumergue, P. 1888. Recherches anatomiques et physiologiques sur les
infusoires ciliés. Ann. Sci. nat. (b) Zool., 1: 1-140.

Forest, H. E. 1879. The natural history and development of the Vorticellidae.
Mid]. Nat., 2:

Gelei, G. von. 1937. Ein neues Fibrillensystem im Ectoplasma von Paramecium.
Arch. Protistenk., 89: 133-62.

Gelei, J. von. 1925. Ein neues Paramaecium aus der Umgebung von Szeged,
Paramaecium nephridiatum n. sp. Allatt. Kézlem., 22: 121-59 (résumé
in German, 245-48). |

1926a. Zur Kenntnis des Wimperapparates. Z. Anat. Entw. Gesch., 81:
530-53,

—— 1926b. Cilienstruktur und Cilienbewegung. Zool. Anz. Suppl., 2: 202-
i:

—— 1926c. Sind die Neurophane von Neresheimer neuroide Elemente? Arch.
Protistenk., 56: 232-42.

— 1927. Eine neue Osmium-Toluidinsmethode fiir Protistenforschung.
Mikrokosmos, 20: 97-103.

—— 1929a. Sensorischer Basalapparat der Tastborsten und der Syncilien bet
Hypotrichen. Zool. Anz., 83: 275-80.

—— 1929b. Uber das Nervensystem der Protozoen. Allatt. Kézlem. (Zool.
Mitt.) , 26: 186-90.

—— 1932a. Die reizleitenden Elemente der Ciliaten in nass hergestellten
Silber-bwz. Goldpraparaten. Arch. Protistenk., 77: 152-74.

—— 1932b. Ein Neue Goldmethode fiir Ciliatenforschung und eine neue
Ciliate: Colpidium pannonicum. Arch. Protistenk., 77: 219-30

—— 1933. Uber den Bau die Abstammung und die Bedeutung der sog. Tast-
borsten bei den ciliaten. Arch. Protistenk., 80: 116-27.

— 1934. Der Cytopharynx der Paramecien. Matemat. és termész. értesit6,
Is 750-50!

—— 1934. Die Differenzierung der Cilienmeridiane der Ciliaten und der
Begriff des Richtungsmeridians. Matemat. és termész. €rtesit6, 51: 632-44.

—— 1934, Das Verhalten der ectoplasmatischen Elemente des Parameciums
wahrend der Teilung. Zool. Anz., 107: 161-77.

CILIATE FIBRILLAR SYSTEMS 265

—— 1934. Die Vermehrung der Sinneshaare von Emplotes wahrend des
Teilungsprozesses. Zool. Anz., 105: 258-66.

— 1934. Eine mikrotechnische Studie ber Farbung der subpellicularen Ele-
mente der Ciliaten. Z. wiss. Mikr., 51: 103-78.

— 1934. Der feinere Bau des Cytopharynx von Paramecium und seine
systematische Bedeutung. Arch. Protistenk., 82: 331-62.

—— 1935. Eine neue Abanderung der Klein’schen trockenen Silbermethode
und das Silberliniensystem von Glaucoma scintillans. Arch. Protistenk.,
84: 446-55.

—— 1935. Der Richtungsmeridian und die Neubildung des Mundes wahrend
und ausserhalb der Teilung bei den ciliaten. Biol. Zbl., 55: 436-45.
— 1935. Historisches und Neues wtber die interciliaren Fasern und ihr

morphologische Bedeutung. Z. Zellforsch., 22: 244-54,

— 1935. Colpidium glaucomaeformae n. sp. (Hymenostomata) und sein
Neuronensystem. Arch. Protistenk., 85: 289-302.

—— 1936-37. Der schraubige K6rperbau in der Ciliatenwelt im Vergleich zu
Symmetrieverhaltnissen der vielzelligen Tiere. Arch. Protistenk., 88:
314-38.

— 1937. Pori secretorii am Ciliatenkérper. Biol. Zbl., 57: 175-87.

—— 1938. Schraubenbewegung und Ko6rperbau pei Paramecium. Arch.

Protistenk., 90: 165-77.

and P. Horvath. 1931. Eine nasse Silber-bzw. Goldmethode fur die

Herstellung der reizleitender Elemente bei den Ziliaten. Z. wiss. Mikr.,

48 9-29.

Grave, C., and C. O. Schmitt. 1925, A mechanism for the codrdination and
regulation of ciliary movement as revealed by micro-dissection and cyto-
logical studies of ciliated cells of molluscs. J. Morph., 40: 479-515.

Greeff, R. 1870-71. Untersuchungen tiber den Bau und die Naturgeschichte
der Vorticellen. Arch. Naturgesch., I-II Abt., 36: 37.

Griffin, L. E. 1910. Explotes worcesteri sp. nov. I. Structure. Philipp. J. Sct.,
a: Col S52:

Haeckel, E. 1873. Zur Morphologie der Infusorien. Jena. Z. Naturgesch.,
7: 516-60.

Hall, R. F. 1923. Morphology and binary fission of Menozdium incurvum.
Univ. Cal. Publ. Zool., 20: 447-76.

Hammond, D. M., and C. A. Kofoid. 1936. The continuity of structure and
function in th neuromotor system of Explotes patella during its life cycle.
Proc. Amer. phil. Soc., 77: 207-18.

Hammond, J. C. 1935. Physiological dominance as a factor in ciliary codrdina-
tion in the Protozoa. Ohio J. Sci., 35: 304-6.

Hartmann, M. 1925. Allgemeine Biologie. Eine Einleitung in die Lehre vom
Leben. Jena.

Heidenhain, M. V. 1899. Beitrige zur Aufklirung des wahren Wesens der
faserformigen Differenzierungen. Anat. Anz., 16: 97-131.

266 CIEIATE FIBRIELARYSYSTEMS

— 1911. Plasma und Zelle. Jena.

Heidenreich, E. 1935. Ptychostomum lumbriculi n. sp. Arch. Protistenk., 85:
205-D):

Higgins, H. T. 1929. Variations in Nyctotherus found in frog and toad tad-
poles and adults. Trans. Amer. Micr. Soc., 48: 141-57.

Hofker, J. 1928. Das neuromotorische Apparat der Protozoen. Tijdschr.
Nederlandsch. 1:34-38.

Horvath, J. 1938. Eine neue Silbermethode fiir die Darstellung des Stiitz-
gitters und der erregungsleitenden Elemente der Ciliaten. Z. wiss. Mikr.,
Dole

Jacobson, Irene. 1931. Fibrillaére Differenzierungen bei Ciliaten. Arch. Protis-
tenk., 75: 31-100.

Johnson, H. P. 1893. A contribution to the morphology and biology of the
stentors. J. Morph., 8: 467-562.

Kate, C. G. B. ten. 1926. Uber das Fibrillensystem der Ciliaten. Dissertation
Univ. Utrecht. Zutphen.

—— 1927. Uber das Fibrillensystem der Ciliaten. Arch. Protistenk., 57: 362-
426.

—— 1928. Uber das Fibrillensystem der Ciliaten. 2. Das Fibrillensystem der
Isotrichen. Arch. Protistenk., 62: 328-54.

Kidder, G. W. 1933a. On the Genus Ancistruma Strand (Ancistruma Mau-
pas). I. The structure and division of A. mytili Quenn. and A. isselz
Kahl. Biol. Bull. 64: 1-20.

—— 1933. Studies on Conchophthirus mytili De Morgan. I. Morphology
and division. Arch. Protistenk., 79: 1-24.

— 1934. Studies on the ciliates from fresh water mussels. I. The structure
and neuromotor system of Conchophthirus anodontae Stein, C. curtus
Engl., and C. magna sp. nov. Biol. Bull. 66: 69-90.

Kirby, Harold. 1932. Protozoa in termites of the Genus Amztermes. Parasitol-
Ogy, 24: 289-304.

Klein, B. M. 1926a. Uber eine neue Eigentiimlichkeit der Pellicula von
Chilodon uncinatus Ehrbg. Zool. Anz., 67: 160-62.

—— 1926b. Ergebnisse mit einer Silbermethode bei Ciliaten. Arch. Protistenk.,
56: 243-79.

— 1927. Die Silberliniensysteme der Ciliaten. Ihr Verhalten wahrend
Teilung und Conjugation, neue Silberbilder, Nachtraige. Arch. Protistenk.,
58: 55-142.

—— 1928. Die Silberliniensysteme der Ciliaten. Weitere Resultate. Arch.

Protistenk., 62: 177-260.

1929. Weitere Beitrage zur Kenntnis des Silberliniensystems der Ciliaten.

Arch. Protistenk., 65; 183-257.

1930. Das Silberliniensystem der Ciliaten. Weitere Ergebnisse. IV. Arch.

Protistenk., 69: 235-326.

CILIATE FIBRILLAR SYSTEMS 267

1931. Uber die ZugehGrigkeit gewisser Fibrillen bzw. Fibrillenkomplex

zum Silberliniensystem. Arch. Protistenk., 74: 401-16.

—— 1932. Das Ciliensystem in seiner Bedeutung fiir Locomotion Coordina-
tion und Formbildung usw. Ergebn. Biol., 8: 75-179.

—— 1933. Silberliniensystem und Infraciliatur. Eine kritische Gegenuberstel-
lung. Arch. Protistenk., 79: 146-69.

—— 1935. Die Darstellung des Silberlinien oder neuroformativen Systems
nebst Griindsatzlichem zur Silbermethodik. Z. wiss. Mikr., 52: 120-57.

—— 1936. Wirkung von Schlangengiften auf Leben und Silberliniensystem
von Infusorien. Arch. Protistenk., 87: 299-313.

—— 1936. Beziehungen zwischen Maschenweite und Bildungsvorgingen im
Silberliniensystem der Ciliaten. Arch. Protistenk., 88: 1-22.

—— 1937. Uber die Eigenk6rplichkeit des Silberliniensystems. Arch. Protts-
tenk., 88: 188-91.

—— 1937. Regionire Reaktionen im Silberlinien- oder neuroformativen Sys-
tem der Ciliaten. Arch. Protistenk., 88: 192-210.

—— 1938. Miss-bzw. Doppelbildung am Silberliniensystem von Ciltaten.
Arch. Protistenk., 90: 292-98.

Kofoid, C. A., and R. F. MacLennan 1932. Ciliates from Bos mdicus Linn.
II. A revision of Diplodinium Schuberg. Univ. Cal. Publ. Zool., 37:
3-152:

Kolliker, A. 1845. Die Lehre von der tierischen Zelle. Z. wiss. Bot., 1: 2.

—— 1864. Icones histiologicae 1. Leipzig.

Koltzoff, N. K. 1903. Uber formbestimmende elastische Gebilde in Zellen.
Biol. Zbl., 23: 680-96.

—— 1906. Die Spermien der Decopoda, als Einleitung in das Problem der
Zellengestalt. Arch. mikr. Anat., 67: 364-571.

—— 1909. Studien iiber die Gestalt der Zelle. I]. Untersuchungen tuber das
Kopfskelett des tierschen Spermiums. Arch. Zellforsch., 2: 1-65.
— 1912. Studien tiber die Gestalt der Zelle. II]. Untersuchungen tber die

Kontraktilitat des Vorticellenstiels. Arch. Zellforsch. 7: 344-423.

Kihne, W. 1859. Untersuchungen tiber Bewegung und Verinderungen der
contractilen Substanz. Arch. Anat. Physiol., 1859: 748-835.

—— 1859. Uber sogenannte idiomuskulare Kontraktion. Arch. Anat. Physiol.,
1859: 418-20.

Lachmann, J. 1856. Uber die Organization der Infusorien, besonders der
Vorticellen. Arch. Anat. Physiol., 1856: 340-98.

Lang, A. 1901. Lehrbuch der vergleichenden Anatomie der wirbellosen Tiere.
(Protozoa). 2. Aufl., Abt. 1. Berlin.

Lebedew, W. 1909. Uber Trachelocerca phoenicopterus (ein marines Infusor).

Arch. Protistenk., 13: 70-114.

Leiberman, P. R. 1929. Ciliary arrangement in different species of Paramecium ——~

irans, Aimer, Micr. Soc, 48: 1-11. AGS

i % LY ee
LSSYAvDS
la LO"

4
¥

ty Ga >

RAR

2
oN

268 CILIATE FIBRILLAR SYSTEMS

Leydig, F. 1883. Untersuchungen uber Anatomie und Histologie der Tiere.
Bonn.

Lieberkihn, N. 1857. Beitrage zur Anatomie der Infusorien. Arch. Anat.
Physiol., 1857: 20-36.

Lucas, M. S. 1934. Ciliates from Bermuda sea urchins. I. Metopus. J. R. mict.
Soc., 54: 79-93.

Lithe, M. 1913. A. Lang, Handbuch der Morphologie der wirbellosen Tiere.
1. Protozoa. Berlin.

Lund, E. E. 1933. A correlation of the silverline and neuromotor systems of

Paramecium. Univ. Cal. Publ. Zool., 39: 35-76.

1935. The neuromotor system of Oxytricha, J. Morph., 58: 257-77.
Lynch, J. E. 1929. Studies on the ciliates from the intestine of strongylocentro-
tus. 1. Entorhipidium Gen. nov. Univ. Cal. Publ. Zool., 33: 27-56.

—— 1930. Studies on the ciliates from the intestine of Strongylocentrotus. Il.
Lechriopyla mystax, Gen. nov., sp. nov. Univ. Cal. Publ. Zool., 33:
307-50.

McDonald, J. Daley. 1922. On Balantidium coli (Malmsten) and Balantidium
suis (sp. nov.), with an account of their neuromotor apparatus. Univ.
Cal. Publ. Zool., 20: 243-300.

MacDougall, Mary S. 1928. The neuromotor system of Chlamydodon sp. Biol.
Bull., 54: 471-84.

MacLennan, R. F. 1935. Dedifferentiation and redifferentiation in Ichthy-
ophthirius. 1. Neuromotor system. Arch. Protistenk., 87: 191-210.
MacLennan, R. F., and F. H. Connell. 1931. The morphology of Expoterion

pernix Gen. nov., sp. nov. Univ. Cal. Publ. Zool., 36: 141-56.

Maier, H. N. 1903. Uber den feineren Bau der Wimperapparate der In-
fusorien. Arch. Protistenk., 2:73-179.

Maupas, E. 1883. Contribution a l’etude morphologique et anatomique des
infusoires ciliés, Arch. zool. exp. gén., (2) I: 427-644.

Merton, H. 1932. Gestalterhaltende fixierungsversuche an besonders kon-
traktilen Infusorien nebst Beobachtungen tiber das verhalten der lebenden
Myoneme und Wimpern bei Stentor, Arch. Protistenk., 77: 491-521.

Metschnikoff, E. 1863. Untersuchungen tiber den Stiel der Vorticellen. Arch.
Anat. Physiol., 1863: 180-86.

—— 1864. Nachtragliche Bemerkungen tiber den Stiel der Vorticellen. Arch.
Anat. Physiol., 1864: 291-302.

Meyer, A. 1920. Analyse der Zelle. Jena.

Neresheimer, E. R. 1903. Die Hohe histologischer Differenzierung bei hetero-

trichen Ciliaten. Arch. Protistenk., 2: 305-24.

1907. Nochmals tiber Stentor coeruleus. Arch. Protistenk., 9: 137-38.
Parker, G. H. 1929. What are neurofibrils? Amer. Nat., 63: 97-117.
Pensa, A. 1926. Particolarita strutturale di alcuni protozoi cigliati in rapporto

con la contratilia. Monit. zool. ital., 37: 165-73.

Peschkowsky, L. 1927. Skelettgebilde bei Infusorien. Arch. Protistenk., 57:

aii

CILIATE FIBRILLAR SYSTEMS 269

Pickard, E. A. 1927. The neuromotor apparatus of Boveria ternedinidi Nelson,
a ciliate from the gills of Teredo navalis. Univ. Cal. Publ. Zool., 29:
405-28.

Powers, P. B. A. 1933. Studies on the ciliates from sea urchins. II. Envtodiscus
borealis (Hentschel). Behavior and morphology. Biol. Bull., 65: 122-36.

Prowazek, S. 1903. Protozoenstudien. III. Ewplotes PEN, Arb. zool. Inst.
Univ. Wien., 14: 81-88.

Pitter, A. 1904. Die Reizbeantwortung der ciliaten Infusorien. Z. allg.
Physiol., 3: 406-54.

Raabe, Zdz. 1932. Untersuchungen an einigen Arten des Genus Concho ph-
thirus Stein. Bull. Int. Acad. Cracovie, Cl. Sci. Math et Nat. Ser. B. Sci.
Nat. (II) (Zool.) 1932 (8/10): 295-310.

Ray, Harendranath. 1932. On the morphology of Balantidium sushilii n. sp.,
from Rana tigrina Daud. J. R. micr. Soc., 52: 374-82.

Rees, C. W. 1921. The neuromotor apparatus of Paramecium. Amer. Nat.,
55: 464-68.

—— 1922. The neuromotor apparatus of Paramecium. Univ. Cal. Publ. Zool.,
20: 333-64.

—— 1930. Is there a neuromotor apparatus in Diplodinium ecaudatum? Sci-

ence, 71: 369-70.

1931. The anatomy of Diplodinium medium. J. Morph., 52: 195-215.

Reichenow, E. 1929. Doflein, Lehrbuch der Protozoenkunde. Sth ed., Jena.

Rosenberg, L. E. 1937. The neuromotor system of Nyctotherus hylae. Univ.
Cal. Publ. Zool., 41: 249-76.

Roskin, G. 1915. La Structure des myonémes contractiles de Stevtor coeruleus.
Tirage a part des memoires scientifique des Chaniavsky Université de
Moscou. 1:

— 1918. Sur la structure de certains elements contractiles de la cellule.
Arch. russ. d’Anat.,

—— 1923. Die Cytologie der Kontraktion der glatten Muskelgellen. Arch.
Zellsforsch., 17: 368-81.

Rouget, C. 1861. Sur les phenomenes de polarisation qui s’observent dans
quelques tissus vegetative et des animaux. J. Physiol. Path. gén., 5:

Schewiakoff, W. 1889. Beitrige zur Kenntnis der holotrichen Ciliaten. Bibli-
oer.) zo0lf> Hd, 1-77.

Schmidt, W. J. 1924. Die Bausteine des Tierk6rpers im polarisierten Lichte.
Bonn.

Schréder, O. 1906a. Beitrage zur Kenntnis von Campanella umbellaria. Arch.
Protistenk., 7: 75-105.

—— 1906b. Beitrige zur Kenntnis von Epistylis plicatilis. Arch. Protistenk.,
TN) 3-S5.

—— 1906c. Beitrage zur Kenntnis von Vorticella monilata. Arch. Protistenk..
7: 395-410.

— 1907. Beitrage zur Kenntnis von Stentor coeruleus und Stentor roeselii

Ehrbg. Arch. Protistenk., 8: 1-16.

270 CILIATE FIBRILEAR SYSTEMS

Schuberg, A. 1890. Zur Kenntnis des Stentor coeruleus. Zool. Jahrb., Abt.
Anat. Ontog., 4: 197-238.

—— 1905. Uber Cilien und Trichocysten einiger Infusorien. Arch. Protistenk..,
6: 61-110.

Seifriz, W. 1929. The contractility of protoplasm. Amer. Nat., 63: 410-34.

Sharp, R. G. 1914. Diplodiminum ecaudatum with an account of its neuro-
motor apparatus. Univ. Cal. Publ. Zool., 13: 42-122.

Siebold, C. Th. von. 1849. Uber einzellege Pflanzen und Tiere. Z. wiss. Zool.,
Le 270:

Siebold, C. Th. von, and H. Stannius. 1845. Lehrbuch der vergleichenden
Anatomie, H. 1. Berlin.

Stein, F. 1854. Die infusionsthiere auf ihre Entwicklung untersucht. Leipzig.

— 1859. Der Organismus der Infusionsthiere nach einigen Forschungen in
systematischer Reihenfolge bearbeitet. I. Abtheilung Allgemeiner Theil
und Naturgeschichte der hypotrichen Infusionsthiere. Leipzig.

— 1867. Ibid., 11. Abtheilung Naturgeschichte der heterotrichen Infusorien,
pp. 140-352. Leipzig.

Stevens, N. M. 1891. Studies on ciliate Infusoria. Proc. Calif. Acad. Sci., 3:
1-42.
Studitsky, A. N. 1932. Uber die Morphologie, Cytologie u. Systematik von
Ptychostomum chattoni Rossolimo. Arch. Protistenk., 76: 188-216.
Taylor, C. V. 1920. Demonstration of the function of the neuromotor ap-
paratus in Explotes by the method of microdissection. Univ. Cal. Publ.
Zool., 19: 403-70.

—— 1929. Experimental evidence of the function of the fibrillar system in
certain Protozoa. Amer. Nat., 63: 328-45.

Turner, J. P. 1933. The external fibrillar system of Evp/otes, with notes on
the neuromotor apparatus. Biol. Bull., 64: 53-66.

Verworn, Max. 1889. Protisten-Studien. Jena.

Visscher, J. P. 1927. A neuromotor apparatus in the ciliate Dileptus gigas.
J. Morph., 44: 373-81.

Worley, L. G. 1933. The intracellular fibre systems of Paramecium. Proc. nat.
Acad. Sci. Wash., 19: 323-26.

Wrisberg, H. A. 1765. Observationum de animalculis infusoriis satura, Got-
tingae.

Wrzesniowski, A. 1877. Betrage zur Naturgeschichte der Infusorien. Z. wiss.
Zool., 29: 267-323.

Yabroff, S. W. 1928. A modification of the Da Fano technique. Trans. Amer.
micr. Soc., 47: 94-95.

Yocom, H. B. 1918. The neuromotor apparatus of Emplotes patella. Univ.
Cal. Publ. Zool., 18: 337-96.

Zon, Leo. 1936. The physical chemistry of silver staining. Stain Tech., 11:
53-65.

CHAPTER: V.

MOTOR RESPONSE IN UNICELLULAR ANIMALS
SO MASA:

RESPONSES consist of changes in structure, composition, form, or move-
ment in organisms, which, in turn, are correlated with changes in the
constituents of the environment or the organisms. Responses are found
in all living systems and are among the most fundamental distinguish-
ing characteristics of life. Motor responses consist of changes in tate
or direction of movement of organisms or their constituents. They
facilitate the control of the environment by the organisms involved and
are consequently of great importance to them. Knowledge concerning
these responses and their relation to the factors correlated with them
makes it possible to control the activities of organisms, it throws light
on the distribution of pain and pleasure (consciousness) which pro-
foundly affects the attitude of man toward his fellow creatures, and it
illuminates the processes involved in instincts and in learning. Such
knowledge is therefore very valuable.

The unicellular organisms are in many respects extraordinarily favor-
able for the study of the more fundamental characteristics of these re-
sponses. They are relatively simple in structure. Many of them can be
readily procured and maintained in great numbers, and the factors in
their environment can be accurately controlled or changed as desired;
and, in addition, details concerning the responses can be readily seen.
Moreover, the motor responses in these organisms are very favorable
for the study of adaptation, as pointed out long ago by Jennings (1906).

In the following pages are presented the more important facts in hand
concerning the motor responses of the rhizopods, flagellates, ciliates, and
colonial forms to light, electricity, and chemicals. These are presented
with the view of encouraging the use of these organisms in further work
on various biological problems. Important results have also been ob-
tained on responses to contact, temperature, and gravity, but limitation in
space prevents the consideration of these.

272 MOTOR RESPONSES

RESPONSES TO LIGHT
A. RHIZOPODS

Many of the rhizopods respond to light and some orient if they are
exposed in a beam of light. Some of the responses are correlated with
the rate of change in intensity, others are not. They are fundamentally
the same in all the species which have been investigated, but they have
been more thoroughly studied in Amoeba proteus than in any of the
other species. The following considerations therefore refer largely to
this species.

A, proteus consists of a thin elastic outer membrane, the plasmalemma,
a central relatively fluid granular mass, the plasmasol, surrounded by a

Figure 93. Camera sketch of horizontal optical section of Amoeba proteus. Ps, plas-
masol; Pg, plasmagel; Pl, plasmalemma; HC, hyaline cap; Pgs, plasmagel sheet; L,
Liquid layer; S, region of solation; G, region of gelation. (After Mast, 1926.)

relatively solid granular layer, the plasmagel, and a thin fluid hyaline
layer between the plasmagel and the plasmalemma (Fig. 93). During
locomotion, the plasmalemma is attached to the substratum and to the
adjoining plasmagel; the plasmagel at the posterior end is transformed
into plasmasol, which flows forward to the anterior end and is there
transformed into plasmagel. The forward flow of the plasmasol is due
to contraction of the plasmagel at the posterior end and expansion at the
anterior end, owing to difference in its elastic strength in these two re-
gions. In some species surface tension is probably also involved in locomo-

MOTOR RESPONSES 212

tion. In A. mira, for example, the anterior portion consists of a thin
sheet of hyaline cytoplasm in close contact with the substratum (Hopkins,
1938). The surface tension at the water-substrate interface is probably
greater than the combined surface tensions at the cytoplasm-substrate and
cytoplasm-water interfaces. This would result in a spreading of the cyto-
plasm over the substrate (like oil over water) wherever the two are in
contact, i.e., at the anterior end of the organism.

With reference to a marine amoeba, Pantin (1923-31) contends that
the cytoplasm at the anterior end swells and extracts water “from the
posterior protoplasm of the amoeba itself, and that this will cause a
stream from the posterior to the anterior end.”’ Presumably he holds that
the cytoplasm shrinks there and gives it off. However, that would neces-
sitate absorption of water during gelation at the anterior end, and elimina-
tion during solation at the posterior end, which is contrary to what is
ordinarily observed in the process of gelation and solation of gels.

Marsland and Brown (1936) suggest that the forward flow is due to
increase in volume during solation at the posterior end, and decrease
in volume during gelation at the anterior end. They give no direct evi-
dence in support of this suggestion, but simply say that “The magnitude
of these volume changes in relation to the observed rate of flow is prob-
lematical.”’

Responses of Amoeba to light are therefore probably due to localized
changes in (1) the elastic strength of the plasmagel, (2) the rate of
transformation of plasmasol to plasmagel and vice versa, or (3) the
firmness, extent, or region of attachment to the substratum (Mast, 1923,
1926a,) 193 ba);

Shock-reactions —Engelmann (1879) long ago observed that if
strong light is flashed on an amoeba, movement stops suddenly, but that
if the intensity is gradually increased, movement continues. This response
therefore depends upon the rate of change of light intensity. Such re-
sponses are usually designated ‘‘Schreckbewegungen,” or shock-reactions.
They are closely correlated with adaptation. The shock-reaction in
Amoeba produced by light varies greatly. It may consist merely of mo-
mentary retardation in streaming in a localized region in a pseudopod, of
total cessation throughout the entire animal with reversal in direction of
streaming after recovery, or of any one of an endless number of modifica-

274 MOTOR RESPONSES

tions between these extremes. The character of the response is correlated
with the amount of light received, as well as with the rate of reception.
There is no fixed threshold and the “‘all-or-none” law does not apply
(Mast, 1931a).

If an amoeba is intensely illuminated for a very short time only, move-
ment does not cease until some time after the light has been cut off.
The period between the beginning of illumination and the response ts

Figure 94. Curves showing for Amoeba proteus the

: relation between luminous intensity, reaction time
\ (RT), stimulation period (SP) and latent period (LP),
and a hyperbola (H). Note that the curve for re-

action time simulates a hyperbola, but that the curve

for the stimulation period does not. This shows that

the amount of light energy necessary for response

varies with the intensity and that the Bunsen-Roscoe

law does not apply. (Modified after Folger, 1925.)

-— .
-_--—-

UMINOUS INTENSIT

known as the “reaction time’’; the time illumination must continue, the
“stimulation period’; and the time it need not continue, the “latent
period” (Folger, 1925). There are therefore two processes involved in
producing this response. The first occurs only in light, the other in light
or in darkness. The action of light probably results in the formation of
a substance which acts to produce, independent of light, another sub-
stance which induces the response.

MOTOR RESPONSES 275)

After an amoeba has responded to rapid increase in illumination,
some time must elapse before it will respond again to the same increase
in illumination. There is therefore a refractory period, a period during
which the amoeba recovers from the effect of the stimulation. During
a part of this period the amoeba may remain either in light of the same
intensity, such as that which induced the response, in light of lower
intensity, or in darkness; but during the remainder of the period it must
be in light of lower intensity or in darkness. There are therefore two
processes which occur during the refractory period, one (1 to 2 minutes)
which proceeds with or without any change in luminous intensity, and
another (10 to 20 seconds) which proceeds only if the intensity is de-
creased. These processes result in the production of the physiological state
which existed before the exposure; that is, in recovery (Folger, 1925).

The latent period and the amount of light energy required to induce
cessation of movement vary with the intensity of the light used (Fig. 94).
Figure 94 shows that as the intensity increases, the latent period increases
rapidly from about one second at 500 + meter-candles to a maximum
of about 6 seconds at 1,000 ++ meter-candles, and then decreases gradu-
ally to about 0.75 seconds at 11,000 + meter-candles; and that the light
energy required to induce cessation of movement decreases from about
7,000 ++ meter-candle seconds at 500 -+ meter-candles to a maximum of
about 24,000 -— meter-candle seconds at 1,500 + meter-candles, and
then increases to about 30,000 + meter-candle seconds at 11,000 +
meter-candles. These results are, however, only rather crude approxima-
tions. They were obtained by a method of calculation which yields results
with a large probable error and they have not been confirmed. The data
are, however, sufficiently accurate to substantiate Folger’s conclusion
that the Bunsen-Roscoe law does not hold.

This work should be repeated, and the latent period established by
direct observation in all luminous intensities, instead of by calculation.
This is especially desirable since recent experience makes it possible to
select specimens of A. proteus in which the responses are much more
consistent than they were in those used by Folger.

No explanation has been offered for the mode of variation in the
latent period, with variations in luminous intensity during the period of
stimulation. However, it has been suggested that the variation in the

276 MOTOR RESPONSES

amount of light energy required to induce cessation of movement is due,
at least in part, to adaptation (Mast, 1931a). For if the light is rapidly
increased and then held, streaming soon begins again, t.e., the organism
recovers from the effect of the increase in light. In other words, it be-
comes adapted (Mast, 1939; Folger, 1925). This shows that the effect
of rapid increase in light is eliminated while the organism is continuously

Figure 95. Camera drawings of Amoeba sp. illustrating the response to localized
illumination. Rectangular areas, regions of high illumination; arrows, direction of proto-
plasmic streaming; dotted lines in B, C and D, positions and forms shortly after the
illumination of the parts indicated; , nucleus; v, contractile vacuole. E and F, same
specimen; F, form and direction of streaming assumed by E after the anterior end had
been illuminated for a few minutes. (After Mast, 1932.)

exposed to the light. It also indicates that there are two opposing processes
involved, i.e., that increase in light induces certain changes in the or-
ganism and that internal factors tend continuously to oppose and to
eliminate these changes. If this is true, the more rapidly a given amount
of light is received, the less time there is for recovery, and consequently
the greater will be the effect of a given quantity of light. This probably
accounts for the increase in the amount of light energy required (with
decrease in intensity) when observations are made in weak light; but it

MOTOR RESPONSES ZF,

does not account for the increase in the amount required (with increase
in intensity) if the observations are made in strong light.

The quantity of light energy required to induce cessation of movement
depends upon the chemical composition of the surrounding medium. In-
crease in HCL, for example, causes an increase in the quantity of light
required. On the other hand, an increase in CO, causes decrease in the
quantity required. In solutions of KCI, CaCl,, and MgCl,, respectively,
the quantity of energy required increases as the salt concentration de-
creases, but in solutions of NaCl there is no consistent correlation be-
tween the quantity of energy and the concentration of the salt. In gen-
eral, the quantity of energy required appears to vary directly with the
viscosity of the cytoplasm (Mast and Hulpieu, 1930). The observations
made by these authors extended, however, over only a very limited range
of environmental variation. The conclusions reached are therefore not
applicable to wide ranges of variations in the environment (Mast and
Prosser, 1932).

Increase in the illumination of any localized region of an amoeba re-
sults in an increase of the thickness of the plasmagel in this region (Fig.
95). Increase in the illumination of the entire amoeba results in an
increase in the thickness of the plasmagel at the tip of the advancing
pseudopods. In turn, this causes a cessation of movement (shock-reac-
tion).

The shorter waves of light are more efficient in inducing this response
than the longer waves (Harrington and Leaming, 1900; Mast, 1910).
According to Inman, Bovie, and Barr (1926), ultra-violet light is prob-
ably more efficient than visible light. Although the distribution in the
spectrum of stimulating efficiency has not been precisely ascertained,
Folger (1925) maintains that it is not closely correlated with tempera-
ture. He did not thoroughly investigate the problem, however.

Kinetic ves ponses.—lf an amoeba is kept for some time in very weak
light it becomes inactive; if the light is then increased, the organism
gradually becomes active again. This response is similar to the response
to change in temperature. It is primarily correlated with the magnitude
of the change, not with the rate of change in intensity. It is probably due
to the effect of light on the rate of transformation of gel to sol and vice
versa. This type of response occurs also in D7fflagia (Mast, 1931c), but

Ona HT CO
% @

15000 m.c.

Rate of locomotion, micra per minute

26600 m.c.

7.5 15 DIS les) 15 22.5 30
Time in light in minutes
Figure 96. Relation between adaptation to light of different intensities and rate of
locomotion in Amoeba proteus. Each point in the figure represents the average for one
measurement on each of from fourteen to twenty-three specimens. The time in light
is the time from the beginning of movement after exposure until the measurements were
made. (After Mast and Stahler, 1937.)

MOTOR RESPONSES 249

the observations on it should be repeated and extended under carefully
controlled conditions.

Mast and Stahler (1937) made a thorough study of the relation be-
tween luminous intensity and rate of locomotion in A. proteus. They
found that if dark-adapted amoebae are exposed to light, the rate of
locomotion gradually increases to a maximum and then remains con-
stant; that the time required to reach the maximum decreases from 15
minutes at 225 meter-candles to a minimum of 7 minutes at 15,000
meter-candles, and then increases to 30 minutes at 40,000 meter-candles;
and that the rate of locomotion at the maximum increases from 128.8 ++
10.8 micra per minute at 50 meter-candles to 219.3 -+ 11.4 micra per
minute at 15,000 meter-candles, and then decreases to 150.2 + 8.5
micra per minute at 40,000 meter-candles (Fig. 96). They present evi-
dence which indicates that the increase in rate of locomotion with in-
crease in light intensity is due to the action of the longer waves, and that
the decrease in rate in intensities beyond the optimum is due to the action
of the shorter waves. This action of light on rate of locomotion 1s similar
to the action of temperature. It is probably due to changes in the rate of
sol-gel and gel-sol transformations. If this is true, both of these trans-
formations must be augmented by the longer waves and retarded by the
shorter.

Orientation —Davenport (1897) found that A. proteus orients fairly
precisely in a beam of direct sunlight and that it is photonegative, but
he did not ascertain the processes involved in orientation.

Mast (1910) demonstrated that if an amoeba is unilaterally illu-
minated, pseudopods develop more freely on the shaded side than on
the illuminated side, and that this results in gradual turning from the
light (Fig. 97). He concludes that orientation is due to retardation in
the formation of pseudopods on the more highly illuminated side, owing
to increase in the thickness of the plasmagel on this side caused by the
gelating effect of light.

There is some evidence which indicates that A. proteus is photopositive
in very weak light (Schaeffer, 1917; Mast, 1931a). More carefully con-
trolled observations concerning this are highly desirable.

It is possible that the kinetic responses in Amoeba are due to changes
in the rate of sol-gel transformations at the anterior end, and gel-sol

280 MOTOR RESPONSES

transformations at the posterior end. Moreover, the shock reactions ap-
pear to be associated with rapid local increases in the sol-gel transforma-
tion. If the views concerning the process of orientation as presented

—oo
3:52 3:49 3:48 P.M.
3:54
<¢———
iF
—
3257°5
Ga
3:55
3:57
a

3:58°5
kK 05 mn. a]

Figure 97. Camera outlines representing different stages in the process of orientation
in Amoeba proteus. 1, Amoeba oriented in light I’; 2-9, successive positions after ex-
posure to light /, time indicated in each. Arrows represent the direction of streaming
of protoplasm in the pseudopods. In those which do not contain arrows there was no
perceptible streaming at the time the sketch was made. / and /’ direction of light; mm,
projected scale. (After Mast, 1910.)

above are correct, it is obvious that orientation is the result of shock
reactions rather than kinetic reactions.

B. HEAGELLATES

Response to rapid changes in the intensity of light is very widespread
among the flagellates, and many of them orient fairly precisely. The
processes involved are essentially the same in all. Ezglena is representa-
tive of those which orient, and Peranema trichophorum is representative
of those which do not.

Euglena rotates continuously on its longitudinal axis as it swims. The
flagellum extends backward along the ventral or abeyespot surface. This
causes continuous deflection of the anterior end toward the opposite

MOTOR RESPONSES 281

surface, resulting in a spiral course. Its direction of movement is changed
by the shifting of the distal end of the flagellum from the surface of the
body so as to increase the angle between it and the surface. This in-
creases the deflection of the anterior end (Fig. 98).

Shock reaction and aggregation —Engelmann (1882) observed that if
the intensity of the light in a field in which euglenae are swimming about
at random is rapidly decreased, they stop suddenly, then turn and pro-

A B

Figure 98. A. Diagrams showing the position of the flagellum as seen in a viscid
medium; a, when Exglena is swimming forward in a narrow spiral; 5, when swerving
sharply towards the dorsal side; c, when moving backwards. B. Dotted area, shows the
position of the moving India-ink particles. a, when Evglena is swimming forward in a
narrow spiral; 5, when swerving toward the dorsal side during a shock-movement.
(After Bancroft, 1913.)

ceed in various directions. He designated the response as a ‘‘Schreck-
bewegung” (fright movement, or shock reaction), because the re-ori-
ented organisms gave the impression of having been frightened. It was
found that if the intensity is slowly changed this response does not
occur. It is therefore dependent upon the rate of change in intensity.

He says that if there is a spot of relatively strong light in the field,
it acts just like a trap; owing to random movements, the euglenae get
into this spot, but as they reach the boundary on the way out, the rapid
reduction in intensity induces the shock reaction and consequently pre-
vents their exit.

Under some conditions the euglenae respond to rapid increase in in-

282 MOTOR RESPONSES

tensity and aggregate in a spot of relatively weak light in the field
(Mast, 1911).

Orientation —lf euglenae are exposed in a beam of light, they usually
swim toward or away from the source of light, 1.e., they may be either
photopositive or photonegative.

Verworn (1895) postulated that if the euglenae are not directed to-
ward or away from the light, so that one side is more strongly illuminated
than the other, the flagellum beats more effectively in one direction
than in the opposite, that this causes the euglenae to turn until both sides
are equally illuminated, and that the flagellum then beats equally in op-
posite directions and the organism moves directly toward or away from
the source of light.

The above hypothesis is, in principle, essentially the same as that
formulated by Ray (1693), in reference to orientation in plants, and
later accepted by de Candolle (1832). According to this idea, the effect
of light on the activity of the motor mechanism, or upon the photo-
receptors connected with it, is dependent upon the intensity (not upon
change of intensity) of the illumination. The light acts continuously
after orientation has been attained, as well as during the process of
orientation. During the process of orientation, the illumination on op-
posite sides 1s unequal, which results in quantitatively unequal action
in the motor mechanism; but after orientation, it is equal on opposite
sides, and consequently the action of the mechanism on opposite sides
is equalized. Verworn applied this theory to ciliates as well as flagellates.
In his earlier work, Loeb (1890) strenuously opposed the theory out-
lined above, accepting Sachs’s “‘ray-direction theory” as the alternative.
He adopted it later (1906), however, and applied it to higher animals,
introducing the idea that the action of the locomotor appendages is
quantitatively proportional to the intensity of the light on the photo-
receptors connected with them. He maintained that this is due to the
effect of light on muscle tonus. This theory has been designated the
“difference of intensity theory,” the ‘‘continuous-action theory,” the
“tropism theory,” and ‘““Loeb’s muscle-tonus theory” (Mast, 1923).

Engelmann (1882) demonstrated that only the anterior end of
Exglena is sensitive to changes in luminous intensity. Jennings (1904)
contends that because of this, all turning from the light results in a
reduction of illumination, whereas all turning toward the light results

MOTOR RESPONSES 283

in an increase in illumination of photosensitive substance. The photoposi-
tive specimens consequently turn until they face the light, whereas photo-
negative specimens turn until they face in the opposite direction. When
the stimulus which induces turning ceases, the organisms continue either
directly toward or from the light.

Mast (1911) made a very intensive study of the process of orientation
in a species of Evglena which crawls on the substratum but continuously
rotates on the longitudinal axis as it proceeds. This Evglena orients very
precisely in light, it has a well-developed eyespot, and it moves so slowly
that the different phases of its responses can readily be followed in
detail. It is therefore very favorable for the study of the process of
orientation.

If the intensity of the light is rapidly decreased in a beam in which
specimens are proceeding toward the source of light, they stop suddenly
and bend in the middle toward the abeyespot surface until the two halves
form nearly a right angle; then they begin again to rotate on the longi-
tudinal axis; and, while rotating, they gradually straighten and proceed
once more toward the light source. If the intensity of the light is in-
creased, or if it is slowly decreased, there is no perceptible response. The
cessation of movement and the bending are therefore dependent upon
the rate of decrease in the intensity of the light in the field, i.e., it 1s a
shock reaction. The decrease in the intensity of light in the field neces-
sarily results in decrease in intensity of light on all the substance in the
field; it therefore must cause decrease in the illumination of the photo-
sensitive substance. The response, then, is dependent upon the rate of
decrease in the light on the photosensitive substance.

If the direction of the beam of light is changed through 90° without
alteration in intensity, the specimens oriented in it are illuminated
laterally. Those in which the eyespot surface faces the light after the
direction of the rays has been changed, stop at once. They bend in the
middle toward the abeyespot surface, then rotate, and gradually
straighten to resume their crawling movements. Those in which the eye-
spot surface does not face the light after the direction of the rays has been
changed, do not respond to the changed direction of the rays, until, in
the process of rotation on the longitudinal axis, this surface faces the
light; then they also stop, bend, rotate, straighten, and proceed. Thus
they continue until, in the process of rotation, the eyespot surface again

284 MOTOR RESPONSES

faces the light, when they again respond. The gradual straightening
during rotation results in greater deflection of the anterior end toward,

en 0-03 seed

Figure 99. Ezglena sp. in a crawling state, showing details in the process of orienta-
tion; v, contractile vacuole; es, eyespot; 7, 0. direction of light; a-c, positions of
Euglena with light from » is intercepted; c-m, positions after light from » is turned
on and that from o cut off, so as to change the direction of the rays. If the ray direction
is changed when the Ezglena is in position c, there is no reaction until it reaches d.
Then it suddenly reacts by bending away from the source of light to e, after which
it continues to rotate and reaches position f, where it gradually straightens to g, and
rotates to 4, when the eyespot again faces the light and the organism is again stimulated
and bends to 7, from which it proceeds to j, and so forth. If the ray direction is changed
when the Ezglena is at d, it responds at once and orients as described above. If the
intensity from » is lower than that from o the organism may respond at once when the
ray direction is changed, no matter in which position it is. (After Mast, 1911.)

rather than away from the light. Thus the anterior end becomes directed
more and more nearly toward the light source, until an axial position
is reached in which changes in illumination of the eyespot surface, owing

MOTOR RESPONSES 285

to rotation, disappear. The organism 1s then oriented (Fig. 99). The
response induced by changing the direction of the rays, or by rotation in
lateral illumination of uniform intensity, is precisely the same as the
response induced by a decrease in the intensity of the light in the field

Figure 100. Side view of anterior end of Exglena viridis. e, pigmented portion of
eyespot; f, flagellum; e.f, enlargement in flagellum; c.v, contractile vacuole; e.s. eye-
spot surface of the organism; a4.s. abeyespot surface of the organism. (After Wager,
1900.)

without a change in the surface illuminated. The change from illumina-
tion of the anterior end or the abeyespot surface to illumination of the
eyespot surface therefore must, in some way, result in a rapid decrease
in the illumination of the photosensitive substance. How is this brought
about?

Wager (1900) demonstrated that the eyespot in Exglena consists
of a spoon-shaped portion containing red pigment and a small globular
enlargement of one of the roots of the flagellum in the concavity of the
pigmented portion (Fig. 100). The eyespot is situated near the eyespot

286 MOTOR RESPONSES

surface, a short distance from the anterior end, with the convex surface
directed outward and backward. When the anterior end, or the abeyespot
surface, is directed toward the light, the enlargement in the eyespot 1s
fully exposed; but when the eyespot surface faces the light, the enlarge-
ment is in the shadow cast by the pigmented portion. It 1s evident, then,
that rapid change from illumination of the anterior end, or the abeyespot
surface, to illumination of the eyespot surface causes rapid decrease
in illumination of the enlargement in the eyespot, and that if the en-
largement is photosensitive, change in the direction of the rays or rota-
tion on the longitudinal axis has the same effect as decrease in the in-
tensity of the light in the field. It is therefore highly probable that the
enlargement in the eyespot is photosensitive and that the pigmented por-
tion functions in producing changes in intensity of light on it, when the
axial position of the organism changes and when it rotates on the longi-
tudinal axis in lateral illumination. This contention is supported by the
facts that the region of maximum stimulating efficiency in the spectrum
is in the blue for Evglena (Mast, 1917) and that blue is absorbed by the
yellowish-red pigmented portion of the eyespot (Fig. 102).

In photonegative specimens the responses to changes in light intensity
in the field and to changes in the surfaces illuminated are precisely like
those of photopositive specimens, except that the responses are induced
by (1) increase rather than decrease in light intensity and (2) by change
from illumination of the eyespot surface to illumination of the abeyespot
surface.

The process of orientation in free-swimming specimens ts, in prin-
ciple, precisely the same as it is in crawling specimens.

Orientation in Evglena is, then, clearly due to a series of responses
dependent upon the rate of change in the intensity of the light on the
photosensitive substance, which 1s probably situated in the concave sur-
face of the pigmented portion of the eyespot. The light does not act
continuously, and there is no evidence whatever indicating anything in
the nature of balanced or antagonistic action of locomotor appendages
on opposite sides, in accord with the Ray-Verworn theory.

The evidence in hand indicates, in short, that the photosensitive sub-
stance is confined to the concavity in the pigmented portion of the eye-
spot; that rotation on the longitudinal axis results in alternate shading
and exposing of this substance, if the organisms are not directed toward

MOTOR RESPONSES 287

or from the light; that this induces shock reactions which result in
orientation; and that the organisms remain oriented and proceed directly
toward or away from the light, because, after they have attained either
of these two axial positions, rotation no longer produces changes in the
illumination of the photosensitive substance in the eyespot, and they
therefore continue in the direction assumed. In other words, the orienting
stimulus ceases after the organism has become oriented. The organism
then continues directly toward or away from the light because (1)
owing to internal factors, it tends to take a straight course, and because
(2) if for any reason it is turned from this course, the orienting stimulus
immediately acts, and induces shock reactions which bring it back on
its course.

Bancroft (1913) presented evidence against the contention that
photic orientation in Evglena is due to shock reactions and concluded
that it is due to tonus effects brought about by “the continuous action
of the light,” in accord with his conception of Loeb’s tropism theory.
Mast (1914) demonstrated, however, that if the evidence presented
by Bancroft is valid, it proves that his explanation of orientation in
Exglena is not correct. Moreover, the fact that after Evglena is oriented,
the rate of locomotion is practically independent of the luminous in-
tensity (Mast and Gover, 1922) also militates against his explanation.

Orientation in light from two sources —In a field of light consisting
of two horizontal beams crossing at right angles, Evglena orients and
goes toward or away from a point between the two beams. The location
of this point is related to the relative intensity of the two beams in such
a way that the tangent of the angle between the direction of locomotion
and the rays in the stronger beam is approximately equal to the intensity
of the weaker divided by that of the stronger (Fig. 101) (Buder, 1917;
Mast and Johnson, 1932). Buder maintains that this demonstrates that
there is a quantitative proportionality between the stimulus and the re-
sponse. Mast and Johnson conclude that “‘it has no bearing on the prob-
lem concerning the quantitative relation between stimulus and response,”
but that it can be explained on the assumptions that the eyespot is a
photoreceptor and that the stimulating efficiency of light varies with the
angle of incidence.

Wave length and stimulating efficiency —The shorter waves in the
visible spectrum are more efficient than the longer in stimulating Euglena

Theoretical

Observed

Figure 101. Graphs showing the relation between the direction of locomotion ob-
served in a field of light produced by two horizontal beams crossing at right angles
and that demanded by the ‘‘Resultantengesetz.’’ Abscissae, angles between the direction
of locomotion and the direction of the rays in the stronger beam observed with dif-
ferent ratios of intensities in the two beams, ranging from 0 at 0° to 1 at 45°; ordinates,
angles between the direction of locomotion and the direction of the rays in the stronger
beam demanded by the ‘‘Resultantengesetz.”” @ Euglena rubra; © Gonium pectorale; @
Volvox minor; ©) Volvox globator. (After Mast, 1907.) Note that if the observed
direction of locomotion were the same as the theoretical all the points would fall on
the broken line, and that this practically obtains for Evglena but not for Volvox and
Gonium. Note also that for the latter, as the ratio between intensity in the two beams
decreases from 1, the difference between the theoretical and the observed results in-
creases to a maximum, then decreases to zero, after which it increases in the opposite
direction. (After Mast and Johnson, 1932.)

MOTOR RESPONSES 289

and other flagellates. Strasburger (1878) concluded that stimulation is
confined to violet, indigo, and blue in the solar spectrum, with the maxt-
mum in the indigo. Engelmann (1882) maintains that for Euglena the
maximum is in the blue between 470 my, and 490 mu, and Loeb and

| | ALA
ZA | ABISh
MAN I
1)

©}
|
S|
LH
cad

S
=—

a —
is
———

PYLON
RI
FB BSe
=a
ie
=] Eee

S
aoe aS
-—+—
=!
me:

~~
anny

ie)

~
Pes Vee ce Rahss

A
x
LJ
Z|
a
LL
ia
Ef
ll

rsa
amas ays
F Pl Blo N

Be a a se a ll al
BSE Seba Res

rex
es
Je eSB ERReSGERaee

SIAR Sees esas
JASE 2S2eares
JAR SVAe sashes

ARS SRBs

[A
>
f=)

Sisuss!

eS
eS

| |Z
{@
ACies

19%
iE
@| |

Ultra Violet Violet Blue Green Yellow Red

=
>

o
e
e
°o
o

Figure 102. Curves representing the distribution in the spectrum of stimulating
efficiency, constructed from data given in Table 15 (See Mast 1917). A, Pandorina (nega-
tive); B, Pandorina (positive) ; C, blowfly larvae; D, Euglena viridis (negative); E,
Euglena viridis (positive) F, Euglena tripteris (negative); G, Avena sativa (oat seed-
lings). (Constructed from data obtained by Blaauw.) The circles represent points experi-
mentally established abscissae, wave lengths; ordinates, relative stimulating efficiency on
the basis of equal energy. The curves for Eudorina and Spondylomorum, not represented in
the figure, are in position and form essentially like those for Pandorina; the curve for
Chlamydomonas is much like that for blowfly larvae; those for Euglena gracilis, E.
minima, E. granulata, Phacus, Trachelomonas, Gonium, Arenicola, and Lumbricus are
nearly like those for E. viridis and E. tripteris. (After Mast, 1917.)

Maxwell (1910) assert that in the carbon-arc spectrum it is between 460
and 510 my. The unequal distribution of energy in the spectrum was not
considered in these conclusions. Mast (1917) made corrections for un-
equal distribution of energy and ascertained the relative stimulating effi-
ciency of negative and positive orientation at intervals of 10 #2 through-

290 MOTOR RESPONSES

out the visible spectrum. He found that as the wave length increases,
the stimulating efficiency also increases very rapidly from zero at about
410 my to a maximum of 21 arbitrary units at 485 mu, and then de-
creases equally rapidly to zero at about 540 my, (Fig. 102). He holds,
however, that the limits of the stimulating region depend upon the
luminous intensity.

Kinetic responses—Ilf Euglena is subjected for long periods to low
illumination or to darkness, it gradually becomes less active; and if the
illumination is then increased, it gradually becomes more active. The
rate of change in activity varies with the magnitude of the change in
intensity. But this response is never so sudden and abrupt as the shock
reaction. There are therefore two types of responses to light in Exglena,
one depending primarily upon the rate of change in luminous intensity,
the other primarily upon change in the amount of light received. The one
results in orientation and aggregation, the other in change in activity.

Mast and Gover (1922) measured the rate of locomotion in several
different flagellates in different intensities of light and found very little
correlation between rate and intensity. The environmental factors were,
however, not accurately controlled, and adaptation was not considered.
The measurements should therefore be repeated, with the methods used
by Mast and Stahler (1937) in their observations on Amoeba.

Reversal in response —Euglena 1s ordinarily photopositive in weak
light and photonegative in strong light. The orienting response therefore
tends to keep it in light of moderate intensity, indicating that these re-
sponses are fundamentally adaptive. This has not been demonstrated,
however, because the direction of orientation is not specifically correlated
with luminous intensity. For example, euglenae which are strongly photo-
positive in a given intensity of light at room temperature may become
equally strongly photonegative if the temperature is rapidly decreased
10 to 15 degrees, the extent of the requisite decrease depending upon
the state of adaptation (Mast, 1911).

This problem is much in need of thorough investigation. It is a very
important problem because it concerns the biological significance of
response to light in these organisms.

II. Peranema tricophorum.——Peranema is a colorless flagellate with-
out an eyespot. It is usually in contact with the substratum and moves
slowly with the flagellum extending forward (Fig. 103). If the luminous

MOTOR RESPONSES Zo

intensity is rapidly increased, it stops suddenly and then deflects the
anterior end sharply to one side. If the intensity is slowly increased, or if
it is decreased, there is no response. The entire organism is sensitive to

Ha Os Tr 2, oy

Figure 103. Camera drawings illustrating the response of Peranema to contact or to
rapid increase in luminous intensity. Al, normal locomotion; 2, immediately after re-
sponse; 3, 4, recovery from response; B, response to contact with grain of sand, 0, and
recovery. Note that response results in a change in the direction of motion of approxi-
mately 90 degrees. (Mast, 1912.)

light, but the flagellum is most sensitive and the posterior end least sen-
sitive (Shettles, 1937).

Dark adaptation—Mast and Hawk (1936) demonstrated that if
light-adapted peranemae are subjected to darkness, the time required in
light of 2,000 meter-candles to induce the response decreases from 30.95
seconds after 15 minutes in darkness to a minimum of 4.54 seconds after
one hour in darkness, then increases to a maximum of 63.46 seconds

292 MOTOR RESPONSES

after 6 hours in darkness, and then remains nearly constant. As the time
in darkness increases, the sensitivity to light rapidly increases to a maxi-
mum, then decreases to a minimum, before it becomes constant (Fig.
104).

ae. ee
ee
(2 4 6 8 /0 /2 /4

TIME IN DARKNESS IN HOURS

A
9

wy
9

REACTION TIME IN SECONDS
NY
is}

Figure 104. Graph showing the effect of dark-adaptation on sensitivity to light in
Peranema trichophorum. Each point on the curve, except the last two, represents the
average reaction time for from fifteen to seventeen tests. (After Mast and Hawk, 1936.)

Light adaptation—Shettles (1937, 1938) made a much more exten-
sive and thorough investigation of this response. He confirmed the con-
clusions reached by Mast and Hawk (1936). A brief summary of other
results obtained, and the conclusions reached, follows:

If dark-adapted peranemae are subjected to light, their sensitivity to
light increases rapidly to a maximum, then decreases considerably, after
which it remains nearly constant (Fig 105). The reaction time consists

MOTOR RESPONSES 295

of an exposure period and a latent period. With increase in intensity
of illumination, the exposure period decreases, at first rapidly, then
more slowly, until it becomes nearly constant; the latent period increases
to a maximum and then decreases, and the amount of light energy re-
quired during the exposure period increases from 22,970 meter-candle
seconds at 538 meter-candles to 54,315 meter candle seconds at 2,152
meter-candles, and then decreases to 13,498 meter-candle seconds at

INTENSITY
J 2l52m.c.

REACTION TIME [N SECONDS

INTENSITY INTENSITY
86// m.c. 6458 =m.c.

/ e 4
TIME IN LIGHT IN HOURS

Figure 105. Graphs showing rate of light-adaptation. Dark-adapted peranemas were
exposed to light (intensity given in the graph) for the time indicated, then subjected
to darkness one half hour, then exposed to 2,152 m.c., and the reaction time measured.
Each point on the curves represents the average reaction time for ten tests, different
individuals being used in each test. (After Shettles, 1937.)

6,458 meter-candles (Fig. 106). He concludes that ‘the amount of light
energy required to induce a shock-reaction in Peranema varies greatly
with the intensity of the light and that the Bunsen-Roscoe law conse-
quently does not hold.”

The latent period decreases from 39.68 seconds at 10° C. to 24.3
seconds at 30° (15.38 seconds), but the exposure period decreases from
27.87 to 22.97 seconds (only 5.8 seconds). This indicates that there

294 MOTOR RESPONSES

60
WY
=
S 50 5
Q
wh
= 40 4
ly
=
re

Ww
S

a SANe }

FERIOD
EXPOSURE PERIOD”

LIGHT ENERGY IN 10,000 METER CANDLE SECONDS

% 538 107% 2/52 3227 4305 $382 6458 &6// 10764
LUMINOUS INTENSITY IN METER CANDLES

Figure 106. Graphs showing relation between luminous intensity and reaction time,
exposure period, latent period, and energy. Each point on the curves of the reaction
time and the exposure period represents the average for ten tests, different individuals
being used in each test. Each point on the curve of the latent period represents the dif-
ference between the reaction time and the exposure period. Each point on the energy
curve represents exposure period luminous intensity. (After Shettles, 1937.)

are at least two processes involved in the response of Peranema to light.
One of these 1s nearly independent of temperature and is therefore prob-
ably photochemical. The other is closely correlated with temperature and
is therefore not photochemical.

After Peranema has responded in light of a given intensity, it must
be subjected to light of a lower intensity or to darkness before it will
again respond. The time required for recovery varies directly with the

MOTOR RESPONSES 29)

luminous intensity in which the response occurred, and inversely with the
temperature.

Wave length and stimulation Stimulating efficiency of light 1s
closely correlated with wave length. There are two maxima in the spec-
trum, one in the ultra-violet at 302 m1 and one in the visible at 505 my.
The latter is nearly twice as great as the former.

The absorption of light by Peranema in the violet remains nearly
constant as the wave length decreases from 450 my to 325 my, then
increases rapidly and extensively to a maximum at 253 my. The maxi-
mum injuring efficiency is also at 253 my. Injury is therefore closely
correlated with the amount of light absorbed, but stimulating efficiency
is not, for the maximum is at 302 my in place of 250 nz. The processes
involved in stimulation therefore differ from those involved in injury.
Injury, in Peranema, is due to coagulation of the protoplasm, whereas
stimulation is probably not due either to coagulation or to increased
viscosity.

Peranema tesponds very precisely and very consistently. Its move-
ments are very slow and its reaction time long. Since it can be grown
under fairly accurately known environmental conditions in total dark-
ness, it is well suited for quantitative work of a high order.

Cy GClEVAES

Very few of the ciliates respond to light. Only one of these, Stentor
coeruleus, has been investigated extensively.

If the luminous intensity is rapidly increased, this organism stops,
turns toward the aboral surface, and then proceeds. This is a shock
reaction, because if the intensity is slowly increased there 1s no response.
If the intensity is decreased, no matter whether rapidly or slowly, there
is no response. If Stentor is exposed in a beam of light, it orients fairly
precisely and swims away from the light, i.e., it is photonegative. It
rotates on the longitudinal axis as it swims, so that if it is not oriented,
the oral and the aboral surfaces are alternately shaded and illuminated.
The oral surface is much more sensitive than the aboral; therefore every
time that this surface is carried from the shaded to the illuminated side,
the result is the same as an increase in the illumination of the entire
organism, and it consequently responds, i.e., it turns toward the aboral
surface. This continues until it is directed away from the light, and

Figure 107. Stentor coeruleus in the process of orientation. Curved line, spiral course;
arrows m and », direction of light from two sources; a-f, different positions of Stentor on
its course; 0, oral surface; ab, aboral surface. At a the Stentor is oriented in light from
m, n being shaded. If » is exposed and m shaded simultaneously when the Stentor is
in position 4, there is usually no reaction until it reaches ¢ and the oral side faces the
light; then the organism may respond by suddenly stopping, backing, and turning sharply
toward the aboral side (dotted outline), and become oriented at once; or it may merely
swerve toward the aboral side without stopping. At e the oral side is again exposed,
and the organism is again stimulated and it again swerves from the source of light.
This process continues until the oral side is approximately equally exposed to the light
in all positions on the spiral course. If the Stentor is at c when » is exposed, it responds
at once and orients as described above. If the light from » is more intense than that
from m, or if the organism is very sensitive when » is exposed and m shaded, it re-
sponds at once, no matter in which position it is. If it is at 4, it turns toward the source
of light, but now repeats the reaction, successively turning in various directions until
it becomes oriented. (After Mast, 1911.)

MOTOR RESPONSES 297

rotation no longer produces changes of intensity on the opposite sur-
faces (Fig. 107). Photic orientation in S/entor is therefore the result
of a series of shock reactions, as is the case in Evglena. There is no evi-
dence in support of the view that it is the result of a continuous quanti-
tative difference in the activity of the cilia on opposite sides, in pro-
portion to the difference in the illumination of these sides. The process
of orientation in Stentor is therefore not in accord with the Ray-Verworn
theory.

If stentors are exposed in a field of diffuse, non-directive light which
contains a dark spot, they aggregate in this spot. The process of ag-
gregation is, in principle, precisely the same as the process of aggrega-
tion of photonegative euglenae in a dark spot. They reach the dark spot
by random movements. No reaction occurs when the organisms enter the
unilluminated area. However, at the periphery on the way out, as the
light intensity rapidly increases, they stop suddenly, turn sharply to-
ward the aboral surface, and then proceed in a different direction. The
dark spot therefore acts like a trap (Jennings, 1904; Mast, 1906, 1911).

The relative stimulating efficiency of different regions in the spectrum
has not been investigated; no observations have been made on the
quantitative relation between the different phases of the shock reaction,
the state of adaptation, and the extent of change in luminous intensity.
Indeed, very little is known about the body processes involved in stimula-
tion and response.

D. COLONIAL ORGANISMS

Response to light is essentially the same in all of the colonial forms
in which it has been studied, but it has been more intensively investigated
in Volvox globator than in any of the other species.

Volvox is a slightly elongated, globular colonial organism somewhat
less than one mm. in diameter. It consists of numerous cells (zodids),
each of which contains two flagella and an eyespot. The zodids are ar-
ranged in a single layer at the surface of the colonies. The eyespot in each
zodid is directed toward the posterior end of the colony, but those at the
anterior end are much larger than the rest (Fig. 108).

Mast (1927a) presented evidence which demonstrates that the eye-
spots consist of a cup-shaped pigmented portion, a lens-like structure
near the opening of the cup, and photosensitive substance between this

298 MOTOR RESPONSES

and the inner surface of the cup. The evidence also indicates that the
lens-like structure brings the longer incident waves of light to focus in the
wall of the cup; and that the shorter wave lengths, after being reflected
from the inner surface of the cup, are focused in the photosensitive sub-
stance (Fig. 109).

!
!
1
i
'
)
1
i}
'
1
1
'
1
1
1
|
'
1
'
'
'
'
!
'
'
'
'
'
1
i}
'
'
'
'
'
(
1
'
'
1
1
1
'
'
'
'

Figure 108. Camera drawing showing the zodids in about one half of an optical sec-
tion through the longitudinal axis of a colony of Volvox. l-a, longitudinal axis of
colony; a, anterior end; z, zodids, f, flagella; e, eyes. Note that the eyes are located at
the outer posterior border of the zodids and that they become larger as the anterior
end of the colony is approached. (After Mast, 1927.)

Movement, response, and orientation in V. globator have been thor-
oughly studied by Mast (1907, 1926b, 1927b, 1932b). The more im-
portant of the results obtained in this study lead to the following con-
clusions.

Shock reaction.—V olvox colonies rotate on the longitudinal axis as
they swim. This is due to the diagonal stroke of the flagella. In a beam
of light they usually orient and go almost directly either toward or away
from the light, i.e., they may be photopositive photonegative, or neutral.

If, while the colonies are swimming toward the light, the intensity ts
rapidly decreased without any change in the direction of the rays, rota-
tion on the longitudinal axis stops and forward movement increases

MOTOR RESPONSES 299

greatly. On the other hand, if the intensity is rapidly increased, the for-
ward movement stops and the rate of rotation increases. If the colonies
are swimming away from the light, the reverse occurs, i.e., forward move-
ment decreases if the intensity is increased, and increases if it is de-
creased. If the colonies are neutral, there are no such responses to changes
of intensity. These responses consist chiefly, if not entirely, of rapid
changes in the direction of the stroke of the flagella. In other words, a
rapid decrease in the illumination of photopositive colonies changes the

Figure 109. Sketches showing the structure of the eyespot in Volvox and its action
on light entering the pigment-cup at different angles. p, pigment-cup; /, lens; y, yel-
low focal spot; 4, bluish green focal spot; 55, photosensitive substance; large arrows,
incident rays of light. Note that the longer waves of the incident light are brought to
focus in the wall of the pigment-cup and that the shorter waves are brought to focus in
the cup, after being reflected from the inner surface, and then continue in the form
of a concentrated beam of bluish-green light. Note also that the more obliquely the
incident light enters the pigment-cup, the nearer the edge of the cup the yellow focal
spot is located. (After Mast, 1927.)

stroke of the flagella from diagonally backward to straight backward.
An increase in the illumination causes it to change from diagonally back-
ward to sidewise. In photonegative colonies precisely the reverse obtains.
These responses continue for only a few seconds, although if the luminous
intensity is slowly changed they do not occur at all. They are therefore
dependent upon the rate of change in intensity, i.e., they are shock-
reactions which are somewhat similar to those observed in Euglena.
Kinetic res ponses.—If Volvox is kept in weak illumination or in dark-
ness for several hours, it becomes inactive; but if the illumination 1s
afterwards increased, it gradually becomes active again. These responses
consist chiefly, if not entirely, in changes in the rate or the efficiency of

300 MOTOR RESPONSES

the stroke. Changes in the direction of the stroke of the flagella are not
involved. They are relatively slow responses which occur, even if the
luminous intensity is gradually changed. The responses are primarily
dependent upon change in luminous intensity, not upon the rate of
change. Consequently, there are, in Volvox, two different types of re-
sponse: (1) typical shock reactions, and (2) responses which consist
merely in changes in activity.

Holmes (1903) maintains there is no consistent correlation between
luminous intensity and rate of locomotion in Vo/vox. But his methods
did not exclude the effect of adaptation. Further work concerning this
correlation is therefore highly desirable.

Orientation —lf a colony of Volvox in a beam of light is laterally
illuminated, it turns gradually until it is oriented, and then proceeds
either toward or away from the light source. When it is laterally illumi-
nated, the zodids, owing to rotation of the colony on the longitudinal
axis, are continuously transferred from the light side to the dark side, and
vice versa. As the zodids pass from the light side to the dark side, the
photosensitive substance in the eyespots becomes shaded by the pigment
cup. As they pass from the dark side to the light side, this substance be-
comes fully exposed. A rapid decrease in the illumination of the sensitive
substance on the dark side of photopositive colonies induces shock reac-
tions on this side, and the flagellar stroke increases in its backward phase.
A rapid increase in illumination of the sensitive substance on the light
side of a colony induces shock reactions. The latter consist of increase in
the lateral phase of the stroke of the flagella (Fig. 110). This difference
in the direction of the stroke of the flagella causes the colonies to orient
gradually, until they are directed toward the light, after which all sides
are equally illuminated. Rotation on the longitudinal axis then no longer
produces changes in the illumination of the photosensitive substance, and
the shock reactions cease. The Volvox colonies continue directly toward
the light because, in the absence of external stimulation, they tend to
take a straight course. Furthermore, if they are forced out of their course,
opposite sides immediately become unequally illuminated, the intensity
of the illumination of the photosensitive substance in the eyespots
changes, and consequently reorientation occurs.

If photopositive colonies are exposed in a field of light consisting of
two horizontal beams which cross at right angles, they orient and swim
toward a point between the two beams. The location of this point de-

Figure 110. Diagrammatic representation of the process of orientation in Volvox.
A, B, C, D, four zodids at the anterior end of the colony; /-a, longitudinal axis; large
arrows, direction of illumination; small arrows, direction of locomotion; curved arrows,
direction of rotation; f, flagella; e, eyes, containing a pigment-cup represented by a
heavy black line and photosensitive tissue in the concavity of the cup. Note that when
the colony is laterally illuminated, the photosensitive tissue in the eyes on the side
facing the light is fully exposed and the flagella on-this side beat laterally. Those on the
opposite side, shaded by the pigment-cup and the flagella on this side, beat directly
backward. The difference in the direction of the beat of the flagella on these two sides is
due to alternate decrease and increase in the luminous intensity to which the photo-
sensitive tissue in the eyes is exposed, owing to the rotation of the colony on its longi-
tudinal axis—an increase causing, in photopositive colonies, a change in the direction
of the stroke of the flagella from backward or diagonal to lateral; and a decrease, a change
from lateral or diagonal to backward. In photonegative colonies, precisely the opposite
obtains. In photopositive colonies, this results in turning toward and in photonegative
colonies turning from the source of light. In both, the turning continues until opposite
sides are equally illuminated, when changes of intensity on the photosensitive tissue are
no longer produced by rotation and the orienting stimulus ceases. (After Mast, 1926a.)

302 MOTOR RESPONSES

pends upon the relative intensity of the light in the beams. The higher
the intensity in one of the beams in relation to that in the other, the
nearer to the former the point is. If the intensity in the two beams 1s
equal, the point is halfway between them. The colonies are oriented
under these conditions, opposite sides are equally illuminated, both in
reference to intensity and direction of the rays, i.e., the angle of inci-
dence at the surface of the colony. If the intensity in the two beams 1s
not equal, the illumination of the oriented colonies is higher and the
angle of incidence greater on one side than on the other. However, when
a colony is oriented in a field of light, no matter how unequal the in-
tensity from different directions may be, transfer of the zodids from side
to side in consequence of rotation on the longitudinal axis causes no
responses. In other words, the effect of unequal illumination on opposite
sides is equal. This obviously must be correlated with the difference in
the angle of incidence.

Mast (1927a) and Mast and Johnson (1932) demonstrated that the
location of the point of focus in the eyespot varies with the angle of
incidence. By ascertaining the location of these points in the eyespots on
opposite sides of the colonies, in relation to the relative intensity of the
two beams, they calculated the distribution of sensitivity and found that
the photosensitive substance is much more sensitive in the central regions
of the eyespot than at the periphery (Fig. 109). The stimulating eff-
ciency of light, therefore, depends upon the location of the point of
focus; this, in turn, depends upon the angle of incidence. The equal effect
of light on the sides of colonies which are unequally illuminated on op-
posite sides when they are oriented, is therefore due to the fact that the
point of focus in the eyespots is more nearly centrally located on the side
which receives the least light than on that which receives most.

In photonegative colonies the process of orientation is precisely the
same as it is in photopositive colonies, except that decrease in intensity
causes increase in the lateral phase, and increase in the light intensity
increases the backward phase of the stroke of the flagella. In consequence,
the illuminated side moves more rapidly than the shaded side. The
colonies therefore turn away from the light source.

Orientation of Vo/vox in light is the result of qualitative differences
in the action of the locomotor appendages on opposite sides. These dif-
ferences are due to shock reactions induced by rapid change in the 1n-

MOTOR RESPONSES 303

tensity or the location of the light in the photosensitive substance in the
eyespot, by virtue of colony rotation on the longitudinal axis. It should
be noted that the responses observed are not the result of quantitative
differences due to continuous action of the light. The explanation offered
is therefore not in accord with the Ray-Verworn theory.

Wave length and res ponse.—The distribution of stimulating efficiency
in the spectrum for Volvox (Laurens and Hooker, 1920) and Gonium
(Mast, 1917) is essentially the same as it is for Evglena; but for the
closely related forms Pandorina and Spondylororum (Mast, 1917) the
maximum is at 535 my in place of 485 my, and the effective region ex-
tends from this wave length much farther in either direction than it does
for Euglena, Gonium, and Volvox (Fig. 102). The orange, pigmented
portion of the eyespot in these forms is opaque in reference to the light
of all those regions of the spectrum which have the highest stimulating
efficiency. The distribution of stimulating efficiency for these forms con-
sequently supports the conclusions reached concerning the structure of
the eyespots, the distribution of photosensitive substance in them, and
their function in the process of orientation.

Threshold.—Mast (1907), on the basis of quantitative results, con-
cludes that the minimum difference in light intensity on opposite sides
of a colony which is necessary to induce a response varies greatly with
the physiological state of the colony; but that with colonies in a given
physiological state, the response varies directly with the intensity, and
the ratio is nearly constant, 7.e., nearly in accord with the Weber-Fechner
law. His observations, however, covered such a small range (2-27 meter-
candles) and the probable error in the results is so large that further
observations concerning this relation are highly desirable.

Reversal 1n res ponse.—V olvox is usually positive in weak, and nega-
tive in strong light. However, the reverse obtains under some conditions.
It may be positive, negative, or neutral in every condition of illumination
in which orientations occurs. If it is positive, a shadow on the photo-
sensitive substance in the eyespots causes a change in the direction of the
stroke of the flagella of the zodids from diagonal to backward. A flash of
light on this substance causes a change of stroke from diagonal to side-
wise. If the colony is photonegative, the reverse obtains; and if it is
neutral, there is no response unless the changes in luminous intensity are
great.

304 MOTOR RESPONSES

Reversal in the direction of orientation from positive to negative is
therefore due to internal changes of such a nature that shock reactions
which were produced by decrease are produced by increase in the il-
lumination of the photosensitive substance. The shift from negative to
positive is due to the reverse. The nature of the response to light in
Volvox depends upon the state of adaptation and upon the intensity of
the illumination. If Volvox is fully adapted in a given intensity, it be-
comes positive if the intensity is increased or negative if it is decreased.
If the colony is not fully adapted, it becomes negative if the intensity ts
increased or positive if it is decreased.

The time required for colonies of Volvox to become negative or posi-
tive after the luminous intensity has been changed (the reaction time)
depends upon the degree of adaptation and the extent of the change.
If colonies which have been subjected first to strong light (1-2 hours)
and then to a variable period in darkness are exposed to strong light,
the time required to become positive (reaction time) increases with in-
crease in the length of the period in darkness (dark adaptation) from
0.04 minutes (with 2 minutes in darkness) to a maximum of 0.52 min-
utes (with 16 minutes in darkness) and then decreases to 0.18 minutes
(with 25 minutes in darkness). If the colonies are kept longer in strong
light and are then subjected to darkness, the reaction time decreases to
a minimum and then increases as the time in darkness increases. If they
are left in darkness until they are fully dark-adapted, and are then ex-
posed to light of different intensities, the reaction time (as the intensity
increases) decreases from 29 minutes in 5.24 meter-candles to a mini-
mum of 0.098 minutes in 7.5 meter-candles, and then increases to 0.358
minutes in 62,222 meter-candles. The energy required to make the colo-
nies positive varies directly with the light intensity, over the whole range
tested. Over most of the range this variation is nearly proportional to the
variation in intensity. No satisfactory explanation of this relation is avail-
able.

If colonies are kept in a given intensity or in darkness, they become
adapted, 7.e., they lose the ability to respond to light. Their responsive-
ness is regained if, after dark adaptation, the intensity is changed. The
processes associated with adaptation and those induced by change in il-
lumination are therefore antagonistic. The rate of these antagonistic
processes varies greatly, depending upon the magnitude of the change

MOTOR RESPONSES 305

in intensity. For example, if dark-adapted colonies are exposed to light
of 22,400 meter-candles for 0.05 minutes then returned to darkness, it
takes 20 minutes or more in darkness to eliminate the effect of the light.
This indicates that, under these conditions, the processes which occur in
light proceed at least 400 times as fast as the reverse processes which
occur in darkness.

To account for the phenomena described, it is necessary to postulate at
least three interrelated processes, some of which must be directly corre-
lated with light in such a way that change in illumination of very short
duration can cause complete reversal in the nature of the response. It is
altogether probable (1) that some of these processes are photochemical
reactions; (2) that others are dependent upon the results of these; and
(3) that all are closely correlated with the physiological state of the
organism as a whole (Mast, 1932b). The evidence now available clearly
indicates that such simple processes as those postulated by Mast (1907)
in his first publication dealing with this problem, and those postulated
by Luntz (1932) are very inadequate (Mast, 1932b).

A considerable number of other facts have been established concerning
reversal in Volvox and related forms. For example, increase in tempera-
ture of hydrogen-ion concentration, and some anesthetics (especially
chloroform) cause photonegative colonies to become strongly photo-
positive. However, they usually remain positive only a few moments,
then become negative again (Mast, 1918, 1919). There is also a very
interesting correlation between reversal in light and response to elec-
tricity, in that photopositive colonies always swim toward the cathode
and photonegative colonies toward the anode (Mast, 1927c). These
facts show that reversal in light is not due to direct action of environ-
mental factors. They also indicate that it is correlated with the rate of
metabolism; but there is no clue to the nature of the processes involved.

RESPONSES TO ELECTRICITY
A. RHIZOPODS

All the rhizopods which have been investigated (Amoeba, Pelomyxa,
Difflugia, Arcella, Actinosphaerium, and others) respond to electricity.
Kithne (1864) and Engelmann (1869) observed that if they are sub-
jected to a series of induction shocks (alternating current), streaming
in them stops and they then round up. Verworn (1895), from observa-

306 MOTOR RESPONSES

tions on rhizopods in a direct current, maintains that immediately after
the circuit is closed, there is marked contraction at the anodal side and
then movement toward the cathode, and that if the current is strong
enough, disintegration begins on the anodal surface of the organism.

Greeley (1904), in referring to Amoeba, says that ‘‘on the anodal
side of the cells the protoplasm is coagulated .. . and on the cathode side
it is liquefied.” Bayliss (1920) maintains, however, that the current
causes only gelation. According to the careful observations of Luce
(1926), hyaline blisters appear on pseudopods oriented with their longi-
tudinal axis perpendicular to the direction of the current. With the aid
of superior optical apparatus, he observed the transformation of these
blisters into pseudopodia. Since there was no indication of gelation, the
phenomenon must have been due to a liquefaction of the plasmagel at
the cathodal surface.

More details concerning response of rhizopods to electricity were ob-
tained by Mast (1931b) in observations on A. proteus in direct and
alternating currents of various intensities. The results obtained are as fol-
lows.

Direct current.——In direct current of low density, movement continues
no matter how the amoebae are oriented in the field, but the formation
of pseudopods is inhibited on the anodal side, resulting in gradual turn-
ing toward the cathode. In stronger currents movement ceases imme-
diately after the circuit is closed, then in a few moments one or more
pseudopods appear on the cathodal side and movement continues directly
toward the cathode. In still stronger currents there is marked contraction
on the anodal side immediately after the circuit is closed. This is soon
followed by disintegration which begins at this side.

If the anterior end of the amoeba faces the cathode when the current
is made, there is, in the lowest density that produces an observable effect,
merely a slight momentary increase in the rate of flow in the plasmasol
immediately back of the hyaline cap. No change in the rate of flow ts
seen elsewhere. If the current is stronger, this increase extends back
farther, the hyaline cap disappears, the plasmasol extends to the tip, the
anterior end becomes distinctly broader, and the plasmagel becomes very
thin (Fig. 111). If the current is strong enough, this is followed by
violent contraction at the posterior end, slight contraction at the an-

MOTOR RESPONSES 307

Figure 111. Sketches illustrating the effect of a galvanic current on a monopodal
Amoeba moving toward the cathode. g, plasmagel; s, plasmasol; /, plasmalemma; ¢,
hyaline cap; 4, hyaline layer; —, cathode; +, anode; arrows, direction of streaming. A,
very weak current, B, C, D, progressively stronger current. Note that in a current of
moderate density the hyaline cap disappears and the plasmasol extends to the plasmalemma
and that in stronger current the cathodal end expands and the anodal end contracts and
finally breaks, after which the granules in the plasmasol flow toward the anode, indi-
cating that they are negatively charged. If the surrounding medium is acid, the amoebae
do not break. (After Mast, 1931a.)

terior end, and, finally, by disintegration beginning at the posterior end,
i.e., that which is directed toward the anode.

If the anterior end of the amoeba is directed toward the anode and
the current is weak, there is merely a momentary retardation of stream-
ing at the posterior (cathodal) end. With successively stronger currents,

308 MOTOR RESPONSES

the streaming at this end (1) stops a few moments, then begins again,
and continues in the original direction; (2) it stops a bit longer, then
begins, and continues in the reverse direction, i.e., the plasmasol then
streams toward the cathode at one end and toward the anode at the other
(Fig. 112); (3) the reversal extends to the anodal end, and a new hya-
line cap forms at the original posterior end, which now becomes the an-
terior end; (4) the reversal is followed by marked contraction at the

Figure 112. Sketches illustrating the effect of a galvanic current on monopodal amoebae
moving toward the anode in a weak current. Labels are the same as in Figure 111. Note
that the direction of streaming reverses, that it begins at the cathodal end and proceeds
toward the anodal end, and that this results in movement in opposite directions at the
two ends during one phase in the process of reversal. (After Mast, 1931a.)

anodal end, which is followed by partial or complete disintegration, al-
ways beginning at the anodal end. The fact should be emphasized that
no matter how extensive a reversal in the direction of streaming may be,
it always begins at the cathodal end of the amoeba.

When the contraction at the anodal end begins, the hyaline layer in
this region becomes thicker here and there, resulting in the formation
of several small blisters and in numerous minute papilla-like foldings in
the plasmalemma (Fig. 111). As contraction continues, some of the
blisters, containing fluid and a few granules in violent Brownian move-

MOTOR RESPONSES 309

ment, round up and are pinched off; and others together with the
plasmagel break, after which granules in the plasmasol stream out and
proceed rapidly toward the anode. This continues until frequently there
is nothing left intact except a crumpled membranous sac, the plasma-
lemma. The plasmagel changes entirely into plasmasol and is carried
away. The fact that the granules are carried toward the anode shows that
they are negatively charged.

McClendon (1910) came to the same conclusion concerning the gran-
ules in the eggs of frogs and the cells in root tips of onions. But Heil-
brunn (1923) says: ‘Particles in the interior of living cells bear a posi-
tive, whereas the particles in the surface layer have a negative charge.”

If a small amount of HCl is added to the culture solution, the amoeba
does not disintegrate, regardless of the current strength. Its plasmagel
turns distinctly yellow at the anodal end immediately after contraction
begins, after which it increases in thickness until the entire amoeba has
solidified and is dead. If the current is broken before more than about
one-fourth of the amoeba has gelated, the gelated portion is usually
pinched off. The rest of the amoeba then proceeds normally.

If the amoeba is moving toward the anode when the current is made,
streaming of the plasmasol reverses before it stops at the anodal end.
This behavior demonstrates conclusively that the effect of the current
begins at the surface directed toward the cathode. The fact that before
the reversal occurs, the thick plasmagel at the cathodal end is replaced
by a very thin plasmagel sheet and a hyaline cap strongly indicates that
the first effect of the current is solation of the plasmagel at the cathodal
surface. This conclusion is supported by the facts that if the anterior end
is directed toward the cathode when the current is made, the plasmagel
sheet disappears entirely, the anterior end enlarges, and the plasmasol
extends to the plasmalemma. It is also true that if the current passes
through the amoeba in a direction perpendicular to the longitudinal
axis, the forward streaming stops, and pseudopods are formed on the
cathodal side.

The contraction of amoeba at the anodal end, and the increase in the
thickness of the plasmagel—especially in specimens directed toward the
anode—seem to show that the current causes gelation at the anodal sur-
face.

The facts that the end directed toward the cathode enlarges, that the

310 MOTOR RESPONSES

opposite end decreases in size, breaks up, and becomes yellow just as do
amoebae killed in an acid solution, and the granules in the amoeba then
move toward the anode, all indicate (1) that the granules bear a negative
charge, (2) that water in the amoeba is carried toward the cathode, and
(3) that the plasmagel at the anodal surface becomes acid.

In very accurately controlled observations on A. proteus, in direct
current, Hahnert (1932) found that there is at first a momentary in-
crease in rate of locomotion and then a gradual decrease. He also noted

25
20_* ahr

: (@) AS} am
15 (@) () 4 LY)
— ()

0 ©)
@ ~ Ore
10 8s
Oe

| B

ORLA | 2050, WEE 4 OG) AB Oy 5 eee |

Figure 113. Graphs showing comparative effect of different densities of current on the
rate of locomotion. Abscissae, time in minutes, (A) before and (B) during the passage
of current; ordinates, apparent rate of locomotion. To obtain actual rate in millimeters
per minute, divide the apparent rate by 85. (After Hahnert, 1932.)

that the rate of decrease varies directly with the current density (Fig.
113), and that the time required to induce cessation of movement in
specimens directed toward the anode varies nearly inversely with the
square of the current density, “the time-intensity relation being nearly
in accord with the equation 7\/¢— (x/) = K in which 7 is the intensity
of the current and ¢ its duration.”

Alternating current —If an active Amoeba mounted in culture fluid is
subjected to a weak alternating current, locomotion ceases at once. The
pseudopods are then retracted partially or entirely, and the animal be-

MOTOR RESPONSES ey

comes somewhat rounded. It remains in this condition a few moments,
then a pseudopod appears on one of the sides between the two surfaces
facing the poles and projects at right angles to a line connecting the two
poles. Almost immediately after the first pseudopod begins to form, an-
other usually appears on the opposite surface and extends in the opposite
direction. Thus the amoeba becomes oriented perpendicularly to the
direction of the current, i.e., at right angles to the direction in which
orientation occurs in a direct current. These two pseudopods usually con-
tinue to stretch out in opposite directions until the amoeba becomes
greatly elongated. Then one is withdrawn, and the amoeba continues in
the opposite direction, soon moving out of the field (Fig. 114).

These pseudopods usually contain no hyaline cap and no plasmagel
at the tip, and the plasmagel elsewhere is very thin. Sometimes such a
large portion of the distal end is without plasmagel that the plasmasol
very definitely streams back at the surface. If the circuit is opened shortly
after these pseudopods have begun to form, no change in movement 1s
seen; if it is closed again, there is still no response. This indicates that
pseudopods in which the plasmagel is very thin, or absent, do not re-
spond to electricity; and that the response to electricity consequently is
due to its action on the plasmagel.

If the current is stronger, movement ceases and the pseudopods re-
tract just as they do in weak currents. The contraction which follows
the retraction of the pseudopods is much more marked, especially on
the surfaces directed toward the poles. Here the plasmagel fairly shrinks
up and becomes yellowish in color. This is apparently precisely what
occurs at the anodal end of an amoeba subjected to the action of a direct
current. If the circuit is opened immediately after the violent contraction
has occurred, the amoeba soon recovers. But the two masses of plasma-
gel that have become yellowish are usually pinched off. An irreversible
transformation takes place in them, which results in the death of a por-
tion of the cytoplasm. Occasionally, however, these masses, especially if
they are relatively small, are taken into the plasmasol and are there di-
gested.

If observations are made under an oil immersion objective on a speci-
men in an alternating current, the plasmagel can be seen to contract and
become yellowish. Fluid is squeezed out on either side of the organism
and the adjoining plasmalemma is thrown into folds and papillae of

312 MOTOR RESPONSES

various sizes. The latter are filled with hyaline substance containing a
few scattered granules which exhibit violent Brownian movement, show-
ing that the substance in which they are suspended is a fluid with low
viscosity. Some of the folds and papillae round up and pinch off, to

Figure 114. A series of camera sketches of an Amoeba, showing the effect of an
alternating current. A, before current was made; B-F, successive stages after it was
made; g, plasmagel; s, plasmasol; /, plasmalemma; /, hyaline layer; c, hyaline cap;
arrows, direction of streaming; double headed arrows, direction of the current. Note
that the Amoeba orients perpendicularly to the direction of the current, that the plasmagel
in the pseudopods is at this time very thin or absent, that the plasmagel contracts violently
at the surface directed toward the poles, that blisters are formed on these surfaces, and
that the Amoeba eventually breaks here and then disintegrates. If the surrounding medium
is acid, the Amoeba does not break and disintegrate. (After Mast, 1931a.)

form spherical bodies filled with granule-containing fluid observed in
the folds (Fig. 114).

If the current is strong enough, the plasmagel usually breaks after
it has thus contracted. The plasmasol flows out and the entire amoeba
soon disintegrates and dissolves. Sometimes breaks occur in the pseudo-

MOTOR RESPONSES 313

pods before there is much contraction, and then the plasmasol flows out
through these. As the plasmasol flows out, it collects about the amoeba;
and the granules and fluid in it do not stream toward the poles as they
do in direct current. Cataphoresis and electroendosmosis are thus neutral-
ized, owing to the reversal in the direction of flow of the current.

There is no change in the responses if acid is added to the culture fluid.
However, instead of disintegrating as usual, the plasmasol coagulates
after the amoeba breaks up.

The essential phenomena observed in the effect of the alternating
current on Amoeba appear to be: (1) mild contraction in extended
pseudopods, beginning at the tip; (2) violent contraction on the two
surfaces facing the poles, with the formation of blisters in these regions;
(3) formation of highly fluid pseudopods between these two surfaces;
and, finally, (4) rupture at the surfaces directed toward the poles, fol-
lowed by disintegration of the organism.

Contraction in the pseudopods, as was repeatedly observed, occurs
first in those directed toward the poles and last in those in which the
longitudinal axis is perpendicular to the direction of the current. In-
deed, there is some indication that there is no contraction at all if the
axis is actually perpendicular to the direction of the current. Contraction
in the pseudopods under these conditions results in retraction which,
in all respects, appears to be the same as retraction of pseudopods in
normal locomotion. The retraction appears to be due to increase in the
elastic strength of the plasmagel, owing to reversible gelation of ad-
joining plasmasol. It may be concluded, then, that the first effect of the
alternating current is reversible gelation of the plasmasol adjoining the
plasmagel at the tip of the pseudopods directed toward the poles. This
results in an increase in elastic strength of the plasmagel in this region,
and in a retraction of the pseudopods. The contraction at the surfaces
facing the poles is, in the beginning, doubtless due to the same phe-
nomena; but the facts (1) that the plasmagel in this region later changes
in color, (2) that it does not become thicker, (3) that fluid is squeezed
out of it, and (4) that it is killed and then breaks, show that contraction
here is associated with profound changes in the plasmagel itself. These
changes result in such marked decrease in the strength and elasticity of
this structure that it breaks readily. These changes are also associated
with simultaneous increase in fluidity of the plasmasol, as indicated by

314 MOTOR RESPONSES

the structure of the pseudopods which form at this time. The evidence
presented above shows that the described changes are brought about by
the action of the current.

Alsup (1939) measured the time required in alternating current and
in light respectively to cause cessation in streaming (reaction time) and
that required for recovery after this response (recovery period). He
found in both that the reaction increased as the recovery period decreased.
This indicates that after the plasmasol has gelated, owing to the action
of electricity or light, and then solated, owing to the recovery processes,
it no longer gelates so readily as it did.

Alsup also found that subminimum exposure to an alternating cur-
rent followed by a subminimum exposure to light, or vice versa, may
induce a response, indicating that the effects of these two agents are
additive.

In order to account for the essential phenomena observed in amoebae
when subjected to the action of an alternating current, it is then neces-
sary to explain reversible gelation of the plasmasol adjoining the plasma-
gel on the sides of the organisms facing the poles. It should be noted
that the gelation of the plasmasol is followed by changes in the plasmagel
in the adjoining regions—changes which result in violent contraction,
loss of fluid, decrease in elasticity, and rupture, and by increase in the
fluidity of the plasmasol.

Mechanics of response.—In rhizopods all of the responses to direct
current appear to be due primarily to solation at the cathodal surface,
followed by gelation at the anodal surface. The question then arises as
to what causes this.

If an electric current is passed through a culture solution containing
amoebae, the negative ions in the solution and in the amoebae move to-
ward the anode, whereas the positive ions migrate toward the cathode.
If the surface layers of the amoebae are semipermeable, as they un-
doubtedly are, there will be an accumulation of positive ions (e.g., Na)
on the inside, and negative ions (e.g., Cl) on the outside of the surface
of the amoebae directed toward the cathode. Positive ions will accumulate
on the outside and negative ions on the inside of the surface directed
toward the anode (Ostwald, 1890). The positive ions will, however,
unite with the hydroxyl ions of the water, forming bases (e.g., NaOH);
and the negative ions will unite with the hydrogen ions of the water,

MOTOR RESPONSES ZiLD

forming acids (e.g., HCl). The cathodal surface layer of the amoebae
should therefore become alkaline on the inside and acid on the outside.
The anodal surface layer should react in the opposite manner.

Numerous observations were made on specimens stained with neutral
red and subjected to direct current under various conditions. No differ-
ence whatever was observed in the color of different regions in any of
these specimens. The neutral red staining in A. proteus is, however,
confined to granules and vacuoles. It was observed that if the specimens
are crushed, the color of these granules and vacuoles does not immedi-
ately change, in accord with the hydrogen-ion concentration of the solu-
tion in which they are immersed. It is obvious, then, that the fact that
no difference in color was observed in the vacuoles and granules does not
prove that the hydrogen-ion concentration of the cytoplasm was the
same. Moreover, there is indirect evidence which indicates that it was
not the same.

Kihne (1864) long ago observed in certain epidermal cells of the
leaves of Tradescantia subjected to a galvanic current that the ends of
the cells directed toward the cathode become alkaline, and that those
directed toward the anode become acid. These cells contain a natural
indicator which is bluish in neutral solutions, red in acid solutions, and
green in alkaline solutions.

Habenicht (1935) came to the same conclusion in experiments on the
effect of the galvanic current on cylinders of egg white.

Mast (1931b) repeated and extended Kihne’s experiments and ob-
tained results which confirm his contentions. It may then be assumed with
considerable confidence that when an amoeba is subjected to a direct
current, the hydrogen-ion concentration in the cytoplasm decreases at
the cathodal end and increases at the anodal end.

Edwards (1923) demonstrated that if an alkaline solution is locally
applied to the surface of an amoeba, the plasmagel in this region dis-
integrates; and that if acid is applied, it becomes thicker, owing to gela-
tion of the adjoining plasmasol. This has been confirmed indirectly by
Pantin (1923), Chambers and Reznikoff (1926), and others. If, then,
the direct current produces a decrease in hydrogen-ion concentration at
the cathodal end and an increase at the anodal end, one would expect
the plasmagel to become thinner at the cathodal end and thicker at the
anodal end. This is precisely what was observed. And if the elastic

316 MOTOR RESPONSES

strength of the plasmagel varies directly with its thickness, as is doubt-
less true, this would result in formation of pseudopods at the cathodal
surface. This has been confirmed by observation. Since streaming toward
the cathode begins at the cathodal surface before it does at the anodal
surface, movement toward the cathode must be due primarily to the
solation of the plasmagel at the cathodal surface. The accumulation of
positive ions at this surface therefore must produce the solation. But this
obviously does not account for the disintegration of the entire organism.
Neither does it account for the violent contraction preceding disintegra-
tion at the anodal side. It will be remembered that violent contraction
and disintegration beginning at the anodal surface were observed only
in relatively strong currents. Furthermore, the reactions were observed
to occur only after large pseudopods develop and begin to advance to-
ward the cathode.

An amoeba disintegrates only if the direct current applied is sufh-
ciently strong. After the circuit is closed, there is, on the inner surface of
the plasmagel or in the plasmagel, an accumulation of positive ions at
the cathodal side, and of negative ions at the anodal side. The former
produces a decrease in the elastic strength of this layer, which results in
the formation of a pseudopod directed toward the cathode—a pseudopod
in which the plasmagel extends to the plasmalemma. Local disintegra-
tion occurs at first; but, as the current continues, more and more of the
plasmagel in this pseudopod disintegrates. The accumulation of nega-
tive ions and consequently of hydrogen ions at the anodal end causes a
thickening of the plasmagel, as well as gelation of the adjoining plas-
masol. This results in violent contraction and finally in the rupture ob-
served at the anodal end.

Cataphoresis and electroendosmosis are probably also involved. The
granules in Amoeba are negatively charged in relation to the fluid. The
fluid consequently tends to flow from the anodal toward the cathodal
end. This would facilitate contraction at the former and expansion at the
latter end, which is precisely what was observed. There is, however, some
evidence to indicate that transfer of water is of little importance in the
rupture and disintegration at the anodal end. This will be presented later.

It is therefore fairly clear how, in a direct current, substances accumu-
late locally; and how this can produce most of the processes associated
with the responses of amoebae in it. But in an alternating current the

MOTOR RESPONSES RT

situation is quite different. There is an equal movement of all substances
in opposite directions. Consequently there can be no accumulation of
different substances in different parts of the organism, unless there is
some process which makes the movement in one direction greater than
that in the other.

The essential phenomena observed in Amoeba as a consequence of
exposure to alternating current may be summarized in the order of their
appearance. A mild contraction begins first at the tips of extended pseudo-
podia. This is followed by violent contraction on the two surfaces fac-
ing the poles (blisters appear on these surfaces). Then highly fluid
pseudopods form between these two surfaces. Finally, the surfaces di-
rected toward the poles rupture and the organism disintegrates.

Numerous observations with the best lens system obtainable were
made on the movement of microscopic particles, both in the field of the
alternating current and in the amoebae in this field. There was no indi-
cation of a drift of these particles nor of their accumulation in any part of
the organisms. It therefore is evident that cataphoresis and electroendos-
mosis cannot be involved in the observed contraction. It seems necessary,
then, to conclude that the phenomenon of contraction is associated with
the movement of the ions produced by the electric current; and, further,
that ion movements are accompanied by processes which result in the
accumulation of ions in certain regions of the organism.

Dixon and Bennet-Clark (1927) and others maintain that alternat-
ing current causes increase in the permeability of the plasmamembrane
in cells. If so, then may not the contraction observed in the plasmagel be
due to the action of substances which enter from the surrounding medium,
since localized accumulation of ions at the surface of the amoeba 1n-
creases its permeability owing to the action of the current?

Two facts suggest that the contraction of Amoeba during exposure to
alternating current cannot be due to the entrance of substances from the
outside. In the first place, the contractions are known to occur in both
alkaline and acid solutions. Secondly, alkaline solutions tend to produce
solation in the plasmagel, thus decreasing its elastic strength. Possibly
the accumulation of ions in or near the plasmagel causes the contrac-
tion.

It is well known that the positive ions, Na, K, Ca, and others ordi-
narily pass through membranes more readily and more rapidly than the

318 MOTOR RESPONSES

negative ions, SO,, PO,, NO,, and others. In a structure like the plas-
magel, the movement of ions is undoubtedly hindered. Nevertheless,
the positive ions may move farther from their initial positions toward
the pole than do the negative ions. It may be assumed that the nega-
tive ions tend to remain in the plasmagel, whereas the positive ions
tend to leave it and return again as the direction of the current reverses.
If the return movement of the positive ions is inhibited, there may be
a momentary preponderance of negative ions within the plasmagel,
and of positive ions in the adjoining substances, i.e., in the plasmasol,
the hyaline layer, and the plasmalemma. If the movement of the posi-
tive ions away from the negative ions is extensive enough, the negative
ions remaining in the plasmagel will unite with the hydrogen ions of the
water surrounding them, to form acids. The hydroxyl ions thus lib-
erated will, owing to the fact that they pass rapidly and freely through
tissues, move out and unite with the positive ions, to form bases in the
substance adjoining the plasmagel. It is possible that this union retards
the return movement of the positive ions during the next reversal in the
direction of the current. Consequently the effect would be cumulative,
gradually increasing the acidity within the plasmagel and the alkalinity
of the substance on either side.

The increase in acidity in the plasmagel would produce gelation in this
layer and probably also in closely applied plasmasol. This would in-
crease the thickness and elastic strength of the plasmagel. Contraction
would be the result. If the accumulation of negative tons were great
enough, the increase in acidity in the plasmagel would cause irreversible
coagulation (death), accompanied by violent contraction and dehydra-
tion, thus making the coagulated plasmagel so brittle that it would break
readily. The increased alkalinity in the plasmasol and plasmalemma
would tend to make the former more fluid, and it would tend to break
up the latter. The postulated action of the current is precisely in accord
with the observations. It also accounts for the fact that no response was
observed in pseudopods which contained no plasmagel. According to the
explanation offered, an accumulation of ions occurs in or near the plas-
magel; there could be no action in structures which have none.

Moreover, on the basis of this hypothesis, it is possible to account
for the well-known fact that in many organisms the effect of a current
varies inversely with the frequency of reversal. It is necessary only to as-

MOTOR RESPONSES 319

sume that the higher the frequency of reversal, the more restricted the
movements of the ions toward the poles, and the shorter the period of
separation of positive from negative ions; and that the shorter this period,
the more restricted the union of the negative ions with hydrogen tons,
and the positive ions with hydroxyl ions. The more restricted these unions,
the less the increase in acidity in the plasmagel, and the less the increase
in alkalinity in the plasmasol, and the less the stimulating and the in-
jurious effect. The hypothesis, then, that the action of the electric cur-
rent on organisms is due to localized increase in acidity and alkalinity
in different regions of the cell is in full accord with the fact that the
effect in alternating currents varies inversely with the frequency.

What bearing has all this on the problem concerning the observed con-
traction at the anodal side of amoebae subjected to direct current?

Carlgren (1899), as previously stated, holds that this is due to elec-
troendosmotic extraction of water by the current, owing to negative
charge of the solid substance. However, the fact that the same phe-
nomenon occurs in alternating current, in which electroendosmosis 1s neu-
tralized, strongly indicates that Carlgren’s conclusion is not valid. It also
seems to show that the anodal contraction in direct current must be due,
as appears to be the case in alternating current, to the action of the cur-
rent on the movement of ions.

The evidence presented in reference to the effect of both direct and
alternating current on A. proteus indicates that the assertion of Bayliss
(1920), Weber and Weber (1922), Taylor (1925), and others that
electricity gelates cytoplasm, is misleading, for it shows that if an electric
current causes gelation in a cell, it probably always causes simultaneous
solation, each being confined to a portion of the cell.

It was demonstrated above that this does not obtain for light. The
implication frequently found in the literature that the action of electricity
on protoplasm is the same as the action of light, appears therefore to be
erroneous.

Heilbrunn and Daugherty (1931) found that if ammonium hydroxide
or chloride is added to the culture fluid, A. protews becomes anopositive.
They maintain that the ‘‘protoplasmic granules” are ordinarily positively
charged and are consequently carried (cataphoretically) toward the
cathode, and that ammonium hydrate or chloride causes a change in
the charge to negative and a consequent reversal in the direction in which

320 MOTOR RESPONSES

they are carried. They contend that the contact of these granules with
the inner surface of the plasmagel causes it to liquefy, and that this
results in the formation of a pseudopod which is directed toward the
cathode if the granules are positively charged and toward the anode if
they are negatively charged. These authors say:

As the granules move either toward cathode or anode, they must tend to
break down the thixotropic gel on the side toward which they move. . .
If this gel is liquefied in any local region, such a region becomes pushed
out to form an advancing pseudopod.

These are interesting views, but they obviously do not account for
the direction of movement of pseudopods in alternating current (Fig.
114), nor for the direction of movement of pseudopods under some
conditions in direct current (Fig. 112). For this would require cata-
phoretic movement of the granules perpendicularly to the direction of
the current in the former, and in opposite directions at opposite ends
of the amoeba in the latter. Moreover, the fact that the granules stream
toward the anode after the amoeba disintegrates, indicates that they are
normally negatively (not positively) charged, and that this charge is
consequently not involved in the cathopositive response. How, then, can
the observed reversal in the direction of orientation be explained?

The cathopositive response is probably brought about as described
above. The ammonium hydrate or chloride added to the culture fluid
probably results in the liquefaction of all the plasmagel, and conse-
quently in free movement toward the anode of the negatively charged
granules suspended in it. According to this view, the reversal in the
direction of galvanic orientation is due to liquefaction of the plasmagel
caused by the ammonium compounds used, not to change in the electric
charge on the “protoplasmic granules.”

B, FLAGELLATES

Some of the flagellates orient very precisely in a direct current. Some
are cathopositive, others anopositive, and still others both or neutral,
depending upon the environmental conditions (Verworn, 1889; Pearl,
1900; Bancroft, 1913). Verworn maintains that orientation is brought
about by differences in the effective stroke of the flagellum in opposite
directions. Pearl believes that it is the result of typical avoiding reac-

MOTOR RESPONSES BAL!

tions, whereas Bancroft thinks that it is identical with the process of
orientation in light. Further details concerning the processes involved
are much desired. Moreover, it is noteworthy that some of the flagel-
lates have proved to be excellent material for quantitative study of the
relation between stimulus and response.

C. CILIATES

The responses to electricity have been more intensively and extensively
studied in the ciliates than in any of the other groups of Protozoa.

Jennings (1906) presents an excellent review of all the earlier in-
vestigations concerning these responses. He maintains that the results
obtained show the following:

The principal feature in the response of all of the different species
studied consists of reversal in the direction of the effective stroke of the
cilia on the cathodal surface. In those species in which other ciliary
actions are only slightly or not at all involved (e.g., Paramecium), this
results in direct turning until the anterior end is directed toward the
cathode. The organism then moves toward this pole. The extent of the
cathodal surface affected varies directly with the strength of the current.
If the current is strong enough to produce reversal over more than half
of the surface of the Paramecium, it swims backward toward the anode.
A still stronger current causes marked swelling of the anterior end and
contraction of the posterior end, changes which are followed by disin-
tegration beginning posteriorly (Fig. 115). The effect of the reversal
on the cathodal surface is variously modified by the normal action of
the cilia in other regions, in such a way that ‘‘with different strengths of
current, and with infusoria of different action systems, this results some-
times in movement forward to the cathode; sometimes in movement
forward to the anode; sometimes in cessation of movement, the anterior
end continuing to point to the cathode; sometimes in a backward move-
ment to the anode; sometimes in a position transverse to the current, the
animal either remaining at rest or moving across the current.”

Jennings holds that all the responses of the ciliates to electricity are
due to stimulation at the cathodal surface, resulting in local reversal in
the direction of the effective stroke of the cilia on this surface.

Bancroft (1906), in his experiments with Paramecium, observed that
if certain salts (especially potassium, sodium, or barium salts) are added

322 MOTOR RESPONSES

to the solution, the organisms will swim forward toward the anode. He
maintains that this is due to stimulation at the anodal surface, and that
consequently it is an exception to Pfliiger’s law.

It is well known that some salts cause paramecia to swim backward,

Figure 115. Progressive cathodic reversal of the cilia and change of form in Para-
mecium as the constant electric current is made stronger. The cathode is supposed to lie

at the upper end. The current is weakest at 7, where only a few cilia are reversed; 2-6,
successive changes as the current is gradually increased. (After Statkewitsch, 903.)

owing to the forward stroke of all the cilia (Jennings, 1899; Mast and
Nadler, 1926; Oliphant, 1938). Obviously, if under such conditions
there is reversal in the direction of the stroke at the cathodal surface,
the paramecia will turn and swim forward toward the anode (Mast,

MOTOR RESPONSES 323

1927c). If this reaction obtains, the forward swimming toward the
anode is in full accord with Pfliiger’s law.

Kamada (1929) made a study of the correlation between the effect
of many different salts on reversal from forward to backward swimming,
and the direction of orientation in a direct current. He maintains that
some salts which induce forward swimming toward the anode do not
induce backward swimming. He consequently supports Bancroft’s views.

Kamada (1931) also maintains that in paramecia which are ano-
positive there is ciliary reversal at the anodal surface, in place of the
cathodal, and that with increase in current density this is modified in
various ways. The evidence he presents is, however, by no means con-
clusive. Further observations are therefore needed.

Paramecia are most sensitive if the anterior end is directed toward the
cathode, less sensitive if it is directed toward the anode, and least sensi-
tive if the longitudinal axis is perpendicular to a line connecting the two
poles (Statkewitsch, 1903; Kinosita, 1936). The same holds for Spv-
rostomum (WKinosita, 1938a).

Statkewitsch (1907) subjected to direct and to alternating currents
paramecia which had been stained with neutral red. He maintains that the
stained structures in them became violet (acid) in weak currents and
distinctly yellowish (alkaline) in strong currents. He apparently did not
observe any difference in the color at the two ends. In Kinosita’s (1936)
experiments with paramecia stained with either neutral red or Nile blue
sulphate, the color of opposite ends differed. He says that the changes
in color observed show that the paramecia become acid at the cathodal
and alkaline at the anodal end, but that the alkaline portion rapidly ex-
tends forward and soon includes the entire body.

There is consequently a diversity of opinion concerning the effect of
the electric current on the hydrogen-ion concentration of the cytoplasm in
Paramecium. This also obtains for other cells, since, as previously stated,
Kine maintains that cells of Tradescantia become acid at the anodal end
and alkaline at the cathodal end. Mast confirmed this, but could observe
no difference in this respect between the two ends in Amoeba. It is there-
fore obviously desirable to have further observations concerning this
problem, for it is theoretically very important.

Kinosita measured the time required to make the anodal end alkaline

324 MOTOR RESPONSES

in different current densities, extending over a wide range. He maintains
that the results indicate that 7\/f— a) ==K. This equation is similar
to the one obtained by Kamada in observations on the destruction of the
surface membrane in paramecia by a direct current. It is also related to
the one obtained by Hahnert (see above) in his observations on the
cessation of streaming in Amoeba in a constant current.

Internal processes involved in response.—Several theories have been
formulated to account for the responses of ciliates to an electric current.
Loeb and Budgett (1897) contend that these are in reality the result of
responses to changes in the chemical constitution of the environment
produced by the electric current. Pearl (1901) asserts that the direction
of the stroke of the cilia is specifically correlated with the direction of
protoplasmic streaming directly below the surface. Coehn and Barratt
(1905) hold that the movements are purely cataphoretic; Bancroft
(1906) maintains that the galvanic responses are due to local changes
in the calcium content of the tissue in relation to that of other ions,
especially monovalent cations. Although Carlgren (1899) lays especial
stress on localized changes in water content within the organisms, due
to endosmotic streaming. Some assert that the movements are due to
direct action of the electric current on the cilia, others that they must
be due to action on a codrdinating center. Ludloff (1895), Verworn
(1895), and Koehler (1925) postulate functional division of the organ-
isms into anterior and -posterior halves, such that one responds in one
way that the other responds in another way. The views of Nernst (1899),
Lucas (1910), Lillie (1923), and others regarding stimulation in higher
organisms would lead to the idea that local changes in permeability of
the surface membrane is the all-essential in controlling the movements
of the lower organisms in an electric field.

The relation between these hypotheses and the facts established has
been very illuminatingly discussed by Jennings (1906) and Koehler
(1925). Both conclude that the facts in hand are not adequately ac-
counted for by any of the hypotheses presented. Jennings contends, as
previously stated, that the most important perceptible characteristic of
the response is reversal in the direction of the stroke of the cilia on the
cathodal side. Koehler holds that the processes involved cannot be solely
dependent upon surface phenomena, that somehow the current results
in a division of the organism into cathodal and anodal portions which

MOTOR RESPONSES 2045)

function differently, owing to different internal factors. But neither Jen-
nings nor Koehler offers any explanation of how the responses are reg-
ulated.

Among the most important of the known facts concerning the re-
sponses in Protozoa to an electric current are those discovered by Lud-
loff in observations on Paramecium. Ludloff (1895) found, as has been
abundantly confirmed, that when the circuit is closed, the direction of
the stroke of the cilia on the surface of the paramecia directed toward
the cathode reverses; but that if the longitudinal axis of the organisms
is directed obliquely to the direction of the current, reversal occurs on

Figure 116. Paramecium showing reversal in the direction of the stroke of the cilia
in a galvanic current. A, weak current; B, strong current; +, anode, —, cathode. (After
Ludloff, 1895.)

all sides of the end of the body nearest the cathode, extending to a line
around the body produced by passing a plane through it at right angles
to the direction of the current. The extent of the portion of the body
on which such reversal occurs depends upon the strength of the current:
the stronger the current, the larger the portion affected (Fig. 116).
The fact that the cilia in different regions on the same side of the
paramecia are not always affected equally by the current seems to show
that the responses observed cannot, as Koehler points out, be due to
direct action on the cilia or to surface phenomena alone; for if they were,
all the cilia on either side should act alike, with the possible exception

326 MOTOR RESPONSES

of those in the oral groove. If this is true, it is evident that the responses
in this form must be associated with internal changes.

The fact that in specimens with the longitudinal axis directed ob-
liquely to the direction of the current, the entire end nearest the cathode
is affected, and the fact that the size of the portion affected varies directly
with the strength of the current, indicates not only that the current re-
sults in a functional division of the organism (as maintained by Ludloff,
Verworn, and Koehler), but also that whatever the factors involved
may be, they act on a structure which is well distributed through the
entire body and which is located some distance below the surface—
probably the neuromotor apparatus. For only in a structure which is
some distance from the surface, could a current produce the same changes
in the distribution of substances on the anodal and the cathodal sides
of the portion affected, resulting in reversal in the direction of the
stroke 1n all the cilia on this portion.

It is well known that momentary reversal in the stroke of the cilia
can be induced in Paramecium by almost any sudden environmental
change (Jennings, 1906), and that more prolonged reversal can be in-
duced by transfer from culture fluid to distilled water or from distilled
water to solutions of monovalent cation salts, but not usually by transfer
to solutions of bivalent cation salts (Mast and Nadler, 1926; Oliphant,
1938). These changes, therefore, produce the same result as is produced
by an electric current on the cathodal surface, indicating similarity in
action.

Greeley (1904) maintains that paramecia drift toward the anode, i.e.,
that they are negatively charged, indicating that there is a negative layer
at the surface. Statkewitsch (1903) observed that if one end is directed
toward the anode and the other toward the cathode, the former shrinks
and the latter swells, indicating that the more solid substance is negative
in relation to the more fluid substance. If all this obtains, a direct cur-
rent will result in a decrease in the concentration of the positive, or an
increase in the concentration of the negative ions on the cathodal side of
the surface of the organism and on each semipermeable structure within.
There will also be a decrease in the concentration of the negative, or an
increase in the concentration of the positive ions on the opposite side. As
a result, water will drift toward the cathodal side, and the solid particles
will drift in the opposite direction.

MOTOR RESPONSES 27,

Since reversal in the stroke of the cilia begins on the cathodal side,
it would seem that it must be associated either with an increase in the
concentration of the negative ions or with a decrease in the concentration
of the positive ions, on the cathodal side of the semipermeable structures
on this side of the organisms. Either a decrease of polarization or an
increase in the water content of the cytoplasm may be involved. If this
is true, then transfer from culture fluid to distilled water and from dis-
tilled water to solutions of monovalent cation salts should, since this re-
sults in reversal of the stroke of all the cilia, produce similar changes
in the concentration of ions or water. In other words, the transfer from
culture fluid to distilled water should produce a decrease in the con-
centration of positive ions on the outside of the semipermeable struc-
tures, or an increase in the water content at the surface. A transfer from
distilied water to solutions of monovalent cation salts should produce
like changes. Whether or not this obtains is at present unknown, but one
would expect it to obtain, if, as is frequently asserted, permeability is
increased by monovalent cation salts.

D. COLONIAL ORGANISMS

No detailed observations have been made on the response to electricity
of any of the colonial organisms except Volvox. Volvox orients very
precisely in direct current. It swims toward the cathode under some condi-
tions and toward the anode under others. Carlgren (1899) maintains
that reversal in the direction of orientation is correlated with the dura-
tion of exposure to the current. Terry (1906) and Bancroft (1907)
contend that it is correlated with the intensity of the light received and
the duration of exposure to it. Mast (1927c) found that Volvox swims
toward the cathode when it is photopositive, and toward the anode when
it is photonegative, i.e., that the response to electricity is specifically cor-
related with the response to light.

In photopositive colonies in which rotation on the longitudinal axis
is inhibited by means of pressure, the flagella on the cathodal side stop
beating immediately after the circuit is closed. They remain inactive 4.5
to 6 seconds and then begin to beat again (Fig. 117). If the circuit
is now opened, those on the anodal side stop for a few moments and
then beat again. If the colonies are swimming and rotating on the longi-
tudinal axis in the normal way, but are not proceeding directly toward

328 MOTOR RESPONSES

either pole when the current is made, flagellar inactivity on the cathodal
side is continuous, owing to the continuous transfer of the zooids from
the anodal to the cathodal side. The colonies therefore turn toward the
cathode until they face it directly, and the transfer of zodids from side
to side ceases.

Orientation.—In photonegative colonies precisely the opposite occurs.
The flagella on the anodal side stop beating after the circuit is closed,

Figure 117. Sketch showing in a stationary photopositive colony of Volvox the effect
of a galvanic current on the currents of water produced by the flagella. A, outline of
colony oriented in light; B, same colony immediately after the circuit was closed; a,
anterior end; straight arrows, direction of illumination; curved arrows, currents pro-
duced by the flagella; +, positive pole; —, negative pole. (After Mast, 1927.)

and the colonies turn toward the anode until they face it and then swim
toward it (Mast, 1927c). :

Galvanic orientation is consequently correlated with photic orientation,
but the processes involved differ, for, as previously stated, photic orienta-
tion is due to a change in the dzrection of the stroke of the flagella on
opposite sides, while galvanic orientation is due to decrease or cessation
in the activity of the flagella on one side.

Electric charge on the colonies —Galvanic orientation in Volvox also
differs from that in Paramecium, for while the one is due to decrease or
cessation in activity on one side, the other, as previously stated, is due to
reversal in the direction of the effective stroke of the cilia on one side.

Since a given colony of Vo/vox may be either photopositive or photo-
negative in the same environment, the difference in response to the light

MOTOR RESPONSES 329

in this environment must be due to changes in the colony itself. Since
photopositive response is specifically correlated with cathopositive re-
sponse, and photonegative response with anopositive response, the differ-
ence in the response to electricity must be due to like changes in the
colony. The only difference observed in the colonies in connection with
the response to light concerns the electric charge. Referring to this, Mast
(1927c) says:

Most of the photopositive colonies observed drifted toward the anode
and most of the photonegative ones drifted toward the cathode, indicating
that the former were negatively and the latter positively charged. However,
owing to the negatively charged glass bottom of the aquarium in which
the observations were made, there was produced in the solution near the
bottom an endosmotic current of water toward the cathode and this current
produced at the upper surface a current in the opposite direction, i.e., toward
the anode. In making the observations it was impossible to ascertain pre-
cisely the location of the colonies in relation to these currents, resulting fre-
quently in uncertainty as to whether the drift was due to cataphoresis or
to endosmosis. The results obtained are consequently somewhat equivocal.

These observation should therefore be repeated under more favorable
conditions, for the results are of fundamental importance in the analysis
of the mechanics of the response to electricity, as will be shown pres-
ently.

Mechanics of response.—The outstanding characteristics of the re-
sponses to the electric current of the colonial forms, exemplified in
Volvox, consist in momentary decrease in the action of the flagella, cor-
related with the direction and the density of the current and the nature
of the response to light. In photopositive colonies this occurs in such a
way that the flagellar activity decreases on the cathodal side after the
current is made. In photonegative colonies the activity decreases on the
anodal side, continues a few seconds, and then increases again. But if
the current is broken, it decreases on the opposite side, continues a few
seconds, then begins again. The extent of the region affected under all
conditions varies directly with the density of the current.

The action of the current must be due to movement of ions, parti-
cles, or fluid in the colonies or the surrounding solution, and to differ-
ences in the responses in photonegative and photopositive colonies to
differences in the effects produced by the movements of these substances.

330 MOTOR RESPONSES

There is considerable evidence which indicates that when colonies are
photopositive and cathopositive, they are negatively charged; and when
they are photonegative and anopositive they are positively charged
(Mast, 1927c). If this obtains, the movement of ions in photopositive
colonies in a direct current would result in decrease in the negative ions

it |

+ +
++ ++
>
+
+
>~
>~
+
++
>

>
+t ++
'

a
>
>

Figure 118. Diagrams illustrating the effect of direct current on the distribution of
ions in colonies of Volvox and their response. —, negative charge on colony; +, positive;
o, no charge; A, anode; K, cathode; large arrows, beam of light; small arrows, direction
of movement of colonies in the beam of light; curved arrows, direction the colonies turn
after the current is made and broken respectively. Note that there is in negatively charged
colonies, after the circuit is closed, a decrease in potential at the cathode, and a decrease
at the anode after it is opened, that the opposite obtains for positively charged colonies,
and that there is no change in potential in neutral colonies. (Modified after Mast, 1927b.)

on the cathodal side. In turn, this would cause decrease in potential and
increase in permeability here. It would also result in increase in the
negative ions at the anodal side and increase in potential and a decrease
in permeability there (Fig. 118). The movement of ions in photonega-
tive colonies would result in increase in positive ions, in the potential,
and increase in permeability on the cathodal side. The reverse would

MOTOR RESPONSES 3)

occur on the anodal side. The ion movement in photoneutral colonies
would result in increase in positive ions, and increase in potential and
decrease in permeability on the cathodal side. On the anodal side there
would be equivalent increase in negative tons and increase in potential
and decrease in permeability. After the current 1s broken, the change
in distribution of ions, and its effect, would be precisely opposite in all
respects) :( Figs 99))r

If the decrease in flagellar activity is due to local decrease in polariza-
tion and increase in permeability, it accounts for the observed direction
of movement of photopositive and photonegative colonies in a direct
current. And if the decrease in flagellar activity is correlated with rate
of change in these characteristics, it also accounts-for the fact that the
decreased activity induced by making or breaking the current continues
only a few seconds, for the change in polarization and permeability
undoubtedly lasts but a few seconds.

How does the fact that strong currents cause decrease in the activity
of the flagella simultaneously on all sides of the colonies, harmonize with
this view?

It is well known that a galvanic current will produce cytolysis if it is
strong enough, and that cytolysis is associated with increase in perme-
ability and decrease in polarization. It is therefore not difficult to see
that such a current could cause decrease in polarization simultaneously
on all sides.

It is probable, however, that the processes involved in galvanic stimula-
tion, resulting in orientation, are not the same in all organisms. For
example, Ludloff (1895) and Statkewitsch (1903) found that in a
galvanic current the fluid in the body of Paramecium is carried endosmoti-
cally toward the surface, on which the stroke of the cilia reverses, 1.e.,
in the specimens which swim forward toward the cathode, there is, if
the current is strong enough, contraction at the anode and expansion
at the cathode end. In Volvox precisely the opposite obtains. In the one,
stimulation appears to be associated with increase, in the other, with de-
crease in water content. In both, however, as pointed out above, it ap-
pears to be associated with decrease in polarization.

In general, it may be said that a galvanic current usually induces in
the lower organisms chemical and physical changes which differ at op-

332 MOTOR RESPONSES

posite sides, the one facing the anode, the other the cathode; and that
either or both of these two sets of changes may result in what is usually
called a response, although not necessarily an orienting response. Thus
while the motor responses in these organisms usually occur at the catho-
dal side when the circuit is closed, Kihne (1864), Verworn (1895),
and McClendon (1911) observed in Amoeba and other rhizopods con-
traction at the anodal side. In some of these organisms, the anodal con-
traction appears to be involved in streaming toward the cathode; but
there are other anodal responses which obviously have nothing to do with
locomotion. For example, Loeb and Budgett (1897) assert that there is, in
Amblystoma, copious secretion of mucus on the anodal side. Moore
(1926) obtained bioluminescence and contraction on the anodal side of
the Ctenophores, Mvemiopsis, and Berde. Lyon (1923) and Lund and
Logan (1925) observed, in Noctdluca, a sort of contraction first at the
anodal side and later also at the cathodal side, and sometimes the reverse.

In all of the organisms referred to above, except Noctiluca, the
anodal responses differ radically from the cathodal responses. This is
very evident from the results of observations on Amoeba, in which it
can be clearly seen that, after the circuit is closed, there is first local
liquefaction of the plasmagel on the cathodal side, then contraction,
and finally cytolysis on the anodal side. In Vo/vox, however, the response
on the cathodal side in colonies which are positive to the cathode, is, in
all perceptible characteristics, precisely the same as the response on the
anodal side in those which are positive to the anode. Here, then, is an
actual reversal in the action of the current, i.e., an actual reversal of
Pfliger’s law. Closing the circuit apparently produces the same effect
on the anodal side of colonies in certain physiological states as it does
on the cathodal side of colonies in other physiological states.

These physiological states are, as set forth above, specifically associated
with those involved in reversal in the direction of photic orientation. As
demonstrated above, these are dependent upon illumination, tempera-
ture, and chemicals in the environment, which apparently control the
electric charge carried by the colonies and the bodies within them. What
is much needed now is a more comprehensive study of these charges,
in relation to the chemical and the physical content of the environment
as well as the character of the responses of the colonies.

MOTOR RESPONSES 333

RESPONSES TO CHEMICALS
A. RHIZOPODS

None of the rhizopods except Amoeba has been studied with refer-
ence to motor responses to chemicals. Many of the observations made
on Amoeba are so indefinite that further work under more carefully con-
trolled conditions is highly desirable. In this work the large form,
Pelomyxa carolinensis, which can now be readily procured, would doubt-
less be very favorable.

Using a capillary pipette, Edwards (1923) applied various chemicals
locally to the surface of active specimens of A. proteus. With a few of
these chemicals he obtained fairly definite results, which lead to the
following conclusions:

If an alkali comes in contact with the side of an active amoeba, stream-
ing stops and a local protuberance is formed at the point of contact. If
the solution is weak, the protuberance develops into a normal pseudopod,
which continues indefinitely toward the source of the solution. If it is
strong, the protuberance breaks at the tip and the central portion of the
amoeba flows out, leaving nothing but a crumpled membrane.

If an acid is applied, streaming stops and a similar protuberance is
formed but it does not become large and does not develop into a nor-
mal pseudopod. If the acid is weak, the streaming soon begins again
and the protuberance gradually disappears. If it is strong, the protuber-
ance is very small and pseudopods form on the region opposite the point
of application.

If acid is applied after a rupture in the surface of an amoeba has been
produced by local application of an alkali and after the central portion
begins to flow out, the flow immediately stops and the amoeba soon
proceeds normally. These and other facts show that alkalies cause the
cytoplasm of Amoeba to solate and that strong acids cause it to gelate;
but what is involved in the formation of a protuberance by weak acid
at the region of application is not clear.

A strong solution of sodium chloride results in formation of pseudo-
pods opposite the region of application; a weak solution results only in
the formation of a protuberance in the region to which it is applied.
Thus Amoeba is negative to a solution of this salt if it is strong, and
positive if it is weak. This indicates that the former induces gelation of

334 MOTOR RESPONSES

the cytoplasm, and the latter, solation. Whether or not this obtains for
other salts has not been ascertained.

Strong alcohol produces a blister at the point of application, followed
by formation of pseudopods on the opposite surface.

The effect of all these substances is correlated with the kind of chem1-

220

Gel/Sol Ratio

/40

Rate of Locomotion (Mu/min)

Figure 119. The relation between rate of locomotion, gel/sol ratio, and hydrogen-ion
concentration in a balanced salt solution. A, Ai, rate of locomotion and gel/sol ratio in
solutions containing salts in concentrations given in Table 2; B, B,, rate of locomotion
and gel/sol ratio in salts in concentration five times as great as those given in Table 2.
Curve A, mean rates of movement of images of amoebae given in Table 2; B, means
of the rates of movement of seventeen to twenty-four individuals for from five to seven
minutes each in each hydrogen-ion concentration; Ai and B,, mean gel/sol ratios cal-
culated from results obtained in measurements made on seventeen to twenty-four indi-
viduals in each hydrogen-ion concentration as described above. (After Pitts and Mast,

1933.)

cals, with their concentration in the medium surrounding the amoebae,
and with the length of exposure to them.

If these conclusions are valid, one would expect amoebae in a culture
medium to aggregate in regions which are alkaline or which contain
relatively little salt. Hopkins (1928) made some observations concern-
ing the former. He put specimens into a drop of solution which was

MOTOR RESPONSES 335

pH 7.1 and then joined this drop with other drops which were respec-
tively pH 6 and 8. He found that the amoebae at the border between
the drops formed pseudopods which protruded toward the drop added,
no matter whether it was alkaline or acid. This indicates that amoebae in

Rate of Locomotion (Nu/min)

0/2°Y /0G//99

Figure 120. The relation between rate of locomotion, gel/sol ratio, hydrogen-ion con-
centration, and sodium-ion concentration. Each of the curves represents a series of ex-
periments conducted at constant sodium-ion concentration. The four solid curves are
based on the measurement of the rate of locomotion of an average of 14.8 different
specimens for an average of 88.1 minutes in each hydrogen-ion concentration tested.
The two broken curves are based on the measurement of the gel/sol ratio in an average
of 19 different individuals in each hydrogen-ion concentration tested. (After Pitts and
Mast, 1934b.)

a neutral solution tend to aggregate, either in acid or alkaline regions.
The observations made were, however, not extensive enough to war-
rant any definite conclusions. There is, then, nothing definite known
concerning the relation between differences in the chemical composition
of the medium and aggregation in any of the rhizopods, and but little
concerning the action of chemicals in relation to changes in direction of

336 MOTOR RESPONSES

movement. On the other hand, there are some very definite results con-
cerning the relation between the rate of locomotion and the chemicals in
the surrounding medium, especially hydrogen ions.

Rate of locomotion and H-ion concentration —Hopkins (1928) ob-
served that A. protews, in an ordinary hay culture fluid, is inactive if the
fluid is neutral, but active if the fluid is either acid or alkaline, and that
the rate of locomotion is maximum and nearly equal at about pH 6.5

Gel/Sol Ratio

a ae
Od :
at Te
001 MN
005
62 68 74 60
Al

Rate of Locomotion (Mu/min)

30 I6

Figure 121. The relation between rate of locomotion, gel/sol ratio, hydrogen-ion con-
centration, and calcium-ion concentration. Curve ©, mean rate of locomotion in 0.001 M,
based on measurements on an average of 22.1 different specimens for 11.6 minutes in
each hydrogen-ion concentration tested; curve @, mean rate of locomotion in 0.005 M,
based on an average of 18.7 different specimens for a total average of 96.6 minutes in
each hydrogen-ion concentration tested; curve @, mean gel/sol ratio in 0.001 M, based
on measurements on an average of 21.3 different specimens in each hydrogen-ion con-
centration tested; curve ©), mean gel/sol ratio in 0.005 M, based on measurements on
a total average of 14.6 different specimens in each hydrogen-ion concentration tested.
(After Pitts and Mast, 1934b.)

and pH 8 respectively. Mast and Prosser (1932) confirmed these results.
Pitts and Mast (1933), in a much more thorough study on A. proteus,
demonstrated that the inactivity at neutrality is correlated with the rela-
tion between the amount of sodium or potassium and calcium present;
and that the relation between activity and the concentration of hydrogen
ions varies greatly with the kind, the concentration, and the proportion
of salts in the surrounding medium. Some of the results obtained in this
study are presented in figures 119, 120, 121, and 122. These figures
show the following:

MOTOR RESPONSES 357

In a balanced salt solution the activity is minimum at neutrality and
maximum on either side; but the activity at any given hydrogen-ion
concentration varies with the salt concentration (Fig. 119). In sodium
or calcium salt solutions (Table 2) the maximum rate of locomotion
is nearly as high as in a balanced salt solution. Furthermore, the rate at

Rate of Locomotion(u/min)
ONeY /OC//25

a7 53 49 pH 65 7 77

Figure 122. The effect of adding calcium in different concentrations to 0.005 N sodium
solutions, on the relation between hydrogen-ion concentration, rate of locomotion, and
gel/sol ratio. The solid curve for the solutions containing calcium 0.001 M is based
on measurements of the rate of locomotion, on an average of 14.75 different specimens
for a total average of 89.9 minutes; that for calcium 0.0005 M, on an average of 18.6
different specimens for a total average of 95 minutes; and that for calcium 0.001 M, on
an average of 20.5 different specimens for a total average of 104.6 minutes in each
hydrogen-ion concentration tested. The broken curve for the solution containing cal-
cium 0.001 M is based on measurement of the gel/sol ratio, on an average of 19.8
different specimens; that for calcium 0.0005 M, on an average of 17.9 different speci-
mens; and that for calcium 0.0001 M, on an average of twenty different specimens in
each hydrogen-ion concentration tested. (After Pitts and Mast, 1934c.)

any given hydrogen-ion concentration varies with the salt concentration,
although the relation between the rate and the hydrogen-ion concen-
tration in the one differs greatly from that in the other, and there is no
indication of inactivity at neutrality in either (Figs. 120, 121). If cal-
cium salt is added to a solution of sodium salt, the activity decreases
greatly at neutrality (Fig. 122). The ratio between the amount of plas-
magel and the amount of plasmasol in Amoeba varies with the hydro-
gen-ion concentration; but the rate of locomotion is not specifically cor-
related with this ratio.

338 MOTOR RESPONSES

TABLE 2: RATE OF LOCOMOTION OF A. PROTEUS IN SODIUM AND
CALCIUM SALT SOLUTION*

Composition of the solution used to ascertain the effect of hydrogen-ion concentration
on rate of locomotion and gel/sol ratio in a balanced salt solution. To obtain different
hydrogen-ion concentrations, the acid and the alkaline components were mixed in dif-
ferent proportions.

Note that the concentration of Na, K, Ca, and Mg remains constant, no matter what
the proportion of the two components is.

In the second series of experiments the concentration of all the salts was increased
five times (after Pitts and Mast, 1933).

Acid Component Alkaline Component Molar Ratio
NaH:PO, 0.00150N NaOH 0.00150N Na 60
KH2PO, 0.00010N KOH 0.00010N K 4
CaH4(POs.)2 0.00010N Ca(OH), 0.00010N (Gat Dy
MgCl. 0.00005N MgCl, 0.00005N Mg I

* After Pitts and Mast, 1933, by permission of the Journal of Cellular and Comparative Physiology,
Wistar Institute of Anatomy and Biology.

Mechanics of response —Concerning the action of chemicals produc-
ing these responses on Amoeba, Pitts and Mast make the following state-
ments:

It is obvious that a substance in the environment may influence processes
which occur in a cell either by entering the cell and acting directly on
substances in the cell, or by acting on the surface of the cell in such a way
as to retard or facilitate the passage into or out of the cell of other sub-
stances which, owing to their presence or absence, induce alterations in
internal processes. . . .

It may be assumed, then, either (1) that the gel/sol ratio depends upon
the entrance of salts into the cell and reaction between these and internal
substances, and that the rate of entrance of salts varies with the hydrogen
ion concentration; or (2) that the gel/sol ratio varies with the rate of en-
trance of hydrogen and hydroxyl ions into the cells and reaction between
these and internal substances, and that the rate of entrance of these ions
varies with the concentration of the salts and the hydrogen ions; or (3) that
the gel/sol ratio depends upon the exit of substances from the cell, e.g.,
water, and that this depends upon the hydrogen ion concentration, and the
salt concentration and the kinds of salts present. Let us now attempt to
ascertain if the processes in question are in accord with any of these groups
of assumptions. .

If they are in accord with the first of these assumptions, the gel/sol
ratio must vary directly or indirectly with the amount of salt that enters the
cell and this must vary directly or indirectly with the hydrogen ion con-

MOTOR RESPONSES be)

centration. If it varies indirectly with the amount of salt that enters and
this varies either directly or indirectly with the hydrogen ion concentra-
tion practically none of the results obtained are in accord with the assump-
tions. If it varies directly with the amount of salt that enters and this varies
directly with the hydrogen ion concentration the results obtained with
balanced salt solutions and some others are in full accord with the assump-
tions; but the assumption that the entrance of salts varies directly with
hydrogen ion concentration is not in harmony with the results obtained by
practically all who have investigated this problem. Moreover, the assump-
tions do not account for the independence or inverse variation between hydro-
gen ion concentration and gel/sol ratio in calcium solutions. . . .

If they are in accord with the second of the three assumptions made above,
the gel/sol ratio must depend upon the hydrogen ion concentration with-
in the cell and this must vary with the concentration of the salts and the
hydrogen ions in the surrounding medium. If it varies indirectly with the
hydrogen ion concentration of the surrounding medium and directly or
indirectly with the salt concentration, few if any of the results obtained
are in accord with the assumptions. If it varies directly with the hydrogen
ion concentration and the salt concentration of the surrounding medium,
the results obtained with balanced salt solutions and some others are in ac-
cord with the assumptions, but those obtained with calcium solutions are
not. There is, moreover, no evidence which indicates that the hydrogen ton
concentration within Amoeba varies appreciably with variation in the hydro-
gen ion and the salt concentrations of the surrounding medium (Chambers,
1928)... |

In accord with the third group of assumptions, the gel/sol ratio must vary
with the rate of exit of substances from the cell, and this must vary d1-
rectly or indirectly with the salt and the hydrogen ion concentration, and it
must also vary with the kind of salts present in the environment. Without
entering upon a detailed analysis of the correlation between these assump-
tions and the results under consideration, it is evident that, no matter what
combination is selected, there are between them and the results inconsist-
encies of the same nature as those presented above. .

It is consequently obvious that the results in hand cannot be consistently
explained by any one of the three groups of assumptions made, and that
there must be a fairly complicated interaction between the various factors
involved. If this is true, the statement, without qualification, that any given
factor facilitates gelation or solation is obviously so incomplete that it is
without value. This conclusion is in full harmony with that reached by
Mast and Prosser (1932). ...

In reference to the relations between rate of locomotion and kind of
salts, salt concentration and hydrogen ion concentration, the results ob-
tained are, on the basis of any one of the groups of assumptions considered

340 MOTOR RESPONSES

above, even less explicable than are those in reference to the gel/sol ratio.
The outstanding difficulty here concerns the remarkable decrease in rate
of locomotion as neutrality is approached either from the acid or from
the alkaline side. The results presented show that this decrease occurs in
balanced solutions; that is that it is specifically correlated with the Na/Ca
ratio; that the higher this ratio within the limits of the concentrations
tested, the greater the decrease; and that it does not occur in solutions con-
taining only one salt... .

The results show that if calcium is added to a solution containing only
sodium salts, the rate of locomotion in the alkaline range increases greatly,
with but little change in the acid range and in the region of neutrality; and
that if sodium is added to solutions containing calcium salts, the rate de-
creases greatly in the region of neutrality, with but little change else-
wheres: 2)

The questions now arise as to why addition of calcium to solutions con-
taining only sodium salts causes great increase in rate of locomotion in the
alkaline range, and why addition of sodium to solutions containing only
calcium salts causes great decrease in the rate in the region of neutrality.

Similar questions have arisen in reference to the bimodal curves obtained
by a number of other investigators in plotting the rate of various physiological
processes against hydrogen ion concentration, e.g., by Robbins (1926) and
Farr (1928) im various processes in plants; by Ephrussi and Neukomm
(1927) in the resistance to heat in the eggs of a sea urchin; by Hopkins
(1928) in the rate of locomotion in Amoeba; by Mast (1928) in the rate of
assumption of stellate forms in Amoeba; by Eisenberg-Hamburg (1929) in
the rate of increase in water content in infusoria; by Chalkley (1929) in
water content and gel/sol ratio in Amoeba; and (1930a, 1930b) thermal
death rate in Paramecium, by Chase and Glaser (1930) in rate of locomo-
tion in Paramecium, and by Mast and Prosser (1932) in rate of locomotion
in Amoeba. ...

Only a few of these investigators attempted to elucidate the phenomenon.
Mast and Prosser (1932), as previously stated, concluded that it is cor-
related with salt concentration. We have already considered this view.
Robbins (1926) contends that the hydrogen ion concentration, at which the
median minimum in the plant processes studied occurs, coincides with the
isoelectric point of the principal proteins in the plant. Farr (1928) main-
tains, however, that this view is not tenable. He found in observations on
the relation between hydrogen ion concentration and rate of growth in root
hairs of collards that the hydrogen ion concentration at which the median
minimum occurs varies greatly with salt concentration and he concludes that
it therefore cannot be specifically correlated with the isoelectric point of
any given protein in the organism... .

In reference to Amoeba, the constancy of a median minimum rate of

MOTOR RESPONSES 341

locomotion at pH 7.0 in various solutions indicates some fixity of mechanism
determining this median minimum. This mechanism might be correlated
with the behavior of the membrane in the neighborhood of an ampholyte
isoelectric point near neutrality in accord with the view of Robbins (1926).
But arguing against such an isoelectric point are the pertinent facts that,
(1) the median minimum is lacking in solutions of single salts, though
locomotion in the dilute solutions occurs at hydrogen ion concentrations in
which it is usually found; (2) the difference between the maximum and
the median minimum rate of locomotion depends on the sodium/calcium
ratio and only slightly if at all on the total salt concentration; (3) the
cations have marked effect on the rate of locomotion on the acid side of
the neutral point as well as on the alkaline side; (4) the anions (so far
as chloride and phosphate are concerned) have little effect down as far
in the acid range as the observations were made... .

We are at present unable to suggest a satisfactory explanation for this
median minimum. Whatever the cause of it may be, the relation between
the rate of locomotion in Amoeba and the factors in its environment is doubt-
less fairly complex, for it is probable that these factors influence locomotion
in it by their action on the surface, affecting adhesiveness and other prop-
erties of the surface, as well as by their action on permeability of the sur-
face membrane (Mast, 1926a).

B. MARINE AMOEBAE '

Pantin (1923-31) made observations on the relation between the rate
of locomotion in a marine amoeba and various chemicals. He found that
as the hydrogen-ion concentration increases, the rate increases rapidly
from zero at pH 10 to a maximum at about pH 8, and then decreases
rapidly to zero at pH 5.5. He observed no indication of decrease in
activity at neutrality. He maintains that the rate is closely correlated with
the relative concentration of sodium, potassium, magnesium, and cal-
cium salts, that more than one of these salts is necessary for locomotion,
and that calcium is required in all combinations. He maintains that cal-
cium functions primarily in the contractile mechanism, and the others in
the regulation of permeability. The evidence presented in support of
these conclusions is, however, not unequivocal.

Oxygen is necessary for locomotion in Amoeba (Hulpieu, 1930; Pan-
tin, 1930) but only in very low concentrations. Pantin maintains that
none is immediately necessary, and that it functions in recovery some-
what as it does in the contraction of muscles. This, however, has not been
demonstrated.

342 MOTOR RESPONSES

Ce GILTARES

The most prominent response of the ciliates to chemicals consists in
reversal in the direction of the effective stroke of the locomotor cilia,
and consequent backward swimming. This reversal may be so brief that _
it results in scarcely perceptible backward movement, or it may continue
for several minutes. It has been investigated in some detail in Paramecium,
but not in any of the other ciliates. The rate of locomotion in all the
ciliates is doubtless correlated with the chemical composition of the
surrounding medium, but no measurements concerning this correlation
have been made in any of them.

The reversal in the direction of the stroke of the cilia of paramecia
in response to chemicals extends to the entire surface of the body, ex-
cept the oral groove. It consequently results in backward swimming. It
is the same as the reversal induced by contact and by rapid changes in
temperature or osmotic concentration, and it is usually followed by turn-
ing toward the aboral surface and forward movement in a new direction,
i.e., it usually constitutes the first stage in the avoiding reaction.

Jennings (1906) describes in detail how this response results in aggre-
gation of paramecia in regions which contain ineffective chemicals in re-
lation to those in adjoining regions, e.g., regions which are slightly
acid, surrounded by regions which are slightly alkaline. If this obtains,
he says, paramecia do not respond as they enter the acid region, but do
when they reach the edge of this region and are about to enter the alkaline
region. They consequently remain in the acid region. As more paramecia
enter, owing to random movements, an aggregation is formed.

He maintains that these responses usually result in aggregations in re-
gions which are favorable for the organisms, but that there are exceptions.
That is, he holds that these responses are, in general, adaptive. However,
he has made no suggestions concerning the processes in the organism in-
volved in producing these responses.

Merton (1923) found that sodium and potassium salts induce reversal
in Stentor and that calcium and magnesium do not, but he offers no
suggestions concerning the nature of the action of the former nor the
cause of inaction of the latter.

Mast and Nadler (1926) ascertained the effect of fifty-six different
chemicals on the direction of the effective stroke of the cilia in Parame-
cium. They maintain that all of the monovalent cation salts and hydrates

MOTOR RESPONSES 343

tested (thirty-one), except (NH,),SO, and NH,C,H,0.,, induce rever-
sal; but that none of the bivalent and trivalent cation salts tested (nine-
teen), except CaHPO, and MgHPO,, induce it. Also that Ba(OH).,,
H,PO, and H,C.O, induce reversal, while HCL and lactose do not.

These authors contend that the duration of reversed action is closely
correlated with the concentration of the salts; that it varies with the kind
of salt at any given concentration; and that bivalent and trivalent cation
salts neutralize the effect of monovalent salts.

Concerning the physiological processes involved in reversal in ciliary
action, they make the following statement:

Copeland (1919, 1922) and Grave and Schmitt (1925) demonstrated
that the cilia in higher forms are closely connected with nerve fibers and that
their action is in all probability controlled by nerve impulses. The results
obtained in the investigations of Yocum (1918), Taylor (1920), Rees
(1922), Visscher (1927) and others on the neuromotor apparatus in the
protozoa, indicate that the action of the cilia in these forms is similarly con-
trolled. If this is true, the question arises as to how environmental changes
which have been observed to induce reversal in ciliary action influence
the neuromotor apparatus. This may be conceived to be either through
chemical changes produced in the receptors or elsewhere in the organism
or through changes produced in the electric potential at the surface, or in
the permeability and the consistency of the surface layer. .

The results obtained by Mast and Nadler indicate that reversal in ciliary
action is largely dependent upon the cations, that it is induced by monovalent
but usually not by bi- and tri-valent cation salts, and that it depends upon the
concentration of the salts... .

It is well known that adsorption of the cations is usually relatively greater
in bi- and tri-valent than in monovalent cation salt solutions and that the
adsorption varies with the concentration of the salts in the solution. This
seems to indicate that the difference in the action of the monovalent and
the bi- and tri-valent cation salts and the difference in the action of different
concentrations of the monovalent cation salts is at least in part associated
with differential adsorption of the ions in the various solutions, resulting
in changes in the electrical potential at the surface of the paramecia, which
directly or indirectly produce impulses in the neuromotor apparatus which
pass to the cilia and influence their action. The facts, however, that Ba(OH),
produces reversal while BaCl,, CaCl,, MgCl, do not, that H,C,O, and
H,PO, produce reversal while HCI does not, that CaHPO, and MgHPO,
produce reversal while Ca,(PO,), and Mg(PO,), do not, all indicate
that there must be other factors involved in reversal aside from differential
adsorption of the cations.

344 MOTOR RESPONSES

Oliphant (1938) made a much more extensive and thoroughly con-
trolled study of the effect of monovalent and bivalent cation salts on
ciliary reversal in Paramecium.

The results obtained support the contentions of Mast and Nadler
(1926) that the monovalent salts induce reversal, whereas bivalent ca-
tion salts do not, and that the duration of the reversed action varies
with the kind and the concentration of the salts; but they do not support
their contentions as to the nature of this variation. The results show that
the effect of the salts is due primarily to the action of the cations, and
that the anions have little, if any, effect. They show that the order of
effectiveness of the cations is K>Li>Na>NH,, and that the duration
of their effect varies inversely with the temperature.

Oliphant (1938) cites work which indicates that in Paramecium,
Amoeba, Actinosphaerium, Spirogyra, root hair of Trianea, and eggs of
Arbacia, monovalent cations induce increase, and that bivalent cations
decrease the viscosity of the cytoplasm (Spek, 1921; Heilbrunn, 1923,
1931, Cholodnyj, 1923; Weber, 1924). He concludes that this indicates
that reversal in ciliary action is correlated with increase in viscosity of
the cytoplasm, and contends that this conclusion is supported by the fact
that “reversal in response to temperature occurs only at temperatures
almost immediately lethal,” i.e., at temperatures which cause marked
increase in viscosity. He holds that the action of the cilia is controlled
by the neuromotor apparatus and that increase in viscosity produces im-
pulses in this structure which cause reversal in ciliary action, but he thinks
that changes in electric potential, in permeability, in the consistency of
the surface layer, or in the chemical composition of the receptors or
other structures in the organism may be involved.

It is obvious from the above discussion that there is still much to
be learned concerning the processes involved in the responses of the
ciliates to chemicals.

LITERATURE CITED

Alsup, Fred W. 1939. Relation between the responses of Amoeba proteus
to alternating electric current and sudden illumination. Physiol. Zool.,
12; 35-95.

Alverdes, F. 1923. Der Sondercharakter der von den Ciliaten gezeigten Gal- .
vanotaxis. Pfliiger’s Archiv ges. Physiol., 198: 513-42.

MOTOR RESPONSES 345

Bancroft, F. W. 1906. The control of galvanotropism in Paramecium by
chemical substances. Univ. Cal. Publ. Physiol., 3: 21-31.

—— 1907. The mechanism of the galvanotropic orientation in Volvox. J. exp.
Zool., 4: 157-63.

—— 1913. Heliotropism, differential sensibility, and galvanotropism in
Euglena. J. exp. Zool., 15: 383-428.

Bayliss, W. M. 1920. The properties of colloidal systems. IV. Reversible
gelation in living protoplasm. Proc. roy. Soc., B, 91: 196-201.

Buder, J. 1917. Zur Kenntniss der phototaktischen Richtungsbewegungen.
Jb. wiss. Bot., 58: 105-220.

Carlgren, O. 1899. Uber die Einwirkung des konstanten galvanischen Stromes
auf niedere Organismen. Arch. Anat. Physiol. Lpz., Physiol. Abth.,
49-76.

Chalkley, H. W. 1929. Changes in water content in Amoeba in relation to
changes in its protoplasmic structure. Physiol. Zodl., 2: 535-74.

——1930a. Resistance of Paramecium to heat as affected by changes in hydro-
gen ion concentration and in inorganic salt balance in the surrounding
medium. Pub. Hlth. Rep., Wash., 45: 481-89.

—— 1930b. On the relation between resistance to heat and the mechanism of
death in Paramecium. Physiol. Zodl., 3: 425-40.

Chambers, R. 1928. Intracellular hydrion concentration studies. I. The relation
of the environment to the pH of the cytoplasm and of its inclusion bodies.
Biol. Bull., 55: 369-76.

— Chambers, R., and P. Reznikoff. 1926. Micrurgical studies in cell physiol-
ogy. I. The action of the chlorides of Na, K, Ca, and Mg on the proto-
plasm of Amoeba proteus. J. gen. Physiol., 8: 369-401.

Chase, A., and O. Glazer. 1930. Forward movement of Paramecium as a func-
tion of the hydrogen-ion concentration. J. gen. Physiol., 13: 627-36.
Cholodnyj, N. 1923. Zur Frage uber die Beeinflussung des Protoplasmus
durch mono und bivalente Metallionen. Beih. Bot. Zbl., 39: 231.
Coehn, A., and W. Barratt. 1905. Uber Galvanotaxis vom Standpunkte des

physikalischen Chemie. Z. allg. Physiol., 5: 1-9.

Copeland, M. 1919. Locomotion in two species of the gastropod genus
Alectrion with observations on the behavior of pedal cilia. Biol. Bull.,
37: 126-38.

—— 1922. Ciliary and muscular locomotion in the gastropod genus Polznices.
Biol. Bull., 42: 132-42.

Davenport, C. B. 1897. Experimental Morphology. Vol. 1, New York, 280 pp.

De Candolle, A. P. 1832. Physiologie vegetale. Paris.

Dixon, H. H., and T. A. Bennet-Clark. 1927. Responses of plant tissues to
electric currents. Sci. Proc. R. Dublin Soc., 18: 351-72.

Edwards, J. G. 1923. The effect of chemicals on locomotion in Amoeba. J.
exp. Zool., 38: 1-43.

346 MOTOR RESPONSES

Eisenberg-Hamburg, E. 1929. Recherches comparatives sur le fonctionnement
de la vacuole pulsatile chez les infusoires parasites de la grenouille et
chez les infusions d’eau douce. Influence de la pression osmotique, des
electrolytes et du pH. Arch. Protistenk., 78: 251-70.

Engelmann, T. W. 1869. Beitrige zur Physiologie des Protoplasm. Pflug. Arch.
ges. Physiol., 2: 307-22.

—— 1879. Uber Reizung contraktilen Protoplasmus durch plotzliche Beleuch-
tung. Pflug. Arch. ges. Physiol., 19: 1-7.

—— 1882. Uber Licht- und Farbenperception niederster Organismen. Pflug.
Arch. ges. Physiol., 29: 387-400.

Ephrussi, Boris, and Alex Neukomm. 1927. Resistance a la chaleur des oeufs
d’oursin (Paracentrotus lividus, Lk). Protoplasma, 2: 34-44.

Farr, C. H. 1928. Studies on the growth of root hairs in solutions. IV. The
pH molar rate relation for collards in calctum chloride. Amer. J. Bot., 15:
6-31.

Folger, H. T. 1925. A quantitative study of reactions to light in Amoeba. J.
exp. Zool., 41: 261-91.

Grave, C., and F. O. Schmitt. 1925. A mechanism for the codrdination and
regulation of ciliary movement as revealed by microdissection and cyto-
logical studies of ciliated cells in mollusks. J. Morph., 40: 479-513.

Greeley, A. W. 1904. Experiments on the physical structure of the protoplasm
of Paramecium and its relation to the reactions of the organism to thermal,
chemical and electrical stimuli. Biol. Bull., 7: 3-32.

Habenicht, Wilhelm. 1935. Die Wirkung des galvanischen Stromes auf den
Eiweisszylinder nach Du Bois-Reymond. Protoplasma, 22: 321-36.
Hahnert, W. F. 1932. A quantitative study of reactions to electricity in Amoeba

proteus. Physiol. Zool., 5: 491-526.

Harrington, N. R., and E. Leaming. 1900. The reactions of Amoeba to light
of different colors. Amer. J. Physiol., 3: 9-16.

Heilbrunn, L. V. 1923. The colloid chemistry of protoplasm. I. General con-
siderations: IH. The electrical charges of protoplasm. Amer. J. Physiol.,
64: 481.

Heilbrunn, L. V., and K. Daugherty. 1931. The action of sodium, potassium,
calcium, and magnesium on the protoplasm of Amoeba dubia. Physiol.
Zodl., 4: 635.

Holmes, S. J. 1903. Phototaxis in Volvox. Biol. Bull., 4: 319-26.

Hopkins, D. L. 1928. The effects of certain physical and chemical factors on
locomotion and other life processes in Amoeba proteus. J. Morph., 45:
97-119.

— 1929. The effects of the substratum, divalent and monovalent cations on
locomotion in Amoeba proteus. J. Morph., 48: 371-83.

—— 1938. The vacuoles and vacuolar activity in the marine Amoeba, Flabel-
lula mira Schaeffer and the nature of the neutral red system in Protozoa.
Biodyn., No. 34: 1-22.

MOTOR RESPONSES 347

Hulpieu, H. R. 1930. The effect of oxygen on Amoeba proteus. J. exp. Zool.,
56: 321-61.

Inman, O. L., W. T. Bovie, and C. E. Barr. 1926. The reversal of physiologi-
cal dominance in Amoeba by ultraviolet light. J. exp. Zool., 43: 475-84.

Jennings, H. S. 1899. Studies on reactions to stimuli in unicellular organisms.
Il. The mechanism of the motor reactions of Paramecium. Amer. J.
Physiol., 2: 311-41.

—— 1904. Contributions to the study of the behavior of lower organisms.

Publ. Carneg. Instn., No. 16: 256 pp.

1906. Behavior of the Lower Organisms. New York, 366 pp.

Kamada, T. 1928-31. Control of galvanotropism in Paramecium. J. Fac. Sa.
Tokyo Univ., 2: 295 123239) 285-307.

——— 1934. Some observations on potential differences across the ectoplasm
membrane of Paramecium. J. exp. Biol., 11: 94-102.

Kinosita, H. 1936. Electric stimulation of Paramecium. Jour. Fac. Sci. Tokyo
Univ., 4: 137-94.

—— 1938a. Electric stimulation of Spirostomum. Jour. Fac. Sci. Tokyo Univ.,
D2 71-105,

—— 1938b. Electric stimulation of Paramecium with two successive subliminal

current pulses. J. cell. comp. Physiol., 12: 103-17.

1939. Electrical stimulation of Paramecium with linearly increasing cur-

rent. J. cell. comp. Physiol., 13: 253-61.

Koehler, O. 1925. Galvanotaxis. Handb. d. Norm. u. Pathol. Physiol., 11:
1027-49.

Kiihne, W’. 1864. Untersuchungen iiber das Protoplasma und die Contractilitat.
Leipzig, 158 S.

Laurens, H., and H. D. Hooker, 1920. Studies on the relative physiological
value of spectral lights. II. The sensibility of Volvox to wave-lengths of
equal energy content. J. exp. Zool., 30: 345-68.

Lillie, R. S. 1923. Protoplasmic Action and Nervous Action. Chicago, 417 pp.

Loeb, J. 1890. Der Heliotropismus der Tiere und seine Uebereinstimmung
mit dem Heliotropismus der Pflanzen. Wurzburg, 118 pp.

— 1906. The Dynamics of Living Matter. New York, 233 pp.

Loeb, J., and S. P. Budgett. 1897. Zur Theorie des Galvanotropismus. IV.
Mitt. Uber die Ausscheidung elektropositiver Ionen an der ausseren
Anodenflache protoplasmitischer Gebilde als Ursache der Abweichungen
vom Pfliigerschen Erregungsgesetz. Pfliig. Arch. ges. Physiol., 66: 518-34.

Loeb, J., and S. S. Maxwell. 1910. Further proof of the identity of helio-
tropism in animals and plants. Univ. Cal. Publ. Physiol., 3: 195-97.

Lucas, K. 1910. An analysis of changes and differences in the excitatory process
of muscles and nerves based on the physical theory of excitation. J.
Physiol., 40: 225-49.

Luce, R. H. 1926. Orientation to the electric current and to light in Amoeba.
Anat. Rec., 32: 55.

348 MOTOR RESPONSES

Ludloff, K. 1895. Untersuchungen uber den Galvanotropismus. Pflig. Arch.
ges. Physiol., 59: 525-54.

Lund, E. J., and G. A. Logan. 1925. The relation of the stability of protoplas-
mic films in Noctéluca to the duration and intensity of an applied elec-
tric potential. J. gen. Physiol., 7: 461-71.

Luntz, A. 1931-32. Untersuchungen uber die Phototaxis. Z. vergl. Physiol. I,
14: 68-92; II, 15: 652-78; III, 16: 204-17.

Lyon, E. P. 1923. Effects of Electricity on Noctiluca. Proc. Soc. exp. Biol. N.Y.,
20: 284-85.

McClendon, J. F. 1910. On the dynamics of cell division. I. The electric
charge on colloids in living cells in the root tips of plants. Archiv. f.
Entw. mech. d. Organismen. 31: 80-90.

McClendon, J. J. 1911. Ein Versuch amdboide Bewegung als Folgeerscheinung
wechselnden elektrischen Polarisationszustandes der Plasmahaut zu
erklaren. Pflig. Arch. ges. Physiol., 140: 271-80.

Marsland, D. A., and D. E. S. Brown. 1936. Amoeboid movement at high
hydrostatic pressure. J. cell. comp. Physiol., 8: 167-78.

Mast, S. O. 1906. Light reactions in lower organisms. I; Stentor coeruleus. J.

exp. Zool., 3: 359-99.

1907. Light reactions in lower organisms. II. Volvox. J. comp. Neurol.,

ti 99-180:

—— 1910. Reactions in Amoeba to light, J. exp. Zool., 9: 265-77.

—— 1911. Light and the Behavior of Organisms. New York, 410 pp.

—— 1912. The reactions of the flagellate Peranema. J. Anim. Behav., 2:
91-97.

—— 1914. Orientation in Evglena with some remarks on tropisms. Biol. Zbl.,
34: 641-74.

—— 1916. The process of orientation in the colonial organism, Gonimm
pectorale, and a study of the structure and function of the eyespot. J. exp.
Zoole:202 1-172

—— 1917. The relation between spectral color and stimulation in the lower
organisms. J. exp. Zool., 22: 471-528.

—— 1918. Effects of chemicals on reversion in orientation to light in the

colonial form, Spondylomorum quaternarium. J. exp. Zool., 26: 503-20.

1919. Reversion in the sense of orientation to light in the colonial form,

Volvox globator and Pandorina morum. J. exp. Zool., 27: 367-90.

— 1923. Photic orientation in insects with special reference to the drone-
fly, Eristalis tenax and the robber fly, Erax rufibarbis. J. exp. Zool., 38:
109-205.

—— 1926a. Structure, movement, locomotion and stimulation in Amoeba. J.
Morph., 41: 347-425.

—— 1926b. Reactions to light in Volvox, with special reference to the proc-
ess of orientation. Z. vergl. Physiol., 4: 637-58.

MOTOR RESPONSES 349

—— 1927a. Structure and function of the eyespot in unicellular and colonial
organisms. Arch. Protistenk., 60: 197-220.

—— 1927b. Reversal in photic orientation in Volvox and the nature of photic
stimulation. Z. vergl. Physiol., 5: 730-38.

—— 1927c. Response to electricity in Volvox and the nature of galvanic
stimulation, Z. vergl. Physiol., 5: 740-61.

—— 1928. Factors involved in changes in form in Amoeba. J. exp. Zool., 51:
97-120.

—— 1931a. The nature of response to light in Amoeba proteus (Leidy). Z.
vergl. Physiol., 15: 139-47.

—— 1931b. The nature of the action of electricity in producing response and
injury in Amoeba proteus (Leidy) and the effect of electricity on the
viscosity of protoplasm. Z. vergl. Physiol., 15: 309-28.

—— 1931c. Movement and response in D/fflvgia with special reference to the
nature of cytoplasmic contraction. Biol. Bull., 61: 223-41.

—— 1932a. Localized stimulation, transmission of impulses, and the nature
of response in Amoeba. Physiol. Zodl., 5: 1-15.

—— 1932b. The rate of adaptation to light and to darkness in Volvox glo-
bator. Z. vergl. Physiol., 17: 644-58.

—— 1936. Motor responses to light in the invertebrate animals. Biological
Effects of Radiation (ed. by B. M. Dugger). New York. Pp. 573-623.

—— 1939. The relation between kind of food, growth, and structure in
Amoeba. Biol. Bull. 77: 391-98.

Mast, S. O., and Mary Gover. 1922. Relation between intensity of light and
rate of locomotion in Phacus pleuronectes and Euglena gracilis and its
bearing on orientation. Biol. Bull. 43: 203-9.

Mast, S. O., and Brainard Hawk. 1936. Response to light in Peranema
tricho phorum. 1. Relation between dark-adaptation and sensitivity to
light. Biol. Bull. 70: 408-12.

Mast, S. O., and H. R. Hulpieu. 1930. Variation in the response to light in
Amoeba proteus with special reference to the effects of salts and hydrogen
ion concentration. Protoplasma, 11: 412-31.

Mast, S. O., and P. L. Johnson. 1932. Orientation in light from two sources
and its bearing on the function of the eyespot. Z. vergl. Physiol., 16:
252-74.

Mast, S. O., and J. E. Nadler. 1926. Reversal a ciliary action in Paramecium
caudatum. J. Morph., 43: 105-17.

Mast, S. O., and C. L. Prosser. 1932. Effect of temperature, salts, and hydrogen
ion contentrition on rupture of the plasmagel sheet, rate of locomotion,
and gel/sol ratio in Amoeba proteus. J. cell. comp. Physiol., 1: 333-54.

Mast, S. O., and N. Stahler. 1937. The relation between luminous intensity,
adaptation to light, and rate of locomotion in Amoeba proteus (Leidy).
Biol. Bull., 73: 126-33.

350 MOTOR RESPENSES

Merton, H. 1923. Studien tiber Flimmerbewegung. Pflug. Arch. ges. Physiol.,
198: 1-28.

Moore, A. R. 1926. Galvanic stimulation of luminescence in Pelagia noctiluca.
J. gen. Physiol., 9: 375-79.

Nernst, W. 1899. Zur Theorie der electrischen Reizung. Nachr. v. d. Kgl.
Gesell. Wiss. Gottingen, Math. physik. KI., 104-8.

Oliphant, J. F. 1938. The effect of chemicals and temperature on reversal in
ciliary action in Paramecium. Physiol. Zodl., 11: 19-30.

Ostwald, W. 1890. Elektrische Eigenschaften halbdurchlassiger Scheidewande.
Z; phys. Chem=63\ 71-82.

Pantin, C. F. A. 1923-31. On the physiology of amoeboid movement. J. Mar.
piols AssmiUigiew3> 24-69-SBrit) exp: Biol., e519). "3: aye oe
8: 365-78; Proc. roy. Soc., 105:-538-79.

Pearl, R. 1900. Studies on electrotaxis. I. On the reactions of certain infusoria
to the electric current. Amer. J. Physiol., 4: 96-123.

——— 1901. Studies on the effects of electricity on organisms. II. The reactions
of Hydra to the constant current. A. Jour. Physiol., 5: 301-20.

Pitts, R. F., and S. O. Mast. 1933. The relation between inorganic salt concen-
tration, hydrogen ion concentration and physiological processes in
Amoeba proteus, 1. Rate of locomotion, gel/sol ratio and hydrogen-ion
concentration in balanced salt solutions. J. cell. comp. Physiol., 3: 449-62.

— 1934a. II. Rate of locomotion, gel/sol ratio and hydrogen ion concentra-
tion in solutions of single salts. J. cell. comp. Physiol., 4: 237-56.

—— 1934b. III. The interaction between salts (antagonism) in relation to
hydrogen-ion concentration and salt concentration. J. cell. comp. Physiol.,
4: 435-55.

Ray, J. 1693. Historia Plantarum, 2: 985-1944. London.

Rees, C. W. 1922. The neuromotor apparatus of Paramecium. Univ. Cal. Publ.
Zool., 20: 333-64.

Robbins, W. J. 1926. The isoelectric point for plant tissues and its importance
in absorption and toxicity. Univ. Mo. Stud., 1: 3-60.

Schaeffer, A. A. 1917. Reactions of Amoeba to light and the effect of light
on feeding. Biol. Bull. 32: 45-74.

Shettles, L. B. 1937. Response to light in Peranema trichophorum with special
reference to dark-adaptation and light-adaptation. J. exp. Zool., 77:
215-49.

— 1938. Effect of ultraviolet light and x-rays on Peranema tricho phorum.
J. cell. comp. Physiol., 12: 263-72.

Spek, J. 1921. Der Einfluss der Salze auf die Plasmokolloide von Actinosphae-
rium Eichhorni. Acta Zool. Stock., 2: 153.

Statkewitsch, P. 1903. Uber die Wirkung der Induktionsschlige auf einige
Ciliata, Physiol. russe, 3: 1-55.

— 1907. Galvanotropismus and Galvanotaxis der Ciliata. Z. allg. Physiol.,
6: 13-43.

MOTOR RESPONSES 351

Strasburger, E. 1878. Wirkung des Lichtes und der Warme auf Schwarm-
sporen. Jena. Z. Naturw., n. f., 12: 551-625.

Taylor, C. V. 1920. Demonstration of the function of the neuromotor ap-
paratus in Evplotes, by the method of microdissection. Univ. Cal. Publ.
Zool., 19: 403-470.

—— 1925. Cataphoresis of ultramicropic particles in protoplasm. Proc. Soc.
exp. Biol. N.Y722:153336,

Terry, O. P. 1906. Galvanotropism of Volvox. Amer. J. Physiol., 15: 235-43.

Verworn, M. 1889. Psycho-physiologische Protistenstudien. Jena, 218 pp.

1895. Allgemeine Physiologie. Jena, 615 pp.

Visscher, J. P. 1927. A neuromotor apparatus in the ciliate Dileptus gigas.
J. Morph. and Physiol., 44: 373-81.

Wager, H. 1900. On the eyespot and flagellum of Ezglena viridis. J. linn. Soc.
(Zool.), 27: 463-81.

Weber, E. B., and I. Weber. 1922. Reversible Viskositatserhohung des Cyto-
plasmas unter der Einwirkung des elektrischen Stromes. Ber. dtsch. bot.
Ges., 40: 254-58.

Weber, F. 1924. Plasmolyseform und Protoplasmaviskositat. Ost. Bot. Z., 73:
261.

Yocum, H. B. 1918. The neuromotor apparatus of Ezplotes patella. Univ. Cal.
Publ. Zool., 18: 337-96.

CHAPTER VI

RESPIRATORY METABOLISM
THEODORE LOUIS JAHN

STUDIES OF THE RESPIRATION of the Protozoa have, for the most part,
been fragmentary, and our information on the subject resembles an ac-
cumulation of a number of more or less isolated data, rather than a
unified body of knowledge. This situation is due to a variety of causes,
chief among which is probably the fact that most studies of respiration
have been made by physiologists who chose, among the members of the
animal kingdom, the organisms which seemed to be the most suitable
for a particular type of experiment. From this viewpoint the study of
protozoan respiration has suffered a severe handicap, in that a consider-
able mass of protozoan protoplasm, free from bacteria and other organ-
isms, has not always been easy to obtain, and in that our methods for
measuring very small rates of respiratory exchange have not been nearly
as accurate or as convenient as we might desire. However, with gradual
technical advances, it seems probable that in the near future we shall see
the development of an organized account of protozoan respiration, and
it also appears probable that this development will take place among
investigators who are primarily interested in the Protozoa. Therefore,
it seems advisable to combine a review of data on protozoan respiration
with a discussion of the general problems of respiratory metabolism in
other biological materials, and to outline for the student not thoroughly
trained in the lore of respirometry some of the purposes, methods, and
possible interpretations of such a study.

Among the Protozoa, the intake of oxygen does not require compli-
cated respiratory mechanisms. Apparently diffusion, high rate of water
exchange, and protoplasmic movements (cyclosis, amoeboid streaming,
and ‘‘metabolic’” movements) are sufficient to maintain a suitable level
of O, tension in the protoplasm and to prevent the accumulation of toxic
amounts of CO,. The mechanisms which are responsible for protoplasmic
movements and the high rate of water exchange are more properly treated

RESPIRATORY METABOLISM 353

under the subjects of movement, permeability, and excretion. Therefore
the problem of respiratory metabolism of a protozoan organism, at least
for the present discussion, is easily resolved into a problem comparable
to that of cellular respiration in the Metazoa.

PURPOSES OF STUDYING RESPIRATION

One of the first questions to be considered is: “What can be learned
by studying respiration?’ The measurement of gaseous exchange is not
an end in itself, but it is a tool which, when used singly or in combina-
tion with other tools, may help us to obtain answers to the following
types of questions.

1. What is the rate of energy expenditure of the organism? How does
the metabolic rate (basal and otherwise) of one species compare with
that of another? How does it change during starvation? Or conjugation?
Is this rate dependent upon the O, or CO, tension of the environment?
How is it affected by narcotics? How does it vary with temperature? Or
with other chemical and physical factors of the normal environment?

2. What is the source of this energy? Is it obtained by oxidation of
fats, carbohydrates, or proteins? Or by anaérobic oxido-reductions? Can
this source be shifted by changing the chemical or physical environment
of the organism? What are the intermediate products formed during
oxidation of the substrate? Is oxidation of the substrate complete (1.e.,
to CO, and H,O), or may these intermediate products be excreted by
the cell?

3. What is the mechanism by which energy is obtained from the sub-
states? Is this brought about through the intermediary action of dehydro-
genase, cytochrome, and Warburg’s respiratory enzyme? Or is it brought
about through dehydrogenase and reversible oxidation-reduction systems,
such as yellow pigment or pyocyanine? Or by glutathione? Can these
substances be replaced by artificial oxidation-reduction systems? Can a
shift in this mechanism of respiration be induced by changing the avail-
able substrate? How is this mechanism related to the degree of anaérobio-
sis which the organisms can endure? Is the organism capable of synthesiz-
ing the respiratory enzymes from simple substances, or must they be
obtained from complex outside sources, i.e., from vitamins?

Measurements of the respiratory metabolism of an organism may be
used as indices of the rate at which it uses energy, the substrate from

354 RESPIRATORY METABOLISM

which this energy is derived, and the mechanism by which it ts obtained
from the substrate. In addition to these factors, there is a possibility of
a direct relationship between certain metabolic processes and the patho-
genicity of parasitic forms.

METHODS OF MEASURING AEROBIC RESPIRATION

Methods that have been or might be used for measurement of aérobic
protozoan respiration fall naturally into two groups—those applicable

TABLE 3: SENSITIVITY OF RESPIROMETERS

Nearest Unit to Approximate Sensitivity
Type of Respirometer Which Meniscus in Terms of Scale
Can Be Read* Divisions

Standard Warburg

(Warburg, 1926) o.2 mm. I mm.=1.0-2.0 mm.’ O;
Microsemidifferential

(Duryée, 1936) 9-2 mm. Imm.=o.5 mm.’ Oz
Microdifferential

(Fenn, 1928) 9:1 mm. I mm.=o.3 mm.3 Og
Microdifferential

(Described in text) 9:2 mm. I mm.=o.2 mm.3 Op,
Straight capillary tubes |

(Howland and Bernstein, 1931) oor mm. 0.01 mm.=o.oo1 mm.3 Oy
Straight capillary tubes in closed air

chamber

(Gerard and Hartline, 1934) 9006 mm. 0.006 mm.=0.0013 mm. Op
Microdifferential

(Schmitt, 1933) : micron I micron=0.0005 mm.? Oy
Cartesian diver

(Needham and Boell, 1938) 5.2 mm. I mm.=o.oo1 mm.’ Og

* It is assumed that the meniscus is read to the nearest 0.2 mm., unless otherwise stated by the
authors. In those cases where the author claims greater reading accuracy, the smallest unit of change
which can be detected is given in this column. If stability is adequate, the sensitivity of several models
may be considerably increased by the use of special reading devices.

to concentrated suspensions of organisms, and those applicable to a few
or to single cells. For concentrated suspensions, titration, gas analysis,
and standard manometer methods have been used; and for studies of

RESPIRATORY METABOLISM 595

single protozoan cells, micromanometric methods have been devised
(Kalmus, 1927; Howland and Bernstein, 1931). More recently, still
better micromanometric methods have come into existence, but these have
not yet been applied to the respiration of protozoa. Table 3 gives the
sensitivity of various types of manometers, some of which have not yet
been used to measure the respiration of Protozoa. It should be remem-
bered that whenever a respirometer is made more sensitive to changes
in gas volume produced by organisms, it simultaneously becomes more
sensitive to slight changes produced by the environment (thermal and
barometric effects) and to inaccuracies arising from imperfect design
and construction (ground-glass connections and stopcocks, surface phe-
nomena at the meniscus of manometer fluid, inaccuracies of capillary bore
and so forth). Therefore, stability of the apparatus, on which final ac-
curacy must depend, becomes more and more difficult to obtain. For that
reason the most sensitive types should be reserved solely for those prob-
lems in which concentrated suspensions are undesirable or unobtainable.
By comparison of Tables 3 and 4, it should be possible to determine ap-
proximately the type of respirometer necessary for any one of a variety
of problems.

1.TITRATION METHODS

a. Dissolved O, determinations. For any aquatic animal it is possible
to measure O, consumption by placing the organisms in a closed chamber
filled with water of known O, content and by measuring the amount of
O, left after a definite period of time. For this purpose a modified Win-
kler titration method is usually used (Standard Methods of Water Anal)-
sis, 1936). Special precautions are necessary whenever the animals can-
not be removed from the solution, or if iron is present. This method has
been used by Lund (1918a, 1918b, 1918c) and Leichsenring (1925)
on Paramecium and Col poda.

b. Measurement of CO, production. Production of CO, may be
measured by placing the organisms in a small amount of solution in a
small open container. This is placed inside of a larger closed container
in which an alkali, preferably Ba(OH),, is present. The CO, given off
by the organisms is absorbed by the alkali, which can then be titrated
with acid in the presence of an indicator. This method was recom-
mended by Lund (1918d) for use with Paramecium.

356 RESPIRATORY METABOLISM

The above titration methods are applicable only to quite large num-
bers of organisms. It seems possible that these methods could be im-
proved by the use of accurately controllable microburettes, smaller vol-
umes of liquid, and so forth, but it is doubtful whether they could
be made as accurate and reliable as some of the manometric procedures
discussed below. Also, respiratory quotients are not easily obtained by
titration methods. i

2. GAS ANALYSIS

Soule (1925), Amberson (1928), and Root (1930) have applied the
standard Haldane-Henderson methods of gas analysis to respiration of
the Protozoa. These methods are adequate for use with rather con-
centrated suspensions and pressures, and possess certain definite ad-
vantages when O, or especially CO, tension is being varied experimen-
tally and would have to be determined separately if manometric methods
were used. Whenever gases other than CO, are evolved by an organism,
gas analysis seems to be the only satisfactory method of measurement.
The details of gas analysis methods are discussed by Peters and van
Slyke (1932). Soule (1925), for studies of the metabolism of Lessh-
mania tropica and Trypanosoma lewisi, used gas analysis, supplemented
by readings of an insensitive manometer, the purpose of which was
principally to indicate when gas exchange was taking place (method
described in detail by Novy, Roehm, and Soule, 1925).

3. STANDARD MANOMETRIC METHODS

The principle of the manometric method is somewhat as follows:
the organisms, in a suitable immersion medium, are placed in a closed
flask large enough so that a considerable air space is present. The flask
is connected to a capillary manometer tube partially filled with a liquid.
Alkali may be present in a separate small container inside of the flask.
If so, then CO, is absorbed, and the amount of O, consumed may be
measured by means of the movement of fluid in the manometer tube
as changes in volume (Haldane, Thunberg, Winterstein, Duryée, and
Dixon types), or in pressure at a given volume (Warburg), or as the
resultant of simultaneous changes in both (Barcroft differential type).
Manometric methods, although very simple in outline, are filled with
pitfalls for the inexperienced investigator, and a careful reading of the

RESPIRATORY METABOLISM Soy),

excellent treatise of Dixon (1934) is recommended. In this publication
the theory and the more common forms of the apparatus are described
in detail.

The Barcroft differential type can be made sufficiently small and
sensitive for the study of respiration of Protozoa, when moderately con-
centrated mass cultures are available. A manometer designed by Dr.
T. C. Evans (modified from that of Bodine and Orr, 1925), which
has been in use in this laboratory for some time, seems to be quite
suitable. It resembles the standard Barcroft (Dixon, 1934, p. 37) ex-
cept that the two stopcocks are replaced by one double stopcock, which
insures the simultaneous opening and closing of both flasks. The cups
may be relatively small (about 5 cc.), and the U-shaped portion of
the capillary (0.3 mm. bore) is placed at an angle of about twenty
degrees from the horizontal. This design combines a high degree of
sensitivity with ruggedness and dependability. The Duryée (1936) modi-
fication of the Thunberg-Winterstein principle and the Fenn (1928)
form of microdifferential respirometer also seem to have a sensitivity
adequate for moderately concentrated suspensions. Schmitt (1933) has
devised an extremely sensitive form of microdifferential manometer, in
which the gain in sensitivity and accuracy is due chiefly to an elaborate
reading device and a system of temperature control which is stated to
make meniscus movements of as little as one micron both detectable and
significant.

4, CAPILLARY MANOMETER

The use of capillary tubes for measuring respiration of single protozoan
cells was introduced by Kalmus (1927, 1928a). This method was
improved by Howland and Bernstein (1931), who by means of a mictro-
injection device drew small amounts of oil, air, KOH solution, and
water containing an animal into small capillary tubes, so that they were
finally arranged in the following order: oil, KOH, air, animal in water,
oil. As the animal consumed O,, the distance between the oil-water and
the oil-KOH interfaces decreased. This change was measured mictro-
scopically by means of an ocular micrometer and a calibrated mechanical
stage. By using control tubes made in a similar manner but without an
animal, it was possible to correct for slight movements due to thermo-
barometric changes and to osmotic differences between the water and

358 RESPIRATORY METABOLISM

KOH. Gerard and Hartline (1934) have improved the method by
enclosing the tubes in an air tight chamber to eliminate barometric dis-
turbances, and by using a screw micrometer to increase accuracy of
reading.

5. CARTESIAN DIVER ULTRAMICROMANOMETER (NEEDHAM AND BOELL,
1938)

This is an application of the principle of the Cartesian diver for use
as a constant-volume manometer. The ‘‘diver’” chambers are constructed
from capillary tubing and consist of a bulb partially filled with gas,
an open capillary neck, and a solid glass tail to ensure that the diver
floats upright. The diver is placed in a closed chamber partially filled
with a strong salt solution, the specific gravity of which is such that the
diver maintains a position below the surface of the salt solution, and
that a small amount of this solution enters the neck. If the amount of
gas in the diver is changed by a reaction, more salt solution will be
drawn in or forced out of the neck, the specific gravity of the diver
will change, and the level of flotation will also change. By changing
the pressure on the salt solution, the diver may be brought back to any
given level. Therefore, the diver may serve as a constant-volume ma-
nometer, and changes in the amount of gas in the chamber may be calcu-
lated from the changes in the external pressure which are necessary to
maintain the diver at a definite level. This application of the Cartesian
diver was suggested by Linderstrom-Lang (1937), and has been used
for parts of amphibian embryos by Needham and Boell (1938), who
describe the use of this instrument for measurement of O, consumption,
anaérobic glycosis, and respiratory quotient. From Table 3 it may be
noted that this instrument when read only to 0.2 mm. has a sensitivity
as great as those which employ special reading devices, and that it
therefore has the possibility of being made more sensitive.

AEROBIC RESPIRATION

1. THE NORMAL RATE OF RESPIRATION

One fundamental essential in measuring the respiration of any bio-
logical material is that other material, also capable of respiratory ac-
tivity, be absent or very well controlled. This means that bacteria must be
absent, or at least must contribute only a negligible amount to the

RESPIRATORY METABOLISM 359

measured respiration. Various workers have tried removing bacteria
by washing and filtering, or have corrected the figures for O, consump-
tion by running controls of bacteria without Protozoa. Data obtained
by these methods are extremely difficult for a reviewer to evaluate
critically, and usually one may either accept them at face value until
they can be checked with bacteria-free cultures or ignore them entirely.
In the present discussion the tendency has been to accept all data in
which the magnitude of the error is not obviously large, and to point
out possible difficulties involved. In view of the fact that some bacteria
have a respiratory rate per gram many times that of other types of cells
(the rate for Azotobacter is stated by Burk [1937] to be equivalent to
that of a 200-pound man consuming one ton of glucose per hour), the
present viewpoint may be considered far from conservative. In some
cases (e.g., the papers on cyanide insensitivity of Paramecium) the data
on Protozoa seem quite adequate to prove the principal conclusions of
the author, but are not accurate enough to afford detailed comparisons
of respiratory rate. In such cases only the main points (e.g., insensitivity
to cyanide) are given serious consideration. In only a few cases have
investigators used bacteria-free cultures for measurement of respiration
(cf. Table 4).

For comparative purposes in work with metazoan tissues, it is custom-
ary to express oxygen consumption in cubic millimeters (at normal tem-
perature and pressure denoted as N.T.P.) per hour per milligram of dry
weight of the tissue (symbolized by Qo,). In the protozoan literature,
where the rate of O, consumption is expressed in absolute units, this unit
is sometimes the Qo, but is more usually mm® per hour per organism,
principally because the counting of organisms is simpler than measuring
dry weight. Some authors use the symbol Qo, for O, consumption per
1,000,000 or per 100,000,000 organisms (e.g., von Fenyvessy and
Reiner, 1928; Hall, 1938), but it seems preferable to avoid confusion
by retaining this symbol for its original meaning and using a new symbol
for consumption per 1,000,000 organisms, perhaps Qo, as used in Table
4. However, since not dry weight, nor wet weight, nor number of or-
ganisms affords the possibility of comparing the oxygen consumption per
unit of respiring protoplasm, these discrepancies are not as important as
one might at first suppose. In the case of flagellates such as Astasia and
Chilomonas, in which a high percentage of the weight may be in the

360 RESPIRATORY METABOLISM

form of carbohydrate reserves, either dry or wet weight would be a poor
index of the amount of respiring protoplasm. If one wishes to compare
the rate per unit of protoplasm, the best index is probably the nitrogen
content, because protein and other nitrogen-containing compounds are
not ordinarily stored as reserve food. However, in the case of Protozoa
which secrete nitrogen-containing tests, this criterion might also be
very poor. Therefore any comparison of the absolute rate of different
species, even after all differences in technique, immersion fluid, and
physiological condition of the animal have been overcome, must usually
be made with reservations, or at least with an adequate understanding of
the limitations involved. Some authors have chosen to calculate O,
consumption in terms of cubic millimeters of organisms, but the errors in-
herent in the methods of packing the animals for measurement (usually
centrifuging) or in calculating volume from linear dimensions are too
great to allow close comparison of data for different types of organisms.
However, for an extended series of experiments on the same or very
similar organisms, the use of dry weight (A. Lwoff, 1933) or of volume
measurements (Elliott, 1939) seems to be entirely satisfactory.

If concentrated suspensions of organisms are used, the rate of shaking
should be carefully controlled. The importance of this factor is demon-
strated by the data of Hall (1938). If ammonia is produced by an
organism, it is necessary to maintain acid within the respiring chamber
in order to obtain true values of O, consumption. This very important
procedure is discussed by Specht (1935).

Another question which arises in expressing results in absolute form
is that of measuring basal metabolism, i.e., the metabolism of rest. In a
mammal, for instance, there are certain well-defined limitations of condi-
tions under which O, consumption may be termed a measurement of
basal metabolism. In a protozoan it is more difficult, if at all possible,
to apply these criteria, and in all known measurements we have a sum
of the total metabolic processes, i.e., of those to which we refer as “basal,”
those due to movement of the organism, and, if the medium is nutrient,
those due to the manufacture of reserve food material and to growth.
The energy expended for each of these purposes will probably vary
with the species, the physiological state, and the environmental condi-
tions.

If the metabolic substrates of an organism undergo complete oxida-

RESPIRATORY METABOLISM 361

tion, we are able, by means of measurements of O, consumption, to
determine directly the amount of energy available to the organism. If
oxidation is incomplete, a further knowledge of the oxidation products
is necessary. It is usually assumed, unless we have knowledge to the
contrary, that oxidation is complete in the a€robic Metazoa (cf. intes-
tinal nematodes, von Brand and Jahn, 1940). Among the bacteria and
also among the Protozoa this is not always true, even in the presence of
normal O, tension. However, since carbohydrate cleavage and intra-
molecular oxidation, even in the presence of O,, may be considered an
anaérobic process, that question will be discussed under ana€robiosis.

From studies on the heat of combustion, we know that complete
oxidation of glucose yields 677,000 calories per gram molecule, or about
3,700 calories per gram. Complete combustion of protein yields 5,700
calories per gram, and fats yield 8,000-9,000 calories per gram. There-
fore, if we know the substrate being oxidized and the rate at which O,
is consumed, we can calculate the energy made available by oxidation.
According to the equation

CHO: = GOs GiCO, | 6 H,O —- 677,000 cal.

one gram molecule of glucose requires six gram molecules of O,. The
volume of O, consumed (at N.T.P.) is 6 & 22.4 liters, or 134.4 liters.
The ratio of O, consumed to calories released is 134 liters/677,000
calories, or about one calorie for each 200 mm? of O, consumed. Similar
calculations may be made for fats and proteins.

Table 4 contains most of the known data for respiratory rates for
the Protozoa which can be expressed in absolute terms—either as mm*
O, per organism per hour, or mm’ O, per gram dry or wet weight per
hour. Similar tables are given by von Brand (1935) and Hall (1938).

2. THE EFFECT OF O, TENSION ON O, CONSUMPTION

For many types of biological material it has been quite well established
that, under usual experimental conditions, O, consumption is inde-
pendent of O, tension, within very wide limits (exceptions cited by
Tang, 1933, McCoy, 1935). Recently, however, Kempner (1936, 1937)
demonstrated that this is not true for several species of bacteria, for
human leucemic leucocytes, for red blood cells of man, fowl, and allt-
gator, and for pine needles if CO, is present or if the temperature is

362 RESPIRATORY METABOLISM

above 25° C. With these same materials in CO,-free alkaline media
below 25° C., O, consumption was independent of O, tension. The
effect of O, tension apparently varied with pH, CO, tension, salt con-

TABLE 4: MEASUREMENTS OF PROTOZOAN RESPIRATION

3
M*mi O) per aoe
Hour per Tem- Bac-
: as per mg. Dry ; :
Species Million Weizh perature, | teria- Investigator
eight
C free
Qu, Qo,
Paramecium caudatum 120 (CO) De No | Barratt (1905)
140 Tis No_ | Lund (1918c)
2,250 Re No | Zweibaum (1921)
3,900 19° No_ | Necheles (1924)
5,600 Dp No_ | Kalmus (1928b)
500 Mtg ae No | Howland and Bern-
stein (1931)
Paramecium multimicro-
nucleatum THODT Dre No | Mast, Pace, and Mast
(1936)
Colpidium campylum 200 24.0° No | Pitts (1932)
112.5 19.8° Yes | Hall (1938)
Colpidium colpoda 200 17.0° No | Wachendorff (1912)
200 No | Peters (1929)
Colpoda sp. 600-1, 200] 19.7° No_ | Adolph (1929)
Glaucoma piriformis 35 22m Yes | M. Lwoff (1934)
Blepharisma undulans (0.5)* 20.8° No_ | Emerson (1929)
Spirostomum ambiguum 2,590 25.0° No_ | Specht (1935)
Strigomonas oncopelti 0.4 62 28.0° Yes | A. Lwoff (1933)
Strigomonas fasciculata 0.4 55 28.0° Yes | A. Lwoff (1933)
Leptomonas ctenocephalus 0.3 40 28.0° Yes | A. Lwoff (1933)
Trypanosoma equiperdum 0.05 37.0° Yes | Von Fenyvessy and
Reiner (1928)
Chilomonas paramecium 17-26 25.0° Yes | Mast, Pace, and Mast
(1936, 1937)
Astasia sp. 2, 400 Yes | Jay (1938)
Khawkinea halli 2,050 Yes | Jay (1938)
Actinosphaerium eichhornii, 1, 100 20.0° No | Howland and Bern-
stein (1931)
Amoeba proteus (0.2)* 20.0° No_ | Emerson (1929)

* Not Qoo but mm* O. per hour per mm%.

tent, and temperature. Since O, consumption of the yellow pigment of
respiration (see below) varies with O, tension, it would not be sur-
prising to find a similar relationship in organisms with this mechanism.
However, this is not always the case (Schlayer, 1936). Clarification of

RESPIRATORY METABOLISM 363

the relationship between O, tension and O, consumption in Protozoa
will apparently require much more data than is now available. A dis-
cussion of the theoretical relationship between oxygen tension and oxygen
consumption is given by Marsh (1935).

For Protozoa, the available evidence indicates that within wide limits
O, tension has little or no effect on the rate of O, consumption for
Paramecium and Col poda, and that it does have an effect on Spirostomum.
Lund (1918a) found that the rate of O, consumption for Paramecium
was independent of O, tension between 0.04 cc. and 2.2 cc. O, per
137 cc.—a 55-fold range. This was determined by placing thick sus-
pensions of Paramecium in stoppered bottles and measuring the dis-
solved O, content of the water by the Winkler method, until the animals
died. Lund’s conclusion was confirmed by Amberson (1928), who
placed the organisms in a closed vessel, in contact with an atmosphere
of known O, content. By gas analyses he demonstrated a uniform rate
of O, consumption, with O, partial pressures which varied from
50 to 220 mm. Hg, and only a slight decrease (about 20 percent) at
pressures as low as 11 mm. Hg. Adolph (1929) found that the O,
consumption of Colpoda did not vaty significantly with O, tension
between 155 and 750 mm. Hg. In a single experiment at 4-8 mm. Hg,
O, consumption decreased to 31 percent of its previous value. However,
Adolph did find that low O, tension (40 mm.) was correlated with
smaller size of the progeny of cultures. Specht (1935) measured the
respiration of Spirostomum in pure oxygen, in air, and in 0.5 percent
O, in N,. He found that O, consumption in these gases was in the
ratio of 151 to 100 to 71, and that CO, production was in the ratio of
175 to 100 to 70.

When considering the effect of low O, tensions on O, consumption
for any of the larger Protozoa, one should consider the O, tension at
various points within the organism as well as at the surface. This can
be calculated by the diffusion equations of Harvey (1928) and others,
on the assumption that the rates of cyclosis and water exchange are
low. The O, tension at the center of an ellipsoid which is consuming
O, uniformly throughout its substance, will be zero when the shortest
radius
Be

364 RESPIRATORY METABOLISM

where D is the diffusion coefficient of O,, c is the O, tension at the
surface, and A is its rate of O, consumption. In the case of a cylinder
(e.g., Spzrostomum) the factor 5 should be 4. For Colpoda, Adolph
(1929) calculated the value of “a” to be 148 y at atmospheric O,
tension, and 72 y at 40 mm. Hg partial pressure of O,. In large ciliates
this factor might be important at low O, tensions, even with a rather
low rate of O, consumption.

3. EFFECT OF CO, TENSION ON O, CONSUMPTION

Root (1930) showed that when CO, tension was raised from 1 mm.
Hg to 15-20 mm. Hg, the respiration of Paramecium increased slightly
(less than 15 percent), and it was believed that this increase might be
caused by increased activity of the organisms. As the CO, tension was
increased above 60 mm. Hg, O, consumption decreased continuously
to about 40-60 percent of the control when CO, tension reached 220-
360 mm. Hg. Similar experiments on fertilized Arbacia eggs did not
show increase at low concentrations, and all CO, tensions greater than
30 mm. Hg produced a decrease to less than 40 percent of normal.
Paramecium apparently was much more resistant to increase of CO,
tension than Arbacia. For both Paramecium and Arbacia only slight
effects were obtained with HCl, at pH values comparable to those
present during the CO, experiment (4.5 to 7.5 for Paramecium).

From the information available, it is not possible to determine the
mechanism of action of CO, on protozoan respiration, and results with
other organisms are few and variable. Apparently CO, is not involved
as an inhibitor or accelerator of any of the known mechanisms of
respiration (to be discussed below), and at present we can only say
that the effect on respiration seems to be indirect, and that the results
are not due to pH changes in the external fluid. However, internal pH
changes, as suggested by Root (1930), might account for the effect.
This is an explanation comparable to that given by Jahn (1936) for
the effect of the lack of CO, on growth of Chilomonas and bacteria.
It is well established that certain bacteria will not grow in the absence
of CO,. Jahn (1936) studied the effect of CO,-free media on growth
of Chilomonas and Colpidium and found a distinct inhibition with
Chilomonas and none with Colpidium. The explanation was offered
that the inhibition of growth might be caused by inadequate intracellular
buffering in those species which were affected by lack of CO,, and that

RESPIRATORY METABOLISM 365

organisms whose normal environment is high in CO, might depend
more on CO, buffering than those the normal environment of which
is low in CO,. (For possible application of this idea to culture of in-
testinal forms, see Jahn, 1934).

4, THE EFFECT OF THE PHYSIOLOGICAL STATE ON ©; CONSUMPTION

It seems as if the effect of various factors which influence the physio-
logical state of an organism may be reflected in measurements of O,
consumption. Factors which have been investigated for the Protozoa are
starvation, age of the culture, and conjugation.

Lund (1918c) starved Paramecium in tap water and noted an ap-
preciable decrease in respiration during the first twenty hours. This was
simultaneous with the disappearance of deutoplasmic food reserves from
the protoplasm. Upon feeding starved animals with boiled yeast sus-
pensions, the rate of oxygen consumption could be increased two to
three times. This increase was independent of cell division. Leichsenring
(1925) demonstrated a decrease of 23 percent after twenty-four hours of
starvation and 29 percent after seventy-two hours.

The effect of the age of the culture on O, consumption was first
studied by Wachendorff (1912), who found that for Colpidium colpoda
the O, consumed per organism diminished from 191 mm* per hour
the first day to 151 mm* the tenth day, and to 59 mm‘* on the thirtieth
day. Reidmuller (1936) reported a higher rate of O, consumption for
young cultures of Trichomonas foetus than for older cultures. Twenty-
four to forty-eight-hour-old cultures consumed 4.3 mm? O, per mm‘* of
organisms, sixty-hour cultures, 2.8 mm,? and three-day-old cultures only
0.81 mm* O,. Andrews and von Brand (1938) reported a decrease
in sugar consumption per organism for this species, with increasing age
of the culture, and their data indicate that the differences observed by
Reidmuller were real, in spite of objections to the method used be-
cause of the possibility of hydrogen or methane evolution.

Zweibaum (1921) studied the rate of O, consumption of Paramecium
caudatum in relation to conjugation. He found that the rate just before
conjugation was about 0.73 mm? O, per thousand organisms per hour.
During conjugation this rate rose to 3.4 mm*/1,000/hour, and immedi-
ately after conjugation decreased to about 0.73. During the first eight
or nine days following conjugation, the rate rose slowly to 2.0
mm?/1,000/hour, and remained at this value from four to five months.

366 RESPIRATORY METABOLISM

5. THE EFFECT OF TEMPERATURE ON © CONSUMPTION

Data concerning the effect of temperature on O, consumption are not
numerous, and in most cases are incomplete. Barratt (1905) determined
the CO, production of Paramecium at various temperatures and found
that the rate at 27°-30° C. was more than twice that at 15° C. Wachen-
dorff (1912) found that C. col/poda respired about four times as fast at
17° as at 7° C. Leichsenring (1925) demonstrated that Paramecium,
when transferred from a temperature of 20° C. to one of 35° C., showed
a respiratory increase of 35 percent, and that when transferred to a
temperature of 15°, 10°, 5°, 0° C. respiration was decreased 30 per-
cent, 34 percent, 50 percent, and 58 percent respectively. These effects
were not completely reversible. The data of Kalmus (1928b) showed
a Q,, value (temperature coefficient) of 1.5 for Paramecium respira-
tion between 23° and 32° C. A. Lwoff (1933) found a Q,, value of 2.1
between 13° and 23° C., and about 1.5 between 23° and 32° C. The
temperature characteristic (1 value) was 9,830 calories for the range 13°
to 34° C. —_ Lwoff also calculated a j value of 21,350 for the synthesis
of respiratory enzyme (oxidase, see below) by the organism between
18° and 31.5° C., and a value -of —52,000 between 31.5° and
SOME

6. THE EFFECT OF ANESTHETICS AND POISONS ON O, CONSUMPTION

The effect of various toxic agents (e.g., KCN, CO, N;H, arsenite,
urethanes, and so forth) which are supposed to exert a specific effect
on the normal functioning of certain respiratory enzymes, will be dis-
cussed in connection with the mechanism of respiration. The effect of
other anesthetics on respiration has not been extensively studied. Leich-
senring (1925) found that ethylene and nitrous oxide had no effect on
the respiration of Paramecium, and that ether and chloroform produced
decreases of as much as 25 percent (after two and one hours respec-
tively). The effect was reversible with ether, but not with chloroform
if the exposure was more than one-half hour. Co/poda was more sensitive
to these substances than Paramecium. Von Fenyvessy and Reiner (1928)
found no decrease in respiration of Trypanosoma equiperdum when one-
percent Germanin was added. This was surprising, in view of the fact
that Germanin is very toxic for trypanosomes 7” vivo.

RESPIRATORY METABOLISM 367

7. THE EFFECT OF NUTRITIVE SUBSTANCES AND OTHER MATERIALS

The effect of various nutritive substances on Protozoa has been demon-
strated by a number of investigators, but the criteria used are usually
growth or the accumulation of food reserves, and not respiration. In
some cases it is possible to determine that the substance is oxidized
directly (e.g., glucose), or that it contributes toward the synthesis of
the respiratory enzyme (A. Lwoff, 1933). These data will be dis-
cussed below. The effect of various substances on the respiration of
Paramecium was studied by Leichsenring (1925), who found that cap-
rine, glutamic acid, peptone, and aminoids increased respiration 12-18
percent; that glycocoll and succinic acid increased respiration 8-9 per-
cent; and that tyrosine and cystine produced little effect. Lactose gave
an increase of 16 percent, and other sugars and polysaccharides gave in-
creases of 3-10 percent. Thyroxin gave an increase of 13 percent. No
explanation was made of the mechanism of these effects.

Mast, Pace, and Mast (1936) found that Chilomonas grew well,
formed considerable starch but little fat, and consumed 0.17 mm* of O,
per 10,000 organisms per hour, in a solution of MgSO,, NH,Cl,
K,HPO,, Na-acetate, and silicon. When sulphur was omitted, the starch
remained constant, fat accumulated, O, consumption decreased, and the
animals finally died. When acetate was omitted, the organisms decreased
in size, starch and fat decreased, and O, consumption decreased to 0.07
mm?/10,000/hour. When both sulphur and acetate were omitted, starch
decreased to zero, fat accumulated, O, consumption decreased, and the
organisms died. These authors conclude that starch is normally changed
to fat, that sulphur induces oxidation of fat, thereby increasing respira-
tion and preventing accumulation of fat, and that fat oxidation is
probably associated with a cystine-cysteine mechanism (see glutathione,
below). Mast and Pace (1937) reported a 25 percent increase in res-
piration of Chilomonas when Na,SiO, was added to inorganic media.
This was supposedly caused by the catalytic action of Si on organic
syntheses.

8. EVOLUTION OF GASES OTHER THAN CO,

The possibility that Protozoa evolve gases other than CO, was first
shown by Cook (1932) for the flagellates of termites (Termopszs neva-
densis). A gas which was not absorbable by hydroxide was evolved by

368 RESPIRATORY METABOLISM

normal termites, under anaérobic conditions. However, this gas was not
evolved if the intestinal flagellates had been removed by oxygenation,
and it was suggested that it might have been evolved by the flagellates.
The evidence is incomplete, however, in that oxygenation probably also
changed the bacterial flora. Witte (1933) observed the production of
gas bubbles by Trichomonas foetus. This was confirmed by Andrews and
von Brand (1938), who found that this gas was not absorbable by
alkali and that when mixed with O, it burned with indications of ex-
plosiveness. Final identification was not made. The formation of gas
vacuoles has been reported for several organisms, but in most cases there
is little evidence regarding the identity of the gas. Bles (1929) believed
that the gas vacuoles of Arcella contained O,,.

The chlorophyll-bearing flagellates, of course, might give off O, in
the presence of strong light, because of photosynthesis, and this might
also be true of the ciliates which harbor zodchlorellae. There is con-
siderable indirect evidence that this is true, but no direct measurements
are available.

INVESTIGATIONS WHICH CONCERN THE SOURCE OF ENERGY

Whenever an oxidizable material is subjected to complete combustion,
the ratio of CO, given off to O, consumed will vary with the type of
material. This ratio (CO,/O,) is called the respiratory quotient (R.Q.).
Carbohydrates (relatively rich in oxygen) have an R.Q. of 1.0; fats
(relatively poor in oxygen) have an R.Q. of about 0.71; and proteins
have an R.Q. of 0.83 if the nitrogen is eliminated as urea, and 0.93 if it
is eliminated as ammonia. By measurements of the respiratory quotient
we may obtain an index of the type of material which is being oxidized
by the organism, at least as to whether it is predominantly carbohydrate
or predominantly fat. If the excreted nitrogenous material can be identi-
fied and measured, the amount of protein and consequently the amounts
of carbohydrates and fats consumed can be calculated. Under these con-
ditions the measurement of R.Q. becomes more significant.

These interpretations are based on the assumptions that complete
oxidation of metabolic substrates is the sole cause of gaseous exchange,
and that gases other than O, and CO, are not involved. If these assump-
tions are unsound, then any interpretation of the R.Q. is necessarily
more difficult. If carbohydrate is being converted into fat within the

RESPIRATORY METABOLISM 369

organism for purposes of storage, the R.Q. may reach a value of 1.4.
If fat or protein is being converted to carbohydrate, there will be a cor-
responding tendency for the R.Q. to be lowered. A high value for the
R.Q. can also arise whenever CO, is removed from a compound with-
out the consumption of O,, or whenever an oxygen debt accumulates
(during heavy exercise). Whenever an oxygen debt is being removed,
the R.Q. may fall to extremely low values (during rest after exercise).
Unusual values of R.Q. may be obtained if substrates other than carbo-
hydrates, fats, or proteins are being consumed. In the case of very rapid
protozoan growth on substrates of organic acids, the R.Q. should vary
with the oxygen content of the molecule being oxidized (acetic acid,
1.0; proprionic acid, 0.85; butyric acid, 0.80).

The respiratory quotient of an organism may be calculated with almost
any of the manometric methods described above, if separate measure-
ments are made with and without a CO,-absorbing alkali in the respira-
tory chamber. In this case, one set of readings (with KOH) will give a
measure of the O, consumed, and the other set (without KOH) will
be an index of the difference between CO, given off and O, consumed.
From this the R.Q. may be calculated, provided no NH, is evolved and
no CO, is retained in the immersion fluid. If these complications arise,
suitable modifications may be introduced. The use of somewhat more
complicated manometer chambers allows measurements of simultaneous
O, consumption and CO, production to be made on the same material
(Dixon, 1934). For the Protozoa this method seems preferable because,
in addition to its usual advantages, it prevents the results from being
affected by the possible secretion of ammonia (Specht, 1935) and
other bases (e.g., sodium carbonate from oxidation of Na-acetate, Jahn,
1935a). However, even this method does not correct for the possible
evolution of hydrogen or methane (cf. Trichomonas foetus, Andrews
and von Brand, 1938).

For the ciliates, several measurements of R.Q. have been made. Wa-
chendortf (1912) reported R.Q. values of about 0.3 for Colpidium, but
in view of later developments it seems as if this material should be re-
examined with more modern methods. Emerson (1929) studied the
respiration of Blepharisma undulans and found an R.Q. slightly less
than 1.0. Daniel (1931) obtained an R.Q. of 0.84 for Balantidium colt,
but the possible effects of bacteria were not well controlled. Amberson

370 RESPIRATORY METABOLISM “

(1928) reported an R.Q. of 0.69 for Paramecium, and Root (1930)
in a number of experiments obtained an R.Q. value of 0.62. Root also
found that the R.Q. varied somewhat irregularly with changes in CO,
tension. However, there was a definite trend toward high R.Q. values
in media of high CO, tension, and the average R.Q. at 238-423 mm.
Hg. was 1.43. This apparently was caused by a decrease in O, consump-
tion (see above) without a corresponding decrease in CO, production,
thereby giving a high R.Q. According to Root, “It is possible that the
suppression of oxidations under these conditions results in the produc-
tion of acid metabolites which drive out carbon dioxide from the bi-
carbonate contained in the cells and in the surrounding medium.” Similar
experiments on Arbacia eggs did not show an increase in R.Q., and it
was assumed either that acid substances were not produced or that they
were rapidly converted into a non-acid form and did not accumulate
in appreciable amounts. Apparently CO, tension is a factor which should
be considered when making measurements of R.Q. However, if ex-
periments are conducted with standard manometric techniques, this factor
is probably not important.

Specht (1935) measured the R.Q. of Sprrostomum in manometers,
both with and without the presence of acid in a side arm of the manom-
eter flask. He found an R.Q. of 0.24 without the acid and 0.84 when
acid was present. This discrepancy was explained as being caused by
the elimination of NH, by the organisms, and the value of 0.84 is
therefore accepted as more nearly correct. However, it was also demon-
strated that the R.Q. was 0.98 in an atmosphere of O,, and that this
value was not affected by the presence or absence of acid. Apparently
NH, is not produced at high O, tension. These experiments indicate
very clearly that ammonia secretion is a possible source of error in
measurements of protozoan respiratory quotients, and therefore one
should be suspicious of the validity of low R.Q. values unless adequate
precautions have been taken against the ammonia error.

For the free-living flagellates, very low values of R.Q. have been
reported. Jay (1938) found R.Q. values of 0.34 and 0.56 for Astasia
and Khawkinea, respectively. Mast, Pace, and Mast (1936) reported
R.Q. values of 0.28 to 0.37 for Chilomonas, and 0.72 for Paramecium
multimicronucleatum under similar conditions. The possible explana-
tions mentioned by Jay for the low R.Q. value are conversion of pro-

RESPIRATORY METABOLISM 371

tein to carbohydrate, or incomplete oxidation of carbohydrate. The ex-
planation of Mast ef a/. is that carbohydrate was being synthesized from
carbon dioxide. This explanation is based on previous nutritional studies,
but these are open to question (review, Hall, 1939). One obvious possi-
bility is that CO, may be retained in the immersion fluid, but Mast
and his coworkers obtained only a slightly higher R.Q. value when the
bound CO, was liberated by acid (single experiment only). In these
cases the explanations offered must be considered as only tentative, until
the possibilities of NH, production and CO, retention are positively
eliminated. Mast and his coworkers also reported for Chilomonas that
under certain conditions starch was converted to fat, and that fat oxida-
tion could be decreased by depriving the organism of sulphur. However,
values obtained for the R.Q. were variable and showed no definite cor-
relation with these conditions.

Values of the R.Q. reported for members of the family Trypano-
somidae are within the normal range. Soule (1925) obtained an R.Q.
of 0.84-0.91 for Lesshmania tropica and 0.74-0.89 for Trypanosama
Jewisi in blood agar medium. When glucose was present, the R.Q. rose
to 0.95 for L. tropica and 0.94 for T. lew7s7. Novy (1932) reported res-
piratory quotients of 0.93 to 1.0 for T. Jew7s7, L. tropica, L. donovani, L.
infantum, Strigomonas oncopelti, S. culicidarum, S. culicidarum var.
ano phelis, S. lygaeorium, S. media, S. muscidarum, and S. parva, when
grown on glucose-blood agar. When grown on glycerol-blood agar or
plain blood agar, the R.Q. was about 0.8 to 0.87 for the four species of
Leishmania. Von Fenyvessy and Reiner (1924) found an R.Q. of 0.60
for Trypanosoma equiperdum in diluted blood. A. Lwoff (1933) ob-
tained R.Q. values of 1.0 for Strigomonas oncopelti and S. fasciculata,
and a value of 0.88 for Leptomonas ctenoce phali.

Apparently the only R.Q. measurement on a rhizopod is that of
Emerson (1929) on Amoeba proteus, which gave a value slightly less
than 1.0.

Another method, in addition to that of the respiratory quotient, which
might be used as an index of the source of energy in an organism is
the calorific quotient. Since the ratio of heat produced to oxygen con-
sumed differs with carbohydrate, fat, and protein (3.5, 3.3, and 3.2,
respectively), it is possible to measure heat production and O, con-
sumption and to use this ratio as an index of the substrate being utilized.

S72 RESPIRATORY METABOLISM

However, because of the small differences in the ratios and because of
the complications of the technique of heat measurement, the calorific
quotient has not been found to be very useful in determining the energy
source for Metazoa (Needham, 1931), and apparently has not been
tried for Protozoa.

INVESTIGATIONS WHICH CONCERN THE MECHANISM OF RESPIRATION

1. GENERAL THEORY

For a general consideration of the mechanism of respiration, the
reader is referred to the monographs of Meldrum (1934) and Holmes
(1937), to standard textbooks of general physiology, to several excel-
lent discussions in recent volumes of the Annual Review of Biochemistry,
and to the forthcoming volume of the Cold Spring Harbor Symposia in
Quantitative Biology (Vol. VII). The present discussion of the mecha-
nism of respiration will include only those portions of a bare outline
which are necessary for an understanding of the data and interpretations
which are to follow.

The first step in oxidation of a substrate is the removal of hydrogen
from the substrate molecule, and the addition of this hydrogen to any
other molecule which will serve as a hydrogen acceptor. After de-
hydrogenation is accomplished, the resulting molecule is supposed to be
very unstable and easily undergoes oxidation by molecular oxygen, to
form CO, and water. The enzymes necessary for these final stages in
respiration are not well known, but the enzymes and respiratory pig-
ments responsible for dehydrogenation and the subsequent transfer of
the hydrogen to O, with formation of water are listed below. (Any
distinction between respiratory enzymes and pigments is purely arbi-
trary. )

(1) Dehydrogenases are enzymes which bring about activation of the
substrate, so that it may be oxidized by oxygen or intermediate hydrogen
acceptors such as cytochrome. These enzymes are highly specific, in that
they react with only one or a few substrates. Dehydrogenases are divided
into two groups: anaérobic dehydrogenases, which cannot reduce molecu-
lar O, in the presence of their substrates, and aérobic dehydrogenases
which can do so. Cytochrome and cytochrome oxidase are important
factors in the completion of oxidation by anaérobic dehydrogenases.

RESPIRATORY METABOLISM BS,

(2) Cytochrome is a group of pigments or enzymes, which in the
living cell are oxidized under aérobic, and reduced under anaérobic
conditions, but which cannot be oxidized directly by molecular O,. These
serve as hydrogen acceptors for anaérobic dehydrogenase systems.

(3) Oxidase—The term oxidase includes all enzymes which are
capable of performing oxidations in the presence of molecular oxygen.
To this group belongs the respiratory enzyme of Warburg, which ts
perhaps identical with the oxidase of cytochrome, which in turn is
also referred to as indophenol oxidase because of one method of detect-
ing its activity. This enzyme brings about the oxidation of reduced
cytochrome by molecular oxygen, and water is supposed to be oxidized
to hydrogen peroxide during the process. Aérobic dehydrogenases are
sometimes classified as oxidases.

(4) Catalase is an enzyme present in aérobic organisms and usually
absent in anaérobes. This enzyme converts hydrogen peroxide to water
and molecular oxygen, and its place in the respiratory chain is given
below.

(5) Peroxidases are enzymes which in the presence of an oxidizable
substrate convert hydrogen peroxide to water and activated oxygen,
thereby causing oxidation of the substrate. The exact rdle of peroxidases
in cellular respiration is not understood. The peroxidases are iron com-
pounds, and other iron compounds, such as cytochrome and methemo-
globin, exhibit some peroxidase-like activity.

(6) Yellow respiratory pigment, or enzyme, is a flavo-protein capable
of reversible oxidation and reduction which may be reduced in a re-
action involving oxidation of substrate (through the intermediary action
of a co-enzyme) and which can then be reoxidized in the presence of
molecular O, (with formation of H,O,) or other hydrogen acceptors.

(7) Glutathione is an amino-acid complex capable of reversible oxt-
dation and reduction, and which may act as a hydrogen acceptor through
the reduction of an -S-S- group to two -SH groups (cysteine to 2
cysteine) which are auto-oxidizable in the presence of molecular O,.

The details of how these substances function in the living cell are
subject to considerable controversy, but for our present purpose we may
regard the general outline for the first four items listed above as follows:

(1) substrate + 2 oxidized cytochrome ———==
dehydrogenase

oxidized substrate +- 2 reduced cytochrome

374 RESPIRATORY METABOLISM
(2) 2 reduced cytochrome + O, == 2 oxidized cytochrome + H,O,

oxidase

(3) H,O, == H,O+ 4 O,

catalase

The substrate may be activated by “anaérobic’’ dehydrogenase, and
it is then oxidized by cytochrome, the cytochrome itself being reduced
in the process (equation 1). Cytochrome is, in turn, oxidized by an
oxidase system which may be identical with Warburg’s respiratory en-
zyme (equation 2). During this process H,O, is formed and is then
broken down to water and molecular O, by catalase (equation 3).

The oxidase and catalase systems are inhibited by the presence of
HCN and H,S, and the oxidase system is also inhibited by CO. In the
presence of any of these reagents, reaction (1) can proceed but not
reactions (2) or (3). Therefore all of the cytochrome becomes reduced,
and respiration by means of this mechanism is stopped. The dehydrogen-
ase systems are inhibited by narcotics (e.g., the urethanes), by warm-
ing and cooling, and these agents leave all of the cytochrome in the
oxidized state. These two general methods of treatment, therefore, may
be used as tools in studying the above respiratory mechanisms. There are
also a€robic dehydrogenases which, in addition to activating the sub-
strate, can react directly with molecular oxygen without the mediation
of cytochrome and oxidase. Respiration which is brought about by this
type of system is not supposed to be affected by HCN.

It is possible to demonstrate that in some systems certain reversible
oxidation-reduction indicators (e.g., methylene blue) can replace the
cytochrome-cytochrome oxidase system, and that in this capacity the
action of these indicators may or may not be affected by HCN and CO
(e.g., grasshopper embryos, Bodine and Boell, 1937; Escherichia coli,
Broh-Kahn and Mirsky, 1938). If HCN and methylene blue are added
to such a respiratory system and inhibition does not occur, the ensuing
reactions might be visualized as follows:

oxidized substrate

(4) substrate + methylene blue
dehydrogenase

+ leuco-methylene blue
(5) leuco-methylene blue + oxygen = methylene blue ++ H,O,

Since catalase is inactivated by HCN, the hydrogen peroxide presumably

RESPIRATORY METABOLISM 3715

accumulates and in E. co/7 cultures can be measured experimentally. In
anaérobes which normally do not possess catalase, this mechanism might
explain the bacteriostatic effect of oxidation-reduction indicators.

It is also known that there are certain pigments of bacteria and yeasts
(e.g., yellow enzyme, or pyocyanine) which are capable of bringing
about a similar result, and other naturally occurring oxidation-reduction
indicators have been described (echinochrome, hermidin, and pigments
from B. violaceus and Chromodoris zebra) which apparently might func-
tion in a similar fashion. The reactions involving yellow pigment and
its coenzyme may be indicated as follows:

(6) substrate + coenzyme oxidized substrate + reduced
coenzyme
(7) reduced coenzyme + yellow pigment = coenzyme +-
leuco-yellow pigment
(8) leuco-yellow pigment + oxygen = yellow pigment + H,O,
(9) H,O, = H.0+ 40, |

catalase

dehydrogenase

In this case only the action of catalase is prevented by HCN, and there-
fore H,O, accumulates. In the absence of O, the leuco-yellow enzyme
may be oxidized by other substances (e.g., by methylene blue). The
yellow enzyme has been found to be a combination of protein and
vitamin G, and it is believed that while this sort of system is present
in a€robic organisms, it assumes its greatest importance in anaérobic
species. In anaérobic organisms (yellow enzyme can be prepared from
bottom beer yeast or lactic acid bacilli) we have, then, a respiratory
system which is quite independent of cytochrome and Warburg’s oxidase,
and which therefore is insensitive to HCN and CO. Perhaps when we
say that the respiration of a given species is cyanide insensitive, we may
be inferring that that species has a respiratory system more suited to
anaérobic conditions (temporary or otherwise). Under anaérobic condi-
tions the leuco-yellow pigment is probably oxidized by substances other
than molecular oxygen, and H,O, is not formed. The known respiratory
enzymes of bacteria are summarized by Frei (1935) and Stephenson
(eae

The relationships between the various respiratory enzyme systems

376 RESPIRATORY METABOLISM

are not so well known nor so clear-cut as, for the sake of clarity and
brevity, they have been made to appear in the above outline. Whenever
we conclude, on the basis of the action of certain reagents on respiration,
that one respiratory mechanism is very important and that another is
not, we should do so only with certain mental reservations, and the
conclusions should not be considered final, but merely indicative.

2. EXPERIMENTS WHICH CONCERN THE CYTOCHROME-CYTOCHROME
OXIDASE SYSTEM OF HYDROGEN ACCEPTORS

We have, through the action of HCN and CO on respiration, a tool
for determining how much of the respiratory activity of a given organism
is carried on by means of the cytochrome-respiratory enzyme system and
how much is not. Respiration which is not cyanide and CO sensitive
may be due to aérobic dehydrogenases, or to anaérobic dehydrogenases
plus an enzyme of the yellow-pigment type or perhaps to the action
of peroxidases. Such analyses have been made for several types of bio-
logical material. It has been determined that respiration of some cells
is extremely sensitive to HCN (e.g., yeast, B. col, most bacteria, and
mammalian tissues), while that of others is quite resistant (Chlorella,
Paramecium, Sarcina, Pneumococcus, B. acidophilus, Streptococcus,
Staphylococcus); also, the same organism may differ in sensitivity at
different periods during its life history (grasshopper eggs, Robbie, Boell,
and Bodine, 1938). One technical precaution which should be ob-
served in cyanide experiments is the use of a KOH-KCN absorbing
fluid for CO, (van Heyningen, 1935). By the selection of the proper
KOH-KCN mixture, the osmotic transfer of KCN through the air from
the experimental material to the KOH solution can be prevented. This
is apparently one possible source of error in all work on the effect of
cyanide on protozoan respiration—that none of the authors has used
balanced KOH-KCN solutions.

Among the Protozoa the effect of cyanide has been studied on several
ciliates and flagellates. It is quite well established that the respiratory
mechanism of Paramecium is insensitive to cyanide (Lund, 1918b; Shoup
and Boykin, 1931; Gerard and Hyman, 1931), and the work of Shoup
and Boykin (1931) shows that the addition of iron salts does not
increase respiration and that very little or no iron is present in Para-
mecium. These results may be interpreted to mean that the cytochrome-

RESPIRATORY METABOLISM Sigh

respiratory enzyme system plays no part in the respiration of Paramecium.
Petets(1929) obtained no inhibition with M/500 KCN on Colpidinm
colpoda. The data of Pitts (1932) for C. campylum shows that less than
20 percent of the respiration is cyanide sensitive and that this depression is
only temporary, and that while still in cyanide the respiration may rise
to a rate which is as much as 25 percent above normal and drop again
to 80 percent of the normal rate. M. Lwoff (1934) found that the
respiration of Glaucoma piriformis in peptone solution was depressed as
much as 80 percent during the first half hour, but that by the third
hour the rate had returned to normal (M/1,000 KCN), or almost
normal (15 percent below in M/450 KCN), and then decreased. In a
weaker KCN solution (M/4,500) this latter decrease did not occur.
The organisms were able to live twenty-four hours in M/450 KCN
and eight days in M/1,000, but they did not divide. In M/2,000 and
M/5,000 KCN multiplication of the organisms occurred slowly. In
glucose-Ringer solution, M/450 KCN did not inhibit, but produced an
acceleration of as much as 36 percent during the first half hour. The
conclusion to be drawn from these data is that the ciliates, as far as we
know, are relatively insensitive to the action of cyanide, and we might
consider the temporary inhibitions produced in some cases as secondary
effects rather than direct effects upon the respiratory mechanism (cf.
another explanation mentioned below). It would be interesting to rein-
vestigate the effect of KCN on some of these ciliates by the use of
balanced KCN-KOH solutions. It seems possible that the data, especially
in the case of the temporary effects, may be complicated by the loss of
KCN from the experimental solution to the KOH solution.

Among some of the flagellates, however, there seems to be quite a
different respiratory mechanism. A. Lwoff (1933) found that M/3,000
KCN inhibited respiration of Str7gomonas oncopeltz 90 percent, of
S. fasciculata 83 percent, and of Leptomonas ctenocephali 95 percent.
With M/1,000 KCN, both species of Str7gomonas were inhibited 90
percent, and L. ctenocephali 95 percent. The latter species was extremely
sensitive and was inhibited 92 percent by M/20,000. Growth was also
decreased in those concentrations which inhibited respiration, and the
organisms were killed only by much greater concentrations. M. Lwoft
(1934) reported an inhibition of 90 percent for Polytoma uvella, and
Jay (1938) an inhibition of 60-65 percent for Khawkinea and Astasia

378 RESPIRATORY METABOLISM

at a concentration of M/100 KCN. Von Fenyvessy and Reiner (1928),
however, reported no effect with 0.1 percent KCN (M/65) on either
oxygen consumption or acid production of Trypanosoma equiperdum in
glucose-bicarbonate-Ringer solution.

These results demonstrate that the respiratory mechanisms of various
Protozoa are probably not the same. The respiratory mechanisms of some
Protozoa seem to resemble those of Ch/orella and Sarcina, while those
of other species resemble the mechanisms of yeast and mammalian and
other tissues. This question is one which should be studied carefully
in a wide variety of organisms, and with a wide concentration range
of cyanide solutions. The taxonomic position of the Protozoa should
make such an investigation doubly interesting. It would also be of
interest to know if the cyanide insensitivity of Paramecium 1s still main-
tained in the presence of glucose and other substances, or if an apparent
change in the respiratory mechanism is brought about by the presence
of glucose. Emerson (1929) found that the respiration of Chlorella was
cyanide sensitive only in the presence of glucose; Gerard (1931) found
that glucose had no effect on the cyanide sensitivity of Sarcima, but
M. Lwoff (1934) found that the respiration of Glaucoma was accelerated
by HCN when glucose was absent. These divergent results should have
a final explanation in terms of the respiratory or other metabolic mecha-
nisms involved.

An alternative theory to the supposed coexistence of cyanide sensitive
and insensitive fractions in the normal cell is that all normal respiration
is CN sensitive, and that in the presence of CN an entirely new
respiratory mechanism is called into existence. This interpretation would
indicate that among many bacteria, algae, and ciliates (but not among
certain flagellates) there is a greater adaptability of the respiratory
mechanism than among the more specialized cells of the Metazoa. Such
generalizations are probably premature, but it does seem possible that
aérobic protozoa which can live anaérobically for considerable periods
of time might have a dual respiratory mechanism.

It has been demonstrated for several biological materials that respira-
tion sensitive to cyanide is also sensitive to CO (because of CO-inhibi-
tion of cytochrome oxidase), and that respiration insensitive to cyanide
is not depressed and may even be stimulated by CO (literature cited
by Bodine and Boell, 1934). A. Lwoff (1933) showed that the KCN

RESPIRATORY METABOLISM ous)

sensitive fraction (90 percent) of S. fasciculata respiration was also
sensitive to CO. In an 80/20 mixture of CO/O, inhibition was 61 percent,
in 95/5 mixtures 85 percent, and in 98/2 mixtures 90 percent. Values
of K for the Warburg-Negelein equation

A CO

== anes

where A is O, consumption in the CO/O, mixture and A, is O, con-
sumption in the control, varied from 2.58 in 80 percent CO to 5.3 in
98 percent CO. M. Lwoff found that carbon monoxide (2-5 percent O,
in CO) produced the same effect on Glaucoma as KCN in both peptone
and in glucose-Ringer solutions. In peptone there was a marked inhibi-
tion for the first half hour and then a return to normal or almost normal,
and in glucose-Ringer there was an increase of 20 percent. Reidmuller
(1936) reported no appreciable effect of CO on O, consumption for
Trichomonas foetus in 95/5 mixtures of CO and O,. The effect of KCN
was not investigated. The effect of CO on other Protozoa should be in-
vestigated together with the effect of cyanide. Recently azide (HN;)
has been found to have an effect on respiration which is similar to but
not identical with that of HCN and CO (Keilin and Hartree, 1936; and
others), and it would be interesting to make comparisons of these re-
agents on protozoan material.

For the purpose of inhibiting the cytochrome-cytochrome oxidase sys-
tem, CO is apparently much more specific then HCN or azide. This is
especially true if inhibition accurs in the dark but not in the presence
of bright light, because the inactive compound formed by CO and
cytochrome oxidase is dissociated upon illumination into CO and active
oxidase. The reversibility upon illumination of CO inhibition has not
been investigated for Protozoa.

The distribution of cytochrome among the Protozoa is a relatively
untouched subject. A. Lwoff (1933) found two absorption bands in
Strigomonas fasciculata, one at 530, and another rather broad band at
555 my. These bands disappeared upon passage of O, through the solu-
tion. Upon addition of KCN no other bands became visible, and the
question arises as to whether the 555 band was the b and c bands of cyto-
chrome or the b band of the hemochromogen, as has been found for
various bacteria. Lwoff found the 555 band also in S$. oncopelti, Glau-

380 RESPIRATORY METABOLISM

coma piriformis, and Exglena gracilis. By treating G. piriformis and
Polytoma uvella with sodium hydrosulphite and pyridine, he obtained
the bands of pyridine-hemochromogen. The observations on Glaucoma
are especially interesting because respiration is KCN and CO insensitive,
and the explanation of the KCN and CO experiments therefore needs
further clarification. Perhaps these organisms contain both cytochrome
and a KCN insensitive system (e.g., yellow pigment) which may func-
tion interchangeably. This would explain their adaptability to both aérobic
and anaérobic conditions, the presence of cytochrome and KCN and CO
insensitivity, and perhaps also the somewhat oscillatory character of
Colpidium and Glaucoma respiration in the presence of KCN (Pitts,
1932; M. Lwoff, 1934). This, of course, is pure speculation. However,
the possibility of any discrepancies in the supposed parallelism between
CN, CO, and HN, insensitivity and the absence of cytochrome should
warrant an intensive investigation. Reidmuller (1936) was unable by
spectroscopic methods to find either cytochrome or hemochromogen in
Trichomonas foetus, and this result should be expected because of the
CO-insensitivity of Tr7chomonas respiration.

3. EXPERIMENTS WHICH CONCERN OTHER SYSTEMS OF HYDROGEN AC-
CEPTORS

If we assume that the Warburg-Keilin system is not present in the
ciliates, then we must seek another respiratory mechanism. Is this to be
found in the action of glutathione? According to M. Lwoff (1934), the
effect of arsenious acid on respiration offers a tool for detecting the action
of glutathione because it is not supposed to affect the Warburg-Keilin
respiratory system and because it does combine with -SH groups, thereby
inhibiting the normal functioning of glutathione. In Glaucoma piriformts
M. Lwoff found that M/1,900 arsenious acid (neutralized sodium ar-
senite) inhibited 75-80 percent of the respiration and that M/1,150
inhibited 90 percent. The organisms moved slowly in M/400 to M/2,000
and remained alive more than thirteen days in M/6,000, but did not
multiply. The inhibition of respiration was entirely reversible (recovery
in 1 1/2-2 hours from M/2,000). Monoiodoacetic acid, at least in some
cases, is supposed to be similar in its action to arsenious acid, that is, it
combines with -SH. (In other cases its action may be different, e.g., in
the prevention of lactic-acid production from glycogen in muscle extracts

RESPIRATORY METABOLISM 381

in which glutathione is not present.) Therefore we may use it as an ad-
ditional indicator in detecting the action of glutathione in respiration.
M. Lwoff found that monoiodoacetic acid produced 61-82 percent in-
hibition of respiration of Glaucoma in concentrations of one part in
121,000 to 77,000, while the ciliates appeared normal and moved slowly.
These results are interpreted to indicate that glutathione seems to be
quite important in the respiration of Glaucoma. Another interpretation
which has been used for work on other material (Korr, 1935; Cohen and
Gerard, 1937) is that arsenites inhibit dehydrogenases (Szent-Gyorgyi
and Banga, 1933). It is interesting that the accepted dehydrogenase in-
hibitors (urethanes, see below) do not result in as great an inhibition
with Glaucoma as atsenites and monoiodoacetic acid.

Another respiratory mechanism which might exist among the cyanide-
insensitive Protozoa is the yellow pigment found in yeast and other an-
aérobic organisms. It seems as if an investigation of the distribution of
enzymes of this type should be made among the Protozoa, especially with
those species in which respiration proves to be cyanide insensitive. Since
a large number of Protozoa are presumably facultative anaérobes, it
might be possible to poison the normal aérobic mechanism and study the
anaérobic mechanisms under various conditions, as has been done for
Escherichia coli by Broh-Kahn and Mirsky (1938).

4. INHIBITION OF THE DEHYDROGENASE SYSTEM

The dehydrogenases are apparently a part of the respiratory chain in-
volved in several of the enzyme mechanisms. Therefore one would ex-
pect any substance which inhibits the dehydrogenases to inhibit
respiration. Such is the case with the urethanes. M. Lwoff (1934) found
that in the respiration of Glaucoma one percent methyl urethane pro-
duced 9 percent inhibition of respiration, 2 percent inhibited 38 percent,
2.5 percent inhibited 52 percent, and 3.5 percent inhibited 55-63 percent.
Ethylurethane in concentration of 1.66 percent inhibited 44 percent, and
2 percent inhibited 57-61 percent. Propylurethane in 0.5 percent solu-
tion produced an inhibition of 47 percent. The ciliates appeared normal,
movement was slow, but the effect on respiration was reversible. There-
fore we may conclude that dehydrogenase systems are probably involved
in the respiration of Glaucoma. It might be interesting to try the com-
bined effects of urethane and arsenious acid, in order to obtain evidence

382 RESPIRATORY METABOLISM

for or against the idea that both dehydrogenase and glutathione are part
of the same respiratory chain.

5. SYNTHESIS OF RESPIRATORY ENZYMES—VITAMINS

After we have determined that a certain organism has a certain type of
respiratory system, the question arises of how the enzymes are formed.
Is the organism capable of synthesizing them from relatively simple
compounds, or must certain prosthetic groups be present in the nutritive
substrate? For certain flagellates this question has been answered very
definitely by A. Lwoff (1933). Strigomonas oncopelti, S. fasciculata, and
Le ptomonas ctenocephali have respiratory systems which are 90 percent
dependent upon cytochrome (as demonstrated above with KCN and
CO). It was found that S. oncope/ti could live indefinitely in peptone
solutions without the addition of hematin compounds. For the other
two flagellates, hematin compounds were found to be necessary. L.
ctenocephali would not grow unless rabbit blood (or an equivalent
amount of hematin) were present in concentrations of one part to 1,200.
S. fasciculata showed growth in blood dilutions as great as 1/1,000,000,
and within limits the amount of growth was directly proportional to the
amount of blood. Hemoglobin disappeared rapidly from the culture
medium, and it apparently was being used to form more respiratory
enzyme. When small amounts of blood were added, the Qo, increased
linearly for several hours, until apparently all of the hematin was con-
vetted into respiratory enzyme; then the Qo, remained constant. This
constant level varied with the amount added. It was found that for each
gamma of blood (between one and 5 gamma) added to 1 mgm. of
flagellate (dry weight) the Qo, was increased about 3 units. After 8.5
hours 5 gamma of blood raised the Qo, from 19.5 to 37.0. Blood could
be replaced with hematin, prohemin, and protoporphyrin, but not by cyto-
chrome C nor by a wide variety of synthetic hematin and porphyrin com-
pounds, chlorophyll, peroxidase, or active iron (Lwoff, 1938). Ap-
parently only the porphyrin compound which contained the vinyl (-CH
= CH.) radical (protoporphyrin) was effective. Deuteroporphyrin,
which differs from protoporphyrin in having hydrogen in place of the
vinyl groups, was not effective. More recent investigations of the chem-
ical structure of cytochrome indicate that the vinyl groups may be neces-
sary for linking the prosthetic group (iron porphyrin) to the protein

RESPIRATORY METABOLISM 383

portion of the cytochrome molecule through a pait of sulphur atoma.
Lwoff calculated that each flagellate required 520,000 molecules of pro-
toporhyrin in order to bring the Qo, to 55, and that each organism must
contain about 700,000 molecules of protoporphyrin before division
would take place.

Although cytochrome C was ineffective alone, the action of proto-
porphyrin was increased when cytochrome was present (A. Lwoff, 1936).
A. Lwoff (1933) found that the organisms had absorption bands at
555 and 530 mn, and this indicated the presence of cytochrome. There-
fore we may conclude that protoporphyrin is necessary for the building
of (1) cytochrome or (2) cytochrome oxidase. Since cytochrome C can-
not be substituted for protoporphyrin, it seems as if the reaction proto-
porphyrin > porphyrin C is irreversible and that protoporphyrin is neces-
sary for the synthesis of something other than cytochtrome—probably
cytochrome oxidase. On the assumption that all of the iron is used to
build the respiratory enzyme, we may calculate the rate of catalysis: one
gramatom of iron at 28° carries 4.83 grammolecules of O, per second.
For yeast, Warburg obtained a value of 100. Therefore, on the basis of
these assumptions, it seems as if the respiratory enzyme of yeast is 20.8
times as active as that of S/rigomonas. It has been demonstrated by M.
Lwoft (review, A. Lwoff, 1938) that hematin is necessary for the
growth of S. muscidarum, S. culicidarum vat. ano phelis, L. tropica, La
donovani, L. agamae, L. ceramodactyli, and Schizotrypanum cruzi, as well
as for the organisms discussed above. The mechanism of its action has
not been intensively studied, but presumably it may serve a purpose
similar to that it serves in S. fascculata.

We may conclude from the above experiments that protoporphyrin 1s
necessary for the normal metabolism and growth of Strigomonas fascicu-
Jata and that the organism is not capable of its synthesis. Therefore proto-
porphyrin may be considered a vitamin. Comparable examples are known
for other organisms: e.g., cholesterol for Trichomonas columbae, T.
foetus, and Eutrichomastix coluborum; aneurine (vitamin B,) for
Glaucoma pivriformis, S. oncopelti, S. fasciculata, S. culicidarum, cet-
tain bacteria, and fungi; pyrimidine and thiazol (parts of the aneurine
molecule) for Polytomella caeca and Chilomonas paramecium; ascorbic
acid for Schizotrypanum cruzi; and lactoflavine, nicotinic acid, and
phospho-pyridine-nucleotides for various bacteria. In all cases where the

384 RESPIRATORY METABOLISM

function of these essential compounds is known or indicated, they seem
to be necessary for the formation or at least for the normal functioning
of the respiratory enzymes (review, A. Lwoff, 1938). Recent work on
the supposed function of most of these substances has been reviewed by
Burk (1937) and Stern (1938).

6. THE DETECTION OF OXIDASE, PEROXIDASE, AND CATALASE

No extensive investigation of isolated enzyme systems of the Protozoa
has been made. Certain enzymes, especially oxidase and peroxidase, are
supposed to react with certain stains so that the position of the enzyme
may be located in the cell. The methods used involve the Nadi,
Dopa, benzidine-H,O,, and pyronine-gnapthol-H,O, reactions (methods
given by Roskin and Levinsohn, 1926; Guyer, 1936; McClung, 1937).
These reactions are important in studying vertebrate blood and nerve
cells, but apparently no correlation has been made of the presence of
“oxidase” or “peroxidase” granules, detectable by these methods, and
the respiratory mechanisms discussed above, and it has not been demon-
strated that these reactions are specific for oxidase or peroxidase. Several
observations have been made on the Protozoa (Roskin and Levinsohn,
1926; Bles, 1929), and when more is known about the respiratory
mechanisms of the Protozoa it might be possible to correlate the results
of staining and of manometric methods. However, such attempts are
omitted in the present discussion. Perhaps a certain degree of localization
of the enzymes could be obtained by centrifuging the organisms and mak-
ing activity tests on cell fragments, as has been done for peptidase in
marine ova (Holter, 1936).

The presence of catalase can be detected by adding hydrogen peroxide
to a cell suspension and measuring (chemically or manometrically) the
oxygen evolved. Burge (1924) studied the catalase action of Paramecium
and Colpoda and found that it was decreased by ether and chloroform,
but not by ethylene or nitrous oxide. A much more accurate method was
used by Holter and Doyle (1938), who found that the average catalase
activity per individual of Frontonia, Paramecium, and Amoeba was in
the ratio 190:30:5. Considerable variation was found between different
cultures and even between different individuals from the same culture.
Reidmuller (1936) reported only a trace of catalase, no peroxidase, and
no indophenol oxidase for Trichomonas foetus.

RESPIRATORY METABOLISM 385

THE MEASUREMENT OF ANAEROBIC METABOLISM AND GLYCOLYSIS

The measurement of anaérobic metabolism is somewhat more complex
than the measurement of aérobic. The standard criterion of O, con-
sumption does not exist, and the auxiliary criterion of CO, production
indicates only the carbon which is completely oxidized. Sometimes this
may comprise only a small percentage of the total metabolic changes,
and in some cases measurements may be complicated by the presence of
hydrogen, methane, and other gases. Therefore we must usually attempt
to trace anaérobic metabolism by measuring changes in the concentration
of several substances in the liquid phase, instead of one or two substances
in the gaseous phase; and this is more difficult. In some cases it is possible
to give an organism a known substrate, for example, carbohydrate, and
to measure the decrease in the quantity of the substrate and the increase
in the amount of decomposition products at various intervals. From
carbohydrate decomposition these may be alcohols, aldehydes, and or-
ganic acids. From protein decomposition we might expect a wide variety
of nitrogen-containing amino-acid fragments, and by deaminization of
amino acids a wide variety of organic acids may be produced. From de-
composition of lipoids we may expect products somewhat similar to
those from carbohydrates. Methods for the final identification of these
compounds usually take one into the field of microanalytical biochemistry
(see Peters and van Slyke, 1932; Friedemann, 1938; and publications
by von Brand, Reiner, and others, cited below). The identification of
the acid formed is important in any study of the energetics of anaérobic
carbohydrate metabolism, because the processes which yield the various
acids release quite different amounts of energy. For many purposes,
however, it is customary, if not adequate, to measure acid production by
manometric measurement of the amount of CO, which is released from a
bicarbonate buffering system, as CO, or stronger acids are produced by
the organism. This gives an index of acid production, but leaves us
ignorant of the nature of the acid. Changes in total titratable acidity or
alkalinity are also used, and it seems as if, under certain conditions,
accurate titration curves might be obtained which would give a fair index
of the kind and amount of acids present.

In metazoan metabolism it is usually assumed that oxidation of glu-
cose is preceded by a molecular rearrangement which results in the
formation of lactic acid.

386 RESPIRATORY METABOLISM

Glucose = 2 lactic acid + 43,000 cal. (A H)
2 lactic acid 4. 60, > 6CO, + 6H,O + 634,000 cal. (A H)

The first reaction is referred to as glycolysis, or cleavage. Glycolysis is
reversible, and it occurs under both aérobic and anaérobic conditions, but
the rate of the reverse reaction (lactic acid + glucose) is very much less
under anaérobic than under a€robic conditions. Consequently, in some
tissues (or in the tissue medium) lactic acid may accumulate aérobically,
but usually it accumulates only during anaérobiosis. If lactic acid does
tend to accumulate, it can be measured by allowing it to displace CO,
from a bicarbonate immersion medium (usually glucose-bicarbonate-
Ringer). If it is assumed that the CO, given off by oxidation is equal
to the O, consumed, then the amount of lactic acid can be calculated as
the “excess CO,,” 1.e., the CO, evolved in addition to that released by
oxidation. This is expressed as QO for the unit one cmm. of CO, per
mg. dry weight of tissue per hour. (Older authors use Q2, which is
easily confused with Q¢o,, the respiratory CO,, and recent German au-
thors use QO: for the same quantity.) If O, is replaced by N,, all of the
CO, evolved must come from glycolysis, and the unit is expressed as
Qe (or QR, or QX’). For comparative studies on various organisms,
it has been found to be useful to calculate the Meyerhof quotient (M.Q.),
which is defined as

Qo.

This is an index of the amount of lactic acid reconverted to glucose per
unit of oxygen consumption, 1.e., a measure of the resynthesis of glucose.
Recent work on the interpretation of Meyerhof quotients is reviewed by
Burk (1937). Recent investigations of glycolysis in vertebrate tissue
indicate that glucose is converted into pyruvic acid, part of which 1s
oxidized and part of which is resynthesized into glucose, and that lactic
acid is a step in the resynthesis, rather than the end product of glycolysis.
It seems as if this may also be true for Protozoa.

OCCURRENCE OF ANAEROBIOSIS AND GLYCOLYSIS

Many Protozoa live in media which are almost if not entirely devoid of
oxygen. Examples are those which inhabit the bottom of stagnant ponds

RESPIRATORY METABOLISM 387

(especially if a considerable amount of decaying organic matter, and
consequently hydrogen sulphide, is present), those which are found in
sewage-disposal plants, those which appear near the bottom of putrid
laboratory cultures, and those which inhabit the lumen of the lower in-
testine of Metazoa. These organisms, because of the characteristics of
their environment, are deprived of one of the chief sources of energy
available to other animals—the reduction of molecular oxygen, and they
must be able to obtain energy by other methods, such as molecular re-
arrangements (e.g., glucose to lactic acid) or oxido-reductions (€.g.,
glucose to CO, and alcohol). A summary of the early theories of an-
aérobic fermentations is given by Slater (1928), and a review of the
data pertaining to anaérobic life of Protozoa and other invertebrates 1s
given by von Brand (1934). Some of the anaérobic Protozoa seem to be
obligatory anaérobes and are quickly killed by aération (e.g., Tre pomonas
agilis, Lackey, 1932). Therefore one might expect them to have a type
of metabolism comparable to those of the anaérobic bacteria.

Other organisms, such as certain intestinal forms, are certainly not
strict anaérobes, but are facultative, or amphibiotic. Measurements of the
intestinal gases (reviewed by von Brand and Jahn, 1940) and of the oxt-
dation-reduction potential of the digestive tract (Jahn, 1933a) indicate
that the lumen of the intestinal tract is largely devoid of oxygen. How-
ever, organisms which live at the surface of the epithelium (e.g., Gzar-
dia) and within the villi, and especially those such as Endamoeba histo-
lytica and Balantidium coli which invade the tissue, do have access to
molecular oxygen. The O, tension of the environment of the rumen in-
fusoria of ruminants must be extremely variable, but, for considerable pe-
riods of time, almost devoid of oxygen. The question then arises as to
what kind or kinds of respiratory mechanisms are present in these faculta-
tive organisms. The same question arises with such organisms as Para-
mecium, Which are normally aérobic but can withstand lack of oxygen
for a relatively long period of time. Do the facultative anaérobes of the
phylum Protozoa have respiratory mechanisms comparable to those of
bacterial facultative anaérobes? This question, although interesting and
suggestive, is unanswerable at present because we know nothing about
the respiratory mechanisms of anaérobic Protozoa, and not very much
about those of bacteria. However, recent investigations indicate that
among bacteria the respiratory mechanism of the strict anaérobes is

388 RESPIRATORY METABOLISM

probably different from the anaérobic mechanism of the facultative an-
aérobes (Broh-Kahn and Mirsky, 1938).

There is considerable evidence that carbohydrate decomposition takes
place in Protozoa under anaérobic conditions. It was found by Piitter
(1905) that the glycogen content of Paramecium decreased under an-
aérobic conditions. He also found that Paramecium poor in glycogen
could live anaérobically for a considerable length of time, probably at
the expense of albumen. A. Lwoff (1932) found that Glaucoma piri-
formis could live three days without oxygen only if sugar were present.
M. Lwoff (1934) obtained a value of 10 for the QN: of G. pirtformis in
peptone broth (Qo, == 35). Emerson (1929) found that under an-
aérobic conditions 80 mm* of Blepharisma released 12.5 mm.* CO, per
hour from a bicarbonate buffer mixture; negative results were obtained
with Amoeba proteus. Zhinkin (1930) demonstrated that the glycogen
content of Stentor decreased under anaérobic conditions and that visible
fat increased. Upon exposure to O, that fat disappeared. This apparent
conversion of glycogen to fat was observed in experimental cultures and
under natural conditions in winter when the O, content of ponds was
negligible, but it did not occur in experimental cultures in the presence
of light because of the photosynthetic action of zoéchlorellae. Some data
of this type was also obtained for Prorodon teres and Loxodes (Zhinkin,
1930) and for Paramecium (Pacinotti, 1914). The possible changes
which take place in the glycogen content of intestinal amoebae and
ciliates should also be investigated in this connection (see discussion by
von Brand, 1934).

The trypanosomes are a group of organisms which live under con-
ditions of high O, tension, but they apparently have a high degree of
anaérobic metabolism (glycolysis). At least, they use much more sugar
than they could possibly oxidize with the O, which they consume, and
apparently the amount of acid produced by glucose destruction does not
differ much under aérobic or anaérobic conditions. According to the data
of von Fenyvessy and Reiner (1924), the O, consumption for a billion
trypanosomes (T. equiperdum) suspended in diluted blood was about
0.07 mg. per hour. The sugar consumed under similar conditions (Yorke,
Adams, and Murgatroyd, 1929) was about 5 mg. Since complete oxida-
tion of 5 mg. of sugar requires about 5 mg. of O,, it appears as if the
major portion. of the sugar destruction was anaérobic. This is discussed

RESPIRATORY METABOLISM 389

by von Brand (1934, 1935). On the basis of an assumed R.Q. of one,
von Fenyvessy and Reiner (1928) subtracted the Qo, from the rate of
CO, evolution from a bicarbonate solution and found that the resulting
value was equal to the QN:. Therefore the rate of glycolysis (acid pro-
duction) is the same in O, or N, and is independent of O, consumption.
The data of von Fenyvessy and Reiner showed that the amount of CO,
evolved when the organisms were in bicarbonate-glucose-Ringer was so
high under aérobic conditions that apparent R.Q. values of 1.7 to 3.6
were obtained. Therefore we must conclude that glucose —> acid conver-
sion is very high in T. eguiperdum. According to the experiments of von
Brand, Regendanz, and Weise (1932), this acid production is appar-
ently not a true glycolysis, because lactic acid could not be detected in the
medium by chemical methods. However, if we consider lactic acid to be
a step in the resynthesis of pyruvic acid to glucose (see above), the ab-
sence of lactic acid may merely mean that resynthesis does not occur. In
this case the Meyerhof quotient should be zero. It was shown by Reiner
and Smythe (1934) and Reiner, Smythe, and Pedlow (1936) that
aérobic sugar destruction by T’. equiperdum was as follows:

1 glucose — 1 glycerol 4 1 pyruvic acid
1 glycerol + O, — 1 pyruvic acid +. 2H,O

Apparently lactic acid and CO, were not produced. For T. Jew/s7, under
aérobic conditions, the end products were identified as succinic, acetic,
and formic acids, ethyl alcohol, and carbon dioxide.

It has been suggested that the large amount of acid produced by
trypanosomes might be the mechanism by which toxic effects are pro-
duced. This possibility was investigated for T. evans by Kligler, Geiger,
and Comaroff (1929), who analyzed the blood of infected rats and
concluded that death was caused by lactic acid acidosis. In subsequent
publications (Kligler, Geiger, and Comaroff, 1930; Geiger, Kligler, and
Comaroff, 1930) they reported the measurement of glycolysis of T.
evansi, having obtained even higher values (9.2 mgm./billion/hour)
than Yorke, Adams, and Murgatroyd had obtained for T. equiperdum.
Von Brand, Regendanz, and Weise (1932) measured the glucose, lactic
acid, and alkali reserve of animals infected with T. gambiense, T. brucei,
and T’. equiperdum, and found no evidence of low glucose, high lactic
acid, or low alkali reserves, and therefore no support for the acidosis

390 RESPIRATORY METABOLISM

theory of death. Von Brand (1933) measured the rate of sugar destruc-
tion for various trypanosomes, and obtained high values (8.0 mgm. /bil-
lion/hour) for the pathogenic trypanosomes T. bruce7, T. gambiense,
T. rhodesiense, and T. congolense, and very low values for the nonpatho-
genic T. /ew7s7 (about 1.4), and still lower values for the pathogenic
Schizotrypanum cruzi. The results of these investigators indicate that
although sugar destruction and formation of acid may be a contributing
factor, it will not explain all of the pathological effects of the trypano-
somes. This is reviewed by von Brand (1938).

It is interesting to compare the high aérobic glycolysis rate of trypano-
somes with that of malignant tumors. The Warburg quotient (aérobic
glycolysis/Qo,) for normal tissues is usually less than 0.3 (except retina
and placenta), while that for benign tumors is about one, and that for
malignant tumors is 3.1-3.9 (review, Needham, 1931). The Warburg
quotients calculated from the data of von Fenyvessy and Reiner (1928)
for T. eguiperdum are 0.78 to 2.67. It has been found that KCN may
change the Warburg quotient of chick embryos from 0.1 to 3.4, but
the quotient for T. equiperdum, in the presence of KCN, showed no
significant change (0.78 and 0.80 in two experiments). These compari-
sons may be taken to indicate that the relative glycolytic rate of the
trypanosomes 1s different from that of normal tissues and resembles in
certain respects that of benign or malignant tumors, but the fact that
pyruvic and other acids are formed by trypanosomes instead of lactic
acid invalidates this comparison.

Meyerhof quotients were calculated by A. Lwoff (1933) for S¢r7-
gomonas fasciculata, S. oncopelti, and Leptomonas ctenocephali and
were found to be 1.20, 1.38, and 0.125 respectively. The first two are
within the normal range of metazoan tissues (Needham, 1931), but
that of Leptomonas is very low. Values of the M.Q. calculated from the
data of von Fenyvessy and Reiner (1928) on trypanosomes are approxi-
mately zero, indicating no reversal of glycolysis, and this conclusion
agrees with the chemical equations given above.

WuHy ARE ANAEROBES ANAEROBES, AND AEROBES AEROBES?

One question which arises in any treatment of anaérobiosis is, “Why
does oxygen prevent growth of obligatory anaérobes?”’ There are several
explanatory theories:

RESPIRATORY METABOLISM 59

1. Oxygen is directly lethal to the cell.

2. Anaérobes do not contain catalase and therefore are incapable of
destroying the toxic H,O, which is formed by reduction of oxygen (see
equations given above).

3. Growth of anaérobes is dependent upon the presence of a low
oxidation-reduction potential in the medium, the attainment of which
is prevented by oxygen.

4. O, forms a loose chemical complex with the respiratory system of
obligatory anaérobes, and thereby inhibits its activity.

The relative merits of these theories, as applied to bacteria, are dis-
cussed by Hewitt (1936) and Broh-Kahn and Mirsky (1938). The first
theory is certainly not true for those anaérobic organisms which will
grow under anaérobic conditions after exposure to oxygen. The second
theory is supported by considerable evidence, in that most anaérobes do
not contain catalase, and in that some bacteria (e.g., pneumococci) will
grow aérobically until they are killed by the accumulation of H,O, re-
sulting from their metabolism (so-called “suicide” of cultures). How-
ever, some anaérobes do contain catalase, and apparently it has not been
definitely demonstrated that strict anaérobes consume O, in order to
produce H,O,, or even that obligatory anaérobes do produce H,O, (Broh-
Kahn and Mirsky). The theory, however, might still be applicable to
organisms such as penumococci and hemolytic streptococci, and to
Escherichia coli in the presence of HCN and methylene blue. In these
cases appreciable amounts of H,O, can be detected.

Among the Protozoa we have very little evidence of the relative merits
of the first two of these theories. It is shown by the work of Cleveland
on termites and on xylophagous cockroaches (Cleveland, Hall, Sanders,
and Collier, 1934, include citations of earlier papers) that the symbiotic
Protozoa which inhabit the digestive tracts of these organisms are prob-
ably strict anaérobes. At least the O, tension of their normal environment
is extremely low, and they are rapidly killed by appreciable quantities
of molecular O,. Cleveland found that at 23° C. the time necessary for
death of the symbionts of termites was an inverse function of oxygen
tension (e.g., in Termopsis, all Protozoa were dead in 72 hours at one
atmosphere, in 30 minutes at 3.5 atmospheres). This is apparently due
to an increase in O, concentration in the digestive tract, with increased
O, pressure in the atmosphere, and this could easily be explained on the

392 RESPIRATORY METABOLISM

basis of either of the above two theories. Additional evidence, however,
might be gained for the second theory from the fact that one atmosphere
of O, is more toxic at low temperatures (4-5° C.) than at high (23-
25° C.), and that four atmospheres of O, are more toxic at high tempera-
tures than at low. The greater solubility of O, at 4-5° C. can account for
the greater toxic effect with one atmosphere pressure. However, the re-
verse effect at four atmospheres O, pressure must, as suggested by Cleve-
land, be connected in some manner with metabolic processes. Super-
ficially, at least, these results seem to be explicable on the basis of the
second of the above theories, i.e., the organisms grew more rapidly and
produced more H,O, at the higher temperatures. An examination of the
protozoa for catalase, or of the digestive contents of oxygenated insects
for H,O,, might yield pertinent information.

The data of Cleveland (1925) on the toxicity of oxygen for the in-
testinal Protozoa of earthworms, salamanders, frogs, and goldfish, are
possibly open to this explanation, but here also we lack experimental
evidence. Where such defaunation procedures failed, as in the rat, we
might assume that the O, tension of the digestive tract was not raised
in a manner comparable with that which occurred in the smaller or-
ganisms, or that the Protozoa present were more resistant. The former
theory seems much more probable. (For review of the chemistry of the
intestinal contents, see von Brand and Jahn, 1940.)

The theory that the growth of anaérobes is dependent upon a low
oxidation-reduction potential in the medium was proposed by Quastel
and Stephenson (1926), and has gained considerable support among
bacteriologists (review, Hewitt, 1936) and some dissent (literature
cited by Broh-Kahn and Mirsky, 1938). Positive evidence consists mainly
of the facts that (1) during the growth phases of anaérobic cultures,
especially the sporulating anaérobes, much lower oxidation-reduction
potentials are produced than during the growth phases of cultures of
aérobes; and (2) anaérobic forms do not start growing until the
potential is quite low (E, < + 100 mv.). The first type of evidence
does not help to distinguish between cause and effect, and there are
some exceptions to the general trend. Most of these, however, are due
to the fact that the organism is only one factor which tends to determine
the E, of the medium; the chemical composition of the medium certainly
determines, to a great extent, what potentials may be attained. The second

RESPIRATORY METABOLISM 393

type of evidence is well founded in fact—anaérobes do not grow in media
of high E,, value. However, if the E, value of a suitable medium is low-
ered through displacement of air with H, or N,, or by various chemical
reagents, or by the growth of an aérobic organism, then the ana€robic
forms are capable of growth. According to this theory, anaérobes and
aérobes differ in their ability to grow at various points along the E, scale,
in a manner comparable to that which is exhibited by various acido-
philic and basiphilic forms in growing at various points along the pH
scale. Of course, there are intermediate-range and wide-range forms in
respect to both pH and E,. The fact that the toxic effects of lack of O,,
or of supernormal O, tensions, are not equal in all species supports this
idea, but these data, of course, are subject to other interpretations.

Investigations of the rdle of oxidation-reduction potentials among the
Protozoa have never passed the preliminary stages. The possible im-
portance of such a study was pointed out by Jahn (1933b, 1934), in
connection with experiments on -SH compounds, on the toxic action of
methylene blue and on possible relationships with auto- and allelocataly-
sis. Measurements of the E, of Chilomonas cultures (Jahn, 1935b), of
hay infusions (Efimoff, Nekrassow, and Efimoff, 1928), and of digestive
contents (Jahn, 1933a) have been made. However, until more data be-
come available, most of these results are difficult to interpret. It was de-
termined that Chilomonas would grow in mixtures of NaSH and H,O,
only if the concentrations of these were balanced so that the medium
just failed to reduce methylene blue. This might indicate a microaéro-
philic tendency for Chilomonas. Other experiments with Chilomonas
indicated that it could live, but could grow only slowly, however, in
media in which methylene blue was reduced. Neither of these ideas is
contradictory to its known habits in laboratory cultures. It was also
demonstrated (Jahn, 1935b) that casein-acetate broth cultures of C/v-
lomonas, when exposed to the air, developed potentials of -20 mv. at
pH 7.55, a point at which methylene blue is about half reduced. The
chief difficulty in interpreting experiments pertaining to the effect of Ey
on growth is that it is necessary to change O, tension in order to change
E,,. This makes an experiment containing only one variable seemingly im-
possible to execute, and the theory, therefore, has not been amenable
to experimental approach.

The fourth theory of the effect of O, on anaérobes—that of an inac-

394 RESPIRATORY METABOLISM

tivation of the respiratory mechanism of obligatory anaérobes by O,—
is mentioned by Broh-Kahn and Mirsky (1938), but at present is
unsupported by experimental evidence. The bacteriostatic effect of dyes
might also be interpreted to mean that these have an inactivating effect
on the respiratory mechanism.

In connection with the effect of oxygen on anaérobes, it should be
mentioned that the converse problem also exists. Why do aérobes die in
the absence of oxygen? On this question there has been considerably
less discussion in the literature than on the former. It can be seen from
the respiratory equations given above that activation of the substrate
and partial oxidation can proceed without O,. Even in the presence of
O, a considerable destruction of substrate, in some forms (e.g., trypano-
somes, cited above), seems to be incomplete. Poisoning of the aérobic
cell under anaérobic conditions is supposedly caused by the accumulation
of toxic products of carbohydrate cleavage or of incomplete oxidation,
or by the conversion of all of the respiratory pigment to the reduced
state. In facultative anaérobes the former, and in obligatory aérobes the
latter theory seems more probable. The observation of Fauré-Fremiet,
Léon, Mayer, and Plantefol (1929) that Paramecium withstands lack of
O, longer at 4° C. than at higher temperatures, is open to either inter-
pretation. The data of Piitter (1905), which indicate that Paramecium
can live longer under anaérobic conditions when the ratio of volume of
medium to cells is higher, can be explained on the basis of accumulation
of toxic products. What these toxic products are probably depends to a
large extent upon the organism, the substrate, and the conditions of the
experiment, and the most likely possibilities include lactic and lower
fatty acids.

OXIDATION-REDUCTION POTENTIAL VERSUS RESPIRATION
AND GROWTH

Another question which arises is why we might or might not expect
the oxidation-reduction potential of the medium to affect the respiration
and growth of the organism. It is obvious from the outline of the respir-
atory processes given above, and from many other types of data, that
oxidation-reduction phenomena are involved in respiration. The respira-
tory pigments and perhaps also the respiratory enzymes are reversible
oxidation-reduction systems. Therefore their action should be affected

RESPIRATORY METABOLISM 395

by the potential of the medium in which they are found, that is, by the
oxidation-reduction potential of protoplasm, and they, in turn, must
to a large extent determine this potential. If we were to mix numerous
half-reduced, completely reversible oxidation-reduction indicators in a
homogeneous solution, an equilibrium would be reached, and the po-
tential attained would depend upon the E, values of the substances and
upon their relative amounts. Substances with E, values far from the result-
ing E,, value of the solution would be either completely reduced or com-
pletely oxidized, and would be unable to contribute much toward oxidizing
or reducing small amounts of added materials unless the E, of the mixture
were appreciably changed.

Obviously such a simple system is not present in protoplasm. Because
of the presence of irreversible systems and of the continual introduction
of new substrate and the removal of certain end products, a true equi-
librium is never attained. Also, the colloidal nature of protoplasm makes
possible the existence of different E, values in different phases of the
substance, and the differential adsorption of oxidized and reduced ma-
terial at interfaces may produce a potential different from that in any of
the phases. Therefore the term ‘‘oxidation-reduction potential of proto-
plasm” may be without any interpretable significance (for discussion, see
Jahn, 1934; Korr, 1938). But there must certainly be a significance to
the Eo values of the respiratory pigments, and the possibility of an indi-
vidual expression of these values may be maintained by the polyphasic
nature of protoplasm. The oxidation-reduction potential, which can be
measured with indicators, is probably an index of the potential developed
by one or more of these pigments (for summary of such measurements,
see Chambers, 1933; Cohen, 1933). It is known that the apparent E,
value of protoplasm, as measured by indicators, varies with the E, of the
external medium when the external O, tension is changed. Therefore,
why cannot the E, of the external medium determine the degree of re-
duction of the respiratory pigments and therefore the rate of respiration?
This mechanism might be used to explain the inhibition which is pro-
duced by oxidation-reduction indicators in cultures of bacteria (Dubos,
1929) and in cultures of Chilomonas (Jahn, 1933b). One difficulty in
predicting what reactions would occur in protoplasm, even if we had a
thorough knowledge of the oxidation-reduction systems involved, 1s the
fact that such knowledge can tell us only what reactions might or might

396 RESPIRATORY METABOLISM

not occur if all of the reactions were reversible. Since many of the re-
acting substances are changed irreversibly and since the rates of reactions
are dependent not only upon Eo values but upon enzymes, knowledge of
E, and E, values cannot indicate what reactions will occur.

Much of this discussion of oxidation-reduction potentials is pure
speculation, but it is the type of speculation (often unexpressed) which
has spurred investigators to a study of the naturally occurring oxidation-
reduction systems, of the apparent oxidation-reduction potential of proto-
plasm, and of the E, values developed in bacterial and protozoan cultures
(reviews, Needham and Needham, 1927; Wurmser, 1932; Chambers,
1933; Clark, 1934; Hewitt, 1936). Interpretations of the data have not
always been as fruitful as one might expect, and one is led at times to
suspect that the modes, if not the points of attack on the problem, are in
need of revision. However, since the necessity of some such relationship
as Outlined above seems sound, it is more probable that merely the time
for the harvest has not yet arrived. Clark (1934) estimated that another
half century will be necessary for the solution of these problems.

Another means by which the oxidation-reduction potential of the
medium is supposed to affect metabolism is described by the surface catal-
ysis theory suggested by Quastel (1930), Kluyver (1931), and others
(discussed by Hewitt, 1936). It is suggested that many oxidative pro-
cesses of bacteria take place at the surface of the cell (Quastel, 1930), and
it seems as if for these reactions the E, of the medium would be more im-
portant than that of the protoplasm. It is also very probable that oxida-
tion-reduction enzymes are merely surface catalysts, which produce their
effect by nature of intense interfacial electrical fields (Kluyver, 1931).
These fields might be affected by the potential of the medium, whether
they occur at the cell surface or within the protoplasm. An intriguing
speculation would be that the respiratory pigments are distributed among
the various phases of protoplasm, and that the enzymes are actually the
interfaces of the emulsion. Unfortunately, such ideas are difficult to
check experimentally. However, since oxidation-reduction enzymes are
proteins which in all probability exert their catalytic properties through
surface action, it is possible that the catalytic interfaces of the cells, dis-
cussed by Kluyver (1931), are merely the surfaces of the protein mole-
cules.

RESPIRATORY METABOLISM 397
LITERATURE CITED

Adolph, E. F. 1929. The regulation of adult body size in the protozoan
Col poda. J. exp. Zool., 53: 269-312.

Amberson, W. F. 1928. The influence of oxygen tension upon the respiration
of unicellular organisms. Biol. Bull., 55: 79-91.

Andrews, Justin, and Th. von Brand. 1938. Quantitative studies on glucose
consumption by Trichomonas foetus. Amer. J. Hyg., 28: 138-47.

Barratt, J. O. W. 1905. Die Kohlensdureproduktion von Paramecium aurelia.
Z. allg. Physiol., 5: 66-72.

Bles, E. 1929. Arcella. A study in cell physiology. Quart. J. micr. Sca., 72:
527-648.

Bodine, J. H., and E. J. Boell. 1934. Respiratory mechanisms of normally de-
veloping and blocked embryonic cells (Orthoptera) . J. cell. comp. Physiol.,
Bis eis,

—— 1937. The action of certain stimulating and inhibiting substances on the
respiration of active and blocked eggs and isolated embryos. Physiol.
Z00\.,. 10:5 245-572

Bodine, J. H., and P. R. Orr. 1925. Respiratory metabolism. Biol. Bull., 48:
1-14.

Brand, Th. von. 1933. Studien tiber den Kohlehydratstoffwechsel parasitischer
Protozoen. II. Der. zuckerstoffwechsel der Trypanosomen. Z. vergl.
Physiol., 19: 587-614.

— 1934. Das Leben ohne Saurstoff bei witbellosen Tieren. Ergebn. Biol.,
10:37-100.

—— 1935. Der stoffwechsel der protozoen. Ergebn. Biol., 12:161-220.

—— 1938. The metabolism of pathogenic trypanosomes and the carbohydrate
metabolism of their hosts. Quart. Rev. Biol., 13: 41-50.

Brand, Th. von, and T. L. Jahn, 1940. Chemical composition and metabolism
of Nematode parasites of vertebrates, and the chemistry of their en-
vironment. In An introduction to Nematology (ed. by J. R. Christie).
(In press.) Baltimore.

Brand, Th. von, P. Regendanz, and W. Weise. 1932. Der Milchsduregehalt
und die Alkalireserve des Blutes bei experimentellen Trypanosomeninfek-
tionen. Zbl. Bakt., I. (Orig.) 125: 461-68.

Broh-Kahn, R. H., and I. A. Mirsky. 1938. Studies on anaerobiosis. I. The
nature of the inhibition of growth of cyanide-treated E. coli by reversible
oxidation-reduction systems. J. Bact., 35: 455-75.

Burge, W. E. 1924. The effect of different anaesthetics on the catalase content
and oxygen consumption of unicellular organisms. Amer. J. Physiol.,
69: 304-6,

Burk, Dean. 1937. On the biochemical significance of the Pasteur reaction and
Meyerhof cycle in intermediate carbohydrate metabolism. Some Funda-
mental Aspects of the Cancer Problem. Occ. Publ. Amer. Ass. Adv. Sci.

298 RESPIRATORY METABOLISM

Chambers, R. 1933. An analysis of determinations of intracellular reduction
potentials. Cold Spring Harbor Symp. Quant. Biol., 1: 205-13.

Clark, W. M. 1934. The potential energies of oxidation-reduction systems and
their biochemical significance. Medicine, 13: 207-50.

Cleveland, L. R. 1925. Toxicity of oxygen for Protozoa in vivo and in vitro.
Biol. Bull. 48: 455-68.

Cleveland, L. R., S. R. Hall, E. P. Sanders, and Jane Collier. 1934. The wood-
feeding roach Cryptocercus, its Protozoa, and the symbiosis between
Protozoa and roach. Mem. Amer. Acad. Arts Sci., 17: 185-342.

Cohen, Barnett. 1933. Reactions of oxidation-reduction indicators in biological
material, and their interpretation. Cold Spring Harbor Symp. Quant. Biol.,
Ls 24223)

Cohen, R. A., and R. W. Gerard. 1937. Hyperthyroidism and brain oxida-
tions. J. cell. comp. Physiol., 10: 223-40.

Cook, S. F. 1932. The respiratory gas exchange in Termopsis nevadensis. Biol.
Bull. 63: 246-57.

Daniel, G. E. 1931. The respiratory quotient of Balantidinm coli. Amer. J.
Hyg., 14: 411-20.

Dixon, M. 1934. Manometric methods as applied to the measurement of cell
respiration and other processes. London.

Dubos, R. 1929. The relation of the bacteriostatic action of certain dyes to
oxidation-reduction processes. J. exp. Med., 49: 575-92.

Duryée, W. R. 1936. A modified microrespirometer. Z. vergl. Physiol., 23:
208-13.

Efimoff, W. W., N. J. Nekrassaw, and Alexandra W. Efimoff. 1928. Die
Einwirkung des Oxydationspotentials und der H-Ionenkonzentration auf
die Vermehrung der Protozoen und Abwechselung ihrer Arten. Biochem.
Zi, 1972 VO5"0s

Elliott, A. M. 1939. A volumetric method for estimating population densities
of Protozoa. Trans. Amer. micr. Soc., 58: 97-99.

Emerson, R. 1929. Measurements of the metabolism of two protozoans. J.
gen. Physiol., 13: 153-58.

Fauré-Fremiet, E., C. Léon, A. Mayer, and L. Plantefol. 1929. Recherches sur
le besoin d’oxygéne libre. L’oxygéne et les mouvements des _para-
mécies. Ann. Physiol. Physicochim. biol., 5: 633-41.

Fenn, W. O. 1928. A new method for the simultaneous determination of
minute amounts of carbon dioxide and oxygen. Amer. J. Physiol., 84:
110-18.

Fenyvessy, B. von, and L. Reiner. 1924. Untersuchungen uber den respira-
torischen Stoffwechsel der Trypanosomen. Z. Hyg. Infektkr., 102: 109-
169)

—— 1928. Atmung und Glykolyse der Trypanosomen. H. Biochem. Z., 202:
75-80.

RESPIRATORY METABOLISM 399

Frei, W. 1935. Atmungssysteme der Bakterien. Zbl. f. Bakt., 134: 26-35.

Friedemann, T. E. 1938. Metabolism of pathogenic bacteria. J. Bact., 35:
527-46.

Geiger, A., I. J. Kligler, and R. Comaroff. 1930. The glycolytic power of
trypanosomes (Trypanosoma evanst) in vitro. Ann. Trop. Med. Parasit.,
24: 319-27.

Gerard, R. W. 1931. Observations on the metabolism of Sarcina lutea. II.
Biol. Bull., 60: 227-41.

Gerard, R. W., and H. K. Hartline. 1934. Respiration due to natural nerve
impulses. A method for measuring respiration. J. cell. comp. Physiol.,
4: 141-60.

Gerard, R. W., and L. H. Hyman. 1931. The cyanide sensitivity of Para-
mecium. Amer. J. Physiol., 97: 524-25.

Guyer, M. F. 1936. Animal micrology. Chicago.

Hall, Robert H. 1938. The oxygen-consumption of Colpidium campylum.
Biol. Bull., 75: 395-408.

Hall, R. P. 1939. The trophic nature of the plant-like flagellates. Quart. Rev.
Biol., 14: 1-12.

Harvey, E. N. 1928. The oxygen consumption of luminous bacteria. J. gen.
Physiol., 11: 469-75.

Hewitt, L. F. 1936. Oxidation-reduction potentials in bacteriology and bio-
chemistry. 2d ed. (London County Council) London,

Heyningen, W. E. van. 1935. The inhibition of respiration by cyanide. Bio-
chem; J).,, 29° 2036-39:

Holmes, Eric. 1937. The metabolism of living tissues. London.

Holter, H. 1936. Studies on enzymatic histochemistry. XVIII. Localization of
peptidase in marine ova. J. cell. comp. Physiol., 8: 179-200.

Holter, H., and W. L. Doyle. 1938. Studies on enzymatic histochemistry.
XXVIII. Enzymatic studies on protozoa. J. cell. comp. Physiol., 12: 295-
308.

Howland, R. B., and A. Bernstein. 1931. A method for determining the oxy-
gen consumption of a single cell. J. gen. Physiol., 14: 339-48.

Jahn, Theo. L. 1933a, Oxidation-reduction potential as a possible factor in
the growth of intestinal parasites in vitro. J. Parasit., 20: 129.

—— 1933b. Studies on the oxidation-reduction potential of protozoan cul-
tures. I. The effect of —SH on Chilomonas paramecium. Protoplasma, 20:
90-104.

—— 1934. Problems of population growth in the Protozoa. Cold Spring Har-
bor Symp. Quant. Biol., 2: 167-80.

—— 1935a. Studies on the physiology of the euglenoid flagellates. VI. The
effects of temperature and of acetate on Evglena gracilis in the dark.
Arch. Protistenk., 86: 251-57.

— 1935b. Studies on the oxidation-reduction potential of protozoan cul-

400 RESPIRATORY METABOLISM

tures. II. The reduction potential of cultures of Chilomonas paramecium.
Arch. Protistenk., 86: 225-37.

— 1936. Effect of aeration and lack of CO, on growth of bacteria-free
cultures of protozoa. Proc. Soc. exp. Biol. N.Y., 33: 494-98.

Jay, George, Jr. 1938. Respiration of Astasia sp. and Khawkinea halli. Anat.
Rec., 72 (Suppl.): 104.

Kalmus, H. 1927. Das Kapillar-Respirometer: Eine neue Versuchsanordnung
zur Messung des Gaswechsels von Mikrodrganismen. Vorlaufige Mit-
teilung demonstriert an einem Beispel: Die Atmung von Paramecium
caudatum. Biol. Zbl., 47: 595-600.

—— 1928a. Die Messung der Atmung, Garung und CO,-Assimilation kleiner
Organismen in der Kapillare. Z. vergl. Physiol., 7: 304-13.

— 1928b. Untersuchungen tiber die Atmung von Paramecium caudatum.
Z. vergl. Physiol., 7: 314-22.

Keilin, D., and E. F. Hartree. 1936. On some properties of catalase hematin.
Proc..toy. Soc. By i213 173-91.

Kempner, W. 1936. Effect of low oxygen tension upon respiration and fer-
mentation of isolated cells. Proc. Soc. exp. Biol. N.Y., 35: 148-51.
— 1937. Effect of oxygen tension on cellular metabolism. J. cell. comp.

Physiol., 10: 339-64.

Kligler, I. J., A. Geiger, and R. Comaroff. 1929. Susceptibility and resistance
to trypanosome infections. VII. Cause of injury and death in trypano-
some infected rats. Ann. Trop. Med. Parasit., 23: 325-35.

— 1930. Effect of the nature and composition of the substrate on the develop-
ment and viability of trypanosomes. Ann. Trop. Med. Parasit., 24: 329-
45.

Kluyver, A. J. 1931. Chemical activities of microorganisms. London.

Korr, I. M. 1935. An electrometric study of the reducing intensity of lumi-
nous bacteria in the presence of agents affecting oxidations. J. cell. comp.
Physiol., 6: 181-216.

— 1938. Oxidation-reduction potentials in heterogeneous systems. J. cell.
comp. Physiol., 11: 233-45.

Lackey, J. B. 1932. Oxygen deficiency and sewage Protozoa: with descriptions
of some new species. Biol. Bull., 63: 287-95.

Leichsenring, J. M. 1925. Factors influencing the rate of oxygen consumption
in unicellular organisms. Amer. J. Physiol., 75: 84-92.

Linderstrom-Lang, K. 1937. Principle of the Cartesian diver applied to
gasometric technique. Nature, 140: 108.

Lund, E. J. 1918a. Relation of oxygen concentration and the rate of intra-

cellular oxidation in P. caudatum, Amer. J. Physiol., 45: 351-64.

1918b. Rate of oxidation in P. cawdatum and its independence of the

toxic action of KCN. Amer. J. Physiol., 45: 365-73.

—— 1918c. III. Intracellular respiration, relation of the state of nutrition of

RESPIRATORY METABOLISM 401

Paramecium to the rate of intracellular oxidation. Amer. J. Physiol.,
ASE SET

—— 1918d. A simple method for measuring carbon dioxide produced by
small organisms. Biol. Bull., 36: 105-14.

Lwoff, A. 1932. Recherches biochimique sur Ja nutrition des Protozoaires.
Monogr. Inst. Pasteur, 1932.

—— 1933. Die Bedeutung des Blutfarbstoffes fiir die parasitischen Flagellaten.
Zbl. Bakt. Abt. 1 (Orig.), 130: 498-518.

—— 1938. Les Facteurs de croissance pour les microdrganismes. Premier
Congrés des Microbiologistes de Langue francaise, 4 Paris.

Lwoff, M. 1934. Sur la respiration du Cilié Glaucoma piriformis. C. R. Soc.
Biol. Paris, 115: 237-41.

Marsh, G. 1935. Kinetics of an intracellular system for respiration and bio-
electric potential at flux equilibrium. Plant Physiol., 10: 681-97.

Mast, S. O., and D. M. Pace. 1937. The effect of silicon on growth and respi-
ration in Chilomonas paramecium. J. cell. comp. Physiol., 10: 1-14.
Mast, S. O., D. M. Pace, and L. R. Mast. 1936. The effect of sulfur on the
rate of respiration and on the respiratory quotient of Chilomonas para-

mecium. J. cell. comp. Physiol., 8: 125-39.

McClung, C. E., Ed. 1937. Handbook of microscopical technique. New York.

Meldrum, N. U. 1934. Cellular Respiration. London.

Necheles, H. 1924. Unpublished experiments. Cited by Kestner and Plant,
in WWinterstein’s Handbuch der vergleichenden Physiologie. Bd.
2/2.

Needham, Joseph. 1931. Chemical embryology, Vol. II. Cambridge Univ.
Press, London.

Needham, Joseph, and E. J. Boell. 1938. Metabolic properties of the regions
of the amphibian gastrula. Proc. Soc. Exp. Biol. N.Y., 39: 287-90.
Needham, J., and D. M. Needham. 1927. The oxidation-reduction potential

of protoplasm: a review. Protoplasma, 1: 255-94.

Novy, F. G. 1932. Respiration of microorganisms. J. Lab. clin. Med., 17: 731-
47.

Novy, F. G., H. R. Roehm, and M. H. Soule. 1925. Microbic respiration. I.
The compensation manometer and other means for study of microbic
respiration. J. Infect. Dis., 36: 109-67.

Pacinotti, G. 1914. Infusorien, welche Glykogen in Fett umwandeln. Boll.
Soc. eustach. No. 3.

Peters, J. P., and D. D. Van Slyke. 1932. Quantitative clinical chemistry Vol.
II. Methods. Baltimore.

Peters, R. A. 1929. Observations on the oxygen consumption of Colpidium

Pitts, R. F. 1932. Effect of cyanide on respiration of the protozoan, Colpidimm
campylum, Proc. Soc, exp. Biol. N.Y., 29: 542.
Putter, A. 1905. Die Atmung der Protozoen. Z. allg. Physiol., 5: 566-612.

402 RESPIRATORY METABOLISM

Quastel, J. H. 1930. The mechanism of bacterial action. Trans. Faraday Soc.,
26: 853-64.

Quastel, J. H., and M. Stephenson. 1926. Experiments on “‘strict’’ anaerobes.
I. The relation of B. sporogenes to oxygen. Bio-chem. J., 20: 1125-37.

Reiner, L., and C. V. Smythe. 1934. Glucose metabolism of the Trypanosoma
equiperdum in vitro. Proc. Soc. exp. Biol. N.Y., 31: 1086-88.

Reiner, L., C. V. Smythe, and J. T. Pedlow. 1936. On the glucose metabolism
of trypanosomes. J. biol. Chem., 113: 75-88.

Riedmuller, L. 1936. Beitrag zum kulturellen Verhalten von Trichomonas
foetus. Zbl. Bakt. (Orig.), 137: 428-33.

Robbie, W. A., E. J. Boell, and J. H. Bodine. 1938. A study of the mechanism
of cyanide inhibition. I. Effect of concentration on the egg of Me/anoplus
differentialis. Physiol. Zo6l., 11: 54-62.

Root, W. S. 1930. The influence of carbon dioxide upon the oxygen con-
sumption of Paramecium and the egg of Arbacia. Biol. Bull., 59: 48-62.

Roskin, Gr., and L. Levinsohn. 1926. Die Oxydasen und Peroxydasen bei
Protozoa. Arch. Protistenk., 56: 145-66.

Schlayer, C. 1936. The influence of oxygen tension on the respiration of
pneumococci (Type I). J. Bact., 31: 181-90.

Schmitt, F. O. 1933. The oxygen consumption of stimulated nerve. Amer. J.
Physiol., 104: 303-19.

Shoup, C. S., and J. T. Boykin. 1931. The insensitivity of Paramecium to
cyanide and effects of iron on respiration. J. gen. Physiol., 15: 107-18.

Slater, W. K. 1928. Anaerobic life in animals. Biol. Rev., 3: 303-28.

Soule, M. H. 1925. Microbic Respiration. II]. Respiration of Trypanosoma
lewisi and Leishmania tropica. J. Infect. Dis., 36: 245-308.

Specht, H. 1935. Aerobic respiration in Spirostomum ambiguum and the
production of ammonia. J. cell. comp. Physiol., 5: 319-33.

Standard Methods of Water Analysis. 1936. New York.

Stephenson, M. 1939. Bacterial Metabolism, London.

Stern, Kurt G. 1938. The relationship between prosthetic group and protein
carrier in certain enzymes and biological pigments. Cold Spring Harbor
Symp. Quant. Biol. 6: 286-300.

Szent-Gyorgyi, A., and I. Banga. 1933. Uber das Co-ferment der Milchsauer-
oxydation. Z. phys. Chem., 217: 39-49.

Tang, Pei-Sung. 1933. On the rate of oxygen consumption by tissues and
lower organisms as a function of oxygen tension. Quart. Rev. Biol., 8:
260-74.

Wachendorff, T. 1912. Der Gaswechsel von Colpidinm colpoda. Z. allg.
Physiol., 13: 105-10.

Warburg, O. 1926. Uber den Stoffwechsel der Tumoren. Berlin.

Witte, J. 1933. Bakterienfreie Ziichtung von Trichomonaden aus dem Uterus
des Rindes in einfachen Nahrbéden Zbl. Bakt. Abbt. I. Orig., 128:
188-95.

RESPIRATORY METABOLISM 403

Wurmser, R. 1932. La Signification biologique des potentiels d’oxydoréduc-
tion: Biol. Reva 7350-81.

Yorke, W., A. R. D. Adams, and F. Murgatroyd. 1929. Studies in Chemo-
therapy I. A method for maintaining pathogenic trypanosomes alive in
vitro at 37°C. for 24 hours. Ann. trop. Med. Parasit., 23: 501-18.

Zhinkin, L. M. 1930. Zur Frage der Reservestoffe bei Infusorien. Z. Morph.
Okol. Tiere, 18: 217-48.

Zweibaum, J. 1921. Richerche sperimentali sulla coniugazione degli Infusori.
I. Influenze della coniugazione sull’ assorbimento dell’ O, nel Para-
mecium caudatum. Arch. Protistenk., 44: 99-114.

CHAPTER Vil

THE CONTRACTILE VACUOLE

J. H. WEATHERBY

INTRODUCTION

SINCE THE FIRST description of the protozoan contractile vacuole, prob-
ably made by Spallanzani in 1776, few structures in these organisms have
received such intensive investigation. Unfortunately, solutions of many
of the perplexing questions which have arisen as a result of these studies
are not yet at hand. Indeed, much of the more recent work has given rise
to entirely new questions which are no less insistent in their demands
for answers than were the earlier ones. In the literature claims and
counterclaims are abundant; important discoveries have been made only
to be discarded because of lack of confirmation, or, in some instances,
because of direct contradiction. In view of the somewhat confused state
of the evidence concerning contractile vacuoles a re-survey of some of
the more important questions seems to be in order.

Probably the first question asked by the first investigator to see a con-
tractile vacuole was ‘“What is its function?’ Needless to say, this first
investigator did not learn the answer, and, in the opinions of many, the
most recent investigation probably does not supply the complete answer.
Following this question there have been others hardly less interesting.
Is it essential to life? Is it a permanent structure, or does it arise anew
at the beginning of each new cycle? Does it always occupy the same
position in the organism with respect to other structures? Is the vacuole
surrounded by a permanent membrane? Is its discharge to the exterior
through a permanent excretory pore? If there is no pre-formed excretory
pore, how may one explain the formation even of a temporary pore, and
once formed how is it closed again? What natural forces operate to expel
the contents of the vacuole? There are many other equally interesting
questions, but only a few can be considered at this time.

The most promising order for discussing these problems seems to be

THE CONTRACTILE VACUOLE 405

to deal first with those pertaining to origin of the vacuole—to see, if
possible, just where this organelle comes from, if it does not exist in
the cell as a permanent structure. Then, having traced its origin, questions
dealing with structure and function will follow in more logical order.
An attempt will be made to follow this general plan, but the very nature
of the subject will necessitate digressions from time to time.

While a conscientious review of the literature has been attempted, it
is quite possible that important publications have been overlooked. It is
hoped that this will prove not to be true, not only for the sake of com-
pleteness, but also for the sake of giving credit where credit is due. The
author herewith offers his apologies to anyone whose labors have not
been acknowledged.

THE ORIGIN OF CONTRACTILE VACUOLES

Metcalf (1910) noticed in amoeba of the proteus type that the vacu-
ole is surrounded by a layer of granules of the same approximate size
and appearance as the “microsomes” of the general cytoplasm. When
the vacuole is of moderate size, these granules form a layer on its surface
one granule thick; when the vacuole is fully distended, as just before
systole, there are spaces between the granules; but when the vacuole is
small the layer may be several granules thick. At systole the vacuole
usually collapses completely, and the granules may be seen clumped to-
gether in the region of the cytoplasm previously occupied by the vacuole.
The new vacuole arises in the midst of these granules, and is formed by
the fusion of several small vacuoles. According to Metcalf, who reported
observations which sometimes lasted for as long as several hours on a
single organism, the vacuole never arises in any other part of the body
under normal conditions, except among the granules which surrounded
it before its last contraction. From these observations he concludes that
the granules are associated in some way with the origin and the function
of the vacuole, and for this reason calls them “‘excretory granules.” How-
ever, he decides that the granules are not essential for life, since most
of them, together with the vacuole, may be removed from an Amoeba
by operation without a fatal result. Under these conditions a new vacuole
develops, although there are few if any granules to be seen surrounding
it when it first appears. Metcalf reaffirmed his statement concerning these
observations in 1926,

406 THE. CONTRACTILE: VACUOLE

Mast (1926) agrees with Metcalf concerning the frequent presence

of granules around the vacuole, but does not interpret this as indicating
a physiological association between them. This opinion is based on his
having observed vacuoles functioning perfectly normally without the
presence of a single granule in the immediate vicinity of the vacuole.
To these granules Mast applies the name “‘beta granules,” to distinguish
them from others of a different nature which are also present in the
cytoplasm. Mast and Doyle (1935) reinvestigated the relationship be-
tween granules and vacuole. By centrifuging amoebae it is possible to
cause stratification of various cytoplasmic constituents. Organisms treated
in such a manner can be operated on so as to remove all or any desired
portion of almost any one of the constituents, including these granules.
It was found by Mast and Doyle that removal of all or most of the gran-
ules resulted in the death of the organism. Removal of fewer granules
caused a decrease in pulsation frequency of the vacuole, which was di-
rectly proportional to the relative number of granules removed; that is,
pulsation frequency was found to be directly proportional to the number
of granules remaining. Removal of the contractile vacuole alone resulted
in the prompt formation of another. Concerning this same question,
Mast (1938, p. 312) more recently states:
The beta granules around the contractile vacuole vary greatly in number
and the layer of substance in which they are embedded varies greatly in
thickness, without any apparent variation in the function of the vacuole.
These facts indicate that neither the granules nor the layer of substance 1s
involved in the function of the contractile vacuole, at least not directly.

Howland (1924a) found that there is no concentration of granules
on the surface of the vacuole in Amoeba verrucosa, but she considers
it likely that the vacuole arises from the coalescing of small hyaline
globules, which in turn are derived from the dissolving of granules.
In any case, Howland traces the ultimate origin of the vacuole back to
granules in somewhat the same manner that Metcalf does, although in
A. verrucosa these granules are probably dispersed throughout the cyto-
plasm. On the other hand, Haye (1930) found in fixed and stained
preparations of A. verspertilio essentially the same relationship between
granules and vacuole as described by Metcalf and later by Mast; that is,
the filled vacuole is more or less covered by granules, and after systole
the new vacuole arises in the midst of these granules.

THEJGONTRACTILE) VACUOLE 407

Hall (1930a) studied the cytoplasmic inclusions in Tr7chamoeba after
osmic and silver impregnation. In a few instances he observed the adher-
ence of blackened globules to the outer surfaces of vacuoles. At first
glance these appeared to be vacuoles with heavily impregnated walls,
but close observation revealed the granular or globular nature of the
blackened material.

It must be remembered that these granules are not confined to the im-
mediate vicinity of the contractile vacuole, but usually are scattered
throughout the entire cytoplasm as well. If the origin of the vacuole is
associated with and dependent on the presence of these granules, then one
would expect other parts of the organism to be at least potentially capable
of giving rise to vacuoles, since some granules are present in other
parts. That such a phenomenon actually occurs in Amoeba has been ob-
served by various authors, among whom are Day (1927), as well as
Howland and Mast and Doyle. In this connection it 1s interesting to note
that Dimitrowa (1928) was able to induce formation of extra vacuoles
in Paramecium caudatum by interfering mechanically with the normal
function of those already present. These extra vacuoles usually appeared
to be entirely normal, although in a few instances there were no radial
canals. The customary number of vacuoles was restored at fission by
failure of the organism to form new ones if two extra ones had been in-
duced, or by the formation of one new vacuole if only one had been in-
duced artificially. In the event that there were three extra vacuoles, one
daughter cell received three and formed a single new one when it in turn
divided.

Haye (1930) investigated eight species from two orders of flagellates.
In Phacus pleuronectes he found that the walls of contractile vacuoles
contain lipoid granules which are arranged in a net-like fashion. In
Euglena pisciformis and Trachelomonas hispida the surfaces of both the
reservoir and the vacuoles show a granular structure. In Persdinium
steinii no granules were observed, nor were accessory vacuoles seen, ex-
cept in organisms obtained from a laboratory aquarium in which con-
ditions were thought to have been abnormal. A differentiated plasma
zone, reminding one of the ‘excretory plasma” of fresh-water Protozoa,
was noted around the pusule. In the wall of the pusule of P. divergens
were observed lipoid granules similar to those in the wall of the contrac-
tile vacuole of fresh-water Protozoa. Besides the two sac pusules, a col-

408 THE CONTRACTILE VACUOLE

lecting pusule with daughter vacuoles and numerous accessory vacuoles
were observed in Phalacroma sp. In Goniodoma sp. there were, besides
a sac pusule, a collecting pusule with daughter pusules and an accessory
vacuole. Only one large pusule was noted in Ceratzum hirundinella. \n
both orders of flagellates Haye believes that emptying of accessory vacu-
oles is accomplished by diffusion through the walls into the contractile
vacuole, rather than by coalescence with it.

Hall (1930b) found that in Menoidium, stained according to the Da
Fano silver method, the contractile vacuole is formed by the fusion of
several smaller vacuoles arising near the gullet.

The mode of origin of contractile vacuoles has been studied in a
greater variety of ciliates than in either rhizopods or flagellates, and in-
formation on this subject is proportionally more abundant. Taylor
(1923) observed in Explotes that the vacuole (V,), in its final form
immediately before contraction, is the result of the fusion of several
smaller vacuoles, and that these smaller vacuoles (designated as group
V.) in turn are formed by the fusion of still smaller vacuoles (group
V,). The smallest vacuoles in the series are thought to arise as the result
of the dissolving of granules, or to arise de novo. Thus Taylor suggests
granules as a possible source of vacuolar fluid, and he observed formation
of the vacuole by the fusion of several small accessory vacuoles. King
(1933), who studied Ezp/otes after impregnation with osmic acid, found
that the smallest visible accessory vacuoles (V,) have their origin at the
distal ends of a very large number of collecting canals, located just under
the ectoplasm on the dorsal surface of the ciliate. These canals radiate
like a sun-burst from the vicinity of the vacuoles, and seem to end blindly
in the protoplasm of the organism. These canals have a diameter of ap-
proximately 0.5 micron at their distal ends, and become relatively much
narrower as they pass away from the region of the vacuoles. The canals
are not visible in living organisms, but may be clearly demonstrated by
proper impregnation with osmic acid. On the basis of information now
available, it is difficult to tell whether the canals described by King and
the granules mentioned by Taylor represent different interpretations of
the same structures, observed under different conditions, or whether the
canals merely provide a means for the transport of fluids which have
originated in more distant parts of the body as a result of the activity
of granules.

THE CONTRACTILE VACUOLE 409

Of particular interest are the observations of MacLennan (1933) on
the Ophryoscolecidae, ciliates from the stomachs of cattle. The cycle of
the contractile vacuole was studied in both living and fixed material, in-
cluding the following genera: Ophryoscolex, Epidinium, Ostracodinium,
Polyplastron, Eudiplodinium, and Metadinium. In all these genera the
contractile vacuole is formed by the coalescence of small accessory vacu-
oles, just as in Ezplotes, as described by Taylor and also by King, and
in Amoeba, as described by Metcalf. These accessory vacuoles arise from
the dissolving of granules which are found in sharply defined regions
around the contractile vacuole in Exdzplodinium and Metadinium, in a
narrow dorsal strip of the ectoplasm in Ostracodinium, and in the whole
ectoplasm in Ophryoscolex and Epidinium. If one may be permitted to
assume that the canals and granules in Evp/oftes are identical, then the
mode of origin of the contractile vacuole in this form is quite similar to
that described by MacLennan for the Ophryoscolecidae; and in its funda-
mental features it also resembles that reported for amoebae as well as for
some of the flagellates.

In addition to the flagellates previously mentioned, Haye (1930) also
studied representatives of thirteen genera from four orders of ciliates.
In Opalina dimidiata, Isotricha prostoma, Spirostomum ambiguum, and
Nyctotherus cordiformis little was observed which suggests the mode
of origin of contractile vacuoles. Except for the fact that the walls of
the canals were found to contain lighter and darker zones—probably
because of the presence of lipoid granules—little was observed in Para-
mecium caudatum which may be associated with origin of vacuoles or
their contents. Rod-shaped entosomes were found closely packed about
the wall of the vacuole in Lionotus fasciola. In Stentor polymorpha the
wall of the vacuole is very delicate and shows only here and there a
granular structure; several secondary vacuoles are usually present. In
Blepharisma undulans, Balantidium entozoén, Polyplastron multivesi-
culatum, Ostracodinium gracile, and O phrydium versatile are to be found
granules (entosomes) within the wall, or closely associated with the
wall, of the contractile vacuole. The vacuole in Epistylis plicatilis is
formed from numerous secondary vacuoles; no granules or entosomes are
to be observed.

Von Gelei (1933) states that the vacuole system in Spathidium con-
sists of a primary vacuole, located usually in the posterior end of the

410 THE CONTRACTILE VACUOLE

organism. Around this are one or two rows of smaller secondary vacuoles,
which fuse and give rise to a new primary vacuole following systole.
Whether or not these secondary vacuoles originate from granules was
not ascertained by von Gelei. Essentially the same relationship between
primary and secondary vacuoles in Ble pharisma was described by Moore
(1934), who made the further statement that excretory granules could
not be observed. Both Wenrich (1926) and King (1928) found the
vacuoles of P. trichium to be vesicle-fed, although neither author men-
tioned the origin of these vesicles. Day (1930) found that the vacuolar
fluid reaches the elongated canal of Spirostomum by the fusion of small
vacuoles with the canal throughout its entire length. A similar source
of fluid in canals of P. caudatum was also reported, but in neither in-
stance was the origin of the accessory vacuoles mentioned.
Fauré-Fremiet (1925) observed the filling of contractile vacuoles in
several species of Vorticella by the discharge of small vesicles into the
vacuole. These vesicles originate in the wall of the vacuole, and cor-
respond to the “mural vacuoles’’ described by Haye (1930) for Cam-
panella, Chilodon, Dogielella, and some of the Ophryoscolecidae. Nas-
sonov (1925) also investigated Chilodon and Dogielella, and found
structure and mechanism of filling to be somewhat different from that
described by Haye. According to Nassonov, vacuoles in these forms do
not appear to have the thick walls described by Haye, nor even to have
any sort of membrane, but lie directly in the cytoplasm. However, there
is a strongly osmiophilic structure closely associated with them, which,
for Chilodon at least, and possibly also for Dogzelella, may be mistaken
for a thick wall or membrane under certain conditions. In both forms the
osmiophilic structure remains essentially unaltered in appearance after
collapse of the vacuole. Nassonov observed the origin of accessory
vacuoles (the mural vacuoles of Haye) in these osmiophilic structures,
and believes them to contribute to the filling of the contractile vacuole.
Many authors hold that in certain Protozoa, typified by Paramecium
caudatum, the question of origin of the contractile vacuole does not arise,
since in these forms the vacuole system is a permanent structure. This
view 1s not universally accepted, as will be pointed out later. But, whether
permanent or temporary, there still exists a no less fundamental question
as to the origin of the fluid which finds its way into these organelles. If
the origin of this fluid is associated with granules in many diverse organ-

THE CONTRACTILE VACUOLE 411

isms, as much of the evidence implies, then it would be somewhat un-
expected if such granules are not to be found distributed generally
throughout the Protozoa. MacLennan called attention to the fact that
many investigators have demonstrated a more or less solid membrane
surrounding vacuoles in a variety of Protozoa, these demonstrations
having been made by osmic-acid impregnation. Most of these workers,
according to MacLennan, used the warm method of impregnation advo-
cated by Nassonov. Hirschler showed that this method tends to produce
overimpregnation, resulting in the production of a heavy black band or
membrane in what is actually a granular zone. MacLennan found this
to be true in the Ophryoscolecidae, while impregnation by the cold
method shows this same region to be granular, a condition which can be
seen in living material. He further calls attention to the fact that figures
showing solid impregnation of the vacuolar walls of Chilodon and
Dogielella, published by Nassonov, of Balantidium by Bojewa-Petrus-
chewskaja, and of the Cyclopostheiidae by Strelkow, indicate a marked
granular roughening of the outer margin of the osmiophilic layer. He
takes this to indicate that what has been interpreted by these authors as
a solid membrane may, in fact, be only the result of overimpregnation
of a granular zone. Among the Ophryoscolecidae alone MacLennan found
various degrees of aggregation of these granules—from virtually none to
a very pronounced aggregation—around the contractile vacuole. He fur-
ther showed that localization of the origin of contractile vacuoles in these
forms is correlated with the degree of aggregation of the granules.

Of interest in this connection are the observations of Lloyd and Scarth
(1926) on the origin of vacuoles in Spirogyra. These authors found that
in sufficiently high concentrations even the most innocuous plasmolytes
may by themselves cause subsidiary vacuoles to arise in the cytoplasm. It
is not only by plasmolytes that this effect is produced however, but also
by other more readily penetrating substances such as the narcotics, chloro-
form, and ether, and by very low concentrations of salts. But without
any artificial influence, similar vacuoles may form in normal cells. Their
‘constant occurrence was demonstrated in the gametes during conjugation
in Spirogyra, and their excretory function in the taking up of water from
the central vacuole and its discharge to the exterior in typical ‘‘contrac-
tile” fashion was proved. The authors state that these vacuoles originate
from peculiar “‘lecithin-like” bodies already present in the cytoplasm.

412 THE CONTRACTILE VACUOLE

Scarth and Lloyd (1927) claim that the vacuolar wall arises from the
“‘kinoplasm” of Strasburger. They observed a reciprocal quantitative re-
lation between kinoplasm and mitochondria. The activity of kinoplasm
resembles that of lecithin, which is abundant in mitochondria. On the
basis of this resemblance, they conclude that water at least may accumu-
late in the vacuoles without the visible interaction of any other struc-
ture.

The observations and opinions reported in the foregoing pages, while
somewhat contradictory at times, point to two general conclusions con-
cerning the origin of contractile vacuoles. First, in the great majority
of forms, perhaps in all forms, fluid reaches the contractile vacuole
through the fusion of small vesicles, or accessory vacuoles, with the con-
tractile vacuole or its filling canals. The vesicles arise within what often
appears to be the wall of the vacuole; the accessory vacuoles usually
originate at a greater or less distance from the contractile vacuole, and
coalesce to form the latter. Second, vesicles originate within walls of
vacuoles which have been shown in many instances to be granular in
nature or to be intimately associated with granules; accessory vacuoles
have been reported by various authors as originating among granules
which may be closely associated with the vacuole, or occasionally re-
moved some distance from it. Thus it appears that in spite of their great
variety of shapes and general appearances under the microscope, con-
tractile vacuoles originate in a remarkably similar manner in all forms
so far investigated with this problem in mind.

In certain instances authors have reported the absence of granules in
the vicinity of the contractile vacuole, and from this have concluded that
granules are not concerned in the origin of vacuoles. In this connection
it must be remembered that in certain forms, e.g., Euplotes, some of
the Ophryoscolecidae, and apparently in others as well, the accessory
vacuoles which ultimately give rise to the contractile vacuole originate
at some distance from the ultimate site of the final vacuole. Also, gran-
ules frequently are visible only after osmium or silver impregnation.
Keeping in mind the greater vulnerability of negative evidence, one is
justified in the thought that perhaps a reéxamination of organisms for
which the absence of granules in the vicinity of the vacuole has been re-
ported, may reveal the presence of granules in other parts of the body,
either scattered or in aggregates. Such scattered granules, which are known

THE CONTRACTILE VACUOLE 413

to exist in some amoebae, may be the site of origin of new vacuoles when
the function of the original vacuole is disturbed by artificial means or
removed by operation. While such granules have not been demonstrated
to be scattered about in the cytoplasm of P. caudatum, their presence
would explain the origin of extra vacuoles in this form, when func-
tion of the original vacuoles is interfered with mechanically, as reported
by Dimitrowa. The origin of new vacuoles at fission would have a similar
explanation, since, as proposed by Dimitrowa, during fission the greater
abundance of metabolites would impose a necessity on the organism
essentially similar to interference with normal function. After fission,
when the daughter cells are smaller than the parent cell was immediately
prior to fission, and the metabolic rate is lowered, there no longer exists
a stimulus for the formation of extra vacuoles, and the daughter cells
appear quite normal, with the usual number.

THE STRUCTURE OF CONTRACTILE VACUOLES

The question of the structure of the contractile vacuole and its as-
sociated parts has occupied the attention of protozodlogists for many
years. As a result the main question has been broken up into several
parts, each concerned with a limited phase of this main question. Is the ~
vacuole surrounded by a permanent membrane? Is its discharge to the
exterior through a permanent excretory pore? If there is no permanent
pore, how may one explain the formation even of a temporary pore, and
once formed how is it closed again? Is the vacuole a permanent structure,
or does it arise anew at the beginning of each new cycle?

The dispute as to the presence or absence of a permanent membrane
surrounding the vacuole began over a hundred years ago, and continues,
with little to indicate that is will end within the near future. According
to Taylor (1923), to whom we may refer for a more detailed account
of the history of this question, the following investigators have written
in support of the idea of a permanent membrane: Ehrenberg, Siebold,
Claparéde, Lachmann, Degen, and Stempell. Those who believe that
the vacuole possesses no permanent wall are: Dujardin, Meyen, Stein,
Wrzesniowski, Perty, Schmidt, Zenker, Maupas, Rhumbler, Biitschli, Lan-
kester, and Khainsky. Taylor himself holds this view, at least for Ez-
plotes. Without reflecting unfavorably in any way on the researches of
those who worked on this subject prior to 1900, or possibly as late as

414 THE CONTRACTILE VACUOLE

1920, one must admit that only limited importance can be attached to
their opinions. Unquestionably most of these investigators were careful
observers, and expressed opinions only after due consideration of all the
factors which they were able to recognize. But the microscope of today
is a far different instrument from that of a hundred years ago, or even
fifty years ago; and chemical procedures, particularly those dealing with
colloids, have undergone extensive development. However highly one
may regard this or that early investigator, the fact remains that none
could have been better than the tools with which he worked, and ad-
mittedly the tools were poor. Consequently, the author maintains that
it is neither unkind nor unappreciative to propose that these various early
opinions be considered mainly as of historical interest, and of little
worth in settling the question as to the presence or absence of permanent
membranes, or of any kind of membrane for that matter, around con-
tractile vacuoles. The employment of the best of modern instruments
and techniques leaves the question in an unsatisfactory state.

Before presenting the more recent evidence concerning this question
of membranes, perhaps it will not be unwise to present briefly the more
fundamental question of what constitutes a membrane. Most textbooks
either avoid the issue more or less completely or describe the structure
and properties of the artificial membranes so often used in the laboratory
for experimental purposes. Although reliable information concerning
living membranes is scant, there is sufficient evidence to justify the divi-
sion of membranes into two types: morphological membranes and physi-
ological membranes. Morphological membranes are permanent struc-
tures which are frequently visible in living material viewed through the
microscope, and usually may be demonstrated more or less clearly by
suitable staining techniques. Apparently they consist mainly of a reticu-
lum, or framework, which is described by some authors as being com-
posed largely of protein. Such membranes are usually thought to possess
an appreciable amount of rigidity, and to serve primarily as supporting
structures. Free permeability in both directions is usually assigned to
them.

Physiological membranes are entirely different from morphological
membranes in many important respects. Usually they are considered to
be so thin as to be invisible even with the highest magnification. They
may possess a certain degree of rigidity, but probably much less than

THE (CONTRACTILE VACUOLE 415

morphological membranes with which they are often associated in liv-
ing material. Semipermeability, or more properly selective permeability,
is a property of all living physiological membranes. Colloid-chemists,
as well as many physiologists, are agreed that a physiological membrane
is simply a phase boundary, an interface between two different fluids.
In order that such phase boundary may be more or less permanent, it
is necessary that the two phases be only slightly miscible at most—the
more complete the immiscibility the more nearly perfect and permanent
the membrane. A very wide variety of molecules show polar phenomena;
that is, the two ends of the molecules are electrically and chemically
different. This results in orientation of molecules with respect to one
another and to various other molecules, in much the same manner that
a compass needle becomes oriented with respect to the magnetic poles of
the earth. This phenomenon of orientation is associated with organic
acids, alcohols, aldehydes, lipoids, fats, proteins, and many other so-
called “physiological” compounds. Thus at the interface of the two-
phase system, oil-water, the oil molecules (glycerol-esters of fatty acids)
become oriented in such a manner that the hydrocarbon ends of the
molecules project into the oil phase, whereas the glycerol ends project
into the water. Fat molecules undergo much more nearly perfect orienta-
tion than water molecules, although with the latter there appears to be
a certain degree of orientation. Such an aggregation and packing to-
gether of oriented polar molecules at an interface represents a physio-
logical membrane. Since protoplasm contains a variety of polar molecules,
the membrane formed between protoplasm and water is composed of
various types of molecules apparently arranged in the form of a mosaic.
The thickness of such a membrane has not been definitely established.
Some authors maintain that it is only a single molecule thick, or at most
only one or two milli-micra thick, but at least one author (Peters, see
Clark, 1933, p. 40) has advanced a theory according to which the cell
is composed of a three-dimensional protein mosaic, with the molecules
in the interior of the cell oriented on the surface film. Since the interior
of many cells is known to be fluid, the structure must be regarded as an
orientation rather than as an anatomical skeleton. This theory of Peters’s
agrees fairly well with certain evidence concerning the action of drugs
on cells. Without entering into the question as to how far orientation
extends beneath the surface layer, suffice it to say that it is well established

416 THE CONTRACTILE VACUOLE

that orientation of surface molecules occurs at the interface between two
different phases (provided, of course, that at least one phase contains
polar molecules), whether the system is composed of oil and water or
protoplasm and water. The converse of this is equally true; since the
_ivery existence of this oriented layer depends on the presence of two
different phases, the removal of one phase necessarily results in disintegra-
tion of the membrane. Whether or not this surface layer of oriented
molecules actually comprises the true physiological membrane may be
subject to debate, but the importance of such a membrane is obvious,
since it not only separates the organism from its surroundings but at
the same time provides the only means of communication between the
interior and the exterior of the cell.

If one accepts the idea of a physiological membrane as a layer of
oriented molecules at a phase boundary, as outlined briefly above, then
it necessarily follows that any cell vacuole which contains a fluid differ-
ent from cytoplasm must be surrounded by such a membrane. In the
light of the information available at the present time, membranes around
protozoan contractile vacuoles probably should be considered as tem-
porary; although it is not inconceivable that in some forms the new vac-
uole may be formed with such rapidity, in the midst of oriented mole-
cules remaining after systole, that dispersion of the membrane does not
have time to occur before the second phase is present again. If such condi-
tion obtains, then the membrane may be considered as having a greater
or less degree of permanence. There are numerous references to such
physiological membranes in the literature on contractile vacuoles, so it
appears that the idea has gained rather wide acceptance. The controversy is
not so much concerned with such membranes as with the presence of
absence of morphological, and hence permanent, membranes.

Before leaving the subject of physiological membranes, there are sev-
eral phenomena which may be discussed profitably with this concept in
mind. Repeatedly authors speak of the coalescence of accessory vacuoles
to form contractile vacuoles. Taylor (1923) refers to this, and considers
coalescence to be due to a reversion of the gel state of the surrounding
film to the sol state. He further states that vacuoles are surrounded by
highly viscous boundaries of endoplasm, and that the consistency of the
papilla pulsatoria strikingly resembles that of the endoplasmic boundaries.
Without raising the question as to whether or not coalescence of accessory

THE CONTRACTILE VACUOLE 417

vacuoles and the simultaneous rupture of vacuole wall and papilla pul-
satoria represent sol-gel reversibility, it is obvious that exactly these
phenomena must be anticipated, on the basis of the concept of physiologi-
cal membranes such as described above. When two accessory vacuoles lie
touching each other, the cytoplasmic phase of the two-phase system is
pushed aside, at the same time removing the basic forces on which the
presence of these membranes depends. In the absence of these forces,
the membranes disintegrate at the site of contact, and the two vacuoles
fuse into one. Likewise, when the filled vacuole comes in contact with
the papilla pulsatoria, one phase (the cytoplasmic phase again) is pushed
aside, the membranes disintegrate at the site of contact, and the contents
of the vacuole are discharged to the exterior. After discharge the two-
phase system is again established, since there is cytoplasm on one side of
the pore (within the organism) and water on the other (outside the
organism), whereas prior to discharge there was water on the outside as
well as a fluid composed chiefly of water within the vacuole. The papilla
is formed in this manner from the vacuole wall, which readily accounts
for the similarity noted by Taylor.

Other phenomena which can be explained in like manner by the pres-
ence of physiological membranes are easy to find in the Protozoa. The
ingestion of food by Amoeba is essentially the result of fusion of the
walls of the organism after they have been extruded around the food
particle in such a manner as completely to enclose it. The two-phase
system exists as long as there is water on one side of the cell membrane
and cytoplasm on the other; but when the engulfing process is complete
and cell membrane is in contact with cell membrane with no water
separating the two portions, the membrane disintegrates at the site of
contact, and continuity of cell structures as well as of the vacuole mem-
brane is established. The food-vacuole membrane persists as long as
there is water within to maintain the two-phase system. In a similar man-
ner one may explain the readiness with which an amputated fragment
of an amoeba unites with the parent body when the two portions come
together, although it has no bearing on the fact that a fragment from a
diverse strain is refused. It has also been observed that occasionally an
amoeba attempts to engulf a relatively large organism, such as P. cauda-
tum, but is unable to accomplish this completely. The Paramecium is
squeezed in two, apparently, with half inside the amoeba and half out-

418 THE CONTRACTILE VACUOLE

side. Calculations have been made of the physical force necessarily ex-
erted by the amoeba to accomplish this, but they have not taken into
account some of the properties of physiological membranes, such as spon-
taneous disintegration when membrane comes into direct contact with
membrane. The adherence of conjugating organisms may be dependent,
likewise, on these same properties of membranes. While it is interesting
to speculate on such matters, it must be admitted that these remarks on
feeding amoebae are purely speculative, with little other than superficial
observation to support them.

Concerning the presence of a permanent (morphological) membrane
surrounding the contractile vacuole, there are diverse opinions. These
diverse opinions apply not only to different species but even to the same
species. Howland (1924a) found that the contributory globules as well
as the vacuole may be removed from the organism to the surrounding
water, where they retain their identity for an indefinite period of time.
This may be taken to indicate a considerable degree of permanence of the
vacuole wall, such as would be possessed by a morphological membrane,
although Howland 1s of the opinion that these vacuole membranes are
temporary. A temporary physiological membrane, formed of oriented
molecules in a compact layer, may be expected to retain its identity for
an appreciable period of time before dispersion of the molecules occurs,
but it hardly seems probable that this “appreciable period of time’’ can
be more than a few minutes. Day (1927) expresses the opinion that the
vacuole wall in A. proteus is a ‘condensation membrane,” or gel, disap-
pearing with each contraction. By ©
likely implies such a structure as has been described above as a physiologi-
cal membrane, so that the two terms may be taken as synonymous. Con-
cerning A. proteus, Mast (1938, p. 307) states: “At the surface of the
contractile vacuole under the layer of substance containing the beta gran-
ules, there is a layer or membrane about 0.5 micron thick which is opti-
cally well differentiated from the adjoining substance on either surface,
for under favorable conditions a line indicating an interface can be clearly
seen at both these surfaces.’’ Whether this membrane is a permanent
structure or is formed anew with each successive vacuole was not sug-
gested by Mast. After an examination of fixed, stained, and sectioned
material, Haye (1930) concluded that vacuole membranes are lacking
in Amoeba, although it is probable that this author referred to morpho-

condensation membrane” Day very

THE CONTRACTILE ‘'VACUOLE 419

logical and not to physiological membranes, since these latter cannot
be demonstrated in this manner.

Among the flagellates, Nassonov (1924) found an osmiophilic mem-
brane surrounding the vacuole in Chilomonas paramecium. Haye (1930)
could distinguish no vacuole wall in the Euglenoidina, but in many other
flagellates examined by him distinct walls were visible in stained ma-
terial.

Among the ciliates, morphological membranes are reported in a great
variety of organisms by many investigators. Nassonov (1924) reports
osmiophilic walls for the vacuoles in Paramecium caudatum, Lionotus
folium, Nassula laterita, Campanella umbellaria, Epistylis gallea,Z ootham-
nium arbuscula, and Vorticella sp. Fauré-Fremiet (1925) confirmed
the findings of Nassonov, using several species of Vorticella, in which
osmiophilic walls were observed, even after collapse of the vacuole.
Young (1924) concludes from studies on P. caudatum stained with iron
hematoxylin that the vacuole system is a permanent and continuous struc-
ture. King (1928) arrived at essentially the same conclusion concerning
the vesicle-fed system of P. trichium. Wenrich (1926) observed definite
vacuole walls in P. ¢richium stained with Mayer's hemalum or Heiden-
hain’s iron-alum-hematoxylin. Concerning this Wenrich states (p. 89):

It was somewhat surprising to find how distinctly the vacuolar walls showed
in fixed and stained specimens. The relative thickness of the wall is note-
worthy and it usually appears to be laminated. In sectioned material the walls
contained strands of more or less intensely staining material, suggesting
the presence of contractile fibers.

Von Gelei observed osmiophilic walls in P. caudatum (1925, 1928)
and in Spathidium giganteum (1935). Haye (1930) found thin vacuole
walls in the following forms: Blepharisma undulans, Lionotus fasciola,
Ophrydium versatile, Stentor polymorphus, Spirostomum ambiguum,
Balantidium entozo6n, and Isotricha prostoma. Thick walls were ob-
served in Campanella, Chilodon, Dogielella, Paramecium, and the Oph-
ryoscolecidae. As previously mentioned, Nassonov (1925) examined
Chilodon and Dogielella after osmium impregnation and concluded
that the vacuoles possess no membranes, but lie directly in the cytoplasm.
In these latter organisms, structures considered by Nassonov to be the |
Golgi apparatus surround the vacuole in such a manner that they may be
mistaken for vacuole walls in certain preparations.

420 THE’ CONTRACTILE VACUOLE

The presence of definite vacuole walls or morphological membranes
around the vacuoles of ciliates is not accepted by all authors. Thus, Taylor
(1923) believes the vacuole in Ezp/lotes to disappear completely at sys-
tole, and to be replaced by an entirely new structure. If a morphological
membrane were present, this could hardly obtain, although Taylor was
able to distinguish a ‘‘highly viscous boundary’’ of endoplasm surround-
ing the vacuole. Moore (1931) was unable to cause osmication of the
vacuole wall in Blepharisma undulans, using the technique of Nassonov,
and from this she concludes that the vacuole lacks a permanent wall.
This opinion was again expressed (1934) after further observation.
The findings of Moore afte in opposition to those of Haye, who re-
ported thin vacuole walls in the same species. Day (1930) concludes
from his observations on Paramecium caudatum, Spirostomum ambi-
guum, and S. teres that vacuoles in these forms are temporary structures
which disappear at systole. King (1935, p. 564) found that:

The permanent components of the contractile vacuole system in Paramecium
multimicronucleata include the pore with its discharging tubule, and the
feeding canals, each made up of a distal excretory portion, an ampulla and
an injection tubule. . . . The membrane of the contracting vacuole is a

temporary structure, disappearing at systole. The pore is closed by the remnant
of the old vacuole which ruptures at the next systole.

Essentially the same was found in P. aurelia. These observations were
made on material osmicated at 38° C. It is somewhat surprising that
the vacuole proper of the contractile vacuole systems in P. multimicro-
nucleata and P. aurelia is a temporary structure, replaced anew after each
contraction, whereas that of the closely related species P. caudatum is
commonly believed to be a permanent structure. Perhaps the stainable
vacuole wall described by Young for P. cavdatum represents the same
kind of material which King believes closes the excretory pore follow-
ing systole in P. multimicronucleata and P. aurelia.

After examining both living and stained material, Fortmer (1926)
concludes that the membrane of the vacuole in Protista is a temporary
structure, which, after fulfilling its purpose, closes the excretory pore
during the period of diastole. He further believes that all surface layers
sharing in the excretion process have the property of fusing together again
merely on contact.

The present state of the knowledge concerning the presence or absence

THE CONTRACTILE VACUOLE 421

of membranes around contractile vacuoles is exceedingly unsatisfactory.
Aside from the fact that many investigators admit the probability of,
or in some instances the necessity for, a physiological membrane of the
type described above as composed of molecules oriented and more closely
packed together at the phase boundary, very little of a positive nature is
known. It is difficult to understand how a vacuole without any sort of
membrane can retain its identity in cytoplasm with which its contents ap-
pear to be freely miscible. That most of the cellular contents are freely
miscible with water is indicated by the fact that discharge of the cell
contents into the surrounding water, following rupture of the cell wall,
is soon followed by dispersion of most of the cytoplasm into the sur-
rounding medium; usually only granules, of one sort or another, re-
main to indicate the original position of the extruded material. If the
vacuole content is mostly water, a belief quite generally if not universally
held, how can a vacuole ever be formed in the absence of any kind of
membrane to prevent this water from flowing back into the cytoplasm as
rapidly as it is mobilized?

As indicated above, a great many investigators have demonstrated
structures which were interpreted as vacuole “walls.” In some material
these walls were visible in living organisms as layers of substance, opti-
cally different from substances on either side of it. In other material the
walls were visible only after fixation and staining. One is more or less
obliged to accept an author’s description of structures in material ex-
amined by him; but an observation sometimes is subject to two entirely
different interpretations. This is clearly shown by MacLennan (1933) in
his work on the Ophryoscolecidae, as pointed out earlier. In several in-
stances the presence of morphological membranes is claimed by various.
authors, on the basis of observations on what may have been overim-
pregnated material. Because of the extreme thinness of physiological
membranes, it is doubtful if they ever can be demonstrated visually, but
evidence obtained from the study of other colloidal systems indicates that
they almost certainly exist.

THE FUNCTION OF CONTRACTILE VACUOLES

During the years that have intervened since the discovery of the con-
tractile vacuole, an extensive literature concerning its function (or func-
tions) has accumulated. Excellent reviews of this literature have been

422 THE CONTRACTILE VACUOLE

published from time to time (Howland, 1924b; Day, 1927; Lloyd,
1928). Therefore no attempt will be made to present another review
at this time, except in so far as the works to be mentioned have a direct
bearing on one or the other of the two functions generally conceded to
be most probable.

Of the various functions assigned to the contractile vacuole those of
excretion of metabolic waste products and regulation of hydrostatic pres-
sure within the cell have received most frequent support. Some authors
prefer to limit “metabolic waste products’ to nitrogenous substances,
although others include carbon dioxide as well. In view of the scarcity
of evidence bearing directly on the subject, it hardly seems advisable at
this time to distinguish between different kinds of metabolic wastes. On
the other hand, if one is to understand excretion to mean the expulsion of
any sort of waste material from the organism, then the function was defi-
nitely established as excretory when Stokes (1893), and later Jennings
(1904) proved the discharge of the vacuole to the exterior. But such a
generalization offers little satisfaction.

Probably the earliest suggestion that the vacuole is an excretory organ-
elle was made by Stein and Schmidt (see Kent, 1880, p. 69), who
stated that “the functions discharged by the contractile vacuole are ex-
cretory and correspond most nearly with that of the renal organs of the
higher animals.” Griffiths (1888) made the statement, based on his
own experiments, that the vacuole performs the function of a kidney,
and that its secretions are “capable of yielding microscopic crystals of
uric acid.”” As material for these experiments he used Amoeba, Parame-
cium, and Vorticella. In describing these experiments, Griffiths says (p.
132)

After the addition of alcohol minute flakes could be distinctly seen floating
in the fluid of certain vacuoles. Bearing in mind the murexide reaction, there
is every reason to believe that these flakes are nothing more or less than
minute crystals of uric acid.

These experiments were repeated many times, generally with positive
results, indicating the presence of uric acid. At times, however, the vacuole
was found not to contain the slightest trace of uric acid. Howland
(1924b) repeated these experiments using Paramecium, Centro pyxts,
and Amoeba, but always with negative results. However, uric acid was
found in cultures of Paramecium and Amoeba by Howland, and the

TEECCON TRAC TILE) VACUOLE 423

concentration was observed to be roughly proportional to the age of the
culture. From this she concludes that uric acid is excreted by these forms,
though probably not by the vacuole.

Experiments of Nowikoff (1908), Shumway (1917), and Riddle
and Torrey (1923), in which the effects of thyroid feeding and the re-
sponse of Paramecium to thyroxin were observed, offer further though
indirect evidence in favor of the excretory function. Flather (1919)
found that epinephrine, posterior pituitary extract, and pineal gland ex-
tract produce similar results—an acceleration in pulsation frequency, and
a dilatation of the vacuole. Since these drugs cause diuresis in vertebrates,
the action on vacuoles may be interpreted as resembling stimulation of
excretion.

Weatherby (1927) found that urea is excreted by Paramecium cauda-
tum, but was unable to detect urea in the fluid of the contractile vacuole |
by means of the micro-injection of his own modification of the xanthydrol
reagent of Fosse (1913). This reagent yields positive results with dilu-
tions of urea as great as one part in 12,000. Calculations based on the
volume of fluid eliminated by vacuoles and the quantity of urea excreted
by known numbers of organisms in mass cultures indicate that the con-
centration in fluid of the vacuole would be of the order of one part in
2,000 or 3,000, if all the urea were excreted via this route. It therefore
appears that at most only a small part of the total urea is excreted in this
manner. After removal of the fluid from the contractile vacuole of
Spirostomum by means of micro-manipulation apparatus, and subsequent
hydrolysis with urease, Weatherby (1929) found urea to be present in
the vacuolar fluid in a concentration of about one part in 100,000. Cal-
culations of the rate of excretion of urea by known numbers of Sperosto- |
mum in mass cultures indicate that this amount of urea accounts for only
about one percent of the total urea excreted.

Parnas (1926) concludes from observed differences in pulsation fre-
quency that the vacuole is mainly excretory in marine Protozoa, and both
excretory and osmotic-pressure-regulatory in fresh-water forms. The ex-
cretory function is accepted apparently without reservation by von Gelet
(1925, 1928), who homologizes the various parts of the vacuole system
in Paramecium with the vertebrate kidney, ureter, bladder, and urethra,
although he admits the possibility that this system may aid in removing
excess water from within the organism. In Paramecium, von Gelet states

424 THE CONTRACTILE VACUOLE

that the vacuole removes approximately ten times as much water as is
taken in with food, a fact which he fails to correlate with his claim of a
predominantly excretory function. Day (1927) suggests that vacuoles in
Amoeba originate in “the fusion and coalescence of ultramiscroscopic
droplets of soluble katabolic waste which may include water of osmosis.”’
He observed that conductivity water increases size, number, and pulsation
frequency of vacuoles. Essentially the same observations and conclusions
were extended by him to Paramecium and Spirostomum (1930). Mac-
Lennan (1933) observed in the Ophryoscolecidae that granules accumu-
late around the vacuole during the early part of diastole and then are
gradually reduced in number. The formation of accessory vacuoles in
these granular regions involves a solution of granules in the vacuolar
fluid. He suggests this as a possible method for the elimination of kata-
bolic wastes. Since he found the pellicle of these organisms to be rela-
tively impermeable, MacLennan believes an excretory function to be all
the more probable in these forms, since the vacuole is the only visible
means for the removal of wastes. Adolph (1926) found that no change
of external conditions alters significantly the rate of elimination of fluid
by vacuoles of Amoeba, and from this concludes that water is not elimi-
nated merely because it has unavoidably diffused into the body.
Dimitrowa (1928) observed (as mentioned earlier) that mechanical
interference, as by pressure on the cover glass, induces the development
of extra vacuole systems in Paramecium. In most instances these vacuoles
assumed normal structure, size, and pulsation frequency, although in some
cases there were actively pulsating vacuoles with no radial canals. Dimit-
rowa explained the formation of extra vacuoles, as well as of those nor-
mally formed at fission, by the assumption that if for one reason or an-
other the excretory organs become inadequate to remove wastes, extra
organs are formed. If one vacuole is rendered ineffective by mechanical
interference, then another is formed to take over its function. Likewise,
since metabolism is thought to be increased during fission, new vacuoles
are formed to care for the increased production of wastes. Extra vacuoles,
induced artificially, obviate the necessity for the formation of a like
number at fission, since an ample excretory function is already present.
Somewhat contradictory evidence has been presented by various authors
concerning the nature of nitrogenous end products of metabolism in the
Protozoa. As previously mentioned, Griffiths (1888) reported uric acid

LAE IGONTRACTILE: VACUOLE 425

in the vacuolar fluid of several forms. Howland (1924b) was unable to
confirm this, but found uric acid in mass cultures of Amoeba and Parame-
cium. Weatherby (1929) found urea to be excreted by Paramecium and
Spirostomum, but detected no ammonia nor uric acid; ammonia, as well
as a questionable trace of uric acid, were found to be excreted by Didz-
nium. Specht (1934) found that Spzrostomum exctetes ammonia, the
amount being augmented by lack of oxygen and minimized by abundance
of it. Weatherby noticed that cold aqueous extracts of many substances
commonly used in culture media (hay, wheat, barley, rye, oats, malted
milk, beef extract, blood fibrin, and blood albumen) yield positive tests
for uric acid, and suggested this as a possible source of the uric acid
found by Howland in cultures of Paramecium and Amoeba. Lwoff and
Roukhelman (1926) found amino-nitrogen as well as additional nitro-
gen, which they report as ammonia plus amide-nitrogen, in pure cultures
of Glaucoma. No urea nor uric acid was present. Doyle and Harding
(1937) analyzed the food (in the form of Psewdomonas) supplied Glau-
coma, and found that most of the nitrogen present was excreted as am- |
monia approximately six hours after ingestion of food. No urea was
detected.

If the contractile vacuole is active in excretion of nitrogenous wastes,
as is frequently maintained, then one would expect it to be able to ex-
crete certain dyes which had been injected into the cytoplasm. Many at-
tempts doubtless have been made to demonstrate such a phenomenon,
but few accounts of such experiments are to be found in the literature.
Apparently negative results have discouraged publication. A personal com-
munication from one investigator reports complete failure to demon-
strate elimination of dyes by way of the contractile vacuole, although
the dyes used in these experiments are known to be excreted readily by
the kidney of higher forms. Howland and Pollack (1927) found that
Picric acid, injected into the cytoplasm of Amoeba dubia, is picked up
and excreted by the contractile vacuole.

Ludwig (1928) studied gaseous metabolism in Paramecium, and
found that the amount of oxygen dissolved in water taken with food is
insignificant, compared with the respiratory requirement of the organism.
For the satisfaction of the oxygen requirement, there must be a quantity
of water, saturated with oxygen, equivalent to 260 to 30,000 times the
amount taken in through the gullet. Oxygen intake must also occur

426 THE CONTRACTILE VACUOLE

through the cell surface. The amount of water expelled by the vacuole
corresponds within reasonable limits to that necessary for the excretion
of carbon dioxide, if it is assumed that this water is saturated with the
gas. From this Ludwig concludes that the vacuole is of special significance
not only in the regulation of osmotic pressure within the cell, but also
in excretion of carbon dioxide.

Evidence bearing directly on the excretory nature of the vacuole func-
tion is exceedingly scant, and for the most part negative. The reason
for the relatively few observations is immediately apparent to all who
have attempted experiments of this nature. Perhaps a more thorough
investigation of the nature of nitrogenous waste products in other Protozoa
will suggest more effective methods for answering the question, by reveal-
ing other chemicals which may be detected more readily.

Hartog (1888), Degen (1905), Zuelzer (1910), Doflein (1911),
and others maintain that the contractile vacuole is concerned primarily
in the regulation of hydrostatic pressure within the cell, or the preven-
tion of overdilution of the cell contents by water taken into the cell
in feeding as well as through the cell membrane by osmosis. Harvey
(1917) found that Noctiluca, which normally lives near the surface of
sea water, sinks when transferred to diluted sea water, but ultimately
rises to the surface again. Meanwhile, expansion takes place, owing
to the taking up of water by the organism. This passage of water from
exterior to interior is from a region of higher concentration to one of
lower concentration, and therefore contrary to the laws of osmosis in
simple systems. When organisms are transferred from diluted sea water
to pure sea water, they shrink, vacuoles are formed, and these appear to
discharge to the exterior in somewhat the same manner as contractile
vacuoles. This appears to aid in reéstablishing the normal salt concentra-
tion within the organism. Hance (1917) made extensive observations
on a race of Paramecium possessing extra contractile vacuoles. He found
that these animals cannot withstand immediate immersion in water
containing 0.5 percent sea salt, but can be acclimated gradually to this
concentration. The number of vacuoles is not reduced by this treatment,
but the pulsation frequency is reduced. This response may be taken
to indicate a decreased rate of entry of water into the cell, presumably
because of the higher external osmotic pressure; but Hance observed
also an increased viscosity and toughness of the pellicle, which may indi-

THE CONTRACTILE VACUOLE 427

cate that a decreased permeability of the cell wall is partly responsible.

Herfs (1922) investigated the effects of changes in tonicity of the
external medium on several kinds of organisms, both free-living fresh-
water forms and parasitic forms. He found the pulsation frequency in
Paramecium to be decreased to about one-fourth the normal when the
organism is transferred from fresh water to 0.75 percent NaCl solution.
Lower concentrations of salt produce less marked changes. Gastrostyla
Steini’ showed essentially the same reaction, except that organisms kept
for about fourteen days in one-percent NaCl solution were found to
contain no contracting vacuoles. With Gastrostyla the vacuole seems to
disappear at a NaCl concentration of 1.1 percent to 1.3 percent, and
to reappear at a concentration of about 0.5 percent. The pulsation fre-
quency of Nyctotherus cordiformis, an intestinal parasite of the frog,
was found to vary between wide limits, presumably because of cor-
responding variations in the water content of the medium. Graded pulsa-
tion frequencies were observed zz vitro when the exterior medium varied
from tap water to one-percent NaCl solution. Opalina ranarum, which
possesses no contractile vacuole, can adapt itself to relatively wide varia-
tions in tonicity of the exterior medium without developing a vacuole,
if the changes are made gradually. From this Herfs was led to doubt
whether or not a vacuole is necessary for the prevention of overdilution
of the cytoplasm. He noted further that the lack of a vacuole in O palina
goes hand-in-hand with the lack of a cell mouth; whereas Nyctotherus
possesses both a cell mouth and a vacuole. From this he assumes that it
is the water taken in through the mouth that is pumped out by the
vacuole. Herfs does not seem to be altogether consistent in this idea,
since he further states, as his opinion, that in ordinary cases at least
the water taken in through the entire cell surface is of decisive signifi-
cance for the appearance of the vacuole. With respect to the adaptation
of organisms possessing no vacuoles to variations in tonicity of the ex-
terior medium, Herfs seems to have overlooked as an explanation, the
possibility of an interchange of salts between cell and medium, a pos-
sibility which will be mentioned again later.

Eisenberg (1926), assuming the volume of Paramecium to be ap-
proximately equal to that of an ellipsoid of rotation having the same
dimensions, found that the two vacuoles discharge a volume of liquid
equal to that of the organism in 20 minutes, 51 seconds. The average

428 THE CONTRACTILE VACUOLE

amount of fluid expelled in three experiments, each of 10 minutes’ dura-
tion, was 45,000 cubic micra; and the average amount of water taken
into the organism with food was 11,700 cubic micra, or about one-
fourth the total amount expelled. In animals not feeding, the entire
amount of fluid expelled entered the body otherwise than with food.
Basing his theory on the work of Nirenstein as well as on his own
observations, Eisenberg concludes that water penetrates the body by way
of the peristome, even when a food vacuole is not in process of forma-
tion. It was further observed that an increase in the osmotic pressure
of the exterior medium results in a decrease in pulsation frequency, and
that equi-osmotic solutions of different chemicals may cause different
degrees of slowing.

Fortner (1926) concludes, largely on the basis of theoretical con-
siderations, that the vacuole operates for the preservation of vital cell
turgescence, since there must be an accumulation of water in the proto-
plasm because it is surrounded by a membrane impermeable to water
and aqueous solutions.

Eisenberg (1929) investigated the relationship between the osmotic
pressure and the pulsation frequency of the vacuole in Balantidium
entozoon. He found that the frequency of the formation of vacuoles
depends on the osmotic pressure, and is all the greater the more the
pressure is reduced below that of the usual environment. A pulsation
frequency accelerated by the removal of the organism to a medium
of lower osmotic pressure does not remain accelerated, but returns to
normal after a certain period of time. The rapidity and extent of this
return to normal are proportional to the osmotic pressure of the medium.

Frisch (1935) was unable to adapt Paramecium caudatum and P.
multimicronucleata to sea water, the organisms dying when the concen-
tration reached 40 percent. However, among other marked changes in
the organisms was a pronounced decrease in pulsation frequency of the
vacuoles.

Day (1930) concludes from his observations on Spirostomum and
Paramecium that the vacuole is a hydrostatic organelle, which functions
also in elimination of metabolic wastes. He found conductivity water
to increase the size, number, and rate of pulsation of vacuoles. The
lowering of the temperature of the culture medium slows the organisms
and retards the contraction rate of vacuoles, while the raising of the

THE CONTRACTILE VACUOLE 429

temperature increases movement and pulsation frequency.

Kitching (1934) found that the rate of output of fluid from the
contractile vacuole of a fresh-water peritrich ciliate is decreased to a
new steady value immediately, when the organism is placed in a mixture
of tap water and sea water. The rate of output returns to its original
value immediately, when the organism is replaced in tap water. Pulsa-
tion is stopped when the medium contains more than 12 percent of sea
water. Transference of marine peritrich ciliates from sea water to
mixtures of sea water and tap water leads to an immediate increase in
body volume, to a new and generally steady value. Return of the organ-
isms to pure sea water results in an immediate return of body volume
to normal, or less. When the concentration of sea water is less than
75 percent, the pulsation rate increases, and then generally falls off
slightly to a new steady value which is still considerably above the normal
in sea water. The maximum sustained increase in rate observed by Kitch-
ing was 80-fold. From these observations it is concluded that the vacuole

is probably a regulator of hydrostatic pressure in the fresh-water Protozoa, |

but in those marine Protozoa which possess vacuoles the functions remain
obscure.

Hyman (1936) believes that the vacuole in Amoeba vers pertilio serves
to discharge water which has necessarily entered the cell from a hypotonic
medium.

One of the most remarkable instances of adjustment of a protozoan to
abnormal media is shown in the experiments of Hopkins (1938) on the
marine amoeba, Flabellula mira. He found that this amoeba can be
cultured in any concentration, from sea water diluted twenty times with
fresh water to sea water concentrated ten times by evaporation. It never
forms contractile vacuoles such as are typical for fresh-water Protozoa.
The food vacuoles, when extruded from the cell, contain large quantities
of water as well as fecal material. The rate of elimination of fluid by
means of these vacuoles is inversely proportional to the concentration
of the medium, and directly proportional to the volume of the amoeba.
When the concentration of the medium is decreased, the organism swells
at first, and then shrinks to its original volume. During shrinkage,
elimination of fluid by food vacuoles does not nearly account for the
volume loss. If the concentration of the medium is increased, the amoeba
shrinks at first, and then swells to its original volume. Only a small

430 THE CONTRACTILE VACUOLE

incfease in concentration is necessary to cause shrinkage, indicating an
osmotic value for the cytoplasm, after adjustment, only slightly above
that of the medium. Hopkins concludes from these observations that
when the medium is either diluted or concentrated, the organism auto-
matically loses or gains osmotically active substances to or from the
medium respectively, in such proportion that when adjustment is com-
pleted the osmotic value of the cytoplasm is but slightly higher than
that of the medium, and that this is accomplished independently of the
action of vacuoles. Herfs, whose observations on Opalina have been
described previously, may find such an explanation applicable to the
unexpected behavior of this organism. It is interesting to speculate as
to whether or not such adjustment to external osmotic-pressure differences
as postulated for Flabellula, and possibly Opalina, represents the most
primitive type of mechanism for this type of adjustment with Noctiluca
(see Harvey 1917), which develops contractile vacuoles when the tonic-
ity of the external medium is greatly reduced, occupying a position inter-
mediate between Flabellula and those forms which possess vacuole sys-
tems.

In spite of the quite extensive literature dealing with the question,
one is obliged to admit that virtually nothing has been proved beyond
question concerning the function or functions of contractile vacuoles. Car-
bon dioxide and nitrogenous wastes of one sort or another are undoubtedly
excreted by Protozoa. It is reasonable to suppose that at least a part of these
highly soluble wastes finds its way into the fluid of the vacuole and is
excreted in this manner. Many authors hold that it is not only reasonable
to suppose this, but that it is unreasonable to suppose that it does not
occur. But, be that as it may, the contractile vacuole certainly has not
been proved an organelle whose main function is excretion of metabolic
wastes. Likewise, there is indisputable evidence that many fresh-water
Protozoa show a decreased pulsation frequency when the tonicity of the
exterior medium is increased; and there is equally valid evidence indicat-
ing that the reverse occurs when many marine and parasitic Protozoa
are transferred to a medium having a decreased tonicity. One may regret
the fact, but it is none the less true, that these observations prove
nothing more than the bare statement which describes the observations.
They strongly suggest that the vacuole operates to prevent excessive
dilution of the cytoplasm, or to regulate osmotic pressure within the

THE CONTRACTILE VACUOLE 431

cell, but beyond this the interpretation is subject to criticism. As stated
by Calkins (1926), these supposed functions are not necessarily exclu-
sive, and the possibility still exists that other functions, as well as
these, are performed by the contractile vacuoles.

CONTRACTILE VACUOLES AND THE GOLGI APPARATUS

Few publications within recent years on the general subject of con-
tractile vacuoles have aroused as much interest or stimulated as much
constructive research as that of Nassonov (1924), in which he sug-
gests that the vacuole in Protozoa is homologous with the Golgi ap-
paratus in metazoan cells. Neglecting for the moment the ultimate status
of this proposed homology, one must admit that this article is responsi-
ble, either directly or indirectly, for valuable work which otherwise
might have been delayed indefinitely.

Before attempting a discussion of the literature bearing on this pro-
posed homology, a few words concerning the general nature of the
Golgi apparatus may be of benefit. Its discovery in 1899 is attributed
to the man whose name it bears. For approximately twenty-five years
after the first description of such a structure its actual existence was
doubted by many competent cytologists. Demonstration of the Golgi
apparatus in most cells requires a somewhat rigorous treatment of the
tissue with various chemical agents, some of which may reasonably be
suspected of leaving in the cytoplasm chemical or physical changes of
such a nature as to be visible after the Golgi technique, when in reality no
such structures exist, pre-formed, in the cell. The problem is probably
complicated even further by the multiplicity of forms and shapes which
the Golgi apparatus is observed to assume in different cells. At present
there seems to be little doubt but that such structures exist, pre-formed,
in most cells. Many investigators go so far as to state that the Golgi
apparatus is one of two or three cytoplasmic constituents which are in-
variably present in all cells, both plant and animal. If this is true, then
it is probable that the rdle of the Golgi apparatus in the life history ©
of the cell is of very great importance.

Demonstration of the Golgi apparatus, or Golgi bodies as the struc-
tures are frequently called, depends on the reduction of certain metallic
compounds to the free metals, the compounds most frequently used con-
taining either osmium or silver. The reduced metal results in blackening

432 THE CONTRACTILE VACUOLE

of the structure. It has been observed that treatment of stained material
with turpentine or hydrogen peroxide results in the bleaching of most
structures other than the Golgi apparatus, which may have been black-
ened by the procedure; Golgi bodies resist even prolonged bleaching
effects of these agents. Structures which normally are blackened by the
Golgi technique, or any of its modifications, are not blackened if the
cell or tissue is first subjected to alcohol or dilute acetic acid. For these
reasons, and others which need not be mentioned here, the Golgi ap-
paratus is thought to be composed largely of lipoid substances.

Together with the Golgi apparatus, mitochondria are generally con-
ceded to be invariably present in all cells. These structures, variously
called chondriome, chondriosomes, cytomicrosomes, and so forth, are
frequently present in the form of short rods or ovoid granules, although
the shape is not constant for different types of cells. Some authors main-
tain that besides the Golgi apparatus and mitochondria a third invariable
cytoplasmic constituent, the vacuome, is also present. Whether invari-
ably present or not, the vacuome is at least frequently found in cells.
The literature dealing with these structures is exceedingly confusing,
owing largely to the lack of a uniform nomenclature. Repeatedly several
authors have written of the same structure under different names, or
different structures under the same name. The lack of standard techniques
also contributes to the confusion. One of the most commonly used tech-
niques for differentiating between the Golgi apparatus and mitochondria
is staining of the tissue with a mixture of neutral red and Janus green;
mitochondria readily stain with the latter, and some authors maintain
that the Golgi apparatus is stainable with neutral red. The vacuome also
stains readily with neutral red, and on this basis it has been proposed
that the two structures, Golgi apparatus and vacuome, are identical.
Others have found within the same cell neutral-red stainable inclusions
which are not osmiophilic, and osmiophilic inclusions which are not
neutral-red stainable; so it appears that the two structures are not identi-
cal in all cells, but in some exist as separate entities. Furthermore, it is
sometimes claimed that osmiophilic bodies (the Golgi apparatus) are
derived from mitochondria (Janus-green stainable, but not osmiophilic).
If this latter is true, then one might expect to find in occasional cells
structures which are both osmiophilic and Janus-green stainable, although
such a situation has not come to the attention of the author. Unfortu-

THE CONTRACTILE VACUOLE 433

nately, no method or group of methods has been devised for the identifi-
cation of these structures, which is acceptable to all concerned. However,
for the protozoan Golgi apparatus it is generally conceded that in most
instances it exists as granules, globules, spherules, short rods, or ovoid
structures; but there appear to be many exceptions. These bodies reduce
certain osmium and silver compounds to the free metals, thereby causing
a blackening of the structures which resists bleaching with turpentine
and hydrogen peroxide. Most stains commonly used in cytological studies
are ineffective, although in some instances neutral red is found to stain
some structures which answer other requirements for the true Golgi
apparatus. Except for the occasional positive reaction to neutral red, the
protozoan Golgi apparatus reacts in a manner practically identical with
that of the metazoan Golgi apparatus.

Nassonoy (1924) demonstrated the presence of osmiophilic mem-
branes around the vacuoles in Paramecium caudatum, Lionotus folium,
Nassula laterita, Campanella umbellaria, Epistylis gallea, Zoothamnium
arbuscula, V orticella sp., and Chilomonas paramecium. These membranes
he found to be permanent structures, merely collapsing at systole of the
vacuole—not disappearing, to be reformed anew during the next period
of diastole. In Paramecium the vacuole system was found to consist of
a thin-walled reservoir and filling canals, the latter composed of the
short injection canal, the ampulla, and the distal section. The distal
section Nassonov found to be surrounded by a specially differentiated
plasma, from which hypertonic fluid is secreted into the lumen of the
canal. This hypertonicity results in the passage of water into the canal,
and ultimately into the vacuole. The vacuole wall in Paramecium is
considered not to take part in secretion, but to serve only as a temporary
reservoir or bladder. In other forms which possess no filling canals, the
osmiophilic vacuole wall is considered capable of performing the secre-
tory function as well. The formation of small droplets of fluid within
this wall was sometimes seen to occur, following partial systole of the
vacuole.

Subsequent observations by Nassonov (1925) on Chilodon and
Dogielella necessitated a modification of the original view so as to
include conditions which were not observed in the organisms men-
tioned in the earlier article. In Chilodon the osmiophilic material appears
as a heavy black ring, although this ring is not always complete. If

434 THE CONTRACTILE VACUOLE

fixation occurs at diastole, the vacuole appears to lie within the ring.
The ring does not collapse at systole, but remains more or less un-
changed. The vacuole is believed to be formed by the flowing together,
or coalescing, of small droplets (Sammelvacuolen) which form within
the substance of this ring. In Dogielella the osmiophilic material is in
the form of a ring around the vacuole, resembling, as Nassonov de-
scribes it, the rings around the planet Saturn. On contraction of the
vacuole, the ring remains essentially unaltered, showing a certain amount
of elasticity. The vacuole seems to arise as a result of the coalescing of
numerous droplets of fluid, just as in Chilodon. These two forms, as well
as many others described by other authors, represent a separation of the
Golgi apparatus from the vacuole, although the close functional associa-
tion remains. Nassonov’s conception of this close functional association
is expressed in a third publication (1926), in which he states that the
Golgi apparatus serves as a mechanism for collecting certain materials
from the cell substance and preparing them in such a way that they
can be discharged from the cell by the vacuole. To do this the Golgi
apparatus need not be a part of the vacuole system, nor even in direct
contact with it. This conception represents an important departure from
the first, in so far as morphology is concerned, but does not alter the
essential physiological relationship. Further evidence that such is the
function of the metazoan Golgi apparatus was obtained from experiments
in which the dye, Trypan blue, was injected into mice. On examination
of sections taken from the livers and kidneys of these mice, it was found
that the dye was concentrated in that region of the cells of the liver
and of the convoluted tubules of the kidney in which the Golgi apparatus
is situated. Distribution of mitochondria in these cells was found to be
quite different, indicating that these structures are not intimately assoct-
ated with the collection of the dye.

Some authors summarily reject the idea of a relationship between
vacuole and Golgi apparatus, solely on the ground that the wall of the
vacuole proves not to be osmiophilic. However, some of these same
authors present evidence which supports the idea of a physiological
relationship, even though the actual identity of the two structures is dis-
proved. Nassonov himself was among the first to demonstrate that by
no means all contractile vacuoles have osmiophilic walls, but this does

DHE ICONTRACTILEE VACUOLE 435

not alter the possibility of such a functional relationship as he sug-
gested.

Brown (1930) found that the Golgi apparatus of Amoeba proteus
is the characteristic protozoan type of globules and spherules, with
clear centers and dark rims. From a central focus these spherules appear
under the microscope to be crescent-shaped structures. He suggests that
the minute vacuoles which occur in the endoplasm of Amoeba are as-
sociated in some way with these crescent-shaped structures, and that
they unite to form the contractile vacuole. Brown further suggests this as
the reason that the vacuole in this form is not blackened by osmic acid,
as it is in Paramecium.

Hall (1930a) found small globular inclusions in Trichamoeba which
are osmiophilic, and which resist bleaching by either hydrogen peroxide
or turpentine. These inclusions are similar in size and distribution to
those which are stained vitally by neutral red. In material impregnated
by the Kolatchev method, the contractile vacuoles are not blackened. In
material prepared according to the Mann-Kopsch method, small globules,
similar to those seen in the Kolatchey material, are blackened. These
globules likewise resist bleaching by turpentine and hydrogen peroxide.
In the Mann-Kopsch material, small vacuoles—two, three, or more in
number—are blackened in many amoebae. In a few instances a number
of blackened globules were seen adherent to the wall of the contractile
vacuole, which, on casual examination, gave the appearance of a vacuole
with blackened walls. Hall suggests that in material less effectively
bleached, such a condition might easily be mistaken for heavily impreg-
nated vacuoles. Nigrelli and Hall (1930) report the presence of small
osmiophilic and neutral-red stainable granules in Arcella vulgaris.

Mast and Doyle (1935) apply the name “‘beta granules’ to small
structures, usually spherical but sometimes ellipsoidal or rod-like in
shape, which have a diameter of about one micron. These granules are
distributed more or less uniformly throughout the cytoplasm, except at
the surface of the contractile vacuole, where they tend to become con-
centrated in a layer. Aggregation of granules on the surface was described
by Metcalf (1910), as previously mentioned. These granules, accord-
ing to Mast and Doyle, are stained vitally by Janus green, but only on
the surface, indicating that they have a differentiated surface layer similar
to that in mitochondria. In addition to beta granules, these authors in-

436 THE CONTRACTILE VACUOLE

vestigated other more or less spherical cytoplasmic inclusions, which
they call “cytoplasmic refractive bodies.” The outer layers of these
bodies are readily stained by neutral red and osmium, whereas the central
portions react negatively to osmium and stain but faintly with neutral
red. Apparently these are the same structures studied by Brown, who
believes them to give rise to minute vacuoles which are precursors to
contractile vacuoles. The beta granules are not blackened by osmium.
Many of these granules are usually situated close to the contractile
vacuole, while others are scattered throughout the entire cytoplasm. The
pulsation frequency of the vacuole is proportional to the number of
beta granules remaining after some have been removed by operation,
indicating a close relationship between granules and vacuole function.
Removal of most of these granules results in the death of the organism.

The relationship between the contractile vacuole and cytoplasmic in-
clusions in Amoeba is puzzling. One would be inclined to accept, at
least tentatively, the idea of the origin of vacuoles in the beta granules
which surround it, were it not for the fact that in a variety of other
Protozoa the vacuole has been seen to originate as minute droplets in the
region of the cell occupied by osmiophilic granules. Yet in A. proteus
the granules among which the vacuole apparently arises are not osmio-
philic nor stainable by neutral red, but are stainable by Janus green. It
might be suggested that the situation in Azzoeba is the reverse to what
it appears to be in other Protozoa, but such a suggestion offers no satis-
faction. A more likely explanation lies in the uncertainty of identification
of these cytoplasmic inclusions. Some authors (e.g., Hall, 1930a) consider
the vacuome, which is neutral-red stainable, identical with the Golgi
apparatus, which is osmiophilic; this Hall observed to be true in Trich-
amoeba. Others (e.g., MacLennan, 1933) have identified both neutral-
red stainable and osmiophilic granules as separate structures within the
same organism. Apparently Dunihue (1931) finds the same in Para-
mecium. Further, MacLennan observed that the only granules in Evdz-
plodinium which can be impregnated with osmium are those found in
the vacuolar region; yet in a study of living material, it was shown that
this region is composed of granules which originate in the surrounding
ectoplasm. Therefore, these granules, as they assemble in the vacuolar
region, undergo some change, either chemical or physical or both, which
makes them osmiophilic. It has been suggested at one time or another

THE CONTRACTILE VACUOLE 437

that the Golgi apparatus is derived from mitochondria. Until this puz-
zling situation is clarified, it seems necessary to assume that in some
organisms neutral-red stainable granules (vacuome), osmiophilic gran-
ules (the Golgi apparatus), and Janus-green stainable granules (mito-
chondria) exist as separate and distinct entities, whereas in others the
Golgi apparatus may be combined with one or the other of the two
remaining types of granules. Hirschler (1924) found only one kind
of lipoid body in Gregarina and Spirostomum, and suggested that these
represent a primitive type of organism in which Golgi apparatus and
mitochondria are combined in a single type of granule. Until the identity
and function of the various types of granules in A. proteus have been
investigated further, it is difficult to arrive at any reasonable conclusion
concerning the relationship of the contractile vacuole to them.

Hirschler (1927) examined a variety of organisms after fixation and
staining with several dyes, as well as impregnation with osmium and
silver. From these studies he concludes that both Golgi apparatus and
mitochondria are present in Bodo lacertae, Lophomonas blattarum, L.
striata, Try pano plasma dendrocoeli, Entamoeba blattae, Monocystis agilis,
Trypanoplasma helicis, Diplocystis phryganeae, Gregarina polymorpha,
and Clepsidrina blattarum. In these organisms the Golgi apparatus and
the mitochondria were shown to have the same staining reactions as
corresponding structures in metazoan cells.

Hall (1929) found osmiophilic granules which resist bleaching with
hydrogen peroxide in Peranema trichophorum, in Menoidium and Eu-
glena (1930b); in Chromulina sp., Astasia sp., and Chilomonas para-
mecium (1930c); and in Stylonychia (1931). Hall and Dunihue (1931)
found similar granules, or globules, in Vorticella. In many of these ex-
periments two or more methods of osmium impregnation, as well as
silver impregnation, were used. In some of them the osmiophilic bodies
were found to be stainable with neutral red also. In some species the
wall of the contractile vacuole was found to be osmiophilic after pro-
longed osmication, but generally this was readily bleached by hydrogen
peroxide or turpentine. Janus green and neutral red were used as vital
stains for several organisms; in these the osmiophilic granules were
identified as the neutral-red stainable material, whereas smaller granules
were stained with Janus green.

Fauré-Fremiet (1925) observed in several species of Vortzcella es-

438 THE ‘CONTRACTILE. VACUOLE

sentially the same type of structure as that described by Nassonov. The
vacuole wall was found to be in the form of a ring deeply blackened
by osmium. Following systole, the vacuole collapses, but the wall re-
mains quite evident. Small vesicles or droplets appear within the thickness
of the wall, fuse together, and thus give rise to the new vacuole. On
the other hand, Finley (1934) demonstrated, by means of recognized
osmium and silver-impregnation techniques, discrete globular inclusions
in the cytoplasm of Vorticella convallaria, V. microstoma, and V. cam-
panula. These globules were readily distinguishable from the rod-shaped
mitochondria by staining with a mixture of Janus green and neutral red,
the globules reacting positively to neutral red and negatively to Janus
green, whereas with mitochondria the reverse was true.

Moore (1931) found distributed through the entire endoplasm of
Ble pharisma globules with osmiophilic cortices and osmiophobic centers.
These structures resist bleaching with turpentine. Only in instances of
overimpregnation is the contractile vacuole blackened in this form, al-
though paramecia, mixed with the Blepharisma uniformly show black-
ened vacuole systems. Where impregnation of the vacuole is produced
in Ble pharisma, it is readily bleached with turpentine. No evidence was
noted by Moore that in these osmiophilic globules lay the origin of the
contractile vacuole. In a later investigation, Moore (1934) found that
the secondary vacuoles do not empty their contents into the primary
vacuole, and thus contribute to its filling; but as contraction of the
primary vacuole occurs, the secondary vacuoles move into the place it
had occupied, where they coalesce to form a new primary vacuole. No
“excretory granules” were observed, but in the earlier work Moore de-
scribed osmiophilic globules scattered throughout the cytoplasm. On
the basis of these observations, Moore rejects the Nassonov homology for
Ble pharisma.

King (1933) found in Ezplotes that the vacuoles termed group V,
by Taylor (1923) have their origin at the distal ends of a very large
number of collecting tubules, located just under the ectoplasm on the
dorsal surface of the organism. The presence of these tubules was demon-
strated by impregnation with osmium. King believes that these tubules,
or canals, like those in Paramecium, are responsible for collection of
fluid which ultimately reaches the contractile vacuole.

In a comprehensive series of observations on the Ophryoscolecidae,

THE CONTRACTILE VACUOLE 439

MacLennan (1933) found that the osmiophilic granules contribute di-
rectly to the formation of accessory vacuoles, which in turn form the
contractile vacuole. With respect to the possible function of the vacuole,
he states (p. 236):

The vacuolar region found in these ciliates shows definite evidence of the
elimination of materials by means of the vacuolar fluid and corresponds to
the secretary region or “region of Golgi’ in gland cells. The nature of the
materials eliminated by the vacuolar region was not determined in this in-
vestigation. Since, however, the pellicle in the Ophryoscolecidae has been
shown to be relatively impermeable and since the vacuolar region is the
only demonstrable path by which materials are constantly being passed to the
exterior, it is likely that the katabolic wastes of these ciliates are eliminated
by this organelle rather than by direct diffusion through the pellicle.

Dunihue (1931) found that the vacuole system in Paramecium cauda-
tum is osmicated only after the neutral-red stainable globules. These
globules and Janus-green stainable elements, he believes, represent the
vacuome and chondriome (mitochondria) respectively. King (1935)
noted a “specialized excretory protoplasm’? surrounding the feeding
canals in P. multimicronucleata, but denied that this material is ho-
mologous with the Golgi apparatus of metazoan cells.

The opinions of von Gelei (1925, 1928) concerning the structure
and function of the contractile vacuole in Paramecium are of special
interest. He described essentially the same structures in stained Para-
mecium as those mentioned by Nassonoy. The zone of specialized plasma
around the distal portion of the canals, particularly, was described in
detail, and an excretory function assigned to it. This specialized plasma
von Gelei calls “‘nephridial plasma,” its excretory function being implied

by its name. This “excretory” function von Gelei believes is entirely dif- |

ferent from the ‘‘secretory’”’ function assigned by Nassonov, when the
latter considers the specialized plasma to be the Golgi apparatus. This
disagreement appears to be imaginary rather than real, since an analysis
of their respective views indicates that the two authors observed struc-
tures which are identical in practically every respect; but to describe the
function, they selected different words. Moreover, these different words,
when translated into terms of physiological processes, are practically iden-
tical. Von Gelei pictures two different arrangements of the deeply stain-
ing material in this zone of specialized plasma; in one the stained ele-

440 THE CONTRACTILE VACUOLE

ments are in the form of short rods, which lie with their long axes at
right-angles to the long axis of the filling canal, in much the same manner
as the bristles of a test-tube brush are arranged with respect to the wire
handle to which they are attached; and in the other, these elements are in
the form of a net surrounding the filling canal. It is interesting to note
that this net-like arrangement is commonly seen in the Golgi apparatus
of many metazoan cells, as well as in Dogielella. Von Gelei (1933) ob-
served in Spathidium giganteum that not only the contractile vacuole and
the smaller vacuoles in its immediate vicinity possess osmiophilic walls,
but also others further removed. One can but wonder if this represents the
origin of contractile vacuoles by the coalescence of secondary vacuoles,
which have arisen in more or less remote parts of the organism.

In Monocystis agilis and M. ascidiae, Hirschler (1924) identified two
kinds of lipoid bodies. The smaller of these he considers mitochondria,
the larger the Golgi apparatus. In Gregarina polymorpha, G. blattarum,
and Spirostomum ambiguum only one kind of lipoid body was ob-
served. From this Hirschler concludes that the latter are representatives
of a more primative state, in which lipoid bodies are not yet differenti-
ated into mitochondria and Golgi apparatus.

In most instances in which description of structures are given in sufh-
cient detail, and in which organisms have been subjected to a variety
of stains as well as osmium and silver impregnation, very strong evi-
dence has been presented to indicate that contractile vacuoles derive the
fluid which they expel to the exterior from granules which are osmio-
philic, argentophilic, and sometimes neutral-red stainable. In some cells
the osmiophilic granules are aggregated around the vacuole or in that
part of the cell in which the vacuole ordinarily arises; this is usually
associated with the origin of the vacuole in a restricted portion of the
cell. In other cells the osmiophilic granules are dispersed to a greater
or less extent, sometimes apparently uniformly throughout the cytoplasm,
this is usually associated with at least the potential origin of vacuoles in
almost any part of the cytoplasm. Evidence bearing on the subject indi-
cates that these osmiophilic granules may represent at least one type of the
“excretory granule” so frequently mentioned in the literature.

Several authors have reported osmiophilic substances in the form of
relatively broad bands, or rings, which may or may not be in direct
contact with the vacuole wall. Most authors seem to agree that the usual

THE ‘CONTRACTILE VACUOLE 441

form for the protozoan Golgi apparatus is that of granules, globules,
or short rods. Overimpregnation of a granular region, which occurs when
the process is carried out at too high temperatures, has been shown to
produce heavy black bands, or rings, in certain organisms. This fact
suggests the possibility that the Golgi apparatus may be of the usual
form, even in those organisms in which the band, or ring, type has
been observed. Identification of osmiophilic substances answering the
known criteria for the Golgi apparatus has been extended to include
representatives of the four classes of Protozoa: Mastigophora, Sarcodina,
Sporozoa, and Ciliata. This substantiates the idea that the Golgi apparatus
is a cytoplasmic inclusion of all living cells.

CONCLUSION

In spite of the multiplicity of claims, counter claims, theories, and
suggestions, a few generalizations seem to be established well enough to
indicate at least some of the fundamental processes associated with ac-
tivity of the contractile vacuole. It is not intended that these shall be
accepted as proved beyond question, but rather that the evidence points
in their direction more consistently than in any other. Further investiga-
tion may necessitate a complete revision of opinion concerning these
processes, but in the light of the information available at the present
time the following conclusions seem to be justified.

1. Contractile vacuoles originate as a result of the activity of certain
cytoplasmic inclusions, which may be aggregated in the immediate vicin-
ity of the vacuole in some species, or distributed more or less generally
throughout the cytoplasm in others. Temporary contractile vacuoles are
formed by the fusion or coalescence of small accessory vacuoles, which
in turn originate by the fusion of still smaller accessory vacuoles, the
last and smallest vacuoles being formed in or associated with the cyto-
plasmic inclusions mentioned above. More or less permanent contractile
vacuoles (e.g., those of Paramecium) receive fluid as small droplets, or
accessory vacuoles which fuse with some portion of the filling canals;
these droplets originate in or on cytoplasmic inclusions in the same man-
ner as those mentioned above.

2. On the basis of known physicochemical laws and processes, it is
necessary to postulate the existence of a physiological membrane sur-
rounding the contractile vacuole. In some organisms, particularly those

442 THE CONTRACTILE VACUOLE

possessing more or less permanent vacuole systems, these organelles ap-
pear to be surrounded by morphological membranes.

3. Direct evidence concerning the function of contractile vacuoles is
almost entirely lacking. Indirect evidence indicates that in fresh-water
forms the vacuole protects the organism against excessive dilution of its
cytoplasm. In marine and parasitic forms such a function would seem to
be largely superfluous, although even in these the elimination of at
least a small quantity of water by some mechanism appears to be neces-
sary. Direct evidence indicating the presence of waste products of me-
tabolism in the vacuolar fluid is very scant, although, in those forms
possessing relatively impermeable surface structures, the vacuole is the
only visible means by which such wastes may be passed to the exterior.

4. In some Protozoa three types of cytoplasmic inclusions have been
identified, in others only two types. In all Protozoa so far examined with
this in view, at least some of the inclusions are osmiophilic. In some oth-
ers these osmiophilic inclusions are also stainable by neutral red, but not
by Janus green. Osmication of certain Protozoa by one technique or an-
other frequently shows more than one type of inclusion to be osmiophilic,
but generally one of these resists bleaching by hydrogen peroxide or
turpentine more completely than the others. Such inclusions are generally
recognized as the Golgi apparatus. By comparing living organisms with
those stained vitally with various dyes, as well as with others impregnated
with osmium or silver, identity of the Golgi apparatus and the cyto-
plasmic inclusions concerned with the origin of the contractile vacuole
has been established for many forms. The usual form of protozoan Golgi
apparatus is granular, globular, or rod-like. In a few species (Para-
mecium, Dogielella, Chilodon, and others), it frequently appears as a
network, while in others (Lonotus, Nassula, Campanella, and others)
it is in the form of a thick ring, or membrane, surrounding part or all
of the vacuole. Evidence has been presented which indicates that in some
of these, if not all, a granular structure has been overimpregnated, this
causing it to assume the appearances mentioned. It therefore appears that
fluid which is expelled from the organism by the contractile vacuole
originates as droplets in association with the Golgi apparatus, although
the Golgi apparatus is not necessarily in intimate contact with the vacu-
ole. Concerning the origin of secretions in metazoan gland cells, Bowen
(19297 pai )estates::

THE CONTRACTILE VACUOLE 443

Secretion is in essence a phenomenon of “granule” or droplet formation.
Starting with a single such secretory droplet about to be expelled from the
cell, we find it possible to trace its origin step by step to a minute vacuole,
which has thus from the beginning served as a segregation center for a
specific secretion-material. The primordial vacuole is found to arise in that
zone of the cell characterized by the presence of the Golgi apparatus, and
the evidence indicates, if it does not demonstrate, that the primordial
vacuole arises through the activity of the Golgi substance and undergoes a
part at least of its development in contact with, or imbedded in, the Golgi ap-
paratus.

The idea of Nassonov, as developed in 1925 and 1926, as well as that
of MacLennan (1933), concerning the origin of protozoan vacuoles
could hardly be expressed more exactly.

The outstanding features of contractile vacuoles, taken collectively,
then, do not lie in differences among them, but rather in similarities,
both morphological and physiological. Another fundamental link in the
kinship between all cells seems to be established by the apparent homol-
ogy, both structural and functional, between the protozoan and the meta-
zoan Golgi apparatus.

LITERATURE CITED

Adolph, E. F. 1926. The metabolism of water in Amoeba as measured in the
contractile vacuole. J. exp. Zool., 44: 355-81.

Bowen, R. H. 1929. The cytology of glandular secretion. Quart. Rev. Biol., 4:
299-324 and 484-519.

Brown, V. E. 1930. The Golgi apparatus of Amoeba proteus Pallas. Biol. Bull.
59: 240-46.

Butschli, O. 1887. Die Contraktilen Vacuolen. Bronn’s Thierreich, Protozoa,
1: 1411-59.

Calkins, G. N. 1926. The biology of the protozoa. Philadelphia.

Clark, A. J. 1933. Mode of action of drugs on cells. London.

Day, H. C. 1927. The formation of contractile vacuoles in Amoeba proteus.
J. Morph., 44: 363-72.

—— 1930. Studies on the contractile vacuole of Spzrostomum and Para-
mecium. Physiol. Zool., 3: 56-71.

Degen, A. 1905. Untersuchungen uber die Contractile Vacuole und die
Wabenstruktur des Protoplasmas. Botan. Ztg., 63: 160-202.

Dimitrowa, Ariadne. 1928. Untersuchungen iiber die tiberzahligen pulsier-
enden Vakuolen bei Paramecium caudatum. Arch. Protistenk., 64: 462-
They,

Doflein, F. 1911. Lehrbuch der Protozoenkunde.

“tt THE CONTRAGIILE, VACUOLE

Doyle, W. L., and J. P. Harding. 1937. Quantitative studies on the ciliate
Glaucoma. Excretion of ammonia. J. exp. Biol., 14: 462-69.

Dunihue, F. W. 1931. The vacuome and the neutral red reaction in Para-
mecium caudatum. Arch. Protistenk., 75: 476-97.

Eisenberg, E. 1926. Recherches sur le fonctionnement de la vesicule pulsatile
des Infusoires dans les condition normales et sous I’action de certains
agents experimentaux. Arch. Biol. Paris, 35: 441.

—— 1929. Recherches comparatives sur le fonctionnement de la vacuole pulsa-
tile chez les Infusoires parasites de la grenouille et chez les Infusoires
d’eau douce. Influence de la pression osmotique, des electrolytes et du pH.
Arch. Protistenk., 68: 451-70.

Fauré-Fremiet, E. 1925. La Structure permanent de l’appareil excréteur chez
quelques vorticellides. C. R. Soc. Biol. Paris, 93: 500-3.

Finley, H. E. 1934. On the vacuome in three species of Vorticella. Trans.
Amer. micr. Soc., 53: 57-65.

Flather, M. D. 1919. The influence of glandular extracts upon the contractile
vacuoles of Paramecium caudatum. Biol. Bull., 37: 22-39.

Fortner, H. 1926, Zur Frage der diskontinuierlichen Excretion bei Protisten.
Arch. Protistenk., 56: 295-320.

Fosse, R. 1913. Sur I’Identification de l’urée et sa précipitation de solution
extrément diluées. C. R. Acad. Sci. Paris, 137: 948-57.

Frisch, J. A. 1935. Experimental adaptation of fresh water ciliates to sea
water. Science, 81; 537.

Gelei, J. von. 1925. Nephridialapparat bei Protozoen. Biol. Zbl., 45: 676-83.

1928. Nochmals tber den Nephridialapparat bei den Protozoen. Arch.

Protistenk., 64: 479-94.

—— 1933. Wandernde Exkretionsvakuolen bei den Protozoa. Arch. Protis-
tenk., 81: 231-42.

Griffiths, A. B. 1888. A method of demonstrating the presence of uric acid
in the contractile vacuoles of some of the lower organisms. Proc. roy. Soc.
Edinb., 16: 131-35.

Hall, R. P. 1929. Reaction of certain cytoplasmic inclusions to vital dyes and
their relation to mitochondria and Golgi apparatus in the flagellate, Pera-
nema trichophorum. J. Morph., 48: 105-18.

—— 1930a. Cytoplasmic inclusions of Trichamoeba and their reaction to vital
dyes and to osmic and silver impregnation. J. Morph., 49: 139-51.
—— 1930b. Cytoplasmic inclusions of Menoidium and Euglena, with special
reference to the vacuome and ‘“'Golgi apparatus” of Euglenoid flagellates.

Ann. Protist., 3: 57-68.

—— 1930c. Osmiophilic inclusions similar to Golgi apparatus in the flagel-
lates, Chromulina, Chilomonas, and Astasia. Arch. Protistenk., 69: 7-22.

—— 1931. Vacuome and Golgi apparatus in the ciliate, Stylonychia. Z. Zell-
forsch., 13: 770-82.

THE GONTRAGHEE VACUOLE 445

Hall, R. P., and F. W. Dunihue. 1931. On the vacuome and food vacuoles
in Vorticella. Trans. Amer. micr. Soc., 50: 196-205.

Hance, R. T. 1917. Studies on a race of Paramecium possessing extra con-
tractile vacuoles. J. exp. Zool., 23: 287-333.

Hartog, M. M. 1888. Preliminary notes on the functions and homologies of
the contractile vacuole in plants and animals. Rep. Brit. Assoc. Ady. Sci.,
Bath, pp. 714-16.

Harvey, E. B. 1917. A physiological study of specific gravity and of lumines-
cence in Noctiluca, with special reference to anesthesia. Pap. Tortugas
Lab., 11: 237-53.

Haye, Ans. 1930. Uber den Exkretionsapparat bei den Protisten, nebst Be-
merkungen tiber einige andere feinere Strukturverhaltnisse untersuchten
Arten. Arch. Protistenk., 70: 1-86.

Herfs, A. 1922. Die pulsierende Vakuole der Protozoen, ein Schutzorgan
gegen Aussiissung. Arch. Protistenk., 44: 227-60.

Hirschler, J. 1924. Sur une Méthode de Noircissment de l’Appareil de Golgi.
C. R. Soc. Biol. Paris, 90: 893.

— 1927. Studien uber die sich mit Osmium schwarzenden Plasmakom-
ponenten einiger Protozoenarten, Z. Zellforsch., 5: 704-86.

Hopkins, D. L. 1938. Adjustment of the marine amoeba, Flabellula mira
Schaeffer, to changes in the total salt concentration of the outside medium.
Biol. Bull 7525337 a(@Abstr:):

Howland, R. B. 1924a. Experiments on the contractile vacuole of Amoeba
verrucosa and Paramecium caudatum. J. exp. Zool., 40: 251-62.

— 1924b. On excretion of nitrogenous waste as a function of the contractile
vacuole, J. exp. Zool., 40: 231-50.

Howland, R. B., and H. Pollack. 1927. Expulsion of injected solute by the
contractile vacuole of Amoeba. Proc. Soc. exp. Biol. N.Y., 25: 221-22.

Hyman, Libbie H. 1936. Observations on Protozoa: The impermanence of
the contractile vacuole in Amoeba verspertilio. Quart. J. micr. Sci., 79:
43-56,

Jennings, H. S. 1904. A method of demonstrating the external discharge of
the contractile vacuole. Zool. Anz., 27: 656-58.

Kent, W. S. 1880. A Manual of the Infusoria. London.

Khainsky, A. 1910. Zur Morphologie und Physiologie einiger Infusorien
(Paramecium caudatum) auf Grund einer neuen histologischen Methode.
Arch. Protistenk., 21: 1-60.

King, R. L. 1928. The contractile vacuole in Paramecium trichinm. Biol. Bull.,
DDI 9-69:

—— 1933. Contractile vacuole of Explotes. Trans. Amer. micr. Soc., 52:
103-6.

—— 1935. The contractile vacuole of Paramecium multimicronucleata. J.
Morph., 58: 555-71.

446 THE CONTRACTILE, VACUOLE

Kitching, J. A. 1934. The physiology of contractile vacuoles: osmotic relations.
J. exp. Biol., 11: 364-81.

Lachmann, K. F. 1891. Uber einiger neu entdekte Infusorien und ttber Con-
tractilen Blasen bei den Infusorien. Verh. Naturh. Ver. preuss. Rheinl.,
16: 66-68, 91-93.

Lloyd, F. E. 1928. The contractile vacuole. Biol. Rev., 3: 329-58.

Lloyd, F. E., and G. W. Scarth. 1926, Origin of vacuoles. Science, 63: 459-60.

Ludwig, W. 1928. Der Betriebsstoffwechsel von Paramecium caudatum. Arch.
Protistenk., 62: 12-40.

Lwoff, André, and Nadia Roukhelman. 1926. Variations de quelques formes
d’azote dans une culture pure d’infusoires. C. R. Acad. Sci. Paris, 183:
156-58.

MacLennan, R. F. 1933. The pulsatory cycle of the contractile vacuoles in
the Ophryoscolecidae, ciliates from the stomach of cattle. Univ. Cal.
Publ. Zool., 39: 205-50.

Mast, S. O. 1926. Structure, movement, locomotion, and stimulation in
Amoeba. J. Morph., 41: 347-425.

—— 1938. The contractile vacuole in Amoeba proteus (Leidy). Biol. Bull.,
74: 306-13.

Mast, S. O., and W. L. Doyle. 1935. Structure, origin, and function of cyto-
plasmic constituents in Amoeba proteus with special reference to mito-
chondria and Golgi substance. Origin and function based on experimental
evidence; effect of centrifuging on Amoeba proteus. Arch. Protistenk.,
86: 278-306.

Maupas, E. 1883. Contribution a I’Etude Morphologique et Anatomique des
Infusoires Ciliés. Arch. zool. exp. gén., 2 serie, 1: 427-664.

Metcalf, M. M. 1910. Studies upon Amoeba. J. exp. Zool., 9: 301-31.

—— 1926. The contractile vacuole granules in Amoeba proteus. Science, 63:
523-24.

Moore, Imogene. 1931. Reaction of Blepharisma to Golgi impregnation
methods. Proc. Soc. exp. Biol. N.Y., 28: 805-6.

—— 1934. Morphology of the contractile vacuole and cloacal region of
Blephavisma undulans. J. exp. Zool., 69: 59-104.

Nassonoy, D. 1924. Der Exkretionsapparat der Protozoen als Homologen
des Golgischen Apparats der Metazoazellen. Arch, mikr. Anat. 103:
437-82.

—— 1925. Zur Frage tiber den Bau und die Bedeutung des lipoiden Exkre-
tionsapparates bei Protozoa. Z. Zellforsch., 2: 87-97.

——— 1926. Die physiologische Bedeutung des Golgi Apparats im Lichte der
Vitalfarbungsmethode. Z. Zellforsch., 3: 472-502.

Nigrelli, R. F., and R. P. Hall. 1930, Osmiophilic and neutral-red-stainable
inclusions of Arcella. Trans. Amer. micr. Soc., 49: 18-25.

Nowikoff, M. 1908. Uber die Wirkung des Schildrusen Extrakts und einiger
anderer Organstoffe auf Ciliaten. Arch. Protistenk., 11: 309-26.

THE GONTRACTILE VACUOLE 447

Parnas, J. K. 1926. Allgameines und Vergleichendes des Wasserhaushalts.
Handbuch. d. norm. u. pathol. Physiol., 17, correlationem 3: 137-60.
Riddle, M. C., and H. B. Torrey. 1923. The physiological response of Para-

mecium to thyroxin. Anat. Rec., 24: 396.

Scarth, G. W., and F. E. Lloyd. 1927. The réle of kinoplasm in the genesis
of vacuoles. Science, 65: 599-600.

Shumway, W. 1917. Effects of thyroid diet upon paramecia. J. exp. Zool., 22:
529-62.

Specht, Heinz. 1934. Aerobic respiration in Spirostomum ambiguum and
the production of ammonia. J. cell. comp. Physiol., 5: 319-33.

Stempell, W. 1914. Uber die Funktion der Pulsierende Vacuole und einen
Apparat zur Demonstrationen derselben. Zool. Jahrb., Abt. Allg. Zool.
Physiol. Tiere, 34: 437-78.

Stokes, A. C. 1893. The contractile vacuole. Amer. mon. micr. J., 14: 182-88.

Taylor, C. V. 1923. The contractile vacuole in Evplotes: an example of the
sol-gel reversibility of cytoplasm. J. exp. Zool., 37: 259-90.

Weatherby, J. H. 1927. The function of the contractile vacuole in Paramecium
caudatum ; with special reference to the excretion of nitrogenous com-
pounds. Biol. Bull., 52: 208-18.

— 1929. Excretion of nitrogenous substances in Protozoa. Physiol. Zodl.,
2: 375-94,

Wenrich, D. H. 1926. The structure and division of Paramecium trichinm
Stokes. J. Morph., 43: 81-103.

Young, R. A. 1924. On the excretory apparatus in Paramecium. Science, 60:
244,

Ziilzer, Margaret. 1910. Uber den Einfluss des Meerwassers auf die pul-
sierende Vacuole. Roux Arch. Entw. Mech. Organ., 29: 632-40.

CHAPTER WAIT

THE TECHNIQUE AND SIGNIFICANCE OF CONTROL
IN| PROTOZOAN CULTURE

GEORGE W. KIDDER

INTRODUCTION

DURING THE LAST FEW YEARS there has come to be an appreciation of
methods of culturing Protozoa which will permit the investigator to_
determine the conditions under which his study is being made. Studies
of populations and the various interesting and important factors in-
volved, mass physiology, nutrition, and numerous other phases of cellu-
lar activity may be profitably dealt with by the student of the Protozoa
only when he can be sure that the effects noted are due to the conditions
under investigation. The science of protozodlogy has passed through the
phase of “pure-mixed” methods of culture. This term simply means
that a single strain of Protozoa is grown in association with a chance
combination of other microdrganisms, usually bacteria. Many valuable
and thought-provoking contributions, based upon this method, have
been made, and these contributions have paved the way to the more pre-
cise evaluations of the present.

In the culture of practically any species of Protozoa, it may be safely
said that the bacteria as a group offer the most serious obstacle to con-
trolled conditions. Experimental modifications of factors such as nutritive
materials, temperature, oxygen or carbon dioxide tensions, oxydation-
reduction potentials, and so forth, may produce effects, but whether these
effects are the result of changes in protozoan activity per se, or are sec-
ondary through the change of activity of the bacteria, is usually nearly
or totally obscure. These facts are recognized, and there has been built
up a body of literature reporting progress in methods which will allow
for the control or, better still, the elimination of bacteria. Numerous 1n-
vestigators have succeeded in sterilizing various species of Protozoa and
have made great strides in advancing our knowledge of cellular activities
through the use of pure cultures.

GONTROL OF CULTURES 449

It is the purpose of this section to devote some time to methods or
techniques of protozoan sterilization, in order to bring before the reader
some of the many problems which must be dealt with in work of this
kind. As may be supposed, the nature of such a discussion makes it
necessary to assume at least a rudimentary knowledge of bacteriological
technique. And above all there must be a thorough appreciation of the
potentialities of many different types of bacteria to resist even the most
careful methods of irradication, potentialities which express themselves
in some cases only after prolonged periods of apparent sterility.

In addition to an outline of techniques for sterilization, the question
of acceptable tests for the sterility of cultures will be considered, and
finally some of the problems and conditions arising from the establish-
ment of sterile Protozoa in culture.

THE PROBLEM OF PROTOZOAN STERILIZATION

1. GENERAL MATERIAL

Protozoa from natural waters, soil, and so forth, are, and have been
throughout their existence, in association with bacteria. This does not
mean that the bacterial flora of their surroundings has remained constant
either as to numbers or types. The flora is probably continually changing.
This very change is one of the most important factors in the succession
of microscopic animals in ponds and streams. The variety of bacterial
types one would expect to encounter in any extended survey of natural
ponds is practically limitless. Therefore it is impossible to do more than
discuss the general factors to be taken into account in dealing with bac-
teria.

To attain successful sterile cultures of Protozoa, it is, of course, de-
sirable to have rather large numbers of healthy organisms with which to
work. It is usually possible to isolate single organisms into fresh infusion
and obtain from fair to good growth. If they are bacteria-feeders (the
great majority of free-living ciliates are), enough food organisms are
brought over in the isolation to insure, at least in a high percentage of
cases, against starvation. As the bacteria multiply they, in turn, are uti-
lized by the Protozoa.

In dealing with ciliates from the wild, a partial substitution method
may be attempted. For a number of species, this method facilitates later
sterilization. If the ciliate to be used will feed on living yeast (and this

450 CONTROE OF CULTURES

can be determined only by experimentation), then cultures may be estab-
lished by suspending yeast cells in spring water, distilled water, or bal-
anced salt solutions, and introducing the desired ciliate. Often abundant
growth will result and after a few subcultures have been made, the ratio
of bacteria to Protozoa will be reduced. When serious attempts at sterili-
zation are then carried out, the yeast cells will be found relatively easy
to eliminate. It should not be supposed that it will be possible to elimi-
nate the contaminating bacteria in this way, as they will be multiplying
slowly all the while. In fact, long-continued cultures of this type are apt
to show a decided increase in non-nutritive bacteria over those which
were present at the start (see Kidder and Stuart, 1939). Therefore it
is advisable to make from one to three subcultures only, and then to start
the sterilization procedure.

In general it can be said that the larger the protozoan the more diffi-
cult it will be to sterilize. This fact becomes apparent from an examina-
tion of the literature, and was noted by Hetherington (1934). Physical
properties likewise play a rdle in ease of sterilization. Holotrichous and
heterotrichous ciliates, possessing large numbers of closely set cilia, are
apt to retain a few of their associated bacteria even after repeated wash-
ing, while hypotrichous ciliates may be washed free of bacteria more
readily. Flagellates, being for the most part smooth in surface, are rela-
tively easy to sterilize. Activity is also important, both as to movement
and metabolism. Highly motile forms may usually be freed of bacteria
more readily than sluggish types. Those possessing a high rate of me-
tabolism tend to utilize or defecate the contents of their food vacuoles
more rapidly than the slow-growing types, and do not tend to carry over
viable spores to contaminate later cultures. Many other characters which
will influence the facility with which sterilization may be accomplished
might be mentioned, but these will become apparent when we examine
some of the procedures.

2. GENERAL METHODS OF STERILIZATION

In order to rid the Protozoa of their associated bacteria, workers have
made use of three principles. The first and most generally useful is simple
washing in sterile fluid. This is the dilution method whereby the bacteria
are diluted out of the solution. The Protozoa must be retained of course,
and a number of different manipulations have been devised to insure this.

CONTROL OF CULTURES 451

The principle of dilution takes for granted that the bacteria either are
suspended in the fluid or that they may be caused to become suspended.

The second principle is one of migration. The Protozoa to be sterilized
are allowed or caused to swim through sterile fluid or semisolid medium
or over the surface of solid medium, !eaving the bacteria behind. This
method has been used with success on a number of different types of
ciliates and flagellates and a few amoebae. The Protozoa may be induced
to migrate laterally by introducing them into one side of a flat dish of
sterile fluid. Or, if they happen to be negatively geotropic, they may
be introduced into the bottom of a vessel of sterile fluid and taken off
at the top. Those that are positively geotropic may be introduced at the
top and taken off at the bottom. Extremely active types may be able to
migrate through a semisolid medium and literally scrape off their adher-
ing bacteria.

Combinations of the above two principles have been used with marked
success, and a number of ingenious pieces of apparatus have been de-
signed to facilitate the manipulations and to reduce the chance of ex-
traneous contamination. These will be described in detail later.

The third principle that has been applied to this problem is that of
bactericidal agents. This method has met with questionable success and
then usually only in cases where resistant phases (cysts) could be ob-
tained. As might be expected, any agent which would kill the bacteria
in a culture would most surely kill trophic Protozoa. It has been shown
many times that the various species of Protozoa are much more suscep-
tible to the usual toxic agents than many of the common bacteria. (An
exception to the foregoing statement is indicated in the work of Brown,
et al., 1933, using X-rays as a sterilizing agent.)

3. SPECIAL METHODS AND MANIPULATIONS

This section will be devoted to a description of the procedures which
have been used by various investigators to rid the different types of Proto-
zoa of their associated bacteria. Considerable pains will be taken to de-
scribe the apparatus used, the manipulations performed, and the results
obtained. It is hoped that by so doing the reader will be able to gain
constructive ideas which will allow him either to utilize one of the de-
scribed methods or to formulate a modification which will meet his needs.

A. Dilution —One of the first reports of the sterilization of Protozoa

452 CONTROL OF CULEURES

by this method is that of Hargitt and Fray (1917), using Paramecium
aurelia and P. caudatum. They experimented with a number of modifi-
cations of the washing technique and followed their results by plating
on nutrient agar. Their first procedure was dilution by centrifugation,
wherein they centrifuged down the paramecia and then quickly with-
drew the supernatant fluid with a sterile pipette. The paramecia were
then covered again with sterile fluid, and the process repeated five
times. At the end of the fifth wash they found that the number of bac-
teria “‘per drop” had decreased from 500 colonies (per plate) in the
first wash to 3 colonies in the fifth. These results were not satisfactory,
however, and the method was abandoned. The authors offer the follow-
ing objections to the method:
a great deal of time was consumed, the wash waters had to be drawn off
immediately after the centrifuge stopped or the paramecia rose in a body and
prevented the removal of the wash water. . . . Another serious drawback
to the centrifuge method is the difficulty of keeping the wash waters free
from contamination by bacteria from the air. The air may contain such
enormous numbers of bacteria that instruments and media which are sterile
to start with will be contaminated unless precautions are taken to prevent
the contact of air bacteria. A sterile pipette laid down on the table is no
longer sterile, a wash water left unprotected is soon contaminated by air
bacteria [p. 435}.
It will become obvious from later discussions of these points that success
of the centrifuge method in the hands of Hargitt and Fray was pre-
vented by two principal faults in their manipulations—too few washes,
and failure to keep their centrifuge and wash tubes plugged at all times.
These authors next attempted to reduce the chances of outside con-
tamination by the transfer method, using watch crystals enclosed within
Petri dishes, and transferring single ciliates through five separate dishes.
They again failed to effect sterility, but succeeded in reducing the num-
ber of colonies ‘“‘per drop” from 2,500 in the first wash to one colony
in the fifth wash. They blame their lack of success with this procedure
upon the fact that the amount of wash fluid was so large that consider-
able time was required to locate the ciliate between transfers.
Successful sterilization was accomplished by transferring individual
paramecia through five successive washes of sterile tap water in sterile
depression slides. The transfer pipettes were sterilized by dry heat be-
fore using, as were the Petri-dish-contained depression slides. The tap

GONTROL OF CULTURES 453

water was autoclaved. They claim to have effected sterilization in a high
percentage of their trials, with a total time consumption per ciliate of not
more than five minutes. If their bacteriological tests are accepted (de-
velopment of bacterial colonies on agar plates, time of incubation not
given) then we can only conclude that they were extremely fortunate
in avoiding ciliates with ingested viable spores.

A chief criticism of the work of Hargitt and Fray was given by Par-
part (1928). He pointed out that most of their sterility tests were con-
fined to the washing fluids and not to the supposedly sterilized paramecia.
In reinvestigating their results, he found that five washes gave sterile
fluid at the end, but that even after ten washes in six out of eight trials
the animals themselves were contaminated. He ascribed this fact to the
probability of the carrying over of viable spores within the vacuoles of
the ciliates. He suggested a simple modification of the method of Hargitt
and Fray, which yielded fifty sterile paramecia out of fifty trials. Instead
of five washes he employed ten, thereby increasing the dilution factor.
Time was allowed (five hours) for the ciliates to void their vacuoles
of possible spores in the fifth wash.

His method ts essentially as follows: A single Paramecium was trans-
ferred with a sterile pipette from a wild culture to a sterile Petri-dish-
enclosed depression slide containing about six drops of washing fluid.
After about one minute, the animal was transferred to the next similar
bath. At the fifth bath the Paramecium was allowed to swim about for five
hours and was then carried through five further washes. All of the manipu-
lations were carried out under a rather elaborate hood to minimize the
possibility of contamination from the air.

This modification of the simple washing method has probably been
more generally used than any other, owing principally to its simplicity
of manipulation. It is admirably adapted to large ciliates which can be
followed with ease under the low powers of the dissecting microscope.
The smaller the organism the more difficult this method becomes. Of
course failure will most surely follow any deviation from absolutely
aseptic technique.

In an attempt further to simplify the technique as outlined by Par-
part, especially regarding the hood under which the transfers were
carried on, the following procedure has yielded extremely satisfactory
results in our laboratory (Kidder, Lilly, and Claff, 1940). Syracuse

454 CONTROL) OF CULGURES

watch glasses are enclosed in cellophane bags, the ends of which are
folded over, and the whole sterilized in the autoclave. After cooling, the
bags are carefully opened and 5 ml. of sterile wash fluid is placed in each
watch glass by means of sterile serological pipettes. The protozoan to
be sterilized is placed in the first bath by means of a micro-pipette
inserted through the open end of the bag. There are three obvious ad-
vantages in this modification, aside from simplicity of apparatus. The
opening of the bag is at the side of the dish and at some distance from
it. The top of the dish and therefore the fluid is never exposed to the
air from above. The same situation is here repeated as obtains when
making tube inoculations in ordinary bacteriological technique, where
the tube is always held at a slant. This system is less dangerous than
one in which the top of a Petri dish must be removed, and obviates
the necessity for a hood or drape. The second advantage is that the ob-
server may follow the movements of the protozoan at all times and may
then readily draw it up in the transfer pipette. This is usually impossible
or difficult when using a Petri dish with the cover in place, as the water
of condensation reduces the visibility markedly. Water does not condense
on the cellophane, and observations are therefore not hampered. The
third advantage is simply one of choice of containers. The Syracuse
watch glasses holding 5 ml. of fluid raise enormously the all-important
dilution factor.

When it is possible to obtain large numbers of Protozoa in heavy con-
centrations, sterilization may be accomplished by centrifugation. This
method, although unsuccessful in the hands of Hargitt and Fray (1917),
has been used to advantage recently (Kidder and Stuart, 1939). It 1s
recommended for use with those species of Protozoa which are so small
as to make them difficult to follow under the powers of a dissecting
microscope. By choosing a washing fluid favorable for the species to be
used and carrying the number of washes far enough, it is usually possible
to recover large numbers of sterile Protozoa after the final wash. The
method which was finally adopted for the sterilization of Colpoda
steinii (see Burt, 1940, for species designation) is quoted below:

After excystment had occurred the ciliates were concentrated by slow
centrifugation and the concentrate removed to a single, sterile, cotton-plugged
centrifuge tube. This ciliate concentrate was diluted with 10 ml. of sterile
distilled water and recentrifuged at a speed which would just throw down

CONTROL OF CULTURES 455

the majority of ciliates in 3 minutes. It was found that the most satisfactory
speed for this purpose was 2000 revolutions per minute. As soon as the
centrifuge stopped the supernatant fluid (9 ml.) was immediately withdrawn
with a sterile 10 ml. pipette and the tube was allowed to stand for about two
minutes in order that the ciliates might swim to the top of the remaining
milliliter of water. With a sterile 1 ml. pipette, 0.5 ml. of ciliate suspension
was withdrawn and placed in an empty sterile centrifuge tube. This suspen-
sion was again diluted, and the process repeated until the ciliates had gone
through an average of fifteen such transfers with the accompanying dilutions
(a dilution factor of approximately 10'). This method entails a great loss
of ciliates but was found necessary inasmuch as, without removal to fresh
tubes with the consequent discarding of the residue, contaminations were
invariable. It was demonstrated that the contaminations resulted from the
fact that ciliates died or became immobilized during centrifugation and were
carried passively to the bottom of the tube with their adhering bacteria. How-
ever, by discarding the dead forms we were able to completely sterilize several
hundred ciliates at each attempt and these gave us the necessary organisms
with which to work [Kidder and Stuart, 1939, p. 332].

The washing fluid which was used in this case was sterile Pyrex-
distilled water. All pipettes were paper-wrapped and autoclaved. The
centrifuge tubes were closed with large cotton plugs and autoclaved.
During centrifugation the cotton was folded over and fastened with a
rubber band, to prevent the plug from being drawn into the tube.

B. Migration —Probably the first report of a technique for obtaining
Protozoa free from bacteria by the utilization of migration was that of
Ogata (1893). He reports that he was able to recover as many as fifty-
two sterile phytomonads (Polytoma uvella) within five to thirty minutes
after the start of the migration. The apparatus he employed was a capil-
lary tube 10 to 20 cm. in length, with a 0.3 to 0.5 mm. bore. This tube
was filled to within one to 2 cm. from the end with a sterile fluid, and
then inserted into a culture of the flagellates and allowed to fill com-
pletely. Care was taken to avoid aid bubbles between the layers. Both
ends of the capillary were sealed by heat and the whole allowed to stand
for from five to thirty minutes. Eventually some of the flagellates were
found to have migrated away from their associated bacteria, and when a
number had collected in the upper end of the tube, this end was broken
off and the flagellates inoculated into nutrient media.

A refinement of the same technique is reported by Stone and Rey-
nolds (1939) for the sterilization of the parasitic flagellate Tv7chomonas

456 CONTROE OF CULTURES

hominis. Their capillary tube was made from a piece of 6 mm. Pyrex
tubing about eight inches in length, which, before sterilization, had been
plugged at both ends with cotton. The capillary was then drawn from one
end of the tube, its tip broken off with sterile forceps, and a series of

()

\c(!

Figure 123. Capillary
tube used for the steriliza-
tion of Trichomonas ho-
minis, The whole tube is
filled with sterile fluid, the
lower end sealed in a flame,
and the Protozoa to be ster-
ilized are layered on to the
fluid at the large end. The
Protozoa eventually migrate
through the capillary por-
tion, but the associated bac-
teria are trapped at the first
or second bend. (Redrawn
from Stone and Reynolds,
1939.)

loops constructed (Fig. 123). All of these manipulations were carried
on with care not to contaminate the outside of the tube, for after the
loops were made the whole tube was filled to within one inch of the top
with sterile fluid (in this case, one part Ringer’s, eight parts horse serum)
by suction, applied to the large end. The capillary end was then sealed
off and the tube, in a vertical position, was incubated forty-eight hours

€ONTROE? OF? CULTURES 457

as a check on sterility. If no turbidity developed, contaminated Ti-
chomonas were layered onto the fluid in the large end of the tube.
Within forty-eight hours many flagellates had migrated down the tube
and could be seen in the last inch or two of the capillary portion. The
authors state that the bacteria failed, for the most part, to migrate past
the first loop, and never passed the second. The last portion of the capil-
lary was cut off and sealed by means of a flame and the cut-off portion
was submerged in tincture of iodine (7 percent) for one hour. Then
one end was grasped in the fingers and the tube held upright to drain.
When dry, pieces of the tube were broken off with sterile forceps and
dropped into selected culture media. The authors state that they have
repeatedly isolated T. hominis bacteria-free by this method, but have not
tested it with other Protozoa.

The above method appears to be applicable to many types of Protozoa,
and should receive serious consideration. The manipulations offer some
difficulty, however, and extreme care will have to be exercised to insure
against outside contamination, especially during the filling of the tube
with the sterile fluid and again during the breaking of the sections of
capillary into nutrient culture media.

Probably the simplest method which takes advantage of the migration
of Protozoa in fluid media is the Petri-dish method. A sterile Petri dish
is partially filled with sterile fluid and placed on the stand of the dissect-
ing microscope so that one edge is under the objective. After all motion
of the fluid has ceased, the Petri dish cover is raised and a drop of con-
centrated protozoan culture is placed very near the edge opposite the
one under the lens. This manipulation must be done with great care,
so that the fluid is disturbed as little as possible. The cover is then gently
lowered and sufficient time (five to ten minutes) is allowed for the
Protozoa to swim to the opposite edge of the dish. The cover is again
raised, and single organisms are picked out with sterile pipettes and
transferred to selected media. Minimum time for the migration is im-
portant, so that none of the highly motile bacteria will reach the area
from which the Protozoa are being taken. Enough Protozoa should be
separated singly in this way to allow for the law of averages. The greater
the motility of the Protozoa, the smaller their size, and the smoother
their bodies, the greater the chance for successful sterilization by this
method.

458 CONTROL OF CULTURES

We have used the above method to sterilize a number of flagellates
(Euglena, Astasia, Chilomonas). With a single migration across a Petri
dish and the selection of twenty-five organisms at each trial, the per-
centage of sterile to contaminated cultures was very high (80 to 90
percent). We used a Plastocoel (transparent) shield over the microscope
and always worked in a draft-free room. Tetrahymena geleii (Furgason,
1940) was also sterilized by a single migration, but with about only 10-
percent efficiency. Although the Tetrahymena were more motile than the
flagellates, they proved harder to rid of their bacteria, probably because
of the tendency of the bacteria to become lodged among the cilia.

Oehler (1919) states that he was able to free various Protozoa of bac-
teria by allowing them to migrate over the surfaces of agar in Petri dishes.
This technique may be applicable to a few types which are able to swim
in a very thin film of moisture.

The utilization of large tubes of sterile fluids for migration purposes
was first mentioned by Purdy and Butterfield (1918). In their studies on
the growth of bacteria in sewage, they state that they obtained on one
occasion sterile Paramecium after allowing the ciliate to swim through
thirty feet of sterile water. They call this the “marathon bath,” but give
no details of its construction, use, or (and this appears to be important
from the practical standpoint) means of sterilization of so long a tube.

Glaser and Coria (1930) carried out a rather exhaustive study on
methods of sterilization. One of the methods they used with success was
the large-tube migration, used with negatively geotropic Protozoa. Their
apparatus consisted of a tube fourteen or more inches long, with one-
fourth inch bore and a fine tapering point. The large end was plugged
with cotton and the whole sterilized in a container. Sterile fluid was
drawn up to within two inches of the top by applying suction to the
large end through a rubber tube. About 2 ml. of contaminated protozoan
culture was then drawn up, and this formed a layer beneath the sterile
fluid. The fine end was sealed in the flame and the tube mounted upright
in a rack (Fig. 124). After periods of time varying from five minutes to
eighteen hours, depending on the species of Protozoa, samples from the
top of the tube contained many organisms which had “washed themselves
free of most other microorganisms” (p. 790). It was usually necessary
to repeat the migration, and this was accomplished by filling a second
tube as before, and then drawing up two inches of fluid from the top

CONTROL OF CULTURES 459

of the first tube. Occasionally a third wash was necessary to render the
Protozoa bacteriologically sterile. Glaser and Coria were successful in
sterilizing three species of ciliates and three species of flagellates by this
method, and later (1935) three other species of ciliates were added to
this list. The identifications of the organisms sterilized are uncertain,
except for the well-known types, Paramecium and Chilomonas.

Another method of migration described by Glaser and Corta in their

Fig. 124. Migration tube.
The tube is filled with a
sterile fluid to within about
two inches from the top.
Protozoa to be sterilized
are drawn up under the
sterile fluid and the small
tip sealed in a flame. The
Protozoa migrate upward
and are taken off at the top.
(Redrawn from Glaser and
Coria, 1930.)

1930 paper was one employing a V shaped tube (Fig. 125), filled with
Noguchi’s semisolid medium. The larger arm of the tube measured 12
cm. in length and had an inner diameter of 28 mm. The smaller arm was
9 cm. in length with an inside diameter of 8 mm. After sterilization, the
tube was filled with 15 ml. of sterile melted medium and this was al-
lowed partially to solidify. Then the contaminated culture was placed at
the bottom of the tube by injection through the small arm with a long,
fine pipette. Air bubbles were excluded. The tube was then allowed to
stand at room temperature for a sufficient time for some of the Protozoa
to reach the top of the large arm, from the surface of which they were re-
covered.

460 CGONTROE OF CULTURES

This method, employing the use of semisolid media, appears to be
applicable for many types of Protozoa. The consistency of the medium
through which the Protozoa migrate seems to favor the removal of bac-
teria, in that vigorous motion is necessary. The bacteria, on the other
hand, would be largely prevented from dispersing far from the point
of inoculation, at least for some time. This method, or some modification
of it, should receive serious consideration from future investigators, in-

Figure 125. V migration
tube for semisolid media.
The Protozoa to be steril-
ized are injected through
the small arm and deposited
at the bottom of the V. They
migrate up through the
semisolid medium and are
removed at the top of the
large arm. (Redrawn from
Glaser and Coria, 1930.)

terested in the problems arising from the use of bacteriologically sterile
protozoa.

C. Combinations of dilution and migration —Cleveland (1928) de-
scribed in detail the various manipulations which he employed to effect
the sterilization of Tritrichomonas fecalis, a flagellate parasitic in the hu-
man intestine. The method which yielded consistently satisfactory results
was a combination of washing and migration. The flagellates to be steril-
ized were concentrated by centrifugation, and the supernatant fluid drawn
off with sterile pipettes. The packed flagellates were than layered onto
the surface of sterile fluid (serum-saline) in centrifuge tubes, and the
centrifuging process repeated. This procedure was continued through
twenty sets of tubes, at which time Cleveland states that the ratio of
Tritrichomonas to bacteria was about fifty to one. This constituted the

CONTROL OF CULTURES 461

dilution part of the technique, and by this part of the procedure alone
he was able to obtain many sterile flagellates. Higher percentages of
sterile flagellates resulted, however, when he added a final migration to
the above washing. After washing, a drop of the packed flagellates was
placed in the center of a large Petri dish filled with sterile fluid. The
Protozoa migrated in all directions, and after various intervals of time
loops of medium, taken two to three inches from the center, were found
to contain ten to fifteen trichomonads. These loops were inoculated di-
rectly into tubes of media and the majority proved to be sterile.

Hetherington (1934) described a much simpler technique, wherein
he alternated the dilution method with migration. He employed Colum-
bia culture dishes in Petri dishes, micropipettes for the transfer of the
organisms, and 10 ml. serological pipettes for filling the dishes. The
procedure was as follows: A drop of concentrated protozoan suspension
was placed in the left margin of the first dish, care being taken not to
disturb the one ml. of sterile fluid. By observing the activity of the Proto-
zoa, it was seen that numbers of them migrated to the right edge of the
fluid. Fifteen to twenty-five Protozoa were picked up in sterile micro-
pipettes and transferred to the left side of a second dish. These were
allowed to migrate. Those Protozoa which migrated were then placed in
a third dish and allowed to remain there for three hours. They were
transferred to the left side of the fourth dish and allowed to migrate,
then transferred to the fifth dish for a second three-hour period. The
sixth and seventh dishes were again used for migration, the Protozoa
from the seventh being placed in culture medium.

By exercising care in the handling of the fluids, the pipettes, and the
covers of the Petri dishes, this method gave excellent results. Spores were
defecated during the two three-hour periods. Especially adherent bacteria
were lost during the migrations and washing, according to Hetherington,
owing to the fact that the medium was nutritive (Bacto yeast extract in
Peter's medium), resulting in the heightened activity of the bacteria.

Recently Claff (1940) has described an apparatus designed to sterilize
negatively geotropic Protozoa, which employs both the principles of
dilution and migration and at the same time reduces the chances of air
contamination. This apparatus consists of six flasks in series (Fig. 126).
The Protozoa are injected into the bottom of flask 1 through a rubber
vaccine cap with a hypodermic needle. From this point on, until they are

462 CONTROL’ OF CUETURES

finally recovered from the sixth flask, they are in an entirely closed sys-
tem. After injection, the Protozoa are allowed to migrate to the narrow
top of the flask and, when large numbers have collected, they are forced
over into the bottom of flask 2 by a volume (1-2 ml.) of sterile medium
from the liter reservoir. After each migration the process is repeated,
and the fluid drained from the system at the top of flask 6 is kept in

Vaccine Porr Vaccine Porr

Figure 126. Migration-dilution apparatus drawn to show construction. All flasks are
filled with fluid and sterilized. After cooling, the Protozoa to be sterilized are injected
through the vaccine port of flask 1. Successive migrations result, and the Protozoa are
finally collected in the test tube from flask 6. (From Claff, 1940.)

sterile test tubes and used as bacteriological controls on the fluid going
before the Protozoa. The first Protozoa collected in the sixth tube are
then separated into the various culture media.

The chief advantages of this apparatus are the simplicity of operation
and the reduction of chance contamination. The whole apparatus ts as-
sembled in a compact unit and can be sterilized partially full of medium,
Completion of the filling of the flasks is carried out after cooling, ex-
treme care being taken to expel all air bubbles. No air bubbles may be
allowed to enter during the injection of the contaminated Protozoa, as
these will practically always rise ahead of the migration and contaminate
every flask in order.

Claff gives experimental evidence for the sterilization of Paramecium,
Tillina, Tetrahymena and Glaucoma in this apparatus. It has been used
on numerous occasions in this laboratory and found to be very satisfac-

GONTROL OF CULTURES 463

tory. Perhaps its chief drawbacks are that it is limited to negatively geo-
tropic organisms and to those types which are relatively powerful
swimmers, and that the flasks must be specially built, as well as the car-
riage, and that the whole is rather expensive. However, in those labo-
ratories in which an extensive program of investigation requiring sterile
Protozoa is being carried on, the purchase or the building of this appara-
tus will be found advantageous.

D. Bactericidal agents—Many methods have been tried in which
agents were employed to kill bacteria without killing the Protozoa under
investigation. Inhibition of bacterial overgrowth has been reported on
numerous occasions by the use of various chemicals (Zumstein, 1900;
Kofoid and Johnstone, 1929; and others) but sterility has rarely been
obtained. Cleveland (1928) states that he used numerous chemicals in
the hope that some would prove less injurious to Tritrichomonas than to
the associated bacteria. His results were entirely negative, and he states
that this type of investigation “appears to be almost a hopeless under-
taking” (p. 256).

We do have, however, a few reports which indicate the possibility of
obtaining sterile Protozoa after treatments with various chemicals which
are more toxic to the bacteria than to the Protozoa. In all cases the bac-
tericidal agents were used on protozoan cysts, not on the trophic forms.
Frosch (1897) was the first to report the sterilization of cysts by chemi-
cal means. By the immersion of old cysts of Amoeba nitro phila in satu-
rated sodium carbonate for a period of three days, Frosch claims to have
killed all the associated non-spore-forming bacteria and to have recov-
ered some of the amoebae. This method was repeated by Walker (1908)
and the results confirmed. Oehler (1924) examined the possibility of
treating ciliate and amoebae cysts with a variety of disinfectants, acids,
alkalies, and salts but these results were far from encouraging. Severt-
zoff (1924) investigated the action of toluene, chlorine, and calcium
sulphide on the cysts of “‘soil amoebae” and found that the cysts were
able to withstand the deleterious effects of these chemicals better than
the associated bacteria (non-spore-forming types). He claims to have ef-
fected complete sterility by the use of calcium sulphide, but was unable
to establish pure cultures from the resulting cysts. Glaser and Coria
(1930) were unable to obtain sterile Evglena proxima by their washing
methods, but succeeded by treating ‘round or encysted stages’ (p. 803)

464 CONTROL OF CULTURES

with a solution of potassium dichromate (1.25 to 2.5 percent) for from
fifteen to thirty minutes. Perhaps the most exhaustive study on the rela-
tive effects of chemicals on bacteria and protozoan cysts was carried out
by Luck and Sheets (1931). They investigated the lethal concentrations
of some eighteen substances on two-day-old cysts of Explotes taylori and
their associated bacteria. They appear to have obtained sterile ciliates in
some cases, when silver nitrate was used in high concentrations of glu-
cose and sucrose. All other substances either were more toxic to the
protozoan cysts than to the bacteria or were uniformly lethal to both.

This line of approach to protozoan sterility does not seem to be too
encouraging. As might well be supposed, the investigator is limited in
any case to cyst-forming types. Even these are not uniformly resistant
to chemical action, so that it appears that little can be learned at this
time from the experiences of others. It seems that the methods referred
to above, while they may have produced the desired results in some cases,
are not to be recommended for routine work. There is always the prob-
ability that any type of protozoan collected from the wild will have as-
sociated with it spore-forming bacteria, which are always highly resistant
to disinfectants. If a cyst-forming type of protozoan is first sterilized by
washing or some other method and established in culture with a single
known bacterium, then some of these disinfectants might be used to ad-
vantage later for obtaining large numbers of sterile organisms for ex-
perimentation.

Of the physical bactericidal agents which have been employed, heat,
used in different ways but always upon cysts of various species, has been
most generally used. Walker (1908) reports that he was able to obtain
sterile “Amoeba intestinalis’ by inoculating an agar plate, first with
concentric rings of B. col7 and then, in the center, with amoebae cysts.
The plate was heated to 70°-75° C. for one hour, which was sufficient to
kill the Bac#llus coli but did not kill the cysts. He then added fresh bac-
teria to the center of the plate and the amoebae excysted, fed, and mi-
grated through the rings of dead B. co/7, Walker states that in their migra-
tion through the dead bacteria they freed themselves of all living bacteria
(by scraping them off?). He was able to recover sterile amoebae at the
periphery of the plate. This report is surprising, in that moist heat was
used, and cysts of Protozoa in general, under these conditions, are usually

killed.

CONTROL OF CUETURES 465

Oehler (1924) reports that he was able to obtain sterile Colpoda
from cysts which had been heated either at 37° C. for six weeks or at
60°-64° C. for several hours. While it is true that certain types of bac-
teria are killed by desiccation for considerable lengths of time, it would
be nothing short of a miracle if only these delicate forms happened to
make up the associated flora from the wild. In this laboratory we have
employed a modification of Oehler’s method for obtaining large numbers
of C. steinii, but a short description of the results will indicate that the
method is not to be relied upon for initial sterilization.

Figure 127. Details of
construction of the sponge-
and-glass plunger for the
collection of cysts. The cen-
ter rod is solid, and a piece
of tubing holds the sponge
down.

We maintain cultures of C. ste/nii, sterilized by centrifugation, as de-
scribed in a preceding section, in suspensions of the common nonspore-
forming coliform bacterium, Aérobacter cloacae. From time to time we
have needed large numbers of sterile ciliates for experimental work,
and these were obtained from cysts collected as follows. A ring of sponge
is placed at the bottom of a glass plunger which will reach well into a
culture tube (Fig. 127). These sponge and glass plungers, with the
top of the rods wrapped in cotton, are placed in test tubes of water and
autoclaved. When cool, the plunger is transferred aseptically to a culture
of Colpoda in Aérobacter. After the food organism is largely depleted,
the ciliates encyst on and within the sponge. The plunger is then with-
drawn and placed in a dry sterile test tube and set aside to dry. At first

466 CONTROL OF CULTURES

it was hoped that prolonged desiccation, which we knew would not
harm the cysts, would cause the death of the bacteria. Tests were made
after one, two, three, and six months (duplicate preparations being
placed by desiccators) by placing the plungers into tubes of sterile ex-
cysting fluid (yeast extract). While the length of time required to show
turbidity increased with the time of drying, viable Aérobacter were pres-
ent in every case. Heating the dry sponge to 80° C. for three hours did
not kill the cysts, nor did it kill the Aérobacter. The heat resistance is
very different in the dry and the wet states, as evidenced by the fact
that one hour of heating at 50° C. is enough to kill a suspension of this
bacterium.

It was found, however, that a simple manipulation could be employed
with eight-months’-old sponge-glass plunger preparation to obtain large
numbers of sterile ciliates. U-shaped tubes were partly filled with yeast
extract and sterilized, and the sponge-glass plunger was inserted aseptt-
cally into one arm. The tube was placed in an upright position and the
minimum time was allowed for the ciliates to excyst. Then, without
disturbing the fluid, the plug from the other arm was lifted and some
of the fluid containing the freshly excysted ciliates was withdrawn. These
ciliates were sterile, provided not too long a time had elapsed since the
sponge was introduced and provided the fluid was not unduly disturbed.
What appears to happen is that the dry bacteria take a considerable time
to become active and to disperse through the medium, while a few sec-
onds after the ciliates leave their cyst walls they migrate to the top of
the fluid. It should be remembered that this method has been used only
when the associated bacteria have been reduced to a single species. By
careful manipulation and by having the conditions just right, it might be
possible to use this method on wild, cyst-forming material, but this 1s
only a conjecture as it has not been done to date.

The possibility of using radiation for sterilization was investigated by
Brown, ef al. (1933). They found that E. taylor7, trophic or cysts, with-
stood a much longer exposure to X-rays (2,110 Roentgen units per
second) than did the associated bacteria (in this case Pseudomonas
fluorescens and B. coli). They were able to obtain many sterile ciliates
by this method. Here again, the success of the method appears to be due
to the specific type of bacteria present, and it is extremely doubtful if,

CONTROL OF CULTURES 467

under the conditions of wild cultures, sterile Protozoa could be obtained
with any regularity by this method.

THE IMPORTANCE OF ADEQUATE STERILITY TESTS

It is, of course, obvious that any method for ridding Protozoa’ of bac-
teria must be carried out under the rigid rules of bacteriological tech-
nique. Bacteria are so varied in form and activity that special pains must
be taken to check the results of any method before the treated Protozoa
may be pronounced sterile. Microscopic examination is of little or no
use, at least until time has been given for any accompanying bacteria
to multiply. We must therefore give any possible contaminant every
conceivable chance to multiply, thereby revealing its presence.

Sterility tests are usually made in two ways, by inoculation into fluid
media and by spreading on nutrient solid media. In the fluid media
(broth) contamination shows itself when the broth becomes turbid. The
turbidity test is sufficient, when the contaminant is such a one as will
distribute itself through the media. In other words, turbidity denotes
contamination. But lack of turbidity does not always denote sterility.
Some bacteria grow very slowly in broth and form small clumps which
sink to the bottom of the test tube, leaving the broth clear. This experi-
ence was reported by Hetherington (1933, 1934) and by Stuart, Kidder,
and Griffin (1939). A macroscopic examination of a tube was not enough
to reveal the presence of these organisms.

Plating, either by pipetting fluid from the culture to be tested or by
streaking the surface of solid media (nutrient agar) with a needle dipped
into the culture, is usually more satisfactory than the turbidity test. The
plates are allowed to incubate, and the surface is examined for bacterial
colonies. The usual contaminants from wild infusions will appear in
from twenty-four to forty-eight hours. But this method has its limitations.
Some bacteria grow very slowly at room temperatures, but well at higher
temperatures. Others are the reverse. Duplicate sets of plates should
always be made, one set to be incubated at room temperature and the
other at temperatures from 30° to 37° C. The time factor should be
carefully considered. The slow-growing types (such as the Mycobac-
tertum reported by Stuart, Kidder, and Griffin, 1939) may not appear
until many days after the inoculation. It is necessary in all tests with agar

468 CONTROL OF GUETURES

plates to keep the plates for at least ten days and it is safer to keep them
two weeks. The plating method is usually a better criterion of conditions
within a culture than the turbidity test, for another reason. When dealing
with ciliates, normally bacteria-feeders, it is often the case that the bac-
teria are eaten out of the media almost as fast as they multiply. This is
more likely to happen when the ciliate is a voracious feeder and multi-
plies rapidly and when the contaminant is one of the slow-growing va-
riety. It should not be supposed, however, that all of the bacteria will
be eaten, although two such cases are on record (Elliott, 1933; Johnson,
1935). Some of the bacteria will almost invariably escape and be carried
along from transplant to transplant. On the solid media, however, the
ciliates do not move about, and colonies of bacteria develop unhampered.

Although they are not prevalent in wild infusions, tests should always
be conducted for anaérobic bacteria. The simplest test and one which
will usually determine their presence or absence is the following: Tubes
containing not over 3 ml. of nutrient broth plus a two to two-and-a-half-
inch layer of paraffin oil, are plugged with cotton and autoclaved for
twenty minutes at fifteen pounds’ pressure. Rubber stoppers are sterilized
at the same time. Immediately after sterilization the rubber stoppers are
fitted into the tubes, which are then allowed to cool. When the broth is
cool, inoculations are accomplished by injecting the material to be tested
through the paraffin oil into the broth, the rubber stopper being im-
mediately replaced. This type of culture will allow even obligatory an-
aérobes to multiply, although not necessarily to the height of their
capacity. Enough growth is obtained, however, to determine the pres-
ence of anaérobic contaminants. The obligatory anaérobic bacteria, it
must be admitted, do not form an important group for our consideration,
as they occur so infrequently. The facultative anaérobes may be detected
by more common procedures.

ESTABLISHMENT OF STERILIZED PROTOZOA IN CULTURE

The sterilization of Protozoa is, after all, only a means to an end. It
is of very little value to the investigator if, after going to the trouble to
rid a species of Protozoa of their associated bacteria, the Protozoa fail to
live. For the most perfect control of a protozoan culture for experimental
work, pure cultures are necessary. This means that the protozoan under
investigation must be established in a medium containing no other living

CONTROL OF CULTURES 469

organism. This is easily accomplished in a number of cases, even to the
establishment in media containing only dissolved proteins. A large num-
ber of flagellates probably exist in nature by the utilization of dissolved
substances and, when sterilized, continue to employ this type of nutri-
tion. Among the free-living ciliates, all species are known to possess oral
openings into which solid foods are drawn. Some of these, however, are
able to live on dissolved proteins in pure culture, e.g., Tetrahymena.
Other types may be able to obtain only a small amount of nutriment
from the dissolved proteins, but are able to feed on nonliving particu-
late matter. This was found to be the case in this laboratory with Glaz-
coma scintillans (unpublished work). When sterilized ciliates were
placed in a wide variety of media containing dissolved proteins, very
little multiplication took place. However, good growth resulted in un-
filtered Yeast Harris, containing quantities of broken-down yeast cells.
Still other types of free-living ciliates appear to be unable to exist with-
out living organisms as food. This may be the case with the true carni-
vores, and here there is an excellent opportunity to make some interesting
studies on the ‘“Zweigliedrige Kulture” without employing bacteria. It
is only necessary to be able to grow the food Protozoa in pure culture
and to supply it to the sterile carnivores. Some work along these lines
has already been started in our laboratory, using pure cultures of Tetra-
hymena as the food organism and studying the effects of such a diet on
G. vorax (Kidder, Lilly, and Claff, 1940). Similar cultures of Stylony-
chia pustulata are being studied by D. M. Lilly (Lilly, 1940), and the
nutritional requirements of a Evglena-feeding Perispira is being investi-
gated by the author and V. C. Dewey in this laboratory. Oehler (1919)
stated that Colpoda steinii was unable to live on dissolved nutrients but
would live on particulate matter, including dead bacteria. Kidder and
Stuart (1939) were unable to confirm Oehler’s results, but found that
C. stesniz was dependent upon living organisms. They remark, however,
that the possibility does exist that some combination of food substances
and conditions, as yet not known, may possibly allow this important
ciliate to reproduce in the absence of living organisms.

It may be inferred from what has already been said that the estab-
lishment of a sterile protozoan in culture is not a routine matter. Of
course the goal is a pure culture, for more precise control is then possible.
With some types it has been found that growth follows when they are

470 CONTROL OF CULTURES

placed immediately in a medium containing dissolved proteins (tryptone,
proteose-peptone, yeast extract, and so forth). These types are the true
saprozoic forms. Others must have particulate matter, so it is best, espe-
cially when dealing with a new type, to inoculate into a wide variety of
media. In this laboratory it is the practice to start our newly sterilized
Protozoa in five different types of media, usually ten isolations into each.
Our standard five types for first tests are 0.1-percent proteose-peptone;
5-percent yeast autolysate; 0.5-percent yeast extract; 0.5-percent malted
milk; and 0.5-percent unfiltered Yeast Harris. It is sometimes necessary
to have quite a range of pH values within the different media, in order
to obtain growth in even one or two of the tubes.

A number of species of Protozoa appear to be dependent upon living
organisms as a source of food. This is true not only of the carnivores,
but seems to hold for a number of bacteria-feeders as well. With the
carnivores it is usually sufficient to observe their diet in nature to decide
upon a suitable food animal. If the food animal can be grown in pure
culture, then the chances are good that it will be possible to establish
the carnivore in “Zwiegliedrige Kulture.” Some carnivores have been
found to be very selective, while others are able to feed on any one of a
number of organisms. Occasionally a natural bacteria-feeder will turn
carnivorous and then can be established without bacteria.

With the obligatory bacteria-feeders the best that can be done, as far
as we now know, is to establish them on a single species of favorable
food bacteria. Here again it is absolutely necessary to start with sterile
Protozoa, as even in the so-called non-nutritive fluids (salt solutions,
distilled water, and so forth) many extraneous bacteria, which are not
favorable as food, will multiply and be continually present from trans-
plant to transplant. Results may be entirely misleading under these con-
ditions, as a number of common bacteria prove to be deleterious to many
Protozoa (see Kidder and Stuart, 1939). One method of setting up cul-
tures containing a single protozoan species in a suspension of a single
species of bacteria is simply to try out a number of known bacteria until
one is found which will support growth. However, some ciliates prove
to be extremely selective, even as to specific bacteria. If poor growth or
no growth results after all the known bacteria have been used, then the
investigator must try to isolate from the wild culture the type of bacteria
upon which the ciliate was originally feeding. This procedure is tedious,

CONTROL OF CULTURES 471

but sometimes necessary. A wild culture is selected in which the Protozoa
under investigation are multiplying rapidly. From the fluid of this cul-
ture, surface-streak plates are prepared and pure cultures of all the
different types of bacteria are obtained. From these pure cultures, sus-
pensions are systematically prepared and inoculated with the sterile
Protozoa. This was the method used by Johnson (1933) in his work on
Oxytricha. Johnson states that his selection was made on the basis of
prevalence, in a thriving wild culture. In other words, the type of bac-
teria found in the greatest abundance he supposed to be the type upon
which the Protozoa were most likely to be feeding. We have found that
this is not always the case. In our work on the ciliate T7/lina (T. canalt-
fera, obtained from Dr. J. P. Turner and described by him in 1937)
we were able to obtain growth on one species only, out of twenty-six
types isolated from a thriving culture. This one species (a Zopfius) was
the least prevalent of all on our plates. The reason for this appears to be
that T7//7na being so very selective, the Zopfws were eaten out of the
culture by the time we took our samples, while the other twenty-five
species were left to multiply. We have found this situation to hold in a
number of cases, so that we are of the opinion that the results obtained
by Johnson were due to the fact that he was dealing with a ciliate which
was not rigidly selective.

The work tending to show that supplementary factors (viz., thiamin
and the like) are necessary for the growth of several Protozoa in pure
culture has been reviewed in a subsequent chapter, but several observa-
tions regarding the same theme may be given here, as they apply to
“Zwiegliedrige Kulture.” These supplements may make the difference
between success and failure to establish a protozoan in bacteria-free cul-
ture. Investigations are now going on in this laboratory on the supple-
ment question, but they are as yet far from complete. Therefore little
can be said as to the exact nature of the substances or factors to be de-
scribed. In order to present this problem clearly to the reader, a descrip-
tion of a typical example will be given.

D. M. Lilly, working in this laboratory, has studied the nutritional
requirements of two hypotrichous ciliates, Stylonychia pustulata and
Pleurotricha lanceolata. Both of these forms are bacteria-feeders in na-
ture, but will also become carnivorous in the presence of other small
ciliates. Sterilization was carried out, with the use of the dilution method,

472 CONTROL OF CULTURES

in Syracuse watch glasses enclosed in cellophane bags, as previously
described. In the case of Stylonychia, it was found possible to establish
them on living yeast cells, suspended in distilled water, in the absence
of any other food material. Sterile ciliates would not live on autoclaved
yeast, however. Sterile ciliates would eat quantities of living Tetrahymena
(taken from agar slants and suspended in distilled water), but would
not divide. Sterile ciliates, placed in suspensions of autoclaved yeast, and
living Tetrahymena grew well and established flourishing cultures. Sterile
ciliates in dead yeast and dead Tetrahymena, failed to multiply. Additions
of none of the known water-soluble vitamins changed the situation. The
inference is, as Lilly points out (1940), that Stylonychia requires, among
other things, two unknown factors—one found in yeast (even after
autoclaving), but not present, at least in sufficient quantities, in Tetra-
hymena; the other what might be called a living factor, present in living
yeast and Tetrahymena. Both of these factors are present in certain favor-
able species of bacteria when the bacteria are alive, but the “living factor”
is destroyed with the death of the bacteria. The so-called living factor
is not a surprising requirement among Protozoa, as experience has shown
that many different types will not live without being supplied with some
type of living organism. The yeast factor seems to belong to the water-
soluble, heat-stabile group, but is not identifiable with any one of the
known B complex. While this factor is present in dried and pasteurized
yeast (Brewer's Yeast Harris), it is not present in sufficient quantities
in Difco dehydrated yeast extract. Concentration and partial purification
of the yeast factor have been carried out, but until this work is further
along we must content ourselves with these few facts.

This example is one of many similar cases and serves to point out that
several conditions must be recognized and fulfilled, if the investigator
is to be successful in establishing sterile Protozoa in culture. The possi-
bility of supplementary factors must be considered before it can be said
of any type that it cannot be grown bacteria-free. Somewhat the same
situation was encountered by Glaser and Coria (1933) in their work on
Paramecium. They finally announced a complicated medium which
proved to be successful, and this medium contained pieces of fresh rabbit
kidney (possibly supplying the living factor during the early growth
phases of the ciliate).

It is not the purpose of this chapter to consider in detail all of the

CONTROL OF CULTURES 473

interesting work which has been done regarding accessory growth fac-
tors and nutritional supplements. A large number of these are consid-
ered in the chapter on pure cultures (Chapter IX). It might be sug-
gested, however, that one of the most fertile fields of protozoan investi-
gation has been opened up with the development of bacteria-free tech-
niques, and our knowledge of unsuspected requirements in the nutrition
of carnivores should be extended greatly in the near future. The possi-
bilities are many along these lines, and therefore considerable time has
been devoted to the methods which will have to be employed in the be-
ginning of any such studies.

LITERATURE CITED

Brown, M. G., J. M. Luck, G. Sheets, and C. V. Taylor. 1933. The action of
X-rays on Explotes taylori and associated bacteria. J. gen. Physiol., 16:
397-406.

Burt, R. L. 1940. Specific analysis of the genus Colpoda with special reference
to the standardization of experimental material. Trans. Amer. Micros.
Soc. (in press.)

Claff, C. L. 1940. A migration-dilution apparatus for the sterilization of
Protozoa. Physiol. Zo6l. (in press. )

Cleveland, L. R. 1928. The suitability of various bacteria, molds, yeasts and
spirochaetes as food for the flagellate Tritrichomonas fecalis of man as
brought out by the measurement of its fission rate, population density,
and longevity in pure cultures of these microorganisms. Amer. J. Hyg.,
8: 990-1013.

Elliott, A. M. 1933. Isolation of Colpidium striatum Stokes in bacteria-free cul-
tures and the relation of growth to pH of the medium. Biol. Bull., 65:
45-56.

Frosch, P. 1897. Zur Frage der Reinziichtung der Amében. Zbl. Bakt., Orig.,
21: 926-32.

Furgason, W. H. 1940. The significant cytostomal pattern of the “Glaucoma-
Colpidium group,” and a proposed new genus and species, Tetrahymena
geleii, Arch. Protistenk. (In press.)

Glaser, R. W., and N. A. Coria. 1930. Methods for the pure culture of certain
Protozoa. J. exper. Med., 51: 787-806.

—— 1933. The culture of Paramecium caudatum free from living micro-
organisms. Jour. Parasit., 20: 33-37.

1935. The culture and reactions of purified Protozoa. Amer. J. Hyg.,
PABA VIMIEP AU

Hargitt, G. T., and W. W. Fray. 1917. The growth of Paramecium in pure
cultures of bacteria. J. exper. Zool., 22: 421-54.

Hetherington, A. 1933. The culture of some holotrichous ciliates. Arch.
Protistenk., 80: 255-80.

474 CONTROL OF CULTURES

—— 1934. The rdle of bacteria in the growth of Colpidium colpoda. Physiol.
Zool. 7: 618-41.

Johnson, D. F. 1935. Isolation of Glaucoma ficaria Kahl in bacteria-free cul-
tures, and growth in relation to pH of the medium. Arch. Protistenk.,
86: 263-77.

Johnson, W. H. 1933. Effects of population density on the rate of reproduc-
tion in Oxytricha. Physiol. Zo6l., 6: 22-54.

Kidder, G. W., D. M. Lilly, and C. L. Claff. 1940. Growth studies on ciliates.
IV. The influence of food on the structure and growth of Glaucoma
vorax, sp. nov. Biol. Bull., 78: 9-23.

Kidder, G. W., and C. A. Stuart. 1939. Growth studies on ciliates. I. The
role of bacteria in the growth and reproduction of Colpoda. Physiol.
Zool., 12: 329-40.

Kofoid, C. A., and H. G. Johnstone. 1929. The cultivation of Endameba
gingivalis (Gros) from the human mouth. Amer. J. publ. Hlth., 19:
549-52.

Lilly, D. M. 1940. Nutritional and supplementary factors in the growth of
carnivorous ciliates. (MS.)

Luck, J. M., and Grace Sheets. 1931. The sterilization of Protozoa. Arch.
Protistenk., 75: 255-69.

Oehler, R. 1919. Flagellaten- und Ciliatenzucht auf reinem Boden. Arch.
Protistenk., 40: 16-26.

—— 1924. Weitere Mitteilungen tiber gereinigte AmGdben- und Ciliaten-
zucht. Arch. Protistenk., 49: 112-34.

Ogata, M. 1893. Uber die Reinkultur gewisser Protozoen (Infusorien). Zbl.
Bakt., Orig., 14: 165-69.

Parpart, A. K. 1928. The bacteriological sterilization of Protozoa. Biol. Bull.,
113-20:

Purdy, W. C., and C. T. Butterfield. 1918. Effect of plankton animals upon
bacterial death rates. Amer. J. publ. Hlth., 8: 499-505.

Severtzoff, L. B. 1924. Method of counting, culture medium and pure cultures
of soil amoebae. Zbl. Bakt., Orig., 92: 151-58.

Stone, W. S., and F. H. K. Reynolds. 1939. A practical method of obtaining
bacteria-free cultures of Trichomonas hominis. Science, n.s., 90:
O1-92:

Stuart, C. A., G. W. Kidder, and A. M. Griffin. 1939. Growth studies on
ciliates. III. Experimental alteration of the method of reproduction in
Colpoda. Physiol. Zoél., 12: 348-62.

Turner, J. P. 1937. Studies on the ciliate Ti/lina canalifera, n. sp. Trans. Amer.
micr. Soc., 56: 447-56.

Walker, E. L. 1908. The parasitic amebae in the intestinal tract of man and
other animals. J. med. Res., 17: 379-459.

Zumstein, H. 1900. Zur Morphologie und Physiologie der Evglena gracilis
Klebs. Jb. wiss. Bot., 34: 149-98.

CHAPTER IX

FOOD REQUIREMENTS AND OTHER FACTORS
INFLUENCING GROWTH OF PROTOZOA
IN PURE CULTURES

Rege. ALT

Ir Is OBVIOUS that the growth of Protozoa is influenced by many differ-
ent factors. The importance of some of these is well recognized and the
relationships to growth are partially understood in a few instances, but
there is little or no detailed information bearing on other factors. Here
and there, investigations have suggested possible solutions to certain
problems, but just as frequently have uncovered new problems which
in turn must be solved in the approach to an understanding of protozoan
growth. The present lack of information extends to such questions as
the list of essential elements, the nature of the simplest organic foods
adequate for various species, “‘growth factor’ or vitamin requirements,
and the combined effects of various environmental factors on growth.
Furthermore, Protozoa in cultures constitute populations and presumably
are subject to general laws of population growth. Hence the final inter-
pretation of many experimental results demands further knowledge of
the behavior of populations.

From the experimental standpoint, several types of protozoan popu-
lations may be distinguished. (1) The pure culture contains a single
protozoan species with no other microérganisms. In most cases such
cultures have been started from pure lines and are thus genetically homo-
geneous. The number of bacteria-free strains now in existence is un-
certain, although an estimate of 100 may be fairly accurate. Many strains
of Phytomastigophora are maintained by Pringsheim (1930), while ad-
ditional species belonging to various groups of Protozoa are to be found
in several other laboratories. (2) The species-pure culture contains a
single protozoan species, usually in pure line, with bacteria, algac, or
other microérganisms as sources of food. Populations of this type have
been maintained on known species of microérganisms (e.g., Ocehler,

476 FOOD REQUIREMENTS

1916, 1919; Philpott, 1928; Geise and Taylor, 1935; D. F. Johnson,
1936; W. H. Johnson, 1933, 1936; Loefer, 1936d) or on mixtures of
bacteria. (3) Mixed populations, as described in the work of Gause
(1935), contain two species of Protozoa feeding on other microérgan-
isms, or perhaps one upon the other. This technique presents interesting
possibilities. (4) Wild populations are mixtures of species as obtained
from natural sources. Such populations have been studied particularly
in relation to succession of species in cultures (e.g., Woodruff, 1912).

The present discussion deals primarily with investigations on pure
cultures, which, with their obvious advantages, afford favorable ma-
terial for the study of many problems. With the exclusion of other
microorganisms, it is possible to control the food supply and to deter-
mine, more accurately than by other methods, the relation of environ-
mental factors to growth. Detailed investigation of metabolic activities
is possible with pure cultures, whereas allowance must be made for
other microorganisms when bacteria-free material is not used. The pure-
culture technique and scrupulous cleanliness of glassware are essential
in studies on food requirements. This is true particularly of investiga-
tions on autotrophic nutrition, since protein contamination, to the ex-
tent of one part in millions, may influence growth. Likewise, pure cultures
are a prerequisite to investigations on specific growth factors, or vitamins.
Some of the methods used by various investigators have been described
elsewhere (Pringsheim, 1926; Hall, 1937a). The technique is not par-
ticularly difficult and, while the preparation of glassware is somewhat
laborious and constant precaution against contamination must be exercised,
the results more than justify the additional time and effort.

Food REQUIREMENTS OF PROTOZOA

Food requirements of the various groups of Protozoa differ in certain
general respects. The chlorophyll-bearing species may utilize carbon
dioxide, while other types require a more complex carbon source. Nitro-
gen requirements also vary. Some forms thrive on ammonium salts or
on nitrates; growth of other species is supported by nothing simpler than
an amino acid; while that of a third group is dependent upon peptones
or comparable protein-cleavage products. On the basis of such criteria,
a number of different methods of protozoan nutrition have been recog-
nized (Lwoff, 1938a; Pringsheim, 1937d). A somewhat simplified classi-
fication (Hall, 1939b) is presented below:

FOOD REQUIREMENTS 477

I. Phototrophic nutrition is characteristic of chlorophyll-bearing
species, which utilize the energy of light in photosynthesis. Some appear
to be obligate phototrophs, while others may be grown in darkness under
suitable conditions. On the basis of nitrogen requirements, several va-
rieties of phototrophic nutrition may be recognized:

(1) Photoautotrophic nutrition is characteristic of species which can
grow in inorganic media; Chlorogonium euchlorum (Loefer, 1934; Hall
and Schoenborn, 1938a) is typical. No obligate photoautotroph is known.

(2) Photomesotrophic nutrition 1s that in which one or more amino
acids serve as nitrogen sources. In Euglena deses (Dusi, 1933b) this
seems to be the simplest possible method of nutrition. Photomesotrophic
nutrition may also be carried on by facultative photoautotrophs.

(3) Photometatro phic nutrition is characteristic of species which grow
in peptone solutions or comparable protein media. Exglena pisciformis
(Dusi, 1933b) has been described as an obligate photometatroph. This
type of nutrition may also be carried on by facultative photoautotrophs
and photomesotrophs.

II. Heterotrophic nutrition is characteristic of species which have no
chlorophyll and hence require an organic carbon source. Some chloro-
phyll-bearing species have been grown in darkness and may, in this sense,
be considered facultative heterotrophs. On the basis of nitrogen require-
ments, three varieties of heterotrophic nutrition may be distinguished.

(1) Heteroautotrophic nutrition involves utilization of inorganic
nitrogen compounds in the presence of an organic carbon source. Poly-
toma uvella (Pringsheim, 1921; Lwoff and Dusi, 1938a) and Astasia sp.
(Schoenborn, 1938) are examples.

(2) Heteromesotrophic nutrition: growth requirements may be satis-
fied by one or more amino acids as sources of nitrogen and carbon.
Growth is usually much more vigorous with an additional carbon source,
such as acetate. Polytomella caeca (Pringsheim, 1937a, 1937c) is repre-
sentative. ;

(3) Heterometatrophic nutrition is characteristic of organisms which
grow in peptone solutions or similar media. Obligate heterometatrophs,
such as Hyalogonium klebsi (Pringsheim, 1937b) and Glaucoma piri-
formzs (A. Lwoff, 1932), cannot be grown in amino-acid solutions or
simpler media. This type of nutrition is exhibited by various holozoic
Protozoa (ciliates, amoebae) which have been grown in pure culture.
Among the parasitic flagellates, certain Trypanosomidae (M. Lwoff,

478 FOOD REQUIREMENTS

1930, 1933a, 1936) have been grown under comparable conditions;
other parasitic flagellates (M. Lwoff, 1929a, 1929b, 1929c, 1929d,
1933a, 1933b, 1937, 1938a; Glaser and Coria, 1935b; Cailleau, 1936a,
1936b, 1937a, 1937b, 1938a, 1938b) apparently require, in addition,
blood, serum, tissue extracts, or special growth factors.

PHOTOAUTOTROPHIC NUTRITION

Photoautotrophic nutrition is generally attributed to the chlorophyll-
bearing plant-like flagellates and is, by definition, limited to this group
of Protozoa. On the other hand, there is no evidence to support the as-
sumption that all chlorophyll-bearing species are photoautotrophic, since
several green flagellates have been grown only in amino acid or peptone
media. Furthermore, in the absence of pure cultures, there is no conclu-
sive evidence that any member of the Chrysomonadida, Heterochlorida,
Cryptomonadida, Dinoflagellida, or Chloromonadida is capable of carry-
ing on photoautotrophic nutrition. While it may be expected that such
flagellates will be found in each of these orders, speculation must re-
main subject to experimental verification.

The known facultative photoautotrophs are: Chlamydomonas agloé-
formis (M. Lwoff and A. Lwoff, 1929), Chlorogonium elongatum
(Loefer, 1934), C. eachlorum (Loefer, 1934; Hall and Schoenborn,
1938a), Haematcoccus pluvialis (M. Lwoff and A. Lwoff, 1929), and
Lobomonas piviformis (Osterud, 1938, 1939), representing the Phyto-
monadida; and Euglena anabaena (Dusi, 1933b; Hall, 1938b), E. gracz-
lis (Pringsheim, 1912; Dusi, 1933a; Hall and Schoenborn, 1939a),
E. klebsii and E. stellata (Dusi, 1933b) and E. viridis (Hall, 1939a),
representing the Euglenidae.

The establishment of autotrophic strains has often encountered difh-
culties, and conflicting results have sometimes been reported for the same
species. Some of the apparent contradictions may be the result of differ-
ences in culture media and in technique. In addition, the technical diffi-
culties may sometimes be augmented by a selective action of inorganic
media, as observed in Evglena (Hall and Schoenborn, 1938b).

The present knowledge of food requirements in photoautotrophic
nutrition is far from complete. In fact, it is not yet possible to list all
the elements which are essential to growth, and little or nothing 1s known
about quantitative food requirements. However, the following elements,

FOOD REQUIREMENTS 479

which are found as general constituents of protoplasm, may be listed as
probably essential to growth: C, H, O, N, P, S, Ca, Fe, K, Na, Mg, Ck
Additional possibilities include Cu, Sr, Al, Mn, Zn, Ni, B, Rb, Ba, Si, Ti,
V, As, Co, and Cr, since these have all been demonstrated in plant or
animal tissues and some appear to be essential to the growth of higher
organisms.

By a process of successive eliminations, it should be possible to deter-
mine which elements are and which are not essential to growth. Such
investigations, however, are entirely dependent upon adequately purified
chemicals. In certain investigations (Hall, 1938b, 1939a; Hall and
Schoenborn, 1939a; Osterud, 1938) analyzed reagents have been used
in the preparation of culture media and, within such limits, the composi-
tion of each medium is known. One of these media (EF) contains the
following elements: C, H, O, N, P, K, Mg, S, Ca, and Cl in appreciable
amounts, and traces (1 & 10 to1 X 10 gm. percc.) of Cu, Ba, Fe, As,
Mn, Na, Zn, and Pb. This medium has supported growth of Evglena
gracilis, E. viridis, E. anabaena, and Lobomonas piriformis. Another
medium (EC) has supported growth of E. gracilis, E. viridis, and Chlo-
rogonium euchlorum. So far as the component elements are concerned,
this medium differs from EF in the absence of Ba, in lower concentrations
of Ca, Cl, Mg, and Mn, and in higher concentrations of P and K. Media
EA and EAB, which have supported growth of E. gracilis, E. viridis, and
C. euchlorum, contain a trace of Al, but no Ba; except for concentrations,
the list of elements is otherwise the same as in EC and EF. Just how
many of the “‘trace’’ elements are actually essential to growth has not
been determined. The omission of Ba from three media and of Al from
two media seems to be of little significance, and the status of these two
elements as essential substances is questionable. By comparable methods
of elimination, it may be possible to determine whether various other
elements are actually essential in photoautotrophic nutrition.

In a few cases there is evidence that particular elements exert signifi-
cant effects on growth. Calcium requirements of Ezglena stellata (Dust,
1933b) are much greater than those of other Euglenidae investigated,
and manganese (Hall, 1937c) has been found to accelerate growth of
E. anabaena. In addition, a few similar observations on heteroautotrophic
flagellates have been reported. For instance, A. Lwoff (1930) has re-
ported that Fe is essential to growth of Polytoma uvella. Similarly, Mast

480 FOOD REQUIREMENTS

and Pace (1935) found that Chilomonas paramecium survived for only
a few transfers in media without S. For example, one S-free line died on
the seventh day and others on the third, while several lines in media
containing S were maintained for from twenty to twenty-four days. An-
other example is that of Hyalogonium klebsu, which requires relatively
large amounts of calcium (Pringsheim, 1937b).

It is possible, of course, that the action of certain elements may not
be specific; in other words, comparable effects on metabolism may be
exerted by several different elements, one of which may be substituted
for another. This possibility should be considered in investigations on
food requirements of photoautotrophs and heteroautotrophs.

PHOTOMESOTROPHIC NUTRITION

Euglena deses (Dusi, 1933b) may be considered an obligate photo-
mesotroph, a flagellate which has lost the primitive photoautotrophic
ability characteristic of various other green flagellates. In addition to
this species, several facultative photoautotrophs among the Euglenidae
are known to carry on photomesotrophic nutrition: E. anabaena (Dust,
1933b; Hall, 1938b), E. gracilis (Dusi, 1933a), E. klebsii, and E.
stellata (Dusi, 1933b). An interesting feature of these Euglenidae 1s
that a particular amino acid may support growth of one species but not
another (Dusi, 1931). For example, phenylalanine was satisfactory for
E. anabaena, E. gracilis, and E. stellata, but not for E. deses and E.
klebsii, while serine was adequate for growth of all except E. anabaena.
Comparable differences were noted for several other amino acids.

Among the Phytomonadia, photomesotrophic nutrition has been dem-
onstrated in Chlamydomonas agloéformis and Haematococcus plu-
vialis (A. Lwoff, 1932), and also in Lobomonas piriformis (Osterud,
1939). In addition, Loefer (1935b) observed, in Chlorogonium elonga-
tum and E. euchlorum, acceleration of growth by glycocoll and several
other amino acids, added separately and in mixtures, to an inorganic
medium and to a salt solution containing sodium acetate.

The growth of photomesotrophic species may be accelerated by the
addition of various carbon sources (e.g., sodium acetate) to an amino-
acid medium. Concerning mineral requirements in photomestrophic
nutrition, nothing is known beyond the fact that amino acids have often

FOOD REQUIREMENTS 481

been added to salt solutions comparable, except for the omission of
inorganic nitrogen, to the media used for photoautotrophic nutrition.

PHOTOMETATROPHIC NUTRITION

Photometatrophic nutrition can be carried on by all of the chlorophyll-
bearing flagellates which have been established in pure culture. Certain
species, such as E. pisciformis (Dusi, 1933b), may prove to be obligate
photometatrophs, although recent observations (Dusi, 1939) indicate
that E. pisciformis should not be so classified. Peptones of one type or
another have usually furnished the food supply, and in at least a few
cases a solution of peptone in distilled water has supported growth. In
addition to peptones, gelatin (Hall, 1938b) may also support growth,
and certain species are known to produce proteolytic enzymes (Mainx,
1928; Jahn, 1931; Hall, 1937b). Many of the flagellates grow well on
agar slants, provided the agar is enriched with a suitable peptone and
sometimes with an additional source of carbon; such cultures are con-
venient for the maintenance of laboratory stocks.

Although various peptone media are satisfactory for all the species
which have been studied, growth may be accelerated by the addition of
salts of certain fatty acids, various carbohydrates, and several alcohols.
Acceleration of growth by carbohydrates has been noted in E. gracilis
(Jahn, 1935b) and in two species of Chlorogonium (Loefer, 1935a).
Fermentation of dextrose by E. proxima was reported by Glaser and
Coria (1930, 1935a), but other workers have failed to note such changes
in cultures of Euglenidae. Furthermore, Loefer (1938b), using Bene-
dict’s colorimetric method, failed to detect utilization of dextrose by
C. elongatum and C. euchlorum, in spite of the accelerating effect on
growth. Acceleration of growth by ethyl alcohol has been reported by
Loefer and Hall (1936) in E. deses and E. gracilis, and similar effects
of several alcohols on the latter species have been described by Provasoli
(1938c). Acceleration of growth by fatty acids, in cultures exposed to
light, has been reported for E. gracilis (Jahn, 1935d) and E. stellata
(Hall, 1937d). Furthermore, macroscopic observations on cultures in
various stock-culture media have indicated such effects in approximately
thirty species maintained in our laboratory. However, most of the quanti-
tative studies on carbon sources have been based upon cultures main-

482 FOOD REQUIREMENTS

tained in darkness (heteromesotrophic and heterometatrophic nutrition) ,
as described below.

Hutner (1936) failed to note acceleration of growth by fatty acids
or carbohydrates in E. anabaena or by carbohydrates in E. gracilis. Since
Hutner’s conclusions apparently were based upon the macroscopic ap-
pearance of his cultures, he may have overlooked effects comparable to
those reported by other workers.

HETEROAUTOTROPHIC NUTRITION

The utilization of inorganic nitrogen compounds in the presence of
acetate or another organic carbon source, has been attributed to several
colorless Phytomastigophora: Chilomonas paramecium (Mast and Pace,
1933), Polytoma uvella (Pringsheim, 1921; Lwoff and Dusi, 1938a),
P. obtusum (Lwoff, 1929b, 1932), and Astasza sp. (Schoenborn, 1938,
1940). The results of Mast and Pace have not been duplicated by Loefer
(1934) nor by Hall and Loefer (1936). Pringsheim (1935a) reported
growth of C. paramecium in an ammonium-salt and acetate medium, but
Lwoff and Lederer (1935) and Pringsheim (1935b) have pointed out
that Pringsheim’s medium contained “‘extract of soil,” without which
the flagellates failed to grow. Hence, Pringsheim did not confirm the
observations of Mast and Pace. More recently, Lwoff and Dusi (1937a,
1938a, 1939b) have grown a strain of this species in an ammonium
acetate medium, but only in the presence of either thiamine or thiazole
and pyrimidine. Since Lwoff and Dusi added organic nitrogen com-
pounds to their medium, application of the term heteroautotro phic to C.
paramecium may be inappropriate. The contradictory results obtained
by various workers with this species have not yet been explained. It is
possible that different strains may vary in their nutritional requirements.
Or it is conceivable that the strain of Mast and Pace was established
through a selective process, similar to that reported in several species
of Euglena (Hall and Schoenborn, 1938b).

The first known instance of heteroautotrophic nutrition in Euglenida
is that described by Schoenborn (1938, 1940) in Astasia sp. This strain
has now passed the nineteenth transfer, so that the peptone carried over
from the original stock culture has been reduced, through serial dilu-
tion alone, to a calculated concentration of less than 1.8 & 10? gm.
perce

According to Pringsheim (1937b), Chlorogonium euchlorum may be

FOOD REQUIREMENTS 483

grown in darkness as a facultative heteroautotroph, provided glucose
caramel is added to the medium. Osterud (1939) has reported growth
of Lobomonas piriformis in an ammonium-nitrate and acetate medium
for three transfers (twelve weeks) in darkness. Likewise, in a medium
similar to that of Osterud, growth of Ezglena gracilis has been observed
(Schoenborn, 1939) through four successive transfers, covering a period
of eighteen weeks. These suggestive observations indicate that certain
chlorophyll-bearing flagellates may retain the ability to grow in inorganic
nitrogen media, even after suppression of photosynthesis.

HETEROMESOTROPHIC NUTRITION

Heteromesotrophic nutrition has been demonstrated in several color-
less Phytomastigophora. The Cryptomonadida are represented by CAzlo-
monas paramecium, which has been grown in an amino-acid and acetate
medium by Mast and Pace (1933) and by Hall and Loefer (1936).
This type of nutrition has not yet been demonstrated in colorless Eugle-
nida, and E. gracilis has been grown in darkness for only a few transfers
in a medium containing asparagin and acetate (A. Lwoff and Dusi, 1929,
1931). Such results are modified by the addition of thiamine, as de-
sctibed below. Several heteromesotrophs have been identified among
colorless Phytomonadida. Pringsheim (1921) found glycocoll an ade-
quate nitrogen source for Polytoma uvella, and comparable results were
later obtained for P. obtusum (A. Lwoft, 1929b, 1932; A. Lwoff and
Dusi, 1934). In addition, P. caudatum var. astigmata (A. Lwoff and
Provasoli, 1935), Polytomella agilis (A. Lwoff, 1935b), and P. caeca
(Pringsheim, 1935, 1937c; A. Lwoff and Dusi, 1937a) seem to be
capable of heteromesotrophic nutrition. On the other hand, the related
species, Hyadlogonium klebsii (Pringsheim, 1937a), appears to be an
obligate heterometatroph. The chlorophyll-bearing phytomonad, Chloro-
gonium euchlorum, has been grown in darkness in an asparagin medium
(A. Lwoff and Dusi, 1935b); likewise, Lobomonas piriformis is capable
of growth under similar conditions in a glycocoll and acetate medium
(Osterud, 1939).

HETEROMETATROPHIC NUTRITION

All of the colorless Phytomastigophora which have been investigated
appear to thrive in simple peptone media, although growth is always
accelerated by the addition of a suitable organic carbon source. In addi-

484 FOOD REQUIREMENTS

tion, a few of the chlorophyll-bearing species have been maintained in
darkness in such media (Jahn, 1935c, 1935d; A. Lwoff and Dusi, 1929,
1931, 1935a, 1935b; M. Lwoff and A. Lwoff, 1929; Loefer, 1934;
Provasoli, 1938b), especially with added acetate. Certain Trypanosomi-
dae, such as Strigomonas oncopelti, have been grown in peptone media
(M. Lwoff, 1930, 1933a, 1936), but growth of other Trypanosomidae
(M. Lwoff, 1929a, 1929b, 1929c, 1929d, 1933a, 1933b) and of Polymas-
tigida (Glaser and Coria, 1935b; Cailleau, 1935, 1936a, 1936b, 1937a,
1937b, 1938a, 1938b, 1939) seems to be supported only by more complex
organic media containing blood, serum, tissue extracts, or particular
growth factors. Several of the ciliates—Colpidium campylum and C.
striatum (Elliott, 1933, 1935b), Glaucoma ficaria (D. F. Johnson,
1935a), G. piriformis (Lwoff, 1924, 1925, 1929a), Paramecium bursaria
(Loefer, 1934b, 1936b, 1936c), and certain Sarcodina—Acanthamoeba
castellanii (Cailleau, 1933, 1934), Mayorella palestinensis (Reich, 1935,
1936), have been grown in peptone media comparable to those which
support growth of the heterometatrophic Phytomastigophora. On the
other hand, Glaser and Coria (1930, 1933, 1935a) have used somewhat
more complex media for several free-living ciliates.

At present little is known of the nitrogen requirements in heterometa-
trophic nutrition, and definite conclusions regarding the saprophilic or
saprogenic nature of particular species are not always possible. Enzymes
which hydrolyze gelatin and casein are produced by C. stratum (Elliott,
1933), by G. piriformis (A. Lwoff and Roukhelman, 1929; Lawrie,
1937), and by Saprophilus oviformis, Trichoda pura, and Chilodon
cucullus (Glaser and Coria, 1935a); hence these ciliates cannot be con-
sidered saprophilic organisms. A. Lwoff (1924, 1925) found that com-
plete peptones were satisfactory for growth of G. piriformis, whereas silk
peptone, gelatin, and fibrin, each supposedly lacking certain amino acids,
were inadequate. Recently, however, several strains of C. campylum have
been grown in the writer's laboratory for twenty-four transfers in silk
peptone media and for eighteen transfers in gelatin media. In a compari-
son of various peptones, Elliott (1935b) noted that C. striatum and C.
campylum grew most rapidly in the peptones containing high percentages
of free amino N. and Van Slyke amino N. Growth of the same ciliates
(Hall and Elliott, 1935) was also accelerated by certain amino acids,
added singly to a medium which supports slow multiplication. As in

FOOD REQUIREMENTS 485

Col pidium, Loefer (1936c) found for P. bwrsaria that the least satisfac-
tory of several peptones were those containing the smallest amounts of
amino N. Hence preliminary partial hydrolysis of peptones appears to
be advantageous, especially in the early growth of ciliate populations.

Nitrogen metabolism of G. piriformis has been investigated by
A. Lwoff and Roukhelman (1929), who have traced the quantitative
changes in total N, peptone N, amino N, ammonia N, and amide N. In
Witte peptone medium, peptone N decreased steadily. Amino N in-
creased for the first two weeks, and then gradually decreased for two or
three weeks; later, a secondary increase sometimes followed the death of
many ciliates. Somewhat comparable results have been reported for
Acanthamoeba castellanii (Cailleau, 1934), although hydrolysis was
always less extensive than in cultures of G. piriformis and much less
ammonia N was produced.

Fermentation of carbohydrates and the acceleration of growth by carbo-
hydrates and other carbon sources have been reported in many species.
Colas-Belcour and A. Lwoff (1925) observed fermentation of dextrose
and levulose by Leptomonas ctenocephali, Leishmania tro pica, and L.
donovani (var. infantum). More recently, M. Lwoff (1936) has reported
fermentation of fourteen carbohydrates by Strigomonas muscidarum and
of a smaller number by S. media and S. parva. These three flagellates
showed specific differences in their fermentation reactions. Likewise,
Cailleau (1937b) has described fermentation of several monosaccharides
and disaccharides by Eutrichomastix colubrorum, and the fermentation of
dextrin, starch, and inulin, as well as some of the simpler carbohydrates,
by Trichomonas foetus and T. columbae. Utilization of dextrose by try-
panosomes (for review, see von Brand, 1938) has been known for some
years. Recently, utilization of dextrose by T. foetws — the strain of Glaser
and Coria (1935b)—has been measured by Andrews and von Brand
(1938), who found that rate of utilization was correlated with growth
rate. Although growth of C. paramecium is accelerated by dextrose (Loe-
fer, 1935a), utilization of the sugar could not be detected (Loefer,
1938b) by means of Benedict’s colorimetric method. Acceleration of
growth by starch has been reported for Polytoma caudatum (A. Lwoft
and Provasoli, 1935), P. obtusum (A. Lwoff and Provasoli, 1937), and
Polytomella agilis (A. Lwoff, 1935b).

Few observations have been reported for the Sarcodina. Fermentation

‘

486 FOOD REQUIREMENTS

of carbohydrates by Acanthamoeba castellanii was not observed by Cail-
leau (1933), who also obtained no evidence that sugars are actually con-
sumed by this species. On the other hand, dextrose accelerates growth
of Mayorella palestinensis (Reich, 1936), maximal effects being pro-
duced by concentrations of 0.5 to 1.0 percent.

In Colpidium campylum and Glaucoma piriformis, Loefer (1938b)
has measured dextrose consumption over a short pH range. In general,
the rate of utilization followed the growth-pH relationship. Fermenta-
tion of carbohydrates has been demonstrated previously in several species.
G. piriformis produces acid from dextrose, levulose, galactose, and mal-
tose (Colas-Belcour and A. Lwoff, 1925), and also from dextrin and sol-
uble starch (D. F. Johnson, 1935b); G. ficaria (D. F. Johnson, 1935b)
ferments the same carbohydrates, with the apparent exception of levulose.
The reactions of C. campylum and C. striatum (Elliott, 1935a) are sim-
ilar to those of G. pirtformis, except for fermentation of mannose and
failure to ferment galactose. Growth of C. campylum is accelerated by
several carbohydrates, in addition to those which are fermented. Ac-
cording to Glaser and Coria (1935a), dextrose and maltose are fer-
mented, and starch and cellulose are hydrolyzed, by Saprophilus oviformts
and Trichoda pura; starch and cellulose are attacked also by Chilodon
cucullus, Paramecium caudatum, and P. multimicronucleatum. Growth of
P. bursaria (Loefer, 1936c) is increased by dextrose, mannose, maltose,
dextrin, and melizitose, while little or no effect is produced by other
carbohydrates. No marked change in pH occurred in any case.

Other carbon compounds known to accelerate growth of heterometa-
trophs include various alcohols and salts of certain organic acids. The
effects of several fatty acids on the growth of C. paramecium have been
compared quantitatively by Loefer (1935a): the greatest acceleration
was produced by acetate, butyrate, and valerate. Recently, Provasoli
(1937a, 1937b, 1938a, 1938b, 1938c) has completed more extensive
investigations on nine colorless flagellates (C. paramecium, Hyalogo-
nium klebsii, Polytoma obtusum, P. caudaltum, P. uvella, P. ocellatum,
Polytomella caeca, Astasia quartana, A. chattoni). Acetate and butyrate
accelerated growth of all, while the effects of other fatty acids varied with
the species. Provasoli has pointed out that the negative results previously
obtained with certain salts probably resulted from their use in toxic
concentrations. The effects of sodium acetate on the growth of several
colorless species had been described previously by A. Lwoff (1929b,

FOOD REQUIREMENTS 487

1931, 1932, 1935a, 1935b, 1938a), who proposed a class of “‘Oxy-
trophes”’ to include organisms showing marked acceleration with acetate.
Acceleration of growth by several alcohols has been reported by
Provasoli (1938c) in Astasia chattont, A. quartana, Polytomella caeca,
Polytoma ocellatum, and Chilomonas paramecium.

For chlorophyll-bearing flagellates maintained in darkness, the effects
of various carbon sources on growth seem to be much the same as for
the colorless species. The growth of several species of Evglena (A. Lwoff
and Dusi, 1929, 1931; Dusi, 1933a; 1933b; Jahn, 1935c, 1935d; Hall,
1937d) is accelerated by salts of certain fatty acids, particularly acetic,
just as in the case of Astasia (A. Lwoff and Dusi, 1936). Jahn (1935d),
in comparing the effects of certain salts on growth of E. gracilis in dark-
ness and in light, found that butyrate and acetate were most effective in
either case, while lactate produced a much greater acceleration in dark-
ness than in light. Succinate was toxic in light, but produced a slight ac-
celeration in darkness. Comparable effects of fatty acids have been re-
ported in several Phytomonadida—Chlamydomonas agloéformis and
Haematococcus pluviatis (M. Lwoff and A. Lwoff, 1929), and Chloro-
gonium elongatum and C. euchlorum (Loefer, 1935a). Accelerating
effects of carbohydrates on the growth of Evglena anabaena in darkness
have also been noted (Hall, 1934).

Elliott (1935b) has described acceleration of growth by acetate and
butyrate in the ciliates Colpidium campylum and C. striatum, the effects
being limited to the pH range 6.5 to 7.5, approximately. The increases
ranged from about 15 percent to 300 percent at different pH values.
Accelerating effects of pimelic acid on C. campylum also have been
reported (Hall, 1939c), but the substance was used in low concentra-
tions and may have been important as a catalyst, rather than as a carbon
source.

TROPHIC SPECIALIZATION

In this brief survey of the food requirements of Protozoa, it has been
pointed out that different methods of nutrition may be exhibited by
different members of the same family or even of the same genus. Such
varying degrees of specialization are particularly interesting, in that they
afford a basis for speculation concerning the evolution of the more
animal-like Protozoa from the plant-like flagellates.

Theoretically, the evolution of animal-like flagellates from chlorophyll-

488 FOOD REQUIREMENTS

bearing facultative photoautotrophs may have proceeded as follows:
(1) Certain flagellates lost the ability to use inorganic compounds as
the sole source of nitrogen, except in the presence of a suitable organic
carbon compound. This type of specialization may or may not have in-
volved the loss of chlorophyll in the beginning. (2) The ability to grow
in inorganic-nitrogen media was lost completely, so that a single amino
acid represented the simplest adequate nitrogen source. (3) The ability
to grow in an amino-acid medium was lost, as the degree of specialization
approached that of the animal-like flagellates.

On the other hand, the existence of heteroautotrophic flagellates,
which can utilize inorganic nitrogen sources without carrying on photo-
synthesis, suggests the possibility that primitive colorless flagellates may
have appeared before the origin of chlorophyll. Chlorophyll, with the
attendant power of photosynthesis, would thus have been acquired dur-
ing the evolution of plant-like flagellates. This hypothesis would gain
additional support from the demonstration of chemoautotrophic nutri-
tion in flagellates, and the suggestive report of such a phenomenon in
Chilomonas paramecium (Mast and Pace, 1933) is particularly inter-
esting. Further evidence may eventually necessitate revision of the cur-
rent view that the chlorophyll-bearing flagellates are the most primitive
of all the Protozoa.

Even in adhering to the concept of a primitive chlorophyll-bearing
stock, it must be admitted that in the known cases of heteroautotrophic
nutrition, the presumed “‘loss’’ of chlorophyll has introduced only one
new food requirement, a simple organic carbon source (e.g., acetate).
A second stage of specialization is represented by such types as the
chlorophyll-bearing Exglena deses and the colorless Polytomella caeca,
each of which requires a simple organic nitrogen source. The third step
in specialization also appears in the Phytomastigophora, and is illus-
trated by the chlorophyll-bearing Exglena pisciformis, described by Dust
(1933a) as an obligate photometatroph, and the colorless Hyalogonium
klebsii, reported to be an obligate heterometatroph (Pringsheim, 1937a).
If the types of nutrition described for these various species are taken
for granted, it must be admitted that the presence of chlorophyll is no
handicap to progressive physiological specialization. Furthermore, the
mere absence of chlorophyll has not necessitated specialization beyond
the first degree, although it may be accompanied by the assumption

FOOD REQUIREMENTS 489

of heteromesotrophic or heterometatrophic nutrition. Hence the case
for primitive chlorophyll-bearing forms as the ancestors of all the
Protozoa may not be so strong as is generally assumed.

The plant-like flagellates as a group, however, furnish a logical start-
ing point for the evolution of other groups of Protozoa. Primitive meth-
ods of nutrition are not the only methods to be observed in Phytomastt-
gophora, and it is obvious that some of these flagellates approach in their
growth requirements the Zodmastigophora, Sarcodina, and Ciliata, repre-
sentatives of which have been grown in peptone media comparable to
those required by E. pisczformis and H. klebsiz. Accordingly, it seems
that, so far as physiological modifications are concerned, the evolution of
animal-like flagellates and other groups of Protozoa from an ancestral
stock of plant-like flagellates could have presented few problems.

SPECIFIC GROWTH FACTORS, OR VITAMINS

A concise definition of the term, growth factor, is not yet available.
The term is now usually restricted to an essential substance which the
organism in question cannot synthesize, or perhaps cannot synthesize
rapidly enough to meet the normal requirements for growth. Such a
growth factor may exert its characteristic effects, even when present in
low concentration. By general agreement, the concept excludes the es-
sential food substances and elements necessary for synthesis of proto-
plasm. While a growth factor may in itself accelerate growth, it is to
be distinguished from nonessential growth stimulants, which also pro-
duce noticeable effects when present in low concentrations.

A survey of the rapidly growing literature reveals that growth-factor
requirements may differ among the species of a single protozoan genus,
and that some species can be grown in media apparently containing no
growth factors, while related types are much more exacting. At present,
there is no sound basis for generalization. Lack of information concern-
ing food requirements makes it impossible in some cases to decide
whether or not a specific growth factor is necessary, and occasionally a
postulated need for growth factors has disappeared after further in-
vestigation. Thus Dusi (1936) suggested that growth of E. viridis in
inorganic media might be impossible without a growth factor, but the
species has since been grown as a photoautotroph (Hall, 1939a). Simi-
larly, Hutner (1936) concluded that a vitamin-like substance is a neces-

490 FOOD REQUIREMENTS

sary constituent of media for the growth of E. anabaena and E. gracilis
in light. The former has been grown in inorganic media by Dusi and by
Hall; the latter, by Dusi and by Hall and Schoenborn. It is obvious,
therefore, that the inorganic food requirements of a given species should
be satisfied, as a prerequisite to the evaluation of specific growth factors.

Aneurin, or thiamine (vitamin B,).—It must be admitted that the
facultative photoautotrophs and heteroautotrophs are capable of synthe-
sizing aneurin, if this substance is actually essential to the growth
of such organisms. Various other Protozoa, however, apparently show a
definite need for aneurin, or for one or both of its constituents.

Among the Cryptomonadida, the thiamine requirements of C. para-
mecium have been investigated by A. Lwoff and Dusi (1937b, 1938a).
In their first publication these workers stated that growth of the
flagellate in asparagin medium is supported by thiamine, or by thiazole
alone. In their later article, they have concluded that for growth in an
ammonium acetate medium, thiamine can be replaced by thiazole and
pyrimidine, but not by either one separately. Without the growth factor,
growth in the control medium was always negative. Recently, A. Lwoff
and Dusi (1938b, 1938c) have shown that these substances are not
specific; so far as C. paramecium is concerned, several thiazoles and
pyrimidines are satisfactory for growth.

In the Euglenida, the existence of photoautotrophic species (E. gracilis,
and others) and the occurrence of heteroautotrophic nutrition (Astasza
sp., Schoenborn, 1938, 1940) seem to belie a need for thiamine in cer-
tain species. Furthermore, Elliott (1937a) observed no accelerating ef-
fect of this substance on the growth of E. gracilis in light. However, it
has been assumed that such flagellates are capable of synthesizing thia-
mine in light, and this hypothesis receives indirect support from re-
ports that the growth of E. gracilis in darkness is possible in an asparagin
and acetate medium only when thiamine (Lwoff and Dusi, 1937c) or
pyrimidine (Lwoff and Dusi, 1938a) is present. On the other hand,
Dusi (1939) has concluded that E. pisciformis requires such a growth
factor even in light, since an asparagin medium containing thiamine (or
both thiazole and pyrimidine) supported growth, while the same medium
without a growth factor was unsatisfactory.

In this connection, it has been noted (Hall, 1938b) that growth
of E. anabaena in light is little, if any, better in an asparagin medium

FOOD REQUIREMENTS 491

than in an ammonium-nitrate medium, the two media differing only
with respect to the nitrogen source. Hence asparagin may actually be a
poor nitrogen source for Euglenidae, even in light, and any growth
stimulant might produce an effect comparable to that noted by Dusi in
E. pisciformis. It should be noted, also, that the serial-transfer technique,
in which Dusi apparently used two-drop inocula, might require an
increase of as much as a hundred times in each transfer, if the original
density of population is to be maintained. In the writer's experience
with E. anabaena, the increase in asparagin medium was never greater
than twenty-five times in any tranfer, and was often less. Hence the
possibility exists that Dusi’s rate of dilution in serial transfers was much
more rapid than the growth of his flagellates in media without growth
factors, and that the use of larger inocula might reveal E. pzsczformzs to
be capable of slow growth in asparagin media.

If growth factors are actually essential, all the chlorophyll-bearing
Phytomonadida which have been investigated appear to synthesize such
substances from the constituents of suitable inorganic media. The color-
less species, P. uvella and P. obtusum (Lwofft and Dusi, 1938a), show
the same synthetic ability in salt solutions to which acetate has been
added. Polytoma ocellatum and P. caudatum (Lwoff and Dusi, 1937b,
1937c) apparently require thiazole for growth in such media, while
Polytomella caeca (A. Lwoff and Dusi, 1937a, 1938a, 1938b, 1938c) re-
quires both thiazole and pyrimidine. A. Lwoff and Dusi (1937a) have
shown that P. caeca grows fairly well in an asparagin medium, and much
more rapidly after the addition of thiamine or of thiazole and pyrimidine.
They have assumed, accordingly, that the growth in asparagin alone was
dependent upon a trace of thiamine in the asparagin itself. The same
interpretation is also applied to several other flagellates, on the basis
of similar evidence. Just as in the case of C. paramecium, several dif-
ferent pyrimidines and thiazoles accelerate the growth of P. caeca (Lwoft
and Dusi, 1938b, 1938c), and several thiazoles are also effective with
P. ocellatum.

Among the Zo6dmastigophora, Strigomonas oncopelti (M. Lwoff,
1937), S. culicidarum, and S. fasciculata (M. Lwoff, 1938b) appear to
require thiamine, which cannot be replaced by thiazole and pyrimidine.
The last two species require hematin in addition to thiamine.

Of the Sarcodina, Acanthamoeba castellanii (A. Lwoff, 1938b) ap-

492 FOOD REQUIREMENTS

parently requires thiamine, or both pyrimidine and thiazole. Accord-
ingly, Lwoff has concluded that this species is capable of synthesizing
this growth factor from the two components, although such a synthesis
has not been demonstrated. With respect to growth-factor requirements,
A. castellanii thus seems to resemble Polytomella caeca and Chilomonas
paramecium.

So far, only a few investigations have been completed on the ciliates.
Hall and Elliott (1935) noted that the addition of yeast extract in low
concentration to a gelatin medium would support growth of Colpidium
campylum and C. striatum, whereas gelatin medium alone was unsatis-
factory. A. Lwoff and M. Lwoff (1937) have since found that Glau-
coma piriformis will grow in a silk-peptone-dextrose medium containing
thiamine, while the control cultures failed in the second or third transfer.
Likewise, Elliott (1937a, 1939) noted a marked acceleration of growth
in C. striatum when thiamine was added to a standard peptone solution
and to a peptone medium autoclaved at pH 9.6. In the latter case, the
controls showed very little growth in the first transfer. Observations of
the Lwoffs (1937, 1938) indicate that G. pzr7formis requires the entire
thiamine molecule, and is presumably unable to synthesize the substance
from the thiazole and pyrimidine constituents. Various other related
compounds cannot be substituted for thiamine.

Other Growth Factors —Vitamin B, (riboflavin) apparently will not
replace thiamine in meeting the growth requirements of Colpidium
striatum, although a moderate acceleration of growth by this factor has
been noted (Elliott, 1939). A vitamin B, concentrate has produced even
less noticeable effects on the growth of the same species (Elliott, 1939).

Vitamin C (ascorbic acid) requirements have been investigated in
several species. M. Lwoff (1938a, 1939) has reported that ascorbic
acid is one of the factors essential to growth of Trypanosoma cruzt,
Leishmania tropica, and L. donovani in cultures; and Cailleau (1938a,
1938b, 1939) has reached the same conclusion for Trichomonas foetus,
Eutrichomastix colubrorum, and T. columbae.

Nicotinic acid and nicotinamide both seem to serve as growth factors
for certain bacteria (for review, see Koser and Saunders, 1938). None
of the investigations on Protozoa has yet been completed.

Hematin has been found essential for the growth of certain Tryp-
anosomidae—for example, Trypanosoma cruzi, Leishmania donovani,

FOOD REQUIREMENTS 493

L. tropica (M. Lwoff, 1938a, 1939), Leptomonas ctenocephali, and
Strigomonas fasciculata (M. Lwoff, 1933a). Protohemin and protopor-
phyrin have been substituted for hematin in the case of T. cruzi. The
significance of such growth factors has been discussed by A. Lwoff
(1934, 1936), who suggested that these substances may enter into the
composition of respiratory catalysts (cytochrome).

Cholesterol_—The investigations of Cailleau (1936a, 1936b, 1937a,
1937b, 1938a, 1938b) indicate that cholesterol and certain other sterols
serve as grown factors for the parasitic Polymastigida, Trichomonas
columbae, T. foetus, and Eutrichomastix colubrorum. The physiological
significance of these substances has not yet been determined for Protozoa.

Extract of soil—aAn aqueous extract of soil has been used extensively
by Pringsheim, who found it to accelerate the growth of a number of the
plant-like flagellates and also to facilitate the growth of certain species
in simple media. Accordingly, Pringsheim has considered this extract
a source of unknown growth factors. The accelerative action has been
verified by A. Lwoff and Lederer (1935), whose results suggest that soil
extracts contain organic nitrogen in concentrations sufficient for growth
of Polytomella agilis. Hence the status of soil extracts as a source of
growth factors is yet to be evaluated.

GROWTH STIMULANTS

Growth stimulants differ from growth factors in that they are not
essential to life. Like growth factors, however, they may be effective
in low concentrations. So far as their relation to Protozoa is concerned,
pantothenic acid and the plant “hormones” (auxins) may be placed in
this category. Elliott (1935c) has shown that pantothenic acid accelerates
the growth of Colpidium campylum, the maximal effect being noted
at pH 6.0. Above 7.0 there was either no acceleration, or else a slight
decrease in growth rate. Similar experiments with Haematococcus pluvi-
alis, within the pH range 4.5 to 8.5, showed no acceleration. Addition of
pantothenic acid to gelatin, gliadin, and zein media, which in themselves
did not support the growth of Colpidiumm (Hall and Elliott, 1935), was
without effect. These results indicate that pantothenic acid is not a
substitute for thiamine.

The effects of several plant hormones, or auxins, on the growth of
Euglena gracilis, Khawkinea halli, and C. striatum have also been in-

494 FOOD REQUIREMENTS

vestigated by Elliott (1938). The growth of E. gracilis was markedly
accelerated at pH 5.6, while effects at lower and higher pH values were
much less significant. In the colorless euglenoid, K. alli, and in C.
striatum no acceleration of growth was observed at any pH. Thus the
effects of the auxins may be correlated with the presence of chlorophyll,
as well as the pH of the medium. Elliott (1937b) has shown further

log of numbers numbers

TIME TIME

Figure 128. Growth phases in a hypothetical population. In the curve at the left,
logarithms of numbers are plotted against time; on the right, numbers are plotted against
time for a comparable population. Successive growth phases are numbered from 1 to 7.

that such acceleration may also be dependent upon light, since there was
no effect on growth of E. gracilzs in darkness.

An accelerating effect of pimelic acid upon the growth of Colpidinm
cam pylum has been noted by Hall (1939c); concentrations ranging from
10° to 10+ gm. per cc. were effective in gelatin and in peptone media.
These results are comparable to the findings of Mueller (see Koser and
Saunders, 1938) with the diphtheria bacillus. Certain preliminary ob-
servations (Hall, 1938a) may indicate a possible growth-factor status
for pimelic acid, but a definite conclusion is not yet warranted and pimelic
acid may be considered, at least for the present, a growth stimulant for
C. campylum.

Glucose caramel, as used by Pringsheim (1937b, 1937c), may also
be classified as a growth stimulant. Pringsheim insists that this substance

FOOD REQUIREMENTS 495

does not serve as a carbon source in his cultures of plant-like flagellates,
and that it should be considered a “growth factor.’ On the other hand,
it has not yet been demonstrated that glucose-caramel is essential to the
life of Protozoa, and until such evidence is available the substance should
not be classified as a growth factor.

GROWTH IN CULTURES AS A POPULATION PROBLEM

The growth of microdrganisms in cultures has been described by
Buchanan (Buchanan and Fulmer, 1928) in terms of seven phases:
(Fig. 128): (1) Initial stationary phase, during which there is no in-
crease in population; (2) lag phase (phase of positive growth accelera-
tion), in which the division rate increases to a maximum; (3) logarithmic
growth phase, during which the maximal rate is maintained; (4) phase
of negative growth acceleration, in which the division rate decreases
steadily; (5) maximum stationary phase, in which the population re-
mains practically constant; (6) phase of accelerated death, in which
the total population begins to decrease; and (7) a so-called logarithmic
death phase, during which the population decreases at a more or less
constant rate.

Little is known about the history of protozoan populations, and com-
plete growth curves seem to have been traced for only two species in
pure cultures—Paramecium bursaria (Loefer, 1936b) and Polytoma
(Provasoli, 1938c). Loefer’s growth curves, comparable to the numbers
curve in Figure 128, show in general the phases recognized by Buchanan.
Since counts were made at intervals of twenty-four hours or more, an
initial stationary phase was not detected in several of the cultures. Total
population histories covered from twenty to forty days in different media.
Provasoli’s curve for Polytoma also shows the general growth phases.
Phelps (1935, 1936) traced Glaucoma piriformis well into the maximal
stationary phase and observed in most cases the first five of the con-
ventional growth phases. On the basis of such evidence, it may be assumed
that the growth of Protozoa in pure cultures follows the general trends
observed in populations of bacteria and yeasts.

More information concerning growth of protozoan populations is
needed, since interpretation of experimental results may depend upon
such knowledge. For example, the addition of a given substance to a

496 FOOD REQUIREMENTS

logarithms of
numbers

TIME

Figure 129. Hypothetical modifications (curves B-G) of the normal growth of a
population (curve A), from the initial stationary to the maximal stationary phase.

certain medium might produce any one of several effects: (1) The
maximal population might be increased without any appreciable effect
on the growth rate; that is, the length of the logarithmic phase might
be increased without any change in the division rate (curve B, Fig. 129),
as compared with that in the control medium (curve A). (2) The
growth rate might be increased without a change in density in the
maximal stationary phase (curve C). (3) Both the growth rate and the
maximal density of population might be increased (curve D). (4) The

FOOD REQUIREMENTS 497

maximal density of population might be decreased, with no appreciable
effect on the early growth rate (curve E). (5) The growth rate might
be decreased, without any effect on the maximal density of population
(curve F). (6) Both the growth rate and the maximal density of popula-
tion might be decreased (curve G). These possibilities will serve as
illustrations. Curves A, B, and E would show no significant differences
in the early histories of the cultures; yet each reaches a different maximal
stationary phase, one higher and one lower than that of the control
(curve A). On the other hand, early examination of cultures A, C, and
F would show significant differences in population density and in growth
rate, although each culture eventually reaches the same maximal density.
Hence without detailed information concerning the behavior of popula-
tions, there is obvious need for caution in interpreting experimental
results.

In an analysis of the conditions which might influence the population-
growth curve, the concentration of available food, the general condition
and density of the initial population, the pH of the medium, the tempera-
ture of incubation, the accumulation of waste products, the oxygen ten-
sion, and the redox potential of the medium—to mention some of the
more apparent factors—all seem to be significant. Although the impor-
tance of such factors may seem obvious, their detailed relationships to
growth are mostly unknown.

THE INITIAL POPULATION

Both the physiological condition and the size of the initial population
may influence the rate of growth. Phelps (1935) has reported that the
length of the initial stationary phase and the lag period bears a rela-
tion to the age of the inoculum. With inocula taken from the logarithmic-
growth phase of a stock culture, the length of the combined lag and
initial stationary phases was usually reduced to zero. With inocula from
older cultures, these two phases were usually detectable and were often
quite pronounced. Obviously, therefore, age and other qualities of the
inoculum must be considered in comparative studies on population growth
and on the effects of environmental factors.

The relation between the initial density of population and the growth
rate in bacterized cultures has been disputed for many years. Robertson
and others have described an allelocatalytic effect, in which the rate of

498 FOOD REQUIREMENTS

population growth is correlated more or less directly with the size of the
initial population. A number of investigators, however, either have noted
no significant correlation, or else have found that the growth rate varies
inversely with the initial density of population (e.g., Woodruff, 1911).
W. H. Johnson (1936, 1937) has pointed out that these results must
be interpreted in relation to the concentration of bacteria in the cultures.
Jahn (1929), who partially eliminated bacteria by growing Ezglena sp.
in inorganic media and washing the flagellates before inoculation, found
that the growth rate varied inversely with the initial density of the popu-
lation. Most of the literature on bacterized cultures has been reviewed by
Jahn (1929), W.H. Johnson (1933, 1936, 1937), and Petersen (1929).

In contrast to the studies on bacterized cultures, very little work has
yet been done with bacteria-free strains. Evidence bearing on the growth
of such populations has been presented by Phelps (1935), Mast and
Pace (1938), Reich (1938), and Hall and Schoenborn (1939b).
Phelps concluded, for Glaucoma piriformis, that the density of popula-
tion at the end of the logarithmic phase is, within wide limits, inde-
pendent of the initial density of population. This may mean that the
logarithmic phase is prolonged in the cultures with small inocula; or
that the growth rate is higher in the cultures with low initial density;
or perhaps that both the growth rate and the length of the logarithmic
phase are increased. Phelps did not consider this question in detail, but
some of his data (ser. I], Figs. 4, 5) indicate, at the end of the loga-
rithmic phase, an average generation time of about four hours in the low-
initial-density cultures and approximately five hours in the cultures
started with larger inocula. Such data suggest an inverse relationship be-
tween the initial density of population and the rate of population growth.

In Chilomonas paramecium (Mast and Pace, 1938) the rate of repro-
duction varies directly with the initial density of population under some
conditions, but inversely under others. Some sort of optimal relationship
is indicated by the findings of Mast and Pace, since the growth rate in-
creased to a maximum with decreasing volumes of medium per flagellate
and then decreased to zero.

Reich (1938) observed that in Mayorella palestinensis the division
rate varies directly with the initial density of population in cultures
started with less than 3,000 amoebae per cc., although the population
“eventually attained is largely independent of the quantity of inoculum.”

FOOD REQUIREMENTS 499

These results are interpreted as supporting Robertson’s concept of allelo-
catalysis, although Reich does not subscribe to the theory of an auto-
catalyst of growth.

In observations on Ezg/ena, Hall and Schoenborn (1939b) have noted
that the population tends to reach a concentration which is more or less
independent of the initial density of population under the conditions
described, and thus that the increase in the population varies inversely
with the initial density of population. These conclusions were based
upon counts made after specified periods of incubation, and population
curves were not traced.

At present it is impossible to correlate the results which have been
obtained with bacteria-free cultures, although the cited observations all
indicate that the initial density of population influences the rate of
population growth. Since the experiments on various species have been
carried out under different conditions and in different media, it is pos-
sible that some of the puzzling contradictions may eventually be traced
to differences in technique, rather than differences in the nature of popu-
lation growth. In fact, different relationships between the density of
population and the rate of growth might reasonably be expected under
different experimental conditions.

GROWTH IN RELATION TO WASTE PRODUCTS

Investigations on so-called waste products of Protozoa have led to con-
flicting opinions. Woodruff (1911, 1913) concluded that waste prod-
ucts inhibit growth of the homologous species, although growth of a
different species may be relatively unaffected by the same substances.
The other extreme is represented by Dimitrowa’s (1932) observation
that the growth of Paramecium caudatum is accelerated by the addition
of small amounts of old medium to the experimental cultures. More
recently, W. H. Johnson and Hardin (1938) have observed no significant
effects of old culture medium on the growth of P. multimicronucleata.

Very little work on this problem has been carried out with pure
cultures. The observations of A. Lwoff and Roukhelman (1929), that
growth of G. périformis ceases long before the food supply is exhausted,
has lent some support to the view that growth may be inhibited by
accumulated waste products. The results of later investigations are not
so readily interpreted. Mast and Pace (1938) have noted that old culture

500 FOOD REQUIREMENTS

medium, in high concentrations, inhibits the growth of Chilomonas
paramecium, while small amounts accelerate growth. Reich (1938) found
that the addition of culture filtrate produced no effect on the growth
of M. palestinensis. However, Reich’s filtrate was obtained from young
cultures (twenty-four-hour cultures in one experiment, for example), and
his technique is not entirely comparable to that of Mast and Pace. Hall
and Loefer (1940), working with C. campylum in peptone medium,
found that the addition of old culture filtrates (one part in ten, to five
parts in ten) markedly increased the population yield, as compared
with that in control cultures. Furthermore, the growth rate, after the
first or second day of incubation, was noticeably higher in the cultures
containing old-culture filtrate. Acceleration of comparable magnitude
was also produced by the addition of aged sterile medium to fresh pep-
tone medium. In view of the latter observation, it now seems impos-
sible to attribute the effects of old-culture filtrates solely to a ‘‘biological
conditioning’ of the medium, or entirely to a product or products
elaborated by the organisms growing in the medium. A basic explana-
tion for these various phenomena is not yet available, and it is possible
that any single explanation may be inadequate. Thus the ‘‘factor” of
Mast and Pace is said to be heat-labile, whereas the effects noted by
Hall and Loefer were produced by culture filtrates and aged sterile
medium which had been sterilized in the autoclave. At any rate, these
results are not only interesting in themselves, but they may also furnish
important clues in untangling the conflicting opinions concerning alle-
locatalytic and autocatalytic phenomena. For instance, some preliminary
observations of the writer have already shown that the “accelerating
factor” in old cultures may have a definite bearing on the growth of
C. campylum, in relation to initial density of population.

GROWTH IN RELATION TO FOOD CONCENTRATION

It seems obvious that, within reasonable limits, the density of a
protozoan population should vary more or less directly with the con-
centration of available food until an optimal concentration is reached,
although the relationship might not be evident in the early history of
the culture. Such a generalization is supported by studies on pure cul-
tures.

Cailleau (1933) noted that peptone concentrations of 3.0 percent sup-
ported abundant growth of Acanthamoeba castellanii, while lower con-

FOOD REQUIREMENTS 501

centrations were much less favorable. In Colpidium campylum, Bond
(1933) observed comparable relationships between growth rate and
concentration of yeast autolysate. Optimal concentrations of peptone for
C. striatum (Elliott, 1935b) lie between 1.0 and 3.0 percent, while for
Glaucoma ficaria (D. F. Johnson, 1935a) the optimum ts about 1.5 per-
cent. In both species of Colpidium the effects were apparent after twenty-
four hours of incubation and became more marked in older cultures.
Loefer’s (1936b) observations on Paramecium bursaria indicate similar
relationships, although the limits are somewhat narrower than for
Col pidium and Glaucoma. For example, one of the peptones tested was
optimal in 0.5-percent solution, whereas no growth of P. bursaria oc-
curred in a concentration of 1.4 percent. Phelps (1936) observed that in
the logarithmic phase the growth rate of G. piriformis was, within wide
limits, practically independent of the food concentration. In later history
of the cultures, however, the relationships were comparable to those ob-
served by Elliott and D. F. Johnson. Rottier (1936b) described, in
Polytoma uvella, a direct relation between the growth rate and the con-
centration of peptone (0.2 to 1.0 percent), and of asparagin (0.2 to 2.0
percent), significant differences being noted after about five days of
incubation.

The effect of a substance added to an adequate medium also varies
with concentration, as would be expected. Johnson (1935a) reported
for Glaucoma ficaria in peptone medium, maximal acceleration by 0.5-
percent yeast extract and inhibitory effects of concentrations above 2.0
percent. In Paramecium bursaria (Loefer, 1936b), the optimal concen-
tration of the same yeast extract was 0.03 percent, and growth was defi-
nitely inhibited in a 0.5-percent solution. Dextrose was most effective for
P. bursaria in a concentration of 0.5 percent. Reich (1936) has obtained
similar results with Mayorella palestinensis. The effects of added sodium
acetate on P. wvella (Rottier, 1936b) vary in the same fashion, the maxi-
mal effect being produced by 0.8-percent acetate. The optimum is much
lower in Euglena stellata (Hall, 1937d), in which 0.05-percent sodium
acetate was most effective in both light and darkness.

GROWTH IN RELATION TO pH OF THE MEDIUM

It has been known for many years that the acidity or the alkalinity of
the medium bears some relation to the growth of Protozoa, and investi-
gations on bacterized cultures have determined the optimal pH and pH

502 FOOD REQUIREMENTS

range for a number of species (for reviews, see Loefer, 1935c; D. F.
Johnson, 1935a). More recently, similar investigations have been carried
out with bacteria-free material.

Dusi (1930) has shown that each of six species of Euglena has a
characteristic pH range in certain media, and that the optimum varies
somewhat for the different species. Jahn (1931), using quantitative
methods, has studied that pH relationships of E. graczlzs in detail, and
similar relationships have been determined for E. anabaena and E. deses
(Hall, 1933a) and for two species of Astasza (Schoenborn, 1936).
Among the Cryptomonadida and Phytomonadida, the pH-growth rela-
tionships of Chilomonas paramecium, Chlorogonium elongatum, and C.
euchlorum have been investigated by Loefer (1935c). Growth of the
two phytomonads, with an optimum slightly above pH 7.0, was more
or less comparable to that of several Euglenidae; C. paramecium, on the
other hand, showed a bimaximal pH-growth curve with peaks at pH
4.9 and 7.0 and an intermediate low point at pH 6.0.

Relationships between growth and pH have also been determined for
several ciliates. Elliott (1933, 1935b) has described the pH ranges and
optima for Colpidium campylum and C. striatum, and has pointed out
that the pH relationships vary with the type of medium. In one peptone
medium (Difco tryptone) a bimodal curve, with peaks at pH 5.5 and
7.5, was noted; in certain other peptone media, a unimaximal pH-growth
curve was observed. The addition of sodium acetate or a carbohydrate
(e.g., maltose) to tryptone medium changed the shape of the curve from
bimaximal to unimaximal. The extent of the pH range also varied with
the type of medium. D. F. Johnson (1935a), in similar fashion, has com-
pared the pH-growth curves of Glaucoma ficaria and G. piriformis in dif-
ferent types of media. Appreciable differences between the two species
were noted, and the pH range and general form of the growth curves
were found to vary with the type of medium, much as in Colpidium.
More recently, Loefer (1938a) has studied the growth rate and general
morphology of Paramecium bursaria in relation to the pH of the medium.
Conditions known to be optimal for the symbiotic Chorella (Loefer,
1936a) did not coincide with those most favorable to the growth of
P. bursaria containing the algae. The pH optimum for the ciliate was
approximately 6.8, and growth occurred within the range 4.9 to about
7.8. The size of the ciliates varied with the pH, but independently of
the growth rate.

FOOD REQUIREMENTS 503

In addition to the presumably direct influence upon growth rate, the
pH of the medium has been found to modify the effects of other fac-
tors. For example, Elliott (1935a) has shown that the maximal acceler-
ating effect of certain carbohydrates on the growth of C. striatum 1s
exerted below pH 7.0, with little or no acceleration above that point.
Some of Jahn’s (1935b) results with E. gracilis in inorganic medium
also seem to show a correlation between pH and the effect of several
carbohydrates. Elliott noted also that the effects of sodium acetate and
- butyrate varied with the pH. The former inhibited growth of C. striatum
more or less completely at pH 6.0 and lower, but produced moderate
acceleration near the neutral point. Butyrate was toxic at pH 6.5 or
below, but showed an accelerating effect at pH 7.0-7.5. Jahn (1934)
has suggested that such effects of acetate and butyrate may be explained
on the basis that only the undissociated organic-acid molecule is toxic.
A. Lwoff (1935a), in reviewing Elliott’s work, stated that acetate and
butyrate inhibit the growth of Colpidium; this is true for only a certain
pH range. Another indirect effect of the pH is the influence on tempera-
ture relationships, as indicated in Jahn’s (1933a) observation that the
susceptibility of E. gracilis to relatively high temperature is lowest at pH
5.0 and greatest above pH 7.0. The growth-accelerating effects of panto-
thenic acid on C. striatum (Elliott, 1935c) and of plant auxins on E.
gracilis (Elliott, 1938) are also dependent upon the pH of the medium.

The evidence already accumulated shows that the pH relationships of
Protozoa are exceedingly complex, and that they vary not only with the
individual species but also with the composition of the medium and with
other environmental conditions. Furthermore, such relationships may
vary with time, since Jahn (1931) has observed that the optimal pH
differs in young and in old cultures of E. gracilis. To some extent, the
pH-growth relationships may be correlated with the activities of enzymes,
which may show characteristic pH optima; for example, the protease of
G. piriformis (Lawrie, 1937) shows maximal activity at pH 6.0. But
this may represent only one of many ways in which growth is related
to the pH of the medium.

OXYGEN RELATIONSHIPS

That oxygen tension of the medium influences growth of Protozoa is
obvious, but relatively little detailed information has been accumulated

504 FOOD REQUIREMENTS

in experimental studies. Observations on the natural occurrence of
Protozoa indicate definite differences in oxygen requirements. Some
species appear to be strict aérobes, others are perhaps comparable to the
microaérophiles among the bacteria, and many intestinal parasites are
probably to be regarded as facultative anaérobes. Investigations on proto-
zoan respiration are discussed in another chapter of this volume (Chap-
ter Vib):

Growth in relation to oxygen requirements has been investigated for
only a few bacteria-free strains, and no attempt has been made to corre-
late definite oxygen tensions with growth rate. G. piriformis, according
to Lwoff (1932), is incapable of growth under anaérobic conditions.
Likewise, Hall (1933b) found that under reduced oxygen tension
(Buchner pyrogallol method), growth of C. campylum was approxt-
mately 50 percent less than in aérobic controls in peptone medium. With
added dextrose, however, growth was greater than in the aérobic con-
trols in peptone medium. The results suggest a certain degree of simi-
larity between C. campylum and the facultative anaérobes among the
bacteria. Phelps (1936) demonstrated that aération of flask cultures
in yeast autolysate produces a much heavier population of G. pirzformzs
than in unaérated flasks. These results are somewhat comparable to the
findings of Jahn (1936), who compared G. piriformis and Chilomonas
paramecium with respect to effects of aération. Growth of the ciliates
was most rapid at first in unaérated flasks, but after three days of in-
cubation the aérated flasks showed heavier populations. In C. para-
mecium, however, growth was consistently more rapid in the unaérated
flasks. Rottier (1936a) has reported that the growth of Polytoma uvella
in flasks is more rapid than in tubes, after approximately forty hours of
incubation. Likewise, aérated tube cultures showed heavier growth than
unaérated ones.

THE REDOX POTENTIAL

As applied to culture media, the redox potential may be considered
an indication of the oxidizing or reducing power of such an oxidation-
reduction system. In other words, the more positive the redox potential,
the more highly oxidized is the medium; the more negative the po-
tential, the more highly reduced will be the medium. In effect, the
potential is a measure of intensity rather than of oxidizing or reducing
capacity, and hence is somewhat analogous to the pH, which gives no

FOOD REQUIREMENTS 505

indication of the amount of acid or alkali necessary to change the re-
action by a given amount. In addition, the redox potential varies with
the pH of the medium. A number of investigators have correlated the
redox potential of culture media with the growth and metabolism of
bacteria, but very little work along this line has yet been done in proto-
zoology.

So far, the only detailed investigations are those of Jahn (1933b,
1935a), who has studied growth of Chilomonas paramecium in relation
to the redox potential of the medium. In his first publication, Jahn
found that growth is accelerated by NaSH, while the addition of H,O,
to a peptone and acetate medium inhibited growth. On the other hand,
relatively rapid growth occurred when both peroxide and a high con-
centration of -SH were added to the medium. These results were ex-
plained on the basis of the redox potential. In his second article, Jahn
traced the continuous changes in the pH and Eh in cultures of C. para-
mecium. The Eh of different media was found to drop as much as
300-460 my. during the first few days of incubation, and Jahn suggested
that such changes may involve not only a lowering of the oxygen ten-
sion but also the accumulation of reducing substances in the medium.
After three to five days, depending upon the type of medium, the Eh
began to rise; this change was attributed to a sharp decrease in the
growth rate of the flagellates, with a corresponding decrease in oxygen
consumption.

So far as the Protozoa are concerned, Jahn’s results indicate that
there is much to be learned concerning detailed relationships between
growth and the redox potential of culture media. The exact effects of
changes in the redox potential are still unknown, and the relative im-
portance of the redox potential and the oxygen tension in different cases
is yet to be determined. Possible relationships to growth have been dis-
cussed by Jahn (1934).

GROWTH IN RELATION TO TEMPERATURE

The importance of temperature relationships is obvious, and rigid
control of temperature is essential in many types of experimental in-
vestigations. The actual relationships between growth and temperature
are undoubtedly complex, since changes in temperature may not only
affect metabolic activities of the organism directly, but may also modify

506 FOOD REQUIREMENTS

the action of other environmental factors. Conversely, changes in vari-
ous environmental conditions may modify the temperature relationships
of a given species.

In one of the few investigations carried out on pure cultures, Jahn
(1935c) has demonstrated an interesting temperature relationship in
Euglena gracilis. In darkness, the optimal temperature for this species
in a peptone medium was about 10° C. When sodium acetate was added
to the medium, not only was growth accelerated, but the optimal tem-
perature was shifted to about 23°, a point approaching the optimum
for growth in light. Another instance in which the temperature rela-
tionships vary with other environmental conditions is represented by
the thermal death time of E. gracilis (Jahn, 1933a), which appears to
be a function of the pH, the greatest resistance to a temperature of 40° C.
being noted at pH 5.0, and a greater susceptibility above pH 7.0 than
below.

GROWTH IN RELATION TO LIGHT AND DARKNESS

Little or nothing is known concerning the relation between light and
the growth of colorless Protozoa. On the other hand, the importance of
light is obvious in the case of the chlorophyll-bearing species, and the
relation to photosynthesis probably accounts for a number of the known
effects of light. Dusi (1937) has noted certain interesting peculiarities
of several Euglenidae. In constant light, E. gracil7s grows well in pep-
tone medium, but poorly in inorganic media. On the other hand, E.
klebsi grows perfectly in inorganic medium under constant illumina-
tion, while E. viridis is incapable of growing under such conditions,
even in peptone medium. No explanation for such specific differences in
light relationships is yet available. An apparent relationship between
light and the optimal temperature for growth has been noted by Jahn
(1935c), who reported that the optimal temperature for growth of E.
gracilis in darkness lies near 10° C., whereas the optimum in light for
the same species is approximately 25°. The presence or absence of light
is also a factor which must be considered in interpreting the effects of
carbon compounds on growth. Thus Jahn (1935d) has observed that
the accelerating effects of several organic acids on the growth of E.
gracilis are relatively much greater in darkness than in light. Succinate,
on the other hand, produced a slight acceleration in darkness, but was

FOOD REQUIREMENTS 507

mildly toxic in light, while oxalate exerted no effect in darkness and a
slight acceleration in light. Hall (1937d) observed also that in E. ste/-
lata tolerance to concentrations of acetate above 0.2 percent was much
less in light than in darkness. Furthermore, Jahn (1936b) has obtained
some evidence that intensity of light may influence the effects of carbo-
hydrates on the growth of E. gracilis in an inorganic medium. Another
instance involving light relationships is the effect of plant ‘“hormones”’
on E. gracilis, in which Elliott (1937b) has shown that growth 1s ac-
celerated in light, but not in darkness.

ACCLIMATIZATION

Acclimatization of Protozoa to various experimental conditions has
been reported in many instances, ranging from acclimatization to toxic
chemicals to the development of resistance to antibodies. A few cases
have been described in bacteria-free cultures of free-living species. Such
a process may occasionally be involved in the establishment of pure cul-
tures, as reported by Elliott (1933) for Colpidium striatum and by
Johnson (1935a) for Glaucoma ficaria. More recently, Loefer (1938c)
has studied the acclimatization of several species (C. campylum, G. pirt-
formis, Chlorogonium euchlorum, Euglena gracilis, and Astasia sp.) to
progressively increased salt concentrations. E. gracilis developed no ap-
preciable tolerance, but, after a series of transfers, the other species
all showed the ability to grow in salt concentrations which were lethal
in the initial exposures. The salinity finally tolerated by C. campylum
was higher than that of ordinary sea water. Further investigations on
acclimatization should prove interesting, and may throw some light on
various experimental results which at present seem very puzzling.

LITERATURE CITED

No attempt has been made to include all the literature on bacteria-free
cultures of Protozoa. Among the papers cited, those which contain good
bibliographies are indicated by an asterisk.

Andrews, J., and T. von Brand. 1938. Quantitative studies on glucose con-
sumption by Trichomonas foetus. Amer. J. Hyg., 28: 138-47.

Bond, R. M. 1933. A contribution to the study of the natural food cycle in
aquatic environments. Bull. Bingham oceanogr. Coll. 4 (Art. 4), 89 pp.

Brand, T. von. 1938.* The metabolism of pathogenic trypanosomes and the
carbohydrate metabolism of their hosts. Quart. Rev. Biol., 13: 41-50.

508 FOOD REQUIREMENTS

Buchanan, R. E., and E. I. Fulmer. 1928. Physiology and biochemistry of
Bacteria. Vol. I. Baltimore.

Cailleau, R. 1933. Culture d’ Acanthamoeba castellanii sur milieu peptone.
Action sur les glucides. C. R. Soc. Biol. Paris, 114: 474-76.

—— 1934. Utilization des milieux liquides par Acanthamoeba castellanit.
C. R. ‘Soc Biol? Paris’ 1G 39729=23;

—— 1935. La Nutrition de Trichomonas columbae en culture. C. R. Soc. Biol.
Paris, 119: 853-56.

—— 1936a. Le Cholestérol, facteur de croissance pour le flagellé Trachomonas

columbae. C. R. Soc. Biol. Paris, 121: 424-25.

1936b. L’ Activité de quelques stérols envisagés comme facteurs de
croissance pour le flagellé Trichomonas columbae. C. R. Soc. Biol. Paris,

122: 1027-28.

—— 1937a. Nouvelles Recherches sur I’activité de quelques stérols con-
siderées comme facteurs de croissance pour le flagellé Trichomonas
columbae. C. R. Soc. Biol. Paris, 124: 1042-44.

—— 1937b.* La Nutrition des flagellés Tetramitides. Les stérols, facteurs
de croissance pour les Trichomonades. Ann. Inst. Pasteur, 59: 137-293.

—— 1938a. Le Cholestérol et I’acide ascorbique, facteurs de croissance pour
le flagellé tetramitide Trichomonas foetus Riedmiiller. C. R. Soc. Biol.
Paris, 127: 861-63.

—— 1938b. L’Acide ascorbique et le cholestérol, facteurs de croissance pour
le flagellé Extrichomastix colubrorum. C. R. Soc. Biol. Paris, 127:
1421-23.

—— 1939. L’Acide ascorbique, facteur de croissance pour le flagellé Tricho-
monas columbae. C. R. Soc. Biol. Paris, 130: 319-21.

Colas-Belcour, J., and A. Lwoff. 1925. L’Utilisation des glucides par quelques
Protozoaires. C. R. Soc. Biol. Paris, 93: 1421-22.

Dimitrowa, A. 1932. Die Férdernde Wirkung der Exkrete von Paramecium
caudatum Ehrbg. auf dessen Teilungsgeschwindigkeit. Zool. Anz., 100:
127-32.

Dusi, H. 1930. Limites de la concentration en ions H pour la culture de
quelques euglénes. C. R. Soc. Biol. Paris, 104: 734-36.

—— 1931. L’Assimilation des acides aminés par quelques eugléniens. C. R.
Soc. Biol. Paris, 107: 1232-34:

—— 1933a. Recherches sur la nutrition de quelques euglénes. I. Euglena
gracilis. Ann. Inst. Pasteur, 50: 550-97.

—— 1933b. Recherches sur la nutrition de quelques euglénes. II. Evglena
stellata, klebsii, anabaena, deses et pisciformis. Ann, Inst. Pasteur, 50:
840-90.

—— 1936. Recherches sur la culture et la nutrition d’Euglena viridis. Arch.
zool. expr. gén., 78 (M. et R.): 133-36.

—— 1937. Le Besoin de substances organiques de quelques eugléniens a
chlorophylle. Arch. Protistenk., 89: 94-99.

FOOD REQUIREMENTS 509

—— 1939. La Pyrimidine et le thiazol, facteurs de croissance pour le flagellé
a chlorophylle, Ezglena pisciformis. C. R. Soc. Biol. Paris, 130: 419-22.

Elliott, A. M. 1933.* Isolation of Colpidium striatum Stokes in bacteria-free
culture and the relation of growth to pH of the medium. Biol. Bull.,
65: 45-56.

—— 1935a. Effects of carbohydrates on growth of Colpidinm. Arch.
Protistenk., 84: 156-74.

— 1935b.* Effects of certain organic acids and protein derivatives on the
growth of Colpidium. Arch. Protistenk., 84: 225-31.

— 1935c. The influence of pantothenic acid on growth of Protozoa. Biol.
Bull., 68: 82-92.

— 1937a. Vitamin B, and growth of Protozoa. Anat. Rec., 70 (Suppl.):
27:

— 1937b. Plant hormones and growth of Evg/ena in relation to light.
Anat. Rec., 70 (Suppl.): 128.

—— 1938.* The influence of certain plant hormones on growth of Protozoa.
Physiol. Zo6l., 11: 31-39.

— 1939. The vitamin B complex and the growth of Colpidinm striatum.
Physiol. Zo6l., 12: 363-373.

Gause, G. F. 1935. Experimentelle Untersuchungen uber die Konkurrenz
zwischen Paramecium caudatum und Paramecium aurelia. Arch.
Protistenk., 84: 207-24.

Geise, A. C., and C. V. Taylor. 1935. Paramecia for experimental purposes
in controlled mass cultures on a single strain of bacteria. Arch. Protistenk.,
84: 225-31.

Glaser, R. W., and N. A. Coria. 1930. Methods for the pure culture of certain
Protozoa. J. exp. Med., 51: 787-806.

—— 1933. The culture of Paramecium caudatum free from living micro-
organisms. J. Parasite., 20: 33-37.

1935a. The culture and reactions of purified Protozoa. Amer. J. Hyg.,
2S VA1=20:

—— 1935b. Purification and culture of Tritrichomonas foetus (Riedmiller)
from cows. Amer. J. Hyg., 22: 221-26.

Hall, R. P. 1933a. On the relation of hydrogen-ion concentration to the growth
of Euglena anabaena vat. minor and E. deses. Arch. Protistenk., 79:
239-48.

—— 1933b. Growth of Colpidium campylum with reference to oxygen re-
lationships. Anat. Rec., 57 (Suppl.): 95.

—— 1934. Effects of carbohydrates on growth of Euglena anabaena vat.
minor in darkness. Arch. Protistenk., 82: 45-50.

—— 1937a.* “Growth of free-living Protozoa in pure cultures.” In Culture
Methods for Invertebrate Animals. Ithaca. Pp. 51-59.

—— 1937b. Certain culture reactions of several species of Euglenidae. Trans.
Amer, mice. Soe. 563-285-387.

510 FOOD REQUIREMENTS

—— 1937c. Effects of manganese on the growth of Euglena anabaena,
Astasia sp. and Colpidium campylum. Arch. Protistenk., 90: 178-84.

1937d. Effects of different concentrations of sodium acetate on growth

of Exglena stellata, Anat. Rec., 70 (Suppl.): 127.

1938a, Pimelic acid as a growth factor for the ciliate, Colpidium
campylum. Anat. Rec., 72 (Suppl.): 110.

—— 1938b. Nitrogen requirements of Evglena anabaena var. minor. Arch.
Protistenk., 91: 465-73.

—— 1939a. The trophic nature of Euglena viridis. Arch. zool. expr. gén.,
80 (N. et R.): 61-67.

—— 1939b. The trophic nature of the plant-like flagellates. Quart. Rec.
Biol., 14: 1-12.

—— 1939c. Pimelic acid as a growth stimulant for Colpidinm campylum.
Arch. Protistenk., 92: 315-19.

Hall, R. P., and A. M. Elliott. 1935. Growth of Colpidium in relation to
certain incomplete proteins and amino acids. Arch. Protistenk., 85:
443-50.

Hall, R. P., and J. B. Loefer. 1936. On the supposed utilization of inorganic

nitrogen by the colorless cryptomonad flagellate, Chilomonas paramecium.

Protoplasma, 26: 321-30.

1940. Effects of culture filtrates and old medium on growth of the
ciliate, Colpidium campylum. Proc. Soc. exp. Biol. N. Y., 43: 128-33.
Hall, R. P., and H. W. Schoenborn. 1938a. Studies on the question of auto-

trophic nutrition in Chlorogonium euchlorum, Euglena anabaena and
E, deses. Arch. Protistenk., 90: 259-71.

—— 1938b. The selective action of inorganic media in bacteria-free cultures

of Euglena. Anat. Rec., 72 (Suppl.) : 129-30.

1939a. The question of autotrophic nutrition in Exglena gracilis.

Physiol. Zo6l., 12: 76-84.

—— 1939b. Fluctuations in growth rate of Euglena anabaena, E. gracilis
and E. viridis and their apparent relation to initial density of population.
Physiol. Zo6l., 12: 201-08.

Hutner, S. H. 1936. The nutritional requirements of two species of Euglena.
Arch. Protistenk., 88: 93-106.

Jahn, T. L. 1929.* Studies on the physiology of the euglenoid flagellates.
I. The relation of the density of population to the growth rate of
Euglena. Biol. Bull., 57: 81-106.

—— 1931. Studies on the physiology of the euglenoid flagellates. III. The
effect of hydrogen-ion concentration on the growth of Ezglena gracilis.
Biol. Bull., 61: 387-99.

—— 1933a.* Studies on the physiology of the euglenoid flagellates. IV.
The thermal death time of Exglena gracilis Klebs. Arch. Protistenk., 79:
249-62.

—— 1933b.* Studies on the oxidation-reduction potential of protozoan cul-

FOOD REQUIREMENTS ul

tures. I. The effect of -SH on Chilomonas paramecium. Protoplasma, 20:
90-104.

—— 1934. Problems of population growth in the Protozoa. Cold Spring
Harbor Symp. Quant. Biol., 2: 167-80.

—— 1935a. Studies on the oxidation-reduction potential of protozoan cul-
tures. II. The reduction potential of cultures of Chilomonas paramecium.
Arch. Protistenk., 86: 225-37.

—— 1935b. Studies on the physiology of the euglenoid flagellates. V. The
effect of certain carbohydrates on the growth of Exglena gracilis Klebs.
Arch, Protistenk., 86: 238-50.

—— 1935c. Studies on the physiology of the euglenoid flagellates. VI. The
effects of temperature and of acetate on Ezglena gracilis cultures in the
dark. Arch. Protistenk., 86: 251-57.

—— 1935d. Studies on the physiology of the euglenoid flagellates. VII.
The effect of salts of certain organic acids on growth of Evglena gracilis
Klebs. Arch. Protistenk., 86: 258-62.

—— 1936. Effect of aeration and lack of CO, on growth of bacteria-free
cultures of Protozoa. Proc. Soc. Exper. Biol. N. Y., 33: 494-98.

Johnson, D. F. 1935a.* The isolation of Glaucoma ficaria in bacteria-free
cultures, and growth in relation to pH of the medium. Arch. Protistenk.,
86: 263-77.

—— 1935b. Fermentation of carbohydrates by Glaucoma and effects of

carbohydrates on growth of two species. Anat. Rec., 64 (Suppl.) : 106-07.

1936.* Growth of Glaucoma ficaria Kahl in cultures with single species

of other microorganisms. Arch, Protistenk., 86: 359-78.

Johnson, W. H. 1933.* Effects of population density on the rate of reproduc-
tion in Oxytricha. Physiol. Zoél., 6: 22-54.

—— 1936.* Studies on the nutrition and reproduction of Paramecium.
Physiol. Zodl., 9: 1-14.

—— 1937.* Experimental populations of microscopic organisms. Amer.
Nat7 135220!

Johnson, W. H., and G. Mardin. 1938. Reproduction of Paramecium in old
culture medium. Physiol. Zodl., 11: 333-46.

Koser, S. A., and F. Saunders. 1938. Accessory growth factors for bacteria
and related microorganisms. Bact. Rev., 2: 99-160.

Lawrie, N. R. 1937. Studies in the metabolism of Protozoa. III. Some prop-
erties of a proteolytic extract obtained from Glaucoma piriformis. Bio-
chem. J., 31: 789-98.

Loefer, J. B. 1934. The trophic nature of Chlorogonium and Chilomonas.
Biol. Bull., 66: 1-6.

—— 1935a.* Effect of certain carbohydrates and organic acids on growth
of Chlorogonium and Chilomonas, Arch. Protistenk., 84: 456-71.
—— 1935b. Effects of certain nitrogen compounds on growth of Chloro-

gonium and Chilomonas. Arch. Protistenk., 85: 74-86.

12 FOOD REQUIREMENTS

—— 1935c.* Relation of hydrogen-ion concentration to growth of Chilo-
monas and Chlorogonium. Atch. Protistenk., 85: 209-23.

—— 1936a. Isolation and growth characteristics of the ‘“‘zoochlorella’” of
Paramecium bursaria. Amer. Nat., 70: 184-88.

— 1936b. Bacteria-free cultures of Paramecium bursaria and concentration
of the medium as a factor in growth, J. exp. Zool., 72: 387-407.

—— 1936c. Effect of certain “peptone’’ media and carbohydrates on the
growth of Paramecium bursaria. Arch. Protistenk., 87: 142-50.

—— 1936d. A simple method for maintaining pure-line mass cultures of
Paramecium caudatum on a single species of yeast. Trans. Amer. micr.
SOC., 552)254-56;

—— 1938a. Effect of hydrogen-ion concentration on the growth and
morphology of Paramecium bursaria. Arch. Protistenk., 87: 142-50.
—— 1938b.* Utilization of dextrose by Colpidium, Glaucoma, Chilomonas

and Chlorogonium in bacteria-free cultures. J. exp. Zool., 79: 167-83.

—— 1938c. Effect of osmotic pressure on the motility and viability of fresh-
water Protozoa. Anat. Rec., 72 (Suppl.) : 50.

Loefer, J. B., and R. P. Hall. 1936. Effect of ethyl alcohol on the growth of
eight protozoan species in bacteria-free cultures. Arch. Protistenk., 87:
#23-50.

Lwoff, A. 1924. Le Pouvoir de synthése d’un protist hétérotrophe: Glaucoma
piriformis. C. R. Soc. Biol. Paris, 91: 344-45.

—— 1925. La Nutrition des infusoires au dépens des substances dissoutes.
C. R. Soc. Biol. Paris, 93: 1272-73.

—— 1929a. Milieux de culture et d’entretien pour Glaucoma piriformis
(cilié) . C. R. Soc. Biol. Paris, 100: 635-36.

—— 1929b. La Nutrition de Polytoma uvella Ehrenberg (flagellé Chlamydo-
monadinae) et le Pouvoir de synthése des protistes hétérotrophes. Les
protistes mésotrophes. C. R. Acad. Sci. Paris, 188: 114-16.

—— 1930. Le Fer, élément indispensable au flagellé Polytoma uvella Ehr.
C. R. Soc. Biol. Paris, 104: 664-66.

—— 1931. La Nutrition carbonée de Polytoma uvella. C. R. Soc. Biol. Paris,
1072 1070372?

——— 1932.* Recherches biochimiques sur la nutrition des protozoaires. Le
pouvoir synthése. Monogr. Inst. Pasteur.

—— 1934. Die Bedeutung des Blutfarbstoffes fiir die parasitischen
flagellaten. Zbl. Bakt., Orig. 130: 498-518.

—— 1935a. L’Oxytrophie et les organisms oxytrophes. C. R. Soc. Biol.
Paris, 119: 87-90.

—— 1935b. La Nutrition azotée et carbonée de Polytomella agilis
(Polyblépharidée incolore). C. R. Soc. Biol. Paris, 119: 974-76.

—— 1936. La Fonction de la protohémin pour les protozoaires et les
bactéries parahémotrophes. C. R. Soc. Biol. Paris, 122: 1041-42.

FOOD REQUIREMENTS Biles

—— 1938a. Remarques sur la physiologie comparée des protistes eucaryotes.
Les Leucophytes et l’oxytrophie. Arch. Protistenk., 90: 194-209.

—— 1938b. La Synthése de l’aneurine par le protozoaire, Acanthamoeba
castellanii. C. R. Soc. Biol. Paris, 128: 455-58.

Lwoff, A., and H. Dusi. 1929. Le Pouvoir de synthése d’Euglena gracilis
cultivée a l’obscurité. C. R. Soc. Biol. Paris, 102: 567-69.

— 1931. La Nutrition azotée et carbonée d’Evglena gracilis en culture
pure a l’obscurité. C. R. Soc. Biol. Paris, 107: 1068-69.

—— 1934. L’Oxytrophie et la nutrition des flagellés leucocophytes. Ann.
Inst. Pasteur, 53: 641-53.

—— 1935a. La Supression expérimentale des chloroplastes chez Euglena
mesnilz, Ann. Inst. Pasteur, 119: 1092-95.

—— 1935b. La Nutrition azotée et carbonée de Chlorogonium euchlorum
a l’obscurité; l’acide acétique envisagé comme produit de |’assimilation
chlorophylliene. C. R. Soc. Biol. Paris, 119: 1260-63.

— 1936. La Nutrition de l’euglénien Astasia chattoni. C. R. Acad. Sci.
Paris, 202: 248-50.

-—— 1937a. La Pyrimidine et le thiazol, facteurs de croissance pour le
flagellé Polytomella caeca. C. R. Acad. Sci. Paris, 630-32.

——— 1937b. Le Thiazol, facteur de croissance pour les flagellés Polytoma
caudatum et Chilomonas paramecium. C. R. Acad. Sci. Paris, 205: 756-58.

——— 1937c. Le Thiazol, facteur de croissance pour Polytoma ocellatum
(Chlamydomonadine). Importance des constituants de l’aneurine pour
les flagellés leucophytes. C. R. Acad. Sci. Paris, 205: 882-84.

—— 1938a. Culture de divers flagellés leucophytes en milieu synthétique.
GoRs See Biol Pans 127: 53-56.

—— 1938b. L’ Activité de diverses pyrimidines, considérées comme facteurs
de croissance pour les flagellés Polytomella caeca et Chilomonas para-
mecium. C. R. Soc. Biol. Paris, 127: 1408-11.

—— 1938c. Influence de diverses substitutions sur l’activité de thiazol con-
sidéré comme facteur de croissance pour quelques flagellés leucophytes.
C. R. Soe: Biol. Paris, 128: 238-41.

Lwoff, A., and E. Lederer. 1935. Remarques sur |’‘‘extrait de terre’ envisagé
comme facteur de croissance pour les flagellés. C. R. Soc. Biol. Paris,
WD) STATS

Lwoff, A., and M. Lwoff. 1937. L’Aneurine, facteur de croissance pour le
cilié Glaucoma piriformis. C. R. Soc. Biol. Paris, 126: 644-46.

—— 1938. La Specificité de l’aneurine, facteur de croissance pour le cilié
Glaucoma piriformis. C. R. Soc. Biol Paris, 127: 1170-72.

Lwoff, A., and L. Provasoli. 1935. La Nutrition de Polytoma caudatum vat.
astigmata (Chlamydomonadine incolore), et la synthése de |’amidon par
les leucophytes. C. R. Soc. Biol. Paris, 119: 90-93.

—— 1937. Caractéres physiologiques du flagellé Polytoma obtusum, C. R.
Soc. Biol. Paris, 126: 279-80.

514 FOOD REQUIREMENTS

Lwoff, A., and N. Roukhelman. 1929. Variations de quelques formes d’azote
dans une culture pure d’infusoires. C. R. Acad. Sci. Paris, 183: 156-58.

Lwoff, M. 1929a. Culture de Leptomonas ctenocephali var. chattoni Laveran
et Franchini, en milieu privés de sang frais: milieux liquides au sange
chauffé. Bull. Soc. Path. exot., 22: 247-51.

—— 1929b. Milieu d’isolement et d’entretien pour Schizotrypanum cruzi
Chagas. Bull. Soc. Path. exot., 22: 909-12.

—— 1929c. Action favorisante du sang sur la culture due Leptomonas
ctenocephali (flagellé trypanosomide). C. R. Soc. Biol. Paris, 99: 472-74.

—— 1929d. Culture de Leptomonas ctenocephali Fanth. (flagellé trypanoso-
mide) en milieu privé de sang frais: les organes stérilisés. C. R. Soc.
Biol. Paris, 99: 1133-35.

—— 1929e. Influence du degré d’hydrolyse des matiéres protéiques sur la
nutrition de Leptomonas ctenocephali (Fantham) in vitro. C. R. Soc.
Biol. Paris, 100: 240-43.

—— 1930. Une Flagellé parasite hétérotrophe: Leptomonas oncopelti
Noguchi et Tilden (Trypanosomidae). C. R. Soc. Biol. Paris, 105: 835-
aie

—— 1933a.* Recherches sur la nutrition des trypanosomides. Ann. Inst.
Pasteur, 515i:

—— 1933b. Remarques sur la nutrition des trypanosomides et des bactéries
parahémotrophes. Le “fer actif’ de Baudisch. Ann. Inst. Pasteur, 51:
707-13.

——— 1936, Le Pouvoir de synthése des trypanosomides des muscides. C. R.
Soc. Biol. Paris, 121: 419-21.

——— 1937. L’Aneurine, facteur de croissance pour le flagellé trypanosomide
Strigomonas oncopelti (Noguchi et Tilden). C. R. Soc. Biol. Paris, 126:
Were

—— 1938a. L’Hématine et l’acide ascorbique, facteurs de croissance pour le
flagellé Schizotrypanum cruzi. C. R. Acad. Sci. Paris, 206: 540-42.

—— 1938b. L’Aneurine, facteur de croissance pour le Strigomonas
(flagellés Trypanosomides). C. R. Soc. Biol. Paris, 128: 241-43.

——— 1939. Le Pouvoir de synthése des leishmanies. C. R. Soc. Biol. Paris,
130: 406-8.

Lwoff, M., and A. Lwoff. 1929. Le Pouvoir de synthése de Chlamydomonas
agloéformis et d’'Haematococcus pluvialis en culture pure, 4 l’obscurité.
C. R. Soc. Biol. Paris, 102: 569-71.

Mainx, F. 1928.* Beitrage zur Morphologie und Physiologie der Eugleninen.
II. Teil. Untersuchungen tiber die Ernahrungs-und Reizphysiologie.
Arch. Protistenk., 60: 355-414.

Mast, S. O., and D. M. Pace. 1933. Synthesis from inorganic compounds of
starch, fats proteins and protoplasm in the colorless animal, Chilomonas
paramecium, Protoplasma, 20: 326-58.

—— 1935. Relation between sulphur in various chemical forms and the

FOOD REQUIREMENTS 515

rate of growth in the colorless flagellate, Chilomonas paramecium. Proto-
plasma, 23: 297-325.

—— 1938. The effect of substances produced by Chilomonas paramecium
on rate of reproduction. Physiol. Zodl., 11: 359-82.

Oehler, R. 1916. Amébenzucht auf reinem Boden. Arch. Protistenk., 37:
175-90.

—— 1919. Flagellaten- und Ciliatenzucht auf reinem Boden. Arch.
Protistenk., 40: 16-26.

Osterud, K. L. 1938. The nitrogen requirements of Lobomonas piriformis.
Anat. Rec., 72 (Suppl.) : 128-29.

—— 1939. The nitrogen and carbon requirements of Lobomonas piriformis.
Anat. Rec., 75 (Suppl.) : 150-51.

Petersen, W. A. 1929. The relation of density of population to rate of re-
production. Physiol. Zodl., 2: 221-54.

Phelps, A. 1935. Growth of Protozoa in pure culture. I. Effect upon the
growth curve of the age of the inoculum and of the amount of the
inoculum. J. exp. Zool., 70: 109-30.

1936. Growth of Protozoa in pure culture. II. Effect upon the growth
curve of different concentrations of nutrient materials. J. exp. Zool., 72:
479-96.

Philpott, C. H. 1928. Growth of Paramecium in pure cultures of pathogenic
bacteria and in the presence of soluble products of such bacteria.
J. Morph., 46: 85-129.

Pringsheim, E. G. 1912. Kulturversuche mit chlorophyllfiihrenden Mikro-
organismen. II. Zur Physiologie der Euglena gracilis. Beitr. Biol. Pfl., 12:
1-48.

—— 1921. Zur Physiologie saprophytischer Flagellaten (Polytoma, Astasia
und Chilomonas) . Beitr. allg. Bot., 2: 88-137.

1926. Kulturversuche mit chlorophyllfihrenden Mikroorganismen.
V. Mitt. Methoden und Erfahrungen. Beitr. Biol. Pfl., 14: 283-312.
— 1930. Algenreinkulturen. Eine Liste der Stamme welche auf Wunsch

abgegeben werden. Arch. Protistenk., 69: 659-65.

—— 1935a. Uber Azetatflagellaten. Naturwissenschaften, 23: 110-14.

— 1935b. Wuchstoffe im Erdboden? Naturwissenschaften, 23: 197.

—— 1937a. Assimilation of different organic substances by saprophytic
flagellates. Nature, 139: 196.

—— 1937b.* Beitrage zur Physiologie saprophytischer Algen und
Flagellaten. 1 Mitt.: Chlorogonium und Hyalogonium. Planta, 26: 631-
64.

—— 1937c.* Beitréige zur Physiologie saprophytischer Algen und
Flagellaten. 2 Mitt.: Polytoma und Polytomella. Planta, 26: 665-91.
—— 1937d.* Beitrige zur Physiologie saprotropher Algen und Flagellaten.
3 Mitt.: Die Stellung der Azetatflagellaten in einem physiologischen

Ernahrungssystem. Planta, 27: 61-92.

516 FOOD REQUIREMENTS

Provasoli, L. 1937a. La Nutrition carbonée du flagellé Polytoma uvella. C. R.
Soc. Biol. Paris, 126: 280-82.

—— 1937b. La Nutrition carbonée du flagellé Polytoma ocellatum. C. R.
Soc. Biol. Paris, 126: 847-49.

—— 1938a. La Nutrition carbonée de l’euglénien Astas7a quartana (Moroft).
GRP Soc Biel Paris 1272 sl-s3:

—— 1938b. Remarques sur la nutrition carbonée des eugléniens. C. R. Soc.
Biol. Paris, 127: 190-92.

—— 1938c. Studi sulla nutrizione dei Protozoi. Boll. Lab. Zool. agr. Bachic.
Milano, 9 (rpr.), 124 pp.

Reich, K. 1935. The cultivation of a sterile amoeba on media without solid
food. J. exp. Zool., 69: 497-500.

—— 1936. Studies on the physiology of Amoeba. I. The relation between
nutrient solution, zone of growth and density of population. Physiol.
Zool., 9: 254-63.

—— 1938. Studies on the physiology of Amoeba. II. The allelocatalytic
effect in Amoeba cultures free of bacteria. Physiol. Zodl., 11: 347-58.

Rottier, P. B. 1936a. Recherches sur les courbes de croissance de Polytoma
uvella. L’influence de l’oxygénation. C. R. Soc. Biol. Paris, 122: 65-68.

—— 1936b. Recherches sur la croissance de Polytoma uvella, L’influence de
la concentration des substances nutritives. C. R. Soc. Biol. Paris, 122:
776-80.

Schoenborn, H. W. 1936. Growth of two species of Astasia in relation to pH

of the medium. Anat. Rec., 67 (Suppl.) : 121.

1938. Growth of Astasia sp. and Exglena gracilis in media containing

inorganic nitrogen. Anat. Rec., 72 (Suppl.) : 51.

—— 1939. Growth of Evglena gracilis on inorganic nitrogen sources in the
absence of light. Anat. Rec., 75 (Suppl.) : 151.

—— 1940. Studies on the nutrition of colorless euglenoid flagellates. I.
Utilization of inorganic nitrogen by As/asia in pure cultures. Ann. N. Y.
Acad. Sci.,-40: 1-36.

Woodruff, L. L. 1911. The effect of excretion products of Paramecium on its

rate of reproduction. J. exp. Zool., 10: 557-81.

1912. Observations on the origin and sequence of the protozoan fauna

of hay infusions. J. exp. Zool., 12: 205-64.

—— 1913. The effect of excretion products of Infusoria on the same and
on different species, with special reference to the protozoan sequence in
infusions. J. exp. Zool., 14: 575-82.

CHAPTERYX

THE GROW Ti OF eiHE -PROTOZOA:

OscaR W. RICHARDS

GROWTH is a fundamental attribute of living organisms, manifested by
a change of size of the individual, or in the number of organisms in a
unit of environment. Negative growth may occur during adverse con-
ditions or in certain dimensions when growth involves change of form.
The analysis of population growth requires knowledge of the environ-
ment, the individuals, and the interactions of each on the other.

Growth is determined by measurement, and the information gained
from any single measure is delimited by the nature of the measuring unit
chosen. Rarely is a single measure adequate for the study of growth,
even though it may be useful for practical application. Analytical studies
require the simultaneous use of as many different measures as are neces-
sary to give a picture sufficiently complete for the analysis. When there
is no change in form, certain dimensions may be related directly, as
length with volume or weight, but in allometric growth the conversion
constants may change during the course of the growing period. These
problems will be illustrated and discussed in this chapter, in so far as
numerical data are available.

METHODS FOR THE MEASUREMENT OF GROWTH

Individual Protozoa have been measured to show growth changes in
length and breadth, but these two dimensions may not permit very exact
calculation of volume if the shape of the animal departs much from
that of a sphere, cube, ellipsoid or other simple geometrical form. The
area of the animal may be calculated by the use of a planimeter, from
an enlarged photomicrograph or a tracing of the outline of the animal.
The softer animals may be gently compressed between a slide and a
cover glass, and the area measured. Multiplication of this figure by
that of the thickness of the preparation gives the volume. The three-
halves power of the area obtained from planimetric measurement may

518 GROWTH

give the volume of some species fairly accurately. This is true for only
a few solids, such as the cube. The method has an error of about 33
percent when used with spherical organisms. Chalkley (1929) measured
the volume of Amoeba by gently drawing it into a capillary tube of
known diameter and calculating the volume from the length of tube
filled plus the two hemispherical ends.

Populations of Protozoa are usually measured by counting a sample
of the population in a Sedgewick-Rafter cell with a Whipple disc in the
eyepiece of the microscope, or with a hemocytometer (cf. Woodruff,
1912; Hall et a/., 1935). The chief source of error of this method de-
pends on how closely the sample represents the population. Care must
be used that none of the animals are lost by sticking to the transfer
pipette and to make sure that all are counted once only. Berkson ef al.
(1935) has given a quantitative treatment of the errors of counting
red blood cells with a hemocytometer, and their evaluation might be
applied to estimates of protozoan populations.

Tippett (1932) has suggested that the mean number may be esti-
mated by counting the squares containing 0, 1, 2, and so forth animals
and using the tables prepared for the Poisson distribution. With Proto-
zoa, greater precision may be obtained by killing the animals before
making the count. Many killing fluids are hypertonic, and animals may
be lost from the osmotic effects of the killing fluid. Jennings (1908)
and others have found that Worcester’s fluid causes little change with
paramecia when a sufficient amount is used to overwhelm the animals.
Hardy’s (1938) method of estimating numbers by comparing with
standards containing a known number of dots might be used when high
precision is not required.

Protozoa may be centrifuged into a tube with a calibrated capillary
bottom, similar to that used by Carlson (1913) for yeast. Elliott (1939)
obtains greater precision and convenience by fusing a hematocrit tube
toa 10 ml. centrifuge tube. When the animals are killed before centrifug-
ing, it is necessary that the volume of the animals not be changed by an
anisotonic killing fluid. Commercially made tubes should be carefully
calibrated, as errors as great as 12 percent have been reported for some
makes of Hopkins vaccine tubes. Solid packing may not be possible with
the usual laboratory centrifuges, but for given conditions constant pack-
ing may be obtained in equal time intervals. If the values are to be used

GROWTH 519

for other than intercomparison, the centrifugal force used should be
stated. The nomogram of Shapiro (1935a) simplifies this computation.
If the distribution of animals of different sizes changes, e.g., just after
a large proportion of them have divided or during endomictic reorgani-
zation, the total volume may not indicate the number present. Size
changes of yeast cells and failure to obtain constant packing have been
reported by Richards (1934). Shapiro (1935b) has discussed the valid-
ity of the centrifuge method with respect to marine ova. Simultaneous
counts and volume determinations of Colpidium campylum have been
made by Bond (1933).

The population density of pigmented forms may be estimated from
the optical density of the suspension, by means of a nephelometer
(Richards and Jahn, 1933). A beam of light is passed through the
suspension and the amount of light absorbed by the organisms is meas-
ured by a photoelectric cell and a microammeter. When I is the micro-
ammeter reading with a given tube and medium and J; is the reading
of the suspension at time ¢, then the optical density, D = Jog I, — log I.
In this way the small variations in transmission of different test tubes
may be canceled out, and it is not necessary to open the tube, a factor
which may be important if the organisms are reared in a bacteria-free cul-
ture.

The optical density depends on the number of organisms present, the
distribution of organisms of various sizes, and their metabolic condition.
It is sometimes difficult to relate measurements with this criterion to
the number of organisms present, because changes in internal cell struc-
ture (e.g., storage products) may alter their transparency. With proper
care and control, the nephelometer may give a useful measure of the
amount of protoplasm present in the population. For technical informa-
tion the following may be consulted: Kober and Graves (1915), Mestre
(1935), Russell (1937), and Miiller (1939). Difficulties in the use
of the method have been summarized by Loofbourow and Dyer (1938)
and by Stier, Newton, and Sprince (1939). Miss Wright (1937) has
measured the turbidity of bacterial suspensions by passing the light
beam through the suspension at right angles to the axis of the photo-
electric cell.

The dry weight of Protozoa may be obtained by filtering them from
the culture medium with filter paper of fine porosity, a sintered glass

520 GROWTH

filter, an alundum crucible, or an asbestos mat; washing them rapidly
to remove the culture fluid, but not to burst the cells, and drying them
to a constant weight. A vacuum desiccator containing sulphuric acid or
phosphorous pentoxide at room temperature may give better results
than a drying oven. This is a difficult method to control, so as to get
consistent results.

Bacteriological methods of diluting and plating are not often ap-
plicable to Protozoa, but may be useful for testing the culture medium
to make certain that it is bacteria-free. Standard texts should be con-
sulted for methods. The ordinary nutrient agar is not a certain medium
for estimating the bacteria found in water, and special media must be
used. The number of colonies on an incubated plate may be less than
the number of bacteria unless care has been used to prevent clumping of
the bacteria. The errors of plate counts have been evaluated by Mattick
et al. (1935) and by Ziegler and Halvorson (1935). Gordon (1938)
has questioned these probability tables.

Other methods which might be useful to protozodlogists are the
measurement of the suspension in terms of viscosity (Shapiro, 1937)
and the determination of the velocity of sedimentation (Nielsen, 1933).
These would be used with killed or nonmotile animals.

THE GROWTH OF INDIVIDUAL PROTOZOA

Simpson (1902) measured the length and breadth of Paramecium
caudatum with an ocular micrometer at a few and at many hours after
fission. Jennings (1908) supplied the first detailed measurements, and
the data from his summary table are plotted in Figures 130 and 131.
At division the animal decreases in breadth and increases in length.
After the separation the increase in both dimensions is increasingly rapid,
then proceeds at a nearly constant relative rate, and finally slows until
the cycle is repeated.

The graphs of the growth are made on arithlog, or semilogarithmic
paper, to facilitate analysis. This equivalent to plotting the logarithm
of the size against a linear time axis. The slope of the growth curve at
any point is the relative rate of growth (dy/ydt). Two growth curves,
parallel to each other, are changing at the same relative rates. When no
change in form occurs, the curves for area and volume will be corre-
spondingly above and have slopes two and three times as great as a
linear dimension.

GROWTH S21

A year later Popoff (1908) measured the growth of P. caudatum by
killing a sister cell immediately and the other member of the pair at a
given time after fission. His results are expressed in micrometer units
which have been converted into microns. The average of the cells killed

Figure 130. Growth in length and in area of Paramecium caudatum. Data: M from
Mizuno (1927), S from Schmalhausen and Synagajewska (1925), E28 and E38 from
Estabrook (1910), J from Jennings 1908), and P from Popoff (1909).

at fission was taken as the standard size at zero time, and the average
differences for the intervals were added successively to obtain the data
plotted in Figures 130, 131, and 132. He measured length (L), breadth
(B), and thickness (T) and computed the volume as — 4xLBT/24,
assuming the form to be ellipsoid, and he also gives similar measure-
ments of the growth of the nucleus and of the nucleocytoplasmic ratio.

Estabrook (1910) investigated the effect of various chemicals on the

522 GROWTH

growth of P. caudatum and some of his control series, with more fre-
quent measurements, are plotted. Measurements of growth in length by
Schmalhausen and Syngajewskaja (1925) and by Mizuno (1927) are
also available for P. caudatum. Mizuno also determined the area of the
animal with a planimeter, from a camera-lucida tracing.

x
x
f=
fa)
<
WW
a
ao

Figure 131. Growth in breadth and thickness (TH) of Paramecium caudatum. Data:
P from Popoff (1909), M from Mizuno (1927), J from Jennings (1908), E21, E28,
and E38 from Estabrook (1910).

All of the investigators took precautions to prevent any change in the
dimensions of the organism from killing the organism. The curves for
growth in length are quite similar. All were grown in hay infusion
medium and the temperature, when stated, was 24-26° C. Mizuno’s ani-
mals grew at first at a more rapid relative rate than the others. Esta-
brook’s and Schmalhausen’s animals were appreciably larger than
Jennings’s, Mizuno’s, and Popoft’s Paramecia. The decrease in the early

GROWTH D29

logarithmic growth is more rapid in the data from Jennings’s measure-
ments; however, his animals continued to grow for a longer period and
reached a slightly greater length than did those measured by Mizuno
and Popoff. The variations may be due to differences in nutrition or to
race. The results are all in terms of averages from different animals,
and do not show the continuous change of size of a given animal. This
type of averaging of cross-sectional data is known to give variation.

The measurements of breadth (Fig. 131) are not as consistent as
those of length. With the exception of one of Estabrook’s series, the
breadth decreases following fission, and growth in this dimension com-
mences later, in the measurements given. Thickness and breadth meas-
urements, plotted from Popoft’s observations, show no change in size
for the first two hours. The lack of agreement of the different series of
measurements suggests differences in the pattern of growth for the
different races. Growth in thickness occurred later than growth in breadth
with Popoff’s P. caudatum.

Growth in area (Fig. 130) is negative during the time that the
breadth is decreasing, after which the increase continues for most of the
cycle at about the same relative rate. Growth in volume, from Popoft’s
calculations (Fig. 132), does not show the early negative phase, because
his animals apparently did not change form during division as the oth-
ers did. With minor fluctuations the increase in volume continues in a
suitable environment until the time of the next fission.

The growth of Paramecium aurelia (Fig. 132), plotted from the meas-
urements of Erdmann (1920), is quite similar to that of P. caudatum.
Erdmann’s three races differed from one another in size. No decrease
in breadth was reported for this species.

The growth in volume of the first individual and its progeny is given
in Figure 132 for the soil amoeba, Hartmanella hyalina (Cutler and
Crump, 1927). The growth curve is very similar to that of P. caudatum.
Ten drawings were made rapidly at each time, and the volume was ob-
tained from the average areas, on the assumption that the animals were
1 thick. The temperature was 21° C. The growth in volume of Amoeba
proteus was measured by Chalkley (1929). The animals were pipetted
until they assumed a spherical form, and the diameters of the cell and
nucleus were then measured. The growth of the Amoeba is slow, and
Chalkley’s observations were not continued until the equilibrium size

524 GROWTH

#43 - 00000 OMITTED

it}
55
>
—]
oO
>

Figure 132. A. Growth in volume of Paramecium caudatum (P), Frontonia leucas
(F), from Popoff (1907, 1908), and of Hartmanella hyalina (H) from Cutler and
Crump (1927). B. Growth in length of P. aurelia. Data from Erdmann (1920).

was reached. The rate of growth is influenced by the number of nuclet
present in the animal.

Popoff (1908) measured the growth of Frontonia leucas in length,
breadth, and thickness, and estimated the volume as the product of
these three factors. The growth of the nucleus was measured similarly.
His data are given in arbitrary size units, which were converted into
micra. The growth of Frontonia was much more variable than Popoft’s
growth data indicated to be the case for Paramecium, except for the
volume changes. The average volume of the cytoplasm and the nucleus
of the control animals killed at fission, was used as the value for zero

GROWTH 525

time. To these values were added successively the average increments, in
order to obtain the values at each time. The sum of the nuclear and the
cytoplasmic volumes is plotted in Figure 132. The increments were
read from Popoft’s summary graph.

The growth in volume of F. /ewcas is more rapid during the first hour;
the rate then decreases, until the relative rate is nearly linear, that ts,
until about one hour before the next division. The rate increases before
division, mainly owing to the increase in nuclear volume. The volume
of the cytoplasm increases continually. The volume of the nucleus de-
creases for the first two hours after division to about 86 percent of its
size at fission, then increases slowly, until it shows the customary rapid
increase in the three hours preceding a new fission.

Entz (1931) has followed the growth of populations and of indi-
vidual dinoflagellates, Ceratium hirudinella, in their natural habitats.
The largest animals were found in April. The highest rate of division
occurred in late June, July, and August, which coincides with the period
of maximum temperature. The growth was measured in three dimen-
sions, and the time, in hours, for the growth stages is: nucleal and cyto-
plasmic division, one; horn regeneration, 2; slim stage, 27; indifferent
stage, 72; compacted stage, 18; total, 120 hours.

Extensive studies have been made of the variation in the sizes of
Protozoa, and this information has been collated by Adolph (1931).
A change in form during growth is reported by MacLennan (1935) for
Ichthyophthirius. This and other Protozoa would furnish excellent ma-
terial for the study of allometric growth. Cf. Huxley (1932), Need-
ham (1934), Teissier (1934), Richards (1935), and Huxley and Teis-
sier (1936). The size of Paramecium bursaria was modified by changing
the pH of the culture medium (Loefer, 1938a). Cell size and nuclear
size in Oxytricha fallax was found by Woodruff (1913a) to be least
during the periods of rapid reproduction, and to become larger as the
division rate decreases. The nucleocytoplasmic ratio was highest during
the period of greatest reproductive activity, and this was interpreted as
an incidental result rather than as a cause of the rate of division.

The first evidence of division is a slight groove encircling the animal,
and separation occurs in Paramecium about one-half hour later (Jen-
nings, 1908). The nucleocytoplasmic ratio increases for the first three
hours, and then decreases for the next three hours (Popoff, 1909).

526 GROWTH

During the first hour, Paramecia are little affected by chemicals, and
food plays no rdle (Estabrook, 1910). Increase in length is predomi-
nant at first, and then the animals fill out in breadth. Negative correla-
tions of length and breadth were found by Mizuno at one, four, and
six hours after fission. Erdmann (1920) found changes in the sizes of
Paramecia which were related to the endomictic rhythm.

Individual growth has been measured in relatively few species, mostly
Infusioria. Data on other forms would be useful for comparison. Growth
studies should be made successively on the same living animals, to avoid
the difficulties of averaging and of using information from different
individuals. A promising method for obtaining the measurements would
be to take pictures of the animal in isolation culture, at frequent and
regular intervals, through the microscope, with a motion-picture camera.
The images could be measured from the film, which would provide a
permanent record for the analysis of the growth.

The growth of individual Protozoa is quite similar to that observed
with other plants and animals. If we disregard the first decrease in
breadth, the growth curves are sigmoid. Sufficient information is not
available to indicate to what extent there is a general change of form
during growth. Many of these studies were not made with a constant
culture fluid. In order to compare the growth patterns of different species,
the culture conditions should be known to be optimal and reproducible.
Such studies would establish standards for the further study of environ-
ment on the individual growth pattern. The application of geometrical
and metabolic considerations, similar to those used by Schmalhausen
and Syngajewskaja (1925) with bacteria, will contribute to the theory
of growth.

THE GROWTH OF COLONIAL PROTOZOA

The colonial Protozoa are believed by Fauré-Fremiet (1930) to
constitute an intermediary step between the unicellular and the multi-
cellular organisms. The colony grows by regular division from a free-
swimming cell, until a size characteristic for the species is reached. When
the environment is unfavorable, the growth is restricted. In favorable
cultures two types of growth are found. In certain species, e.g., Epzstylis
arenicolae and E. Perrieri, the first divisions are dichotomous and equal,
and the mass growth of the colony follows in geometrical progression.
Later, the sister cells divide unequally, and the growth becomes arith-

GROWTH 527

metical, until the final size of the colony is reached. The time interval
between cell divisions also gradually increases.

The growth of Zodthamnium alternans depends on the common
stalk, according to Fauré-Fremiet (1925). The cell initiating the colony
has a given component of granular material which is distributed un-
equally during division and, as it is used up, the growth of the colony
ceases through transformation of material into the stalk. This may be
analogous to the accumulation of nonliving material in multicellular
organisms, a phenomenon which some biologists believe results in the
limited growth of such organisms. The growth curves are sigmoid
(Fauré-Fremiet, 1925, 1930), and the growth is believed to be auto-
catalyzed by the granular material.

PEDIGREE ISOLATION CULTURE AND LIFE CYCLES

The culture of a single cell in a drop of a suitable food and the iso-
lation of one of the divided cells, shortly after division, into a new drop
of medium, effectively maintains the environment constant, and the
growth is then potentially unlimited. With a constant medium and suit-
able bacterial food, the rate of growth is very nearly constant, as has
been demonstrated by Woodruff and Baitsell (1911), Darby (1930,
1930a, 1930b), Beers (1929), and others. If the growth curve for the
sum of the individuals be plotted on arithlog paper, a straight line will
result, because the growth (7) is exponential, y == yoe*t, when o is the
amount of the seeding, or the growth at time, ¢ — O and e, is the
Naperian base. The proportionality constant k — (/n 2)/G.T., or
0.639/G.T. The generation time (G.T.) is the time between divisions.
Tabel 5 summarizes the growth rates under fairly constant conditions for
certain Protozoa.

The study of the variation in cell division has been obscured by the
unfortunate practice of plotting the division rates in the form of a histo-
gram. The bars of the plot commonly give the average division rate for
ten-day periods. The histogram is used properly to show unit events
which have no intermediate values, e.g., the results of tossing dice
which cannot assume a position intermediate between two of the num-
bered sides. The average division rate is no such discrete, mutually ex-
clusive attribute, but may take any fractional value within the limits of
the experiment. An example showing how information on the division

528

GROWTH

TABLE 5. DIVISION RATES OF PROTOZOA WITH CONSTANT CONDITIONS

Reference

Organism Divisions AG Remarks
Chilomonas 3.5/day 24
paramecium o.14/hour 26-30.5 | NaAc-mineral salts
Didinium nasutum | 3.6/day 2I Fed on Paramecia
Euglena gracilis RO was 10 In dark no NaAc
0.47" 23 In dark with NaAc
3.5 day 25 In wheat infusion
Glaucoma pyriformis | 6.86/day 24.2—| Yeast extract
7.65 to 25.2 | Whole yeast+ yeast
8.02/day extract or peptone
5.62/day
Paramecium aurelia | 0.72/day 20+ Lettuce and bacteria
1.2+/day
1.4/day 26.8
2.02/day 28
Paramecium 2.1/day 25? Over 200 days
caudatum 1.8/day 25-28 | 51-day av. Min. salt
+B. subtilis
2.3/day 26 Oaten medium-+bac-
teria
Polytoma uvella 4.4/day 22 Aérated peptone me-
dium
1.85/day 22 Unaérated peptone me-
dium
Stentor coeruleus 2.1-0.7/hour | 18-20 | Modified Peters’ me-
dium-+ ciliates
0.65 /hour 22 Hetherington medium
+Blepharisma
Stylonychia pustulata| 4.5-5/day 25?
3.2/day 24
.7/da Di 2}
3.7/day 5 Re

* Divisions per day, per organism.

From Darby (1930a).

Mast and Pace (1934)
Smith (1938)

Beers (1929)
Jahn (1935)
Sweet (1939)

Hetherington (1936)

Phelps (1934)

Phelps (1934)
Woodruff and Baitsell

(1911a)

Phelps (1934)

Darby (1930b)

W.H. Johnson (1936)
Gause (1934)

Rottier (1936)

Hetherington (1932)
Gerstein (1937)
Darby (1930)

Baitsell (1912) T
Maupas

rate may be obscured by incorrect histogram plots was given by Richards
and Dawson (1927). The changes in division rate may be plotted to
advantage as a running average. The three-day running average is readily
Gal@ulated: slit xn ens oo ee
for the first is ¥, — (2x, ++ x,) /3; the second, x, = (x, + x, - x3) /3;

. X» are the daily rates for ” days, the value

GROWTH 529

and the last is (x,-1-+ 2x,)/3. If further smoothing is desired, a
five-day instead of a three-day running average may be used, and it is
computed in a corresponding manner. Phelps (1934) gives an example
of the running average plot.

In many of the earlier studies, the culture medium was inadequate, and
after a time the division rate approached zero. Unless the animals were
transferred to a favorable medium, the strain then died out. Such a
growth period has been termed a “cycle” by Calkins. During a cycle,
or, with some Protozoa during periods of nearly constant growth, small
fluctuations in the growth rate occur. These minor variations are termed
“rhythms” (Woodruff, 1905; Woodruff and Baitsell, 1911). Rhythms
are associated with cellular reorganization (endomixis). The constant
culture of Didinium nasutum without rhythms led Beers (1928) to be-
lieve that rhythms were due to food, temperature, and the condition of the
culture medium. Rhythms may have a function in some species and be
merely effects of the environment in others.

That the Calkins cycle depends on the adequacy of the culture medium
has been demonstrated by a number of experimenters, e.g., Woodruff
and Baitsell (1911a), Mast (1917), Beers (1928b), Darby (1930a),
and Gerstein (1937). A medium that may be adequate for a few weeks
may not be suitable for long periods. Dawson’s Paramecium and Ble pha-
v7sma showed gradual negative trends during the three years of the cul-
ture. At this rate the cycle would not end for several years (Richards
and Dawson, 1927) and, in the meantime, some slight change in the
medium might reverse the trend by supplying the cultural inadequacy,
thus prolonging the cycle. Competition may bring out more rapidly the
effects of the environment with populations than with individuals. The
study of Protozoa, maintained for some time in an effectively constant
culture medium, should add materially to our knowledge of growth.
Peters (1901) gives useful methods; yeast techniques are summarized
by Richards (1934).

Dawson kept pedigreed isolation cultures of Histrio com planatus,
Blepharisma undulans, and a mutant P. avrelia for three years. A sta-
tistical analysis of the division rates removed the long-time trends, and
established a seasonal cycle, with a maximum division rate in the sum-
mer and a minimum rate in winter (Richards and Dawson, 1927). The
statistical methods used were those used in economics in the study of

530 GROWTH

cyclic phenomena. Further study suggested that the seasonal cycle was
associated with sunlight (Richards, 1929). The pigmented Blepharisma
followed, more closely than the others, the seasonal variation of radiant
energy. A recent graphic method of Spurr (1937) could be used to
advantage in the analysis of seasonal cycles in the division rate of Pro-
tozoa. Properly controlled studies should be made to determine just how
much effect light has, over a considerable period of time, on the growth
of Protozoa. Such seasonal effect appears reasonable, as it is known that
the reproductive cycles of some birds and other animals are initiated
by the increased amount of light during the early part of the year.

Conjugation restricts variation, which aids in survival during ad-
verse conditions, according to Pearl (1907). Endomixis occasioned
large variations in size, which Erdmann (1920) believed aided in sur-
vival. She advised that attempts at selection be made during or immedi-
ately after endomixis. Changes that aid in survival of a species through
an unfavorable period are important in population studies; they might
even effect the growth of the individual, and they deserve further in-
vestigation by protozodlogists. Selection of rapidly dividing Amoeba
proteus by Halsey (1936) did not produce a permanent race of rapidly
dividing individuals. Burnside (1929) failed to change the size of
Stentor coeruleus by fragmenting animals with large and small amounts
of nuclear material. When the animals regenerated, the regulatory pro-
cesses produced the same sized biotype.

Variations in the cell, at the time of division or during periods of
intercellular reorganization, may aid in the adaptation of the cell to a
new environment and may account for the success or failure of investi-
gators in acclimating an organism to life in a synthetic liquid culture
medium. Hegerty (1939) has shown that young Streptococcus lactis,
at the end of the lag period and just before the period of logarithmic
growth, can produce new enzymes which permit the use of a new sub-
strate, to which the bacteria could not adapt themselves at any other
period of their life cycle. Do comparable changes occur in Protozoa?
If so, pure culture methods would be facilitated.

Thus the nature of the life cycles, as demonstrated by the earlier in-
vestigators (M. Robertson, 1929), may now be studied effectively by
physiological methods, as well as by post-mortem cytology. The de-
tailed discussion of reproduction must be left for consideration in the

GROWTH 531

other chapters. The critical periods of binary fission, conjugation, and
intercellular reorganization are important to the study of growth, and
further information on these phenomena will facilitate our understand-
ing of growth.

PROTOZOAN SUCCESSIONS: NONLABORATORY

The variety of Protozoa and the numbers of each vary in time, and
the abundance of individuals is usually inversely correlated with the
diversity of kinds. At Geneva, Roux (1901) found the largest variety
of species in January and in October and found that in the same loca-
tions there was considerable variation at the corresponding time in two
successive years.

The sequence of Protozoa on sewage filtration beds (New Jersey)
was followed by Crozier (1923) and Crozier and Harris (1923). A
maximum number of rhizopods was found in August and of ciliates in
May-June and November-December. Paramecium had a sharp maxi-
mum in December-January, Vorticella in late December and in May,
and Co/poda in the first third of the year. The sequence was attributed
to the amount of anaérobiosis and to the formation and sloughing of
the film. In this environment the abundance was directly correlated with
the diversity of types.

Noland (1925) found the sequence of Protozoa related to the tem-
perature, oxygen, and carbon-dioxide concentrations in natural ponds.
The hydrogen-ion concentration was not believed to be a controlling
factor. Most of the Protozoa found were not those usually studied in the
laboratory, but when samples were transferred to the laboratory, Col-
poda cucullus, Glaucoma pyriformis, and Paramecium caudatum ap-
peared, showing that these animals may thrive better in the laboratory
than in natural habitats.

Changes in the concentration of Protozoa in a Philadelphia pond
were followed for a year by Wang (1928), who measured also the
temperature, oxygen concentration, pH, and relative amount of dis-
solved gases. The surface forms showed the greatest variation, which
was believed due to the dissolved oxygen, depending on temperature
and on the activity of the plants. A marked increase of acidity could be
a limiting condition. The maximum number of forms was found in
September-October. Since the amount of sunlight was greatest at this

532 GROWTH

time and the temperature was not at a maximum, Richards (1929)
has suggested that sunlight may have had more effect than temperature
on numbers. The kinds of Mastigophora and Infusoria were inversely
correlated with the abundance of individuals during the seasonal varia-
tions.

Coe (1932) found Protozoa attached to cement blocks suspended in
the Pacific Ocean at La Jolla, from June to October. Protozoan sequences
and numbers have been used by Lackey (1938a) for the study of sewage
pollution of streams.

Sufficient information is not yet available to explain the sequence of
protozoan population growths, or the declines and succession by other
species in nature. Many of the factors are interrelated, since the solubility
of dissolved gases is a function of temperature, and the oxygen produc-
tion of aquatic plants depends on the amount of light. The solution of
these ecological problems promises to be of considerable practical value
to man, as well as an aid in the elucidation of the growth processes.

PROTOZOAN SUCCESSIONS: LABORATORY

Cultures maintained in the laboratory are more readily followed than
those in natural habitats, and there are many records of the growth of
populations of Protozoa, their decline and succession by a compar-
able growth of another species. Woodruff (1912) reported that near
the surface of mass cultures the sequence was monads, Colpoda,
Hypotrichida, Paramecium, Vorticella, and then Amoeba. The sequence
of increase and disappearance was identical with appearance, except that
the Amoeba advanced from the sixth to the fifth, and then to the fourth
place. A definite succession was not apparent at the center or the bottom
of the cultures, and a second cycle was rarely observed. The maximum
rise and fall was about equal, but the final disappearance might be long
delayed. The differences in the relative potential of division were be-
lieved to establish the sequence, which was determined by the food and
waste products secreted by the animals. The waste products were shown
to be toxic, and the toxicity was species-specific and did not effect other
species (Woodruff, 1913b). No relation was found between the titrable
acidity and the sequence of the Protozoa (Fine, 1912). The acidity
was related, rather, to the activities of the bacteria present.

Fifteen series of two-liter cultures, made to imitate natural conditions,

GROWTH 533

were followed by Eddy (1928). Counts were made with a Sedgewick-
Rafter cell, but the results were not published, beyond general statements
of sequence and dominance. Light had no effect on the sequence. Tem-
perature exerts its influence by way of the bacteria serving as food for
the Protozoa. Oxygenation of the culture increased the growth, espe-
cially at the bottom of the culture. Too great concentrations of carbon
dioxide were deleterious and could be buffered by including soil in the
culture. The sequence was effected by the quantity and type of the in-
fusion material. Dominance of a species was believed to depend on favor-
able growing conditions for that species, rather than on the rate of re-
production (cf. Woodruff, above).

Unger (1931) has listed the sequence of Protozoa for two years in
five cultures started from five different plants.

Laboratory cultures, not restricted to a single species, show a regular
series of population growths and declines for different species. The na-
ture of the culture, its bacterial flora, and the reproductive potential of
each species regulate the period of intensive growth, and the accumulat-
ing excretion products of the animals bring about the decline of the
population. The growth cycle of a species may modify the medium so
that it becomes favorable for the growth of the next following species.
Limiting conditions are oxygen and carbon-dioxide concentrations, pH,
and temperature, and these will be discussed later. Remarkable flower-
ings of Algae and Protozoa in the ocean and in lakes have been re-
ported and are apparently on a more intense scale than occurs in labora-
tory cultures. Some bacteria are inadequate as food sources; others are
poisonous for some Protozoa; and the rise of a population of these
bacteria would eliminate the susceptible Protozoa in the culture. Poisonous
bacteria have been reported by Hargitt and Fray (1917) and by Kidder
and Stuart (1938). The Protozoa commonly studied in the laboratory
are apparently less frequently found in natural habitats. The growth of
protozoan populations in mixed mass cultures is different from that of
most other organisms, as no equilibrium is reached and maintained;
instead, extinction seems to be the rule.

AUTOCATALYSIS AND ALLELOCATALYSIS

The theories of T. B. Robertson have greatly influenced the study
of growth, and the first of these has been concerned primarily with the

534 GROWTH

growth of Protozoa. Robertson (1923) believed that the growth of an
organism, or of a population of organisms, was awtocatalytic, because the
growth curves were sigmoid and could be fitted by the equation for a
monomolecular, autocatalytic, chemical reaction. The slowest chemical
reaction in the growth process was believed by Robertson to be the con-
trolling master reaction for the process which established the form of the
growth curve, and this could be discovered from the shape of the
growth curve. His particular choice of chemical reaction was not sat-
isfactory, and later he and other investigators have found difficulties
which have, for the most part, led to the abandonment of the autocata-
lytic theory. Cf. Robertson (1923), Snell (1929), Jahn (1930), Kava-
nagh and Richards (1934).

The sigmoid nature of the growth curve is the inevitable result of the
regular geometrical increase during the time that the environment 1s
favorable, and the slowing of this increase when the environment be-
comes unfavorable as a result of the growth in it (decrease of foodstuffs
and accumulation of excretion products). As long as the environment 1s
maintained effectively constant, the rate of growth is constant and the
growth curve is exponential. However, it eventually becomes impossible
to maintain this constancy, and the growth is thus ultimately arrested.
In this sense Bernard’s “milieu interieur’’ is part of the environment.
The granular material which Fauré-Fremiet believes to limit the growth
of some colonial Protozoa is one of the few reported examples of limita-
tion in growth which apparently follows the appearance of a single
substance. Such a substance might be considered a catalyst in the Robertson
sense. Teissier (1937) has questioned this conclusion, and Snell’s (1929)
objections are also applicable. Such substances, however, are rare.

Allelocatalysis, according to Robertson (1924a), is “‘the acceleration
of multiplication by the contiguity of a second organism in a restricted
volume of medium.’ Robertson reported (1921b) that two Enchelys
farcimen, ot two Col pidium colpoda in a drop of culture medium divided
more rapidly than twice the division rate of one individual in an en-
vironment of equal volume. It was shown later that his Colpidium was
Colpoda cucullus. Other publications followed, reporting that some un-
known substance, the allelocatalyst, stimulated cell division, and Robert-
son believed this was formed during nuclear division and effected the
permeability of the cells.

GROWTH 535

Cutler and Crump (1923), using Colpidium, were unable to con-
firm Robertson; and Greenleaf (1924) failed to demonstrate allelocataly-
sis with Paramecium aurelia and P. caudatum, and with Pleurotricha
lanceolata. Peskett (1924) could not demonstrate allelocatalysis with
yeast. Robertson (1924) attributed their failures to the fact that they
had not washed their cultures free from the catalyst present in the me-
dium from which the cells were removed for inoculation. Cutler and
Crump (1925) and Peskett (1925) repeated their work, but were un-
able to demonstrate allelocatalysis either with washed or unwashed cul-
tures.

Yocom (1928) found the division rate of Oxytricha higher in cul-
tures of four drops of medium than in ten-drop cultures, and attributed
the difference to an allelocatalyst. Petersen (1929) found that division
of P. caudatum was accelerated in volumes of culture of 0.83 ml., but
not in volumes of less than 0.21 ml. Dimitrowa (1932) obtained better
growth in “conditioned” medium which had previously supported the
growth of Paramecium than in medium which had not been “‘condi-
tioned.” Colpidium campylum grew better when some sterile filtrate
from an old culture was added to a synthetic medium, according to Hall
and Loefer (1938). Garrod (1936) reported that small inocula of
Staphylococcus aureus did not grow in broth, but that large inoculations
would grow. Mast and Pace (1937, 1938b) give evidence in support of
an unknown substance produced in cultures, which, in low concentra-
tions, stimulates the growth of Chilomonas paramecium, but which in
high concentrations retards the growth of the animals. A soil amoeba,
Mayorella, grown bacteria-free in mass cultures by Reich (1938), di-
vided less when the initial populations were small. His data, replotted
in the form of Figure 134, shows that the populations were proportionate
to the seeding in rate of growth, within the large errors of observation,
and do not support the allelocatalytic theory.

Yeast populations grew at the same rate when the inoculation was
varied from 5 to 8 & 10° cells per ml. (Clark, 1922); and from 12 to
1,200 cells per cu. mm. (Richards, 1932). Peskett (1927) found no
difference when one yeast cell was introduced into volumes from 0.008
to 40 cu. mm. Meyers (1927) failed to demonstrate allelocatalysis with
P. caudatum and found that conditioning the medium lessened the
growth of the animals. Increasing inoculations of Glaucoma up to

536 GROWTH

70,000 times gave no allelocatalysis (Phelps, 1935). Darby (1930)
maintained that allelocatalytic effects were due to the pH of the medium,
and Jahn (1933) believed them due to the oxidation-reduction poising
of the medium. When the medium was optimum, there would be no
increased rate of reproduction; but if the medium was suboptimum,
two or more organisms might modify it enough to permit growth whereas
one organism could not do so and would grow slowly or fail to survive.

Johnson and Hardin (1938) reported that medium conditioned by
the growth of Psewdomonas fluorescens inhibits the reproduction of
Paramecium micronucleatum. With the saline medium, used old-cul-
ture medium was as efficient as fresh medium. The difference between
these and Woodruff’s conclusions may be due to the effects of mixed
bacteria in the natural medium used by Woodruff. Kidder (1939) stud-
ied the effect of conditioning with a bacteria-free Colpidium campylum
culture in proteose-peptone, dextrose broth. He believes that there is an
accelerator and an inhibitor in the conditioned medium for growth.
These were separated by absorption and filtration. Caution should be
exercised in the use of filtered media, as some kinds of filters make the
filtrate toxic (Richards, 1933).

Sweet (1939) reinvestigated the volume seeding relation, using Ew-
glena gracilis, and found that seedings of one and two individuals grew
better in four drops of about 0.05 ml. each and inoculations of four and
eight individuals in slightly larger, five-drop environments. This au-
thor’s methods and technique illustrate survival, rather than growth,
and while a volume effect of the environment was found, her results
did not support the Robertson theories.

The observations of these investigators and others focused attention
on the suitability of the culture medium and suggested that the allelo-
catalytic effects found by some biologists and discredited by others might
be explained on this basis. Woodruff’s (1911) demonstration that the
waste products limited growth was recalled and clarified some of the
volume effects on growth, wherein the yield of cells depended on the
volume of the culture medium rather than on the size of the inocula-
tion. Johnson (1933) explained allelocatalytic effects on the relation
of the bacterial food concentration to the number of Protozoa in the
culture. An allelocatalytic effect on P. caudatum and on Moina macro-
copa was found with a high nutrient concentration, and the reverse of

GROWTH 5), 7

this with media of low nutrient concentrations (McPherson, Smith, and
Banta, 1932).

Another possible interpretation depends on the presence or absence
of essential elements, both organic and inorganic, or on vitamin or
hormone-like effects. This field has hardly been touched, and investi-
gations here may clear up many problems concerning the nutritional
requirements and the responses of the organisms to various culture
fluids. The present tendency is to look in this direction for an under-
standing of variations in the reproductive rate, rather than to attribute
them to special allelocatalysts. Cf. Elliott, (1936), Hammond (1938),
Koser and Saunders (1938), Hall (1939), and other chapters of this
book.

Another explanation of the effect of the volume of the culture on the
reproduction rate of the organisms might be that in larger volumes the
organisms use more energy swimming about, which would leave less
energy for reproduction. This view could be tested by the use of cine-
photomicrographic films in measuring the amount of activity of animals
in large and small isolation cultures, and correlating this figure with
the rate of multiplication. The relation might be different in rich and
in poor nutrient media and, if so, this would elucidate some of the
contradictory observations in the literature.

NUTRITION AND GROWTH

Protozoa (Ciliophora) feed naturally on bacteria, and with mixed
population of both it is difficult to analyze the growth. Maupas recog-
nized this difficulty in the nineteenth century and recommended that
Pasteur’s methods be applied to the pure culturing of Protozoa. How-
ever, for some time little was done, other than to insure a uniform and
adequate supply of bacteria in the medium by cross culturing.

Hargitt and Fray (1917) isolated and identified a number of bacteria
from protozoan cultures and endeavored to grow Paramecium on pute
cultures of bacteria, but found that no single species of bacteria was as
satisfactory food as mixed cultures. Bacillus subtilis was the nearest satis-
factory single species. Some species of bacteria were found to be toxic
to the paramecia, and other poisonous bacteria have been reported by
Kidder and Stuart (1938). Phillips (1922) extended the work of Har-
gitt and Fray and was unable to find a single species of bacteria suit-

538 GROWTH

able for the maintenance of P. aurelia. She concluded also that the
paramecia could not live on dissolved substances, but were dependent
on particulate food. Glaucoma ficaria was grown on a number of single
species of bacteria, yeast, and flagellates by D. E. Johnson (1936).
B. prodigiosus was the most satisfactory food organism. The results
depended largely on the food being small enough for ingestion.

Recent studies have been directed toward determining the food ele-
ments required by Protozoa and toward devising synthetic media in which
the Protozoa could be grown in bacteria-free, pure cultures. While it
it not possible to separate studies on growth and nutrition except for con-
venience, this chapter will be limited to studies occupied primarily with
the analysis of growth. The broad problem of nutrition will be covered
elsewhere. Different species have different nutritional requirements,
and the failure of some protozodlogists to realize this fully has led to
confusion in the literature on growth. Very few data are available which
give the growth of the bacteria, as well as that of the Protozoa, present
in mixed cultures. Considerably more labor would be involved in secur-
ing this information, but the methods have been worked out and the
information gained would justify the work. It is now possible to grow
pure cultures of a variety of Protozoa in bacteria-free synthetic media.
Some of the nutrient conditions limiting growth will be examined briefly.

Tolerance to changes of osmotic pressure was found by Loefer (1938),
in attempts to adapt fresh-water Protozoa to artificial sea water, to be
limited. Yocom (1934) was more successful. Loefer (1939) found
that tolerance to diluted Van Hoff solution developed over several
generations. Changes in the oxidation-reduction potential have been
measured in Chilomonas paramecium caltures by Jahn (1933), and his
results suggest that when the medium is poised at the optimum rH,
growth will be most rapid.

The increased growth of Protozoa at the surface of mass cultures
shows their sensitivity to oxygen. Aération will often extend the growth
to deeper levels. Inadequate amounts of oxygen limit the growth of
Polytoma uvella, and sufficient oxygen must be provided before the
effects of other nutrients may be evaluated (Rottier, 1936; Mond,
1937). Reich (1936) believes oxygen concentration more important
in Amoeba cultures than acidity. Jahn (1936) aérated bacterta-
free cultures of Glaucoma pyriformis and Chilomonas paramecium in
a hydrolyzed casein medium with air, and air freed of carbon dioxide.

GROWTH De)

The Chilomonas grew best in unaérated cultures and not so well in the
cultures aérated with CO, free air. The Glaucoma grew equally well
with and without CO,, but better than in unaérated cultures. Jahn believes
that CO, is necessary to some organisms to avert the weakening of the
buffer systems within the cell. The anaérobes are believed less sensitive
to CO, removal because the amino acids and other weak acids may re-
place the carbonic acid. The lag period in bacterial growth varies with
the CO, concentration (Walker, 1932), and increased production is
associated with physiological changes in the bacterial cells (Huntington
and Winslow, 1937; Gladstone et al., 1935). Similar effects should be
watched for in protozoan populations.

Temperature has long been known to affect growth. Woodruff and
Baitsell (1911b) found that the Q,, for the cell division of P. aurelia
was 2.7, over a range of 21.5° to 31.5° C., and that the optimum range
for them was 24° to 28.5° C. Individual pedigree cultures and mass cul-
tures were measured by Mitchell (1929) over a range of 12° to 27° C,,
and the thermal increment (1) for cell division was found to be 23,000
calories. A lag was found in the isolation cultures, and a method is given
for calculating the division rates from data covering several days. Pos-
sibly with a different culture medium the lag might have been avoided
or changed. Daniel and Chalkley (1933) found y to equal 16,500 for
the whole division process of Amoeba proteus (4° to 30° C). For nu-
clear division ,, equals 16,600 (4° to 35° C.); for cytoplasmic division,
20,500" (11° to 20°C )R75300 {21° to. 26° C:); prophase 11,700, and
anaphase 20,200 (13° to 26° C.). The increments suggest that oxida-
tive processes control cell division.

Jahn (1935) found a maximum growth rate for Euglena gracilzs,
grown in a hydrolyzed casein medium at 10° C., but the addition of
sodium acetate changed the temperature of maximum growth to 23°.
Motility and the occurrence of encystment and palmella stages were
related to the temperature and food. Smith (1938) reported that CAz/o-
monas paramecium grew in a sodium acetate-mineral salts medium from
9.5° to 35° C., with an optimum range of 26° to 30.5° C. Prolonged
exposure to the lower temperatures decreased the resistance of the ani-
mals to the cold. Adaptation to changed temperature required at least
forty-eight hours. The synthesis of fat and starch is a result of tempera-
ture and in turn may control the division rate.

The chlorophyll-containing Protozoa vary in their light requirements.

(S61) uosuyof Qt auojdAry +qx4 +9ywy Aa 6°9-0'F stusofiskd pumompjs

($€61) uosuyof Qt 2uojdAIE+qA+sw | 4:9 puer’$| 6°g-0'F piuvoay puomnn] sy

(9£61) uoqBuTIeyIAPY St-¥t dt+qa ol siusofiukd puomps

(9t61) ryanig £°g-0'9 ‘ds pjkasousp5

(161) uyel 9°9 ydo awAzuq S1jt9p43 Duasng

({L61) Te £°6z di 10 ax ok g LoS sasap puajsng

(€€61) Tey $°6z d 10 FJA+SW g°9 o'g-S°S Duapqvun Duajsng

(4t61) sizag 96-0°S ‘ds wmuipiq

{ (4261) rar0yy S-Lpues:g| $:6-°S snqjnond ppodjor

{ (2861) sq o'9 o'g-S'b "ds wntpidjop

(961) ryanig — $°g-0°9 ‘ds wniptdjon

(961) ONTW Sz auojdAry, | ¢-Lpueg'S| 9°g-o0'F winqpt4as wnip1djor

J2j207] 74s1] ut sinoy oL Qt sqyes auo0jd Ary 9°8 9'°g-'F wmniuosp43aq wWn1U0s0401Y4)

J2j207] 34SI] ur sinoy oL Qt sqyes auojd Ary GL 9°g-+ + wnojyona wn1w0s040]4

(ogt6r1) 12J20T 1ST] ur sinoy o4 Qt sqyes auoyd Ary SL 9°g'F wnqpsu0}2 wmtuoso0.01YyD)

(gt61) a0eg pure yseyy KE 33e}908 Sz]es MUesIOUT 8°9 0°8-g'F wumniaupsvd spuowof1y

(9t61) tyanig ¢°4-1°4 £°L-9°9 ‘ds snadapiyduy
aouasafary syapmay eeu xUNipayy wnuidd aBuny wWs1UD34C)
VOZOLOUd JO HLMOUD AHL NO NOILVYLNADNOD NO]-Nd90NdAH AO LOPAAY AHL “9 ATAavVEe

(9t61) A10s215
(ot61) Aqieq
{(6z61) Aqieq
(<t61) u0jSUTISYIZ}Y

{(¥z61) s1apuneg
{(4z61) earoyy

4(9t6r) tyanag

| (0%61) souof
(ot61) Aqieq

(F<61 ‘1€61) sdjayg
| (0t61 ‘6z61) Agieq
(¥z61) siapuneg
(9t61) ry3nIg

4(9t61) tyynIg

qurzIOd wt O17e1 yeO

wnu1jdo ou
2]qesoary asury

“(C€61) uosuyof worry 4

OT-QI

‘jAduieo
‘I-+S12}2g PIyIpoW

‘quojdad=q ‘y9vI}x9 ysvaA =X ‘ses [eIOUIW—SW x

gh
0°g pue Lg

mt
Tose Jo

ome)
is eS

LL-6"S
ok

0'g-9°L

0°g-0'9

¥-L-S-9

syigow snjqajo1qQ

pipjnasnd piyskuojKag

snajn41309 10131

wnnsiqup wnwojsoudg
‘ds wnwojsouds

‘ds vjXdoisv)g
PIVINUOANUUINW WNIIIULDADY
wn PpNvyI wmnidIWPLDg

DY2INY wnNiDaUP4VY

‘ds wn1.aupivg

‘ds pakydojoyy

542 GROWTH

Euglena gracilis can be grown in the dark for extended periods. Without
light, some of the organisms require more complicated food substrates,
and the experiments demonstrating this have been summarized by Hall
(1939). The amount of sunlight may exert a seasonal effect on the
division rate of Protozoa (Richards, 1929). Reflected light stimulated
multiplication of Paramecium caudatum in the red, but had a depres-
sing action in the violet, according to Zhalkovskii (1938). Filtered,
transmitted light had a greater depressing effect than reflected light.
The difference was believed to be due to the polarizing effect of the re-
flected light. Heritable changes in the size and form of Chilodon un-
cinatus have been produced by McDougall (1929) with ultra-violet
radiation. Giese (1939) found that ultra-violet of 2,654A injured the
nuclear material of P. caudatum and that the damage was less readily
repaired than was the damage to the cytoplasm caused by 2,804A.

The ease with which the acidity of the culture medium may be meas-
ured is responsible for a considerable volume of information (Table 6).
The early measurements of Peters (1904, 1907) and Fine (1912) were
made by titration. The advent of simple methods for the measurement of
the hydrogen-ion concentration was welcomed by the protozodlogists, and
Bodine (1917) related the old and the new methods. Pruthi (1926)
found a sequence in hay infusion of Holphyra, Plagiopyla, Col pidium,
Am phileptus, and Paramecium. The first two do not persist beyond pH 7.5
and the paramecia did not appear before the pH reached 7.0. Mass cultures
and some synthetic media change during the growth of the organisms, and
the change in pH of mixed cultures is probably more the result of bacterial
action than that of the Protozoa. Eddy (1928) believed that the changes
in pH were not of importance in themselves, but rather the result of other
effects. Phelps (1931) attributed the changes to the food supply, and
Johnson (1935,1936) stressed the effect of bacterial action. The ac-
celerative effects of stimulants depend on the acidity of the medium,
and there now seems no question but that there is an optimum pH range
for different media and Protozoa, and that beyond the optimum range
growth is less and may be entirely inhibited. Elliott (1935b) found that
sodium acetate stimulated Colpidium at pH 6.8-7.5, and butyrate at a
pH less than 7.0. The size of P. bursaria has been shown by Loefer
(1938a) to depend on the acidity of the tryptone and proteosepeptone
culture media.

GROWTH 543

A usable source of C, H, O, N, Ca, K, P, and Na is probably required
by all Protozoa. Inorganic media have been used by Hall and Schoen-
born (1938) to separate strains of flagellates, by choosing media in
which one strain will survive and others perish. Some Protozoa can ob-
tain nitrogen from nitrates or ammonium salts, while others require
amino acids, proteoses, or peptones. Sodium acetate, glycerate, or glyc-
erophosphate are among the simplest carbon sources required by the
nonphotosynthetic organisms. Loefer (1935) has summarized the carbo-
hydrate requirements. The growth of Chilomonas paramecium requires
sodium acetate, magnesium, sulphur, and silicon (Mast and Pace, 1933),
and vanadium and copper increase the rate of growth (Bowen, 1938).
Colpidium needs phosphate and a minimum three-carbon source, ac-
cording to Peters (1920). Potassium and magnesium may be omitted
from glass cultures, but are required when quartz vesseis are used.
Uranium salts cannot be substituted for potassium (Peters, 1921). The
addition of pimelic acid to a glycerine-dextrose medium permitted growth
of Colpidium (Hall, 1938b). Bacteria-free P. bursaria grew in propor-
tion to the concentration of the culture medium (Loefer, 1938d).

Polytoma grows better when aneurine (synthetic B,) and thiazol
compounds are present (Lwoff and Dusi, 1937, 1938); and trypano-
somes need hematin and cholesterol. Ameoba and Paramecium grow bet-
ter in the presence of sulfhydryl, and this may be a general requirement
of Protozoa. Hammett (1929) obtained an increased growth of Parame-
cium, although it was not proportional to the SH content. Hall (1938)
found that manganese stimulated the growth of E. anabaena, but failed
to stimulate Astasza sp. and Colpidium campylum.

Culture media have been improved by the addition of yeast extract
for Uroleptus, Dallasia, P. bursaria, Pleurotricha, and Stylonychia, and
Gregory (1925-28) found that the stimulation or depression of the
division rate of Uroleptus mobilis depended on the age of the culture.
Beef extract has proved a suitable food for mixed cultures. Plant hormones,
indoleacetic, indolebutyric, and indoleproprionic acids increase the
gtowth of chlorophyll-containing Protozoa, while pantothenic acid
stimulated those tested by Elliott (1935a, 1938) which did not have
chlorophyll. Mottram (1939) reported that 3 : 4 benzpyrene is a growth
stimulant for Paramecium.

Beers (1928a, 1928c) grew two parallel lines of Didinium nasutum

544 GROWTH

on well fed and on starved Paramecium, and showed that the inadequate
growth of the Didinium restricted to a diet of starved Paramecium was
due to a qualitative deficiency, rather than to a shortage of food. Mond
(1937) reported that Infusoria grown in known concentrations of
Bacillus coli and B. subtilis grow in a linear relation to the available
food. The same amounts of bacteria were used for each division of the
Infusoria. Such studies will permit the determination of the amounts
of energy used for the growth process and for maintenance of life. When
enough data become available, Wetzel’s (1937) methods may be used
and the resulting data would aid in evaluating his theory of growth.

The knowledge of the nutritional requirements of the Protozoa ts
increasing rapidly and suitable methods for growing bacteria-free pure
culture of a number of species are now available. It will be difficult to
decide what is the optimum culture medium for a given species. The
lack of trace elements may appear only after a period of years. Super-
optimal media will give an increased rate of growth which may not be
best for the species (McCay, 1933). Pearl’s (1928) generalization that
the length of life is inversely correlated with the rate of living must be
remembered when experimental conditions are devised either to yield a
maximum amount of Protozoa in a given time, or to provide an op-
portunity for the study and perpetuation of the species under the most
favorable conditions.

THE GROWTH OF POPULATION

Adequate measurement of population growth should include the fol-
lowing information, as well as the number of organisms present at a
given time, per unit of environment: food concentration; the concentra-
tion of excretion products, pH, rH, oxygen, and carbon-dioxide concen-
trations; temperature; the amount of light, when light-sensitive organ-
isms are used; and the effects of other species, when mixed populations
are used, on the species measured and on its environment. Few studies
approach this degree of completeness. The earlier studies made no attempt
to measure the food concentration, when bacteria were the main source
of organic food.

A few Protozoa inoculated into a limited amount of an adequate
culture medium, soon begin to increase in numbers and continue to do so
until a maximum yield is produced. The course of the population growth

GROWTH 545

may be divided into the following phases: (1) a stationary period, (2)
a lag period of increasing rate of growth, (3) a logarithmic period of
constant relative rate of growth, (4) a period of declining rate of growth,
(5) a period of equilibrium of numbers, and finally (6) a period of de-
clining numbers. The duration of these phases, and even the presence or
absence of some of them, depends on the age of the inoculation and the
nature of the environment. The stationary and the lag phases may be
eliminated when the inoculation has been taken from a culture during
the logarithmic period. Very large inoculations may exhaust the food
supply or make the environment toxic from the excretion products,
before any appreciable growth can take place.

The understanding of population-growth studies may be clarified by
the aid of a hypothetical example, Figure 133. If ten organisms were
seeded into a limited amount of a suitable culture medium from a
population in the logarithmic phase of population growth, they would
grow at a constant rate, doubling their number at the end of each
generation time (curve A). If the conditions of growth were identical,
the rate of growth of the inoculum would be the same as it was in the
parent population. After a time the environment will no longer be
effectively constant, and the rate of growth will decrease. This may come
about by the lapse of more time between generations (curve B), or by
only a part of the animals being capable of reproducing (curve C). It
is apparent that if there had been different periods of increasing genera-
tion time, or if different numbers had been permitted to reproduce, it
would have been possible to make curves B and C coincide at all points.
Therefore, it is not possible to decide from the shape of the growth
curve alone, the cause of the slowing of the growth rate. A third posst-
bility, which would give a curve of the same general shape, would be
a selective encystment or the death of some of the animals, which would
reduce the number of individuals capable of reproduction. Such con-
siderations emphasize again the necessity of information from the use of
more than one criterion for the analysis of growth.

The stationary or equilibrium period, when the population does not
change in numbers, is indicated by the curve D and the period of de-
clining population by curve E. The stationary period is usually a dynamic
equilibrium wherein the birth and death rates balance each other, but it
could be static if all of the cells encysted or became otherwise inactive.

NUMBER

vo)

Figure 133. Hypothetical curves to illustrate phases of population growth. Cf. text.

GROWTH 547

The period of decline will depend on the nature of the environment and
the rate of growth. It may show a phase of increasing death rate, a
logarithmic death rate, or a decreasing death rate, or these phases may
follow one another.

Had the inoculum been taken from an old culture which had reached
the equilibrium or the period of decline, then there might have been
a stationary period (F), followed by a period of increasing rate of
growth (G), which would be followed by a constant relative rate (H),
shown by the curve becoming parallel with the A curve. It is advan-
tageous to know and to take into account these phases, in experiments with
populations of unicellular organisms. The duration of the stationary
and the lag phases will vary with the age of the inoculum and the effect
of the previous unfavorable environment of them. Populations from
old cells often provide considerable variation. Whenever possible, expert-
ments should be made during the logarithmic period, to insure unt-
formity of material.

The detailed shape of the growth curve is often not known, because
of infrequent measurements. If the Protozoa divided synchronously at
the end of the generation time the curve would be like curve I.

The difference between the number of organisms in a population,
shown by curves B or C, and the number theoretically possible, shown
by the extension of curve A, is a measure of the inadequacy of the
culture medium. The difference between the expected maximal number,
curve D, and the number at a given time measures the potential growth
yet to be achieved. The environmental resistance may be expressed as one
minus (the potential growth divided by the expected number). This
type of analysis, in terms of the logistic equation, has been made by
Gause (1934) for the population growth of P. caudatum, and his in-
structive graph should be examined by all students of population growth.
For information on the mathematics of growth, Pearl (1925), Jahn
(1930), Richards and Kavanagh (1937) may be consulted. Protozodlo-
gists have not used mathematical methods to any extent. Park (1939)
also reviews Gause’s analysis. Similar growth studies of other protozoan
populations, besides presenting local data, should contribute to the general
understanding of growth.

Buchanan and Fulmer (1928) have reviewed the literature of the
growth of bacterial populations; Richards (1934) yeast populations;

548 GROWTH

Jahn (1934) protozoan populations; and for other animals, Pearl
(1925), Gray (1929), Gause (1932), Johnson (1937), Hammond
(1938), and Park (1939) may be consulted for reviews and bibliog-
raphy.

Jones (1928) followed the population growth of an inoculation of
200 P. multimicronucleatum in 70 cultures at 80° F., with counts of
0.5 ml. samples made periodically. The pH of the medium was also
measured. A maximum crop of 10° paramecia were obtained in 700 ml.
cultures. Growth stopped when the pH decreased to 5. With hay-flour
infusions, two cycles of growth were found (1930). The first was
terminated by the high acidity; when the acidity returned to about
pH 7, the second growth cycle commenced. During a three-day period
Jones (1937a) found that the number having died at the close of the
first growth cycle exceeded the maximum number present during the
second period. The death of the animals was believed to be due to toxic
excretion products, which were neutralized by the materials liberated from
the cytolysis of the dead animals. Death was apparently disruptive, as no
intact dead animals were observed. With large one-gallon cultures, the
decline of the populations was related to the decline of food; and, by
periodically renewing the food, the cultures could be maintained for
four years.

The growth of Evglena gracilis in mass cultures was used by Jahn
(1929) to test the allelocatalytic theory of Robertson. The organisms
were derived from a single cell isolation and grown in an autotrophic
mineral-salts medium, with temperature and light controlled. Jahn’s
larger inoculations gave a population growth with two cycles (Fig. 134).
No evidence for allelocatalysis was obtained. The relative rates of growth
were computed (Jahn, 1930) and found to give a decreasing sigmoid
curve. Jahn emphasized the difference between the absolute rate of
growth (dy/dt) of the total number and the relative rate of growth
(dy/ydt), or division rate, of the organisms, without entering into the
discussion of the relative growth rate as such.

Phelps (1935) measured the population growth of bacteria-free G.
pyriformis in 700 ml. cultures of a mineral salts-yeast extract medium in
one-liter flasks. The length of the stationary and the lag phases were
proportional to the age of the seeding, and seeding from populations
in the logarithmic phase gave no stationary or lag phases. Increasing

4
sak >
Xe ayeaX—-K2 -

NUMBER _IN 0.5 ML.

NUMBER..

Figure 134. A. Population growth curves plotted on arithlog coordinates of Exglena
(E, and E,) from Jahn (1929) ; Paramecium aurelia (PA), P. caudatum (PC), Stylony-
chia pustulata (SP) from Gause (1934); and M. Mayorella palestinensis from Reich
(1938). B. Population growth curves plotted on Cartesian coérdinates (same data).

550 GROWTH

initial densities up to 70,000 times failed to show any allelocatalytic
effect. The number of animals present at the end of the logarithmic
phase was independent of the number in the seeding. In comparing
the phases of Glaucoma population growth with those of bacterial and
yeast populations, he found the following differences: the initial station-
ary and lag phases in G/awcoma populations are much shorter, in propor-
tion to the optimum generation time; the stationary phase is independent
of the size of the seeding; and the change from the logarithmic to the
equilibrium phase of growth is more abrupt. No period of decreasing
population size appeared within 120 hours.

Changing from yeast extract to yeast autolysate increased the yield
(Phelps, 1936). The rate of growth was found to be independent of the
food concentration within wide limits, but the total number of animals
was proportional to the amount of food. The concentration of excretion
products did not inhibit the growth until very great population densities
were reached. This again is quite different from yeast cells, which are
adversely effected by low amounts of excretion and fermentation prod-
ucts. A more favorable food medium and the use of aération flasks,
as well as differences in the species of animals used, may account for
the lesser effect of waste products observed by Phelps than by Wood-
ruff (1911). The G. pyriformis used by Phelps is identified now as
Tetrahymena glaucomiforma.

The growth of populations of Colpidium campylum was measured
by Bond (1933). With small amounts of yeast autolysate, the growth
was slight and the lag period was greatly prolonged. With greater
amounts of food, the equilibrium population was greater, the logarithmic
phase was longer, the rate of growth greater, and the transition from
the logarithmic phase to the equilibrium phase of the growth curve less
abrupt. Bond’s evidence suggests that the yield of animals depends
more on the amount of food available than on an inhibitory effect of
excretion products.

Gause (1934) presented the growth of a population of P. caudatum
on an oatmeal infusion, with bacteria. He fitted the S-shaped growth
curve with the logistic equation, and his analysis of the curve has been
mentioned before. The growth curve of Stylonychia pustulata, Fig. 134,
illustrates rapid growth, with a short equilibrium phase, followed by
a period of negative growth leading to a lower equilibrium level. The

GROWTH Ba!

second equilibrium level decreased slightly from the eighth day to the
sixteenth day, when a second and shorter growth cycle commenced. The
second cycle passed through a brief equilibrium period and then de-
clined to about the same level as that which followed the first growth
cycle. Population growth curves are given for S. mytdllus, P. aurelia and,
in a later monograph (1935), for Glaucoma scintilans, Didinium nasu-
tum, Bursaria truncatella, and P. bursaria. Some of these will be dis-
cussed in the next section. One set of data is interesting from the view-
point of population growth, that for P. awrelia and P. caudatum, grown
separately in a standardized medium which was changed every twenty-
four hours (Fig. 134). The equilibrium phases showed that there
were over twice as many P. awrelia produced as P. caudatum. Gause then
measured the sizes of the animals and computed the mean volume of
each and the total volume of population. The volume curves showed that
very nearly the same volume of protoplasm was produced by each
species, with the same medium and conditions of culture.

P. caudatum was grown in a balanced salt medium, with one unit of
concentration, and with five units’ concentration of bacteria, by Johnson
(1935). The growth curves are sigmoid and show no stationary phase
and only a short lag phase. The equilibrium number was maintained
with no decline for seven days. The number of animals produced in the
greater concentration was more than five times the number in the lesser
concentration. In the lower concentration a single animal divided more
times than did a group of animals, while in the greater concentration a
group divided more rapidly, for about three days, when the population
figure from the single animal seeding passed the group curve to reach
a higher equilibrium level.

Mond’s (1937) estimates of both the bacteria and the infusorian
populations point the way to more adequate studies of protozoan growth.
Populations of Colpoda duodenaria were maintained in aération flasks
for four months by Taylor and Strickland (1938). By continuous feed-
ing, densities of 6 > 10° per milliliter were produced. The size of the
population fluctuated with the amount of food available and could be
modified as the experimenters wished. Over the whole period the num-
ber of Protozoa produced from a given amount of food was constant.
Excretion products did not limit the growth, but the continuous aération
may have ameliorated the effects of the waste products, so that the
conditions are not comparable with unaérated cultures.

Doe GROWTH

The growth curves of protozoan populations are sigmoid and te-
semble closely in form those of other populations. The growth curves
of some Protozoa show all phases. The growth of different Protozoa
depends on environmental conditions, and for details the reader should
consult the original publications. The size of a protozoan population
depends primarily on the amount of available food. Waste products
do not limit the growth, as they do with yeast populations, and are in-
hibitory only in very dense populations. However, yeast populations
contain more organisms than the protozoan populations—Paramecium
(Jones, 1928) 10°; Glaucoma (Phelps, 1936) 7.25 & 10°; Colpoda
(Taylor and Strickland, 1938) 60 10°; yeast (Richards, 1932) 335
X 10° per ml.; bacteria (Steinhaus and Birkeland, 1939) to 2.5 x 10°
—and laboratory populations of yeast are far less dense than those
produced in aérated and cooled commercial fermenters. The total volume
of protoplasm (number of individuals, x mean size) should be con-
sidered, and metabolic rates known, when comparing populations of
different organisms. Under identical conditions P. awrelia and P. cauda-
tum produced nearly the same total volume, although there were over
twice as many of the smaller P. aurelza.

So far no selective mortality has been reported for protozoan popula-
tion growth, although this is well known in yeast populations. The
decline may occur because fewer of the Protozoa reproduce or it may be
due to a slowing of the rate of cell division. Jahn (personal communica-
tion) believed the latter true for his Evg/ena populations. Jones reported
a disruptive mortality in his P. multimicronucleata populations. No
evidence of differences in the sizes and their distribution among Protozoa
—which would reveal how homogeneous the populations are from time
to time during the population growth—has been given in recent studies,
with carefully controlled conditions (e.g., bacteria-free cultures, on syn-
thetic media). Can Protozoa become resistant to an unfavorable medium
and remain abie to reproduce? Is encystment always governed by food
concentration (Taylor and Strickland, 1938), or do other factors have a
role? To what extent can an equilibrium population be maintained by
en- and excystment? The lack of information on these and many other
problems should attract more students of physiology and of growth to
protozodlogy.

GROWTH 99

THE STRUGGLE FOR EXISTENCE

The mathematical analysis of the question of survival by Volterra,
Lotka, Haldane, and others has established certain principles. Gause
(1934, 1935) has contributed to both the experimental and the theoreti-
cal advancement of the subject. The mathematical analyses are compli-
cated, even though in the state of first approximations, and the interested
reader should consult the original articles. Cf. Lotka (1925, 1934),
Kostitzin (1934), Gause (1934, 1935). Chapman (1931) gives a
translation of part of Volterra’s work. Protozoan populations have been
used to test the hypothesis, and some of the experiments of Gause are
here summarized to illustrate the beginning of a quantitative attack on
the problems of struggle for existence and survival of the fittest.

Separate and mixed populations of Paramecium caudatum and Sty-
lonychia mytilus wete grown on an oatmeal infusion inoculated with
B. subtilis. Neither species grew as well in mixed populations, but the
influence of Stylonychia on Paramecium is about forty times as great
as the effect of the latter on the former. With more food, provided by
mixed, wild bacteria, Paramecium grew to about the same level in mixed
populations as it did in pure population. Stylonychia grew only to about
half the number when competing in the same environment with Para-
mecium as it would have alone, and its population soon declined, while
that of the Paramecium maintained itself despite the competition.

Paramecium caudatum and P. aurelia may be grown together, and
will compete for the same food. It is necessary to make comparisons
in terms of volume of protoplasm, as discussed in the previous section.
In mixed populations the growth curves for the two populations are
quite similar for the first eight days, after which the P. aurelia popula-
tion continues to grow, while that of the P. caudatum declines, reaching
the point of extinction in about sixteen days. P. caudatum has an ad-
vantage in a greater coefficient of geometrical increase, but requires 1.64
times as much food as P. awrelia. Consequently, the greater rate of growth
is a liability in competition. P. aurelia is less affected by excretion prod-
ucts, as it can live twice as long in the presence of a strong concentration
of waste excretion products as P. caudatum. With the amount of food
available and the medium used, only the P. awrelia could survive the
competition of the mixed population. Glaucoma scintillans, growing in
competition with P. awrelza, will survive when the latter perishes.

554 GROWTH

A more complicated series of experiments was made on P. aurelia or
P. caudatum and P. bursaria with food supplied by bacteria and yeast.
The P. busaria could eat the yeast, but the two other species could not.
Varying equilibria of populations could be established, depending on
the initial concentrations of the four organisms. In this case the com-
petition is in different niches.

Populations of predators and prey are interesting and have been studied
in epidemiology, notably by Ross and Lotka working with the malarial
parasite. A simpler case, of less personal interest to man, is the compett-
tion of mixed populations of bacteria, Paramecium, and Didinium nasu-
tum. The latter consumes a Paramecium every three hours. In such a
mixed population, Gause found that at first both the Paramecium and
the Didinium populations grew, but later the didinia ate all of the para-
mecia and then promptly starved. With medium with sediment in which
some of the paramecia moved about and thus were not available as food
for the didinia, the didinia ate the available paramecia and then starved
while the remaining paramecia grew. Another experiment utilized Bur-
sarta truncatella, which preyed on P. bursaria.

The experiments may be grouped in three classes: (1) two species in
the same ecological niche, competing for the same food; (2) two species
in different niches, competing for the same food; or (3) two species,
one eating the other. Gause (1935) has given mathematical analyses
of the equilibria, depending on the variables involved. Much progress
has been made in this phase of biological science, even though it 1s
less than a quarter of a century old, and well-planned experiments or
heuristic theoretical analysis may be expected to contribute to an under-
standing of the growth of the Protozoa, to ecology, and to historical
(evolutionary) biological science.

LITERATURE CITED

Adolph, E. F. 1931. The regulation of size. Springfield, Ill. 235 pp.

Allee, W. C. 1934. Recent studies in mass physiology. Biol. Rev., 9: 1-48.

Beers, C. D. 1928a. The regulation of dietary insufficiency to vitality in the
ciliate Didinium nasutum. J. exp. Zool., 51: 121-33.

1928b. Rhythms in infusoria with special reference to Didinium
nasutum. J. exp. Zool., 51: 485-93.

—— 1928c. Some effects of dietary insufficiency in the ciliate Didininm
nasutum. Proc. Nat. Acad. Sci., Wash., 14: 132-37.

GROWTH IID

—— 1929. On the possibility of indefinite reproduction in the ciliate
Didinium nasutum without conjugation or endomixis., Amer. Nat., 63:
125-29:

Berkson, J., T. B. Magath, and M. Hurn. 1935. Laboratory standards in
relation to chance fluctuations of the erythrocyte count as estimated with
the haemocytometer. J. Amer. statist. Ass., 30: 414-26.

Bodine, J. H. 1917. Hydrogen ion concentration of protozoan cultures. Biol.
Bull., 41: 73-77.

Bond, R. M. 1933. A contribution to the study of the natural food-cycle in
aquatic environments. Bull. Bingham oceanogr. Coll., 4: 1-89.

Bowen, W. J. 1938. The effects of copper and vanadium on the frequency of
division. Biol. Bull., 75: 361. (Cf. also Amer. J. Physiol., 1939, 126:
Aes)

Buchanan, R. E., and E. I. Fulmer. 1928. Physiology and biochemistry of
bacteria. Vol. I. Baltimore.

Burnside, L. H. 1929. Relation of nuclear size to body size in Stentor
coeruleus. J. exp. Zool., 54: 473-83.

Carlson, Tor. 1913. Uber Geschwindigkeit und Grésse der Hefevermehrung
in Wirze. Biochem. Z., 57: 313-35.

Chalkley, H. W. 1929. Changes in water content of Amoeba in relation to
change in its protoplasmic structure. Physiol. Zo6l., 4: 535-74.

— 1931. The chemistry of cell division. II. The relation between cell
growth and division in Amoeba proteus. Publ. Hlth. Rep. Wash., 46:
1736-54.

Chapman, R. N. 1931. Animal ecology. New York.

Clark, N. A. 1922. Rate of formation and yield of yeast in wort. J. phys.
Chem., 26: 42-60.

Coe, W. R. 1932. Season of attachment and rate of growth of sedentary
marine organisms at the pier of the Scripps Institution of Oceanography,
La Jolla, Calif. Bull. Scripps Instn. Oceanogr. tech. 3: 37-86.

Crozier, W. J. 1923. On abundance and diversity in the protozoan fauna of
a sewage ‘‘Filter.”’ Sczence, 58: 424-25.

Crozier, W. J., and E. S. Harris. 1923. Animal population of a sewage
sprinkling filter. Stat. Rep. N. J., 503-16.

Cutler, D. W., and L. M. Crump. 1923. The rate of reproduction in artificial
culture of Colpidium colpoda. Bio-chem. J., 17: 878-86.

1924. The rate of reproduction in artificial culture of Colpidinm

col poda. Bio-chem. J., 18: 905-12.

—— 1925. The influence of washing upon the reproduction rate of Col-
pidium col poda. Bio-chem. J., 19: 450-53.

—— 1927. The qualitative and quantitative effects of food on the growth
of soil Amoeba. Brit. J. exp. Biol., 5: 155-65.

Daniel, G. E., and H. W. Chalkley. 1933. The influence of temperature on

556 GROWTH

the process of division in Amoeba proteus (Leidy). J. cell. comp.
Physiol., 2: 311-27.

Darby, H. H. 1930a. The experimental production of life cycles in ciliates.
J. exp. Biol., 7: 132-42.

—— 1930b. Studies on growth acceleration in Protozoa and yeast. J. exp.
Biol., 7: 308-16.

Dimitrowa, A. 1932. Die fordernde Wirkung der Paramaecium caudatum.
Ehrbg. auf dessen Teilungsgeschwindigkeit. Zool. Anz., 100: 127-32.

Eddy, S. 1928. Succession of Protozoa in cultures under controlled conditions.
Trans. Amer. micr. Soc., 47: 283-339.

Elliott, A. M. 1935a. The influence of pantothenic acid on growth of Proto-
zoa. Biol. Bull., 68: 82-92.

1935b. Effects of certain organic acids and protein derivatives on the

growth of Col pidium. Arch. Protistenk., 84: 472-94.

1936. Nutritional studies on Protozoa. Proc. Minn. Acad. Sci., Rept.

6 pp.

— 1938. The influence of certain plant hormones on growth of Protozoa.
Physiol. Zo6l., 11: 31-39.

—— 1939. A volumetric method for estimating population densities of
Protozoa. Trans. Amer. micr. Soc., 58: 97-99.

Entz, G., Jr. 1931. Analyse des Wachstums und der Teilung einer Popula-
tion sowie eines Individuums des Protisten Ceratium hirudinella unter
den natiirlichen Verhialtnissen. Arch. Protistenk., 74: 311-61.

Erdmann, R. 1920. Endomixis and size variations in pure bred lines of
Paramecium aurelia. Roux Arch. EntwMech. Organ., 46: 85-148.

Estabrook, A. H. 1910. Effect of chemicals on growth in Paramecium. J. exp.
Zool., 8: 489-543.

Fauré-Fremiet, E. 1925. La Cinétique du développement. Paris.

1930. Growth and differentiation of the colonies of Zoothamnium

alternans. Biol. Bull., 58: 28-51.

Fine, M. S. 1912. Chemical properties of hay infusions with special reference
to the titratable acidity and its relation to the protozoan sequence. J. exp.
ZOO 2A265-8ile

Garrod, L. P. 1936. Allelocatalysis. J. Path. Bact., 42: 535-36.

Gause, G. F. 1932. Ecology of populations. Quart. Rev. Biol., 7: 27-46.

—— 1934. The struggle for existence. Baltimore.

—— 1935. Verifications expérimentales de la théorie mathématique de la
lutte pour la vie. Act. Sci. Indust., No. 277. Paris.

Gerstein, J. 1937. The culture and division rate of Stentor coeruleus. Proc.
Soc, €xp: Biol. Ni¥-4137; 210=1de

Giese, A. C. 1939. Ultraviolet radiation and cell division, I. Effects of 4 2654
and 2804 A upon Paramecium caudatum. J. cell. comp. Physiol., 13:
139-50.

GROWTH Spy

Gladstone, G. P., P. Fildes, and G. M. Richardson. 1935. Carbon dioxide as
an essential factor in the growth of bacteria. Brit. J. exp. Path., 16: 335-
48.

Gordon, R. D. 1938. Note on estimating bacterial populations by the dilution
method. Proc. nat. Acad. Sci. Wash., 24: 212-15.

Gray, J. 1929. The kinetics of growth. Brit. J. exp. Biol., 6: 248-74.

Greenleaf, W. E. 1924. Influence of volume of culture medium and cell
proximity on the rate of reproduction of Protozoa. Proc. Soc. exp. Biol.
ING Ye, 2ill 405263

Gregory, L. H. 1925. Direct and after effects of changes in medium during
different periods in the life history of Uroleptus mobilis, Biol. Bull., 48:
200-8.

— 1926. Effects of changes in medium during different periods in the
life history of Uroleptus mobilis, Biol. Bull., 51: 179-88.

— 1928. The effects of changes in medium during different periods in
the life history of Uroleptis mobilis and other Protozoa. Biol. Bull., 55:
386-94.

Hall, R. P. 1933. On the relation of hydrogen-ion concentration to the growth
of Exglena anabaena vat. minor and E. deses. Arch. Protistenk., 79: 239-
48.

1938a. Effects of manganese on the growth of Euglena anabaena, Astasta
sp. and Colpidium campylum. Arch. Protistenk., 90: 178-84.
1938b. Pimelic acid as a growth factor for the ciliate, Colpidium

campylum. Anat. Rec., 72 (Suppl.) : 110.

1939. The trophic nature of the plant-like flagellates. Quart. Rev. Biol.,

14: 1-12.

Hall, R. P., D. F. Johnson, and J. B. Loefer. 1935. A method for counting
Protozoa in the measurement of growth under experimental conditions.
Trans. Amer. micr. Soc., 54: 298.

Hall, R. P., and J. B. Loefer. 1938. Effect of the addition of old culture
medium on the growth of Colpidinm campylum. Amat. Rec., 72
(Suppl.) : 50.

Hall, R. P., and H. W. Schoenborn. 1938. The selective action of inorganic
media in bacteria-free cultures of Evglena. Anat. Rec., 72 (Suppl.) : 129.

Halsey, H. R. 1936. The life cycle of Amoeba proteus Pallas, Leidy and of
Amoeba dubia Schaeffer. J. exp. Zool., 74: 167-203.

Hammett, F. S. 1929. Chemical stimulants for growth by increase in cell
number. Proto plasma, 7: 297-322.

Hammond, E. C. 1938. Biological effects of population density in lower
organisms. Quart. Rev. Biol., 13: 421-38; 14: 35-59.

Hardy, A. C. 1938. Estimating numbers without counting. Nature, 142: 255-
56.

Hargitt, G. T., and W. W. Fray. 1917. The growth of Paramecium in pure
cultures of bacteria. J. exp. Zool., 22: 421-55.

558 GROWTH

Hegerty, C. P. 1939. Physiological youth as an important factor in adaptive
enzyme formation. J. Bact., 37: 145-52.

Hetherington, A. 1932. The constant culture of Stentor coeruleus, Arch.
Protistenk., 76: 118-29.

—— 1936. The precise control of growth in a pure culture of a ciliate
Glaucoma pyriformis. Biol. Bull., 70: 426-40.

Huntington, E., and C.-E. A. Winslow. 1937. Cell size and metabolic activity
at various phases of the bacterial culture cycle. J. Bact., 33: 123-44.

Huxley, J. S. 1932. Problems in relative growth. London.

Huxley, J. S., and G. Teissier. 1936. Terminology of relative growth. Nature,
137: 780-81.

Jahn, T. L. 1929. Studies on the physiology of the Euglenoid flagellates. I.
The relation of the density of population to the growth rate of Evglena.
Biol. Bull., 57: 81-106.

—— 1930. Studies etc. II. The autocatalytic equation and the question of
an autocatalyst in the growth of Evglena. Biol. Bull., 58: 281.

—— 1933. Studies on the oxydation-reduction potential of protozoan cul-
tures. I. The effect of -SH on Chilomonas paramecium. Protoplasma,
20: 90-104.

—— 1934. Problems of population growth in the Protozoa. Cold Spring
Harbor Symp. Quant. Biol., 2: 167-80.

—— 1935. Studies etc. VI. The effects of temperature and of acetate on
Euglena gracilis caltures in the dark. Arch. Protistenk., 86: 251-57.
—— 1936. Effect of aeration and lack of CO, on growth of bacteria-free

cultures of Protozoa. Proc. Soc. exp. Biol. N. Y., 33: 494-98.

Jennings, H. S. 1908. Heredity, variation and evolution in Protozoa II. Proc.
Amer. phil. Soc., 47: 393-546.

Johnson, D. E. 1936. Growth of Glaucoma ficaria Kahl in cultures with
single species of other organisms. Arch. Protistenk., 86: 359-78.

Johnson, W. H. 1933. Effects of population density on the rate of reproduc-
tion in Oxytricha. Physiol. Zo6l., 6: 22-54.

—— 1935. Isolation of Glaucoma ficaria Kahl in bacteria-free cultures and
growth in relation to the pH of the media. Arch. Protistenk., 86: 263-77.

—— 1936. Studies in the nutrition and reproduction of Paramecium. Physiol.
Zool., 9: 1-14.

—— 1937. Experimental populations of microscopic organisms, Amer. Nat.,
7-20:

Johnson, W. H., and G. Hardin. 1938. Reproduction of paramecium in old
culture medium. Physiol. Zodl., 11: 333-46.

Jones, E. P. 1928. Population curves of Paramecium. Proc. Pa. Acad. Sci.,
2% O27 =28,

—— 1930. Paramecium infusion histories. I. Hydrogen ion change in hay
and hay-flour infusions. Biol. Bull., 59: 275-84.

GROWTH r po

Kavanagh, A. J., and O. W. Richards. 1939. The autocatalytic growth curve.
Amer. Nat., 68: 54-59.

— 1937a. The unusual mortality which characterizes a Paramecium cul-
ture. Anat. Rec., 70 (Suppl.) : 39.

— 1937b. The potential longevity of a Paramecium culture. Anat. Rec.,
70 (Suppl.) : 39.

Kidder, G. W. 1939. The effects of biologically conditioned medium on the
growth of Colpidium campylum. Biol. Bull., 77: 297-98.

Kidder, G. W., and C. A. Stuart. 1938. The role of chromogenic bacteria
in ciliate growth. Biol. Bull., 75: 336.

Kober, P. A., and S. S. Graves. 1915. Nephelometry (Photometric Analysis) .
I. History of method and development of instruments. J. industr.
Engng. Chem., 7: 843-47.

Koser, S. A., and F. Saunders. 1938. Accessory growth factors for bacteria and
related microorganisms. Bact. Rev., 2: 99-190.

Kostitzin, V. A. 1934. Symbiose, parasitisme et évolution. Act. Sci. Indust.,
No. 96. Paris. 47 pp.

Lackey, J. B. 1938a. Protozoan plankton as indicators of pollution in a
flowing stream. Publ. Hlth. Rep. Wash., 53: 2037-58.

—— 1938b. A study of some ecologic factors affecting the distribution of
Protozoa. Ecol. Monogr., 8: 501-27.

Loefer, J. B. 1935. Effects of certain carbohydrates and organic acids on
growth of Chlorogonium and Chilomonas. Arch. Protistenk., 84: 456-71.

—— 1938a. Effects of hydrogen ion concentration on the growth and
morphology of Paramecium bursaria. Arch. Protistenk., 90: 185-93.

—— 1938b. Effect of osmotic pressure on the motility and viability of
fresh-water Protozoa. Anat. Rec., 72 (Suppl.) : 50.

—— 1938c. Growth of Chlorogonium tetragonium at different hydrogen-
ion concentrations. Anat. Rec., 72 (Suppl.) : 129.

—— 1938d. Bacteria-free culture of Paramecium bursaria and concentration
of the medium as a factor in growth. J. exp. Zool., 72: 387-407.
—— 1939. Acclimatization of fresh-water ciliates and flagellates to media

of higher osmotic pressure. Physiol. Zodl., 12: 161-72.

Loofbourow, J. R., and C. M. Dyer. 1938. Relative consistency of weights
and counts in determining microorganisms by photoelectric nephelom-
eters. Studies Inst. Divi Thomae, 1: 129-35.

Lotka, A. J. 1925. Elements of physical biology. Baltimore.

1934. Théorie analitique des associations biologiques. Act. Sci. Indust.,
No. 187. Paris.

Lwoff, A., and H. Dusi. 1937. La Pyrimidine et le thiazol, facteurs de
croissance pour le Flagellé Polytoma caeca. C. R. Soc. Biol. Paris, 126:
630-32.

—— 1938. Culture de divers flagelles leucophytes en milieu synthétique.
C. R. Soc. Biol. Paris, 127: 53-56.

560 GROWTH

McCay, C. M. 1933. Is longevity compatible with optimum life? Science,
77: 410-11.

MacDougall, M. S. 1929. Modifications in Chilodon uncinatus produced by
ultraviolet radiation. J. exp. Zool., 54: 95-109.

MacLennan, R. F. 1935. Dedifferentiation and redifferentiation in Ichthyoph-
thirius. 1. Neuromuscular system. Arch. Protistenk., 86: 191-210.

McPherson, M., G. A. Smith, and A. M. Banta. 1932. New data with pos-
sible bearing on Robertson’s theory of autocatalysis. Anat. Rec., 54
(Suppl.) : 23.

Mast, S. O., and D. M. Pace. 1933. Synthesis from inorganic compounds of
starch, fat, proteins and protoplasm in the colorless animal Chilomonas
paramecium, Protoplasma, 20: 326-58.

—— 1935. Relation between sulphur in various chemical forms and the rate
of growth in the colorless flagellate Chilomonas paramecium. Proto-
plasma, 23: 297-325.

—— 1937. The relation between the number of individuals per volume of
culture solution and rate of growth in Chilomonas paramecium. Anat.
Rec., 70(Suppl.) : 40.

—— 1938a. The relation between the hydrogen ion concentration of the
culture medium and the rate of reproduction in Chilomonas
paramecium. J. exp. Zool., 79: 429-31.

—— 1938b. The effects of substances produced by Chilomonas paramecium
on the rate of reproduction. Physiol. Zodl., 11: 360-82.

1938c. The relation between the age of the cultures from which

Chilomonas is taken and the rate of reproduction in fresh culture fluid.
Anat. Rec., 72 (Suppl.) : 62.

Mattick, A. T. R., J. McClemont, and J. O. Irwin. 1935. The plate count
of milk. J. Dairy Sci., 6: 130-47.

Mestre, H. 1935. A precision photometer for the study of suspensions of
bacteria and other microorganisms. J. Bact., 30: 335-58.

Meyers, E. C. 1927. Relation of density of population and certain other factors
to survival and reproduction in different biotypes of P. cawdatum. J. exp.
Zool., 49: 1-43.

Mitchell, W. H., Jr. 1929. The division rate of Paramecium in relation to
temperature. J. exp. Zool., 54: 383-410.

Mizuno, F. 1927. Sur la croissance du Paramecium caudatum. Sci. Rep. Tohuku
Univ., 4th Ser., 2: 367-81.

Mond, J. 1937. Réaction d’entretien et ration de croissance dans les popula-
tions bactériennes. C. R. Acad. Sci. Paris, 205: 1456-57.

Mottram, J. C., 1939. An increase in the rate of growth of paramecium sub-
jected to the blastogenic hydrocarbon 3:4-benzpyrene. Nature, 144: 154.

Miller, R. H. 1939. Photoelectric methods in analytical colorimetry. Industr.
Engng. Chem., 11: 1-17.

Needham, J. 1934. Chemical heterogony and the ground-plan of animal
growth. Biol. Rev., 9: 79-109.

GROWTH 561

Nielson, N. 1933. A method for determining the velocity of sedimentation of
yeast. C. R. Lab. Carlsberg, Nr. 19.
Noland, L. E. 1925. Factors influencing the distribution of fresh water ciliates.
Ecology, 4: 437-52.
Park, T. 1939. Analytical population studies in relation to general ecology.
Amer. Midl. Nat., 21: 235-55.
Pearl, R. 1907. A biometrical study of conjugation in Paramecium. Biometrika,
213-078
—— 1925. Biology of population growth. New York.
— 1928. The rate of living. New York.
Peskett, G. L. 1924. Allelocatalysis and the growth of yeast. J. Physiol.,
SO} SSxiit:
—— 1925a. Studies on the growth of yeast. I. Influence of the volume of
culture medium employed. Bio-chem. J., 19: 464-73.
—— 1925b. Studies etc. IJ. A further note on allelocatalysis. Bio-chem. J.,
19: 474-76.
— 1927. Studies etc. III. a further study on the influence of volume of
media employed. Bio-chem. J., 21: 104-10.
Peters, A. W. 1901. Some methods for use in the study of Infusoria. Amer.
Nat., 35: 553-59.
— 1904. Metabolism and division in Protozoa. Proc. Amer. Acad. Arts.
Sci., 39: 441-516.
— 1907. Chemical studies on the cell and its medium. Amer. J. Physiol.,
17: 443-77 ;-18: 321-46.
Peters, R. A. 1920. Nutrition of the Protozoa. J. Physiol., 54: L.
1921. The substances needed for the growth of a pure culture of
Colpidium colpoda. J. Physiol., 55: 1-32.
Petersen, W. A. 1929. Relation of density of population to rate of reproduc-
tion of Paramecium caudatum. Physiol. Zodl., 2: 221-54.
Phelps, A. 1931. Effects of H-ion concentration on the division rate of
Paramecium aurelia. Science, 74: 395-96.
— 1934. Studies on the nutrition of Paramecium. Arch. Protistenk., 82:
134-63.
1935. Growth of Protozoa in pure cultures. I. Effect upon the growth
curve of the age of the inoculum and of the amount of the inoculum.
J. exp. Zool., 70: 109-30.
—— 1936. Growth etc. II. Effect upon the growth curve of different con-
centrations of nutrient materials. J. exp. Zool., 72: 479-96.
Phillips, R. L. 1922. The growth of Paramecium in infusions of known
bacterial content. J. exp. Zool., 36: 135-83.
Popoff, M. 1908. Experimentelle Zellstudien. I. Arch. Zellforsch., 1: 246-
asthe:
1909. Experimentelle Zellstudien. I]. Arch. Zellforsch., 3: 124-80.
Pruthi, H. S. 1926. On the hydrogen ion concentration of hay infusions with

562 GROWTH

special reference to its influence on the protozoan sequence. Brit. J.
exp. Biol., 4: 292-300.

Reich, K. 1936. Studies on the physiology of Amoeba. I. The relation be-
tween nutrient solution zone of growth and density of population.
Physiol. Zo6l., 9: 254-63.

—— 1938. Studies etc. I. The allelocatalytic effect in Amoeba culture free
of bacteria. Physiol. Zodl., 11: 347-58.

Richards, O. W. 1929. The correlation of the amount of sunlight with the
division rate of ciliates. Biol. Bull. 56: 298-305.

—— 1932. The second cycle and subsequent growth of a population of
yeast. Arch. Protistenk., 78: 263-301.

—— 1933. The toxicity of some metals and Berkefeld filtered sea water to
Mytilus edulzs. Biol. Bull., 65: 371-72.

—— 1934. The analysis of growth as illustrated by yeast. Cold Spring Harbor
Symp. Quant. Biol., 2: 157-66.

—— 1935. Analysis of the constant differential growth ratio. Pap. Tortugas
Lab., 29: 173-83.

Richards, O. W., and J. A. Dawson. 1927. The analysis of the division rate
of ciliates. J. gen. Physiol., 10: 853-58.

Richards, O. W., and T. L. Jahn. 1933. A photoelectric nephelometer for
estimating population density of microdrganisms. J. Bact., 26: 385-91.

Richards, O. W., and A. K. Kavanagh. 1937. The course of population growth
and the size of seeding. Growth, 1: 217-27.

Robertson, M. 1929. Life cycles in the Protozoa. Biol. Rev., 4: 152-79.

Robertson, T. B. 1921a. Experimental studies on cellular multiplication. II.
The influence of mutual contiguity upon reproductive rate and the part
played therein by the “x-substance’’ in bacterial infusions which stimu-
lates the multiplication of Infusoria. Bio-chem. J., 15: 612-19.

-—— 1921b. The multiplication of isolated Infusoria. Bio-chem. J., 15: 595-
Glie

—— 1922. Reproduction in cell communities. J. Physiol., 56: 404-12.

——— 1923. The chemical basis of growth and senescence. Philadelphia.

—— 1924a. The influence of washing on the multiplication of isolated
Infusoria and upon the allelocatalytic effect in cultures initially contain-
ing two Infusoria. Aust. J. exp. Biol. med. Sci., 1: 151-73.

—— 1924b. Allelocatalytic effect in cultures of Colpidium in hay infusion
and in synthetic media. Bio-chem. J., 18: 1240-47.

—— 1927. On some conditions affecting the viability of cultures of Infusoria
and the occurrence of allelocatalysis therein. Aust. J. exp. Biol. med. Sci.,
4: 1-23.

Rottier, P.-B. 1936a. Recherches sur les courbes de croissance de Polytoma
uvella, L'influence de l’oxygénation. C. R. Soc. Biol. Paris, 122:
65-67.

GROWTH 563

—— 1936b. Recherches sur la croissance de Polytomella uvella. L’influence
de la concentration des substances nutritives. C. R. Soc. Biol. Paris, 122:
776-79.

Roux, J. 1901. Faune infusorienne des eaux stagnantes des environs de
Genéve. Mém. Inst. nat. genév., Vol. 19. 142 pp.

Russell, J. 1937. A photoelectric cell circuit with a logarithmic response. Rev.
sci. Instrum., 8: 495-96.

Schmalhausen, I., and E. Syngajewskaja. 1925. Studien tber Wachstum und
Differenzierung. I. Die Individuelle Wachstumskurve von Paramecium
caudatum. Roux Arch, Entwkmech. Organ., 105: 711-17.

Shapiro, H. 1935a. Nomogram for centrifugal speed. Industr. Engng. Chem.,
U3 235

—— 1935b. The validity of the centrifuge method for estimating aggregate
cell volume in suspensions of the eggs of the sea-urchin Arbacia
punctulata. Biol. Bull., 68: 363-77.

—— 1937. The viscosimeter method for determination of cell concentration
in suspensions of living cells. Anat. Rec., 70 (Suppl.) : 110.

Simpson, J. Y. 1902. The relation of binary fission to variation. Biometrika,
1: 400-7.

Smith, J. A. 1938. Some effects of temperature on the reproduction of
Chilomonas paramecium. Biol. Bull. 75: 336-37.

—— 1939. Effects of temperature on starch & fat in Chilomonas. Coll. Net,
1473-30:

Snell, S. D. 1929. An inherent defect in the theory that growth rate is con-
trolled by an allelocatalytic process. Proc. nat. Acad. Sci. Wash., 15:
274-81.

Spurr, W. A. 1937. A graphic method for measuring seasonal variation.
J. Amer. statist: Ass., 32: 281-89.

Steinhaus, E. A., and J. M. Birkeland. 1939. The senescent phase in ageing
cultures and the probable mechanisms involved. J. Bact., 38: 249-61.

Stier, T. J. B., M. I. Newton, and H. Sprince. 1939. Relation between the
increase in opacity of yeast suspensions during glucose metabolism and
assimilation. Science, 89: 85-86.

Sweet, H. E. 1939. A micro-population study of Evglena gracilis Klebs in
sterile, autotrophic media and in bacterial suspensions. Physiol. Zodl.,
12 173-200.

Taylor, C. V., and A. G. R. Strickland. 1938. Reactions of Colpoda duodenaria
to environmental factors. I. Some factors influencing growth and
encystment. Arch. Protistenk., 90: 396-409.

Teissier, G. 1928. Croissance des populations et croissance des organismes.
Ann. Physiol. Physicochim. biol., 4: 342-86.

——— 1934. Dysharmonies et discontinuités dans la croissance. Paris.

—— 1937. Les Lois quantitatives de la croissance. Paris.

Tippett, L. H. C. 1932. A modified method of counting particles. Proc. roy.
Soc., A137: 434-46.

564 GROWTH

Unger, W. B. 1931. The protozoan sequence in five plant infusions. Trans.
Amer. micro. Soc., 50: 144-53.

Walker, H. H. 1932. Carbon dioxide as a factor affecting lag in bacterial
cultures. Science, 76: 602-4.

Wang, C. C. 1928. Ecological studies of the seasonal distribution of Protozoa
in a fresh water pond. J. Morph., 46: 431-78.

Wetzel, N. C. 1937. On the motion of growth. XVII. Theoretical foundations.
Growth, 1: 6-59.

Woodruff, L. L. 1905. An experimental study on the life history of

hypotrichous Infusoria. J. Exp. Zool., 2:585-632.

1911. The effect of excretion products of Paramecium on its trate

of reproduction. J. exp. Zool., 10: 557-81.

1912. Observations on the origin and sequence of the protozoan fauna

of hay infusions. J. exp. Zool., 12: 205-64.

1913a. Cell size, nuclear size and the nuclear cytoplasmic relation during
the life of a pedigreed race of Oxytricha fallax. J. exp. Zool., 15: 1-22.
1913b. The effect of excretion products on the same and on different
species with special reference to the protozoan sequence in infusions.

J. exp. Zool., 14: 575-82.

Woodruff L. L., and G. A. Baitsell. 1911a. The reproduction of Paramecium
aurelia in a “constant” culture medium of beef extract. J. exp. Zool.,
Lis 135-42.

—— 1911b, The temperature coefficient of the rate of reproduction of
Paramecium aurelia. Amer. J. Physiol., 29: 147-55.

—— 1911c. Rhythms in the reproductive activity of the Infusoria. J. exp.
Zool., 11: 339-59.

Wright, E. V., and H. Kersten. 1937. An apparatus for measuring turbidity
of bacterial suspensions. J. Bact., 34:581-83.

Yocom, H. B. 1928. The effect of the quantity of culture medium on the
division rate of Oxytricha. Biol. Bull., 54: 410-16.

—— 1934. Observations on the experimental adaptation of certain fresh
water ciliates to sea water. Biol. Bull., 67: 273-76.

Zhalkovskii, B. G. 1938. The differences in biological action of transmitted
and reflected light. I. Experiments with Paramecium caudatum. Bull.
biol. exp. med. U. R. S. S. 493-95. From Chem. Abstr., 1939, 33: 2544.

Ziegler, N. R., and H. O. Halvorson. 1935. Application of statistics to prob-
lems in bacteriology. IV. Experimental comparison of the dilution method,
the plate count, and the direct count for the determination of bacterial
populations. J. Bact., 29: 609-34.

GHAPTER XI

THE RES GYGLE OF THE PROTOZOA

CHARLES ATWOOD KOFOID

INTRODUCTION

THE ORGANISM has the fourth dimension of time. In the course of its
life cycle, its three spatial dimensions change. The fourth changes also,
interacting with the three. It may be measured by metabolic rate, by struc-
tural results of growth, or by organismal cyclic changes which follow
one after the other in sequences. These may be regular, interrupted,
repeated, or in some other way responsive to or dependent upon internal
environmental conditions, or to external conditions, such as changing
quantity or quality of food supply; rise or fall in temperature, of
seasonal origin, or due to migration; inciting or deterrent chemical or
physical factors, such as pH, intensity and duration of light, and changes
of host.

The Protozoa* differ from the Metazoa because of their smaller size
and the resulting more highly significant and potent surface-volume re-
lations, as these affect the rate and intensity of the impact of environ-
mental factors upon the organism and the changes they initiate and
induce. It is therefore to be expected that the Protozoa will be relatively
more susceptible to the modification of the individual and to the dis-
tortion and interruption of its normal life cycle than are the Metazoa,
thus obscuring and complicating the evidence of the existence of life
cycles among them. The factors of time, volume, and season enter more
or less definitely into the life cycles of Metazoa such as hydroids, flukes,
tapeworms, crustaceans, insects, and tunicates. Among the Protozoa, on
the other hand, the time units required for the various cyclic changes
may be very brief, and these changes very often have little or no de-
pendence upon cosmic cycles, with the result that the evidence of their

* Assistance in preparing this chapter, rendered by the personnel of Work Projects
Administration Official Project No. 65-1-08-113, Unit C1, is acknowledged.

566 THE EIBESCYGLE

occurrence and continuity is more difficult to organize than is that for
cycles in the larger, longer-lived Metazoa.

Furthermore, a certain hesitancy about life cycles in the Protozoa
has arisen historically because of the fact that skilled workers in this
field have been caught in error by reason of the difficulties above noted.
Following upon Biitschli’s (1876) and Hertwig’s (1889) fundamental
analyses of conjugation in the Ciliata and its resemblance to maturation
and fertilization in other organisms, there arose a Munich school of
protozodlogists whose labors brought forth an array of protozoan life
cycles fitted to the metazoan pattern. Under the brilliant leadership of
Fritz Schaudinn, most of the major groups of Protozoa were subjected
to this pattern of analysis, with resulting marvelous conformity to type.
Some of these, notably those of Trypanosoma, Endamoeba, and Mastv-
gella, have not stood the test of subsequent critical reexamination. Others,
such as those of Plasmodium, Coccidium, and Paramecium, have, on the
other hand, survived and have proved the validity of the basic assump-
tion that there are life cycles in the Protozoa, though not necessarily
all of the same type.

The life cycle in the Metazoa starts with the diploid or polyploid
zygote, a unicellular stage whose genes, derived from the haploid
gametes, determine the characters of all of the varied subsequent stages
unfolded in the ensuing life cycle. This cycle in many instances is marked
by indirect development with one or more larval stages, followed by
metamorphosis into the adult, sexual maturity, gametogenesis, senes-
cence, and death. In other instances the development is direct, with
adolescence replacing metamorphosis. In both types asexual repro-
duction may intervene at different periods in embryonic, larval, and
even adult life, giving rise by budding, binary and multiple fission, and
sporulation to two or many different functional individuals, all with
the original genetic constitution. Parthenogenesis may also intervene and
alternate with normal sexual reproduction. There is often considerable
change in the external appearance of the successive stages, as in larva,
pupa, and imago of the Lepidoptera, though a striking similarity, even
continuity, may occur in various organ systems from stage to stage.

The stages occurring in the metazoan life cycle are brought about
by the processes of cleavage, gastrulation, organogenesis and histogenesis,
growth, adolescence or metamorphosis, gametogenesis, senescence, and

THEY BIBE(CYGLE 567

death. Asexual reproduction may be interjected into the midst of any
one of these processes, resulting in from one to many repeated genera-
tions of functional individuals. Not infrequently these individuals are
heterogonous, with marked differences in structure from the parent, as
for example in the larval stages of the Trematoda.

This alternation of sexually and asexually produced generations is
widely distributed in the living world, ranging from some of the lower
algae to the Quints. The ease with which regeneration occurs after
mutilation and with which experimental asexual multiplication of func-
tional individuals may be imposed upon the genetic individual is indica-
tive of the fundamental organic basis of asexual reproduction, perhaps
as a corollary of the still more fundamental capacity of growth on the
part of the organism.

The Protozoa, from the evolutionary point of view, are of excep-
tional interest among the phyla, since it is among these primitive organ-
isms that most of the basic biological properties, structures, and functions
of the organism have had their evolution. Within these microcosms all
of the basic functions of living must be performed. As one surveys
their diversities and complexities of pattern, one is impressed with the
evidence that among these minute organisms, adapted to so many eco-
logical niches and exhibiting so many types of behavior, a vast deal
of evolutionary experimentation has been enacted. It is among the
Protozoa and Protophyta that the following have been evolved: nuclear
structure, sex, sexual dimorphism, sexual reproduction, mitosis, chromo-
somes, gametogenesis, histogenesis, multicellularity, sex and somatic
cells, asexual reproduction by the various methods of binary and multiple
fission, budding and sporulation, and the beginnings of the organization
of organ systems. Varying combinations and sequences of these evo-
lutionary accomplishments are exhibited in the diverse patterns of life
cycles to be detected among the Protozoa. Cycles of comparable type,
in some instances apparently independently of one another, have emerged
to a varying degree in the different classes and orders of Protozoa.

These cycles fall into two major groups. The first is the simpler and
the more primitive. It consists merely of recurrent rhythms of homo-
geneous asexual reproduction, in which mitosis produces a multicellular
(== multinuclear) body of from two to many cells, forming a plas-
modium, coenobium, sporocyst, or cyst. Fission of binary, multiple, or

568 THEO IRE ICYGLE

budding type breaks up this body into functional individuals of the
ancestral type. Although nuclear division is essential in the accomplish-
ment of this cycle, it does not initiate it. This is shown strikingly in the
Polymastigophora, in which the entire neuromotor complex of centro-
some, blepharoplast, flagella, undulating membrane, and axostyle of
the individual is duplicated by new growth, accompanied by extensive
dedifferentiation of the parental equipment before the nuclear phe-
nomena of mitosis ensue. Asexual reproduction is thus profoundly an
organismal phenomenon involving a rejuvenation of the organelle sys-
tems of the body of the individual.

This type of life cycle seemingly exists without any evidence of sex,
sexual reproduction, or sexual dimorphism. Efforts to establish sexuality
on the basis of the relative size of supposedly male and female individuals
and upon interpretations of behavior are will-o’-the-wisps of wishful
thinking. The only basis is gametogenesis, verified by reduction of the
diploid to the haploid number of chromosomes, and fertilization, with
the resulting return to the diploid state.

The juxtaposition or even fusion of motile individuals among flagel-
lates and rhizopods may occur when adverse conditions or internal states
induce an adhesive periphery; sometimes cannibalistic feeding of rhi-
zopods resembles fusion; and changes in position from divergence to
lateral contact in sister schizonts among Mastigophora resemble conjuga-
tion, all of which evidence is never to be accepted as sexual behavior
unless confirmed by critical cytological evidence.

The not uncommon opinion that sex is an inherent characteristic of
organisms and that sexual reproduction is to be expected in all animals
and plants and even in bacteria, is as yet without convincing cytological
evidence among the more primitive forms. It has, however, been clearly
demonstrated in the Sporozoa, Ciliata, Foraminifera, and the Volvocidae,
all representatives of the more highly evolved Protozoa. The present
evidence, negative though it be, lends support to the view that sex
was evolved in the Protozoa, perhaps independently in the different
classes. It may well be that its origin rests ultimately on differential
metabolism within the species, leading in time to more favorable condi-
tions for permanent fusion of gametes, though this alone makes no
provision for gametogenesis. The fact that some flagellates and rhizo-
pods have an odd number of chromosomes suggests that they are not

THE EEE CYCLE 569

zygotes nor derived from zygotes, but primitive haploids. While it is
to be expected that the cases of critically proved instances of sexual
reproduction will increase in both number and systematic range with
further investigation, even this will be far from establishing the unt-
versality of sexual differentiation among the Protista. Haploid (odd)
numbers of chromosomes in primitive species will still require a solu-
tion. There are three chromosomes in Trichomonas buccalis (Hinshaw,
1926) and five in lodamoeba biitschli, according to unpublished obser-
vations made in my laboratory by Dr. Dora P. Henry.

In the absence of sex and sexual reproduction among primitive Proto-
zoa, this first type of a merely asexual life cycle is the only one feasible.
It is, however, incorporated into the second type of cycle, in which it
alternates in varying irregularity with sexual reproduction and may even
exhibit several forms with structurally different functional individuals
within the same cycle, as in Plasmodium.

ASEXUAL REPRODUCTION IN ALTERNATING BINARY
AND MULTIPLE FIssION (TYPE I)

An example of the first type of the protozoan life cycle among the
Mastigophora is seen in Trichomonas augusta, in which asexual repro-
duction by binary fission prevails, but 1s interrupted at unknown inter-
vals by the formation of an eight or sixteen-celled somatella with a
common cytoplasm, each cell of which has its own neuromotor ap-
paratus. Within this plasmodium paired schizonts, temporarily joined
to each other by the paradesmose, ceaselessly tug at this tether until
they are disunited except by the common cytoplasm. Serial plasmotomy
releases each schizont, to start again the cycle with binary fission. There
is in this type of cycle no clue to sexual reproduction.

Another example from the Rhizopoda is found in Cowncilmania la-
fleuri, usually called Endamoeba coli, in which there is an alternation
between a unicellular free motile phase and a multicellular encysted one.
During the motile phase binary fission prevails, and reversion to the
unicellular condition follows each mitosis. This is interrupted from time
to time by the encysted phase, in which, following reduction in volume,
the body rounds out and secretes about itself an impervious membrane
or cyst wall of elastin, with a differentiated exit pore closed by a plug.
Encystment follows feeding and the accumulation of food reserves, which

Multiple

frssion

Figure 135. Diagram of the life cycle of Trichomonas augusta from the intestine of
the frog, Rana boylei, including an alternation of binary fission of the two-cell soma-
tella and of multiple fission of the eight-cell plasmodium.

THE, LIFES CYCLE 71

in the cyst are stored in a large central glycogen vacuole. The glycogen
is transformed into chromatoidal structures of unknown composition,
staining deeply and formed on the surface of the glycogen vacuole.
These progressively disappear as mitotic divisions ensue.

Soon after encystment is completed, a series of mitotic nuclear di-
visions occur, resulting in two, four, eight, and sixteen-cell stages,
rarely thirty-two-cell, and in one observed instance approximately a
sixty-four-cell stage, thus running the rhythm of normal cell division
in a metazoan egg. Plasmotomy, however, does not attend the nuclear
divisions. Cyst formation, in this instance, serves the function of assimila-
tion and growth. Measurements of cysts in the one, two, four, eight,
and sixteen-cell stages show a slight progressive increase in diameter.

Excystment occurs normally in the bowel, as shown by the occurrence
of cysts free from glycogen or chromatoidals, with reduced numbers of
nuclei from fifteen down. It can also be followed in fresh stools, as
the small mononucleate amoebulae escape singly out of the exit pore.
Excystment is a form of asexual reproduction, of budding, or progres-
sive multiple or serial fission. In this type of life cycle we find an alterna-
tion of a unicellular free phasé with reproduction by binary fission, with
the formation of a multicellular encysted somatella, with reproduction by
multiple fission and a return to the unicellular motile phase.

ALTERNATION OF ASEXUAL AND SEXUAL REPRODUCTION (TYPE II)

The second major type of the protozoan life cycle is that in which
asexual and sexual reproduction alternate. It may or may not be accom-
panied by sexual dimorphism, as exhibited by differential reaction to
aniline stains in Nina, by structural differentiation of gametes in Evmeria,
of gametocytes in Plasmodium, or of conjugants in Vorticella. It seems
probable that sex has become a genetic characteristic of the individual
throughout the whole cycle, in all life cycles having sexual reproduc-
tion, even though structural features indicative of sexual dimorphism
cannot be detected.

From the biological point of view, it is unfortunate that the life
cycles of parasitic Protozoa have been arranged, in illustrations, in se-
quences as parasitic cycles, rather than biological life cycles. They are
usually designated as beginning with the infection of the host, or in
the case of a parasite with two hosts with that of the primary host, or

SF Le cap

&. -Pseudopodium

Motte phase

Binary fission

--4-Peripheral
is chromatin

\-Glycogen

vacuole

Encysted phase

Multiple serial
f/S5/0N

Figure 136. Diagram of the life cycle of Exdamoeba coli (= Councilmania lafleuri,
Kofoid and Swezy, 1921) from the intestine of man, including an alternation of binary
fission in the motile phase and of budding, or serial multiple fission, of the eight-cell
encysted somatella.

THE sLIBEP Gy CLE Bile,

with that of the more significant host, for example, with the infection
of man in the case of Plasmodium. The psychological effect of this is
to deflect interest from the significant biological aspects of such cycles.

In order to follow these life cycles in their true biological sequences,
we have rearranged them and will now proceed to discuss three of the
most widely known ones, viz., those of Ezmeria, Plasmodium, and Para-
mecium.

THE LIFE CYCLE OF Esmeria schubergi

The life cycle of Evmeria schubergi, a parasite in the intestinal epi-
thelium of Lithobius forficatus, is a typical one with an alternation of
asexual and sexual reproduction, and of a sexual phase with asexual
ones. In this cycle no less than five different structural types of functional
individuals appear, each with a distinctive pattern of shape, size, struc-
ture, and activity. Four of the five appear but once, but one is subject
to numerous repetitions under favorable conditions.

As rearranged, the biological cycle begins with the zygote formed in
the lumen of the intestine of the host by the fusion of a flagellated
spermatozoan with a yolk-laden egg, recently emerging from an intestinal
epithelial cell of its host. Even before the pronuclei fuse, the fertilized
egg forms a fertilization membrane and secretes a cyst wall around its
spherical body (Fig. 137, 1). Two nuclear divisions bring the organism
to the cleavage stage of a four-celled somatella, the sporoblast. There-
upon there ensues the first asexual reproduction, when this somatella
divides into four unicellular spores. Unlike their spherical parent, these
functional individuals are ellipsoidal, and they, too, secrete about their
respective bodies a resistant ellipsoidal spore wall.

There then ensues the second asexual reproduction, when each spore
cell divides into two spindle-shaped, naked unicellular sporozoites, re-
tained within the spore case and the enveloping cyst wall of the sporo-
blast. At about this stage of the cycle, the sporocyst with its eight sporo-
zoites in four spores, is discharged from the intestine of its host, and
further development ceases until this infective stage is eaten by a Litho-
bius. Here the digestive fluids unstopper the cyst, the sporozoites are
released from the spores, each escapes singly through the pore and enters
an epithelial cell of the intestine of the host where it develops as a
trophozoite, changing in pattern from a spindle shape to a spherical one.
The organism at this stage is devoid of any special protecting cover and

Crow th

EDD

Sere
3 Asexual
reproduction

cycle
xN

és ARES
\ Be A
ut

2”? Asexual
reproduction

Lt Asex ual
reproduction

Figure 137. Diagram of the alternating sexual and asexual reproduction in the life
cycle of Eimeria schubergi, from the intestine of Lithobius forficatus. For convenience,
the first two asexual divisions within the sporoblast cyst wall are figured in the sexual
cycle. They are transitional to the repeated multiplicative multiple fissions attending the
infection of the intestinal cells of the host. Owing to post-zygotic reduction, both phases
are haploid except for the diploid zygote. (Modified from Schaudinn, 1900.)

HEE LiPEAG VCLE 575

grows into a somatella of about sixty-four cells, in a rhythm of repeated
mitoses.

Then follows the third asexual reproduction, in which the organism
divides by plasmotomy into motile unicellular spindle-shaped merozoites,
similar in size and pattern to the sporozoites. These in turn infect other
intestinal epithelial cells, and this phase of the cycle is repeated an un-
known number of times so long as susceptible host cells are available
for infection. This phase ends the asexual part of the life cycle, as
gametogenesis approaches, except in the male.

The sexual phase is marked by developing sexual dimorphism among
the merozoites. Presumably sex is determined at fertilization, and all
functional individuals derived from one zygote will accordingly be of
one sex only, and the myriapod host must have acquired an infection by
spores of each sex, in order that both male and female gametes of
Eimeria, fertilization, and spore formation may ensue in the intestine.
The sexual dimorphism of the gametocytes is determined by two factors,
the metabolic rate and probably also the chemical nature of the food
reserves on the one hand, and the structure and number of gametes pro-
duced on the other. Both male and female gamete mother cells grow
to the size and spherical form of the trophozoite, but do not run its
type of rhythm of cleavage, mitoses, and plasmotomy. The female 1s
early differentiated from the male by the internal elaboration of spheri-
cal granules of food reserves or yolk, whereas in the male none appear.
This functional and structural dimorphism is accompanied by a differ-
ence in nuclear behavior. In the male there appear to be as many as six
successive mitoses, as in the trophozoite, producing up to sixty-four
gametes. These are elongated slender, deeply staining bodies, largely
of nuclear substance, with one trailing flagellum and a second one lat-
erally attached to the anterior half of the body and free posteriorly.

The female gamete mother cell, on the other hand, undergoes no divi-
sions, and transforms directly into the egg, though indications of metab-
olic activity appear in deeply staining spherules adjacent to the parasite
in the host’s cytoplasm. This absence of divisions in the female gameto-
cyte, and their superabundance in the male, not only emphasizes a
metabolic contrast, but also on cytological grounds offers cytological
difficulties to the existence of maturation in these phases. These ob-
stacles, which Schaudinn (1900) left unresolved, were removed by the

576 THE AEIBPENGY GEE

discovery by Jameson (1920)—later extended by Dobell (1925), Na-
ville (1931), Yarwood (1937), and Noble (1938)—that the matura-
tion division takes place in the first division of the zygote and that, aside
from the diploid zygote itself, the rest of the cycle is a haploid one.

It is obvious that in the case of the male there is a fourth asexual
reproduction by multiple fission of a sixty-four-celled somatella, and
that this does not occur in the female.

This life cycle is typical in having an alternation of sexual and asexual
reproduction upon which are superposed certain features, in part adap-
tive and in part more fundamentally a part of the cycle. The first of
these features is the building up of multicellular somatellas numbering
respectively four, two, + sixty-four (x n), and + sixty-four in male
cells only, prior to multiple fission. The body thus formed is temporary,
lacking both nervous and hormonal mechanisms of integration to insure
the maintenance of interacting relations. The adaptive aspect lies in the
fact that these multiplicative reproductions make possible, with the least
expenditure of individuals, the quick utilization of the food supply in
the host’s intestinal cells.

This cycle from the cytological point of view, as well as the general
biological one, is atypical in the animal kingdom, though less so in the
plant kingdom, in that only the zygote is diploid and all of the rest of
the cycle is haploid. The fact that other Coccidiomorpha are known to
have the same limitation and that this subclass has affinities with the
flagellates, in some of which an odd number of chromosomes are known,
suggests that the primitive Protozoa are haploid and that the diploid
phase, like the polyploid, is a secondary evolutionary acquisition, de-
pendent, in part at least, on the union of individuals or gametes in
sexual reproduction. Thus both sex and sexual reproduction have had
their origin in the Protozoa. The limitation of the diploid phase to the
zygote only in Evmeria thus has a basic evolutionary significance.

Another feature of this life cycle which also has a basic significance is
the fact that every one of the four phases of asexual reproduction results
in the formation of a somatella of from two to sixty-four cells, and that
the sexual phase also leads to a four-celled somatella. This evidence
clearly indicates that these Protozoa are as truly multicellular, as are the
early stages of the Metazoa. They undergo asexual reproduction as do
Metazoa, from Porifera to Quints, but with this difference: that the

U shicn
\/

27 Asexual” Wy
Reproduction Jy 14
Pa f & Wh [r
IN tS

1502] Asexual (2
Reproduction
Para

Min

)

eS NWS

GO
Hy SX
W We X=
ee uu NV

/N MAN
SeSe52 /N ANOPHELES

Figure 138. Diagram of the life cycle of Plasmodium vivax, parasitic in Anopheles
and in the red cells of man. For convenience, the asexual reproductions of the sporoblast
are figured in the sexual cycle, since they are preparatory to the repeated multiple fissions
of the asexual cycle in man. By analogy with E/meria, maturation is post-zygotic and
both sexual and asexual phases are haploid except for the zygote. There is possibly an
asexual reproduction. (Modified from Schaudinn, 1902.)

578 THE LIFE ICYCEE

units into which they split are single cells, instead of flagellated cham-
bers or axial organizers.

THE LIFE CYCLE OF Plasmodium vivax

A second example of the same general pattern, with added speciali-
zations due to parasitism in two hosts, is found in the malarial parasite,
Plasmodium vivax, with the sexual and one (or two?) asexual phases
in the mosquito, Anopheles, and oft-repeated merogony in the red cells
of the blood of man, and a second asexual phase in the gamete mother
cells of the male only on transfer of these cells to a lower temperature
than that of the blood of man, as on a microscope slide or in the stomach
of the mosquito.

THE LIFE CYCLE OF Paramecium caudatum

The life cycle of Paramecium caudatum makes a definite evolutionary
advance in the Ciliata in two mutually interdependent features. The
first is the differentiation of sex and somatic cells in the same individual,
and the second is a permanent multicellular condition of two cells, de-
rived from an undifferentiated eight-celled cleavage stage.

The original description of the cell, the selection of its name, the
focusing of attention on total cleavage, with plasmotomy in embryology,
rather than upon mitosis, all have combined to emphasize the separation
of one cell from another by a wall or dividing structural boundary. These
are all minor considerations. On the other hand, the significance of
derivation, continuity in time, physiological functions, and above all of
genetics, focus attention on the nucleus and the cytoplasm associated
with it or brought in in the normal sequence of growth, fertilization,
appropriation, or experiment under its control. These are all major con-
siderations. In this modern sense it is biologically medieval to refer, as
do many textbooks and other works, to Paramecium as a unicellular
organism. It is biologically quite as logical to call a whale unicellular.
Both start their cycles as one cell and both achieve multicellularity. No
great biological significance attaches to the particular number of cells
in the multicellular body, except during maturation. The significant
achievement is the differentiation of sex and somatic cells. One of the
primary distinctions in function, as well as in embryological origin, in
the multicellular metazoan is this differentiation.

Cc
uU
V

Asexual cycle
xN

Binary fission

Fertilizatio

|
I

Figure 139. Diagram of the life cycle of Paramecium caudatum exhibiting an alterna-
tion of asexual reproduction, or binary fission of the two-cell somatella, and sexual re-
production with mutual fertilization of conjugants. The first and second divisions of the
eight-cell somatella, or exconjugant, are for convenience included in the diagram of the
sexual cycle, since they are preparatory to the asexual phase.

580 THE LIRE, GYGLE

The life cycle of Paramecium is further complicated by the fact of
conjugation and mutual fertilization of the conjugants. Sexual dimor-
phism is not evident between the conjugants as a whole, but appears in
the behavior of the gamete nuclei. The migrant one is assumed to be
male because of its motility, and the resident one female because of the
lack of this quality. Dimorphism of the conjugants is structurally evi-
dent in Vorticella, in which the males are small and the females large.

The biological life cycle in Paramecium starts with the zygote, formed
from the body of a conjugant by the fusion of the two haploid nuclei,
one from the immigrant male gamete and the other the resident nucleus
of the egg. The cleavage nucleus thus formed utilizes the cytoplasm of
the egg, as in the Metazoa, with only a small amount from the male
gamete. The old macronucleus in each continues (Fig. 139, I-IV) to
disintegrate and is soon entirely metabolized into cytoplasm as food.
This is the death of the soma of the conjugant, the future of which ts
henceforth under a new genetic control. There then ensue three succes-
sive mitotic divisions (Fig. 139, II-VI), representing the cleavage of
the egg to an eight-celled somatella, when cleavage abruptly stops (Fig.
139, VI) and differentiation into four somatic and four sex cells occurs
by the enlargement of the nuclei of the former and an increase in their
chromatin. The four sex cells do not all survive. Three of them dis-
integrate at once, leaving a somatella of five cells, four somatic and one
sex cell. Then begins asexual reproduction which in two peculiar binary
fissions distributes the four macronuclei among the four daughter schi-
zonts, with an accompanying division of the sex cell or micronucleus
at each of the two fissions. In the diagram these two asexual fissions have
been included in the sexual cycle, since they are necessary to restore the
organism to the pattern in which regular asexual reproduction prevails.
They otherwise belong in the asexual period.

The precise period in which maturation occurs in the sexual cycle ts
perhaps undetermined. It has been generally assumed that it occurs in
the first two divisions of the micronucleus in the conjugant, in which
case its third division would be an asexual reproduction of the gamete.
This view does not rest upon exact chromosome count. The occurrence
of post-zygotic maturation in the Sporozoa suggests the possibility of its
occurrence in the Ciliata also. This view is further supported by the death
of three of the post-zygotic sex nuclei and in Paramecium by the un-

THES PIRE CYCLE 581

necessary third mitosis of the pronuclei in the conjugants. The evidence
for chromosome reduction in the conjugants (Calkins and Cull, 1907)
is inconclusive, because of the small size and the large number of the
chromosomes in the three divisions prior to the formation of the zygote.
A cytological examination of chromosome number during conjugation, in
some ciliate with a small number of large chromosomes, may throw
light on this problem. Both pre and post-zygotic alternatives should be
explored.

The asexual cycle proper of Paramecium is one of oft-repeated simple
binary fission, prior to which the two-celled somatella may grow, and
by nuclear division become a four-celled one for a brief period. This
cycle is one of indefinite duration.

This survey and interpretation of life cycles among the Protozoa ex-
hibit the basic similarity of this fundamental characteristic of organ-
isms among them to those emergent among the Metazoa. Peculiar to
the Protozoa is the absence of sexual reproduction in the life cycles of
the more primitive forms, among which it appears that they live a
haploid life and that sexual reproduction has not as yet been evolved.
Rare cases among Metazoa and the Metaphyta of the seeming absence
of sexual reproduction are obviously secondary phenomena, but this
interpretation is less defensible for the primitive Protozoa.

In the higher Protozoa, as in the Metazoa, the life cycle includes
maturation, fertilization, cleavage to a multicellular stage, histogenesis
of organelles, asexual reproduction with resulting functional individuals
of differing structure in the different asexual phases, sexual dimorphism,
adolescence, gametogenesis, senescence, and death.

The emphasis so generally placed upon the unicellular phase of the
Protozoa, as against all Metazoa, tends to obscure their basic similarity
in life cycle to that of the Metazoa, and thus to minimize the biological
significances of the varied evolutionary accomplishments which have oc-
curred in this primitive phylum. Similarities in biological phenomena
are the bases on which an integrated concept of the evolution of life
can be erected.

LITERATURE CITED
Biitschli, Otto. 1876. Studien tiber die ersten Entwicklungsvorginge der

Eizelle, die Zelltheilung und die Conjugation, Abhl. senckenb. naturf.
Ges., 10: 213-452, 15 pls.

582 THE LIE FeYCLe

Calkins, G. N., and Sarah W. Cull. 1907. The conjugation of Paramoecium
aurelia (caudatum). Arch. Protistenk., 10: 375-415, pls. 12-18.

Dobell, Clifford. 1925. The life-history and chromosome cycle of Aggregata
eberthi, Parasitology, 17: 1-136, 6 pls., 3 figs. in text.

Hertwig, Richard. 1889. Uber die Conjugation der Infusorien. Abh. bayer.
Akad. Wiss., Math.-Natur. KI., 17: 151-234, 4 pls.

Hinshaw, H. C. 1926. On the morphology and mitosis of Trichomonas
buccalis (Goodey) Kofoid. Univ. Cal. Publ. Zool., 29: 159-74, 1 pl.
2 figs. in text.

Jameson, A. P. 1920. The chromosome cycle of gregarines, with special refer-
ence to Diplocystis schneideri Kunstler. Quart. J. micr. Soc., London,
64: 207-66, pls. 12-15.

Kofoid, C. A., and Olive Swezy. 1915. Mitosis and multiple fission in
trichomonad flagellates. Proc. Am. Acad. Arts and Sci. Wash., 51:
290-371, 8 pls., 7 figs. in text.

— 1921. On the free, encysted, and budding stages of Councilmania
lafleuri, a parasitic amoeba of the human intestine. Univ. Cal. Publ. Zool.,
20: 169-98, pls. 18-22, 3 figs. in text.

Naville, André. 1931. Les Sporozoaires (cycles chromosomiques et sexualité) .
Mem. Soc. Phys. Genéve, 41: 1-223, 3 tables, 150 figs. in text.

Noble, E. R. 1938. The life cycle of Zygosoma globosum, sp. nov., a gregarine
parasite of Urechis caupo. Univ. Cal. Publ. Zool., 43: 41-66, pls. 7-10,
3 figs. in text.

Schaudinn, Fritz. 1900. Untersuchungen uber Generationswechsal bei
Coccidien. Zool. Jb. Abt. Anat., 13: 199-292, pls. 13-16.

— 1902. Studien tber krankheitserregende Protozoen. II. Plasmodium
vivax (Grassi and Feletti) der Erreger des Tertianfiebers beim Menschen.
Arb. GesundAmt. Berl., 19: 169-250, pls. 4-6.

Yarwood, Evangeline A. 1937. The life cycle of Adelina cryptocerci sp. nov.
a coccidian parasite of Cryptocercus punctulatus. Parasitology, 29: 370-90,
pls. 15-19, 1 fig. in text,

CHAPTERS XIE

FERTILIZATION IN PROTOZOA

JOHN P. TURNER

MUCH HAS BEEN WRITTEN on the phenomena which accompany fertili-
zation in the Protozoa. For detailed analyses of this literature the reader
is referred to texts by Minchin (1912), Doflein-Reichenow (5th Ed.
1929), and Calkins (1933). The aim of this chapter is to give a bird’s-
eye view of the subject, to present some of the more significant facts al-
ready discovered, and last and most important to point out the need for
investigation to determine those facts and principles still awaiting dis-
covery.

If we consider sex to be essentially the formation of gametes and
the fusion of those gametes in the fertilization process, we are using the
term sex in a somewhat broader sense than if we limit it to the differ-
ence or distinction between the two sexes. In the Metazoa these phe-
nomena seem to be fairly uniform for all groups; consequently, when
the basic principles of the process are understood for one animal, those
same principles may be applied to all the higher animals. Until very
recently, however, the Protozoa were thought to belong in a different
category, and one did not apply to them the general laws which were
considered applicable to all other animals.

With recent discoveries, more and more of these preconceived dif-
ferences have disappeared and we are now faced with the question of
how close we can draw the analogies in sex phenomena between the
Protozoa and the Metazoa. In other words, are the fundamentals of
sex, 1.e., maturation of gametes and fertilization, common to all animals,
both metazoan and protozoan? If so, how similar or how dissimilar are
the processes, and if not, just how do they differ? No final answer can
be given to these questions in our present state of knowledge, but con-
siderable evidence may be pointed out that is extremely significant.

In the Metazoa fertilization is accomplished by a small, active micro-
gamete (spermatozo6n) penetrating and fusing with a large, nonmotile

584 FERTILIZATION

macrogamete (ovum). The difference in appearance between the male
and the female gametes, as well as between the two kinds of animals
which produce them, is so clearly recognizable in most cases that we have
come to think of sex in terms of the differences between maleness and
femaleness. Among the Protozoa we find some species which also show
a clear differentiation between male and female gametes, even though
the sex differences between the organisms producing them are not so
apparent. The protozoan organism is unicellular, and in many cases this
single cell produces both male and female gamete nuclei in a kind of
hermaphroditism (e.g., wandering and stationary pronuclei in ciliates).
This complication makes the homologies between Metazoa and Proto-
zoa less easily understandable.

In a great many Protozoa there is no apparent differentiation between
gametes, yet their formation and fusion are accompanied by the same
fundamental processes as is the case with differentiated gametes. Isog-
amous reproduction, therefore, is considered a sexual process.

The difference between the individual and the gamete is not always
clear in Protozoa. Perhaps the most primitive kind of sexual phenomena
is exemplified by two Protozoa, apparently identical to the vegetative
forms, coming together and fusing in a fertilization process. According
to Dobell (1908), this occurs in the flagellate Copromonas subtilis.
Nuclear “reduction” occurs after partial fusion of the cell bodies and
before nuclear fusion (Fig. 140).

COPULATION

GAMETIC MEIOSIS AND FERTILIZATION

Copulation, the complete and permanent fusion of gamete cells, is
the type of sexual activity found generally in the Plasmodroma. In cases
in which the parent organism gives rise to specialized cells which per-
form in fertilization, the process is known as fertilization by union of
gametes, or simply gamogamy. Both isogamy, the union of similar
gametes, and anisogamy or heterogamy, the union of dissimilar gametes,
are found in this group. In cases in which gametes are as extremely dis-
similar as spermatozoa and ova, the union is sometimes referred to as
odgamy. In cases in which the organism itself fuses with another or-
ganism in permanent union, the whole organism functions as a gamete
and the process is called hologamy.

FERTILIZATION 585

In all of these cases, presumably, maturation of nucleus occurs
to prepare it for union with its mate. In most cases maturation takes
place in the last two divisions of the nucleus, before formation of the
fusion nuclei or pronuclei. These two meiotic divisions are similar to
those characteristic of spermatogenesis and odgenesis in Metazoa (see
Sharp, 1934). The result of meiosis is the halving of the chromosome
number to the haploid condition, so that fusion of the two haploid

Figure 140. Copromonas subtilis in hologamous copulation. A, vegetative form; B,
two individuals beginning to fuse anteriorly; C, cytoplasmic fusion well under way,
nuclei in heteropolar, second ‘‘reduction” division; D, zygote with synkaryon and single
flagellum. (After Dobell, 1908.)

gametes will reéstablish the diploid number which is characteristic of
the species.

In the Mastigophora, syngamy has been described in very few forms
except among the Phytomastigina, in which it seems to be the general
rule. A typical case of hologamous copulation was described by Dobell
(1908) in the colorless phytomonad Copromonas subtilis (Fig. 140).
Two organisms which appear identical to each other and to ordinary
vegetative forms come together and partially fuse. This partial union
evidently acts as a stimulus to the nucleus of each gamont, for it pro-
ceeds to undergo two “maturation divisions’ before fusing with its mate.
Of the two products of the first progamic division, one degenerates and

586 FERTILIZATION

the other divides again into two very unequal parts. The smaller de-
generates and the larger is the functional pronucleus, which fuses with
the pronucleus of the other member of the pair. Superficially, this type
of reduction is strikingly similar to polar body formation in metazoan
odgenesis, although the actual reduction in chromosome number was
not established by Dobell. The assumption that the two nuclear divisions
preceding syngamy are reduction divisions seems reasonable, consider-
ing the almost universal occurrence of two divisions in the maturation
of gamete nuclei. However, we are not justified in concluding that re-
duction in chromosome number occurs, unless positive determinations
can be made of the chromosome number before and after reduction. In
Copromonas there is obvious reduction in the amount of chromatin
when all but one product of the two divisions disintegrate; but reduc-
tion in chromosome number has not been demonstrated, although it
must occur somewhere in the life cycle, if chromosomes exist in this
species. From the genetic angle, this is a critical point and should be
determined if possible. After syngamy the zygote may encyst or it may
develop directly into a vegetative form.

Hologamous fertilization has been reported in a few other members
of the Plasmodroma, but is not confined to this group if Brumpt’s
(1909) description of fertilization in the parasitic ciliate Balantidium
coli is correct. In this case two individuals come together and are in-
vested by a common membrane, as in pseudoconjugation of gregarines.
But the two balantidia then fuse completely and permanently. Other
workers have not supported Brumpt’s description and, although the
details differ with species and author, Jameson (1927), Scott (1927),
and Nelson (1934) agree that conjugation and not copulation is the
form of sexual union found in this ciliate.

The occurrence of fertilization has been reported for a number of
flagellates, but in very few cases has the evidence been convincing ex-
cept for the Phytomonadida. Goldschmidt (1907) gave a detailed de-
scription of a sexual cycle in the animal flagellate Mastzgella, similar
in type to those described for Arcel/a and other Rhizopoda. If substanti-
ated, another close link between the Pantostomatida and the Rhizopoda
will be established. According to Goldschmidt, vegetative forms de-
velop into macrogametocytes and microgametocytes, the nuclei of which
give off chromatin into the cytoplasm. These chromidia in turn produce

FERTILIZATION 587

secondary nuclei, each of which appropriates some cytoplasm, forms a
gamete, and undergoes reduction. Active flagellated macrogametes seek
out and fuse with nonmotile microgametes, in contrast to the usual
method. The zygote retains the flagellum and reproduces its monad-like
self for several generations by fission. Then the offspring develop into
the adult vegetative Mastzgella.

Heterogamy seems to be clearly established in this amoeboid flagellate
by the differences in the activity of the gametocytes as well as in the dif-
ferences in size and motility of the gametes. Another point of interest
is the pedogenic reproduction of the zygote. The interpolation of this
asexual cycle into the life cycle bears a strong similarity to sporogony
in Plasmodium and other Sporozoa. In both cases it is the asexual multi-
plication of the zygote before the adult stage, or trophozoite, is formed.

A point of particular interest to cytologists and geneticists alike is
the origin of the gamete nuclei from chromidia. Not only here but also
in a number of the Sarcodina, the origin of gamete and vegetative nuclei
from chromidia has been reported. If the chromidial origin of nuclei
is a fact, what of the genetic and structural continuity of the chromo-
somes and of the genes? Must we accept Hartmann’s (1911) ‘“‘poly-
energid”’ interpretations that the nucleus is really an aggregate of many
small nuclei, each with its sphere of influence, and that chromidia repre-
sent the scattered little nuclei or energids? At present we can only specu-
late. The problem is one of fundamental significance and is in great
need of further investigation.

Chatton (1927) described a case of gametic meiosis in the flagellate
Paradinium poucheti which is ‘exactly comparable in its progress and
complexity’’ to spermatogenesis in certain insects. Included in his out-
line of this process are leptotine and pachytine stages, and diakinesis
with tetrad rings and crosses. It is surprising that such highly developed
processes should be found in such a primitive flagellate, although similar
stages are not uncommon in ciliates and also occur in some Sporozoa
and Sarcodina. The wide distribution of typical meiotic phenomena indi-
cates that they are fundamental in nature, and it is probable that proc-
esses of comparable nature also occur in all forms in which fertilization
takes place. Meticulous examination with improved techniques will
throw much light on this question.

In the Dinoflagellata a few cases of syngamy have been reported, but

588 FERTILIZATION

most of them are too fragmentary or are supported by too little evidence
to be discussed here.

An extremely interesting case has recently been reported by Diwald
(1938) in Glenodinium lubiniensiforme. In this form, four flagellated
isogametes are produced by the subdivision of each parent protoplast,
and these gametes will copulate only with gametes of certain other
clones. The obvious question is, of course, what is the nature of the
difference between these ‘“-” and ‘‘—”’ strains, a difference which in-
hibits their copulating among themselves or stimulates them to copulate
with gametes of the other strain? This problem is yet to be solved, but
it is similar to that found in Paramecium aurelia by Sonneborn (1937)
and in P. bursaria by Jennings (1938). Diwald states that after fertili-
zation the zygotes rest, then germinate and undergo two reduction divi-
sions to form a ‘‘tetrad”’ of four potential individuals, only one of which,
however, usually persists. This is the only described case of zygotic
reduction outside the Telosporidia, with the possible exception of the
amoeba Sappinia diploidea (see p. 595 below). A reasonable doubt re-
mains, however, as to Diwald’s interpretations. He gives no chromo-
some counts that would support his contention, and any assumption of
chromosome reduction not based on determinations of chromosome
numbers before and after reduction, especially in such an unusual case,
is Open to serious question. Dinoflagellates are not popular subjects for
cytological investigation at the present time, but perhaps the work of
Diwald will stimulate further research in this group.

In Ceratium hirundinella fertilization is accomplished in a way simi-
lar to that of the filamentous algae, according to the description of Zeder-
bauer (1904). Two flagellates come together, the protoplasm of each
extrudes from the lorica and makes contact with that of the other. The
two masses now copulate, forming a zygote outside the loricas. Zeder-
bauer observed these protoplasmic fusions only in the living state, so
his account leaves much to be desired in the way of cytological details
on which to base sound conclusions.

Chatton and Biecheler (1936) have more recently reported fertili-
zation by slightly anisogamous gametes in the parsitic form Coccidinium
mesnili.

In Noctiluca scintillans (miliaris) gamete formation has been re-
peatedly reported. In recent accounts Pratje (1921) could find no

FERTILIZATION 589

conclusive evidence for copulation, while Gross (1934) described copu-
lation of isogametes. It seems odd that the life history of this abundant
and spectacular species should still be a matter of such uncertainty.

Sexual reproduction is widespread among the Phytomonadida. These
plant-like flagellates illustrate so nicely the gradations in sexual develop-
ment and differentiation that they have been favorite material for class-
room instruction. 2

Chlamydomonas is a non-colonial genus among the species of which
both isogamy and heterogamy are found. In C. Stemi, according to
Goroschankin (1891), the flagellate divides into many isogametes within
a cyst. The gametes fuse, beginning at their flagellated ends, and zygotes
are formed which develop into resistant cysts. In C. brauniz, the same
author (1890) describes gametes of different sizes fusing in anisogamous
copulation. Besides being definitely though not pronouncedly smaller,
the microgamete is slenderer and more pyriform than the macrogamete.
In still another species the differentiation is still more striking. Some
individuals of C. coccifera (Goroschankin, 1905) are transformed di-
rectly into large nonmotile, egg-like macrogametes, while others divide
into relatively very small, flagellated, sperm-like microgametes.

Chlamydomonas, therefore, illustrates possible stages in the evolution
of sex from isogamy, in which the gametes are smaller than vegetative
individuals but otherwise similar; through early differentiation of gam-
etes, wherein the gametes are only slightly though clearly differentiated
in size and therefore exhibit the very beginnings of anisogamy, to ex-
tremely well-differentiated heterogamy or odgamy, in which the micro-
gametes and macrogametes are almost typical sperms and eggs. Only one
more fundamental advance has been made in the evolution of sex in the
Metazoa, and that is the differentiation of the adult forms into male and
female individuals. Structural developments for the production and care
of offspring belong to a different category.

It is unfortunate that so little is known of the maturation processes
in Chlamydomonas. It is not known where reduction occurs, much less
what the nature of the chromosomes and their behavior in reduction are.
Some investigators assume that because of the way in which the zygotes
of Chlamydomonas, Gonium, Pandorina, and so forth, behave at divi-
sion, reduction is zygotic. Dangeard (1898) found no nuclear reduction
taking place before fertilization in Chlamydomonas and suggested that

590 FERTILIZATION

it occurs during the division of the zygote. Pascher (1916), in his work
on Mendelian inheritance in Chlamydomonas, presented genetic evidence
that reduction is zygotic—that is, reduction in chromosome number
occurs in the first two divisions of the zygote, which produce four
swarmers. This would mean that only the zygote is diploid and that all
other stages in the life cycle are haploid (see p. 611 below).

Here, indeed, is a peculiarly promising opportunity for the correla-
tion of cytological and genetic evidence of chromosome behavior, if
only the cytological data were available. Meiosis, not complicated by
subsequent fusion of gametes, and the attendant bringing together of
homologous chromosomes would offer some interesting possibilities.

Another excellent example of a series of organisms exhibiting pro-
gressively advancing stages in the evolution of sex is found in the colonial
phytomonad flagellates. This series is so well known that it is usually
discussed even in textbooks on general biology.

At one end of the series is Goniwm pectorale. At certain times the
sixteen cells making up this flat colony function as gametocytes by pro-
ducing isogametes, which copulate in pairs to form zygotes. The gametes
may vary somewhat in size, but the manner in which they copulate—
apparently at random with any of the others—indicates that the slight
variation in size of the gametes is without significance. In Stephano-
Sphaera pluvialis, a colony of eight cells, the gametes are all identical.
The chief advance which these isogamous Volvocidae exhibit over the
Copromonas type of fertilization is that the gametes are different from
the vegetative form. In other words, vegetative or asexual forms may
be distinguished from sex cells.

In Pandorina morum (Pringsheim, 1869), a subspherical colony of
sixteen cells, two distinct sizes of gametes are produced, and two combt-
nations are possible. Small gametes may fuse with other small gametes,
and small gametes may fuse with large ones. Large gametes, however,
never fuse with other large ones. Here, then, in a single species is ex-
hibited both isogamy and heterogamy, for the failure of the large gam-
etes to fuse with each other indicates that the size difference is significant.
The critical factor may be the size itself, or it may be some less obvious
factor associated with size.

It might be argued from this that primitive heterogamy is associated
with hermaphroditism. The same colony produces both large and small

FERTILIZATION BION

gametes; so, if the size difference means a step in the direction of male-
ness and femaleness, then the colony is monoecious or hermaphroditic.
This obviously leads to the conclusion that, in the evolution of sex in
these forms, gametes became differentiated into male and female types
before the parent organisms did. The fact that a larger percentage of
primitive Metazoa exhibit hermaphroditism than do the higher forms
lends weight to the assumption that this is a general truth.

In Exdorina elegans differentiation of gametes has become very
marked, and in Volvox the series is climaxed by such extreme differentia-
tion between the microgametes and the macrogametes as is seen in meta-
zoan sperms and eggs. Furthermore, the vegetative cells of the Volvox
colony are comparable to the somatic cells of the Metazoa, while rela-
tively few cells of the colony carry on the germ line. Another advance
seen in Volvox is that some species have developed the dioecious
condition, wherein some colonies produce only microgametes and others
produce only macrogametes.

Among the Infusoria are found a few cases in which gametes are
formed that unite in complete and permanent fusion. This process is
therefore copulation, rather than the usual ciliate conjugation (see p.
617). In most cases of copulation in ciliates, the gamonts undergo re-
peated divisions, which result in the production of numerous small
“microgametes’’ which copulate with each other. Thus copulation of
gametes has been described for Trachelocerca phoenicopterus (Lebe-
dew, 1909), in the Opalinidae (Neresheimer, 1907; Metcalf, 1923),
and in Glaucoma (Dallasia) frontata (Calkins and Bowling, 1929).
While these gametes may differ a little in size and be called ‘‘micro-
gametes’’ and “macrogametes’”’ by some, the differences do not appear
to be very significant. In general, they bear considerable resemblance
to the trophic individuals except in size. In the Vorticellidae, however,
gametes are formed which are truly anisogamous and which fuse per-
manently, although cytologically they more nearly resemble anisogamous
conjugants (see p. 621 below).

In the ciliate Metopus sigmoides, Noland (1927) described a sexual
process which is somewhat intermediate between copulation and conju-
gation. Conjugants come together and join anteriorly, but instead of
exchanging pronuclei as conjugants usually do, most of the cytoplasm
and both pronuclei of one member of the pair pass over into the body

592 FERTILIZATION

of the other member, leaving behind only a shrunken remnant of the
donor, which then detaches itself from the recipient and dies. This
process is functionally very similar to that which occurs in the Vorticelli-
dae, while the differences in structural details serve as a connecting link
to typical ciliate conjugation.

Sexual phenomena seem to be fairly common in the Sarcodina, but
they are not so characteristic of the group as was thought by many of
the earlier workers.

In spite of the many reports of sexual stages in Amoeba proteus, sev-
eral recent investigators have failed to observe any type of reproduction
other than fission. Johnson (1930), whose article includes a review
of the literature, believes that parasites and aquatic fungi have led to
many misinterpretations of the life cycle of Amoeba proteus. Liesche
(1938) carried A. proteus through 800 generations and observed no
sexual stages and no cysts. It is quite possible that there is a sexual
stage that occurs only at long intervals, or only under conditions which
ordinarily do not obtain in the laboratory. It is obvious that if sexual
stages occurred very often in this form, it would be reported more
frequently and more convincingly than it has been, in view of the fact
that this species is cultivated and studied so constantly in scores of
biological laboratories. For instance, at the University of Minnesota
A. proteus has been cultivated continuously for nine years, during which
period thousands of observations have been made and several hundred
permanent slides have been prepared from time to time. In spite of
this prolonged search, no sexual stages have ever been found. Binucleate
forms, presumably early dividing stages, have been observed frequently,
but nothing suggesting gamete formation has ever been noted. It is
true that sexual stages could have occurred and escaped observation, for
examinations have not been made daily, but it seems reasonable to sup-
pose that they would have been discovered at some time if they occurred
at any but the rarest intervals.

However, Jones (1928) confirms the earlier work of Calkins (1907)
and others, with descriptions and photomicrographs of gamete forma-
tion by fragmentation of the primary nucleus. He further claims that
fertilization is accomplished by means of flagellated gametes.

A skeptic might point out that his photomicrographs of gametes and
zygotes (his Figs. 13, 15, Plate 11) are strikingly similar to the figures

FERTILIZATION 593

of the parasite Sphaerita in Amoeba limax, as pictured by Chatton and
Brodsky (1909, Figs. 2, 3). The possibility of confusing sporulation of
Sphaerita with gamete formation in Amoeba is not too remote to be
considered; although, as Calkins has pointed out, a parasitologist is in-
clined to see parasites in everything, and the parasite explanation has
probably been over emphasized. The contradictory reports leave us in
the peculiar position of not being very sure of the life cycle of our best
known and most widely used protozodn, “the common laboratory
Amoeba.”

Fertilization processes have been described for a number of other
amoebae, including Pelomyxa palustris (Bott, 1907) and Sappinia
(Amoeba) diploidea (Hartmann and Nagler, 1908). Bott’s account of
fertilization in Pelomyxa is unusual indeed. The nuclei of this multi-
nucleated plasmodium extrude vegetative and generative chromidia into
the cytoplasm. The chromidia form secondary nuclei, which in turn cast
out the vegetative chromatin. The secondary nuclei, which now contain
only generative chromatin, undergo the first maturation division, in
which the chromosome number is reduced from eight to four. In the
second maturation division four chromosomes appear and split, so that
four go to each pole. Now each granddaughter nucleus divides into two
compact masses of chromatin and a vacuole is formed near-by. The
chromatin of the two masses then migrates into the adjacent vacuole,
in the form of minute granules. After receiving the chromatin, the vacu-
ole forms a membrane and becomes the definitive pronucleus of the
gamete. The pronuclei which have arisen in this unique manner ap-
propriate some cytoplasm and wander out as heliozodn-like gametes,
which copulate in pairs to form zygotes. Each zygote grows into a new
multinucleate Pelomyxa.

Aside from the peculiar role played by the vacuole in this maturation
process, which introduces a sort of modified autogamy into the cycle
just before the regular fertilization process, the cycle is worthy of further
examination. Formation of secondary nuclei from chromidia, which in
turn have resulted from the extrusion of chromatin from primary nuclei,
has been described in many Sarcodina, several Sporozoa, at least one
flagellate (Mastigella, see above) and one ciliate (Trachelocerca phoent-
copterus, Lebedew, 1909).

Many protozodlogists remain skeptical of the entire proposition of

594 FERTILIZATION

the chromidial origin of nuclei (Doflein, 1916). Kofoid (1921) says
“The evidence thus far presented of the de novo chromidial origin of
protozoan nuclei is wholly inadequate to establish this hypothesis.” Intra-
cellular parasites are held responsible for some of the misinterpretations.
More recent investigations have clearly refuted at least some of the
earlier reports of the chromidial origin of nuclei. The reports of Myers
(1935, 1938) and of Le Calvez (1938) on Foraminifera are good ex-
amples of this. However, many other reports must be reinvestigated
before we can establish any very firm basis for our views.

Calkins (1933, p. 70) points out that the chromidial net of Arcella
stains green with the Borrel mixture and usually gives a negative re-
action to the Feulgen treatment. This supports Hartmann’s experiments,
in which the chromidia were dissolved out by pepsin, while the chromatin
of the secondary nuclei remained conspicuous. Bélat (1926) believes
this is conclusive evidence that chromidia are not composed of chromatin.
However, Calkins shows that by omitting the strong hydrolysis of the
Feulgen reaction, the chromidia are positively stained and therefore are
composed of chromatin, or at least that nucleic acid is present in them.
Nucleic acid becomes more concentrated in the nuclei, and this may
explain why the nuclei resist pepsin digestion while the residue is dis-
solved. The author can confirm Calkins’s positive results in staining Ar-
cella chromidia with Feulgen. This organism has been stained with
Feulgen at many stages in its life history by omitting strong hydrolysis,
and intense staining of both chromidia and nuclei has resulted.
Chromidia are colored an intense purple in forms containing nuclet,
as well as in forms in which no detectable nuclei are present. It is not
impossible that in the latter forms, some of the larger chromidia are
actually minute nuclei which are lineal descendants by mitosis of the
original nuclei.

According to Elpatiewsky (1907) and Swarczewsky (1908), the life
cycle of Arcella vulgaris is extremely complicated. In addition to several
methods of asexual reproduction, both chromidiogamy and anisogamous
syngamy occur. In chromidiogamy two Arce/la, the nuclei of which are
degenerating into chromidia, come together. The protoplasm of one
passes over into the shell of the other and, after the intermingling of
the chromidia, half of the protoplasm passes back into the first shell,
and the two organisms pull apart. After separation, the chromidia of

FERTILIZATION 595

each individual give rise to the nuclei of amoebulae, which bud off and
grow into new adults. Zuelzer (1904) described chromidiogamy in D/f-
flugia urceolata, but in this case all the chromidia are said to fuse into
a single mass, and the united protoplasmic bodies condense and form a
cyst. New nuclei form from the chromidia.

The significance of chromidiogamy has never been satisfactorily ex-
plained; in fact, the existence of the process itself remains in consider-
able doubt. While it is true that specimens of Arcel/a may frequently
be found in which no typical nuclei are visible and the cytoplasm of
which may contain numerous chromidia, these may be degenerating
forms, and only a thoroughgoing reinvestigation of the life history of
this interesting organism will convince the skeptics or disillusion the
credulous.

In Elpatiewsky’s account of anisogamy, some individuals form macro-
gametes by repeated nuclear division, while others form microgametes.
The gametes are amoebulae, and the difference between male and fe-
male is one of size. After copulation between large and small gametes,
the zygotes grow up into adult arcellae.

A remarkable type of sexual process was described by Hartmann and
Nagler (1908) in Sappinia (Amoeba) diploidea, a binucleate form
(Fig. 141). The active organism contains two nuclei, derived originally
from two parents. It is therefore a kind of adult prezygote. Two such
binucleate amoebae come together and develop a common cyst, but their
bodies do not fuse. In each amoeba the two nuclei now fuse in a long-
delayed fertilization, or karyogamy, after first giving off “vegetative
chromidia.” The cytoplasms of the two amoebae now fuse completely.
Each synkaryon undergoes two “‘reduction divisions,” after which three
products of each degenerate, leaving one reduced nucleus from each
synkaryon. These two are the nuclei of the vegetative form. If these two
divisions are in reality meiotic divisions, the organism lives a haploid
existence, and constitutes the only known case of zygotic reduction in
the Sarcodina. If not, some other interpretation must be found for the
two divisions which follow syngamy. Since chromosome number and
behavior are not known in this form, no satisfactory conclusions may
be drawn. It may be argued, of course, that the two haploid nuclei, lying
close together in the cytoplasm, are the equivalent of one diploid nucleus,
but such speculation must await the positive determination of the chromo-
some behavior.

596 FERTILIZATION

Another noteworthy point here is that if the two amoebae which en-
cyst together are derived from the same parent, the process is a case of
autogamy or pedogamy; if not, it is delayed hologamy.

Figure 141. Sappinia (Amoeba) diploidea. A, the binucleate vegetative form; B, two
such individuals (sister cells ?) encyst within a common capsule and in each amoeba
the two nuclei fuse together; C, the bodies of the two amoebae now unite, and the two
fusion nuclei undergo two “reduction divisions,’ C, D, after which the nuclei lie side
by side, as in A, throughout the vegetative period. (After Hartmann and Nagler, 1908.)

The life history of the Foraminifera has been a subject of controversy
for many years. Since the pioneer researches of Lister (1895), which
were confirmed by Schaudinn (1903) and others, it has been generally
believed that the life history of Polystomellina crispa is fairly repre-
sentative of the group. According to these investigators, alternation of

FERTILIZATION 597

sexual (macrospheric generation — gamont) and asexual (microspheric
generation — agamont) generations occurs, and the two generations
may be distinguished morphologically, chiefly on the basis of the rela-
tive size of the original chamber of the shell. The protoplasm of the
two adult generations was said to fragment, to produce flagellated iso-
gametes from the gamont and agamete amoebulae from the agamont.
Fertilization occurs free in the water, and the zygotes develop into
agamonts, while the amoebulae develop directly into new gamonts. The
nuclei of both the agametes and the gametes were said to arise from
chromidia which are derived from the fragmentation of the primary
nuclei.

In recent studies on the Foraminifera, Myers (1935, 1936, 1938) has
confirmed the earlier work of Lister and Schaudinn, except for the origin
of the gamete and agamete nuclei. In Patellina corrugata, Polystomellina
crispa, Spirillina vivipara and Discorbis patelliformis, the nuclei of all
stages, according to Myers, are derived by an orderly process of mitotic
divisions from preéxisting nuclei. He believes that the chromidia are
“concerned with feeding and metabolic activities’’ and in no case give
rise to nuclei. This is another blow to those who hold to the chromidial
origin of nuclei in Protozoa.

Myers (1935) further states that gametic reduction occurs in Pa-
tellina corrugata and that the haploid number of chromosomes 1s twelve.
These observations differ from those of Schaudinn on the same species.
In P. corrugata and in S. vivipara, the isogametes are amoeboid, but in
D. patelliformis and Polystomellina crispa they are biflagellated, as indi-
cated by the earlier workers.

In some forms, two or more gamonts become more or less closely as-
sociated in a kind of pseudoconjugation known as a syzygy, wherein the
pseudopodia may temporarily fuse with those of close neighbors, while
in other species they may encyst in a common capsule. This intimate
association possibly has a synchronizing effect on gamete formation.

Le Calvez (1938) supports Myers’s contention that gamete nuclei
are not derived from chromidia, but arise by mitotic divisions from pre-
existing nuclei. In Iridia lucida, he states, the secondary nuclei ‘‘disinte-
grate’ by rapid divisions which at first are typically mitotic. Later, be-
cause they are so small and the character so obscure, the mitoses are
recognizable more by the centrosome than by the clarity of the chromo-

598 FERTILIZATION

somes. Concerning the origin of the secondary nuclei, Le Calvez states
that he “has not been able to discover the chain of processes which, from
the disintegration of the vegetative nuclei, lead to the formation of a
well defined micronucleus” (secondary nucleus). He believes that the
hypothesis of generative chromatin ought to be completely abandoned.

In the Actinopoda, sexual reproduction has been reported for both
Radiolaria and Heliozoa, but in only two forms has the process been
reliably described. The classical case is that of Actinosphaerium eich-
hornii (Hertwig, 1898). The multinucleated vegetative individual forms
a ‘mother cyst’ and absorbs all but a few (up to 20) of its nuclei. The
cytoplasm divides into as many primary cysts (cytospore number one)
as there are nuclei. Each primary cyst divides into two distinct secondary
cysts (cytospore number two), the nuclei of which undergo two succes-
sive “reduction” divisions, resulting in one pronucleus and two “polar
bodies’? each. The matured secondary cysts reunite with their sisters as
gametes, and the nuclei fuse to complete fertilization. This is obviously
a type of autogamy. Hertwig’s claim that in both reduction divisions the
chromosomes (numbering between 120 and 150) are divided in the
metaphase seems open to question. If this were true, the divisions would
not be reductional in character, so that the chromosome number would
have to be reduced in some other manner than the usual gametic meioses.

According to Schaudinn (1896), Actinophrys sol undergoes isoga-
mous macrogamy, or hologamy. He stated that two full-grown similar
individuals come together and form a common cyst. The nucleus of
each divides twice, and at both divisions one nuclear product degenerates
and is expelled. The two cells, with their matured pronuclei, then fuse.
The resulting zygote soon divides into two individuals, which later es-
cape from the common cyst as vegetative animals.

The more recent and detailed investigations of Bélat (1923) have
demonstrated in this species a type of sexual activity similar in many
respects to that described by Schaudinn, except for the significant differ-
ence that the two original gametocytes within the cyst are sister cells,
since they are derived by a progamous division of the original gamont
(Fig. 142). The process, therefore, is a type of autogamy (pedogamy )
similar to that occurring in Actinosphaerium, except that in the latter
case the palmella produces several pairs of sister gametocytes. Incipient

Figure 142. Actinophrys sol in autogamous fertilization. A, progamous division of
original gamont; B, the two daughters of this division within a common envelope, their
nuclei showing looping chromatin threads; C, pairing and twisting of thread-like chromo-
somes (left), and shortening and thickening of chromosomes (right); D, first matura-
tion (reduction) division, with bivalent chromosomes on the equatorial plate (left),
and disjunction and separation of homologous chromosomes (right) ; E, second matura-
tion (equational) division, with first “polar bodies” below; F, pseudopodium of 6
gamete making contact with @ gamete; G, fusion of cell bodies; H, fusion of nuclei to
form zygote. (After Bélar, 1923. D is a composite.)

600 FERTILIZATION

heterogamy is seen in Actinophrys sol. When the gametes unite, one of
them sends out a pseudopodial process to the other, to initiate the fusion.
This pseudopodium is formed by only one member of the pair, and the
maturation processes in this one seem to occur a little ahead of those in
the other. These slight differences between the two gametes are inter-
preted as the beginnings of differentiation toward maleness and female-
ness. In rare cases the pseudopodium of the male fails to make contact
with the female, and then the female sends out a pseudopodium which
brings about fusion. The indication here is that whatever the degree of
differentiation of the gametes is, this differentiation is reversible. Perhaps
the potentiality for pseudopodial formation is retained in all gametes, but
only the one completing maturation first ordinarily exhibits it. When
neither gamete succeeds in connecting with its pseudopodium, no sexual
differentiation is demonstrable. In such cases both gametes form par-
thenogenetic cysts.

The most noteworthy phase of gamete formation in Actmophrys sol
is the striking similarity of the meiotic stages to those of the Metazoa.
Following the progamous division of the gamont into the two gameto-
cytes, two maturation divisions occur which reduce the chromosome
number from the diploid forty-four to the haploid twenty-two. In the
prophase of the first maturation division, the chromatin forms into
slender looping threads (leptonema) which pair off (parasynapsis),
become thicker (pachynema), and are obviously twisted around each
other (strepsinema). Then they shorten (diakinesis) into compact
chromosomes on the metaphase spindle, and the two parts of the bi-
valent chromosomes separate in the anaphase, twenty-two univalent
chromosomes going to each pole. One product of this division degen-
erates, and the other undergoes the second maturation division, which
is equational. The twenty-two chromosomes split longitudinally, so that
the pronucleus and the two polar bodies of each gamete have twenty-
two chromosomes.

It seems that this relatively simple heliozo6n has developed a matura-
tion process that is as highly specialized and clear-cut as any found in
the Metazoa. It is probably safe to say that further diligent search will
undoubtedly reveal other species of Protozoa with equally well developed
meiotic phenomena.

FERTILIZATION 601

SPOROZOA

Among the Sporozoa fertilization is almost universally present. As
would be expected in such a heterogeneous group, all kinds of fertili-
zation processes are known. Isogamy, heterogamy of all degrees of dif-
ferentiation, pseudoconjugation, gametic meiosis, zygotic meiosis, and
many other variations of the fertilization process have been described.
Naturally only a few typical examples, illustrating the chief types of
these phenomena, can be mentioned here.

Monocystis, the gregarine parasite in the seminal vesicles of the earth-

Figure 143. Monocystis rostrata. A and B, metaphase and anaphase of early progamous
divisions of pseudoconjugant, eight chromosomes splitting, eight going to each pole; C and
D metaphase and anaphase of last progamous (reduction) division, paired chromosomes
disjoining, four going to each haploid pole. (After Mulsow, 1911.)

worm, illustrates typical pseudoconjugation, gametic meiosis, and isog-
amous fertilization. Two adult gregarines come together and are en-
closed in a common cyst, but do not fuse. This intimate association with-
out protoplasmic union is pseudoconjugation, and the members of this
chaste betrothal are now gametocytes. The nucleus of each gametocyte
divides again and again to form a large number of small nuclei, which
migrate to the periphery and eventually become the gamete nuclei. Ac-
cording to Mulsow (1911), reduction occurs in the last of these divi-
sions before formation of the pronucleus, in Monocystis rostrata. The
earlier mitoses (Fig. 143) show eight thread-like chromosomes which
split longitudinally, eight halves going to each daughter nucleus. In the
last division the eight chromosomes associate in four pairs. In the ana-
phase that follows, members of the pairs separate and pass to different
poles, thus reducing the number of chromosomes from eight to four.
The surface of the gametocyte produces many small buds, each contain-

602 FERTILIZATION

ing a pronucleus. These pinch off as gametes, and the walls between the
associated gametocytes break down, allowing the gametes of one to fuse
with those of the other. Thus cross-fertilization occurs and the process
is isogamous, as there is no differentiation between gametes in this
species.

Calkins and Bowling (1926), working on a species of Monocystis,
have confirmed Mulsow’s interpretations and have furnished additional
critical evidence in support of Mulsow’s belief. They found the early
progamous divisions with the diploid number of chromosomes (ten)
in each daughter plate and also the final progamous divisions with the
haploid number (five) in each daughter plate.

Naville (1927a) has shown that in three types of Monocystis reduc-
tion is gametic. In types “A” and “B” early divisions of the pseudocon-
jugants show eight chromosomes and type “‘C” shows four as the diploid
number. Anaphases of the last two divisions preceding gamete forma-
tion show four chromosomes going to each pole in types A and B, and
two in type C. The next to the last division, therefore, is the reduction
division. The first amphinuclear division is not reductional, and the
sporoblast is diploid during its subsequent development.

Naville (1927b) also showed that in Urospora lagidis reduction of
chromosome number from a diploid four to a haploid two occurs in the
formation of its anisogamous gametes. A noteworthy occurrence here
is that synaptic conjugation of chromosomes takes place in the synkaryon.
The subsequent division of the synkaryon is equational, but the phe-
nomenon serves to illustrate the possibility, in other forms, of an ex-
tremely precocious synapsis being prolonged throughout the life cycle
until the next sexual stage appears, when the two members of the syn-
aptic pairs would separate in a progamic division. Such a condition, if
it exists, would explain in terms of gametic reduction the few known
examples of zygotic reduction. This hypothesis seems worth investigat-
ing. Valkanov (1935) found pairing of chromosomes (synapsis) and
condensation of chromosomes (diakinesis) into rings and crosses, in
the zygotes of Monocystella arndti; and he believes that reduction oc-
curs in the first zygotic division (see p. 613 below), although he was
not able to follow the subsequent behavior of the chromosomes. His
figures show eleven pairs in the zygote and eleven single chromosomes
in the early divisions of the pseudoconjugants. These numbers indicate

Figure 144. Ophryocystis mesnili, Isogamous gamete formation and fertilization. A
and B, two trophic forms attached to ciliated cells of host; C, gamonts pairing in
pseudoconjugation; D, E, F, two nuclear divisions, probably meiotic; G, mature gamont;
H and I, formation of gametes by internal budding; J, K, L, fusion of gametes in
fertilization; M, N, O, divisions of the zygote to form eight sporozoites in the single
spore. (After Léger, 1907.)

604 FERTILIZATION

zygotic reduction, but actual separation of chromosomes in the reduction
division must be observed before the case is considered to be proved.
M. arndti may have zygotic reduction, as the evidence indicates, but Na-
ville’s interpretation may apply to this case, so that gametic reduction
remains a possibility.

Gamete formation by endogenous budding was found by Léger
(1907) to occur in Ophryocystis mesnili (Fig. 144). In this form two
gamonts adhere in pseudoconjugation, and the nucleus to each divides
twice, one product of each division being destined to degenerate. These
are presumably reduction divisions, although cytological evidence for
this is lacking. One product of the two divisions becomes the pro-
nucleus of the single large gamete which is formed inside the gamont
as a loose internal bud. The walls between the gamonts break down
and the two isogametes fuse. The zygote thus formed develops a spore
wall, and eight sporozoites are produced by metagamic divisions.

Whiie isogamy is most frequently observed in the gregarines, as for
instance in the species already named and in Diplocystis schneideri
(Jameson, 1920), Gregarina cuneata (Milojevic, 1925), Actinocephalus
parvus (Weschenfelder, 1938), and others, several species show various
degrees of anisogamy. Species other than Urospora lagidis, already men-
tioned, are Echinomera hispida (Schellack, 1907), Stylocephalus longi-
collis (Léger, 1903), and Nini gracilis (Léger and Duboscq, 1909),
the last of which shows a marked degree of differentiation between the
microgametes and macrogametes. This differentiation approaches that
usually seen in the Coccidiomorpha.

An extreme differentiation of gametes (odgamy) is seen in Esmeria
as well as in other Coccidia and in many Haemosporidia. The type of
syngamy observed by Schaudinn (1900) in E/meria schubergi will serve
to illustrate this group. Here, as in other cases in which accurate chromo-
some determinations have not been made, it is assumed that reduction
is gametic. The sexual phase starts with some of the merozoites develop-
ing into gametocytes, instead of repeating their asexual cycle. It is diff-
cult to explain on a purely environmental basis why some merozoites
repeat the asexual cycle while others, obviously in the same environ-
ment (intestinal epithelium) develop into gametocytes. If external
factors play the chief rdle in determining whether a protozoan will con-
tinue asexual multiplication or enter a sexual phase, then it will be neces-

FERTILIZATION 605

sary to look further for the effecting stimulus in the Coccidiomorpha.

The phenomenon is more easily explained in these forms by the inter-
pretation of Maupas, which has since been developed especially by Cal-
kins, that internal factors play the determining role. This would mean
that when the protoplasm had reached a certain degree of maturity in its
cycle of development, the sexual phase would be initiated, even though
the external conditions remained unchanged.

Whatever the cause, gametocytes appear and are differentiated into
male and female gametocytes. The macrogametocytes are said to eliminate
their karyosomes to accomplish reduction. By this process they are trans-
formed into large, yolk-filled, egg-like macrogametes. The nuclei of the
microgametocytes are said to give off chromidia and then degenerate.
The chromidia condense into a number of clusters to form the nuclei of
small, sperm-like, flagellated microgametes. A macrogamete is found
and fertilized by a microgamete, and the resulting zygote forms an
odcyst. The synkaryon divides twice to produce four sporoblasts, each
of which now develops two sporozoites.

A more thorough cytological study of the cycle may eventually reveal
chromosome reduction taking place in the two divisions of the syn-
karyon, in which case meiosis would be zygotic; or in nuclear divisions
prior to gamete formation, in which case it would be gametic. Karyo-
some extrusion and the formation of gamete nuclei from chromidia
cannot be accepted today as conclusive evidence of meiotic reduction.
If, indeed, no chromosomes are formed in E/meria schubergi, then we
shall be forced to modify our concept of meiosis. Here again we find
urgent need for the application of improved techniques in cytological
studies of a fundamental nature.

The sexual processes in the Adeleidea differ from those of the other
Coccidia in several interesting respects. In Adelina dimidiata, according
to Schellack (1913), two gametocytes of different sizes unite in a pseudo-
conjugation process similar to that of the gregarines. The nucleus of the
microgametocyte divides twice, and one of the nuclei enters and fertilizes
the macrogamete. In this species only one macrogamete is formed and it
is fertilized by one pronucleus of the microgamete in a way similar to
anisogamous conjugation in the Vorticellidae. The peculiar behavior
of the ciliate Metopus sigmoides (see p. 622 below) in conjugation
also resembles the sexual union of A. dimidiata.

606 FERTILIZATION

Fertilization in the Cnidosporidia is typically autogamous, and will
be dealt with under the subject of autogamy. Little is known of the
fertilization phenomena in the Acnidosporidia, but Crawley (1916) de-
scribes gamete formation in Sarcocyst7s murzs, similar in general to
odgamy in Ezmeria except that the microgametocyte gives rise to the
microgametes by a peculiar kind of nuclear fragmentation.

AUTOGAMY

Autogamy, or self-fertilization, is accomplished in several ways in the
Protozoa, but the result in all cases is the fusion of two gamete nuclei,
both of which have been derived from the same parent cell. In some
cases the two pronuclei have been separated by cytoplasmic divisions
into separate cells which later fuse. In other cases the two pronuclei re-
main in the undivided cytoplasm and fuse after casting out part of the
chromatin, with or without visible meiotic reduction.

Whatever benefit there may be to the individual or the race in ex-
ogamy, or cross-fertilization, in the way of renewing the vigor of the
protoplasm and in propagating the race, this benefit is also a property
of autogamy. There is no apparent reason why autogamy in these re-
spects should not be as efficacious as exogamy. In two respects, however,
the processes differ greatly. In the uniparental inheritance of autogamous
individuals, meiosis will shuffle and sort out whatever genes are pres-
ent; and if the allelomorphs are different, the resulting gametes and
offspring will vary in their characteristics. However, the tendency in this
kind of inbreeding would be overwhelmingly toward the production of
homozygous races. Therefore the pronuclei and the resulting offspring
would be less variable than in races in which exogamy brings together
two sets of genes from two different parents, in the production of a
heterozygous individual (see Jennings, 1920). From the genetic and
the evolutionary standpoint, it is evident that exogamy would tend to
produce a more heterozygous race; and, if natural selection is the criti-
cal factor in evolving organisms by the selection of favorable variants,
then the advantage of exogamy over autogamy 1s apparent.

Another difference is the advantage the autogamous species has over
the exogamous species in accomplishing fertilization. It is obvious that
in exogamous species either the sexual organism or the gamete must
seek out and find a mate before syngamy may occur; and if the organ-

Fig. 145. Diagrammatic life cycle of Sphaeromyxa sabrazesi, 1, mononuclear zygote
(sporozoite) ; 2, multinuclear plasmodium (schizogony), large outline represents plas-
modium during subsequent development; 3-6, 17-19, multiplication of diploid nucle;
7, 20, differentiation of nuclei into large and small; 11-12, 21-23, reduction division
in macro- and microgametocytes; 14-15, 25-26, equational division; 16, macrogamete;
29, microgamete; 30, plastogamy; 31, pansporoblast; 32-37, mitoses of haploid nuclei
to form fourteen; 38, two sporoblasts formed; 39, two pronuclei remaining in each
spore; 40, fertilzation within spore. (After Naville, 1930b.)

608 FERTILIZATION

isms are too widely dispersed or too effectively isolated by barriers, there
will be no progeny. This difficulty simply does not exist for autogamous
organisms, as the isolated individual can reproduce itself sexually with-
out recourse to others of its kind.

Several cases of self-fertilization have already been pointed out (Ac#7-
nos phaerium, Actinophrys, Sap pinia?). In these cases fertilization is ac-
complished by the fusion of sister cells, which have undergone nuclear
reorganization (perhaps reduction). This type of autogamy has been
called pedogamy.

Another type of autogamy is common in the Cnidosporidia and is
known best in the Myxosporidia. Here the nucleus divides to form several
nuclei, without division of the cytoplasm. The nuclei then reunite in
pairs, to form amphinuclei.

In the myxosporidian Sphaeromyxa sabrazesi, according to Schroder
(1907, 1910), the plasmodial body contains two kinds of nuclei. Small
areas become differentiated from the surrounding protoplasm. Each area,
or pansporoblast, contains two nuclei, one large and one small. Both of
these nuclei divide to form seven, so that there are fourteen nuclei pro-
duced in each pansporoblast. The pansporoblast divides into two halves,
the sporoblasts, which are destined to become the two spores. The
daughter sporoblasts receive six nuclei apiece, and the other two nuclei
are expelled at the fission of the pansporoblast and degenerate as “‘re-
duction nuclei.”” Of the remaining six in each sporoblast, two form the
capsule and shell, two form the polar capsules, and two presumably one
of each original kind, remain as the pronuclei and later fuse with each
other in autogamous fertilization. More recently Kudo (1926) has
described a somewhat similar case of autogamy in Myxosoma catostomt.

Debaisieux (1924) found six chromosomes in the early mitoses of
the plasmodium of Sphaeromyxa sabrazesi. In later stages he found the
number reduced to the haploid three.

Naville (1930b) is very specific in his account of this species (Fig.
145) and of S. balbianii. In both forms four chromosomes are reduced
to the haploid two in the plasmodium, just before the formation of the
pansporoblast and after the differentiation of the nuclei into large and
small types. The two types he calls macrogametocytes and microgameto-
cytes, because they are surrounded by a zone of condensed cytoplasm. The
union of two of these zones of cytoplasm in plastogamy brings a large

FERTILIZATION 609

and a small nucleus together in the formation of the pansporoblast. A
considerable portion of the life cycle of these organisms is therefore
passed in the haploid state. Naville also describes a similar diploid-
haploid cycle of four and two chromosomes for Myxidium incurvatum.
In this case there are more variations in the method of spore formation,
but reduction from four to two chromosomes occurs in the plasmodium,
as in Sphaeromyxa, and fertilization occurs between the two remaining
nuclei of the spore.

Many variations of this process occur in other forms, but Chloromyxum
leydigi (Naville, 1931) is of particular interest, because of its two hap-
loid-diploid cycles. In this multinucleated plasmodium, the nuclei divide
by mitosis, showing a diploid chromosome number of four. Then a
heteropolar reduction division occurs, producing large and small nuclei
with two chromosomes each. Internal buds are formed, wherein large
and small nuclei fuse in pairs (first union). The difference in size of
these fusing nuclei makes this an anisogamous fertilization. Several divi-
sions of the fusion nuclei follow, each showing the diploid four chromo-
somes. The young plasmodium grows until the advent of spore forma-
tion, which is marked by the appearance of groups of four nuclei, two
large and two small, each with chromosomes again reduced to the hap-
loid two. The two small nuclei degenerate, while the larger two divide
twice, to form a group of eight which become enclosed in a wall. Of
the eight nuclei thus formed, six function in the formation of the spore
complex and the remaining two fuse in the second fertilization of the
cycle. This second fusion is comparable to fertilization in other forms,
but the first fusion of nuclei in the plasmodium is a secondary develop-
ment interpolated in the life cycle. Its significance is a matter of specula-
tion. The phenomenon is actually a double autogamy and is difficult
to harmonize with meiotic processes of either Metazoa or other Protozoa.
Little is known of chromosome behavior in other Cnidosporidia.

Among the Actinomyxida and Microsporidia, autogamous fertiliza-
tion is said to occur in a manner broadly similar to that of the Myxo-
sporidia. One member of the Actinomyxida, Gayenotia sphaerulosa, has
been shown by Naville (1930a) to undergo chromosome reduction in
the second of three gametogenic divisions. In this case the development
of the pansporoblast occurs as described in the case of other Actino-
myxida. The two nuclei of the sporozoite divide mitotically, forming

610 FERTILIZATION

two large central germinal cells which ultimately give rise to eight male
and eight female gametes respectively, and two smaller peripheral cells
which divide again to form the four enveloping cells of the cyst wall.
Of the three gametogenic divisions, the second reduces the chromosome
number from the diploid four to the haploid two. The gametes are
differentiated on the basis of size, the male gametes being smaller than
the female. The eight microgametes unite with the eight macrogametes
to form eight zygotes and reéstablish the diploid condition.

The origin of the two nuclei in the sporozoites is not known, but it
is presumably by division of an original single nucleus. As the male and
female gametes are produced by a single original sporozoite, it may be
regarded as a hermaphroditic animal. This condition is rare in the Proto-
zoa except in ciliate conjugation, in which the two pronuclei produced
by one conjugant behave differently and in a few cases are morphologi-
cally differentiated (see p. 622 below).

While a number of other life cycles of Cnidosporidia have been
worked out, knowledge of the fertilization process is fragmentary and
data on chromosome behavior in meiosis are almost entirely lacking.
Descriptions of “reduction” generally refer to the loss of chromatin
by the degeneration of some of the nuclei which are sisters of the func-
tional pronuclei. In other cases the extrusion of the karyosome 1s inter-
preted as reduction. Such loss of nuclear elements is not to be confused
with reduction in the chromosome number from the diploid condition
to the haploid. It is possible that the two processes are similar in func-
tion, but until such time as that is demonstrated, we are not justified
in assuming that one is the equivalent of the other.

Autogamous fertilization has also been described for a few ciliates.
According to Buschkiel (1911), the parasitic form Ichthyophthirius
multifiliis becomes encysted and the micronucleus divides twice produc-
ing four, of which two degenerate while the remaining two fuse au-
togamously. Fermor (1913) described a reorganization process within
the cyst of Stylonychia pustulata, wherein the old macronuclei degenerate
and the micronuclei fuse and produce a new nuclear complex. The evi-
dence in support of these two cases is not conclusive.

Diller (1936) has recently given a detailed account of autogamy in
Paramecium aurelia. Vhe process is similar to conjugation except that
there is no pairing nor cross-fertilization. The macronucleus disintegrates

FERTILIZATION 611

during the process, as in conjugation and endomixis, and the micro-
nucleus produces two pronuclei, as in conjugation. The pronuclei fuse
autogamously and the synkaryon divides twice to form four nuclei; two
are macronuclear An/agen which separate at the first fission, and two be-
come micronuclei. Diller challenges the very existence of endomixis
in this species, on the grounds that stages of autogamy and irregular
reorganization processes called ‘‘hemixis’’ have been mistaken for endo-
mixis. Certainly this challenge must be met by careful reéxamination of
the studies already made on endomixis (see Woodruff, Chapter XIII;
also Sonneborn, Chapter XIV).

ZYGOTIC MEIOSIS

In nearly all animals that have two parents, the two sets of chromo-
somes that are contributed to the offspring remain in the nuclei of all
cells derived by mitosis from the zygote. This diploid number 1s charac-
teristic of all cells except the gametes, in which case the chromosomes are
separated out again by one or two meiotic divisions, giving to each gamete
one half the diploid number. This haploid number is found only in
the gamete, because the union of gametes reéstablishes the diploid condi-
tion.

There is evidence to show that in a few Protozoa, notably among the
Telosporidia, reduction in chromosome number occurs in the division
of the zygote. This means that the organism lives a haploid existence
and only the zygote is diploid. Bélat (1926) argued that since the com-
plete reduction process is known only in Aggregata and Karyolysus
among Coccidia and in Diplocystis among gregarines, and that in all
these forms reduction is zygotic, therefore reduction in all members
of the two groups is probably zygotic. He suggested that Mulsow (1911)
confused two species in obtaining his results, but the later work of
Calkins and Bowling (1926) and of Naville (1927a) on Monocystis
has made that conclusion untenable. Bélat further points to the frequent
occurrence of odd numbers of chromosomes in these groups as evidence
of the haploid condition, which implies zygotic reduction. Odd chromo-
some numbers could be explained by postulating a supernumerary or
a sex chromosome, or by interpreting each chromosome as in reality a
pair in close and prolonged synapsis; but these assumptions are unsat-
isfying, in the absence of more adequate evidence.

612 FERTILIZATION

In their preliminary report, Dobell and Jameson (1915) gave the
main features of their later detailed descriptions of the life cycles of
the gregarine Diplocystis schneideri (Jameson, 1920) and the coccidian
Aggregata eberthi (Dobell, 1925). In both cases meiotic reduction occurs
in the first division of the zygote, and the organism lives all the rest of
its life as a haploid animal.

In Diplocystis the nucleus of each pseudoconjugant divides many
times, showing the haploid three chromosomes at each division. The
dividing nuclei migrate to the periphery and eventually form club-shaped
gametes, which pinch off from the gametocyte and fuse with those of
the other pseudoconjugant. After a synaptic clumping of bead-like
chromatin threads, six chromosomes appear in the prophase of the zygo-
tic division and three go to each pole. The diploid number of chromo-
somes in the zygote is therefore reduced at the first amphinuclear dtvi-
sion to the haploid three, a number which is also observed in the later
divisions of the sporoblast.

Agegregata eberthi, like other Coccidia, has well differentiated male
and female gametes. The female gametocyte is transformed bodily into
a macrogamete after a complicated nuclear reorganization, which does —
not, however, involve reduction, though a spindle is formed and the
six haploid chromosomes are seen. The nucleus of the male gametocyte
divides by a complicated method, showing six chromosomes at each
division. When the small flagellated microgamete fertilizes the macro-
gamete, a fertilization membrane appears. The diploid number of twelve
chromosomes appears on the spindle of the first zygotic division. Pairing
of homologous chromosomes follows, and the bivalent chromosomes be-
come closely applied to each other. They later disjoin and six go to
each pole, thereby reducing the number to the haploid condition. All
other divisions of the nuclei are mitotic and six chromosomes appear and
are divided at each mitosis.

It is possible that in both Aggregata and Diplocystis disjunction does
not occur in the metaphase of the first zygotic division, and that, instead
of this the bivalent chromosomes divide equationally, with six bivalents
going to each pole. If at each subsequent mitosis the bivalent chromo-
somes divided until just before gamete formation and then disjoined,
then reduction would be gametic instead of zygotic. However, the evi-
dence seems conclusive enough to convince most biologists that in these
two cases meiosis is truly zygotic.

FERTILIZATION 613

In 1898 Dangeard found no reduction taking place during gamete
formation in Chlamydomonas and suggested that it occurs during the
germination of the egg. Pascher (1916) made no chromosome counts,
but presented genetic evidence for zygotic meiosis in Chlamydomonas.

Hartmann and Nagler (1908) indicated that reduction is zygotic in
Sappinia (Amoeba) diploidea, because three nuclei disintegrate, out of
the four that are formed by two zygotic divisions. Diwald (1938) very
recently stated that, because he could see no meiosis in the formation of
the four gametes of Glenodinium lubiniensiforme and because a tetrad
of four potential individuals are produced by two divisions of the zygote,
reduction occurs in the two zygotic divisions.

It does not seem justifiable to base an assumption of zygotic meiosis
on such indirect and questionable evidence. Genetic evidence must be
considered, in the absence of cytological data; but only positive determina-
tion of chromosome number and identification of the stage in the cycle
in which the number is reduced from diploid to haploid can be accepted
as conclusive evidence of this phenomenon.

Valkanov (1935) has presented fragmentary cytological evidence of
zygotic reduction in Monocystella arndti. He shows eleven long chromo-
somes in the early divisions of the pseudoconjugants. In the first zygotic
division, eleven synaptic pairs condense into short, fat Ys and Xs. He
concludes that reduction is zygotic, but his evidence is admittedly incom-
plete, as he was unable to follow the subsequent behavior of the chromo-
somes. Whether this is truly zygotic meiosis or whether the zygotic pair-
ing of chromosomes is a phenomenon similar to that found in Urospora
lagidis (see p. 602 above) remains an open question. The odd number
of chromosomes lends some support to Valkanov’s belief.

Weschenfelder (1938) has just published what appears to be a clear-
cut case of zygotic meiosis in the gregarine Actinocephalus parvus. In
the early nuclear divisions of the pseudoconjugants, four long, rod-
shaped, haploid chromosomes are repeatedly observed. Isogametes bud
off the mother cell and fertilization occurs as in other gregarines. At the
first division of the zygote, eight chromosomes develop from the syn-
karyon as four synaptic pairs. These pairs disjoin in the anaphase and
four go to each pole, reducing the number of chromosomes to the hap-
loid condition again. Subsequent mitoses in the sporoblast reveal the
haploid four chromosomes, now globular in shape, appearing in the
prophase and passing to each pole in the anaphase.

614 FERTILIZATION

Weschenfelder’s observations have confirmed the suspicions of many
protozodlogists that there exist other gregarines besides Diplocystis
schneideri which undergo zygotic meiosis. The problem of inheritance
in these forms presents some interesting possibilities to the geneticist.
All genes possessed by the two parent organisms are passed to the zygote;
therefore, if odcyst (sporoblast) characters can be differentiated and
mated, the immediate and direct effect of those genes may be observed
in the resulting zygote. Furthermore, a haploid organism whose charac-
teristics are controlled by a single set of chromosomes presents a rare
opportunity for unusual genetic and cytological studies.

SIGNIFICANCE OF FERTILIZATION

The causes and effects of fertilization in Protozoa are subjects upon
which a great deal has been written and some significant data obtained.
The three conditions cited by Maupas (1889) as necessary for conjuga-
tion in ciliates are sexual maturity, diverse ancestry, and hunger. All
three of these contributing factors have been supported by evidence
from some later investigations and all three have been discounted by
other investigations. In many cases the investigators have been dealing
with different species of Protozoa. This in itself is probably responsible
for many of the conflicting conclusions that have been reached. As the
evidence accumulates, it becomes increasingly clear that different Proto-
zoa require different conditions for conjugation and copulation, and
that we are not justified in applying to all Protozoa conclusions derived
from one or two or even several species.

Among the flagellates and rhizopods there are many organisms in
which sexual phenomena have never been reported and in which prob-
ably none exists. These forms, then, are able to reproduce indefinitely
by asexual means. Inherently, therefore, protoplasm does not seem to
require sexual union.

At the other end of the sexual scale are found those Sporozoa and
Foraminifera in which the life cycle is an obvious fact, and in which a
sexual stage develops as one sector of that cycle, without which they
could not continue their existence. If generalizations were made from
these two kinds of organisms, there would be contradictions too obvious
to relate.

For similar but less obvious reasons, we may partially account for

FERTILIZATION 615

the different schools of thought regarding the conditions necessary for
conjugation in ciliates, upon which most of this work has been done.

In regard to ancestry, Calkins (1904) found that in Paramecium
caudatum there are fully as many conjugations between closely related
individuals as between individuals of diverse ancestry. He further indi-
cates (Calkins 1933) that similar results have been obtained through
isolation cultures of Didinium nasutum, P. aurelia, P. bursaria, Styl-
nychia sp., Blepharisma undulans, Spathidium spathula, Oxytricha fal-
lax, Chilodonella cucullus, and Uroleptus mobilis. Sonneborn and Cohen
(1936) found that under identical conditions, ‘“The Johns Hopkins
stock R of Paramecium aurelia can invariably be induced to conjugate,”
while “the Yale stock of the same species cannot.’ This difference ap-
pears to be clearly racial. Sonneborn’s (1937) discovery of two ‘“‘sex
reaction types’’ in a race of P. awrelia may throw considerable new light
on this question. Members of one type readily conjugate with those of
the other type, but do not conjugate among themselves.

At first this looked as though something resembling the two sexes of
other organisms had been found in the reaction of one ciliate to another.
However, the discovery by Jennings (1938) of as many as nine sex
reaction types in P. bursaria seems to remove these types from the cate-
gory of sexes and indicates that they are simply strains which will not
inbreed. The significance of these discoveries is not yet clear, but they
do show that in some cases, at least, diverse ancestry is a potent factor
in conjugation.

In regard to the relative importance of external conditions and in-
ternal conditions in ciliate conjugation, we again find contradictory evi-
dence if we generalize from specific instances. The inductive method of
reasoning is certainly stimulating and productive, but its misuse has led
to some unjustifiably broad propositions. There is a rapidly accumulat-
ing array of evidence that external conditions, such as food, tempera-
ture, pH, population concentration, light, seasons, chemicals, condition
of host in some parasitic forms, and so forth, do play an important rdle
in inducing conjugation in some ciliates. However, there is valid evi-
dence to indicate that in some forms, at least, conjugation can be in-
duced only at certain times in the life cycle of the organism—in other
words, only when the protoplasm is sexually “mature” for conjugation.
Calkins (1933, p. 286) states that “One unmistakable conclusion can be

616 FERTILIZATION

drawn from the many diverse observations and interpretations of the
conditions under which fertilization occurs in ciliates, viz., the proto-
plasmic state with which conjugation 1s possible is induced in large part,
but not wholly, by environmental conditions.”

It is a matter of common observation that when conjugation occurs
in mass cultures, all the ciliates do not conjugate, but only a certain
proportion of them. The proportion may vary with the culture and the
species, but in any case if the conditions in the mass culture are favorable
for inducing conjugation in some individuals, why do they not all con-
jugate? The fact that some do and some do not conjugate under condi-
tions that appear to be identical, would indicate the existence of internal
differences.

Calkins (1933, p. 290) summarizes the evidence and concludes “that
environmental stimuli are without effect in producing conjugations un-
less the protoplasm is in a condition where such conjugations are pos-
sible.” Two examples illustrate different phases of this proposition:
Uroleptus mobilis will conjugate only after a period of from five to
ten days after fertilization, and stock R of Paramecium aurelia (Sonne-
born, 1936) will conjugate only in descendants of animals which have
recently undergone conjugation or endomixis. The time factor is ob-
viously different in these two animals, but both clearly indicate a strong
cyclical differentiation which affects conjugation.

For more detailed analyses of this subject, reference should be made
to Calkins’s Biology of the Protozoa (1933) and to Chapter XIV below,
by Sonneborn.

There is, perhaps, even less agreement concerning the effects of con-
jugation than concerning the causes. In some ciliates, e.g., Urole ptus
mobilis (Calkins, 1919), conjugation results in a definite renewal of
vitality, as indicated by an increase in the fission rate. Calkins interprets
this as a fundamental process, which is an integral and normal part of
the life cyle. Woodruff and Spencer (1924) found a similar renewal
of vitality following conjugation in Spathidium spathula, but Woodruff
(1925) interprets this as a rescue process to “meet the emergency of
physiological degeneration induced by environmental conditions which
are not ideal.’’ Beers (1931) shows that conjugation increases vitality in
Didinium nasutum which has been depressed by inadequate feeding, but
that no depression occurs in well-fed animals.

FERTILIZATION 617

In other ciliates, however, conjugation apparently reduces vitality.
Thus in Blepharisma undulans Calkins (1912) found that all excon-
jugants died, although Woodruff (1927) concluded from his investiga-
tions that conjugation in this species accelerates the division rate. Jen-
nings (1913) concluded that conjugation reduced vitality in Paramecium
as indicated by a reduction in the average rate of fission in exconjugants.

At the present time the problem as regards ciliates seems to be: does
increased vitality following conjugation mean that conjugation is a nor-
mal and essential part of the life cycle, or is it merely an emergency
measure called into play when unfavorable environmental conditions
have resulted in physiological degeneration? This problem is not easy
to solve, because it is difficult to know what optimum or even “normal”
environmental conditions are, and the two are probably not identical.
Another complication is that in some species endomixis may be substt-
tuted for conjugation as a revitalizing process.

Another angle from which this problem may be approached is that
of comparison with the plant kingdom. Many plants are able to repro-
duce themselves indefinitely by asexual methods, but at the same time
sexual stages occur which, though not indispensable to their continued
existence, are nevertheless certainly an integral part of their normal
life cycle and valuable to the organism in other ways. In other plants,
sexual processes must occur at regular intervals under “normal” condi-
tions, or the species will die out.

Further investigation may disclose a similar situation in the ciliates,
wherein some ciliates cannot continue to exist without periods of con-
jugation, while in others endomixis may be substituted for conjuga-
tion, and in still others asexual reproduction will carry on the line in-
definitely. The final answer to this problem will come only through con-
tinued investigation.

CON JUGATION

Conjugation has been defined as the temporary union of two proto-
zoan cells for the exchange of nuclear elements. It is a sexual process,
differing from ordinary sexual union in that it is not directly related
to reproduction. Two organisms enter into the relationship and the same
two functional units leave the relationship; no third party—no progeny
—has come into being. The two conjugants have been genetically

618 FERTILIZATION

changed by conjugation into new genetic entities, but this is actually
genetic transformation rather than reproduction. Before the exconju-
gants return to their normal vegetative condition, they undergo one or
more divisions in most cases, but these divisions are reproduction by
binary fission, an asexual phenomenon. Although these divisions are
modified by the previous sexual union, they are none the less asexual
reproduction. |

Conjugation is peculiar to the Ciliata and the process is strikingly
uniform, with but few exceptions, in all ciliates which have been studied.
The general course of the maturation phenomena in conjugation was
first described by Maupas (1889), who studied the process in a number
of ciliates and divided it for convenience into eight stages. Calkins
(1933) states that ‘“With one or two exceptions (Trachelacerca phoene-
copterus, Spirostomum ambiguum, etc.) all of the free living ciliates
thus far described agree in the general course of their maturation phe-
nomena.” Several parasitic species, however, exhibit some important dif-
ferences from the usual course, and recent investigations have revealed
a few interesting deviations among free-living forms.

With a few noteworthy exceptions, the union of two ciliates in con-
jugation takes place longitudinally and symmetrically (Fig. 146). The
first sign of approaching conjugation in a mass culture is frequently
a tendency to agglomerate in dense masses. Individuals appear to stick
together on contact, even though they may separate soon after. Even-
tually, two individuals will adhere side by side, or with ventral surfaces
together, and become more intimately connected in the anterior region.
The extent of union varies from a thin protoplasmic bridge at the time
of cross-fertilization to an intimate fusion of more than half the body
length in other species.

Two individuals of Ezplotes patella will come into contact, spiral
about each other for a few moments, and then apply themselves to-
gether at their left peristomal margins, so that the appearance is similar
to two turtles stuck together by their left ventral halves (Fig. 146). They
swim forward together in a well codrdinated manner, rotating on an axis
which, owing to the symmetry of the pair, is straighter than the spiraling
axis of a single individual. At this stage the pairs are joined only by
their cirri. After remaining together a short time, they may separate and
repeat the process with the same or with other individuals, until finally

FERTILIZATION 619

a union is made which involves an insecure adhesion of the bodies in
the anterior left peristomal region. The peristome is distorted by the
fusion, but the mouth continues to feed until it degenerates in the reor-
ganization process.

While the majority of ciliates become attached along their ventral or
ventro-lateral margins and fuse anteriorly, several exceptions are note-

Figure 146. A pair of Euplotes patella in conjugation. The micronuclei have under-
gone preliminary division and are now in the first meiotic division; the C-shaped macro-
nuclei are beginning to degenerate. (Turner, 1930.)

worthy. Didinium nasutum (Prandtl, 1906; Mast, 1917) and members
of the Ophryoscolecidae (Dogiel, 1925) join end to end anteriorly, the
latter forming an oral chamber by the juxtaposition of the two deep
peristomal pockets. Dogiel states that the conjugants are smaller than the
ordinary forms, owing to special progamic fissions. In Parachaenia myae,
Kofoid and Bush (1936) found conjugants attached by their posterior

620 BERTILIZATION

ends, the anterior ends pointing in opposite directions. Kidder (1933b)
describes the anterior tip of one member of a pair of Ancistruma isseli
uniting asymmetrically with the peristomal groove part way back on its
mate, though the two are of equal size. Miyashita (1927) shows that
radically asymmetrical union occurs in Lada tanishi, in which the micro-
conjugant, smaller than its mate, attaches its anterior end to the posterior
ventral surface of the macroconjugant. In Kzdderia (Concho phthirius)
mytili, Kidder (1933a) shows an almost tandum association, with the
anterior peristomal region of the slightly smaller member joined by a
wide protoplasmic bridge to the aboral surface of the larger member
of the pair. In Dileptus gigas (Visscher, 1927) fusion takes place along
the ventral surfaces of the proboscides, and the mouth remains in evi-
dence during the entire period of conjugation.

The varied methods of joining of the conjugants suggest that the
location of the fusion bridge is not significant. If ciliate conjugation
evolved from a process similar to pseudoconjugation as seen in present-
day gregarines, as many protodlogists believe, it seems reasonable that
one location would serve as well as another, provided the cortex were
not too firm for a protoplasmic bridge to be formed. It is interesting
in this connection to observe that in Explotes patella (Turner, 1930),
which has a rigid cuirass, no true cytoplasmic bridge is formed. The
wandering pronucleus of one conjugant breaks out of the left anterio-
ventral margin of the one conjugant and passes backwards through a
tube formed by the local separation of the applied surfaces of the two
conjugants, and finally enters the cytostome of the other conjugant. This
method of entering the apposed conjugant probably developed simply
because it was easier to penetrate the soft cytostomal membrane than
the rigid cuirass. The mouth-to-mouth migration of the male pronucleus
in Cycloposthium bipalmatum (Dogiel, 1925) is a simpler example of
the same process.

In Polyspira and other members of the Foettingereidae, there occurs
a remarkable combination of conjugation, fission, and chain formation
called “‘syndesmogamie” by Minkiewicz (1912), and recently renamed
“zygopalintomie”’ by Chatton and Lwoff (1935) in their comprehensive
work on the Apostomea. The two conjugants unite by their lateral sur-
faces rather than by the usual ventral method, then proceed to undergo
a series of synchronous, partial, transverse divisions, until a chain of con-

FERTILIZATION 621

jugating zodids is formed, resembling superficially a double tapeworm.
After a time the fissions cease, and conjugation proceeds between mem-
bers of each pair of zodids according to the “‘classical scheme,” although
the nuclear details have not been worked out. Eventually fission is com-
pleted, and the exconjugants soon separate, reorganizing themselves in
the usual way. These fissions of the paired conjugants appear to be
related in kind to the special preconjugation fissions, observed in several
other ciliates, which result in conjugants that may be distinguished from
the vegetative forms chiefly by their smaller size. Specialized conjugants
are observed in Nicollella cteriodactyli and Collinella gundi (Chatton
and Pénard, 1921); the Ophryoscolecidae (Dogiel, 1925); Déleptus
gigas (Visscher, 1927); Balantidium sp. (Nelson, 1934); Nyctotherus
cordiformis (Wichterman, 1937), and in the microgametes of pert-
trichs.

The preliminary division of the micronucleus in Ezplotes charon and
E. patella (Maupas, 1889; Turner, 1930), without fission of the body,
is a modification of this same tendency of the conjugants to differ from
the vegetative forms.

Conjugants of many other ciliates are in some degree smaller than
vegetative individuals, and this may be the result of reduced feeding or
of other factors as yet unknown. It is among the copulating ciliates that
the greatest difference occurs between the vegetative forms and the
mature gametes (see p. 610 above).

Sexual differences are difficult to elucidate in the ciliates because the
picture is confused by two kinds of possible differences. The two con-
jugants may show differences in size, shape, or other characteristics.
These differences between the two conjugants entering the union may
be interpreted as indicating maleness and femaleness. In a number of
forms the differences are slight but fairly constant, as in Miyashita’s
(1927) ‘“‘macroconjugants’ and “microconjugants’” of Lada tanishi. In
the Vorticellidae, on the other hand, the difference in size and behavior of
the microconjugant and the macroconjugant is very striking, far greater,
in fact, than could be explained on the grounds of fluctuating variation.
The small free-swimming form that seeks out and fertilizes the large
sessile form could reasonably be called the male conjugant, and the large
form may be considered a female conjugant. In the Vorticellidae and in
Metopus sigmoides (Noland, 1927), mutual fertilization 1s not accom-

622 FERTILIZATION

plished, because both pronuclei of one member—the microconjugant in
the Vorticellidae—pass over with the cytoplasm into the other conjugant.
One pronucleus of the donor and one pronucleus of the recipient fuse,
to form the functional synkaryon. The other two pronuclei may or may
not fuse, but in either case they eventually disintegrate. In Metopus the
conjugants separate and the remnant of the donor dies; in the Vorti-
cellidae the microconjugant fuses completely with the macroconjugant.
These are obviously fertilization types intermediate between copulation
and conjugation.

The other category of differences is that exhibited between the wander-
ing and the stationary pronuclei which are produced in the same con-
jugant. They are usually considered to be male and female pronuclei
respectively. Here the only apparent difference may be in their behavior,
as is the case in the majority of ciliates studied. In Explotes patella there
is a slight difference in size between the wandering and the stationary
pronuclei, and there is a special zone of cytoplasm which accompanies
the wandering pronucleus in its migration. In Cycloposthium bipalma-
tum, however, Dogiel (1925) has described a spermatozo6n-like wander-
ing pronucleus, which is in striking contrast to the rounded stationary
pronucleus. These illustrations may be considered as representing stages
in the evolution of distinct sexual differences between pronuclei of
ciliates.

If we assume that differences between members of a conjugating pair
indicate sexual differentiation, then we would have male and female
individuals both producing structurally isogamous but functionally an-
isogamous pronuclet, as in Chilodonella (Chilodon ) uncinatus (Enriques,
1908; MacDougall, 1925). In other cases we would see male and
female conjugants both producing pronuclei which are functionally and
structurally differentiated as male and female, as in Cycloposthium (Do-
giel, 1925).

If we consider the differences in behavior and structure between the
wandering and the stationary pronuclei as indicating sexual differences,
then we must consider the parent conjugants as hermaphrodites, and any
differences between conjugants would then be a leaning toward male-
ness or femaleness on the part of an hermaphroditic organism. Viewed
in this light, members of the Vorticellidae have lost their double nature,
and the microconjugant has come to produce only male functional pro-

FERTILIZATION 623

nuclei and the macroconjugant only female functional pronuclei. The
other pronuclei are produced as usual, but fail to develop. Similarly,
in Metopus sigmoides (Noland, 1927) one conjugant contributes all of
its potentialities to the other, in what may be interpreted as a male ani-
mal contributing its life and all its potential gametes to the female.

THE MACRONUCLEUS DURING CONJUGATION

In ciliates, the micronucleus is concerned with sexual activity and
reproduction and is therefore frequently referred to as the generative
or reproductive nucleus and represents the “germ plasm” of the Metazoa.
The macronucleus, on the other hand is concerned with metabolism or
vegetative activity and is considered the trophic or vegetative nucleus
and represents in part the somatoplasm of the Metazoa. The chromatin of
these two types of nuclei is combined in the single nucleus of other cells
and in the one kind of nucleus found in the multinucleate O palina.

The disintegration of the macronucleus at the time of conjugation,
in all ciliates with dimorphic nuclei, represents the death of the soma
and the end of the genetic unit. Differentiation of the new macronucleus
in exconjugants similarly represents the development of the new somatic
individual from the zygote.

In ciliates generally, the old macronucleus shows signs of disintegra-
tion during the maturation divisions, and, by the time of crossing of the
pronuclei, fragmentation or other evidences of disintegration are well
under way. It is during the differentiation of the new macronucleus from
the synkaryon that the most rapid breakdown and the final absorption
of the old macronucleus occur. This is probably due to the withdrawal
from the cytoplasm of all chromatin-building elements by the developing
macronuclear An/lage or “‘placenta.’’ The old macronuclear remnants are
possibly used as “‘fertilizer,” or reserve of chemical elements in about the
right proportion, for replenishing the cytoplasm and maintaining the
equilibrium.

Before disintegration, the old macronucleus exhibits strange activity
in several species of Anoplophrya (Schneider, 1886; Collin, 1909;
Brumpt, 1913; Summers and Kidder, 1936), and in two species of
Chilodonella (MacDougall, 1936). At about the time of crossing of
the pronuclei, the macronucleus in each conjugant elongates and con-
stricts in the middle, as one half pushes across the protoplasmic bridge

624 FERTILIZATION

into the apposed conjugant. This macronuclear exchange results in each
exconjugant possessing half of both macronuclei. Their eventual de-
composition makes the exchange difficult to explain on functional
grounds. Summers and Kidder suggest that it may represent a ‘“‘remi-
niscence of a more primitive protozoan condition before the separation
of trophic nuclear materials from the germinal materials.”

The odd elongations of degenerating macronuclear chromatin into
ribbon or rod-like fragments in Paramecium may be an abortive attempt
at a similar process.

CON JUGANT MEIOSIS

Because the general course of conjugation, as outlined by Maupas
(1889) is followed by the vast majority of ciliates so far studied, it 1s
convenient to use this outline in reviewing the process. His eight stages
are as follows:

Stage A, in which the micronucleus swells and prepares for division;

Stage B, the first meiotic or maturation division;

Stage C, the second meiotic division;

Stage D, the third nuclear division, which produces the pronuclet;

Stage E, that of mutual exchange and the union of pronuclet;

Stage F, the first metagamic (amphinuclear) division;

Stage G, the second metagamic division;

Stage H, subsequent reorganization.

Stages A, B, C, and D are concerned with preparation for syngamy.
This preparation includes meiosis and the formation of pronuclei (see
Fig. 147). Stage E is the climax of the entire process, wherein the act
of fertilization is consummated. Stages F, G, and H are concerned with
the reorganization of the body and the reéstablishment of the usual vege-
tative form.

Among ciliates that normally possess more than one micronucleus
there is little uniformity in the number of nuclei that undergo the two
meiotic divisions. In many forms all micronuclei enter the first meiotic
division. Then all products of this division may divide again, or various
numbers of them may be resorbed (see Calkins, 1933, p. 295). In
Dileptus gigas, however, Visscher (1927) has shown that only one of
the large number of micronuclei undergoes maturation.

Two to eighteen micronuclei have been described as entering the first

—
<—

10 11

Figure 147. Diagram of ciliate conjugation. 1, two ciliates joined ventrally, micro-
nuclei in prophase parachute stage; 2, first meiotic (equational) division; 3, second
meiotic (reduction) division in which the chromosome number is reduced from diploid
to haploid, and the macronucleus begins to degenerate; 4, third maturation division,
involving only one of the four nuclei in each animal, the other three degenerate; 5,
migration of the wandering (¢) pronuclei into the apposed animals, 6 and @ pro-
nuclei of left conjugant stippled to indicate common origin; 6, fusion of wandering
and stationary pronuclei to form synkaryon and restore diploid condition; 7, conjugants
separate, first division of amphinucleus in exconjugants; 8, second amphinuclear division;
9, four nuclei produced by the two amphinuclear divisions, the old macronucleus dis-
integrates; 10, two of the four new nuclei develop into new micronuclei, two into new
macronuclei; 11 and 12, first fission of the exconjugant separates out one micronucleus
and one macronucleus to each daughter cell, reéstablishing the vegetative condition.

626 FERTILIZATION

maturation division of different ciliates. Variation in the number of
nuclei involved in the maturation divisions occurs within the same species,
as well as among different species. As only two pronuclei function in
fertilization, and as these two are known to be sister nuclei in many
ciliates, only one micronucleus really needs to undergo maturation.

STAGES A AND B, THE FIRST MEIOTIC DIVISION

In the earlier accounts of conjugation, the first maturation division
in several ciliates was said to be not greatly different from ordinary
vegetative mitosis. Recent accounts, based on careful cytological studies,
show marked peculiarities in the prophase of the first maturation division.
It seems possible, therefore, that more detailed studies, with improved
techniques, may reveal these distinguishing prophase stages in all ciliates.
The fact that such a careful worker as Maupas failed to observe the
highly characteristic changes that occur in Ezplotes patella (Turner,
1930) lends weight to this possibility. j

In the vast majority of ciliates, the prophase of the first maturation
division is highly characteristic and presages the coming reduction. In
some ciliates the micronucleus takes on the form of a crescent, or comma,
during the prophase, and this appearance is sufficiently characteristic to
be recognized as a general type. Among ciliates that exhibit the crescent
formation are various species of Paramecium and the Vorticellidae.

There 1s little agreement as to the number of chromosomes in Para-
mecium or even as to the method by which the crescent is transformed
into the metaphase spindle. The chromosome number in all species 1s
surely larger than the 8 or 9 given by Hertwig (1889) for P. aurelza,
and probably less than 150, which has been attributed to P. caudatum.
Calkins and Cull (1907) suggested that the 165 or more small chroma-
tin rods or fibers seen in P. caudatum are comparable to the physical
counterpart of the individual genes of higher animals. Aggregates of
these would represent a chromosome in cases where chromosomes are
formed. Perhaps the 32 chromomeres of Exzplotes patella (Turner,
1930) also represent 32 genes, although one would expect this highly
specialized hypotrich to have more genes than the more primitive holo-
trich.

Whatever the nature of the chromatin elements in Paramecium may
be, these investigators, as well as Dehorne (1920), show that the first

FERTILIZATION O27

maturation spindle is formed at right angles to the prophase crescent by
the migration of the division center from the apex of the crescent to the
middle of the crescent, and by the pushing out of the other pole across
the crescent. Earlier workers believed the spindle was formed by a
shortening of the crescent. Dehorne finds no chromosomes at all, but,
instead, a simple convoluted thread.

In a wide variety of other ciliates, the prophase develops a “‘cande-
labra”” (Collin, 1909) or “parachute” (Calkins, 1919) stage. It is
noteworthy that the parachute prophase occurs in most of the ciliates in
which reasonably complete chromosome studies have been made and
reduction definitely located. Kidderia (Concho phthirius) mytili may be
an exception to this, but Kidder (1933a) admits he might have missed
finding it. Tannreuther (1926) describes a simple type of chromosome
formation in Prorodon griseus, in which chromosomes arise directly out
of a central chromatin mass upon the equator of the spindle.

In Explotes patella a typical parachute is formed, which is seen as
a stage in the transformation of resting chromatin into the chromosomes
of the metaphase spindle. The events transpire synchronously in both
nuclei produced by the preliminary, or pre-maturation division of the
micronucleus occurring in this species. Each nucleus swells to several
times its original size, as the faintly granular chromatin becomes more
basophilic, and is arranged in a reticulum filling the nuclear space (Fig.
148). The reticulum condenses in the center and becomes polarized, with
most of the chromatin at one pole. Further condensation forms a dense
club-like structure, which presently loosens up and is transformed into
a parachute, with most of the chromatin forming the “‘cloth”’ at one pole,
the spindle fibers forming the ‘‘rope,” and an endosome at the other pole
forming the ‘‘weight.’’ The chromatin then forms thirty-two discrete
chromatin granules, the chromomeres, which soon migrate to the equa-
torial plate in groups of four. These eight groups of four chromomeres
apparently correspond to the eight diploid chromosomes found in other
stages of the life cycle. In the anaphase of this division, sixteen chromo-
meres pass to each pole, and one may frequently observe them associated
in pairs as loosely connected dumb-bells. The sixteen chromomeres, or
eight dumb-bells, which pass to each pole represent the eight diploid
chromosomes and identify this as an equational division.

In Pleurotricha lanceolata, Manwell (1928) found chromomeres that

628 FERTILIZATION

fuse to form about eighty dumb-bells. Since the diploid chromosome num-
ber is forty in this species, four granules (two dumb-bells) evidently
represent a chromosome, just as they do in E. patella, and forty dumb-
bells pass to each pole in the anaphase.

In Kidderia (Conchophthirius) mytili, Kidder (1933a) found thirty-

Figure 148. Stages in the first maturation division of Euplotes patella. A, early nucleus
with finely granular chromatin; B, chromatin reticulum condensing in center; C, para-
chute stage; D, later parachute showing chromatin granules migrating from upper pole,
endobasal bodies and intradesmose visible; E, metaphase stage with thirty-two chromo-
meres arranged in eight chromosome groups; F, anaphase stage with sixteen chromo-
meres (eight chromosomes) passing to each pole. (Turner, 1930.)

two granules forming on the spindle and sixteen passing to each pole,
as in E, patella. Sixteen is the diploid number in this species, so the
thirty-two granules represent half a chromosome each, although there
is no visible association between the halves.

Gregory (1923) described forty-eight chromomeres appearing in the
prophase and fusing to form twenty-four dumb-bell chromosomes in
Oxytricha fallax. In this case twelve dumb-bells pass to each pole. If

FERTILIZATION 629

twelve were the diploid number, this would correspond exactly to the
condition existing in Ewplotes patella and in Pleurotricha lanceolata.
However, Gregory believes that twelve is the haploid number and that
the separation of the twenty-four dumb-bells into two groups of twelve
each, in the first maturation division, means that this is the reduction
division, a condition unusual in ciliates. This interpretation is weakened
somewhat by the fact that twenty-four dumb-bells are formed in the
prophase of the second maturation division, twelve passing to each pole
in the anaphase. It is possible that the twenty-four dumb-bells which
separate into two groups of twelve each in the first maturation division
are actually tetrads, and that the twelve going to each pole are diads.
This would mean that the joining of the original granules is synaptic
in character and that their passing to the same pole indicates splitting,
or equational division. This explanation is not completely satisfying, in
view of the events of the second maturation division. Each of the twenty-
four dumb-bells which are formed in the prophase of the second division
would have to be derived from one granule of the first anaphase dumb-
bells. If that occurred, then the second maturation division would be
reductional; but if they are formed by the splitting of entire dumb-bells,
as believed by Gregory, then the second division would be equational and
the first would be reductional.

Whichever interpretation is correct, one thing seems clear: in all these
forms the chromosomes of the first maturation division are composed
of a definite number of loosely associated chromomeres. It is in their
method of distribution that interpretations differ, and further investiga-
tion in this field will be welcomed by those interested in meiotic phe-
nomena in the Protozoa.

No parachute stage was found by Noland (1927) in Metopus sig-
moides. Instead, the chromatin forms a spireme, which condenses into a
single large sausage-shaped ‘“‘chromosome”’ on the spindle. This divides,
and one part goes to each pole. The interpretation of this condition is
difficult, because of the obscurity of later stages. From the appearance
of the synkaria in Noland’s drawings, one would judge that there are
two large chromosomes in some, and four in others. If two were the
diploid number, the four would represent splitting for fission. Then the
single chromosome of the first maturation division could be interpreted
as a synaptic pair in close union. This speculation may not be justified

630 FERTILIZATION

by further investigation, but it seems reasonable on the basis of the avail-
able facts and is in line with current theory.

Calkins (1919) describes a peculiar situation in Uroleptus mobilis,
in which two types of metaphase stages are found (Fig. 149), one in
which about twenty-four chromosomes appear and twelve go to each pole,
and another in which eight chromosomes appear and eight go to each
pole in an obviously equational division. Although the number 1s not
strictly homologous, the first type is similar to that which occurs in
Euplotes patella and other species, and the second type is what would
be expected if all chromosomes were compact. Intermediate forms are
conceivable, in which some chromosomes are compact and others are

Figure 149. First maturation spindles of Uroleptus mobilis. A and B, two types of
metaphase stages; C and D, two types of anaphase stages found in this form. Both
types are equational divisions, since the diploid eight chromosomes appear in the sub-
sequent division, in which they are reduced to four. (After Calkins, 1919.)

dispersed as several loosely associated chromomeres. This would explain
many irregular counts, which otherwise seem chaotic.

In Chilodonella (Chilodon) uncinatus a parachute is formed after
the division of an endobasal body, according to MacDougall (1925).
Enriques (1908) failed to see the parachute in the same species, but
described a peculiar rod formation. In MacDougall’s material, four
strands of chromomeres are formed from the spireme of the late para-
chute stage and condense into four dumb-bell chromosomes on the
spindle. This author states that the exact number of granules in each
strand was not determined, but her Figure 23 shows four on each strand.
This is interesting, since four chromomeres to a chromosome has been
found in E. patella (see Fig. 148 E) and other ciliates. The four chromo-
somes then split longitudinally and four halves migrate to each pole, at

FERTILIZATION 631

which point they fuse in pairs, forming diads before entering upon the
resting stage.

A tetraploid strain arose spontaneously in a pure culture of Mac-
Dougall’s Chilodonella uncinatus. Maturation phenomena in the tetra-
ploid form were similar to those of the usual diploid form, except that
there were twice the number of chromosomes in every stage. Investiga-
tions, presented and reviewed in a later article by MacDougall (1936),
show meiotic processes which are similar in six species of Chilodonella.
In all species the diploid number of chromosomes is four. A parachute
stage is followed by the formation and the synaptic pairing of chroma-
tin threads, as in “‘classic leptotine and zygotine’’ stages, which condense
to form the pachytene chromosomes. MacDougall’s descriptions reveal
the striking similarity of meiosis in Chilodonella to the general scheme
of meiosis in the Metazoa. Messiatzev (1924) reported synapsis occur-
ring in the first maturation division and again in a fifth amphinuclear
division of Lionotus lamella, but Poljansky (1926) believes that Mes-
siatzev confused his stages in the latter case.

The small number and the large size of the chromosomes in Chilodo-
nella make this a very favorable form for study of meiotic phenomena. It
seems unfortunate that more of the recent studies that have genetic sig-
nificance were not made on this animal, in which chromosome behavior
is clear-cut and well known, instead of on Paramecium, in which it is
practically impossible to determine any of the significant stages in
meiosis.

STAGE C, THE SECOND MEIOTIC DIVISION

The second meiotic division is the reduction division in all ciliates
thus far studied, except in Oxytricha fallax, according to Gregory
(1923). Prandtl (1906), in his work on Didinium nasutum, was the
first to present conclusive evidence on chromosome reduction in ciliates
when he described reduction from sixteen to eight chromosomes in the
second meiotic division. There seems to be no general rule for the num-
ber of nuclei that enter this division. In the species of Chilodonella
studied by MacDougall (1936), only one nucleus is involved in any of
the three progamous divisions; the other products degenerate.

In perhaps the majority of ciliates both products of the first division
enter the second division. In O. fallax (Gregory, 1923) and in forms

632 FERTILIZATION

with multiple micronuclei such as Uroleptus mobilis (Calkins, 1919), a
variable number of nuclei may divide a second time. In Ezplotes patella
all micronuclear products undergo a second meiotic division. Because
of the preliminary division, there are four in each conjugant. No resting
stage occurs between divisions here, in contrast to MacDougall’s (1936)
account of Chilodonella. The daughter nuclei of the previous division
are still connected by their respective drawn-out nuclear membranes when
the chromatin begins to resolve itself into a reticulum in each nucleus
and the granules on the reticulum condense into eight discrete, ovoid

Figure 150. Second meiotic (reduction) division in Explotes patella. A, eight ovoid
chromosomes appearing on the spindle; B, synaptic pairing and lengthening of chromo-
somes; C, disjunction and separation of homologous chromosomes in the anaphase, four
passing to each pole.

chromosomes (Fig. 150 A). The chromosomes now conjugate in four
pairs, in what is probably a delayed synapsis, elongate somewhat, and
disjoin longitudinally, four haploid chromosomes passing to each pole.

Calkins (1919) described a similar pairing and separation of the
eight chromosomes in Uroleptus mobilis. Tannreuther (1926) presents
evidence of chromosome pairing in the reduction division of Prorodon
griseus, but in most cases where synapsis has been observed, it occurs in
the first meiotic division.

STAGE D, THE THIRD DIVISION, AND THE FORMATION OF PRONUCLEI

In all ciliates thus far studied, a third division occurs. This division
is equational in character and usually involves only one nucleus, while
the rest degenerate. The two products of this division are the pronuclei
which take part in fertilization.

In a few ciliates, two, three, and four micronuclei have been reported
to divide at this stage, but in no case has it been demonstrated that the

FERTILIZATION 633

two functional pronuclei are derived from different spindles. In all cases
in which only one nucleus is involved, and possibly also in those where
two or more are involved, the two pronuclei must be genetically identical
if the third division is equational. In Uroleptus (Calkins, 1919), two or
three nuclei divide, but the two pronuclei are always sister nuclei. As
this occurs in both members of the pair, the exconjugants should theo-
retically be genetically identical. This appears to be the significant feature
in the third maturation division.

Figure 151. Explotes patella. A, B, C, third maturation division, in which the four
haploid chromosomes in A split longitudinally and the halves slip past each other
in B, and four go to each pole in C; D, fertilization nucleus in which the ¢ and @
pronuclei have just joined, but their chromosome groups have not yet mingled.

In Ex plotes patella (Turner, 1930), two nuclei enter the third division.
In each nucleus (Fig. 151) the chromatin reticulum condenses into four
strands of chromatin granules, which condense into four compact sausage-
shaped chromosomes lying lengthwise of the spindle. The chromosomes
split longitudinally, and the halves slip past each other as they migrate
to separate poles in this equational division. The chromosomes are all
lying in the same axis, so that as the chromosomes slip past each other
in the early anaphase, they appear end to end, and the figure might
easily be misinterpreted as a transverse division of chromosomes. It 1s
possible that the descriptions of the transverse division of chromosomes
in the third division, given by Enriques (1908), Calkins (1919, 1930),
MacDougall (1925), and others were based on some such artifact. Cal-
kins points out that if each chromosome represents one gene, the method
of division is of no consequence. This interpretation would doubtless
serve for Paramecium, which has a large number of chromosomes, but it
is hardly conceivable that Chilodonella would have only two pairs of
genes. It seems more probable that the apparent transverse division of
some chromosomes in mitosis is due to our inability to demonstrate by

634 FERTILIZATION

present techniques some of the finer structural changes which occur
within the chromatin mass.

Since Prandtl (1906) first noted a difference in size between the wan-
dering and the stationary pronuclei of Didininm nasutum, slight differ-
ences have been reported in a number of other cases. Calkins and Cull
(1907) showed that this is due to a heteropolar third division, in
Paramecium caudatum, Maupas (1889) was the first to record a struc-
tural difference between pronuclei, when he observed the area of dense
cytoplasm in front of the migrating pronucleus of Ezplotes patella. The
most striking dimorphism appears in Cycloposthium, according to
Dogiel (1925). The wandering pronucleus is spermatozoén-like in hav-
ing an elongated tail. All these differences between pronuclei must be
cytoplasmic in origin, for the nuclei, as has been pointed out, are
genetically identical, if our present concepts are correct.

STAGE E, MIGRATION OF PRONUCLEI AND FERTILIZATION

Migration of the wandering nucleus occurs synchronously in the two
conjugants, so that they generally pass each other in the cytoplasmic
bridge which joins the two conjugants. In Cycloposthium (Dogiel,
1925), the spermatozodn-like male pronucleus passes out of the mouth
of the parent body and into the mouth of the recipient, by way of the
juxtaposed peristomal cavities (Fig. 167).

In Explotes patella (Turner, 1930), the wandering pronucleus breaks
out of the left anterior tip of the parent body, which is pressed into the
peristomal field of its mate (see p. 620, above), passes backward be-
tween the appressed conjugants, and finally enters the cytostomal area of
the recipient. Both pronuclei form spindles as the male approaches the
female, and four chromosomes can be seen in each. As the pronuclet
touch, their membranes dissolve and the two groups of four chromo-
somes mingle, as fertilization is completed and the diploid condition
restored (Fig. 151 D). In Chilodonella, MacDougall (1925) shows
that the two haploid chromosomes are visible throughout the migration
period (Fig. 152), but lose their identity soon after fertilization.

The appearance of the pronuclei at the time of union varies with the
species. In a number of ciliates, they are in the form of a spindle similar
to those of Euplotes, although few show chromosomes. In other ciliates,
the pronuclei are spherical and vesicular at the time of union. In still
others, intermediate conditions have been reported.

FERTILIZATION 635

Figure 152. Chilodonella uncinatus. Migration of the pronuclei across the protoplasmic
bridge. Each pronucleus contains two haploid chromosomes still attached by strands to
their sister halves. Also visible in each conjugant are old and new oral baskets and the
granular remnants of the old macronucleus. (After MacDougall, 1925.)

STAGES F, G, AND H, THE EXCON JUGANTS

The subject of reorganization is dealt with elsewhere in this volume,
but we may consider briefly some of the cytological aspects of the re-
organizing exconjugant.

After fusion of the pronuclei, the fertilization nucleus divides one or
more times, and from the products of division the new micronuclear
and macronuclear elements are formed, while extra products disintegrate.
The number of divisions the synkaryon undergoes before differentia-
tion of the macronuclei and micronuclei in various ciliates is reviewed
by Kidder (1933b) in his work on Ancistruma. Kidder lists eight
species in “group A,” in which the micronucleus and the macronucleus
are differentiated after the first amphinuclear division. To these we may
add Chilodonella cucullulus (Ivani¢, 1933); C. chattoni, C. labiata, C.
caudata, C. faurii (MacDougall, 1936); and N yctotherus cordiformis
(Wichterman, 1937). In other ciliates, differentiation occurs after the
second amphinuclear division. Kidder lists twenty-one species in this
“group B,” which includes a majority of the best-known ciliates. To
this list may be added Balantidium (Nelson, 1934), from the Chim-
panzee.

In about half of these species, all four products remain functional,

636 FERTILIZATION

and in the others two or three develop into micronuclei and macro-
nuclei, while the remaining one or two degenerate.

In Ezplotes patella, the macronucleus and the micronucleus are never
sister nuclei, so that the first amphinuclear division evidently separates
the macronuclear line from the micronuclear line. However, there is no
apparent difference, except one of position, between the two products
of the first division.

In “group C’ Kidder lists twelve ciliates, in which differentiation
occurs after the third amphinuclear division. To this list, which includes
the familiar Paramecium caudatum, may be added Parachaenia myae
(Kofoid and Bush, 1936). The number of products, if any, which de-
generate appears to vary with the author, as well as with the species.
In about half of these species, all nuclei remain functional.

In Paramecium caudatum, four of the eight amphinuclear products
become macronuclei and are distributed by fission to the four grand-
daughters, while four become micronuclei. According to Calkins and
Cull (1907), all of these micronuclei remain functional and are dis-
tributed by fission; but Maupas (1889), Jennings (1920), and Doflein-
Reichenow (1928) indicate that three degenerate, while the fourth
remains as the functional micronucleus and divides at each subsequent
fission, just as it does in P. putrinum (Doflein, 1916).

In some material, both of these schemes are represented in P. cauda-
tum; but, perhaps because the dividing micronucleus is more easily
identified, the latter type is more clearly and unquestionably demon-
strable.

In “group D,” in which differentiation occurs after the fourth amphi-
nuclear division, Kidder lists only P. mu/timicronucleata (Landis, 1925),
Kidderia (Conchophthirius) mytili (Kidder, 1933a), and Bzrsaria
truncatella, according to Prowazek (1899). But Bwrsaria belongs in
“group C,”’ according to Poljansky (1928, 1934).

After the final division of the synkaryon, which separates macro-
nuclear elements from micronuclear elements, the micronuclei return to
the normal condition. This involves shrinkage in size and the restora-
tion of the chromatin to the homogeneous condition. If more than the
normal number are formed, they are separated by the subsequent body
fissions until the normal number is established, as in P. caudatum,
according to Calkins and Cull (1907). If the normal number were

FERTILIZATION 637

differentiated, they divide at every subsequent body division, as in
Euplotes patella.

The macronuclei grow to their normal size and are distributed by
body fissions to the daughter and granddaughter cells, if more than the
vegetative number are formed, as in Paramecium. Thus the ordinary
vegetative form is reéstablished.

During their development, the young macronuclei undergo some
striking and significant changes. Several investigators have reported
marked enlargement of the macronuclear Av/agen in ciliates, since the
early description of the “ball-of-yarn” stage in Nyctotherus cordiformis
by Stein (1867).

Calkins (1930) showed that the macronuclear Anlage of Uroleptus
halseyi at first contains no chromatin, if chromatin be defined as a
nucleic acid containing substance. As the young macronucleus grows,
minute chromatin granules are formed within the matrix and grow in
size and number until the nucleus is filled with large, intensely staining
chromatin granules. Subsequent divisions of this “‘placenta’’ and of the
cell body restore the normal vegetative nuclear complex.

A number of other ciliates show enlargement of the placenta to a size
greater than that of the ordinary condition, after which shrinkage, or
condensation, reduces it to its vegetative size. According to MacDougall
(1925), the young Anlage of Chilodonella uncinata stains very faintly
at first, but more intensely later. The enlargement continues until the
young macronucleus nearly fills the cell.

In Explotes patella (Turner, 1930), as in Uroleptus, the early macro-
nuclear An/age contains little, if any, demonstrable chromatin. After
several hours of growth, a fine chromatin reticulum develops and is
transformed into a broken spireme. The chromatin spireme enlarges
with the nucleus and resembles the “ball of yarn” in Nyctotherus cor-
diformis, as pictures by Stein (1867) and Wichterman (1937), except
that the spireme is more tortuous. Earlier stages resemble those of
Meto pus sigmoides as drawn by Noland (1927), and of Paraclevelandia
simplex (Kidder, 1938) in cystic reorganization. The spireme finally
becomes shorter and very much thicker, as chromatin granules are
formed along its periphery. At this stage a fine thread is discernible run-
ning through the center of the spireme. The granules become scattered
throughout the macronuclear Anlage as the structure of the spireme

638 FERTILIZATION

disappears. The large granular ball thus formed condenses somewhat
and then elongates, starts bending into its normal C-shape, and com-
bines with one or two sizeable remnants of the old macronucleus which
have persisted through the process. When the elongating macronuclear
Anlage approaches the remnants of the old macronucleus, the latter lose
their pycnotic appearance and the chromatin reorganizes itself into dis-
crete, dispersed granules, again resembling the normal condition. The
new and the old portions then unite, end to end, to produce the C-shaped
nucleus of the trophic form, the reconstituted portion forming the pos-
terior portion. Examination of hundreds of exconjugants at this stage
convinces one that the proximity of the elongating Av/age is the influ-
ence which brings about the reorganization of the old remnants. The
old chromatin is so thoroughly reorganized before joining the new
Anlage that it possibly has little more effect on the nature of the
new nucleus than if it had been dissolved and re-formed within the new
nuclear membrane.

Ikeda and Ozaki (1918) first reported fragments of the old macro-
nucleus being incorporated into the developing macronuclear An/age in
Boveria labialis.

Kidder (1933a, 1933b) has described an interesting phenomenon oc-
curring in the macronuclear Anlagen of Kidderia (Concho phthirius)
mytilt and in Ancistruma isseli, At each of the two or three exconjugant
fissions which separate the seven or eight new macronuclei, all un-
separated macronuclei cast out in an orderly manner a sphere of chroma-
tin. According to Kidder, this “may represent the sloughing off of the
germinal chromatin contained in the amphinucleus, a substance that 1s
superfluous for the further activity of a purely trophic cell element
(Reichenow, 1927).” Diller (1928) suggested this as an explanation
of a similar occurrence during endomixis in Trichodina. Chromatin ex-
trusions from developing macronuclear An/agen would appear to be
even more closely analogous to the Ascaris type of chromatin diminution,
as brought out by Boveri, than is the chromatin diminution described
by MacDougall in the division of the macronucleus in Chilodonella.

Until recently the behavior of the macronucleus in conjugation has
been given scant attention. The disintegration of the old macronucleus
and the differentiation, number, and distribution of new macronuclear
Anlagen have been noted, but no great significance has been attached to

FERTILIZATION 639

these processes. It now seems probable that more intensive investigation
of the réle of the macronucleus in conjugation will be extremely profit-
able.

LITERATURE CITED

Beers, C. D. 1931. Some effects of conjugation in the ciliate Didinium
nasutum, J. exp. Zool., 58: 455-70.

Bélat, K. 1923. Untersuchungen an Actinophrys sol Ehrenberg. I. Die
Morphologie des Formwechsels. Arch. Protistenk., 46: 1-96.

—— 1926. Der Formwechsel der Protistenkerne. Ergebn. Zool., 6: 235-
654.

Bott, K. 1907. Uber die Fortpflanzung von Pelomyxa palustris. Arch.
Protistenk., 8: 120-58.

Brumpt, E. 1909. Demonstration du réle pathogéne du Balantidinm coli.
Enkystment et conjugaison de cet infusore. C. R. Soc. Biol. Paris, 67:
103-5.

—— 1913. Etudes sur les Infusoires parasites. I. La Conjugaison
d'Anoplophrya circulans Balbiani, 1885. Arch. parasit., 16: 187-210.

Buschkiel, A. L. 1911. Beitrige zur Kenntnis des Ichthyophthirius multi filis
Fouquet. Arch. Protistenk., 21: 61-102.

Calkins, G. N. 1904. Studies on the life history of Protozoa. IV. Death of
the A series. J. exp. Zool., 1: 423-61.

——— 1907. The fertilization of Amoeba proteus. Biol. Bull., 13: 219-30.

——— 1912. The paedogamous conjugation of Blepharisma undulans. J.
Morph., 23: 667-88.

—— 1919. Uroleptus mobilis, Engelm. 1. History of the nuclei during
division and conjugation. J. exp. Zool., 27: 293-357.

—— 1930. Uroleptus Halseyi Calkins. II. The origin and fate of the macro-
nuclear chromatin. Arch. Protistenk., 69: 151-74.

—— 1933. The Biology of the Protozoa. Philadelphia.

Calkins, G. N., and R. Bowling. 1926. Gametic meiosis in Monocystis. Biol.
Bull., 51: 385-99.

— 1929. Studies on Dallasia frontata Stokes. II. Cytology, gametogamy
and conjugation. Arch. Protistenk., 66: 11-32.

Calkins, G. N., and S. W. Cull. 1907. The conjugation of Paramecium
aurelia (caudatum ). Arch. Protistenk., 10: 375-415.

Chatton, E. 1927. La Gamétogénése méiotique du flagellé Paradininm pouchett.
G. Re AcadsSets Patisnt 85: 553-55.

Chatton, E., and B. Biechler. 1936. Documents nouveaux relatifs aux
Coccidinides (Dino flagellés parasites). La Sexualité du Coccidinium
mesnili n. sp. C. R. Acad. Sci., Paris, 203: 573-76.

Chatton, E., and A. Brodsky. 1909. Le Parasitisme d’une Cytridinee du
genre Sphaerita Dangeard chez Amoeba limax Dujard. Etude compara-
tive. Arch. Protistenk., 17: 1-18.

640 FERTILIZATION

Chatton, E., and A. Lwoff. 1935. Les Ciliés Apostomes. I. Apercu historique
et général; étude monographique des genres et des especes. Arch. zool.
exp. gén., 77, Protistologica L: 1-453.

Chatton, E., and C. Pénard. 1921. Les Nicollellidae. Infusoires intestinaux
des Gondis et des Damans, et le “cycle évolutif’”’ des ciliés. Bull. biol.,
59: 87-153:

Collin, B. 1909. La conjugaison d’Anoplophrya branchiarum (Stein) (A.
circulans Balbiani.). Arch. zool. exp. gén., Series 5, 1: 345-88.

Crawley, H. 1916. The sexual evolution of Sarcocystis muris, Proc. Acad.
nat. Sci. Philad., 68: 2-43.

Dangeard, P. A. 1898. Sur les Chlamydomonadinées. C. R. Acad. Scie., Paris,
127: 736-38.

Debaisieux, P. 1924. Sphaeromyxa sabrazesi Laveran et Mesnil. Cellule, 35:
267-301.

Dehorne, A. 1920. Contribution a l’étude comparée de l’appareil nucleaire
des Infusoires ciliés (Paramecium caudatum et Colpidium truncatum),
des Euglenes et des Cyanophycees. Arch. zool. exp. gén., 60: 47-176.

Diller, W. F. 1928. Binary fission and endomixis in the Trichodina from
tadpoles (Protozoa, Ciliata). J. Morph., 46: 521-61.

— 1936. Nuclear reorganization processes in Paramecium aurelia, with
descriptions of autogamy and “hemixis.” J. Morph., 59: 11-67.

Diwald, K. 1938. Die ungeschlechtliche und geschlechtliche Fortpflanzung
von Glenodinium lubiniensiforme spec. nov. Flora, Jena, 32: 174-92.

Dobell, C. C. 1908. The structure and life-history of Copromonas subtilis.
Quart. J. micr. Sci., 52: 75-120.

—— 1925. The life-history and chromosome cycle of Aggregata eberthi.
Parasitology, 17: 1-136.

Dobell, C. C., and A. P. Jameson. 1915. The chromosome cycle in Coccidia
and gregarines. Proc. roy. Soc., B., 89: 83-94.

Doflein, F. 1916. Lehrbuch der Protozoenkunde. 4th ed., Jena.

Doflein, F., and E. Reichenow, 1928. Doflein, Lehrbuch der Protozoenkunde.
5th ed., Jena.

Dogiel, V. 1925. Die Geschlechtsprozesse bei Infusorien (speziell bei den
Ophryoscoleciden) , neue Tatsachen und theoretische Erwagungen. Arch.
Protistenk., 50: 283-442.

Elpatiewsky, W. 1907. Zur Fortpflanzung von Arcella vulgaris Ehrbg. Arch.
Protistenk., 10: 441-66.

Enriques, P. 1908. Die Conjugation and sexuelle Differenzierung der In-
fusorien. Arch. Protistenk., 12: 213-76.

Fermor, X. 1913. Die Bedeutung der Encystierung bei Stylonychia pustulatg.
Zool. Anz., 42: 380-84.

Goldschmidt, R. 1907. Lebensgeschichte der Mastigamében Mastzgella vitrea
n. sp. u. Mastigina setosa n. sp. Arch. Protistenk., Suppl. 1: 83-168.

FERTILIZATION 641

Goroschankin, J. N. 1890. Beitrige zur Kenntnis der Morphologie und
Systematik der Chlamydomonaden. I. Chlamydomonas Braunii (Mihi).
Bull. Soc. imp. Natur., Moscou, pp. 498-520.

— 1891. Beitrige zur Kenntnis der Morphologie und Systematik der
Chlamydomonaden. II. Chlamydomonas Reinhardii (Dang.). Bull. Soc.
imp. Natur., Moscou, pp. 101-42.

— 1905. Beitrage zur Kenntnis der Morphologie und Systematik der
Chlamydomonaden. III. Chlamydomonas cocifera (Mihi). Flora, Jena,
94: 420-23.

Gregory, L. H. 1923. The conjugation of Oxytricha fallax. J. Morph., 37:
555-81.

Gross, F. 1934. Zur Biologie und Entwicklungsgeschichte von Noctiluca
miliaris, Arch. Protistenk., 83: 178-96.

Hartmann, M. 1911. Die Konstitution der Protistenkerne und ihre Bedeutung
fiir die Zellenlehre. Jena.

Hartmann, M., and K. Nagler. 1908. Copulation bei Amoeba diploidea n.
sp. mit. Selbstandigbleiben der Gametenkerne wahrend des ganzen
Lebenszyklus. S. B. Ges. naturf. Fr. Berl., Nr. 5, 112-25.

Hertwig, R. 1889. Uber die Konjugation der Infusorien. Abh. bayer. Akad.

Wiss. Munchen, II. KI. 17: 1-83.

1898. Uber Kernteilung, Richtungskérperbildung und Befruchtung
von Actinosphaerium eichhornii, Abh. math.-phys. bayer. Akad. Wiss.
Munchen, 19: 631-734.

Ikeda, I, and Y. Ozaki. 1918. Notes on a new Boveria species, Boveria
labialis n. sp. J. Coll. Sci. Tokyo, 40: 1-25.

Ivani¢, M. 1933. Die conjugation von Chilodon cucullulus Ehtbg. Arch.
Protistenk., 79: 313-48.

Jameson, A. P. 1920. The chromosome cycle of gregarines, with special refer-
ence to Diplocystis schneideri Kunstler. Quart. J. micr. Sci., 64: 207-66.

—— 1927. The behaviour of Balantidium coli Malm. in cultures. Parasitology,
19: 411-19.

Jennings, H. S. 1913. The effect of conjugation in Paramecium. J. exp. Zool.,
14; 279-391.

—— 1920. Life and death; heredity and evolution in unicellular organisms.
Boston.

—— 1938. Sex reaction types and their interrelations in Paramecium
bursaria. 1, Il. Clones collected from natural habitat. Proc. Nat. Acad.
Sci. Wash., 24: 112-20.

Jehnson, P. L. 1930. Reproduction in Amoeba proteus. Arch. Protistenk.,

71: 463-98.

Jones, P. M. 1928. Life cycle of Amoeba proteus (Chaos diffluens) with
special reference to the sexual stage. Arch. Protistenk., 63: 322-32.
Kidder, G. W. 1933a. Studies on Conchophthirius mytili De Morgan. II.

Conjugation and nuclear reorganization. Arch. Protistenk., 79: 25-49.

642 FERTILIZATION

—— 1933b. On the genus Avcistruma Strand (= Ancistrum Maupas).
II. The conjugation and nuclear reorganization of A. isseli Kahl. Arch.
Protistenk., 81: 1-18.

— 1938. Nuclear reorganization without cell division in Paraclevelandia
simplex (Family Clevelandellidae), an endocommensal ciliate of the
wood-feeding roach, Panesthia, Arch. Protistenk., 91: 69-77.

Kofoid, C. A. 1921. Symposium on fertilization. Anat. Rec., 20: 223-325.

Kofoid, C. A., and M. Bush, 1936. The life cycle of Parachaenia myae gen.
nov., sp. nov., a ciliate parasitic in Mya arenaria Linn. from San Fran-
cisco Bay, California. Bull. Mus. Hist. nat. Belg., 12: 1-15.

Kudo, R. 1926. On Myxosoma catostomi Kudo 1923, a myxosporidian para-
site of the sucker, Catostomus cammersonii. Arch Protistenk., 56: 90-
fit).

Landis, E. M. 1925. Conjugation of Paramecium multimicronucleata, Powers
and Mitchell. J. Morph., 40: 111-67.

Lebedew, W. 1909. Uber Trachelocerca phoenecopterus Cohn, Ein marines
Infusor. Arch. Protistenk., 13: 70-114.

Le Calvez, J. 1938. Recherches sur les Foraminiféres. I. Développement et
reproduction. Arch. zool. exp. gén., 80: 163-333.

Léger, L. 1903. La Reproduction sexuée chez les Stylorynchus. Arch.
Protistenk., 3: 303-57.

—— 1907. Les Schizogregarines des Trachéates. I. Le genre Ophryocystis.
Arch. Protistenk., 8: 159-202.

Léger, L., and O. Duboscq. 1909. Etudes sur la sexualité chez les grégarines.
Arch. Protistenk., 17: 19-134.

Liesche, W. 1938. Kie Kern- und Fortpflanzungsverhaltnisse von Amoeba
proteus (Pall.). Arch, Protistenk., 91: 135-86.

Lister, J. J. 1895. The life-history of the Foraminifera. Philos. Trans., B.
186: 401-53.

MacDougall, M. S. 1925. Cytological observations on gymnostomatous cilt-
ates, with a description of the maturation phenomena in diploid and
tetraploid forms of Chilodon uncinatus. Quart. J. micr. Sci., 69: 361-84.

—— 1935. Cytological studies of the genus Chilodonella Strand, 1926
(Chilodon Ehrbg., 1838). I. The conjugation of Chilodonella sp. Arch.
Protistenk., 84: 199-206.

—— 1936. Etude cytologique de trois espéces du genre Chilodonella Strand.
Morphologie, conjugaison, réorganisation. Bull. biol., 70: 308-31.
Manwell, R. D. 1928. Conjugation, division, and encystment in Plewrotricha

lanceolata. Biol. Bull., 54: 417-63.

Mast, S. O. 1917. Conjugation and encystment in Didinium nasutum with
especial reference to their significance. J. exp. Zool., 23: 335-59.
Maupas, E. 1889. Le Rejeunissement karyogamique chez les ciliés. Arch.

zool. exp. gén., Ser. 2, 7: 149-517.

FERTILIZATION 643

Messiatzev, J. 1924. The conjugation of Lionotus lamella (in Russian).
Arch. russ. protist., 3, no. 1-2.

Metcalf, M. M. 1923. The opalinid ciliate infusorians. Bull. U. S. nat. Mus.,
120.

Milojevic, B. D. 1925. Zur Entwicklungsgeschichte der Gregarina cuneata
(F. St.), mit besonderer Beriicksichtigung der Entstehung des
Geschlechtskerns. Arch. Protistenk., 50: 1-26.

Minchin, E. A. 1912. An Introduction to the Study of the Protozoa. London.

Minkiewicz, R. 1912. Un cas de reproduction extraordinaire chez Polyspira
Delagei. C. R. Acad. Sci., Paris, 155: 733.

Miyashita, Y. 1927. On a new parasitic ciliate Lada tanishi n. sp., with
preliminary notes on its heterogamic conjugation. Jap. J. Zool., 1: 205-
18.

Mulsow, K. 1911. Ueber Fortpflanzungserscheinungen bet Monocystis
rostrata n. sp. Arch. Protistenk., 22: 20-55.

Myers, E. H. 1935. The life history of Patellina corrugata Williamson, a
foraminifer. Univ. Cal. Publ. Zool. tech., series, 3: 355-92.

—— 1936. The life cycle of Spzrillina vivipara, with notes on morphogenesis,
systematics and distribution in the Foraminifera. J. R. micr. Soc., 56:
120-46.

—— 1938. The present state of our knowledge concerning the life cycle of
the Foraminifera. Proc. nat. Acad. Sci. Wash., 24: 10-17.

Naville, A. 1927a. Le Cycle chromosomique et la meiose chez les Monocystis.
Z. Zellforsch., 6: 257-84.

—— 1927b. Le Cycle chromosomique d’Urospora lagidis (de Saint Joseph).
Parasitology, 19: 100-38.

—— 1927c. Le Cycle chromosomique, la fécondation et la réduction chro-
matique de Chloromyxum leydigi Mingazz. Ann. Inst. océanogr. monaco,
4: 177-208.

—— 1930a. Le Cycle chromosomique d'une nouvelle Actino myxidie:
Guyenotia sphaerulosa n. gen.; n. sp. Quart. J. micr. Sci., 73: 547-75.

—— 1930b. Recherches sur la sexualité les myxosporidies. Arch. Protistenk.,

69: 327-400.

1931. Les Sporozoaires. Mém. Soc. Phys. Genéve, 41, No. 1.

Nelson, E. C. 1934. Observations and experiments on conjugation of the
Balantidium from the chimpanzee. Amer. J. Hyg., 20: 106-34.

Neresheimer, E. R. 1907. Die Fortpflanzung der Opalinen. Arch. Protistenk.,
(Suppl.) 1: 1-42.

—— 1908. Fortpflanzung eines parasitischen Infusors (Ichthyophthirius).
S. B. Ges. Morph. Physiol., Miinchen, 23.

Noland, L. E. 1927. Conjugation in the ciliate Metopus sigmoides C. and
L. J. Morph., 44: 341-61.

Pascher, A. 1916, Uber die Kreuzung einzelliger, haploider Organnismen:
Chlamydomonas. Ber. dtsch. bot. Ges., 34: 228-42.

644 FERTILIZATION

Poljansky, G. 1926. Die Conjugation von Dogielella sphaerii (Infusoria
Holotricha, Astomata). Arch. Protistenk., 53: 407-34.

— 1928. Uber die Konjugation von Bursaria truncatella. Zool. Anz., 79:
D1-58.

— 1934. Geschlechtsprozesse bei Bursaria truncatella O. F. Mill. Arch.
Protistenk., 81: 420-546.

Prandtl, H. 1906. Die Konjugation von Didinium nasutum. Atch.
Protistenk., 7: 229-58.

Pratje, A. 1921. Noctiluca miliaris Suriray. Beitrage zur Morphologie,
Physiologie und Cytologie. I. Morphlogie und Physiologie. (Beobach-
tungen an der lebenden Zelle.) Arch. Protistenk., 42: 1-98.

Pringsheim, E. 1869. Uber Paarung von Schwarmsporen, die morphologische
Grundform der zeugung im Pflanzenreiche. Monatsber. preuss. Akad.
Wissensch., 721-38.

Prowazek, S. 1899. Protozoenstudien. I. Bwrsaria truncatella und thre Con-
jugation. Arb. zool. Inst. Univ. Wien. 11: 195-286.

Schaudinn, F. 1896. Uber die Copulation von Actinophrys sol Ehrbg. S. B.
preuss. Akad. Wiss., 83-89.

—— 1903. Untersuchungen tiber die Fortpflanzung. Arb. GesundhAmt., 19:
547-76.

Schellack, C. 1907. Uber die Entwicklung und Fortpflanzung von Echinomera
hispida A. Schn., Arch. Protistenk., 9: 297-345.

— 1913. Coccidien-Untersuchungen II. Die Entwicklung von Adelina
dimidiata A. Schn., einem Coccidium aus Scolopendra cingulata Latr.
Arb. GesundhAmt. Berl., 45: 269-316.

Schneider, A. 1886. Anoplophrya circulans (Balb.) Tabl. Zool., 1:31-88.

Schroder, O. 1907. Beitrage zur Entwicklungsgeschichte der Myxosporidien.

Sphaeromyxa sabrazesi (Laveran et Mesnil). Arch. Protistenk., 9: 359-81.

1910. Uber die Anlage der Sporocyste (Pansporoblast) bei Sphaeromyxa

Sabrazesi Laveran et Mesnil. Arch. Protistenk., 19: 1-5.

Scott, M. J. 1927. Studies on the Balantidium from the guinea-pig. J. Morph.,
44: 417-65.

Sharp, R. W. 1934. Introduction to Cytology. New York.

Sonneborn, T. M. 1936. Factors determining conjugation in Paramecium
aurelia. 1. The cyclical factor: the recency of nuclear reorganization.
Genetics, 21: 503-14.

— 1937. Sex, sex inheritance and sex determination in Paramecium aurelia.
Proc. nat. Acad. Sci. Wash., 23: 378-85.

Sonneborn, T. M., and B. M. Cohen. 1936. Factors determining conjuga-
tion in Paramecium aurelia. 1. Genetic diversities between stocks or races.
Genetics, 21: 515-18.

Stein, F. 1867. Der Organismus der Infusionsthiere. I]. Abt. Leipzig.

Summers, F. M., and G. W. Kidder. 1936. Taxonomic and cytological

FERTILIZATION 645

studies on the ciliates associated with the amphipod family Orchestiidae
from the Woods Hole district. Il. The coelozoic astomatous parasites.
Arch. Protistenk., 86: 379-403.

Swarczewsky, B. 1908. Uber die Fortpflanzungserscheinungen bei Arcella
vulgaris Ehrbg. Arch. Protistenk., 12: 173-212.

Tannreuther, G. W. 1926. The life history of Prorodon griseus. Biol. Bull.,
51: 303-20.

Turner, J. P. 1930. Division and conjugation in Ezplotes patella Ehrenberg
with special reference to the nuclear phenomena. Univ. Cal. Publ. Zodl.,
35, 193-258.

Valkanov, A. 1935. Untersuchungen tber den Entwicklungskreis eines
Turbellarienparasiten (Monocystella Arndti). Z. Parasitenk., 7: 517-38.

Visscher, J. P. 1927. Conjugation in the ciliated protozoén, Dileptus gigas,
with special reference to the nuclear phenomena. J. Morph., 44: 383-
414,

Weschenfelder, R. 1938. Die Entwicklung von Actinocephalus parvus Well-
mer. Arch. Protistenk., 91: 1-60.

Wichterman, R. 1937. Division and conjugation in Nyctotherus cordiformis
(Ehr.) Stein (Protozoa, Ciliata) with special reference to the nuclear
phenomena. J. Morph., 60: 563-611.

Woodruff, L. L. 1925. The physiological significance of conjugation and

endomixis in the Infusoria. Amer. nat., 59: 225-49.

1927. Studies on the life history of Blepharisma undulans. Proc. Soc.

exp. Biol. N.Y., 24: 769-770.

Woodruff, L. L., and H. Spencer. 1924. Studies on Spathidium spathula.
Il. The significance of conjugation. J. exp. Zool. 39: 133-96.

Zederbauer, E. 1904. Geschlechtliche und ungeschlechtliche Fortpflanzung
von Ceratium hirundinella. Ber. dtsch. bot. Ges., 22: 1-8.

Zuelzer, M. 1904. Beitrage zur Kenntnis der Difflugia urceolata Carter. Arch.
Protistenk., 4: 240-95.

CHAPTER XIII

ENDOMIXIS

LORANDE Loss WOODRUFF

DURING RECENT YEARS the attention of students of the Ciliophora has
been focused increasingly on macronuclear changes during the life of
the individual cell and the life history of the species. This has revealed
that the macronucleus is by no means a relatively passive agent in the
nuclear complex, but rather a product of micronuclear activity which
undergoes various radical transformations in contributing to the “somatic”
functions of the cell until another is provided from the same source.

MACRONUCLEAR REORGANIZATION

As early as 1859, Stein noted a clear band, or Kernspalt, in the mactro-
nuclei of hypotrichous ciliates, but chiefly within the past decade this has

Figure 153. Stages in the progress of the reorganization bands in Aspidisca lynceus
from the center of the horseshoe-shaped macronucleus to its tips. (From Summers, 1935.)

been intensively studied by several investigators who have shown that
Kernspalten are but a part of the regions now called reorganization
bands, reconstruction bands, and so forth, which have an important
function in the transformation of macronuclear substance at the time
of division (see p. 21). Thus in certain cases material visibly passes
from this region into the surrounding cytoplasm, as, for example, in

ENDOMIXIS 647

Uroleptus (Calkins, 1919, 1930), Explotes patella (Turner, 1930), and
As pidisca (Summers, 1935), while in others, such as Ez plotes worcesteri
(Griffin, 1910), “extrusion bodies” are either absent or not described.
Again, in some ciliates reorganization bands may not appear so clear-cut
as in hypotrichous forms. Nevertheless the macronuclear material 1s
eliminated as a residual mass, left between the two parts of a dividing
macronucleus, as in Ancistruma and Conchophthirius (Kidder, 1933a,
1933b), Colpidium, Glaucoma, and Urocentrum (Kidder and Diller,
1934), and Blepharisma (D. Young, 1939); or beside the macronucleus

Fig. 154. Macronuclear dissolution in Blepharisma undulans. A, vegetative individual
with dumb-bell-shaped macronucleus; B, early stage in the formation of the central bulb;
C, central bulb fully formed; D, dissipation of the central bulb almost accomplished.
(From D. Young, 1939.)

during or after cell division, as in Chilodonella (MacDougall, 1936)
and Colpoda (Kidder and Claff, 1938) (Figs. 153, 154).

Variations in the details of such intrinsic macronuclear reorganiza-
tion processes are indeed legion—reaching their climax, perhaps, in one
or more of the types of “hemixis’’ described by Diller (1936)—but the
accumulating data seem to justify the belief that they may be universal
in the ciliates and perhaps represent at once a “‘purification” (Calkins)
of the macronucleus, a regulation of the nucleo-cytoplasmic ratio, and
a contribution of significance to the metabolic activities of the cyto-
plasm. For summaries, reference may be made to Summers (1935) and
Kidder and Claff (1938).

Apparently, intrinsic macronuclear reorganization phenomena dur-
ing the division of the cell suffice, in most species at least, for the con-

648 ENDOMIXIS

tinued well-being of the race, but provisions are also made for the
periodic destruction of the macronucleus and its replacement from the
micronuclear reserve: in some species by endomixis and autogamy, and
probably in all by conjugation.

ENDOMICTIC PHENOMENA

A petiodic replacement of the macronuclear apparatus, without syn-
karyon formation, was described in Paramecium aurelia, and named en-

Figure 155. General plan of the usual nuclear changes during endomixis in Para-
mecium aurelia. A, typical nuclear condition; B, degeneration of macronucleus (chro-
matin bodies not shown) and first division of micronuclei; C, “climax’’: second division
of micronuclei; D, degeneration of six of the eight micronuclei; E, division of the
cell; F, first reconstruction micronuclear division; G, second reconstruction micro-
nuclear division; H, transformation of two micronuclei into macronuclei; I, micro-
nuclear and cell division; J, typical nuclear condition restored. (Constructed from the
description and figures of Woodruff and Erdmann, 1914.)

domixis, by Woodruff and Erdmann (1914). Following their account,
in summary, endomixis in this species involves the resolution of the old
macronucleus into chromatin bodies, which disintegrate in the cytoplasm,

ENDOMIXIS 649

and the transformation of one or two of the products of the micronuclear
divisions into new macronuclei, to reconstitute the normal vegetative
apparatus when distributed by cell division (Figs. 155, 156).
Immediately after this announcement, Hertwig (1914) described
similar phenomena in P. aurelia as parthenogenesis, induced, he believed,
by degenerative changes, and emphasized the fact that in his study of
conjugation in this species (1889), he had noted stages in certain non-
conjugants that were open to a similar interpretation. Thereafter en-

AA MA
iN
Mh ME hi

Figure 156. Possible methods of micronuclear and cell division at the climax of endo-
mixis in Paramecium aurelia. tb is typical. (From Woodruff and Erdmann, 1914, p. 448.)

domixis, or, if one prefers, diploid parthenogenesis, was reported by
many investigators, including Erdmann and Woodruff (1916) in P.
caudatum, Calkins (1915, 1919) in Didinium nasutum and Uroleptus
mobilis, Moore (1924) in Spathidium spathula, Erdmann (1925) in
P. bursaria, Woodruff and Spencer (1923) in P. polycaryum, Klee
(1925) in Explotes longipes, Ivanié (1928, 1929) in Chilodonella
uncinatus, Vorticella nebulifera, Euplotes charon, and E. patella, Man-
well (1928) in Pleurotricha lanceolata, Diller (1928) in Trichodina
sp., Chejfec (1928, 1930) in P. caudatum, Fauré-Fremiet (1930) in
Zoothamnium alternans, Stranghoner (1932) in P. multimicronucleatum,
Tittler (1935) in Urostyla grandis, Kidder (1938) in Paraclevelandia
Simplex, and Gelei (1938) in Paramecium nephridiatum. In most of

650 ENDOMIXIS

these studies, it must be admitted, the authors failed to follow the
sequence of nuclear events in series of pedigreed animals, but fitted their
findings in isolated animals into the picture of endomixis as originally
portrayed. Three of these investigations are of particular significance
at the moment.

Diller (1928), in a study of “binary fission and endomixis in Tricho-
dina from tadpoles,” gives a categorical account of the reorganization
process. He shows the resolution of the macronucleus into chromatin
bodies: ‘In most cases the macronucleus breaks up completely by form-
ing numerous spherical bodies of varying sizes.” And the origin of the
primordium of the new macronucleus is from a residual micronucleus:
“The final eight products of the micronuclear divisions are originally all
apparently similar. Seven of them, however, rapidly differentiate into
macronuclear Anlagen, while one remains the functional micronucleus.”
The process is ‘characterized by the absence of maturation spindles and
synkaryon formation” (Fig. 157).

Stranghéner (1932), in a detailed description of endomixis in Parame-
cium multimicronucleatum, emphasizes the fact that “Im Gegensatz zur
Conjugation bildet der Macronucleus bei der Auflosung keine wurst-
formigen Schlingen,’’ and describes and figures the incorporation of
chromatin spheres from the old macronucleus into the new one (Fig
158)e

Kidder (1938) observes a “nuclear reorganization without cell divi:
sion in Paraclevelandia simplex,’ in which the details of the process
are unique but the end result is the same. The old macronucleus elim1-
nates a large part of its chromatin and the remainder then becomes 1n-
corporated with one product of a single micronuclear division, to con-
stitute the primordium of the new macronuclear apparatus (Fig. 159).

In this codperation between macronucleus and micronucleus, de-
scribed by Strangh6ner and Kidder as endomixis, we seem to have, as it
were, stages in the evolution of macronuclear metamorphosis intermedi-
ate between the intrinsic changes evidenced by reorganization bands,
direct elimination of material, and so forth, and the complete compe-
tence of the micronucleus alone to form a new macronucleus during
endomixis as it has been described in other species.

That intrinsic reorganization is adequate for the continued life of
the race appears to be evident from the study of Dawson (1919) on an

Ta Tb

8a 8b

Figure 157. Diagram of the normal process of endomixis in Trichodina sp. 1, normal
vegetative animal; 2, macronucleus fragmented and disintegrating, and not shown in sub-
sequent diagrams (the micronucleus has migrated to the other end of the body) ; 3,
micronucleus divided the first time; 4, micronucleus divided the second time; 5, micro-
nucleus divided the third time; 6, seven of the nuclei differentiating into macronuclear
Anlagen, while the eighth remains the functional micronucleus which divides before
each cell division; 7a and 7b, daughters resulting from the first cell division and having
four and three macronuclear Anlagen respectively; 8a and 8b, daughters resulting from
the second cell division (the 8b monomacronucleate individual is completely reorganized ) ;
9a and 9b, daughters resulting from the third cell division (growth of the macronucleus
will reconstruct them into normal vegetative individuals). (From Diller, 1928.)

Fig. 158. Endomixis in Paramecium multimicronucleatum, (From Stranghéner, 1932.)

ENDOMIXIS | 653

amicronucleate race of Oxytricha hymenostoma, in which, obviously, a
micronuclear reserve could play no part. Woodruft’s work (1935) on a
race of Blepharisma undulans, without endomixis or autogamy, and
D. Young's findings (1939) on the same race, showing the elimination
of material from the macronucleus during division, support this thesis.

However, these results might be anticipated because several species
have been cultured for long periods without showing any evidence of a

Figure 159. Endomixis in Paraclevelandia simplex. 1, pte-cystic form (the macronucleus
differentiating into degenerating posterior, and reorganizing anterior chromatin; the mi-
cronucleus in prophase) ; 2, later stage (the micronucleus in anaphase; further differentia-
tion of macronucleus) ; 3, telophase of the micronucleus (note the smoothly granular an-
terior half of the macronucleus) ; 4, fusion of daughter micronucleus with reorganized por-
tion of macronucleus (posterior portion of macronucleus shrinking away from the old
nuclear membrane; enlarged condition of the daughter micronuclei quite characteristic).
(From Kidder, 1938.)

reorganization process, either intrinsic or endomictic, in the free-living
animals, and probably none occurs. As examples, reference may be made
to the work on Spathidium spathula by Woodruff and Moore (1924), on
Paramecium calkinsi by Spencer (1924), and on Didinium nasutum
by Beers (1929). In regard to reorganization by endomixis, the culture
of P. caudatum studied by Metalnikov (1937), and the (to date)
thirty-three-year-old culture of P. awrelia at Yale University may be cited,
unless the future should prove that autogamy, to the exclusion of en-
domixis, occurs in these species (Woodruff, 1932).

Thus in the species in which reorganizational phenomena occur, it

654 ENDOMIXIS

appears that intrinsic reorganization, as well as endomixis, meet the nor-
mal exigencies of existence and keep the race on the even tenor of its
way. For significant possibilities of genetic change in heterozygous indi-
viduals, however, reorganization is accompanied by synkaryon forma-
tion, either in autogamy or conjugation.

AUTOGAMY

The first definite statement of autogamy in the ciliates was given in a
brief article by Fermor (1913), who described the degeneration of the
macronucleus and the origin of a new one from a synkaryon of micro-
nuclear origin, during the encystment of Stylonychia pustulata. But the
problem was not emphasized until Diller (1936) described autogamy in
P. aurelia and stated:

I have not been able to confirm the micronuclear behavior which Woodruff
and Erdmann have described for endomixis in P. awrelia. In the failure of
such verification I am inclined to deny the existence of endomixis as a valid
reorganization process. I feel that Woodruff and Erdmann have combined
stages of hemixis and autogamy into one scheme, “‘endomixis,” overlooking
the maturation and syncaryon stages in autogamy.

And in regard to his own earlier description of endomixis in Trichodina
sp., Diller remarks, “It may be that hemixis and exconjugant stages were
lumped together as ‘endomixis’ in this account’ (Figs. 160, 161).

In enthusiasm for the concept of autogamy, it may be well not to
exclude endomixis in P. aurelia—or Trichodina sp.—without careful
consideration, although there is no inclination to deny that autogamy
occurs in P, aurelia, in view of the combined data presented in the
cytological study by Diller and the genetical studies of Sonneborn
(1939a, 1939b, 1939c). However, Sonneborn’s observations were not
made on the Yale race of P. awrelia nor on the mating-type variety which
it represents, because there are as yet no known genes in this variety,
and such tests therefore cannot be made.

In the opinion of the writer, the crucial cytological stages are not
absolutely demonstrated, in part because most of the animals were
taken from mass cultures and relatively few from isolated lines or from
pedigreed lines, and therefore the sequence of events was not determined
from pedigreed serzes. To demonstrate satisfactorily the exact sequence,
it is necessary to follow critically the nuclear behavior in series of pedi-

Figure 160. Nuclear changes during autogamy in Paramecium aurelia. (From Diller,
1936.)

TYPICAL INDIVIDUAL

ihre! |

Figure 161. Hemixis. Diagram of the macronuclear behavior (exclusive of conjugation
and autogamy) in Paramecium aurelia. The macronuclei are represented by large solid
ovals; macronuclear fragments by smaller circles; micronuclei by small round dots;
“Anlagen-like’’ macronuclei by stippled circles. The interrelationships of the various
forms are indicated by arrows. (From Diller, 1936.)

ENDOMIXIS 657

greed animals from day to day, as emphasized by Woodruff and Erd-
mann (1914, pp. 457-72) and Beers (1935).

The study of pedigreed series of animals, for example, precluded, it
is believed, the possibility of “combining stages of hemixis and autogamy
in one scheme.” Indeed, Erdmann and Woodruff (1916), contrasting
endomixis in P. caudatum and P. aurelia, stated that they had ‘some
data which suggest that under certain conditions merely a partial re-
organization, not involving the formation of macronuclear Av/agen,

Figure 162. Climax of
endomixis in Paramecium
aurelia. The old macronu-
cleus is merely in the form
of a membrane from which
the numerous chromatin
bodies have been ejected
and are free in the cyto-
plasm. Eight so-called re-
duction micronuclei. (From
Woodruff and Erdmann,
1914, plate 2.)

may lead, at least temporarily, to the continuance of the life of the line.”
This would appear to be Diller’s ““hemixis.”

On the other hand, ‘overlooking the maturation and synkaryon
stages’ is a different matter, as will be appreciated by anyone who has
worked on the cytology of Paramecium. This may have occurred, even
though Woodruff and Erdmann naturally ‘“‘expected”’ to find autogamy
when they observed the primordia of macronuclei in non-conjugants.
Their inability to find maturation spindles and synkaryon of course led
them to coin the name endomixis for the process. But an equally plausible
explanation, at least in the mind of the writer, as to why these investi-
gators did not find such stages, nor even the paroral lobe, in which the
synkatyon is characteristically located, according to Diller, is that these

658 ENDOMIXIS

did mot occur in their material. None have been observed by other in-
vestigators studying what they interpret as endomixis in various species.
As already stated, none were found by Diller in endomixis in Trichodina.
Certainly none occur in Paraclevelandia simplex, according to the clear-

Figure 163. 1, Autogamy, Woodruff race (gamete nuclei in contact in the paroral
cone at the right; five or six degenerating nuclei are visible; macronucleus in skein) ;
2, autogamy, Woodruff race (synkaryon formation; gamete nuclei enclosed within a
common membrane; paroral cone; no degenerating micronuclei visible; macronucleus in
skein) ; 3, autogamy, isolation, Philadelphia race (synkaryon, in paroral cone, in meta-
phase of first division; no degenerating micronuclei seen; macronuclear skein frag-
menting; a number of macronuclear bodies of various stages of degeneration present in
the cell). Animals 1 and 2 are from mass cultures. (From Diller, 1936.)

cut description of Kidder (1938). So from the latter account alone it 1s
evident that endomictic phenomena actually do occur. Synkaryon forma-
tion is not a necessary antecedent to the formation of a macronuclear
primordium (Figs. 162, 163).

But confining attention to Paramecium aurelia, the fact must be
emphasized that almost the entire picture, and not merely the crucial
detail of the presence or absence of a synkaryon, differs in the endomixis
of Woodruff and Erdmann and the autogamy of Diller. In endomixis,

ENDOMIXIS 659

maturation ‘“‘crescents’’ were not observed, and the elimination of chro-
matin bodies was found to be the typical method of macronuclear de-
struction. Only one among the many hundreds of endomictic animals
studied by Woodruff and Erdmann showed even a slight simulation of
the macronuclear ribbon-formation so characteristic of conjugating ani-
mals, and also of autogamy according to Diller. This single animal, of
the four-thousand-and-eighty-seventh generation, was figured as atypi-
cal. However, eight years later Woodruff and Spencer (1922) found, on
one single day in a subculture from this same pedigreed race at about
the eight-thousand-nine-hundredth generation, several animals with rib-
bon-like degenerating macronuclei. The publication of this exception
brought a protest from Erdmann, who was convinced that conjugation
must have occurred in the subculture.

Now much of Diller’s work has been done on this same Yale race,
and therefore it is clear that ribbon-formation does occur, other than at
conjugation, in this race, under certain conditions. It is not clear how
Diller’s culture conditions differ from those in the Yale Laboratory,
where ribbon-formation has not been observed since the instance in 1922,
referred to above. A clue may be afforded by De Lamater (1939),
who found that different kinds of bacteria in the culture medium of this
same race of Paramecium had marked effects on the macronuclear
changes. It is possible that other types of bacteria or other environmental
changes may underly the differences between endomixis and autogamy.

PERIODICITY OF ENDOMIXIS

Another important point is the rhythmic periodicity of endomixis ob-
served by Woodruff and Erdmann (1914, 1916) and Woodruff (1917a,
1917b), which, according to Diller, is absent in autogamy. He says:
“Under the conditions of my experiments, no regular periodicity in the
incidence of autogamy was evident.”

Woodruff and Erdmann (1914) and Woodruff (1917) definitely
stated that the interendomictic periods in both P. awrelia and P. caudatum
showed some variation in length and furthermore were somewhat modi-
fied by environmental factors, but nevertheless were strikingly periodic—
endomictic periods and interendomictic periods affording the rhythms
in the division rate of pedigreed cultures. And this rhythmicality has
appeared throughout the years in the culture of P. awrelia in the Yale

660 ENDOMIXIS

Laboratory, whenever tests have been made; but now, in the thirty-
third year of its life, with unimpaired vitality, the interendomictic
periods seem to be slightly more variable in length (Fig. 164).

A number of other investigators have studied the question of peri-
odicity in this and other races of P. aurelia and P. caudatum, among them
R. T. Young (1918), Jollos (1916, 1920), Erdmann (1920), Chejfec
(1930), Galadjieff (1932), and, in particular, Sonneborn (1937a).

Figure 164. Graph of the division rate of Paramecium aurelia, line III, subculture IE,
averaged for five-day periods. Endomixis occurred during the periods indicated by an X.
Note that the interendomictic periods exhibit some variation in length, and the final
endomixis shown is deferred. (From Woodruff and Erdmann, 1914.)

The latter compared the endomictic period in the Yale race of
P. aurelia with that of another race under identical environmental condi-
tions. Sonneborn shows that great variations may exist in the interendo-
mictic interval, not only in different races, but even in the same race under
carefully standardized conditions of daily isolation culture.

So in regard to the periodicity of endomixis, it now appears that the
limits of approximately 25 to 30 days and 40 to 50 fissions for P.
aurelia, and 50 to 60 days and 80 to 100 fissions for P. caudatum, as
originally announced, are somewhat too narrow and stereotyped. En-
vironmental and racial factors play a still greater part than these investt-
gators believed. But withal, the endomictic process does recur with con-
siderable regularity when the environmental and genetic factors are
uniform, and so must still be regarded as periodic.

GENETICAL STUDIES ON ENDOMIXIS

Genetical sttidies on endomixis include those by Erdmann (1920),
Jollos (1921), Parker (1927), Caldwell (1933), Kimball (1937,
1939), and Sonneborn (1937b, 1939a, 1939b, 1939c). The results

ENDOMIXIS 661

obtained by Sonneborn (1939), in particular, afford evidence that the
preciseness of the ratios and the segregation of sex or mating types,
following the reorganization process, is quite as regular and exact as
after conjugation. Thus it would appear that autogamy and not endo-
mixis is involved. Indeed, Sonneborn states that in one race of P. aurelia
—not the Yale race nor the mating-type variety which it represents—
the alternative between endomixis and autogamy was tested genetically
by determining the genotypes following reorganization in clones of type
Aa (genes determining mating types). Genetic analysis of the reorgani-
zations showed that all the resulting lines are homozygous, half of them
dominant and half recessive. From any one reorganizing individual both
catyonides are of the same genotype. Thus, under these conditions (mass
cultures at 31° C. and isolation lines at 27° C.), autogamy, not endo-
mixis, takes place.

Accordingly the combination of genetical and cytological data at pres-
ent available justifies the conclusion that autogamy occurs, under cer-
tain circumstances at least, in some races of P. aurelia. Granting this
raises the question whether all the investigations reported on the
physiology and genetics of endomixis actually are on autogamy, because
Paramecium is the form that has been almost universally employed in
such studies. If the accumulated data are really all in regard to autogamy,
then the question is essentially one of name. On the other hand, if both
endomixis and autogamy occur in Paramecium, then, for a time, confu-
sion is worse confounded.

Obviously, at present it is useless to attempt to generalize in regard
to reorganization in Paramecium—that must await far more extended in-
vestigation. However, the personal judgment of the writer, at the mo-
ment, is that both endomixis and autogamy do occur in Paramecium—
an opinion reached, it is believed impartially, from a consideration of
the picture of the micronuclear divisions and the macronuclear destruc-
tion, as he saw them in the original work on the reorganization process
and as demonstrated in the cytological preparations of Diller. Certainly
the two pictures presented are quite different; indeed, in many ways as
different as the stages in endomixis and conjugation appeared in the
original study. And Sonneborn (1939a) remarks: ‘Probably it will be
found that autogamy and endomixis take place in different races or under
different conditions.” The occurrence of these two processes, either in

662 ENDOMIXIS

different races or in the same race, synchronously or otherwise, thus adds
to the known repertoire of the versatile Paramecium.

CONCLUSIONS

A synoptic view of the rapidly accumulating data on macronuclear
reorganization phenomena in the Ciliophora justifies, it is believed, the
statement that these processes include intrinsic reorganization (reor-
ganization bands, and so forth) , coéperation of macronucleus and micro-
nucleus (endomixis), a new macronucleus of micronuclear origin (en-
domixis), and a new macronucleus of synkaryon origin (autogamy and
conjugation). These constitute a series of macronuclear metamorphoses
of increasing complexity, affording progressively greater possibilities for
the organism.

LITERATURE CITED

Beers, C. D. 1929. On the possibility of indefinite reproduction in the ciliate
Didinium without conjugation or endomixis. Amer. Nat., 63: 125-29.

— 1935. Structural changes during encystment and excystment in the ciliate
Didinium nasutum. Arch. Protistenk., 84: 133-55.

Caldwell, L. 1933. The production of inherited diversities at endomixis in
Paramecium aurelia. J. exp. Zool., 66: 371-407.

— 1915. Didinium nasutum: \. The life history. J. exp. Zool., 19: 225-
39.

— 1916. General biology of the protozoan life cycle. Amer. Nat., 50:
257-70.

Calkins, G. N. 1919. Uroleptus mobilis Engelm. I. Renewal of vitality
through conjugation. J. exp. Zool., 29: 121-56.

—— 1930. Uroleptus halseyi Calkins. Il. The origin and fate of the macro-
nuclear chromatin. Arch. Protistenk., 69: 151-74.

Chejfec, M. 1928. On the nuclear reorganization of Paramecium caudatum.
Acta Biol. exp., 2: 89-121.

—— 1930. Zur Kenntniss der Kernreorganizationsprozesse bei Paramecium
caudatum. Arch. Protistenk., 70: 87-118.

Dawson, J. A. 1919. An experimental study of an amicronucleate Oxytricha.
I. A study of the normal animal with an account of cannibalism. J.
exp. Zool., 29: 473-511.

De Lamater, A. J. 1939. Effect of certain bacteria on the occurrence of
endomixis in Paramecium aurelia. Biol. Bull. 76: 217-225.

Diller, W. F. 1928. Binary fission and endomixis in the Trichodina from
tadpoles. J. Morph., 46: 521-52.

—— 1936. Nuclear reorganization processes in Paramecium aurelia, with
descriptions of autogamy and ‘“‘hemixis.” J. Morph., 59: 11-67.

ENDOMIXIS 663

Enriques, P. 1916. Duemila Cinquecento generazioni in un infusorio, senza
coiugazione né partenogenesi, né depressioni. R. Accad. Sci. Bologna,
Ser. VII.

Erdman, R. 1920. Endomixis and size variation in pure bred lines of Para-
mecium aurelia, Roux Arch. EntwMech. Organ., 46: 85-148.

— 1925. Endomixis bei Paramecium bursaria. S. B. Ges. natur. Fr. Berl.,
Jahrgang, 1925: 24-27.

Erdmann, R., and L. L. Woodruff. 1916. The periodic reorganization process
in Paramecium caudatum. J. exp. Zool., 20: 59-97.

Fauré-Fremiet, E. 1930. Growth and differentiation of the colonies of
Zoothamnium alternans. Biol. Bull., 58: 28-51.

Fermor, X. 1913. Die Bedeutung der Enzystierung bei Stylonychia pustulata.
Zool. Anz., 42: 380-84.

Galadjieff, M. 1932. Sur le probleme de l’immortalité des Protozoaires (Vingt
ans de culture de l’infusoire Paramecium caudatum sans conjugation).
Bull. Acad. Sci. U.R.S.S., VII série, Classe Sci. Math. Nat.

Gelei, J. von. 1938. Beitrage zur Ciliatenfauna der Umgebung von Szeged.
VII. Paramecium nephridiatum. Arch. Protistenk., 91: 343-56.

Griffin, L. E. 1910. Ewplotes worcester sp. nov. II. Division. Philipp. J. Sct.,
I SLD=3 6:

Hertwig, R. 1889. Uber die conjugation der Infusorien. Abh. Bayer. Akad.
Wiss. Cl 2ai7e

—— 1914. Uber Parthenogenesis der Infusorien und die Depression-
szustande der Protozoen. Biol. Zbl., 34: 557-81.

Ivani¢, M. 1928. Uber die mit den Parthenogenetischen Reorganisations-
prozessen des Kernapparates verb. Vermehrungscysten v. Chilodon
uncinatus. Arch. Protistenk., 61: 293-348.

—— 1929. Zur Auffassung der sog. bandférmigen Grosskerne bei Infusorien ;
zugleich ein Beitrage zur Kenntnis der sog. parthenogenetischen und
thnen ahnlichen Reorganisationsprozesse des Kernapparates bei Proto-
zoen. Arch. Protistenk., 66: 133-59.

Jennings, H. S. 1929. Genetics of the Protozoa. Bibliog. Genet., 5: 105-330.

Jollos, V. 1916. Die Fortpflanzung der Infusorien und die potentielle Un-
sterblichkeit der Einzellen. Biol. Zbl., 36: 497-514.

—— 1920. Experimentelle Vererbungsstudien an Infusorien. Z. Indukt.
Abstamm.- u. VererbLehre, 24: 77-97.

Kidder, G. W. 1933a. Studies on Concophthirius mytili, 11. Conjugation and

nuclear reorganization. Arch. Protistenk., 79: 25-49.

1933b. On the genus Ancistruma Strand. Il. The conjugation and

nuclear reorganization of A. isseli. Arch. Protistenk., 81: 1-18.

1938. Nuclear reorganization without cell division in Paraclevelandia
simplex, an endocommensal ciliate of the wood-feeding roach, Panesthia.

Arch. Protistenk., 91: 69-77.

664 ENDOMIXIS

Kidder, G. W., and C. L. Claff. 1938. Cytological investigations of Colpoda
cucullus. Biol. Bull., 74: 178-97.

Kidder, G. W., and W. F. Diller. 1934. Observations on the binary fission
of four species of common free-living ciliates, with special reference to
the macronuclear chromatin. Biol. Bull., 67: 201-19.

Kimball, R. F. 1937. The inheritance of sex at endomixis in Paramecium
aurelia. Proc. Nat. Acad. Sci., Wash., 23: 469-74.

—— 1939. Change of mating type during vegetative reproduction in Para-
mecium aurelia. J. exp. Zool., 81: 165-79.

Klee, E. E. 1925. Der Formwechsel im Lebenskreis reiner Linien von Explotes
longipes. Zool. Jarhb., 42: 307-66.

MacDougal, M. S. 1936, Etude cytologique de trois espece du genre Chilodo-
nella strand. Bull. biol., 70: 308-331.

Manwell, R. D. 1928. Conjugation, division, and encystment in Pleurotricha
lanceolata. Biol. Bull. 54: 417-63.

Metalnikov, S. 1937. Le Role et la signification de la fécondation. Scientia,
Milano, Mars, 1937: 167-76.

Moore, E. L. 1924. Endomixis and encystment in Spathidium spathula. J.
exp: Zool) 39: 317237

Parker, R. C. 1927. The effect of selection in pedigree lines of Infusoria.
J. exp. Zool., 49: 401-39.

Sonneborn, T. M. 1937a. The extent of the interendomictic interval in
Paramecium aurelia and some factors determining its variability. J. exp.
Zool., 75: 471-502.

— 1937b. Sex, sex inheritance and sex determination in Paramecium
aurelia. Proc. Nat. Acad. Sci., 23: 378-385.

1939a. Sexuality and related problems in Paramecium. Coll. Net, 14:
77-84.

—— 1939b. Genetic evidence of autogamy in Paramecium aurelia. Anat.
Rec:,. 7585..(Supp!s)

—— 1939c. Paramecium aurelia: mating types and groups; lethal interac-
tions; determination and inheritance. Amer. Nat., 73: 390-413.

Spencer, H. 1924. Studies on a pedigree culture of Paramecium calkinsi.
J. Morph., 39: 543-551.

Stein, F. R. 1859. Der Organismus der Infusionsthiere. I. Abth. Leipzig.

Stranghéner, E. 1932. Teilungsrate und Kernreorganisationsprozess bei Para-
mecium multimicronucleatum. Arch. Protistenk., 78: 302-60.

Summers, F. M. 1935. The division and reorganization of Aspidisca lynceus,
Dio phrys appendiculata, and Stylonychia pustulata, Arch. Protistenk., 85:
175-208,

Tittler, J. A. 1935. Division, encystment and endomixis in Urostyla grandis,
with an account of an amicronucleate race. Cellule, 44: 189-218.

Turner, J. P. 1930. Division and conjugation in Evplotes patella, with special

ENDOMIXIS 665

reference to the nuclear phenomena. Univ. Cal. Publ. Zool., 33: 193-258.

Woodruff, L. L. 1917a. Rhythms and endomixis in various races of Para-
mecium aurelia. Biol. Bull., 33: 51-56.

—— 1917b. The influence of general environmental conditions on the
periodicity of endomixis in Paramecium aurelia. Biol. Bull., 33: 437-62.

—— 1932. Paramecium aurelia in pedigree culture for twenty-five years.
Trans. Amer. micr. Soc., 51: 196-98.

—— 1935. Physiological significance of conjugation in Blepharisma
undulans. J. exp. Zool., 70: 287-300.

Woodruff, L. L., and R. Erdmann. 1914. A normal periodic reorganization
process without cell fusion in Paramecium. J. exp. Zool., 17: 425-517.

Woodruff, L. L., and E. L. Moore. 1924. On the longevity of Spathidium
spathula without endomixis or conjugation. Proc. Nat. Acad. Sci. Wash.,
10: 183-86.

Woodruff, L. L., and H. Spencer. 1922. On the method of macronuclear dis-
integration during endomixis in Paramecium aurelia, Proc. Soc. exp.
Biol. N.Y., 19: 290-91.

—— 1923. Paramecium polycaryum, sp. nov. Proc. Soc. exp. Biol. N.Y., 20:
338-39.

—— 1924. Studies on Spathidium spathula. Ul. The significance of conju-
gation. Jour. exp. Zool., 39: 133-96.

Young, D. 1939. Macronuclear reorganization in Blepharisma undulans.
J. Morph., 64: 297-353.

Young, R. T. 1918. The relation of rhythms and endomixis, their periodicity
and synchronism in Paramecium aurelia. Biol. Bull., 35: 38-47.

CHAPTER XIV.

SEXUALITY IN UNICELLULAR ORGANISMS
T. M. SONNEBORN

AMONG UNICELLULAR ORGANISMS, many different sexual conditions
have long been known; descriptions of these are readily accessible (e.g.,
Calkins, 1926). In recent years, surprising discoveries concerning
sexuality have been reported in some of the commonest and most studied
unicellular organisms, such as Polytoma and Chlamydomonas among the
flagellates, and Paramecium and Euplotes among the ciliates. In this
chapter an attempt will be made to set forth this recent work and to
examine critically the current interpretations of it.

The work on the flagellates began earlier and has been carried fur-
ther than the work on the ciliates; it will therefore be presented first.
Although in both classes of organisms investigations of similar nature
have been pursued on a number of forms (see especially Moewus, 1935a,
1935b, 1935c, 1937a), they have been carried further on Chlamydo-
monas among the flagellates and on Paramecium among the ciliates. As
these show essentially the same general relations as do other species of
the same classes, the following account will be confined in the main to
these two genera.

SEXUALITY IN Chlamydomonas

The recent work on Chlamydomonas has appeared in a series of ex-
tensive and detailed studies by Moewus since 1932 (Moewus, 1933,
1934, 1936, 1937b, 1938a, 1938b, 1939a, 1939b, 1939c; Hartmann,
1932, 1934). From the beginning, its great importance with relation to
problems of sexuality has been apparent and, as the work progressed,
these relations have been repeatedly emphasized by Hartmann, Moewus,
and others. However, in order to approach the facts without theoretical
bias, they will be restated here in a purely descriptive way.

Six species of Chlamydomonas have been most fully investigated: C.
braunit, C. dresdensis, C. eugametos, C. paupera, C. paradoxa, and C.

SEXUALITY 667

pseudo paradoxa. In all species the vegetative cells and gametes have a
haploid set of ten small dot-like chromosomes. Under appropriate con-
ditions, differing in different species and races, the vegetative cells pro-
duce or become gametes that copulate and form a diploid zygote cyst.
(In some species, vegetative cells function as gametes; in others, gametes
differ from vegetative cells.) Under certain conditions, maturation divi-
sions, restoring the haploid condition, take place in the cyst. The reduced
cells emerge from the cyst and each gives rise by vegetative multiplica-
tion to a clone.

THE KINDS OF GAMETIC DIFFERENCES OBSERVED IN Chlamydomonas

The basic problem of sexuality in unicellular organisms is whether
the copulating or conjugating cells regularly differ from each other. As
will appear at once, certain differences are found only in some species or
races, not in others; while other differences seem to be of general oc-
currence.

Morphological differences between copulating cells or gametes.—In
C. coccifera (Moewus, 1937b), the copulating pairs invariably consist
of a large, nonflagellated gamete and a small, flagellated one. In C.
braunii, both copulants are flagellated, but one is always much smaller
than the other. In the remaining species, there is no regular morpho-
logical difference between copulating gametes. Nevertheless, in particu-
lar pairs of at least certain species (e.g., C. ewgametos; Moewus, 1933),
one gamete may be as much as twice as large as the other, while in other
pairs of the same species no size difference appears. All possible kinds of
gamete combinations are found: large with large, large with small, and
small with small. Finally, in species like C. pseudo paradoxa (Hartmann,
1934), the gametes are regularly smaller than vegetative cells, though
the two gametes do not ordinarily differ from each other.

Functional differences between gametes—In C. coccifera (Moewus,
1937b), the large gametes lack flagella, and the small ones retain them.
Consequently, the small gametes are more active and must move toward
the larger ones to accomplish copulation. Further evidence of the greater
activity of the smaller gamete appears during copulation, for its contents
regularly pass into the larger gamete. Less functional differentiation
appears in C. braunii; here both gametes are flagellated and active, but
during copulation the smaller gamete regularly empties into the larger

668 SEXUALITY

one. This same functional difference occurs also in those pairs that differ
markedly in size in C. ewgametos; but not in the usual pairs, in which
the gametes are alike in size (Moewus, 1933). Nor does it appear in
other species in which the gametes are morphologically isogamous. Func-
tional differentiation thus appears to be strictly correlated with morpho-
logical differentiation, occurring only when one gamete is flagellated and
the other not, or when one is much larger than the other. Further, both
morphological and functional differentiation may exist between gametes
in some copulating couples and not in others of the same species (C.
eugametos, Moewus, 1933).

Physiological differences between gametes.—In this section will be
given only the general evidence of physiological differentiation between
gametes, reserving for later consideration the question of the nature of
such differentiation. If copulating gametes are not diverse physiologically,
then any two gametes can copulate with each other; but if they are regu-
larly diverse, there must be at least two kinds of gametes, with copula-
tion taking place between gametes of different types. The basic question,
therefore, is simply whether or not any two gametes of a species can
copulate with each other.

In some species and varieties (C. coccifera, C. braunu, C. paupera,
C. eugametos f. typica and simplex, C. paradoxa, and C. pseudopara-
doxa from Coimbra, Portugal; Moewus, 1933; Hartmann, 1934;
Moewus, 1936, 1937b, 1938a), the answer to this question is simple
and definite. Gametes produced within a clone do not copulate with
each other, but they do copulate with gametes produced by certain other
clones. In these races, therefore, the copulating gametes must always
be physiologically diverse. Moreover, this diversity is not invariably
associated with morphological or functional diversity, for five of the
seven races showing this phenomenon have morphologically and func-
tionally isogamous gametes.

To the same category belongs a race of C. pseudoparadoxa, from
Giessen; but the physiological difference is less apparent, requiring
special methods to bring it to light. In this race, copulation does not
normally occur either between gametes of the same clone or between
gametes of different clones. Gametes of C. pseudoparadoxa are recog-
nizable by their small size. Moewus (in Hartmann’s article, 1934) found
that the noncopulating gametes of the Giessen race could be rendered

SEXUALITY 669

capable of copulating by subjecting them to filtrates from gamete cul-
tures of the Coimbra race of the same species. (See 675 ef seq.
for a further account of these filtrates.) However, filtrates from any one
Coimbra clone would activate some of the Giessen clones, but not all.
The remaining Giessen clones required for activation treatment with a
filtrate from a different Coimbra clone, one which would copulate with
the first Coimbra clone. The Giessen gametes would now copulate only
if a clone activated by the one filtrate was mixed with a clone activated
by the other filtrate. Thus the Giessen clones are of two diverse physio-
logical types, and copulation occurs only between the two types, not
between gametes of the same clone.

In all the races thus far considered, there are regularly physiological
differences between copulating gametes, which are invariably members
of different clones. In the remaining three races investigated by Moewus
(1934, 1938a), C. eugametos f. subheteroica and f. synoica and C.
dresdenis, copulation occurred regularly among gametes of any one
clone. Among these three races, the situation in C. eugametos f. sub-
heteroica is unique. In any culture relatively few cells copulate. If the
cells left over after copulation has ceased in a culture are mixed with
the left overs from cultures of other clones, some of the mixtures will
exhibit typical copulation. Exhaustive analysis of many clones shows
that the same results hold here for the left overs as for entire clones of
the species previously discussed. The left overs of any one clone are
always of the same physiological type, but other clones yield left overs
of a different type. There are just two kinds of clones, differing in the
type of left overs they produce. Left overs of one type copulate only
with left overs of the other type.

From these observations, Moewus (1934) concludes that each clone
produces both types of gametes, but always one in much greater fre-
quency than the other. Some clones regularly produce mostly one kind
of gamete; other clones regularly produce mostly the other kind of
gamete. Copulation then takes place within a clone until all the rarer
type of gametes have found partners, so that all the left-over cells are
of the prevailing type. Moewus (1934) reports that the behavior of
this race can be made to simulate that of those previously discussed by
subjecting the cultures to very dilute formalin or acetaldehyde. With
this treatment, the cultures no longer yield copulation within a clone,

670 SEXUALITY

but the clones are divisible into two physiological types, with copula-
tion taking place only between the two types of clones. Under these
conditions, the type of each clone is identical with the type of its left-
over cells under normal conditions.

These results of Moewus show that different clones of C. evgametos
f. subheteroica produce different kinds of gametes and that in mixtures
of left-over cells and in mixtures of chemically treated cultures copula-
tion is between physiologically diverse types of gametes. His further
conclusion that the copulation occurring under normal conditions within
each clone is also between the same two types of gametes has not been
directly demonstrated, though it appears a reasonable inference. The
possibility that copulation is here taking place between gametes of the
same type has not been excluded. Convincing evidence on this important
question calls for direct tests with “split pairs,” as performed by Kimball
(1939a) on Paramecium (see p. 697).

The other two races showing copulation within a clone, C. ewgametos
f. synoica and C. dresdensis, differ from C. ewgametos £. subheteroica in
three respects: (1) copulation occurs presumably on a much larger scale
within a clone, the proportion of left overs being relatively small; (2)
in different cultures of the same clone the left overs may be of different
types; (3) no environmental means of suppressing or decreasing copula-
tion within the clone has been reported. Otherwise the observations re-
ported for these two races agree with those reported for C. ewgametos
f. subheteroica. Mixture of the left overs from different cultures in all
possible combinations of two shows that in each race there are two kinds
of left-over gametes, with copulation occurring only between the two
kinds. The same uncertainty attaches here to Moewus’s conclusion that
the copulations within a clone are likewise between gametes of different
type. However, the interpretation is here rendered more probable, for it
has been shown that both types are producible within a single clone,
different cultures of the same clone yielding left overs of different type.

The question at issue here is of theoretical importance. Does copula-
tion ever take place between cells that are physiologically as well as
morphologically and functionally identical? The preceding survey of the
conditions in various species and races of Chlamydomonas shows that
morphological and functional differences are frequently lacking, but that
at least in some of these cases physiological differences do exist when no

SEXUALITY 671

others are apparent. The only cases about which a reasonable doubt may
still be entertained are those in which copulation occurs within a clone.
This matter ‘has been intensively studied by Pascher (1931) in C. pau pera
and by Pringsheim and Ondraéek (1939) mainly in Polytoma. Their
observations are in fundamental disagreement with those of Moewus,
leading them to conclude that these forms show copulation without any
physiological sex differentiation. Further, Pringsheim and Ondracek
could not confirm Moewus’s observation that the cells left over after
copulation were unable to copulate with each other because they were
all of one physiological type. They attribute the cessation of copulation
to a change in the chemical conditions in the culture, rendering it un-
suitable for copulation. Appropriate modification of the conditions leads
to resumed copulation. They therefore deny the validity of the left-over
technique for the analysis of the question at issue. The reader is referred
to their article for a detailed criticism of numerous points in Moewus’s
work. Moewus (1940) has replied to these criticisms in an article that
appeared too late for inclusion in this review.

THE NATURE OF THE PHYSIOLOGICAL DIFFERENCES BETWEEN GAMETES
IN Chlamydomonas

As the union of gametes in Chlamydomonas is obviously a sex act,
the physiological differences that usually, if not always, characterize the
gametes may be considered sex differences. This section will set forth the
number and interrelations of these sexes, their chemical characteristics,
and their possible relation to male and female.

The number of sexes and their interrelations —The system of breed-
ing relations in Chlamydomonas was discovered by mixing together, in
combinations of two, cultures of the sexes isolated from the species and
varieties of Chlamydomonas examined. The two species C. paradoxa
and C. pseudo paradoxa constitute one interbreeding system, and the four
species C. engametos, C. paupera, C. braunii, and C. dresdensis consti-
tute another interbreeding system; but these two systems of species will
not breed with each other.

Among the first group of species, Moewus (Hartmann, 1934) found
two sexes in each of two races (from Giessen and from Coimbra) of C.
pseudoparadoxa and in C. paradoxa. In order to discover whether the
two sexes were alike in the three races, they were matched up in all

672 SEXUALITY

possible combinations. As appears in Table 7, no two of the sexes are
exactly alike. For example, the sexes I have designated A and B differ
in that A will copulate with C while B will not; A and C differ in
that they copulate with each other, although they are alike in their re-
actions to the other four sexes; and so on. The direct inference naturally
drawn from these observations is that there are six diverse sexes in this
group of races, and I have therefore designated them by six different
letters. The question of whether such multiple sex systems can be reduced

TABLE 7: BREEDING RELATIONS IN Chlamydomonas paradoxa AND
C. pseudo paradoxa*

of the sexes differ from those used by Moewus.

to two sexes, male and female, will be taken up later. Moewus holds
that they can and designates them otherwise than I have done in Tables
7, 8, and 9.

A similar system of multiple sexes is indicated by the breeding rela-
tions in the second group of species, as shown in Table 8, constructed
from the data of Moewus (Hartmann, 1934; Moewus, 1936, 1937b,
1938a). Two sexes have been isolated in C. sp. (coccifera?), C. braunit,
and dresdensis, six in C. paupera, and eight in C. eugametos. Not all of
these are diverse, however. The two in C. dresdensis are the same as two
in C. paxpera and two in C. eugametos; four in C. engametos are reduci-
ble to two diverse sexes identical with two others in C. paupera; and
the remaining two in C. paupera are identical with two others in C.

SEXUALITY 673

eugametos. Altogether, there are eight diverse sexes designated in the
table by the letters G to O. Two of these occur in C. braunii, two in C.
dresdensis, six in C. paupera, and six in C. ewgametos. Their interrela-
tions are shown in condensed form in Table 8. G copulates with all
others but H, H with all others except G and J, and so on. The breeding
relations in this system of eight sexes is in general similar to the rela-
tions shown by the six sexes of the first group of races (Table 7): any

TABLE 8: BREEDING RELATIONS IN Chlamydomonas sp. (coccifera?),
C. braunii, C. dresdensis, C. eugametos AND C. paupera*

Sx G H J K L M N O
Se a ee ee eee

es | a ee ree
eae | 2 Sees ee eae ae
ae ae ee Se |, TS ae eae ag es
i a areas er ee eee eT
eon Ges: | Le a aa Se ee

| i ee eo) een
Tees Sea ea Sse

* + = copulation; — = no copulation. In each species the sexes found are as follows: in both

C. sp. and C. braunii, sexes G and O; in C. dresdensis, sexes H and N;; in both C. exgametos and
C. paupera, sexes H, J, K, L, M, and N. In C. exgametos sexes H and N occur in form typica, J
and M in form simplex, K and L in both forms subheteroica and synoica. Data from Moewus, 1936,
1937b, 1938a; and Hartmann, 1934. The designations of the sexes in this table differ from those
used by Moewus and Hartmann. Not all combinations between the sexes in different varieties and
species have been reported (e.g., the sexes in C. sp. were tested only with the sexes of C. braunii; and
C. braunii was tested with all others except C. paupera), but every possible combination of sexes
was made with at least one representative of the sexes.

sex copulates with any other sex in the group except that sometimes
copulation will not take place with the sex next above or below it in
the table.

Nature of the sex differences——tIn the preceding section the sexes
were defined in terms of the sexes with which they copulate. Two cul-
tures are of the same sex if they do not copulate with each other and if
each does copulate with all the sexes with which the other copulates. Two
cultures are of different sex if they copulate with each other, or if one

674 SEXUALITY

copulates with one or more sexes that the other does not copulate with.
Thus the primary differences among the sexes lie in these breeding rela-
tions. There are at least two further kinds of sex differences that throw
much light on the nature of the sexes in Chlamydomonas.

The first of these involves the intensity of the mating reaction. It
is known that algal gametes of certain species (including Chlamydo-
monas) form groups as a preliminary to copulation (Fig. 165). When
ripe cultures of gametes that can copulate with each other are mixed
together, the gametes at once form clusters of as many as 100 or more

a = Rb ot Y iam
+ fe %

Figure 165. Group for-
mation in Chlamydomonas,
showing the groups formed
in a mixture of cells dif-
fering in sex.- (From
Moewus, 1933.)

gametes. Within the clusters the gametes pair off, with the result that the
cluster disintegrates into copulating pairs. The size of the initial clusters
is partly determined by the number of gametes per unit of volume. When
this concentration is uniform (e.g., 2 < 10° gametes per cc.), the size of
the clusters depends upon which two sexes are present in the mixture.
Certain combinations of sexes yield groups of 100 or more gametes,
others give groups of but 10 to 20 gametes, others give only pairs, and of
course some do not even give pairs. These four grades of reaction have
been designated as 3, 2, 1, and 0 respectively, and the intensities of the
reaction of the sex mixtures shown in Table 8 are given in Table 9. As
shown in Table 9, the same sex may give different grades of reaction
in mixtures with different sexes: thus G reacts to L, M, N, and O with

SEXUALITY 675

intensity 3, to K with intensity 2, to J with intensity 1, and not at all
to H. Moreover, the weak reaction of G with J is not due to the weak-
ness of J, for J reacts with intensity 3 in mixture with L, while the latter
gives but a weak reaction with N. Consequently the strength of the reac-
tion is not a general characteristic of a given sex, but depends in some
way on the particular combination of sexes. When the sexes are arranged
as in Tables 8 and 9, the differing intensities of reaction fall into a defi-
nite system: the strength of reaction between sexes increases with their

TABLE 9: GRADES OF SEX REACTION IN MIXTURES OF SEXES G TO O FROM
THE Chlamydomonas SPECIES C. braunii, C. dresdensis, AND C.
eugametos (FORMS TYPICA, SIMPLEX, SUBHETEROICA,

AND SYNOICA)

Sex G H I K 1G M N O
D fo) fo) I 2 3 3 3 3
ical fo) fe) fe) I 3 3 3, 3
I I fo) fo) O 3 3 3 3
K 2 I fo) O 2 3 3 3
L 3 3 3 2 ) o I 2
M 3 3 3 3 fo) ) O I
N 3 3 3 4 I fo) fo) fe)
O 3 3 3 3 2 I o o

* Mixtures made from cultures with cells in concentration of 2 x 108 per cc. O = no reaction;

1 = pairs only; 2 = clumps of 10 to 20 cells; 3 = clumps of 100 or more cells. Data from Moewus,
1938a. The designations of the sexes differ from those used by Moewus.

distance apart in the table until the maximum reaction (grade 3) 1s
reached. These quantitative differences in intensity of sex reaction sug-
gest that the fundamental differences among the sexes are also quantita-
tive, a suggestion strikingly confirmed by studies of Moewus on the
chemical basis of the sex reaction, as will now be set forth.

The culture fluid in which ripe gametes are living has been shown,
in a number of algae, to contain material (‘‘sex stuffs’) capable of af-
fecting the sexual behavior of other gametes. In Chlamydomonas,
Moewus (1933, and later) obtained this material free from the or-

676 SEXUALITY,

ganisms that produce it, by means of filtration and centrifugation, and
found it to have two striking effects. Gametes grown in the dark are
incapable of copulating, but treatment with the sex stuff from a ripe
culture of gametes of the same sex rendered them capable of copulation.
It will be recalled that activation of non-reactive gametes by filtrates from
cultures of reactive gametes has previously been referred to (pp. 668-
669) as the method employed in activating the peculiar gametes of the
Giessen race of C. pseudo paradoxa, which are always normally nonreac-
tive. This situation differs from most of the others described by Moewus
in that activation is here brought about by the sex stuff from gametes of
a different sex. Reference to page 672 and Table 7 will show that the
sex stuff from sexes B and E were used to activate gametes of sexes C
and D respectively. Similarly Moewus (1934) states that filtrates from
sex K can activate gametes of sex H. This raises the question of how wide
a range of sexes can be activated by sex stuffs from any one sex. Moewus
(1939a), in a discussion of those sexes which I have designated G to
O, states that each sex can be activated by filtrates only when they are
derived from active gametes of the same sex. The earlier results with C.
pseudo paradoxa and C. eugametos (Hartmann, 1934; Moewus, 1934)
do not agree with this generalization.

The second effect is observed when reactive gametes of one sex are
added to sex stuffs obtained from gametes of certain other sexes. The
introduced gametes form groups or clusters as if they were about to
copulate, but eventually the clusters disintegrate without copulation tak-
ing place. This happens when reactive gametes of one sex are added to
sex stuffs from filtrates of reactive gametes of the other sex in the same
race, as, for example, when gametes of sex J are added to filtrates from
gametes of sex M, for sexes J and M are the two found in C. exgametos
f. simplex. Whether similar effects of one sex stuff are producible on
more than one other sex is not stated. The important point here is that
sex stuffs can induce a sex reaction between cells alike in sex, but cannot
induce them to copulate with each other. This indicates that there are
two distinct processes in the sex act: the agglutinative sex reaction, and
the actual fusion of cells and nuclei. The sex stuffs function in the for-
mer but not in the latter process. The existence of sex differences with-
out sex stuffs (or with sex stuffs in ineffective concentrations) is also
shown by the gametes of C. pseudoparadoxa from Giessen (see p. 668

SEXUALITY 677

above). These observations by Moewus are perhaps subject to a very
different interpretation. The agglutinative reactions observed between
gametes of the same sex are weak and transient. Failure to copulate might
well be due to this, rather than to the absence of an additional factor
such as a sex difference. Similar weak mating reactions between cells of
the same mating type were observed by Sonneborn (1937) in Para-
mecium aurelia after the cells had been in contact with animals of an-
other mating type. As in Chlamydomonas, the mating reaction was tran-
sient and did not lead to copulation. Similar behavior was also observed
by Sonneborn (1938a) when cultures of mating types II and V, belong-
ing to non-interbreeding varieties, were mixed together. Here the mating
reaction occurs between animals of different mating types, and yet they
fail to conjugate. Further, cultures known to belong to two mating
types that will interbreed under favorable conditions will, under other
conditions, give a weak and brief mating reaction without proceeding
to conjugate. In view of these observations, it appears to be still an
open question whether the failure of copulation to take place between
cells of the same sex in Chlamydomonas that have given a weak sex
reaction with each other is due to the weakness of the reaction or to
some other aspect of sex, different from the production of diverse sex
stuffs.

In later publications, Moewus (1938b, 1939a) reported the discovery
of the chemical nature of the sex stuffs in the group of sexes G to O.
The active stuffs for these eight sexes are all diverse percentage combi-
nations of the cis and trans forms of the dimethyl ester of crocetin. The
proportions are as follows:

See ere ee yin 7 JG ae pen heyy mg AR MG

Percentage Cis 95 85 UD 65 35 25 15 a5
Percentage trans 5 1S: 25 35 65 Wp) 85 95

The chemical nature of the sex stuffs aids greatly in understanding
the breeding relations summarized in Tables 8 and 9. The order of
sexes from G to O in the table is in the order of decreasing percentages
of cis and increasing percentages of trans dimethyl crocetin. The dif-
ference in percentage of either cis or trans between any two successive
sexes in the table is always 10 percent, except between sexes K and L
which show a difference of 30 percent. Copulation occurs between any

678 SEXUALITY

two sexes differing by 20 percent or more in the production of either
cis or trans dimethyl crocetin, but not if they differ less than this. Fur-
ther, the intensity of the sex reaction (Table 9), as measured by the size
of clusters, also depends upon the difference in proportions of cis and
trans dimethyl crocetin produced by the two sexes under examination: a
difference of 20 percent results in the formation of pairs only (grade
one reaction); a difference of 30 percent yields clusters of 10 to 20
cells (grade 2); a difference of 40 percent or more yields clusters of
100 or more cells (grade 3).

By introducing capillary tubes filled with known mixtures of cis and
trans dimethyl crocetin into one edge of a drop of culture fluid and
adding gametes of a known sex to the opposite edge of the drop, Moewus
(1939b, 1939c) observed that the gametes aggregated at the open end
of the capillary tube whenever it contained cis and trans dimethyl cro-
cetin in proportions differing from those produced by the gametes by 19
percent or more, but not when the difference was less than this. More-
over, the time required to obtain at the mouth of the tube an aggrega-
tion of from 18 to 22 cells was from 200 to 254 seconds when the dif-
ference in proportions was 20 percent, 140 to 180 seconds when the
difference was 30 percent, and 80 to 109 seconds when the difference
was 40 percent. The speed of aggregation increased with increasing
cis/trans difference to 22 to 37 seconds with a difference of 90 percent;
hence the sex stuffs are chemotactic substances, and the grades of sex
reaction are indices of the speed of chemotaxis. Moreover, in any com-
bination of gametes that will copulate, each sex secretes chemicals that
attract the other and each reacts to the chemicals secreted by the other:
both gametes thus attract and both respond.

INTERPRETATION OF THE SEXUAL PHENOMENA IN Chlamydomonas

The sexual phenomena in Chlamydomonas have been interpreted by
Moewus and by Hartmann in accordance with Hartmann’s (1929) theory
of sexuality. This theory may be formulated in the following series of
propositions:

1. Sex is a universal biological phenomenon.

2. There are always two and only two sexes.

3. These two sexes are always male and female.

4. Male and female are qualitatively diverse.

SEXUALITY 679

5. Every cell has the full Anlagen, or potencies, of both male and
female.

6. These potencies are not localized in any one cell component, but
are general properties of all the living material.

7. The sex manifested by a cell is the result of a weakening or
strengthening of the expression of either the male or female potency.

8. This weakening or strengthening may be determined by outer
conditions, or by developmental conditions, or by genetic factors.

9. The degree of weakening or strengthening depends upon the ef-
fectiveness of the determiners listed in proposition 8.

"10. This quantitative variation results in the appearance of each sex
in a series of strengths called valences.

11. Sexual union takes place only under one or the other of two
conditions: (a) when the gametes differ in sex; i.e., when one manifests
a stronger male than female potency, the other a stronger female than
male potency; (b) when the gametes are alike in sex, but very different
in sex valence; e.g., when one is strong female, the other weak female;
or when one is strong male, the other weak male.

12. Sexual union equalizes or reduces the tension resulting from dif-
ference in sex or sex valence.

The work on Chlamydomonas shows that physiological sex differences
may exist in cases in which morphological sex differences are lacking.
This is most clearly evident in those species and races in which each
clone consists exclusively of one sex type. Here sexual union takes place
only between gametes from different clones, the physiological sex dif-
ference of which has been demonstrated. Moewus and Hartmann fur-
ther hold that similar physiological sex differences distinguish the unit-
ing gametes in species and races manifesting copulation among the
members of a single clone. The evidence for this, drawn from experiments
employing the “‘left-over’’ technique, has been set forth on pages 670-
671, along with the contrary evidence of Pascher and of Prings-
heim and Ondra¢ek. There thus remains some doubt, even within the
genus Chlamydomonas, as to whether sex union is invariably accom-
panied by sex differences.

Hartmann’s contention that sex differences are always male and fe-
male could not at first be applied to Chlamydomonas. Moewus, there-
fore, simply classified the sexes as plus (+) and minus (—). In the

680 SEXUALITY

group of species shown in Table 7, sexes A, B, and C were called +,
sexes D, E, and F, —. The three sexes of each type were assigned arbi-
trary strengths or valences: A and F were assigned a valence of 3; B
and E, 2; and C and D, 1. Copulation was thus held to take place either

TABLE 10: SYSTEM OF MATING RELATIONS IN Chlamydomonas braunii, C.
dresdensis, AND C. eugametos, SUMMARIZING THE OBSERVATIONS AND
INTERPRETATIONS OF MOEWUS, 1937B, 1938A, 1938B, 1939A*

Sex (Later View) Female Male
Sex (Earlier View) = =
Valence 4 3 3 I I 2 3 4
Sex as Designated in
This Review G H J K L M N O
Percentage cis
95/5 |85/15/75/25 65/35)35/65 25/75 15/85) 5/95
Percentage trans
4 | G | 95/5 ch I 2 3 3 3 3
3 | H | 85/15 OF" Or Por | Pray |eea eninge lemma (ame
Female | + |——|—— ————_ $<, ——__ | ———.
2) Je 75/25 Ty fl) 2Op sO wait One nas 9.) 8 alas
re | IS YRS 2 I fe) fo) 2 4 3 3
tr | L | 35/65 3 3 3 2 fo) fo) I 2
2 |M | 25/75 Bo 1 lee Sill Ole lela Masai lame
Male =
3 | ING 157/85 34 S51) (SPB Ba Ko i eome amo
AP OL 5/05 ae elie lie ea me eh)
* The numbers 0, 1, 2, 3 in the body of the table give the intensity of the sex reaction; 0 = no
copulation; 1 = pairs which form directly; 2 = preliminary clusters of 10 to 20 cells; 3 = pre-
liminary clusters of 100 or more cells. Percentage cis/Percentage trans = the proportions of the sex

stuffs, cis and trans dimethyl crocetin, produced by the gamets.

between gametes differing in sex (i.e., between any + and any —), or
between two gametes of the same sex differing by as much as 2 in valence
(for example, between gametes of A and C, both of which are held to
be +, because A is + 3 and C is + 1). Similar interpretations were
put forth for the mating relations summarized in Tables 8 and 9. The
sexes G, H, J, and K were denominated +- in sex, with valences of 4,

SEXUALITY 681

3, 2, and 1, respectively; and the sexes L, M, N, and O were said to be
— in sex, with valences of 1, 2, 3, and 4 respectively. In this group,
Moewus (1937b, 1938a) later held that the sex previously called +- was
female, the one called — male. These designations, together with other
pertinent information on the strength of sex reaction and sex stuffs, are
shown in Table 10. (Identification of male and female in the first group
of races has not yet been reported. )

The remainder of the interpretation is largely genetic and will be dis-
cussed here only insofar as appears necessary for a satisfactory under-
standing of the general phenomena of sexuality and their relation to
the theory of Hartmann. For further details the reader should consult
Chapter XV, “Inheritance in Protozoa,” by H. S. Jennings. Moewus
gives evidence for two series of multiple alleles affecting sex in the
braunii, dresdensis, paupera, eugametos group of species: at one locus
is a series of genes M1, M2, M3, and M4, determining the four valences
of male gametes; at another linked locus is a series of genes F1, F2, F3,
and F4, determining the corresponding four valences of female gametes.
When crossing over takes place between these two loci, nuclei with a
chromosome lacking both an M and F gene die, while those with both
M and F genes survive. In the latter, if the valences are equal, sex is
determined by nongenetic factors, the valence is unchanged, and both
male and female gametes arise within a single clone; but if the valences
are unequal, sex is determined by the gene of stronger valence and the
resulting valence is the arithmetic difference between the valences of the
two genes. In races such as C. exgametos f. subheteroica, in which each
clone is always prevailing of one sex, another pair of genes determines
which sex shall prevail. The genetic relations have not been worked out
so fully in the paradoxa-pseudoparadoxa group of races, but there also
multiple alleles are held to operate. Although evidence as to whether
the + and the — genes are alleles has not been reported, observations
on regular non-disjunction showed that the sex and valence resulting
from the presence of two or more alleles was their algebraic sum.

The various genes affecting sex are considered to be the sex realisators,
in agreement with Hartmann. They are held to operate by acting on the
underlying sexual Anlagen, or potencies, A and G, the genes of dif-
fering valence acting on A and G to different extents. Sexual union then
results when gametes differ in sex or in sex valence by as much as 2.

682 SEXUALITY

Further, the grades of reaction shown in Table 10 are presumably indices
of the magnitude of sex tension between the gametes. Difference of sex
always results in a grade 3 reaction, except between gametes of the
lowest valence. When alike in sex, a difference of 2 in valence is re-
quired for a grade 1 reaction and a difference of 3 for a grade 2 reaction.

Certain features of Moewus’s interpretation are of special interest:
(1) his reduction of what appeared superficially to be many interbreed-
ing sexes to but two, assumed to be qualitatively diverse; (2) his iden-
tification of these two sexes with male and female; (3) his distinction
between unions resulting from (qualitative) difference in sex and those
resulting from (quantitative) difference in sex valence. The evidence
and reasoning involved in these views is set forth in the following.

The original basis for holding that only two sexes are present in each
interbreeding system appears to be partly that the sexes were discovered
in pairs. For example, in the paradoxa-pseudoparadoxa group of species
(Table 7), Moewus found the two sexes here called A and F in C.
paradoxa, B and E in the race of C. pseudo paradoxa from Coimbra, and
C and D in the race from Giessen. Similar pairs of sexes were found in
the other group of species: in C. brauniz, G and O; in C. dresdensis,
H and N; in C. exgametos f. typica, H and N; in C. ewgametos f. sim-
plex, J and M; in C. e~gametos f. subheteroica and f. synoica, K and L
in each. Only in C. paw pera did an exception appear; the six types H, J,
K, L, M, and N were all found together in a single natural source.

From this point on, it appears to be simply assumed that the two sexes
in one race are qualitatively the same as the two in any other race with
which it can interbreed. If this assumption be accepted, then the remain-
ing interpretation follows naturally. For example, if in C. paradoxa
(Table 7) the two sexes A and F are designated + and — respectively,
then in the Coimbra race of C. pseudo paradoxa B must be + and E —,
for B copulates with F (—), not with A (-+); and E copulates with
A (-++) not with F (—). Similarly, C and D in the Giessen race are +
and — respectively. This is clear from their mating relations with B and
E. The exceptional copulations between like-sexed gametes (A- with
C+ and D— with F—) are interpreted as follows: A and C must both
be the same sex (++) because of the mating relations set forth above;
yet they must also be unlike in some sexual way, for they copulate with
each other, though neither will copulate with others like itself; hence

SEXUALITY 683

they must differ in degree of sex, or valence. In a similar way, the eight
types G to O (Table 10) are reduced to two sexes, +- and —,, each ap-
pearing in four valences. Here K and L are recognized as +- and — of
the lowest valence because they give a weaker sex reaction (grade 2) with
each other than do J and M (in C. evgametos f. simplex), or H and N
(in C. dresdensis), or G and O (in C. braunii). Of the four grades of
+ gametes, G is most diverse from K because it gives the strongest
reaction with it; hence G has the highest valence among the ++ gametes.
Similarly, O is the — gamete of highest valence, and H and N are the
next strongest + and — types (for they react less strongly with K and
L than do G and O, while the others do not react at all with them).
This leaves J and M intermediate between H and K and between L and
N; and this is confirmed by their grades of reaction with G.

The identification of -+ and — with female and male (in the ewga-
metos—paupera group of species) is based on differences in morphol-
ogy, activity, and function between the gametes in certain species, and
on the assumed identity of the sex differences in all the species. In C.
coccifera and C. braunii, as set forth on pages 667, 668, the two
kinds of gametes differ markedly in size and behavior during copulation:
the smaller gamete empties into the larger one. Further, in C. cocczfera
the large gametes lack flagella and are nonmotile, while the small gametes
have flagella and are motile. Moewus therefore holds that the large,
nonmotile gametes of C. coccifera are eggs and so female, while the
small, motile gametes are comparable to sperm and so are male. If this
be admitted, then the large and the small gametes of C. brawnii are also
female and male, even though both are flagellated, because in combina-
tions between the two species copulation occurs only between large and
small gametes. On the same grounds, the gametes of isogamous species
are female and male, because of the two physiological kinds of gametes
in C. ewgametos f. typica (types H and N), H will copulate only with
the small gametes of C. brawnii while N will copulate only with the
large ones. Thus the + sex has been identified with female and the —
with male in C. e~gametos f. typica. And, since + and — were assumed
to be the same in all races and species, female and male must be the same
in all races and species. The copulations between female gametes (or
between male gametes) of different races must then be consequences of
difference of sex valence.

684 SEXUALITY

Critique of the works of Moewus on Chlamydomonas.—Attention
should be called to certain difficulties in some of the important features
of Moewus’s interpretations and observations.

1. Identification of +- and — with female and male. Moewus’s iden-
tification of + and — with female and male is based, as set forth above,
on two points: the two sexes in anisogamous species, especially in C.
coccifera, are male and female; the two sexes are the same in all races
and species. The point has already been emphasized that the latter is an
assumption, not a fact of observation. The interpretation of the two
sexes in C, coccifera as male and female is based on the proposition that
female gametes are distinguishable from male gametes by their passive
role in copulation, their larger size, and their nonmotility. Though these
criteria are widely accepted as valid, one may question whether the evi-
dence warrants this. The passive rdle of the “female” gamete in copula-
tion is shown by the fact that the ‘‘male” gamete empties its contents
into the “female” gamete. Nevertheless, the same behavior takes place
in a certain race of C. exgametos, in which Moewus (1933) showed
that it is of no sexual significance for both the ++ and the — gametes
may play either rdle in copulation. The same holds for difference in size:
either the + or the — gamete of this race of C. eugametos may be twice
as large as its mate. The difference in behavior is correlated with the
difference in size, but neither is correlated with sex. One may doubt,
then, whether these two criteria are of sexual significance in C. coccifera,
since they are clearly not significant in C. ewgametos. The difference in
motility is perhaps stronger evidence, for only the + gametes of C.
coccifera are non-flagellated and these are generally considered to be
comparable to eggs. It is important to keep clearly in mind that the use
of the terms male and female for the gametes of all the races and species
of Chlamydomonas rests finally on the single fact that the -+- gametes
of C. coccifera lack flagella. Whether this is sufficient ground for holding
they are female in the same sense as the eggs of higher organisms and
for extending the terms male and female to the gametes in all other
species of Chlamydomonas that interbreed with C. coccifera must be left
to the judgment of the critical reader. The present author, in agreement
with Kniep (1928) Mainx (1933) and others, holds that such facts
constitute too slender a basis to justify an interpretation of such general
theoretical significance.

SEXUALITY 685

2. Reduction of systems of multiple gamete types to two sexes. As
earlier set forth, the reduction of the multiple gamete types in an inter-
breeding system to two sexes is based on the assumption that the two
sexes in any one race or species are fundamentally the same as the two
in any other race. In the case of C. paw pera, in which six types of gametes
were found in the same natural source, it is presumably assumed that
three races, each with the same two sexes, were here living together. It
is important to recognize clearly that this view is based on Hartmann’s
theory; it is not an observation or an induction from observation. Chem-
ical analysis of the sex stuffs shows that reduction of the eight gamete
types in the e~gametos-paupera group of species to two qualitatively di-
verse sexes cannot be made on this basis, for the differences among the
eight sex stuffs are exclusively quantitative. The ‘‘tension” assumed to
bring the gametes together is held to be of two kinds. One kind is purely
chemotactic and due to the sex stuffs; this brings the gametes into con-
tact. It is clearly a quantitative phenomenon, dependent upon differences
in relative proportions of cis and trans dimethyl crocetin. The other kind
of tension determines whether gametes that have been brought into
contact will unite in copulation. The evidence for this, together with
considerations that render the conclusion less certain, was set forth on
page 682. However, if an unknown factor determining union in
copulation exists, it appears to act in the same quantitative way as the
sex stuffs, for copulation takes place between any two gamete types that
produce sex stuffs sufficiently diverse to attract each other. Consequently,
there are no observations justifying or even suggesting the introduction
of the concept of two qualitatively diverse sexes; all the observations
point directly to a system of multiple, quantitatively diverse sexes.

In one respect the preceding account may not fairly represent Moe-
wus’s views. The two sex stuffs may be taken as indices of two qualita-
tively diverse sex tendencies or potencies, cis demethyl crocetin being the
manifestation of the + sex potency, and trans dimethyl crocetin of the
— sex potency. In four of the eight types of gametes, the + sex potency
prevails, for these types produce more cis than trans; and this prevails
to different degrees in each type. In this sense these four types of gametes
may be considered as different strengths or valences of the + sex. Cor-
respondingly, the remaining four types could be considered four diverse
valences of the — sex. This view is in accord with that part of Hart-

686 SEXUALITY

mann’s theory which holds that both sex potencies reside in all kinds of
gametes and that the sex of the gamete 1s simply the potency that prevails.
Thus the qualitative sex difference is not segregated into different gam-
etes and has nothing to do with copulation; all gametes have both quali-
tative sex characters and differ only in the quantitative manifestation of
one or the other. These quantitative differences alone determine copula-
tion and sex reactivity. Conceivably two qualitatively diverse sexes might
exist, one producing only cis, the other only trans dimethyl crocetin. But
these have not been found. The observed gamete types are all quantita-
tively diverse grades of intersexes, some prevailingly +_, others prevail-
ingly —. Viewed in this way, the observations are in accord with part of
Hartmann’s theory.

3. Difficulties in Moewus’s observations. There are certain difficulties
in Moewus’s observations that raise serious questions concerning the
reliablity and accuracy of his reports. Two of these must be mentioned.
The first involves the apparently irreconcilable conflict between observa-
tions of the consequences of non-disjunction of the sex chromosomes in
crosses between C. paupera and C. ewgametos and the later discoveries
of the sex stuffs. Moewus (1939a) reports that copulation takes place
between gametes of the same sex when there is at least a difference of
2 in valence. By definition, gametes of valence 5 would copulate with
gametes of valence 3, but not with gametes of valence 4; and gametes
of valence 6 would copulate with those of valence 4, but not with those
of valence 5. In a series of crosses and back crosses involving C. ewga-
metos £. subheteroica (valence 1) in C. paupera (valence 3), Moewus
(as reported by Hartmann, 1934) obtained through nondisjunction of
the sex chromosomes clones that yielded gametes of valences 5 and 6,
presumably recognized as such through the breeding tests mentioned
above. Moewus (1939a) shows that copulation will take place only when
there is a difference of at least 20 percent in the cis or trans dimethyl croe-
tin produced. Valence 5, by definition, copulates with valence 3; but va-
lence 3 produces 85 percent cis or trans dimethyl crocetin. This leads to
the impossible conclusion that the valence 5 gametes produced 105 per-
cent cis or trans dimethyl crocetin. Similarly, the valence 6 gametes would
be required to produce 115 percent cis or trans dimethyl crocetin. This
apparently irreconcilable contradiction in the reports raises the serious
question of whether the reporting is accurate and reliable.

SEXUALITY 687

The same question has been raised by Philip and Haldane (1939)
from an analysis of data in many experiments by Moewus on crossing
over and segregation in both Chlamydomonas and Protosiphon. These
authors calculated that the chance of getting such close numerical agree-
ment among the 22 experiments analyzed was once in 3.5 >< 10°? trials.
According to them “if every member of the human race conducted a
set of experiments of this type daily, they might reasonably hope for
such a success once in 50,000 million years.’ They suggest that this im-
plies a conscious or unconscious adjustment of observations to fit a
theory and they call for repetition of the experiments by an independent
worker. The failure of Pringsheim and Ondracek (1939) in their at-
tempts to confirm certain parts of Moewus’s work, their numerous criti-
cisms, the criticisms of Philip and Haldane, the internal inconsistencies
in Moewus’s data, and the great theoretical importance of the work, all
make independent repetition of the work an urgent need.

SEXUALITY IN Paramecium AND OTHER CILIATE PROTOZOA

The ciliate Protozoa differ from Chlamydomonas and the flagellates
in their nuclear condition and in some features of the sexual phenomena.
There are two kinds of nuclei: macronuclei and micronuclei. Ordinarily
the macronucleus disappears during the sexual processes and a new one
is formed from a product of the micronucleus. The micronuclei alone
contain recognizable chromosomes and play the leading rdle in the
nuclear changes involved in sexual processes. The vegetative individuals
contain diploid micronuclei that undergo maturation with reduction of
the chromosomes to the haploid condition during mating. In each con-
jugant two reduced nuclei are formed. In most ciliates, both of these
are functional: one remains within the animal in which it is formed and
is known as the stationary pronucleus, or gamete nucleus; the other goes
into the mate of this animal and is known as the migratory gamete
nucleus, or pronucleus. The two nuclei present in each conjugant after
exchange of pronuclei unite to form a synkaryon. Conjugation thus in-
volves a reciprocal fertilization, both conjugants being fertilized, each
by the other. The conjugants then separate and each reconstitutes a new
nuclear apparatus and gives rise to progeny by repeated fissions. (See
Chapter XII. )

The mating process is somewhat different in the peritrichous ciliates.

688 SEXUALITY

Unlike most other ciliates, in the peritrichs the two mates differ greatly:
one is sessile and large, the other is motile and much smaller. Of the
two reduced nuclei formed in each mate, only one is functional: one of
those formed in the microconjugant wanders into the macroconjugant
and unites with one of its nuclei. The other nuclei degenerate, as does
the remainder of the microconjugant. Thus only one individual results
from the mating act and this one then reproduces by repeated fissions.

Obviously the phenomena of sexuality are different in the Peritrichida
from what they are in other ciliates. In the following, attention will be
directed chiefly toward these other ciliates, of which Paramecium is an
example. For both kinds of ciliates, however, the problems of sexuality
are essentially the same: (1) Are the conjugant individuals sexually
diverse? That is, can any two individuals conjugate with each other, or
do the individuals differ morphologically or physiologically so that con-
jugation can occur only between individuals of these different types?
(2) Are the two gamete nuclei, formed in each conjugant, sexually di-
verse? (3) Do conjugants differ from non-conjugants? This question
involves the problem of the ciliate life cycle, with possible periods of
immaturity and maturity.

SEXUAL DIFFERENCES BETWEEN CON JUGANT INDIVIDUALS

As already indicated, in one order of ciliates, the Peritrichida, the
conjugants show a clear-cut differentiation into two sex types. One type,
the macroconjugant, is sessile and large; the other, the microconjugant,
is small and free-swimming. Conjugation takes place only between these
two types, never between two individuals of the same type. In these re-
spects the Peritrichida and a few Holotrichida (e.g. Opalina, Trachelo-
cerca, Ichthyophthirius) differ from all other ciliates.

In Metopus, Noland (1927) observed that although the conjugants
are at first morphologically indistinguishable, only one mate is fertilized
and the other one degenerates. Whether this difference in behavior and
fate of the two conjugants of a pair is determined by preéxisting physio-
logical differences between them, or whether it arises first in the process
of conjugation is not known.

In another order of ciliates, the Oligotrichida, a few species have been
reported by Dogiel (1925) and others to show an equally clear-cut
dimorphism, which is not, however, so clearly or simply viewed as a

SEXUALITY 689

sex difference. In Opisthotrichum, as in the peritrichs, there are large
and small individuals that differ considerably in structure, though both
are motile. About 85 percent of the conjugant pairs include one large
and one small member, 15 percent include two large members, and none
include two small members. The small conjugants are thus sexually
specialized for conjugation with large animals only; but the large type is
only to a slight degree sexually specialized: it conjugates more readily
with the small than with the large type, though it can conjugate with
either type.

Indications of differences between the conjugants in some pairs are
often observed. Doflein (1907) observed differences in size between
the two conjugants in many pairs of Paramecium putrinum, and Mulsow
(1913) observed the same thing in about 70 percent of the pairs of
Stentor. Calkins and Cull (1907) reported frequent differences in via-
bility between the two members of pairs of P. caudatum. Zweibaum
(1922) found that in about 70 percent of the conjugant pairs the two
members differed in the amount of glycogen they contained. These ob-
servers suggested that the larger size, greater viability, and higher
glycogen content were female characters, and the reverse characters male.
On the other hand, Jennings (1911) showed by thorough statistical
analysis that while the two members of a pair did sometimes differ in
their characters, on the whole there was a high degree of assortative
mating, or tendency for like to mate with like; and, further (Jennings
and Lashley, 1913a, 1913b), that after conjugation there was remark-
able agreement in character between the two members of a pair (bi-
parental inheritance), even with respect to vigor and viability. It was
generally held, therefore, that in most ciliates regular or frequent dif-
ferences between the two members of conjugant pairs were lacking.

Two observations made long ago raise the question of whether after
all there might not be, beneath the usual superficial morphological simi-
larity of the conjugants, a deeper-lying physiological difference. In
Chilodonella, Enriques (1908) found that although the two conjugat-
ing individuals are indistinguishable at the start of mating, they become
diverse as mating progresses: the left conjugant changes form so as to
appear shorter, and its mouth migrates to the opposite side of the body.
However, it is not clear whether this is an indication of a prior physio-
logical difference between the mates, or whether it is a direct conse-

690 SEXUALITY

quence of their method of union. The second observation is one made
by Maupas (1889). He observed that in certain species conjugation
never occurred in cultures containing animals all from a single natural
source; it was necessary, in order to get conjugation in a single culture,
to have animals from different natural sources. He concluded that di-
versity of ancestry was a necessary condition for conjugation. Many later
observers found that conjugation occurred abundantly among the progeny
of a single individual and so turned attention away from Maupas’s con-
tention, with its implication of physiological difference between con-
jugants. Until recently it was generally supposed that, in most species of
ciliates, any two individuals of the same species could conjugate with
each other if they were capable of conjugation at all.

The interpretations given the various observations just set forth will
be deferred until the newer knowledge of sexuality in Paramecium has
been outlined. In describing this newer work, there will be mentioned
first the usual typical relations, and later, certain instructive exceptions.
The facts on which the following account is based are to be found in
recent articles by Sonneborn (1937, 1938a, 1938b, 1939a, 1939b, and
1939c), Kimball (1937, 1939a, 1939b, 1939c), Jennings (1938a,
1938b, 1939a, 1939b), Gilman (1939), Giese (1938, 1939), and
Giese and Arkoosh (1939).

In P. aurelia, individuals containing macronuclei descended from one
original macronucleus do not as a rule conjugate with each other. Such
a group of individuals is called a caryonide. Caryonides terminate and
new ones are formed when the macronuclei are destroyed and replaced
by products of the micronucleus, during the reorganization following
conjugation and during endomixis or autogamy. At such times usually
two new macronuclei arise in each reorganizing individual, and these go
into different cells at the first fission. The fact that individuals of the
same caryonide do not conjugate with each other agrees with Maupas’s
observation that closely related individuals do not interbreed. But if
several caryonides are present in the same culture, even though all come
from a single original individual, they may conjugate. This agrees with
the observation of the opponents of Maupas, who found conjugation
within a clone.

When several caryonides are cultivated in different dishes and samples
of each are mixed with samples of each of the others, in some of the

SEXUALITY 691
combinations nothing happens—each individual moves about inde-
pendently of the others; but in other mixtures the animals quickly unite
in large clusters. The animals stick together as they collide in their ran-

”
e : os a © ge. 2
> \ Se TB tye A

Figure 166. The mating reaction in Paramecium bursaria. Upper left, single mating
type with individuals scattered singly. Upper right, the clusters formed six minutes after
mixture of cultures of two different mating types. Lower left, a later stage of the mating
reaction (after five hours). Lower right, the final conjugating pairs as they appeared
twenty-four hours later. (From Jennings, 1939.)

dom movements. Animals not in contact do not attract each other; nor
are they in a specially sticky condition, as has been so often maintained,
for neither caryonide shows the least trace of stickiness until the animals

are mixed, and then only when animals of different caryonides collide.
The clusters begin with just two individuals and build up into larger

692 SEXUALITY

aggregations by the repeated addition of other individuals, as these col-
lide with those already united. In the course of an hour or so, the clusters
break down into conjugating pairs. A detailed account of this mating
reaction (Fig. 166) is given for P. bursaria by Jennings (1939a).

The final pairs always consist of one individual from each of the two
caryonides. When animals of the two caryonides differ in size, each
pair consists of one large and one small animal. In P. bursaria (Jennings,
1938a) the two members of each pair differ in color when a normal green
culture is mixed with one made pale as a result of recent rapid multipli-
cation.

When all possible combinations are made among a group of caryo-
nides, they are classifiable, on the basis of their reactions, into two groups
(Table 11); no two members of the same group will conjugate with
each other, but any two caryonides from different groups will. These

TABLE 11: RESULTS OF MIXING TOGETHER ANIMALS FROM DIFFERENT
CARYONIDES OF STOCK F, Paramecium aurelia*

Caryonides

2b1 | 2b2 | 3a2 | gbr | 4b2 | rb2 | 2ar | 2a2 | 3a1 | sbr | sb2

5 op am: = = = ae ata ate fo ae ieee
oo | 2 ea | ee ee

el ie ee | eee
soll SA 5 ES a A | ee a

* + = conjugation; — = no conjugation. Data from Sonneborn, 1938a. The caryonides 2b1,

2b2, 3a2, 4b1, and 4b2 are of mating type I; caryonides 1b2, 2a1, 2a2, 3a1, 5b1, and 5b2 are of
mating type II.

SEXUALITY 693

two groups are said to be of different mating types, and in one group of
races are designated as I and II. Conjugation occurs between types I and
II, never between individuals of the same type, whether they be mem-
bers of the same or different caryonides. In order to ascertain the type of
any unknown caryonide, some of its animals are mixed with type I and
some with type II; conjugation occurs in one of the mixtures, not in the
other. The type of the new caryonide must then be the same as the type

TABLE 12: THE SYSTEM OF BREEDING RELATIONS IN Paramecium aurelia,
DATA FROM SONNEBORN, 1938A*

Variety I 2 3
Mating Type I tee a IV V VI
I - + - ~ - -
I eee atatee | e
I a = - - — ~
ll - _ - = - —
2 |
IV — — + - - -
Vv ie eS b= = “ S
, |
VI ~ ~ — - + -
* The three varieties (1, 2, and 3) do not interbreed; conjugation occurs only between the two
mating types within each variety. + = conjugation; — = no conjugation.

with which it did not conjugate, different from the one with which it
did. For example, if a culture fails to conjugate with type I, but does
with type II, it is type I.

Sonneborn (1938a, 1939a, 1939b) has analyzed some fifty stocks of
P. aurelia, collected from various regions between Canada and Florida
and from the Atlantic to the Pacific Coast. Nearly all showed a similar
system: in each stock all caryonides were classifiable into one or the other
of two mating types. The few remaining stocks consisted exclusively of
but one mating type: e.g., in stock B all caryonides conjugated with type
II from another stock, none with type I; so stock B consists exclusively
of type I. Studied alone, stock B would be considered non-conjugating,
because it never conjugates among its own members. All so-called non-
conjugating stocks behave like this; they consist of only one mating type

694 SEXUALITY

and conjugate readily when mixed with the proper type from another
stock. Mating types appear to be of universal occurrence in P. aurelia.

Although not more than two mating types occur in any one stock,
more than two must exist in the species, for both mating types in some
stocks fail to conjugate with either of the types in certain other stocks.
Altogether, six different mating types have been found (Table 12).
One group of stocks contains types I and II; a second group contains
types III and IV; a third group, types V and VI. Conjugation takes place
only between the two types in the same group, never between types in
different groups. This sexual isolation of the three groups of stocks
makes them distinct genetical species or varieties; but they appear to be
morphologically alike, all conforming to the description of the taxo-
nomic species P. awvrelia. However, they are physiologically diverse in a
number of ways.

Each mating type is uniquely defined by the type with which it mates.
The mating type of a culture can be ascertained by mixing some of its
animals with standard cultures of each of the six mating types. With one,
and only one, of these it will conjugate. Its mating type is the other one
in the variety with which it mates. For example, if it mates with type V,
it belongs to variety 3 and is of mating type VI.

In P. bursaria, Jennings (1938a, 1938b; 1939a, 1939b) reports some-
what different mating-type relations. Each stock of this species shows as
a rule only one mating type. As nuclear reorganization is extremely rare,
a stock is practically equivalent to a caryonide of P. aurelia. The mating
types fall into three different groups, or genetical species (a fact first
found in P. bursaria), with no conjugation between types in different
groups (Table 13). In group I occur the four mating types A to D; in
group II, the eight types E to M; in group III, the the four types N to
Q. In each group each mating type conjugates with all the other types in
that group. This system of multiple interbreeding types is in marked
contrast to the system of paired types in P. awrelia. To discover the group
to which a new stock belongs, it must be mixed with at least two types
from each of the three groups. It will conjugate with one or both of the
types from one group, not with any of the others. It belongs to the group
with which it conjugates. To discover its mating type, it must now be
mixed with all the types of this group until one is found with which it
will not conjugate. It is then of the same type as this one. For example,

SEXUALITY 695

TABLE 13: THE SYSTEM OF BREEDING RELATIONS IN Paramecium bursaria,
DATA FROM JENNINGS, 1939A*

Variety I II III

Mating
Type

|
|
|
|
|
|
|
|
|
|
|

Sa@)
|| + +{+| 1

|
|
a

+/+]/4+][+]/4+]0]4+]+

ate tee) tes ee et

|
+/+][+)4+]4+]4+]4
42 | ae fae | ae tb |

|
|
|
|
|
|
|
|

—/-|-|-) 4/4
+

pet ae Se =
Ill

+/+] 4+

-|-]-|-|-/-|-|-]-|-|-|-|+|+]-

Q |-|-|-]|-/-|-|-|-|-]-|-|-|+]4+]4]-

* The three groups (or varieties) 1, 2, and 3 do not interbreed; conjugation occurs only among
the four or eight mating types within each variety. -- = conjugation; — = no conjugation.

if it mates with A, B, and C, but not with D, it is of mating type D.

Five of the seven species of Paramecium found in the United States
have been examined for mating types and all have shown them. P.
durelia and P. bursaria have already been discussed. In P. caudatum,
Gilman (1939) finds a system of the same kind as found in P. awrelia:
six mating types occurring in three groups, with only two interbreeding
mating types in each group, and no conjugation between types in dif-

696 SEXUALITY

ferent groups. Sonneborn (1938a, 1939a) found two mating types in
P. calkinsi and three interbreeding types in P. trichium, indicating a sys-
tem of multiple types such as Jennings found in P. bursaria. Giese (1938,
1939) and Giese and Arkoosh (1939) have found mating types in P.
multimicronucleatum and P. caudatum.

Kimball (1939c) found in Ezplotes, one of the hypotrichous ciliates,
a system of mating types like the one in P. bursaria. There are five groups
of non-interbreeding types, with morphological differences between some
of the groups, indicating that these may be taxonomically as well as
genetically different species. In each group occur multiple interbreeding
mating types, six in the group most fully studied, any one of these con-
jugating with any of the other five. The striking agglutinative mating
reaction so characteristic of Paramecium appears to be lacking: conjuga-
tion first occurs several hours after mixture of the different types, and
then pairs form directly without the prior formation of clusters.

In view of our present knowledge, it seems allowable to include Mau-
pas’s (1889) old evidence for the necessity of diverse ancestry as evi-
dence for diversity of mating type. If so, at least four more species must
be added to the list of those in which mating types are known: Stylony-
chia pustulata, Leucophyrs patula, Onychodomus grandis, and Loxophyl-
lum fasciola.

This brings the number of species now known to have mating types
to about a dozen. These belong to six different genera and two different
orders of ciliate Protozoa. It appears, therefore, that mating types will
be found to be widely distributed among the ciliates. The view that any
two individuals of the same species can conjugate with each other, if
capable of conjugating at all, is demonstrably false; on the contrary, in
general, conjugation can take place only between individuals of diverse
mating types.

Are there ever exceptions to this general rule? Does conjugation ever
take place between animals of the same mating type? In nearly all the
species examined in detail, conjugation has been observed in cultures
containing only one caryonide, and, as members of the same caryonide
are presumably of the same mating type, this appears to be conjugation
between animals of the same mating type. Can individuals of the same
caryonide ever differ in mating type? And is this the explanation of these
exceptional conjugations within a caryonide? There is only one method

SEXUALITY 697

(Kimball, 1939a) of answering these questions directly. The two ani-
mals that come together for conjugation must be separated before they
become too tightly united, and the mating types of the two members of
such a split pair must be directly ascertained by placing each of them
separately in standard cultures of the different types, to discover with
which ones they will react sexually. If one reacts only with type I and
the other only with type I, they must be of different types; but if both
react with the same type, then they are alike in mating type.

This problem has been most fully studied by Kimball (1939a, 1939b).
In P. aurelia, he found that conjugation within a caryonide occurred un-
der two very different kinds of conditions. One kind is very common; it
occurs in caryonides genotypically of type I, when the last preceding
caryonide in the direct line of ancestry was of type II. Under these condi-
tions conjugation may occur in the caryonide during the first few days
of its existence. Kimball split some of these conjugant pairs, tested them
directly for mating type, and showed that in each pair one animal was of
type I, the other of type II. Thus both mating types can be present in a
single caryonide, and the mating is between the two types only. Kimball
now obtained clone cultures from the two members of such split pairs
and found in every pair that both cultures were of type I and showed no
further conjugation among their own members. Hence the type II ani-
mals originally present in the caryonide changed to type I. The early oc-
currence of type II was due to the type II character of the immediate
ancestors. This phenotypic or cytoplasmic ‘“‘hang-over”’ fades out, as the
new genotype comes into action. Not all individuals accomplish this at
the same speed, so for a short time some are still type II while others
have completed the change to type I; at this moment conjugation may
occur. A little later all have changed to type I, and conjugation is no
longer possible. Similar “cytoplasmic lag” in the inheritance of other
characters in Paramecium had been reported by both De Garis (1935)
and by Sonneborn and Lynch (1934).

The other type of conjugation within a caryonide is of much rarer
occurrence. In the race of P. aurelia examined by Kimball (1939b), less
than 3 percent of the caryonides showed it. In these, conjugation oc-
curred not only when the caryonide was young, but probably through-
out its whole history. Moreover, any individual in the caryonide gave rise
to progeny that conjugated with each other. Even the members of a split

698 SEXUALITY

pair both gave rise to cultures in which conjugation took place. Never-
theless, Kimball found that the two members of a split pair were always
of diverse mating types at the time they conjugated: one was type I, the
other type II. Hence such caryonides are unstable in mating type. The
type changes back and forth repeatedly; but when conjugation occurs, it
is always between animals of different mating type.

In the Vorticellidae, the invariable morphological and functional dif-
ference between conjugants has already been mentioned. Finley (1939a,
1939b) shows clearly that both types of conjugants not only arise within
a caryonide, but at a single unequal cell division. The macroconjugant
and the microconjugant preduced at this fission can then copulate with
each other or with other similarly differentiated conjugants of the same
caryonide. Here it is obvious that conjugation within a caryonide is never-
theless invariably between different mating types or sexes.

There are, however, a number of known instances of conjugation
within a caryonide which require further investigation. Foremost among
these are species, without morphological difference between the conju-
gants, in which conjugation regularly occurs within a clone or a caryo-
nide. This has been reported as common in P. multimicronucleatum by
Giese (1938, 1939), less common in P. caudatum by Gilman (1939),
and very rare in P. bursaria by Jennings (1938a, 1938b, 1939a, 1939b).
An especially interesting situation is reported for Explotes by Kimball
(1939c). Fluid from a culture of one mating type, added to a culture
of a different mating type, induces the latter to conjugate among them-
selves. Likewise, in mixtures between normal animals of one mating
type and double animals of another type, some of the resulting con-
jugant pairs are unions of singles with singles, a few are doubles with
doubles, though most are, as would be expected, singles with doubles.
The relations here raise the question of whether subjection to fluid from
another mating type makes animals acquire a type corresponding to the
fluid, as Jollos (1926) showed happens in the alga Dasycladus. If so,
it may be difficult or impossible to analyze it satisfactorily, because in
ascertaining the types of members of split pairs they have to be sub-
jected to the very fluid that would change their type. This may be one
of those exasperating problems, like attempting to determine the posi-
tion and the velocity of an electron at the same time, in which the meth-
ods of investigation essentially alter the things being investigated.

SEXUALITY 699

Are any of these observations of conjugation within a caryonide evi-
dence of conjugation between animals identical in mating type? The
direct test has not been made in most cases; but, in the few examples in
which it was made, it was demonstrated that conjugants were always of
different types, in spite of the fact that they were members of the same
caryonide. The evidence is therefore strongly against the occurrence of
conjugation between animals of the same mating type, though final
judgment must await further analysis.

MATING TYPES IN RELATION TO THE MAUPASIAN LIFE CYCLE

According to the well-known theory of Maupas (1889), the ciliate
exconjugant is conceived as being a young individual producing by re-
peated fissions immature cells unable to mate; the cells produced after
many youthful fissions become sexually mature and are capable of con-
jugating; after many more fissions, the cells grow old, losing their power
of conjugating and showing other signs of senescence; and they finally
die. If conjugation occurs during the period of maturity, the conjugants
are rejuvenated and the cycle is renewed. In some ciliates, such as Uro-
leptus mobilis, investigated by Calkins (1920), this Maupasian life
cycle is clearly shown. In Paramecium, however, there are striking spe-
cific and racial differences in presumably so fundamental a matter as the
life cycle.

Many races of P. aurelia (Sonneborn 1937, 1938a) show a definite
period of immaturity: during the first week or two after conjugation,
cultures do not give the mating reaction and cannot conjugate. In a few
more days, the power of conjugating rapidly develops to full strength,
inaugurating a period of maturity. But the organisms remain mature
indefinitely; no period of senescence appears. Only for a day or so during
the periodically recurring processes of nuclear reorganization, are they
unable to conjugate. As soon as reorganization is completed, the mating
reaction reappears in full strength. Why the reorganized cells fail to
begin again with a period of immaturity, as they do after conjugation,
is at present a puzzling and probably a significant fact.

Other races of P. aurelia (Sonneborn, 1938a) not only lack a period
of senescence, but also a period of immaturity: they are able to conjugate
immediately after conjugation. Eight successive conjugations have been
obtained in a period of seventeen days (Sonneborn, 1936). As the

700 SEXUALITY

process of conjugation and nuclear reconstitution require one day, there
could have been only about a day between successive conjugations, a
period in which at most only three or four fissions could take place.

In P. bursaria, Jennings (1939a, 1939b) reports a regularly occurring
period of immaturity. In group I it lasts for from two weeks to several
months; in group II all clones under investigation were still immature at
last reports, eight months after their origin at conjugation. Periods of
immaturity have also been found regularly in P. caudatum by Gilman
(1939) and in Ezplotes by Kimball (1939c). In none of these species
has there as yet been any report that maturity is followed by a period of
senescence, with loss of ability to conjugate. Many of Jennings’s clones
of P. bursaria have been mature for over two years, without loss of sexual
vigor; and in this species endomixis is so rare as scarcely to account for
the results.

Thus age sometimes is and sometimes is not a factor in determining
conjugation; the Maupasian life cycle is not an invariable feature of
ciliate life. Immaturity may be absent, short, or long; maturity may be
coextensive with life, or it may be simply preceded by a period of im-
maturity; or it may be delimited on either side by periods of immaturity
and senescence.

THE ROLE OF ENVIRONMENTAL CONDITIONS IN DETERMINING CON-
JUGATION

Maupas (1889) recognized the importance of environmental condi-
tions in determining conjugation, and most subsequent workers have
been in more or less agreement on this point; but some have carried this
view to the extreme of ascribing to environmental conditions alone the
determination of conjugation. The preceding account has shown that
this cannot always be true, for hereditary and developmental internal
factors have been demonstrated as playing a decisive role in many of the
races and species. Nevertheless, environmental conditions, such as nutti-
tion, temperature, and light, do have marked limiting effects on the
occurrence of conjugation.

In P. aurelia (Sonneborn, 1938a), the mating reaction does not take
place in cultures that are either overfed or completely starved. Inter-
mediate nutritive conditions are most favorable for its occurrence. More-

SEXUALITY 701

over, the cultural conditions must be good in other respects: when dele-
terious bacteria or other unfavorable conditions injure the paramecia,
the mating reaction is weak or lacking.

In variety 1, mating types I and II will react sexually at any tempera-
ture within the range examined, 9° C. to 32° C.; but mating types IH
and IV of variety 2 will not react above 24° C. and types V and VI
of variety 3 not above 27° C.

Similar differences appear in the time of day in which reactions will
occur: variety 1 will react at any time; but variety 2 reacts only between
6 P.M. and 7 A.M., while variety 3 reacts only between 1 A.M. and 1 P.M.
As might be supposed, this periodicity is an effect of the daily alternation
of light and dark. In variety 3, sexual reactivity has been completely
suppressed by exposing the organisms to continuous illumination, and
they have been made to react at all hours by keeping them in continuous
darkness. These effects have been shown (Sonneborn, 1938a) to be due
to the suppression of reactivity by light, not to its stimulation by dark-
ness. Similar diurnal periodicities in mating occur in P. bursaria (Jen-
nings, 1938a, 1939a, 1939b).

The environmental conditions thus determine whether conjugation
will occur when the proper mating types are brought together. Ordinarily
the mating types themselves are hereditary characters (see Chapter XV,
“Inheritance in Protozoa,” Jennings); but in the exceptional unstable
caryonides studied by Kimball (1939b), genetic determination seems
excluded, for the mating types change repeatedly during vegetative re-
production. Here environmental conditions probably determine even the
mating types themselves, and similar relations may be the rule, instead
of the exception, in species in which conjugation within a caryonide oc-
curs regularly. Thus investigations of possible genetic, developmental,
and environmental factors determining conjugation show all to be in-
volved, as might have been expected.

SEX DIFFERENCES BETWEEN GAMETE NUCLEI

Careful observations on the form and behavior of the gamete nuclei
during conjugation were made by Maupas (1889) and by R. Hertwig
(1889). These and nearly all subsequent investigators have agreed that
in most ciliates the two gamete nuclei formed in each conjugant differ in

702 SEXUALITY

behavior: one, the stationary gamete nucleus, remains in the conjugant
that produces it; the other, the migratory gamete nucleus, passes into the
mate and unites with the stationary gamete nucleus located in that ani-
mal. As a rule, the gamete nuclei are morphologically indistinguishable;
but in some species differences in size and form have been reported. The
most extreme example of morphologically different gamete nuclei is in
Cycloposthium (Dogiel, 1925). The spindle resulting in the formation
of the gamete nuclei is heteropolar: one pole, destined to produce the

Figure 167. Conjugation in Cycloposthinm bipalmatum, showing the sperm-like mi-
gratory pronuclei differing from the spherical stationary pronuclei. (After Dogiel. )

stationary gamete nucleus, is larger and rounder than the other smaller
and more pointed pole, destined to yield the migratory gamete nucleus.
The latter arises from the anterior pole of the spindle and develops a
long tail-like appendage at the proximal end, and a small, pointed distal
end, functional in piercing the cuticle in its passage from one mate into
the other (Fig. 167). In other ciliates, lesser differences between the
gamete nuclei have been observed: slight differences in size in Didinium
(Prandtl, 1906), in Paramecium caudatum (Calkins and Cull, 1907),
and in P. multimicronucleatum (Landis, 1925). Calkins and Cull
(1907) concluded that the two gamete nuclei in P. caudatum differ in
their chromatin content, as a consequence of transverse chromosomal
division at the nuclear division which gives rise to them. In most ciliates,
however, no morphological differences between the two gamete nuclei
have been observed.

SEXUALITY 703

SIGNIFICANCE OF THE DIVERSITIES BETWEEN CONJUGANTS AND BE-
TWEEN GAMETE NUCLEI

There is great diversity of opinion regarding the significance of the
observed differences between conjugants and between gamete nuclet.
This is due partly to the variety and complexity of the observed phenom-
ena, and partly to confusion as to the meaning of the concepts employed,
particularly concepts developed primarily with relation to phenomena in
higher organisms. An attempt will be made to summarize the more
prominent views concerning the main types of observed relations and
to set forth some general considerations concerning them.

In some ciliates, the Vorticellidae and a few others, in which the
conjugants are always morphologically diverse and the gamete nuclei
morphologically alike, with fertilization of only the larger conjugant, it
is usually agreed that the conjugants differ sexually. Further, the gamete
nuclei in each conjugant are sometimes said to be of the same sex as the
conjugant. Some authors hold that the sexes here are female and male.

In Chilodonella (see p. 689), in which the conjugants become
morphologically diverse during conjugation and fertilization 1s recip-
rocal, Enriques (1908) concluded that although both conjugants were
functionally hermaphroditic (producing two sexually diverse gamete
nuclei), the conjugants also showed a partial, incompletely developed
sex diversity, for which he devised the term ‘‘hemisexes.’”’

In Opisthotrichum (see p. 689) fertilization is also reciprocal,
involving gamete nuclei with strongly marked morphological sex dif-
ferences. Nevertheless, in a majority of the conjugant pairs, the two
members differ in size. When the two are alike in size, both are large.
Dogiel (1925) interprets these facts as follows: the conjugants are all
functionally hermaphroditic, each producing male and female gamete
nuclei; but the conjugants also show the beginnings of sexual differentia-
tion, the small ones being more differentiated, for they can mate only
with large individuals, while the large ones can mate either with small or
large, though more commonly with the former. The small conjugants are
viewed as considerably differentiated toward the male condition, in spite
of their functional hermaphroditism.

In Paramecium and Euplotes, fertilization is reciprocal, the gamete
nuclei show little or no morphological difference, and the conjugants

704 SEXUALIDY

show no significant morphological differences. Yet the conjugants are
regularly differentiated physiologically into diverse mating types. The
gamete nuclei in these and similar forms are often considered to be
sexually diverse; frequently the migratory gamete nucleus is viewed as
male, the stationary one as female. This introduces the same difficulty
as in Chilodonella and O pisthotrichum. How reconcile sex differences
between the gamete nuclei with the differences between the ‘“‘hermaph:
roditic’” conjugants? Jennings (1939a) inclines toward interpreting
the mating types as manifesting phenomena of self-sterility, or incom-
patibility, of the kind found in certain higher plants (Stout, 1938) and
animals (Morgan, 1938), in the sense that the single clone or caryonide,
like the single self-sterile plant, ordinarily does not fertilize itself. Jen-
nings points out the features in which the two sets of phenomena are
different, as well as those in which they are alike. More recently, Sonne-
born ( 1939¢) has shown that the periodic nuclear reorganization in variety
1 of P. aurelia is regularly a self-fertilization, as maintained by Diller
(1936). Consequently, P. avrela is not self-sterile, but regularly self-
fertile. The failure of individuals of the same mating type to conjugate
with each other is thus not related to any incompatibility between their
gametes, for such appears not to exist. It seems, therefore, more compar-
able to the failure of two individuals of the same sex to unite in copula-
tion. In higher organisms, self-sterility serves to prevent self-fertilization;
in P. aurelia the mating types serve to bring together for cross-fertilization
diverse sex types, each of which regularly undergoes self-fertilization.
The present author, therefore, concludes that the mating-type phenomena
are not properly to be viewed as self-sterility or incompatibility. If by
sexual differentiation is meant the differentiation of the individuals of a
species into diverse kinds, so that mating occurs regularly between dif-
ferent kinds, not between two of the same kind, then the mating types
of Paramecium are diverse sexes. As in O pisthotrichum, the sex differ-
ences between the conjugants are of a different kind from those existing
between the gamete nuclei: one serves to bring together the mates, the
other to bring together their gamete nuclei.

Multiple sex systems, such as those in P. bursaria and Explotes, offer
serious difficulties to those who, like Hartmann (1929), hold there can
be but two sexes. Whether or not one agrees with this contention, the

SEXUALITY 705

work of Moewus (1939a) on Chlamydomonas shows how what appears,
through biological analysis, to be a multiple sex system may be reduced,
through chemical analysis, to a fundamentally dual system. Further in-
vestigation is, of course, required to ascertain whether the multiple sex
systems in ciliates are, in fact, similar in this respect to the system in
Chlamydomonas.

A number of the interpretations of sex relations in ciliates employ
the concepts male and female, as set forth above. Many authors follow
Hartmann (1929), who holds, as has been pointed out on page 678,
that sex differences, wherever found, are always male and female.
The characters by which the female is ordinarily recognized are larger
size, lesser activity, greater storage of nutritive reserves, and egg-like
form; and the male by the corresponding opposed characters. In attempt-
ing to apply these views, however, numerous difficulties are encountered.
In the Vorticellidae, both gamete nuclei in the microconjugant are held
to be male; yet only one of them shows the “male” character of activity
by migrating into the macroconjugant. In O pisthotrichum, the migratory
gamete nucleus has the form of a sperm, but it has the “female” char-
acter of much greater size than the stationary gamete nucleus. The diffi-
culty of using size as an index of femaleness is clearly shown in the work
of Satina and Blakeslee (1930) on certain bread molds. In a number of
strains, two sexes were observed and found to be the same in all strains.
One sex was distinctly larger than the other in each strain, yet the larger
sex in one strain was shown to be identical with the small sex in others.
Geitler (1932) found similar difficulties in identifying the sexes in
diatoms by their activity. These and other difficulties have led Kniep
(1928), Mainx (1933), and others to abandon the concepts of male
and female in unicellular organisms and to view sexual union as brought
about by copulation-conditioning factors, some of which operate to bring
together the cells, others the nuclei. In the present state of knowledge,
this point of view appears to be preferable to one that appeals to such
abstract, ill-defined, and confusing concepts as fundamental maleness
and femaleness.

From this point of view, the conflicts between sexual differentiation
in the gamete nuclei and sexual differentiation in the conjugant indi-
viduals present far less difficulty than from the point of view which

706 SEXUALITY,

requires identification of all sex differences with male and female. There
may be two kinds of copulation-conditioning factors: one functioning in
bringing together the cells, the other in bringing together their nuclei.
In Chlamydomonas and the Vorticellidae, the two kinds of nuclear fac-
tors operate in different kinds of cells; in Paramecium and most other
ciliates, both kinds of nuclear factors operate in each of the kinds of
cells. Thus by abandoning the pure assumption that sex differences,
wherever found, must always be fundamentally the same (male and
female), the conflict between sex differences in nuclei and sex differences
in cells disappears.

LITERATURE CITED

Calkins, G. N. 1920. Uroleptus mobilis Engelm. U1. A study in vitality.

J. exp. Zool., 34: 449-70.

1926. The Biology of the Protozoa. Philadelphia and New York.

Calkins, G. N., and S. W. Cull. 1907. The conjugation of Paramecium
aurelia (caudatum). Arch. Protistenk., 10: 375-415.

De Garis, C. F. 1935. Heritable effects of conjugation between free individuals
and double monsters in diverse races of Paramecium caudatum. J. exp.
Zool., 71: 209-56.

Diller, W. F. 1936. Nuclear reorganization processes in Paramecium aurelia,
with descriptions of autogamy and “hemixis.” J. Morph., 59: 11-68.

Doflein, F. 1907. Beobachtungen und Ideen tiber die Konjugation der In-
fusorien. S. B. Ges. Morph. Miinchen, 23: 107-14.

Dogiel, V. 1925. Die Geschlechtsprozesse bei Infusorien (speziell bei den
Ophryoscoleciden) , neue Tatsachen und theoretische Erwagungen. Arch.
Protistenk., 50: 283-442.

Enriques, P. 1908. Die Conjugation und sexuelle Differenzierung der In-
fusorien. Zweite Abhandlung. Wiederconjugante und Hemisexe bei
Chilodon. Arch. Protistenk., 12: 213-76.

Finley, H. E. 1939. Sexual differentiation in Vorticella microstoma. J. exp.
Zool., 81: 209-29.

—— 1939b, Further observations upon sexual differentiation in Vorticella
microstoma. Anat. Rec., 75, (suppl.), p. 85.

Geitler, L. 1932. Der Formwechsel der pennaten Diatomeen (Kieselalgen).
Arch. Protistenk., 78: 1-226.

Giese, A. C. 1938. Race and conjugation of Paramecium. Physiol. Zoél., 11:
326-32.

— 1939. Studies on conjugation in Paramecium multimicronucleatum.
Amer. Nat., 73: 432-44.

Giese, A. C., and M. A. Arkoosh. 1939. Tests for sexual differentiation in

SEXUALITY 707

Paramecium multimicronucleatum and Paramecium caudatum. Physiol.
Zodl., 12: 70-75.

Gilman, L. C. 1939. Mating types in Paramecium caudatum. Amer. Nat., 73:
445-50.

Hartmann, M. 1929. Verteilung, Bestimmung und Vererbung des Geschlechtes
bei den Protisten und Thallophyten. Handb. d. Vererbungswiss. II.
—— 1932. Neue Ergebnisse zum Befruchtungs-und Sexualitatsproblem.
(Nach Untersuchungen von M. Hartmann, J. Hammerling und F.

Moewus.) Naturwissenschaften, 20: 567-73.

—— 1934. Beitrige zur Sexualititstheorie. Mit besonderer Beriicksichtigung
neuer Ergebnisse von Fr. Moewus. S. B. preuss. Akad. Wiss., Phys.
Math. KI., 379-400.

Hertwig, R. 1889. Uber die Conjugationen der Infusorien. Abh. bayer. Akad.
Wiss. Ll Kile ya toieoa2e

Jennings, H. S. 1911. Assortative mating, variability and inheritance of size
in the conjugation of Paramecium. J. exp. Zool., 11: 1-134.

—— 1938a. Sex reaction types and their interrelations in Paramecium
bursaria. 1. Proc. Nat: Acad: Sci. Wash., 24: 112-17.

—— 1938b. Sex reaction types and their interrelations in Paramecium
bursaria, I. Clones collected from natural habitats. Proc. Nat. Acad. Sci.,
24511 17-20;

—— 1939a. Genetics of Paramecium bursaria. I. Mating types and groups,
their interrelations and distribution; mating behavior and self sterility.
Genetics, 24: 202-33.

—— 1939b. Paramecium bursaria: mating types and groups, mating behavior,
self sterility ; their development and inheritance. Amer. Nat., 73: 414-31.

Jennings, H. S., and K. S. Lashley. 1913a. Biparental inheritance and the
question of sexuality in Paramecium. J. exp. Zool., 14: 393-466.

—— 1913b. Biparental inheritance of size in Paramecium. J. exp. Zool., 15:
193-99.

Jollos, V. 1926. Untersuchungen uber die Sexualitatsverhaltnisse von
Dasycladus clavaeformis. Biol. Zbl., 46: 279-95.

Kimball, R. F. 1937. The inheritance of sex at endomixis in Paramecium
auvelia. Proc. Nat. Acad. Sci. Wash., 23: 469-74.

—— 1939a. A delayed change of phenotype following a change of genotype
in Paramecium aurelia. Genetics, 24: 49-58.

-—— 1939b. Change of mating type during vegetative reproduction in

Paramecium aurelia. J. exp. Zool., 81: 165-79.

1939c. Mating types in Euplotes, Amer. Nat., 73: 451-56.

Kniep, H. 1928. Die Sexualitat der niederen Pflanzen. Jena.

Landis, E. M. 1925. Conjugation of Paramecium multimicronucleata, Powers
and Mitchell. J. Morph., 40: 111-67.

Mainx, F. 1933. Die Sexualitat als Problem der Genetik. Jena.

708 SEXUALITY;

Maupas, E. 1889. La Rajeunissement karyogamique chez les ciliés. Arch. zool.
exp.) gén. (2), 72 149-517.

Moewus, F. 1933. Untersuchungen uber die Sexualitét und Entwicklung von
Chlorophyceen. Arch. Protistenk., 80: 469-526.

—— 1934. Uber Subheterdzie bei Chlamydomonas eugametos. Arch.
Protistenk., 83: 98-109.

—— 1935a. Uber den Einfluss ausserer Faktoren auf die Bestimmung des
Geschlechts bei Protosiphon. Biol. Zbl., 55: 293-309.

—— 1935b. Die Vererbung des Geschlechts bei verschiedenen Rassen von
Protosiphon botryoides. Arch. Protistenk., 86: 1-57.

—— 1935c. Uber die Vererbung des Geschlechtes bei Polytoma Pascheri und
bet Polytoma uvella. Z. indukt. Abstamm. u. VererbLehre, 69: 376-417.

—— 1936. Faktorenaustausch, insbesondere der Realisatoren bei Chlamy-
domonas-Kreuzungen. Ber. dtsh. bot. Ges., 54: 45-57.

—— 1937a. Methodik und Nachtrige zu den Kreuzungen zwischen
Polytoma-Arten und zwischen Protosiphon-Rassen. Z. f. indukt.
Abstamm.- u. VererbLehre, 73: 63-107.

——— 1937b. Die allgemeinen Grundlagen der Sexualitat. Biologe, 6: 145-51.

—— 1938a. Vererbung des Geschlechts bei Chlamydomonas eugametos und
verwandten Arten. Biol. Zbl., 58: 516-36.

——— 1938b. Carotinoide als Sexualstoffe von Algen. Jb. wiss. Bot., 86:
753-03.

—— 1939a. Untersuchungen uber die relative Sexualitét von Algen. Biol.
ZbI., 59: 40-58.

—— 1939b. Carotinoide als Sexualstoffe von Algen. Naturwissenschaften,
2297 OAs

—— 1939c. Uber die Chemotaxis von Algengameten. Arch. Protistenk., 92:
485-526.

—— 1940. Carotinoid. Derivate als beschlecktsbestimmende Stoffe von Algen
Biol. Zbl., 60: 143-66.

Morgan, T. H. 1938. The genetic and physiological problems of self-sterility
in Crona. I and I. J. exp. Zool., 78: 271-334.

Mulsow, W. 1913. Die Conjugation von Stentor coeruleus und Stentor
polymorphus. Arch. Protistenk., 28: 363-88.

Noland, L. E. 1927. Conjugation in the ciliate Metopus sigmoides. J. Morph.,
44: 341-61,

Pascher, A. 1931. Uber Gruppenbildung und ‘‘Geschlechtswechsel’” bei den
Gameten einer Chlamydomonadine (Chlamydomonas paupera). Studien
und Beobachtungen uber die geschlechtliche Fortpflanzung und den
Generationswechsel der Griinalgen. I. Jb. wiss. Bot., 75: 551-80.

Philip, V., and J. B. S. Haldane. 1939. Relative sexuality in unicellular Algae.
Nature, 143: 334.

Prandtl, H. 1906. Die Konjugation von Didinium nasutum, O. F. M. Atch.
Protistenk., 7: 229-58.

SEXUALITY 709

Pringsheim, E. G., and K. Ondracek. 1939. Untersuchungen uber die
Geschlechtsvorginge bei Polytoma. Beih. bot. Zbl., 59A: 117-72.

Satina, S., and A. F. Blakeslee. 1930. Imperfect sexual reactions in homothallic
and heterothallic Mucors. Bot. Gaz., 90 (3): 299-311.

Sonneborn, T. M. 1936. Factors determining conjugation in Paramecium
aurelia. 1. The cyclical factor: the recency of nuclear reorganization.
Genetics, 21: 503-14.

— 1937. Sex, sex inheritance and sex determination in Paramecium
aurelia. Proc. Nat. Acad. Sci. Wash., 23: 378-85.

— 1938a. Mating types in Paramecium aurelia: diverse conditions for
mating in different stocks; occurrence, number and interrelations of the
types: Proc; Amer: Phil Soe, 79:41 1-34.

—— 1938b. Mating types, toxic interactions and heredity in Paramecium
aurelia, Science, 88: 503.

—— 1939a. Sexuality and related problems in Paramecium. Coll. Net, 14:
77-84.

—— 1939b. Paramecium aurelia: mating types and groups; lethal inter-
actions; determination and inheritance. Amer. Nat. 73: 390-413.
— 1939c. Genetic evidence of autogamy in Paramecium aurelia. Anat.

Rec.) 7/5; (suppl) capes):

Sonneborn, T. M., and R. S. Lynch. 1934. Hybridization and segregation in
Paramecium aurelia. J. exp. Zool., 67: 1-72.

Stout, A. B. 1938. The genetics of incompatibilities in homomorphic flowering
plants. Bot. Rev., 4: 275-369.

Woodruff, L. L., and R. Erdmann. 1914. A normal periodic nuclear reorgani-
zation process without cell fusion in Paramecium. J. exp. Zool., 17:
425-518.

Zweibaum, J. 1921. Richerche sperimentali sulla conjugazione degli Infusori.
II. Influenza della conjugazione sulla produzione dei materiali di riserva
nel Paramecium caudatum. Arch, Protistenk., 44: 375-96.

CHAPTER XV

INHERITANCE IN PROTOZOA

H. S. JENNINGS

IN His Genetics of the Protozoa (1929), the author has reviewed some-
what fully the investigations and literature on inheritance in Protozoa,
up to 1929. No attempt is made to repeat here these detailed reviews;
the plan is rather to summarize the present state of knowledge on the
subject. Very great advances have been made since 1929, particularly in
the knowledge of biparental inheritance, largely through the work of
Moewus (1932-38).

The question dealt with in the study of inheritance is: To what extent
and how are the constitutions and characteristics of later genera-
tions affected by the constitutions of their ancestors, particularly by the
constitutions of the*immediate parents? Certain subordinate questions
arise in connection with this: To what extent and how are characteristics
affected by environmental conditions? What are the relations between
environmental modifications and genetic constitution?

By genetic constitution is meant the constitution insofar as it affects
descendants. The genetic constitution is known from studies of multi-
cellular organisms to be embodied in certain genetic materials. These
are, mainly or entirely, found in the chromosomes. General genetics has
shown (1) that in the chromosomes there are great numbers of diverse
genetic materials (known commonly as genes or factors), having dif-
ferent effects on development and characteristics; and (2) that genetic
materials are transferred bodily from parents to offspring.

In the present account the term ‘‘factors’”’ will usually be employed in
place of the term “genes,” since the latter has acquired, of late, certain
doubtful theoretical implications.

Genetic materials have two essential properties: (1) the genetic ma-
terials received from parents affect the development and characteristics
of the descendants; (2) the many different kinds of genetic materials

INHERITANCE 711

(genes or factors) reproduce themselves true to type, in development
and reproduction. Each kind of genetic material assimilates, producing
more material of its own type, and each unit of material, or gene, pro-
duces at division new units like itself.

The Protozoa have chromosomes that are similar to those in other
organisms (see Chapter XII). One question that arises in protozoan
genetics is this: Are there in the Protozoa other genetic materials in ad-
dition to those in the chromosomes, having the two essential properties
just mentioned?

TYPES OF REPRODUCTION AND INHERITANCE

In the Protozoa, as in some other organisms, there are two main types
of relation of offspring to parents:

1. Uniparental reproduction; offspring arise from a single parent, as
in the various types of vegetative reproduction.

2. Biparental reproduction; offspring are formed from the combined
parts of two parents, as in sexual reproduction in the Protozoa by copu-
lation, conjugation, and the like—followed by division.

The two kinds of reproduction differ fundamentally in their relation
to the genetic constitution, or genetic materials (chromosomes and their
genes). In uniparental reproduction, typically each of the genetic mate-
rials of the parent is divided and duplicated, so that the genetic consti-
tutions of offspring are like those of their parents. In biparental repro-
duction, the complex of genetic materials present in each of the two
parents is taken apart, and a new combination is made from parts of
these. The genetic materials of the offspring are a new combination of
those of the two parents.

In consequence of these differences, uniparental and biparental repro-
duction give very different consequences in inheritance. The two will
therefore be dealt with separately.

INHERITANCE IN UNIPARENTAL REPRODUCTION
MATERIAL PROCESSES

Details as to the material processes in uniparental reproduction are
dealt with in other chapters (see Chapters XIII and XIV). The essential
features, for genetics, are that the nuclei divide, each chromosome di-

712 INHERITANCE

vides, each gene divides—one product from each going to each of the
two offspring. In the ciliate Infusoria, the macronucleus not only divides
but is in many cases reorganized (see Chapter XIII). The cytoplasmic
body divides and is to a great extent (or entirely) reorganized. The
general upshot 1s that the constitution of nucleus and cytoplasm is typi-
cally the same in the offspring as in the parent (exceptional conditions
are dealt with in later pages).

INHERITANCE OF CHARACTERISTICS

Clones—All the individuals produced by uniparental reproduction
from a single individual are known collectively as a clone. The general
rule for inheritance in uniparental reproduction {s that all members of
the clone are alike in genetic constitution and in inherited characteristics.
That is, the new individuals (clone) produced from a single parent are
like the parent and like one another in their characteristics, structural and
physiological. Taken together, they form the equivalent of a set of iden-
tical twins.

There are numerous exceptions to this rule of the genetic identity of
parent and offspring in uniparental reproduction, and these are among
the most important and interesting phenomena of genetics. They are
dealt with fully on later pages. But the relation of identity of genetic con-
stitution in parent and offspring holds for perhaps 99.9 percent of all
cases; it is the most striking feature of uniparental reproduction.

Certain manifestations of this principle of identity in genetic consti-
tution between parent and offspring require special consideration:

1. Biotypes. In all Protozoa fully studied, any species consists of a
great number of diverse biotypes—races differing in inherited character-
istics. The different biotypes may differ in size, form, structure, and
physiology (rate of multiplication and the like). Such diverse biotypes
in Paramecium, Difflugia, Arcella, and other Protozoa are described and
illustrated in the present author’s Genetics of the Protozoa (1929).

When individuals of diverse biotypes reproduce uniparentally, as by
fission, the general rule is that each biotype retains its characteristics.
The offspring are like the parents in all conspicuous respects. Thus all
members of a single clone belong to the same biotype and have the same
inherited characteristics. In biotypes of large individuals, each individual

INHERITANCE FAB

produces a clone of large individuals; biotypes of small individuals give
clones with small individuals; rapidly multiplying biotypes produce
rapidly multiplying descendants; and so on. Such inheritance is shown
with respect to vigor or weakness, to resistance and lack of resistance,
and to structural and physiological characteristics of all sorts.

Members of a given biotype, having the same genetic constitution, may
differ in ways induced by different environments, or resulting from dif-
ferent periods in the life of the individual. Such differences are, as a
rule, not inherited in uniparental reproduction (exceptions are dealt with
later). The main classes of non-heritable differences among the indi-
viduals of a single biotype are: age differences; nutritional differences,
and environmental diversities resulting from differences in temperature,
chemical conditions, and the like.

In addition to these, there are in some species non-heritable diversities
of unknown origin between members of the same biotype, the same
clone. Thus in Di fflagia corona, which has a silicious shell bearing spines,
there are within the same clone differences as to the number and size of
the spines borne by the shell. In this case the differences arise at repro-
duction, presumably under the influence of environmental diversities.
They follow the same rule as known environmental differences; they are
not as a rule inherited. If parents with many spines produce descendants,
the mean number of spines in these descendants is the same as in the
descendants of individuals of the same clone that have few spines (excep-
tions noted in later pages).

Thus, as a rule, racial or inherited characters are not altered in unt-
parental reproduction. This is the most striking and obvious feature of
such reproduction. Yet it does not hold absolutely; there are important
limitations and exceptions to this rule. A large proportion of our dis-
cussion of uniparental inheritance will deal with these exceptions. They
are taken up next.

CHANGES IN INHERITED CHARACTERS IN UNIPARENTAL REPRODUCTION

In a number of different categories of cases, inherited differences arise
during uniparental reproduction, so that the members of a single clone
are not all alike in characteristics that are inherited in vegetative repro-
duction. Some of these phenomena are of great interest for general
genetics. They may be classified in various ways.

714 INHERITANCE

AGE CHANGES: SEXUAL IMMATURITY AND MATURITY

Beginning with an individual that has recently conjugated, if the lines
of descent by vegetative reproduction are followed for great numbers
of generations, certain characteristics of the individuals are found gradu-
ally to alter. The offspring produced at different periods differ. In Uro-
leptus mobilis (Calkins, 1919) or in Paramecium bursaria (Jennings,
1939), the individuals are at first sexually immature; they do not con-
jugate under any conditions. This continues for many generations of
vegetative multiplication. The offspring during this period are like the
parents in this respect.

But after many generations have passed, the descendants gradually
become sexually mature. They now conjugate when mixed with indi-
viduals of different mating type. These descendants are thus different
in this respect from their earlier ancestors. In this period their own off-
spring inherit from them the mature condition.

In Paramecium bursaria, and presumably in other species, the mature
condition comes on slowly and gradually. There is for many generations
partial maturity, in which the tendency to conjugate is but slight. The
tendency becomes stronger as generations pass, until full maturity is
reached. The period of full maturity lasts for a great number of genera-
tions, during which the mature condition is inherited in vegetative repro-
duction. Such periods of immaturity and maturity were described fifty
years ago by Maupas (1889) for a number of species of ciliates. In some
species, however, they hardly exist, or the period of immaturity if it
occurs at all is very short. Such is the situation in P. awrelia (Sonne-
born, 1936).

At a late period in the life history, in some species the individuals are
found to become less vigorous as generations pass. They multiply less
rapidly, become “‘depressed,’”’ degenerate. Whether this is an additional
period in the life history, beyond the periods of immaturity and ma-
turity; whether, in other words, it is an age change, constituting a period
of senescence and final senility, or whether it is only a degenerate condi-
tion arising in consequence of living long under unfavorable conditions,
appears as yet unsettled. This period of decline will therefore be con-
sidered in the next section.

In some of the Protozoa, particularly among parasitic forms, in dif-

INHERITANCE VALS,

ferent periods of the life history there are very great differences in form,
structure, and physiology, constituting an “alternation of generations.”
Each condition is transmitted from parent to offspring for many genera-
tions, yet each in time transforms into a later condition.

All these phenomena are commonly thought of as matters of “life
history,” rather than of inheritance. Yet they represent fundamental fea-
tures in those relations of successive generations that are called inherit-
ance. The single cell, reproducing vegetatively, produces a great number
of other free cells that are like itself in their special peculiarities. Later
the character of the cells changes; and again the resulting condition 1s
for a long period inherited in vegetative reproduction. In these respects
the phenomena are like modifications resulting from environmental
action, as shown in later paragraphs.

Are the diverse conditions that are vegetatively inherited in different
periods—such as sexual immaturity and maturity—the result of changes
in chromosomal materials, or changes in the cytoplasm? Dobell (1924)
shows that the chromosomes do not visibly change in the series of
diverse forms passed through in the life history of certain haploid
Sporozoa; throughout all the changes the same set of chromosomes in
the same number are present. Tartar and Chen (1940) have found that
in the period of sexual maturity, in P. bursaria, parts of the individual
consisting only of cytoplasm react sexually. Neither of these observations
proves conclusively that the chromosomal materials are not altered in the
different periods, but they perhaps make it probable that the different
periods in the life history result rather from such interactions between
chromosomes and cytoplasm as must occur in producing the bodily differ-
entiations of a developing multicellular organism.

Whatever the seat of the different inherited conditions in different
periods of the life history, it is clear that the material on which the
different conditions depend must multiply itself, for long periods remain-
ing true to type. An immature individual contains a certain small amount
of the material on which immaturity depends. In ten generations this
material has multiplied to more than a thousand times its original quan-
tity, still remaining immature. Later, having attained the mature condi-

tion, it again multiplies in that condition to thousands of times its
original quantity.

716 INHERITANCE

INHERITED DEGENERATIVE CHANGES RESULTING FROM UNFAVORABLE
CONDITIONS

In many cases, when ciliate Infusoria are cultivated for long periods
in isolation cultures, in which great numbers of successive generations
are produced, the organisms are found in the later generations to decline
in vigor and vitality. This change is progressive; it becomes greater in
later generations. The vital processes become “‘depressed,” slow, inefh-
cient; in particular the rate of multiplication decreases. In time the ani-
mals become degenerate—abnormal in form and structure, reduced in
size. As an index of this decline in vigor and vitality, the changes in the
rate of multiplication are commonly employed. Graphs of the daily num-
ber of fissions show a curve gradually descending from a high point
at the beginning of the isolation culture, to nearly zero at a later period.
A large number of such graphs, based on the work of many different
investigators on many species, are published in the author’s Genetics
of the Protozoa (1929).

It has been held by many investigators that this decline is a matter
of age; that these graphs are curves of senescence. The earlier periods of
immaturity and maturity were believed to be followed inevitably by a
period of senescence. Whether this is, indeed, true for some species is
still uncertain. But for a number of species it has been shown that the
decline need not and does not occur if the conditions are kept entirely
favorable (a summary of investigations on this matter is found in the
author's Genetics of the Protozoa, 1929). In these latter species, there-
fore, the decline and degeneracy are consequences of life under unfavor-
able conditions.

Thus unfavorable environmental conditions, acting for many succes-
sive generations, cause changes in the characteristics of the individuals,
and cause them to produce in the later periods offspring that differ from
those produced in the earlier periods. In the earlier periods, parents and
offspring are vigorous, multiplying rapidly. Later, under the same
environmental conditions as before, parents and offspring are weak,
nonresistant, multiplying slowly. The effects of the unfavorable environ-
ment become cumulative as generations pass, and in vegetative repro-
duction they are transmitted to the offspring.

The inheritance of the depressed condition is demonstrated in the

INHERITANCE TAG

following way. Individuals from the depressed later generations are
cultivated side by side, under the same conditions, with individuals from
the same clone that have not lived under unfavorable conditions. One
of the two sets—the latter—multiplies rapidly and at a high level of
vitality. But the former set, that has lived under unfavorable conditions,
multiplies slowly, at a low level of vitality, in a degenerate condition.

Thus in these Protozoa we find realized what some have held must
occur in mankind: the production of inherited degeneration, by long-
continued bad living conditions.

Discussion of the nature of these changes will be reserved until other
inherited environmental modifications have been considered.

INHERITED ACCLIMATIZATION AND IMMUNITY

The changes in inherited characters induced by unfavorable environ-
mental conditions are not always degenerative in character. In the uni-
cellular organisms, as in multicellular organisms, long exposure to un-
favorable conditions may result in the production of acclimatization or
immunity. In the Protozoa, after removal from the unfavorable condi-
tions, the acquired acclimatization or immunity is inherited in vegetative
reproduction for many generations. Cases are on record in which such
inheritance continued for many months, including hundreds of vegetative
generations.

But in the course of many generations under the favorable conditions,
with the injurious agent no longer present, the acquired immunity
becomes gradually less marked; it slowly decreases, and, finally, in a
sufficiently long period it is lost. But this may not occur until months
after removal from the immunizing agent, during which time the
acquired immunity is inherited.

Such acquisition of inherited immunity is most extensively known in
parasitic Protozoa and in pathogenic bacteria, since in these organisms
it is of medical importance. Detailed accounts of the knowledge in these
fields will be found in the treatise of Taliaferro (1929).

But acquisition and inheritance of acclimatization or immunity occurs
also in free-living Protozoa. A somewhat detailed review and discussion
of investigations in this field will be found in the author’s Genetics of
the Protozoa (1929). Here only brief summaries of some of the more
important investigations in this field can be presented.

718 INHERITANCE

In a famous investigation by Dallinger (1887), published fifty-two
years ago, three species of flagellates were acclimatized, in seven years,
to a high degree of heat. At first the organisims could not tolerate a
temperature higher than 26° C. If subjected to this temperature for some
time, death occurred. By raising the temperature a half degree at a time,
at the average rate of two degrees a month, the tolerance was in the
seven years raised to 70° C.

When the temperature was raised at any period, usually many of the
individuals died. Others lived and multiplied, replacing those that died.
There was thus a selective action of the heat; the individuals that did
not become acclimatized died.

At certain periods, the cytoplasm of the organisms became filled with
small vacuoles, which lasted for a month or more, then disappeared. The
temperature could not be raised further until the vacuoles had disap-
peared. It has been suggested that this vacuolization is a process of getting
rid of water, since it is known that protoplasm containing little water
can tolerate higher temperatures than protoplasms containing much
water.

In cases of acclimatization to high temperatures, it is obvious that all
parts of the organism must be altered. If any part—nucleus, chromo-
somes, cytoplasm—failed to acquire the increased resistance to heat, the
organisms would die.

Extensive experimental studies in this field were carried on for many
years by Jollos (1913, 1920, 1921). He investigated the acclimatization
of Paramecium caudatum to arsenic (solutions of arsenious acid), to
certain other chemicals, and to high temperatures.

The most extensive work was on acclimatization to solutions of arsenic.
It was found that P. awrel7a could not be acclimatized to arsenic, and that
some biotypes of P. cawdatum were likewise refractory. But in other
biotypes of P. caudatum acclimatization was successfully produced.

In the successful experiments, a method combining selective action
with subjection to gradually increasing concentrations of arsenic was
employed. Using as a standard concentration a one-tenth normal solution
of arsenious acid, the animals were first placed in a very weak concen-
tration, the maximum tolerance at the beginning being about one per-
cent of the standard solution. The organisms were left for a long time
(days or months) in a solution too weak to destroy them. Then the con-

INHERITANCE 719

centration was slightly increased, until many were killed. The few that
survived were restored to a weak solution and allowed to multiply.
Again there was an increase in concentration until most died; then a
restoration to a weak solution. As this continued, it was found that the
animals became able to tolerate higher concentrations. In three or four
months, the toleration was thus increased from about one percent to
2.5 percent of the standard solution. In other cases the tolerance was
raised in several months to 5 or 6 percent.

When the organisms that had thus acquired a higher resistance to
arsenic were restored to water containing no arsenic, the increased resist-
ance was for long periods not lost. Tests at intervals showed that they
still retained the higher tolerance to arsenic. The acquired higher resist-
ance lasted in some cases for eight months or more. As the animals were
reproducing at about the rate of one fission daily, the acquired resistance
was inherited for about 250 generations.

But during this time in water the acquired resistance to arsenic gradu-
ally decreased. The rate of decrease was very slow, so that in such a
case as that mentioned above, the tolerance had not returned to its
original low level until after a period of eight months.

The course of events may be illustrated from the history of Jollos’s
clone A of P. caudatum. In this clone the original maximum tolerance
to arsenic was to 1.1 percent of the standard solution. By cultivation in
gradually increasing concentrations for four months, the maximum toler-
ance was raised to 5 percent. Upon restoration to water containing no
arsenic, the tolerance was, in tests for successive periods after the restora-
tion, as. follows: 6 days, 5 percent; 22 days, 5 percent; 46 days, 4.5
percent; 53 days, 4.7 percent; 60 days, 4.7 percent; 75 days, 4.7 percent;
130 days, 4.5 percent; 144 days, 4.2 percent; 151 days, 4.0 percent;
166 days, 3.0 percent; 183 days, 2.5 percent; 198 .days, 1.25 percent;
255 days, 1.0 percent.

Thus the acquired resistance persisted in a very marked degree for
more than five months, but had been entirely lost at the end of eight
and a half months.

Jollos acclimatized Paramecium also to high temperatures, continuing
the experimental cultures in some cases as long as two and a half years.
The tolerance to high temperatures was increased, and the increased toler-
ance lasted in some cases to six months after removal from the high

720 INHERITANCE

temperatures. As in the case of resistance to arsenic, the acquired toler-
ance slowly decreased and finally disappeared when the organisms were
cultivated at moderate temperatures.

To such long-lasting modifications, inherited for many generations
but finally disappearing, Jollos gave the name Dauermodifikationen. This
designation is much employed, even in languages other than German.
Jollos interprets these modifications as affecting only the cytoplasm, not
the chromosomes; a matter to which we shall turn later. Phenomena of a
similar character have been described by Jollos and others in multicellu-
lar organisms. Hammerling (1929) has published an extensive sum-
mary of what are believed to be Dawermodifikationen in many organisms.

Certain additional features of acclimatization in free-living Protozoa
are brought out in the work of Neuschloss (1919, 1920). He investi-
gated the acclimatization of P. cawdatum to certain chemicals, particularly
to quinine, methylene blue, trypan blue, fuchsin; also to arsenic and anti-
mony. He found that he could induce increased resistance in about a
month by gradually subjecting the animals to increasing concentrations
of the substances.

Neuschloss investigated the nature of the changes in the organisms
in the following way. After acclimatizing the organisms to an injurious
substance, two equal samples of that substance were taken, in concen-
tration somewhat greater than that to which the animals were resistant.
To one of these was added a large number of the acclimatized paramecia,
to the other an equal number of the unacclimatized animals. The animals
were in each case left in the samples until death occurred. After death,
the amount of injurious substance that had been removed by the organ-
isms from the solution was quantitatively determined.

In all cases it was found that the acclimatized animals removed from
solution a much greater proportion of the injurious substance than did
the unacclimatized. In the case of the four organic compounds, the per-
centage removed from the solutions by the acclimatized and the unacclima-
tized animals were as follows:

Injurious Acclimatized Unacclimatized
Substance Animals Animals
Quinine 80.0 4.5
Methylene blue SS) 0.5
Trypan blue 54.0 0.75

Fuchsin 60.0 1.00

INHERITANCE 721

The substances removed from solution were found not to exist in the
bodies of the animals. It appears, therefore, that the acclimatized animals
in some way destroy the injurious organic compounds. Presumably they
produce some secretion that has this effect.

In the case of the inorganic poisons, arsenic and antimony, the acclima-
tized animals were found to have acquired the power to transform the
highly injurious trivalent compounds of the two elements into the rela-
tively harmless pentavalent compounds.

The acclimatization was found to be, as a rule, specific for the substance
to which the animals had been exposed. Acclimatization to one of the
organic substances did not increase resistance to the other three. But in
the case of the related inorganic materials arsenic and antimony, acquire-
ment of increased resistance to one induced acclimatization to the other.

INHERITED ENVIRONMENTAL MODIFICATIONS IN FORM AND STRUCTURE

Reynolds (1923) and Jollos (1924) studied extensively the inherit-
ance of certain abnormal forms of the shell in Arce//a; an extended review
of this work is given in the author’s Genetics of the Protozoa (1929).

The work of Jollos shows that these abnormalities are favored by
certain environmental conditions, and perhaps makes it probable that
they originate as environmental modifications. Inheritance of the abnor-
mal conditions continued for many generations.

By selection, the grades or degrees of abnormality could be changed.
By long selection of the most nearly normal individuals, a stock that was
nearly or quite normal could be produced. According to Jollos, the length
of time required to bring the animals by selection back to a normal con-
dition is proportional to the length of time that the ancestors have lived
under the environmental conditions that favor the abnormality.

When the abnormal stock is allowed to multiply without selection,
the proportion of individuals with but slight degrees of abnormality
increases. Jollos holds that this indicates that the abnormal condition is
essentially transient, a Dauermodifikation, in which the cytoplasmic
tendency to abnormality is in time overcome by the nuclear tendency
toward normality. On the other hand, the higher degrees of abnormality
are harmful to the organism, so that natural selection tends to weed these
out, causing the stock to become less abnormal in later generations. See
the extended presentation of the evidence and the discussion on this

722 INHERITANCE

point in the author’s Genetics of the Protozoa (1929, pp. 261-70).

Moewus (1934b) has made extensive studies of inherited environ-
mental modifications in structural characters in Chlamydomonas de-
baryana. This species is known to occur in many varieties differing
slightly in form, size, and structure; Moewus describes and figures twelve
of these. The different varieties are found in nature; also they occur
under different cultural conditions in the laboratory.

Moewus found that he could transform the different varieties one into
another by the use of different culture media. But the transformation did

TABLE 14: ENVIRONMENTAL MODIFICATIONS, Chlamydomonas debaryana;
RELATION OF THE NUMBER OF Days CULTIVATED IN PEPTONE MEDIUM
TO THE NUMBER OF Days IN SALT-SUGAR MEDIUM REQUIRED TO TRANS-
FORM THE ORGANISMS FROM TYPE 1 TO TYPE 5 (FROM Moewus, 1934b)

Daysonibepione Days in Salt-Sugar Medium
T Required for Transformation
as Type I Teper

28 28
140 49
273 133
441 175
567 231
609 379
644 459
672 531
690 534

not occur at once, on transfer to the different culture medium; on the
contrary, many generations in the new culture medium were necessary to
induce the transformation.

An extremely important relation was observed to hold as to the time
required for the transformation. The longer a stock has remained under
conditions producing a given type, the greater the time and the number
of generations required to transform it to a new type, when placed under
new conditions. An example will illustrate the operation of this principle.

Individuals in peptone culture are of type 1. If transferred to a “‘salt-
sugar” infusion, they transform to type 5. If they have been only a few
generations in the peptone culture, the transformation in the salt-sugar
medium occurs very quickly. But if they have lived for a long time in the
peptone medium, a long time is required for transformation to type 5.

INHERITANCE 723

Table 14 from Moewus shows the number of days required for the
transformation to type 5, in relation to the number of days the organisms
had lived in peptone medium. In interpreting this table, it is important
to keep in mind the fact that reproduction occurs at about the rate of
one generation a day, or more rapidly.

In the last examples of Table 14, it required a year and five months
in their new conditions to induce in the organisms the transformation to
type 5. During this period hundreds of generations were produced. The
longer the organisms remain in peptone culture as type 1, the longer it
requires for the new conditions to transform them to type 5.

Moewus presents many other cases illustrating the same principle.

The different types or varieties found in nature usually require a very
long period of culture in a given medium to induce them to transform
to the type characteristic for that medium. Thus a certain stock 4 was
found in nature as type 10, having no papilla. Placed in peptone culture,
it remains as type 10 for 450 generations, then the organisms begin to
transform to type 1, having a papilla; in time all transform to type 1.
The type 10 was seemingly a Davermodifikation in nature; it transforms
to type 1 only after many generations under artificial conditions. Moewus
describes many other cases in which types found in nature resist, for many
generations, changes due to new conditions, but finally transform into
types characteristic for the medium to which they are transferred. Thus
the indications are, as Moewus points out, that many of the diverse types
(‘varieties’) found in nature are in fact long-lasting modifications

resulting from the long-continued action of certain environmental condi-
tions.

VARIATION AND ITS INHERITANCE OCCURRING WITHOUT OBVIOUS
ACTION OF DIVERSE ENVIRONMENTS

In addition to inherited changes produced by diverse environments,
already described, there occur in some cases during vegetative reproduc-
tion inherited changes, without apparent action of diverse environments.
Such cases possibly differ in principle from those described above, illus-
trating the occurrence of inherited variations that are not brought about
by environmental action. If so, they are of much theoretical interest.
On the other hand, they may involve concealed action of diverse environ-
mental conditions, as urged by Jollos (1934). The phenomena are in

724 INHERITANCE

themselves of interest, illustrating certain methods of action not thus far
described. The author’s studies on Difflugia corona may serve as an
example (Jennings, 1916, 1929).

In D. corona (Fig. 168) the individuals differ greatly in a number of
strongly marked structural features. They have a silicious cell, produced

Figure 168. Difflugia corona. Members of four different clones, showing diversities in
characteristics. (After Jennings, 1916.)

at the time of reproduction. The shell bears conspicuous spines, which
differ greatly in number and in length in the different individuals. The
number of teeth surrounding the mouth differs in different cases. The
size of the shell shows wide variation.

Many diverse biotypes occur in nature, each characterized by a definite
combination of characteristics. Some have few spines, some many. Some
have long spines, others short spines. Some have few teeth, others many
teeth. Some are large, others small.

INHERITANCE #25

Even within a single clone there is variation in characters, though much
less than when different clones are taken into consideration. Different
individuals of the same clone may differ greatly in number and length
of spines, slightly in size of the body, very slightly in the number of
teeth.

In vegetative reproduction, the offspring resemble the parent, but
not completely. The general rule is that parent and offspring are alike
in respect to characters that distinguish different biotypes or races, but
they need not be alike with respect to characters in which different indi-
viduals of a clone are diverse. The type of inheritance will be seen from
examples of the numbers of spines in successive generations in certain
clones:

Clone — > —_1—-6

Clone 22 =v 4
Gloners 6 o— o_o —_4— 5 _7 ©
Clone 4: O0—0—0O—O—1—0—0O—1—0O

Thus clones differ from each other in the fact that some usually have
few spines or none, others a slightly larger number, others a still larger
number—though within each clone there is variation. A given biotype,
or clone, is characterized by a certain average number of spines; another
by a different average number. In respect to the average numbers of
spines, many different biotypes may be distinguished.

When from a single clone individuals differing in number of spines
are allowed to multiply until each has produced many descendants, the
usual result is that the average number of spines in the two sets of
descendants is the same. Thus in clone 1 above, the individual with 2
spines and that with 6 spines would, as a rule, produce descendants
having the same average number of spines. The individual differences
within the clone are usually not inherited; the mean differences between
biotypes are inherited.

To what are due the differences in numbers of spines or in other char-
acteristics, between the different members of the same clone, illustrated
above? The shell is produced complete at the time that reproduction
occurs. The individual about to reproduce buries itself in a mass of
soft debris. The protoplasm swells, projects from the mouth of the

726 INHERITANCE

parental shell, and takes on the form of the protoplasmic body. A mass
of silicious particles, which have earlier been collected within the parent,
passes to the surface of the new protoplasmic body and is there molded
into the final form of the shell of the offspring. Meantime the nucleus
has divided and one of the products passes into the young individual.
Parent and offspring now separate.

During this process are formed the spines in their final form, length,
and number. The parent shell may have four spines, the shell of the
new individual five spines or some other number; there may be differences
in the length of the spines. The differences between the characteristics of
parent and offspring might conceivably be due to slight and obscure
differences in the environmental conditions at the time of reproduction.
Or they might be due to diversities in the internal condition or consti-
tution of the individuals at the time of reproduction, however such
diversities had been produced.

Results of long-continued selection From the results thus far set
forth, it appears that the individual diversities within a clone of D/f-
flugia are not usually or strongly inherited. Is there any tendency what-
ever for such inheritance? In other words, does the difference of condi-
tion or constitution that results in diversity of characters ever or in any
degree persist through vegetative reproduction?

This may be tested by long-continued selective breeding within a
clone. Suppose that the basis of selection is the number of spines. From
a single clone two groups are segregated, one containing only individ-
uals with high numbers of spines, the other only individuals with low
numbers of spines. In the former group are retained only descendants
with high numbers of spines; from the second group only descendants
with low numbers of spines. In this way there are obtained in later gen-
erations from the first group individuals descended for many generations
from ancestors all of which had high numbers of spines, and from the
second group individuals descended for many generations from ancestors
with low numbers of spines. Selective breeding of this kind may be car-
ried on with any of the varying characteristics.

When such selection is practiced for many generations, usually for
considerable periods no difference in the offspring of the two groups is
to be discovered. But as the number of generations becomes greater, an

INHERITANCE T2Y.

effect in the direction of selection appears. The offspring of the group
whose ancestors have had high numbers of spines have a greater average
number of spines than the offspring of the group whose ancestors have
had low numbers of spines.

Such results may be illustrated from the clone 326, in which selective
breeding for high and low numbers of spines was carried on for a long
period (see Table 15).

TABLE 15: EARLY RESULTS OF SELECTION FOR LOW AND HIGH NUMBERS
OF SPINES, Difflugia corona (FROM JENNINGS, 1916)

Low Group HicH Group
Beisod Dies Parental Mean Spines Parental Mean Spines
Spines of Descendants Spines of Descendants
I 36 1-4 5.56 5-9 5.46
2 60 1-4 5.13 6-11 5.19
3 60 I-4 : 6-11 5.46
4 120 ih 5.29 4-11 5.79

In the fourth period there is a distinct difference in the two groups.
To test whether this is significant, the fourth period (four months) was
divided into six successive periods, and the numbers of spines in the
offspring of the two groups determined for each. They were as shown
in Table 16.

TABLE 16: LATER RESULTS OF SELECTION FOR Low AND HIGH NUMBERS
OF SPINES, Difflugia corona; NUMBERS OF SPINES IN THE PARENTS (1-5
IN THE Low Group, 7-11 IN THE HIGH GROUP) WITH THE MEAN Num-
BERS OF SPINES ON THEIR OFFSPRING IN SUCCESSIVE PERIODS (FROM JEN-
NINGS, 1916)

PARENTS
I-§ 7-11
Period Offspring
I 5.59 6.11
3) op) 5.71
3 5.49 6.53
4 5.23 ao
5 Helo} 5.38
6 4.63 5-59

728 INHERITANCE

Thus in each of the six periods of the last four months, the parents
with high numbers of spines produced offspring with higher mean num-
bers of spines than did parents with low numbers of spines, though the
differences were not very great.

Selective breeding of this type was practiced also with relation to the
length of the longest spines. The results here show clearly a phenomenon
of much interest, namely inherited variation with “regression toward the
mean’’ of the biotype as a whole. This appeared also in the results of

TABLE 17: INHERITANCE OF SPINE LENGTH, WITH REGRESSION TOWARD
THE MEAN, Djifflugia corona, CLONE 326; MEASUREMENTS OF SPINE
LENGTHS, IN UNITs OF 4 2/3 MICRONS EACH (FROM JENNINGS, 1916)

Parents Mean Lengths of
Length of Longest Number of Offspring Longest Spine in the
Spines, in Units Offspring, in Units

4-6 21 10.38
7-9 | 162 I1.O1

10-12 | 451 11.85

13-15 367 12.90

16-18 129 14.39

19-21 26 14.34

22-24 15 16.34

25 and above 18 17.06

Mean length for all, 12.54

selective breeding with respect to other characters. The results of selection
for length of spines are given in Table 17. (The spines were measured
in units, each of which was 424 microns. )

Table 17 shows that parents with longer spines produce, on the
average, offspring with longer spines, so that there is a distinct tendency
to inheritance in spine length, even within the single clone.

But another relation is equally evident. Parents that are above the
mean of the biotype (12.54) produce offspring that are above the mean,
but not so much above the mean as ate the selected parents. Parents that
are below the mean produce offspring that are below the mean, but not
so much below the mean as are the selected parents. That is, inheritance
of the parental peculiarities occurs, but always with regression toward the
mean of the biotype. On the average, the offspring diverge from the

INHERITANCE 729

racial mean in the same direction as do the selected parents, but not so
far. Only a small part of the selected parents’ divergence from the mean
is inherited by the offspring.

To what is due such inheritance, with regression toward the racial
mean? The interpretation is not clear, but the most natural conception
appears to be the following. The divergence from the racial mean in the
case of the selected parents is partly a matter of environment, partly a
matter of genetic constitution. The latter is inherited by offspring, the
former not. The fact that a part of the parent’s peculiarity is inherited
shows that variation in the genetic constitution itself occurs in vegetative
reproduction and is inherited. It is, however, in large degree masked by
variations due to environmental conditions, and not inherited.

As a consequence of the inheritance of some portion of the parents’
peculiarities, in time the single clone or biotype may by selective breeding
become differentiated into a number of biotypes, differing slightly in
inherited characters. Five such hereditarily different biotypes of D/fflagia
corona, produced through selective breeding from a single one, are
described and figured in the paper of Jennings (1916).

For such heritable change in the genetic constitution two types of
interpretation may be suggested. On the one hand, the heritable changes
may be due to obscure alterations in environmental conditions, so that
these changes are comparable to those in acclimatization or other Dawer-
modi fikationen. On the other hand, the changes may be attributed to
irregularities occurring in the genetic materials—either ‘“‘mutations”’ or
slight irregularities of distribution—when reproduction occurs. At pres-
ent it appears not possible to decide between these two interpretations.

SUMMARY AND INTERPRETATION

To recapitulate, in Protozoa, a number of different types of inherited
changes may occur during vegetative reproduction, giving rise to indi-
viduals or lines of descent that have different characteristics which are
transmitted to descendants in further vegetative reproduction.

First, there are changes that occur in the course of the normal life
history—changes as to sexual maturity, and, in some species, profound
alterations in structural and physiological characters (“‘alternation of
generations’). Each stage in such a life history includes many successive

730 INHERITANCE

generations, during which the distinctive features of that stage (imma-
turity, maturity, or the like) are transmitted to descendants.

Second, there are inherited degenerative changes, resulting from life
under unfavorable conditions.

Third, there are adaptive changes, fitting the organism to a changed
environment—inherited acclimatization, or immaturity.

Fourth, there are inherited changes in form and structure, apparently
neither adaptive nor degenerative, occurring under the influence of spe-
cific environmental conditions.

Fifth, forming perhaps an additional type, there are inherited varia-
tions in form, size, and other characters, that are not obviously due to
environmental conditions.

Some of these five types fall under Jollo’s concept of Dauermodifi-
kationen: changes produced under the influence of environmental condi-
tions, inherited for many generations after removal from those conditions,
but gradually fading out to final disappearance. This includes the third
and fourth types above, and, according to Jollos’s view, also the fifth.
The first type obviously appears not to fall under this concept, and the
second type is not known to disappear upon restoration to a more favor-
able environment.

Most of thest types of modification may disappear or be altered upon
the occurrence of sexual reproduction. In type 1, the condition of matur-
ity changes at sexual reproduction into one of immaturity. In type 2, the
inherited degenerative changes in many cases disappear at sexual repro-
duction (“‘rejuvenescence through conjugation’’), but it is a notable fact
that in some cases they do not. In type 3, the inherited acclimatization and
immunity likewise often disappear at sexual reproduction, though again
in some cases they do not. In types 4 and 5, the effect of sexual repro-
duction is not known.

These inherited modifications, so far as they can be brought under
the concept of Dauermodifikationen, are held by Jollos to have their
seat in the cytoplasm only, the genetic materials of the chromosomes being
unchanged. This is based in the main on the fact that the inherited
modifications grow less and finally disappear, when the organisms are
restored to environments that do not produce the modifications. A change
in the genetic materials of the chromosomes (“‘mutation’’), it is held,
would be permanent; it would not disappear after many generations in

INHERITANCE on

an altered environment. But a change in the cytoplasm, it is urged, would
in the course of time be overcome and dominated by the unchanged con-
stitution of the nucleus, bringing about a return to the original charac-
teristics—as actually occurs. These inherited modifications are, on this
view, essentially transitory conditions, forming no part of the system of
genuinely inherited characters.

The fact that these modifications usually (though not always) dis-
appear at sexual reproduction is likewise held to be due to the fact that
they have their seat in the cytoplasm, though the logic of this is not
entirely clear. As will be shown later, it is the nucleus, not the cytoplasm,
that is directly changed in constitution at conjugation. The disappearance
at conjugation is seemingly attributed to the profound making over of
the cytoplasm that has been believed to occur at conjugation, producing
rejuvenescence. The long-continued inheritance of the modifications dur-
ing vegetative reproduction is held to be an example of cytoplasmic in-
heritance.

If we are here indeed dealing with cytoplasmic inheritance, this
appears to demonstrate that in the Protozoa the cytoplasm partakes of
the essential features of genetic material. These essential features, as
before set forth are: (1) the fact that the material in question affects
the inherited characteristics, and (2) the fact that this material multiplies
true to type. The environmental modifications are, as we have seen, at
times inherited for more than 200 generations after removal from the
conditions that induced them. In 10 generations, the originally modified
cytoplasm has been diluted to one-thousandth of its volume, in any indi-
vidual; in 20 generations, to less than one-millionth of its volume. The
rest of the cytoplasm of the individual is a product of cytoplasmic growth.
Yet the environmental modification still persists. If the cytoplasm 1s the
seat of the modification, the modified cytoplasm must reproduce itself
in its modified condition at fission; otherwise its effect would have dis-
appeared under the great dilution that it has undergone.

On the question as to whether cytoplasmic inheritance indeed occurs
in these organisms, evidence will be presented in the section on biparental
reproduction. There also a method will be indicated by which it may be
determined whether the seat of these modifications is in the cytoplasm
or in the nucleus.

ere INHERITANCE

INHERITANCE IN BIPARENTAL REPRODUCTION

Biparental or sexual reproduction includes the processes that lead to
the formation of new individuals from the united parts of two earlier
individuals. Biparental reproduction in the Protozoa occurs in two main
types, known respectively as copulation and conjugation. Copulation is
characteristic of haploid species; in it two haploid individuals unite com-
pletely to form a diploid zygote, which later divides with reduction, to
form haploid individuals again. This method occurs in the Flagellata.
In conjugation two diploid individuals exchange halves of their nuclei,
including a haploid set of chromosomes; then the two separate and each
continues thereafter to multiply by fission. This is the characteristic
method in the Ciliata.

BIPARENTAL INHERITANCE IN HAPLOIDS: FLAGELLATA

Knowledge of the genetics of flagellates is largely due to the recent
work of Moewus (1932-38).

In flagellates the single motile individual is haploid. In copulation two
such haploid cells unite completely, to form a diploid cell. The two
hapoid cells thus correspond functionally to two gametes, while the dip-
loid cell is the zygote. The zygote is inactive; it secretes a wall about
itself and becomes a cyst. Later, under favorable conditions, the diploid
cyst divides twice by the two “maturation divisions.’ At one of these
divisions chromosome reduction occurs, so that the four cells formed
are haploid. At times additional cell divisions occur before the cells
emerge from the cyst. The cyst wall dissolves and the haploid cells are
freed. Each develops flagella and swims about as a free individual.
On emerging from the cyst, the individuals are often called swarmers
or swarm cells; each is potentially a gamete. These free cells commonly
multiply vegetatively for many generations, the descendants of each
original swarm cell forming a clone.

In any species or variety the haploid individuals or gametes are dif-
ferentiated into two sexes; details as to this will be found in the chapter
on “Sexuality in Unicellular Organisms’’ (Chapter XIV). In some spe-
cies or varieties all members of the same clone are of the same sex, the
different sexes being in different clones (dioecious species or races). In
others both sexes are found among the individuals of a single clone

INHERITANCE 125

C D

Figure 169. Polytoma uvella (A, B) and P. pascheri (C, D); the four races used in
the breeding experiments of Moewus. (After Moewus, 1935b.)

(monoecious species or races). Details as to sex determination will be
found in Chapter XIV; here sex will be dealt with only insofar as it
plays a rdle in the general system of inheritance.

The flagellates exemplify in admirably simple form the system of in-
heritance in haploid organisms. The system is well exhibited in the
studies of Moewus (1935b) on inheritance in Polytoma. In this work

734 INHERITANCE

two species were employed, and of each species there were two varieties.
Polytoma uvella is ellipsoidal in form, with no papilla at the anterior end
(between the places of attachment of the two flagella). In one variety
there is a large eyespot, in the other there is no eyespot (see Fig. 169A,
B). In P. pascher7 the form is a long oval, there is a papilla between the
flagellar attachments, and there is no eyespot. One variety is large, the
other very small (Fig. 169 C, D).

In the four varieties there are thus differences in four pairs of char-
acters. Moewus designates these characters as follows:

Form of body: ellipsoidal, F (as in P. avella)
long oval, f (as in P. pascherz)
Papilla: present, P

absent, p
Eyespot: present, S

absent, s
Size: large, D

small, d

Using these designations, the four varieties may be represented by
formulae as follows:

P. uvella, with eyespot, FpSD
without eyespot, FpsD
P. pascheri, large, fPsD
small, fPsd

Any of these four varieties may be crossed with any other, so that six
different crosses are possible. Each of the varieties has eight chromo-
somes, so that any cross forms a zygote with sixteen chromosomes. From
each zygote arise, by the two maturation divisions, four swarm cells.
These four, after becoming free, multiply vegetatively to form clones.
All the individuals of each clone show the same characteristics as does
the swarm cell from which the clone is derived, so that we may speak
indifferently of the characteristics of each of the four swarm cells, or of
each of the four clones derived from them.

When two varieties that differ in a single pair of characters are crossed,
two of the four swarm cells produced show one of these characters, the
other two the other character. Thus P. wvella with eyespot, S, is crossed
with P. wvella without eyespot, s. Of the four swarmers arising from the

INHERITANCE 735

zygote, two have the eyespot (S), two are without it (s). The cross may
be represented as follows:

SX s=S-+s

It is obvious that the segregation of the two characters must have oc-
curred at the reduction division. One of the sets of eight chromosomes
includes the conditions for producing S, the other the conditions for
producing s.

The results of the crosses in which the parents differ in two pairs of
characters may be exemplified in the mating of P. wve/la without an
eyespot (FpsD) with the large variety of P. pascheri (fPsD) (Fig. 170 A,
B). The two differ in form (F and f), and in presence or absence of the
papilla (P and p). The zygote thus carries both these sets of factors; it is

FpsD
fPsD

The zygote divides into four swarm cells, with reduction. The results
are as follows:

1. From any single zygote two and only two types or combinations are
produced. Two of the four cells are of one combination, two of the
other.

2. Different pairs of types are produced in different cases (Fig. 170).
About half the zygotes yield again the two parental types, FpsD and
fPsD (Fig. 170 C, D). The other half yield FPsD (ellipsoidal with
papilla, Fig. 170 E) and fpsD (oval without papilla, Fig. 170 F).

Thus the characters, form, and papilla are inherited independently.
The factors F and f are in the two members of one pair of chromosomes,
the factors P and p in another pair of chromosomes.

When crosses are made in which the individuals differ in three pairs
of characters (as FpsD x fPsd) or in all the four pairs of characters
(FpSD x fPsd), all the four pairs are found to be independent in their
distribution. From any single zygote only two types of offspring are
produced. But from different zygotes of the three-pair cross, eight dif-
ferent combinations are produced; from those of the four-pair cross,
sixteen different combinations. The different combinations occur in ap-
proximately equal numbers. Thus the factors for the four pairs of char-
acters are distributed in four chromosome pairs, which at reduction are
assorted independently.

Figure 170. Results of a cross between two species of Polytoma. (Combined from
figures of Moewus, 1936) A and B, the two parents ; C and D, types produced by half
the zygotes; E and F, types produced by the remainder of the zygotes.

INHERITANCE Hew!

In consequence of the independent inheritance of these four pairs of
characteristics, there result from crosses certain curious combinations in
which there is lack of harmony in the parts of the individuals. Thus the
eyespot, S, is inherited independently of the size of the cell (D or d).
The eyespot is originally in the large race, D, where its length is about
one-sixth the length of the cell. By crosses it may be transferred to the
small race, d. Here it retains its large size, so that there are produced
small individuals with eyespots about half the length of the individual.

The fact that from any single zygote but two types or combinations
appear among the offspring shows that the reduction of the chromo-
somes must occur at the first division of the zygote. If it occurred at the
second division, there would be in some cases four different types or
combinations from a single zygote.

For suppose that we have a two-factor cross, such as Fp fP. Then
the zygote has the combination FpfP. If reduction occurs at the first
division, in some zygotes the two cells produced are Fp and fP. Each
now divides equationally, giving two cells that are Fp, two that are fP.
In other zygotes the reduction division yields FP and fp; again the second
division yields two cells of each type. In either case but two types are
produced from any one zygote.

But if reduction occurs at the second division, then after the first divi-
sion there are two cells present, both with the combination FpfP. Now
by reduction at the second division, one of these may yield Fp and fP,
the other FP and fp, so that four different combinations would be pro-
duced from a single zygote.

Linkage and crossing over.—Besides the four independent pairs of
characters just described, there are in Polytoma others that are linked
with some of the four. Such a character is length of the flagella. In the
original types, the length of the flagella is proportional to the length of
the body; large cells (A, B, C) have long flagella, small cells (D) have
short flagella (Fig. 169). This proportionality usually holds among the
crosses; those that have large bodies have long flagella, those with small
bodies have short flagella. This indicates either that the factors for size
(D and d) and those for length of flagella are close together in the same
chromosome, and so linked, or that the relation is merely a physiological
one, cells of a given size always producing flagella of length propor-
tional to the size.

738 INHERITANCE

To determine which of these alternatives is correct, Moewus made
1,400 crosses between the large and the small types (D and d). Of these,
1,357 yielded as usual zygotes which gave 50 percent large cells with
long flagella, 50 percent small cells with short flagella. The remaining
73, or 5.2 percent of all, yielded zygotes that gave 50 percent large cells
with short flagella, and 50 percent small cells with long flagella (Fig.
171). In other words, these 73 zygotes gave only cells carrying the new
combinations. It appeared, therefore, that the factors for size of body
and length of flagella are merely linked, through the fact that the two

a b <

Figure 171. New combinations resulting from crossing over. a and c, large cells
with short flagella, b, small cell with long flagella. (After Moewus.)

are close together in the same chromosome. Their distance apart is such
as to yield crossing over in about 5.2 percent of the zygotes. This was
confirmed by making 600 crosses between the new combinations, one
parent having large body cells with short flagella, the other small body
cells with long flagella (Fig. 171). Of the zygotes, 570 gave equal num-
bers that were like the parents, while 30 (or 5 percent) gave equal
numbers of the original combinations—large body with long flagella,
and small body with short flagella. These 30 therefore gave 100 percent
crossovers.

In these cases an extraordinary situation appears, seemingly unique
in crossing over. In all these cases, any zygote that yields any crossover
combination yields exclusively crossovers. The 73 zygotes of the first
1,400 mentioned in the preceding paragraph yielded 50 percent of one

INHERITANCE ey)

of the crossover combinations (large cells with short flagella), 50 per-
cent of the other crossover combination (small cells with long flagella).
The same relations hold for the 30 crossover zygotes out of 600 in the
recrossing of the new combinations; they yield 100 percent crossovers.

Such results can occur only if crossing over takes place between the
two entire chromosomes that are in synapsis, that is, only if crossing
over occurs in the so-called two-strand stage of the synapsed chromo-
some pair. In other organisms, so far as the matter has been analyzed,
crossing over occurs only after the two synapsed chromosomes have
split, so that there are four strands instead of two. Crossing over, then,
occurs between but two of the four strands, with the result that two
strands remain without crossovers. If this were the case in Polytoma, the
zygotes that yield crossovers would yield but two crossover cells and
two that were non-crossovers, in place of yielding only crossovers. The
same situation is found in all the accounts of crossing over in Flagellata
given by Moewus, up to his article of 1938. In this publication he states
that he has observed in Ch/amydomonas the occurrence of crossing over
in accordance with the four-strand schema (details to be given later).

None of the four pairs of characters thus far considered was found to
be linked with sex. That is, any of the alternative characters occurs
equally frequently with either sex. If, therefore, sex depends on a
chromosome pair, it is a fifth pair, not one of the four that carry the
characters above discussed.

In an article of 1936, Moewus presents the results of extensive studies
of inheritance in crosses of Chlamydomonas eugametos with C. pau pera.
Here eleven pairs of characters were distinguishable, some morphologi-
cal, others physiological. In addition, there were sex differences, making
twelve pairs of characters in all.

In the species of Chlamydomonas there are ten chromosomes, so that
some of the twelve pairs of characters must have their factors in the
same chromosome, and in fact Moewus discovered that some of the char-
acteristics are linked. He reports that in the many crosses made, he
analyzed the 8,000 haploid individuals derived from 2,000 zygotes re-
sulting from crosses, and that he obtained 1,024 diverse types in such
proportions as to show that each of the ten chromosomes bears the fac-
tors for one or more pairs of characters. Of most of these combinations
no detailed accounts are given.

Linkage was found to exist between two physiological characters, (1)

740 INHERITANCE

adaptation to acid or alkaline medium, with (2) differences in the num-
ber of cells into which the zygote divides before the cells are set free.
Here, as in the former case, the results are such as to indicate crossing
over in the two-strand stage. Any zygote that yields crossovers yields
nothing but crossovers. Other cases of linkage involved factors for sex;
these will be mentioned in the account of sex inheritance.

Certain general relations may be pointed out in the method of inheri-
tance of non-sexual characters thus far presented, particularly as illus-
trated by Polytoma:

1. The inheritance is the typical Mendelian inheritance for haploid
organisms. The characters manifested in the haploid descendants are
combinations of characters that were manifested in the two parents. No
characters appear in the offspring that were not manifested in the two
parents, that is, no recessive characters occur in haploid organisms. In
them all characters for which factors exist are manifested.

This is, of course, a consequence of the fact that in haploids the
chromosomes are not in pairs, but single. Hence heterozygotes cannot oc-
cur (except in the diploid zygotes). In haploid inheritance, the combina-
tions of characteristics that occur in the offspring depend wholely on the
characteristics manifested in the immediate parents.

2. The inheritance, like that in multicellular organisms, follows the
course that is to be expected if the different pairs of characters depend
on factors present in the different pairs of chromosomes. Independent
segregation, linkage, and crossing over are demonstrated in Protozoa.
Crossing over is, however, as before mentioned, of an exceptional type.

SEX INHERITANCE AND SEX-LINKED INHERITANCE

In any species or race of the flagellates examined by Moewus, there
are two sexes, or mating types. An account of these will be found in
Chapter XIV of the present volume, ‘‘Sexuality in Unicellular Organ-
isms.” Moewus designates the two sexes on his earlier reports as plus
and minus, in later publications as male and female, those earlier called
minus being male, while the plus types are female (Moewus, 1937a).
Here the different types of sex inheritance will be summarized, the ac-
count being based upon the work of Moewus.

In the flagellates investigated, some stocks are dioecious, others
monoecious. Both types may occur in different races of the same species.

INHERITANCE 741

Dioecious races.—tIn these the sexes are in separate clones, all mem-
bers of any one clone being of the same sex. The diploid zygote is
formed by the union of two haploids of opposite sex and from different
clones. By the two maturation divisions, the zygote divides into four
cells. Two of these are always of one sex, two of the other. Thus the
sexes are segregated at the reduction division, as if sex were determined
by a single chromosome pair, one member of which produces one sex,
the other the other sex. Sex is determined by the genetic constitution of
the clone; sex determination is genotypic. Such dioecious stocks occur in
certain races of Chlamydomonas eugametos, of Polytoma pascheri, and
of Protosiphon botryoides.

Subdioecious races—In certain races of Chlamydomonas eugametos
there is found a modification of the dioecious condition. In any single
clone, most of the individuals belong to one sex, a few to the opposite
sex. Some clones are prevailingly plus, others prevailingly minus. A num-
ber of different matings are possible:

1. Plus gamete from a prevailingly plus race, minus gamete from a
prevailingly minus race. Two of the cells from the zygote yield prevail-
ingly plus clones, two yield prevailingly minus clones. The segregation
of “‘prevailingly plus” from “prevailingly minus” therefore occurs at
the reduction division; the difference is genotypic.

2. Plus and minus gametes from a prevailingly plus race. All clones
from the zygote are prevailingly plus.

3. Plus and minus gametes from a prevailingly minus race. All clones
from the zygote are prevailingly minus.

In other crosses, clones from a dioecious race, in which the clones are
exclusively of one sex, are crossed with prevailingly plus or prevailingly
minus clones from subdioecious races. The former will-be spoken of as
“pure” for sex, the latter as ‘‘mixed’’ for sex.

4. Plus gamete from a race pure for sex, minus gamete from a pre-
vailingly minus clone of mixed race. Offspring: two pure plus, two
prevailingly minus mixed.

In other crosses of this type, the results were similar; the “pure”’
condition segregates from the “mixed” condition at the reduction divi-
sion. In general, the two types that unite to produce the zygote separate
again at the reduction division; the differences are genotypic.

The fact that in single clones of the subdioecious races some indi-

742 INHERITANCE

viduals are plus and others minus is held by Moewus to be due to some-
thing in the surrounding conditions; the sex determination within the
clone is phenotypic instead of genotypic, as in the dioecious races. But
in the “prevailingly plus” clones the constitution is such that minus
gametes are not so readily or numerously produced by the conditions as
is the case in “prevailingly minus” clones. Moewus found that subjec-
tion to dilute formaldehyde or acetone causes the zygotes of the sub-
dioecious races to produce gametes of only one sex, that sex which would
have been in the majority if these substances had not been used. The
precise constitution of subdioecious races, together with that of other
types, is considered in a later section.

Monoecious races ——In monoecious races both sexes occur in a single
clone, so that such clones may be spoken of as ‘‘mixed”’ as to sex. This
is the situation in certain races of various species of Chlamydomonas;
in Polytoma uvella, and in somes races of P. pascheri and of Protosiphon
botryoides.

In monoecious stocks obviously the fission of a single individual gives
rise to both sexes. The determination of sex in such stocks is largely or
entirely phenotypic; that is, through external conditions. Such pheno-
typic sex determination is not dealt with in the present chapter.

When from monoecious stocks plus and minus gametes are mated, the
descendant clones are all monoecious, that is, mixed as to sex.

Crosses between dioecious and monoecious stocks —A number of dif-
ferent types of crosses may be made between dioecious and monoecious
stocks, as follows:

1. Plus gamete from a “mixed” (monoecious) clone; minus gamete
from a “‘pure’’ (dioecious) clone (cross of two diverse races of Polytoma
pascher7). Result, two of the four descendant clones are mixed, two pure
minus.

2. Plus gamete from pure clone, minus gamete from mixed clone
(cross of two races of P. pascheri). Result, two clones mixed, two pure
plus.

In these two crosses, two results are notable. (1) The pure condition
segregates from the mixed condition at the reduction division, the two
depending apparently on the two different chromosomes of a pair. The
difference is genotypic. (2) The “pure” condition emerges with the
same sex (plus or minus) as that with which it enters the cross. If the

INHERITANCE 743

plus parent is pure for sex, it is the plus offspring that are pure; if the
minus parent is pure, the minus offspring are pure.

Linkage and crossing over.—Certain crosses yield a small proportion
of exceptional results, which are held to be due to crossing over. Such
are the following:

Polytoma pascheri: pure plus clone by pure minus clone. Result, out
of 2,000 zygotes, 1,843 gave four cells each (as usual), two of which
were pure plus, two pure minus.

The other 157 (7.9 percent) gave but two cells each, and these all
produced clones mixed as to sex.

The results given by the 1,843 zygotes are those to be expected from

ce ae ooo se TC ORAM RT SOE SM TREE EB TE ar SRE SME SE Te

ae |

Figure 172. Diagram of the sex chromosome of Polytoma pascheri plus (upper line,
with factor P), and of P. wvella minus (lower line, with factors P and M). The two
together show the sex pair in the zygote of a cross between the two species.

the principles thus far set forth. How are the 157 exceptional zygotes
to be accounted for?

The exceptions might be produced either by non-disjunction of two
sex chromosomes, or by crossing over between them. If they were the re-
sult of non-disjunction, the 157 individuals would have received both
the plus-producing and the minus-producing chromosomes of the sex
pair; this would account for their mixed sex condition. There would be
a pair of sex chromosomes, in place of the usual single chromosome.
The exceptional individuals would therefore contain nine chromosomes
instead of the eight usual for Polyfoma. But cytological observations
showed that only eight were present. The exceptional cases are there-
fore not the result of non-disjunction.

The alternative explanation is that they are due to crossing over. But
how could crossing over between plus-producing and minus-producing
chromosomes yield clones mixed as to sex?

Moewus concludes that the plus-producing chromosomes must con-

744 INHERITANCE

tain a factor (‘‘realisator’) for the plus sex condition, the minus-pro-
ducing chromosomes a factor for the minus sex condition, and that by
crossing over, both must come into the same chromosome, which there-
fore produces the mixed sex condition. The cells containing such a
chromosome may become either plus or minus, depending on external
conditions.

But if the plus and minus factors may by crossing over come into the
same chromosome, these factors are not alleles; they are not at the same
locus. The condition of the chromosomes may then be represented as in
Figures 172 and 173, in which P represents the plus-producing factor,
M the minus-producing factor.

If crossing over occurs between the P and the M chromosomes, half of

p M

jee. Ge
P

Figure 173. Diagram of the sex chromosomes produced by crossing over between the
chromosomes of Polytoma pascheri plus (light line) and P. zvella minus (heavy line).
The upper chromosome has the factor P from P. pascheri, the factor M from P. uvella.

the resulting chromosomes in this case contain both P and M, while the
other half contains neither. Moewus holds that the cells carrying neither
P nor M die. Thus is accounted for the fact that each of the 157 ex-
ceptional zygotes produces but two cells in place of the usual four. It is
to be noted that here, as in former cases, the results are such as would
be given by two-strand crossing over; a zygote produces either no cross-
over cells or exclusively crossover cells.

From these considerations and others of similar character, Moewus
concludes that in clones mixed as to sex (monoecious races), the sex
chromosome contains both the sex factors—the factor P for producing
the plus sex, and the factor M for producing the minus sex.

Moewus (1936) reached similar results in a cross of Chlamydomonas
paradoxa and C. pseudoparadoxa. Both species are dioecious, any clone
being pure for one sex or the other. When plus clones of one species are

INHERITANCE 745

mated with minus clones of the other, the majority of the zygotes yield
four clones, of which two are pure plus, two pure minus. But in a par-
ticular case, out of 1,000 such zygotes, 117 gave exceptional results, all
the four clones from each zygote being mixed as to sex. These excep-
tions are held to be due to crossing over. One of the haploid parents had
the factor P, the other the factor M in the sex chromosome. By crossing
over, the two factors are brought into one chromosome; the cells that
receive this chromosome yield clones that are mixed as to sex. Half of
the sex chromosomes in which crossing over occurred would lack both
the factors P and M. Moewus holds that the cells that receive such
chromosomes die, while the cells that receive both P and M divide twice
before escaping from the zygote. Thus the zygote produces four cells as
usual, all having the crossover combination P and M. Here, as in former
cases, the results are those characteristic for two-strand crossing over.

Many other cases of crossing over of the sex factors (with results that
require two-strand crossing over) are described by Moewus (1936) in
crosses of C. evgametos and C. paupera—these all indicating that clones
which are mixed as to sex carry a chromosome which has both sex fac-
tors, P and M. Other cases will be mentioned in later paragraphs.

Sex-linked inheritance.—In crosses of the two species of Chlamydo-
monas just mentioned, Moewus observed sex-linked inheritance. The
species C. eugametos has an eyespot, while C. paupera has none. When
C. eugametos of one sex is crossed with C. paupera of the other sex, in
the descendants the gametes that are of the same sex as the C. e~gametos
parent have the eyespot, while those that are of the same sex as the
C. pau pera parent have none. The eyespot is thus linked with sex.

There are, however, a few exceptional cases, due to crossing over, in
which the eyespot no longer goes with the parental sex. The results are
complex and will not here be presented in detail. In all cases the re-
sults, as given by Moewus, are those that would be characteristic for two-
strand crossing over. By analysis of the results, Moewus believes that he
is able to establish the order in the chromosome of the two sex factors
(P, M) and that for eyespot (S), as P-M-S.

Relative sexuality, in crosses between different species——In crosses
between different species, in some cases gametes of like sex may copulate
and yield descendants (see Chapter XIV). Plus gametes from one spe-
cies may unite with either plus or minus gametes from the other species,

746 INHERITANCE

and minus gametes from one with plus or minus from the other.
This phenomenon, known as relative sexuality, is accounted for by
Moewus (following Hartmann) by assuming that the gametes of the
different species differ in the strength, or “valence,” of their sex tendency
and that if two gametes differ sufficiently in the strength of the sex
tendency they copulate, whether of the same or of different sex.

Exemplifying this situation is the fact that any gamete of Polytoma
uvella may copulate with any gamete of P. pascheri, irrespective of the
sex of the gametes. Hence it is held that in one of these species the sex
tendency of the gametes is stronger than in the other species.

The relations as to crossing over described in the preceding section
offer an opportunity for determining whether the sex factors present in
the chromosomes of the two species differ in the strength of the sex
tendency; also for determining in which species the sex factors are
stronger. By crossing over, the plus factor of one species may be brought
into the same chromosome with the minus factor of the other species. It
is then possible to determine which of these prevails, and thus to discover
in which species the sex factor is stronger.

Moewus crossed plus P. pascher7 gametes from a clone pure for sex
with minus P. wvella gametes from a race mixed for sex. According to
the hypothesis, the P. wvel/a gametes, being from a race mixed for sex,
contain a sex chromosome which carries both the plus and the minus
factors, P and M. The P. pascher7 gametes have a sex chromosome con-
taining only the P factor. The situation as to sex chromosomes in the
zygote may therefore be represented as in Figure 172. From this cross
625 zygotes were obtained. Of these, 582 gave the results that are usual
without crossing over. Each zygote gave two clones that are pure plus,
as in the P. pascheri parent, two that are mixed, as in the P. wvella parent.

The remaining 43, or 6.9 percent, gave exceptional results, presumably
due to crossing over. In these, two of the four cells from each zygote
yield pure plus clones, like the P. pascheri parent, while the other two
yield pure minus, unlike either parent. No mixed clones are produced.
How are these results accounted for?

The original condition of the chromosomes is that shown in Figure
172. By crossing over (the break occurring between P and M), the con-
dition shown in Figure 173 is produced. One chromosome contains only
the plus P. wvella factor (P); it is obviously the gametes containing this

INHERITANCE 747

that yield the pure plus clones. The other chromosome contains both the
P from P. pascheri and the M from P. wvella. The gametes that contain
such chromosomes act, as above mentioned, like pure minus gametes.
Therefore the effect of the M P. wvella factor completely overcomes the
effect of the P factor from P. pascher7. The sex factors of P. mvella are
thus stronger 1n sex tendency than those of P. pascheri.

From this the further conclusion is drawn that the gametes of P. wvella
are stronger in sex tendency than the gametes of P. pascheri. Since P.
uvella has only monoecious races (mixed as to sex), these gametes, ac-
cording to the assumption, contain both a strong P and a strong M
factor. It might be anticipated that the two would partly or entirely
counteract each other, leaving the gametes weak or neutral in sex tend-
ency. This, however, does not occur; if Moewus’s assumption is correct,
one of the two fully prevails, the other having no effect, so that the
gametes are strong.

Moewus carried out numerous other crosses of these two species, which
gave results that are in accord with those just set forth. They all indicate
that the P. wvella factors have a stronger sex tendency than the P. pascheri
factors. They agree further with the idea that the P and M factors are in
different loci, and that by crossing over they may be brought into the
same chromosome.

One further result of these experiments is of special interest. In cer-
tain cases there were crosses of two dioecious races (produced by hy-
bridization), in which the gametes were of the same sex (both plus or
both minus). In such crosses between plus gametes, according to the
theory, each gamete contains only the P factor; no M factor is present.
All the offspring in such cases are then of the plus sex, as the theory
would lead one to expect. Similarly, if the two gametes each contain
only the minus factor, all the offspring are of the minus sex.

An elaborate investigation of these matters was carried out by Moewus
(1935a, 1935c) on diverse races of the unicellular alga Protosiphon
botryoides. The relations here, while differing much in details, are con-
cordant with those above set forth for Po/ytoma in the matters of cross-
ing over of sex factors, different strengths of sex tendencies in different
races, segregation at the reduction division, and the like. The results will
therefore not be taken up in detail here. It is to be remarked that in this
species, as in the others, crossing over (as the data are reported by

748 INHERITANCE

Moewus) took place in accordance with the two-strand scheme; any
zygote that gave crossover combinations at all gave only crossover com-
binations.

In Protosiphon an investigation was made on the determination of
sex by external conditions. Protos7phon includes dioecious races, in which
the determination of sex is genotypic, as described in earlier pages;
monoecious races, in which a single clone contains individuals of both
sexes, the determination of sex being mainly phenotypic; and certain
other races that agree in some respects with one type, in some respects
with the other. The phenotypic determination of sex is not here dealt
with.

Of interest for the nature of inheritance are the races which partake
of the features of both types. Such is the race d, described by Moewus
(1935c). At a certain stage in its life history, Protos7phon is a small club-
shaped “‘haplont,’’ containing many haploid nuclei imbedded in a mass
of cytoplasm without cell walls. Such a haplont may be produced by a
zygote resulting from the union of two gametes. In the diploid zygote,
the reduction division occurs and the haploid nuclei multiply, giving rise
to the haplont. Or haplonts may be produced each from a single haploid
swarm cell, which comes to rest on the surface of a solid (as agar) and
produces many nuclei by division of its single nucleus. Haplonts pro-
duced in either of these ways give rise later to swarm cells (gametes).
The nuclei separate, each is surrounded by a small mass of cytoplasm and
each such cell transforms into a flagellate swarm cell. In the race d, as in
dioecious races, all the swarm cells produced by a single haplont are of
the same sex.

But, in the race d, the sex of the swarm cells produced by the haplont
depends on the conditions under which the haplont was produced.
Haplonts grown on acid agar give rise only to plus swarm cells. Those
grown on alkaline agar yield only minus swarm cells. Of those grown
on neutral agar, some yield only plus swarm cells, others only minus
swarm cells. The sex of the swarm cells and of all their descendants by
vegetative fission is determined once for all by the conditions prevail-
ing in the development of the haplont from which they arise. This is
true not only for haplonts produced from single swarm cells, but also for
haplonts produced from zygotes. Since in the zygote the reduction divi-
sion occurs, yet all the four cells resulting from the maturation divisions

INHERITANCE 749

are of the same sex, it is clear that sex segregation does not occur at the
reduction division.

Moewus determined by careful experimentation at exactly what stage
in the development of the haplont sex determination occurs. It is at the
first division of the single nucleus of the swarmer that is dividing to
produce a haplont. If at this time the conditions are acid, the swarm
cells later produced from the haplont are plus; if the conditions at that
time are alkaline, the later swarmers are minus.

In this case, therefore, the sex is determined by an external condition,
and this sex is inherited by all the descendants of the cell in which it 1s
thus determined. The consequences induced by an environmental condi-
tion are inherited.

In his article of 1938, Moewus sums up the conclusions to which his
investigations lead as to inheritance and determination of sex, and par-
ticularly as to the constitution of the sex chromosomes in the different
sex conditions. In pure dioecious races, each sex chromosome carries but
one sex factor, P or M, and sex is segregated at the reduction division.
In different dioecious races, the strength or valence of the sex factors may
be diverse. In monoecious races, the sex chromosome of each individual
carries both sex factors, P and M, in equal strength; which of these shall
prevail in determining the sex of the gamete is decided by external
conditions. In the subdioecious cases, in which any clone is prevailingly
of one sex but produces also a few cells of the other sex, the sex chromo-
somes are held each to contain both sex factors, P and M, but every cell
contains an additional factor which inclines it toward one sex or the
other. The prevailingly plus clones contain a factor which inclines them
toward the plus sex, the prevailingly minus cells a factor which inclines
them toward the minus sex. But the tendencies of these additional fac-
tors are in some cases overcome by special outer conditions, so that a
few cells of the opposite sex are produced.

In addition to these three types, there are other types in which the sex
chromosome carries both the sex factors, P and M, but one of these is
stronger than the other, so that it prevails and determines alone the sex
of the clone. If P is the stronger, the sex of the clone is plus; if M is
stronger, the sex of the clones is minus. Each clone is therefore pure for
sex, and the race is dioecious. Such are called by Moewus complex dioe-
cious races. They are not found in nature, but occur as a result of crossing

750 INHERITANCE

over between dioecious races having sex factors of different strengths.

In this article of 1938, Moewus discusses the difficulties arising in
his data as to crossing over. In all the data presented by him up to this
time, crossing over was reported as occurring in accordance with the two-
strand schema. Any zygote that gave any crossover combination gave
such combinations exclusively. Moewus now concludes that this is not
the normal state of affairs; he characterizes it, indeed, as pathological.
Under certain conditions, he reports, crossing over occurs in the normal
manner, according to the four-strand schema. He promises a future ac-
count of detailed investigations showing this.

Certainly there is great need for clearing up the confused situation as
to crossing over in these organisms. In hundreds of detailed earlier re-
ports, Moewus has given data that are consistent only with two-strand
crossing over. A further serious criticism, based on other grounds, has
been made as to the accuracy of Moewus’s data on crossing over, by
Philip and Haldane (1939).

Aside from this difficulty, the work of Moewus has placed the genetics
of the Protozoa on a new footing. It has brought the phenomena of
inheritance in these organisms into the same system that is manifested in
the Mendelian inheritance of higher organisms. It has brought to light
in the flagellate Protozoa instances of most of the phenomena in such
inheritance, as before known in multicellular organisms.

BIPARENTAL INHERITANCE IN DIPLOIDS: CILIATA

The genetics of diploids is necessarily more complex than that of
haploids. The individuals have during active life two sets of chromo-
somes instead of one. In consequence, in the sexual process two indi-
viduals do not normally unite completely, to form a diploid zygote, as in
the copulation of the flagellates. In the diploid ciliates, two individuals
merely come into intimate contact and exchange pronuclei that contain
each a haploid set of chromosomes, a process known as conjugation. The
two then separate, each carrying two haploid sets of chromosomes, one
from each of the conjugant individuals. Each then multiplies vegetatively,
forming clones, all members of any clone having the same diploid com-
bination of chromosomes. Conjugation thus is like fertilization in higher
organisms, in that it produces new diploid combinations of chromosomes.

Important for the understanding of inheritance in conjugation are the
following:

INHERITANCE foe

1. The macronucleus or macronuclei of each individual disappear,
being absorbed into the cytoplasm.

2. If more than one micronucleus are present, usually all but one are
absorbed and disappear.

3. The single micronucleus divides three times in succession (the
“maturation divisions”). By the first two divisions (Fig. 174, 1 and 2)
four nuclei are produced; of these, three are absorbed and disappear.
The remaining one divides once more into two (Fig. 174, at 3).

Figure 174. The three maturation divisions (1, 2, 3) and the exchange of pronuclei
in the micronuclei during conjugation. A and D are the micronuclei of the two con-
jugants respectively. Reduction at the second division, represented by the separation of
XX and XY. The diagram illustrates the fact that the two diploid nuclei produced
(above) are alike in constitution.

4, The two nuclei produced by the third division are haploid, so that
one of the three maturation divisions is a reduction division.

5. One of the two haploid nuclei (the “migrating pronucleus’’) in
each individual passes over into the other individual, where it unites
with the remaining one (‘‘stationary pronucleus’) of that individual.
Thus there is produced again a diploid nucleus in each individual.

6. This diploid nucleus divides, some of its products becoming large
as macronuclei, others remaining small as micronuclei. These macro-
nuclei and micronuclei are distributed by fission to different individuals,
the exact processes differing in different species.

7. In conjugation, only nuclei are exchanged; each individual retains
its cytoplasm complete. Hence after conjugation each individual has a

752 INHERITANCE

new combination of chromosomes, half derived from each conjugant,
but has the same cytoplasm as before. This gives an opportunity to com-
pare the relative rdles of chromosomes and cytoplasm in inheritance,
since in the ex-conjugants the chromosomal combination is changed,
but the cytoplasm is not.

The only qualification required by the statement that cytoplasm is not
changed is the fact that a minute bit of cytoplasm carrying an aster pre-
cedes the migratory pronucleus into the opposite conjugant. The results
show, as will be seen, that this minute bit of cytoplasm is not effective in
determining inheritance.

The two individuals that conjugate are, in some species at least, physio-
logically differentiated into diverse “‘mating types,’ which play the same
physiological role in bringing about conjugation as do diverse sexes.
For an account of these, Chapter XIV is to be consulted.

Knowledge of biparental inheritance is much less exact and extensive
in the ciliates than in the flagellates. The recent discovery of diverse
mating types furnishes an opportunity for exact analysis; but the novelty
of this discovery has not yet given opportunity for its full investigation.

The earlier work on inheritance in conjugation before the discovery of
mating types, is summarized in the author's Genetics of the Protozoa
(1929). In the early work numerical ratios were not obtained, but qualli-
tative relations of importance were demonstrated. Most generally ex-
pressed, the work showed that conjugation results in the production of
many hereditarily diverse biotypes from the two involved in the conju-
gation. Production of hereditary diversities at conjugation was demon-
strated by the work of Jennings in respect to the following types of
characteristics: rate of fission, rate of mortality, presence of abnormali-
ties. This demonstration of the production of inherited differences at
conjugation was extended in 1930 by the work of Raffel, and in 1932 by
Jennings, Raffel, Lynch, and Sonneborn to various other characteristics,
including size and form, vigor, resistance, and degeneration. It was fur-
ther shown that conjugation causes the descendants of the two members
of a pair to become similar in fission rate, in mortality, in the occurrence
of abnormalities, and probably in size (see Jennings, 1929, pp. 181-85).

Later, more exact work on inheritance in ciliates deals with the inherit-
ance of mating type (Sonneborn, 1937-39; and Jennings, 1938-39)

INHERITANCE HS)

and with the inheritance of size (De Garis, 1935). That on mating type
will be presented first.

INHERITANCE OF MATING TYPE IN Paramecium aurelia

In Paramecium aurelia, according to the work of Sonneborn (1937,
1938, 1939), there are in any variety but two mating types, members of
which unite in conjugation as the two sexes unite in multicellular ani-

Figure 175. The four clones (al, a2, b1, b2) produced from the two ex-conjugants
(a and b) of a pair, in the experiments of Sonneborn and of Jennings.

mals. Three varieties or “groups” are known; individuals of one variety
do not conjugate with individuals of the other varieties.

In Variety or Group 1, the two mating types are known as types I
and II. These unite in conjugation. In studying the types produced by
them, each ex-conjugant after separation was allowed to divide once, and
from each of the products a clone was produced by vegetative reproduc-
tion. Thus four clones were derived from each pair, two from each ex-
conjugant, as shown in Figure 175.

754 INHERITANCE

All members of any one of the four clones belong to the same mating
type, and that type is inherited within the clone until endomixis or a
new conjugation occurs. In a certain experiment described by Sonneborn
(1937), there were fifty-six pairs formed by union of mating types I
and II. The four clones from each pair were constituted as shown in the
accompanying table.

In this table the following relations are seen:

1. In some cases (4 out of 56) all the descendants of the two parents
(which were of diverse type) are of the same type. In these cases the
type of one of the conjugants and its descendants was changed by con-

PARENTS, I X II

Number of pence ne Mating Types of the Four
Pairs iypell anpeul Descendant Clones
I 4 eel
18 3 I Teale
21 2 2 Ie le JOG 3!
13 I 3 I JU JOT, WL
3 4 OE TOG We Tt

jugation. That ts, an individual of type I, receiving a pronucleus from
type II, becomes changed to type II, and vice versa.

2. In some cases the two ex-conjugants of a pair give clones of dif-
ferent mating types.

3. In most cases a single ex-conjugant gives rise to two clones of the
same type.

4. But in some cases a single ex-conjugant gives clones of different
mating type.

Do ex-conjugants that were of a given type before conjugation tend
after conjugation to produce descendant clones of that type, or do they
produce both types with the same frequency? Sonneborn tested this by
mating two clones that were different in appearance, one of each type,
then determining the mating type of the descendants. The results were
as follows:

Of 22 ex-conjugants originally of type I, 5 gave descendants of type
I only, 7 of type II only, and 10 half of type I, half of type II. Of 25 ex-

INHERITANCE eS,

conjugants originally of type I, 4 gave descendants of type II only, 3
of type I only, and 18 half of type I, half of type II.

In summary, there were 47 ex-conjugants, which give 94 descendant
clones. Of these 94, 46 were of the same type as their parent’s before
conjugation, 48 of different type. Thus it is clear that ex-conjugants
originally of a given type produce descendants of both mating types with
equal frequency. The reception in conjugation of a pronucleus from an
individual of different type changes the type as frequently as it leaves it
unchanged. There is no tendency for the descendants to be of the same
type as was the cytoplasmic body from which they are derived (certain
exceptions will be described later).

Of the 47 ex-conjugants just considered, 28 gave two clones of dif-
ferent types, while 19 gave two clones of like type. There is no indica-
tion of a tendency for the two clones descended from a single ex-con-
jugant to be alike in type.

Thus the first fission after conjugation separates the ex-conjugant into
two individuals which may be of different mating type, giving rise to
two clones of different types; or they may be of the same type. Segre-
gation of the diverse mating types (in cases in which it occurs) takes
place at the first fission after conjugation.

What is it that decides the mating type of each clone? Sonneborn 1s
disposed to believe that the segregation is the result of the separation of
the two macronuclei, one macronucleus tending to produce type I, the
other type II. How the two became diverse (if this is the case) is not
known. There appears to be no evidence of a reduction division at this
point, such as might give rise to nuclei differing in chromosomes. It
appears equally difficult to suppose that the two nuclei of the same cell
are subjected to differing conditions, such as to cause one to be of type I,
the other of type II.

Sonneborn (1938) has discovered, however, that the temperature
during conjugation affects the proportion of the types produced in a group
of ex-conjugants. In variety or group 1, higher temperatures cause the
appearance of a greater proportion of type I; in group 3, higher tempera-
tures favor the production of type VI (rather than type V). But it is not
evident how the difference of type in the two products of fission of a
single individual could be induced in any such manner. The segregation
of types at the first fission after conjugation remains a riddle.

756 INHERITANCE

Segregation of mating types at endomixis. —It is of great interest that
at the first fission after endomixis segregation of the mating types may
occur, just as it does after conjugation. The single individual, of a definite
mating type, divides after endomixis into two that may be diverse in
mating type. The nuclear processes in endomixis are not yet fully cleared
up, but are known to be in many respects similar to those which occur
at conjugation. According to the recent work of Diller (1936), the simi-
larity goes so far that there is in endomixis a union of two micronuclei
(presumably haploid), just as occurs in conjugation. In this autogamy
the two micronuclei that unite are of course both from the same indi-
vidual. After their union, the single (diploid) nucleus divides into two,
then into four. Two of these become macronucei and two remain micro-
nuclei, just as in conjugation. The two macronuclei are separated at fis-
sion, just as occurs after conjugation, and the same is true of the micro-
nuclei. As will be seen later, there is genetic evidence that autogamy does
indeed occur at endomixis.

In variety or group 1 of Paramecium aurelia, Kimball (1937) deter-
mined the mating type of the two clones produced by each of 181 indi-
viduals that had undergone endomixis. Of the individuals, 96 gave 2
clones of the same type (both I or both II), while 85 gave two clones of
different types (one type I, one type II). In other respects also the re-
sults of endomixis in relation to the mating types are like those of con-
jugation (see Kimball, 1937).

As a rule, in both endomixis and conjugation the segregation of the
mating types occurs at the first fission after completion of the process.
But in a certain stock there was a small proportion of cases in which
segregation into two mating types occurred at the second fission after
conjugation. In this same stock, cytological examination showed that in
about the same small proportion of cases the ex-conjugants had three or
four macronuclear Anlagen in place of the usual two. It of course re-
quires two fissions to separate these into different individuals. Sonneborn
is disposed to consider this the cause of the occasional segregation of the
mating types at the second fission, in place of the first.

Single-type clones.—There exist clones in which there is no segrega-
tion into different mating types at endomixis. In such clones endomixis
makes no change in the mating type; the clone remains throughout of
the same type (Sonneborn, 1938c). Of twenty-six clones examined by

INHERITANCE 757

Sonneborn, six showed no change of type at endomixis. All these six
were of mating type I.

Such clones may be designated as single-type clones, as compared with
the more common double-type clones, in which from a single clone both
sexes are produced at endomixis.

Crosses between single-type and double-ty pe clones.—Crosses between
the two types (Sonneborn, 1939) show that they are inherited in typical
Mendelian fashion, double type being dominant over single type. The
factor for double type may therefore be designated A, that for single
type a. The two original diploid clones are AA and aa. Crosses of the
two (AA X aa) gave in the 149 pairs examined, all double-type (Aa)
offspring. Mating together these heterozygotes yielded, in 120 pairs, 88
of the dominant double type, 32 of the recessive single type, so that the
results approximate the typical three-to-one ratio (Aa & Aa == AA +
2A + aa). The hybrids Aa were back crossed to the single-type parents
(aa) in 165 pairs; these yielded 88 double-type and 77 single-type de-
scendant clones, an approximation to the expected one-to-one ratio (Aa
aa —=- Aa + aa). Here we have diploid Mendelian inheritance, one of
the characters being recessive.

Genetic evidence of autogamy.—In the further study of the heterozy-
gotes Aa, a discovery of great interest was made. At endomixis, some
of these heterozygotic individuals produce (at the first fission after endo-
mixis) double-type clones, others single-type clones. Is the double-type
clone the heterozygote Aa or the homozygote AA? This was tested by
mating them with the normal single-type individuals aa. All the de-
scendant clones are the heterozygotic double-type Aa. This shows that
the double-type clones produced at endomixis are the homozygotes AA;
correspondingly, the single-type clones produced at endomixis are neces-
sarily aa. From the heterozygotes Aa there are produced at endomixis two
types, both homozygotic: AA and aa.

How is this result brought about? It is the natural consequence of the
occurrence at endomixis of a reduction division with subsequent union
of two of the reduced nuclei (autogamy), as described cytologically by
Diller (1936). The heterozygote nucleus before reduction is Aa. By re-
duction are produced haploid nuclei A and a: by a second division these
give rise to four nuclei A, A, a, a. Three of these haploid nuclei de-
generate, leaving but one, A or a. This remaining nucleus now under-

758 INHERITANCE

goes the third division, yielding in one case two nuclei A and A, in the
second case a and a. These two nuclei now reunite (autogamy), yielding
in the one case the homozygote dominant AA, in the other case the
homozygote recessive aa. Thus after endomixis has occurred in any stock,
the clones are all homozygotes, as Sonneborn points out.

These results are obviously strong genetic evidence for the occurrence
of autogamy at endomixis in Paramecium aurelia.

Certain rare and exceptional conditions in the genetics of the mating
types in P. avrelia are discussed in Chapter XIV, ‘Sexuality in Unicellular
Organisms.”

INHERITANCE OF MATING TYPE IN Paramecium bursaria

In Paramecium bursaria, according to the work of Jennings (1939a,
1939b), there are in one of the varieties four mating types, A, B, C, and
D. Another variety has eight mating types (E, F, G, H, J, K, L, and M),
a third variety four types (N, O, P, and Q) distinct from the four of
the first variety. Inheritance of mating type has been examined only in
the first variety (some of the relations are here published for the first
time).

In variety 1 (as in the other varieties), clones of any one of the types
may conjugate with clones of any of the other types, but not with clones
of their own type Thus in variety 1 six different matings occur, yielding
pairs AB, AC, AD, BC, BD, CD.

Very rarely in this species a single clone, in which all the individuals
belong originally to the same type, may differentiate into two types,
which thereupon conjugate. This phenomenon is parallel to the segrega-
tion of different types from one type at endomixis in P. awrelia. It is
presumably the result of endomixis in P. bursaria; in this species endo-
mixis is known to occur very rarely (Erdmann, 1927). Thus the occa-
sional ‘“‘self-fertilization’’ of a clone is in fact the conjugation of two
diverse mating types. A clone of the mating type D has been observed
to differentiate part of its individuals into the mating type A; these then
conjugate with the D individuals, giving the cross AD. The results of
“‘self-fertilization” may therefore be considered with those of other mat-
ings between two different types.

In studies of mating-type inheritance, four descendant clones are ob-
tained from each pair, two from the first fission of each ex-conjugant,

INHERITANCE 79

as indicated in the diagram of Figure 175. After the attainment of sexual
maturity, the mating type of each of the four clones is discovered by
testing them with standard clones representing the four known mating
types.

The fullest data as yet available are for the cross A & D. Of this cross
the mating types have been determined for the clones descended from
61 pairs, including 175 clones. The 4 original clones did not survive in
all the pairs. They did all survive in 26 pairs (104 clones). The remain-
ing pairs had each but 1, 2, or 3 surviving clones. But in all cases all

TABLE 18: INHERITANCE OF MATING TYPES (THE FouR TYPES ARE
A, B, C, D), Paramecium bursaria

Parents, A X D

Typical Constitution of the Four
Descendant Clones of Each Pair

30 Pairs give type A only A+A+A+A
4 Pairs give type B only B+B-+B+B
4 Pairs give type C only C+C+C+C
23 Pairs give type D only D+D+D+D

clones descended from any pair were of the same mating type. The re-
sults have been summarized as in Table 18.

In Table 18 the following general relations appear:

1. All the descendants of any one pair are of the same mating type,
though the parents were of two different types.

2. Among the descendants of the cross of the two types A and D
occur all four types A, B, C, and D.

3. The majority of the descendant clones are like one or the other of
the two parents in type; only a few differ. In this case 53 of the 61
clones are either A or D (the parental types); only 8 are of types differ-
ent from those of the parents.

4. By conjugation the mating type has become changed in one or
both of the ex-conjugants and their descendants. In the above table, the
type is changed in both ex-conjugants in 8 pairs out of 61; it is changed
in but one of the ex-conjugants in the remaining 53 pairs.

The change in type is due to the exchange of migratory pronuclet,
since the cytoplasm remains unmixed. Individuals of type A, receiving a
micronucleus from type D, are changed to type D in nearly half of the

760 INHERITANCE

cases; to B or C in few cases. In about half the cases they remain un-
changed. Parallel statements may be made for the individuals originally
of type D.

It is clear that although all four mating types are present in the de-
scendants of a cross of but two types, the statistical make-up of the de-
scendant population is influenced by the constitution of the two parents.

TABLE 19: MATING TyPES OF DESCENDANT CLONES FROM 131 Pairs
(INCLUDING 279 DESCENDANT CLONES), IN THE SIX POSSIBLE CROSSES
OF THE FouR MATING TYPES OF VARIETY 1, Paramecium bursaria

Matinc Types OF THE DescENDANT CLONES: NUMBERS

C Number or Pairs YietpiInc Eacu Type
TOSS : elena eae ea Ho 2
of Pairs
IN NOE VID) Like Parents Unlike Parents

ASGB 25 1405} fo) fo) 25 fo)
ASC 6 3 I I 4 2
AeD 61 30 4 HB 53 8
I< 10 ©). 36) fo) fo) 10 fo)
BX D 15 5 8 fo) 2 10 5
Cx D 14 fo) fe) 6 8 14 °
Totals 131 ye ah ay 116 15

The majority of the descendant clones belong to one or the other of the
two parental types. This is evident in the results of all the crosses that
have been made, as shown in Table 19.

As Table 19 shows, of 131 pairs in which the original type of the
conjugants was known, 116, or 88.5 percent, gave descendant clones that
were of the same type as one or the other of the two parent individuals,
while but 15, or 11.5 percent, gave descendants that were of different
mating type from either of the two parents.

What determines the type to which a particular clone belongs? In all
the 131 pairs of Table 19, all surviving clones from any pair (1 to 4
clones per pair, in different cases) were of the same mating type. The
four clones descended from the two members of any one pair were culti-
vated separately and tested separately. The fact that all four turn out to
be of the same mating type shows that the type is determined at the
time that the two ex-conjugants separate and before they divide. It might
be determined at that time either by internal or external conditions. The
fact that in the great majority of cases the descendant clones are of the

INHERITANCE 761

same type as one or the other of the two parents shows that the constt-
tution of the two parents is one important factor in determining the
type of the descendants. But why in the same cross the descendants from
some pairs should be of the same type as one parent, those from other
pairs of the same type as the other parent, while a few are of different
type from either parent—this is as yet quite unknown.

In extremely rare cases the four clones produced by a particular pair
are not all of the same type. Thus in a certain case a pair composed of the
types A X C yielded four clones of the types A, B, B, B. In another case
A X C gave B, D, D, D. The great rarity of such cases indicates that such
results are due to irregularities in the cytological processes, comparable
to cases of non-disjunction of chromosomes.

Immaturity and partial maturity—The relations thus far described
are those that exist after the descendant clones have reached sexual or
reproductive maturity. But for a long period after conjugation, varying
in different clones from a few weeks to more than a year, the descendant
clones are in Paramecium bursaria sexually immature. During this period
the descendant clones do not mate at all, with any of the mating types.
In group 1, if they are mixed with mature individuals of any of the four
mating types A, B, C, D, there is no mating reaction, no formation of
pairs. In this period descendant clones show the characters of no mating
type. Later begins a period of partial maturity, in which a few members
of the clone pair with mature members of certain of the mating types.
During the early part of this period of partial maturity there are in
variety 1 only two mating types instead of four. Some of the descendant
clones form pairs with the types A or B, but not with C or D; others
with C or D, but not with A or B. The former may now be said to con-
stitute the type CD, the latter the type AB. Later these two young types
become further differentiated. Some of the clones that thus far do not
react with A or B acquire the power to react with A, but still do not
react with B; these now belong to the definitive mating type B. Others
acquire the power to react with B but not with A; these belong to the
definitive type A. A similar differentiation occurs among the clones of
the young type CD; some of these become type C, others type D. Thus the
types A and B are closely akin, being for a time one type AB; similarly,
types C and D at first constitute one type, CD. The interest of these
gradual changes in maturity and type during vegetative reproduction has
been emphasized in an earlier section (p. 714 above).

762 INHERITANCE

EFFECT OF THE CYTOPLASM AND ITS RELATION TO
NUCLEAR CONSTITUTION

Certain phenomena in the biparental reproduction of ciliates throw
light on the relative role of the nucleus and the cytoplasm in inheritance.
Such phenomena are shown in the inheritance of a number of different
types of characteristics, but are best seen in crosses of individuals of
different inherited sizes, as set forth in the work of De Garis (1935).

By ingenious methods De Garis obtained in Paramecium caudatum

Figure 176. Change of size resulting
from conjugation of individuals of large
and small races, in the experiments of
De Garis. Upper left, A and B, the
large and small individuals of the pair,
showing their relative sizes. The columns
headed A and B respectively show (read-
ing from above downward) the average
sizes of the descendants of the two at
successive intervals of two days each.
(Diagram based on the measurements of
De Garis, 1935.)

<e,
us

Qpqgnnuenere
on nnnnnqnnn

conjugation between races the members of which differed greatly in size.
The nature of the consequences will best be seen from a typical example.

In a certain case one of the members of a pair (A, Fig. 176) belonged
to a race in which the average length of the individuals was 198 microns,
while the other belonged to a small race with an average length of but
73 microns (B, Fig. 176). The large individual of the pair had thus
about twenty times the bulk of the smaller one.

Before conjugation the large and the small sizes are inherited in the
two races; all individuals of race A are large, all those of race B are small.
The two individuals of the different races conjugate and exchange a
haploid set of chromosomes, then separate. The two are now alike in
their nuclei, but are diverse in their cytoplasm—one having the cytoplasm
of race A, the other that of race B. The ex-conjugant A still has the
large size of race A, the ex-conjugant B the small size of race B.

INHERITANCE 763

The large ex-conjugant A now divides by fission. Its two offspring
grow to the large size usual for race A. The small ex-conjugant B di-
vides; its two offspring grow only to the small size usual for race B. The
offspring divide again, and fission continues at the rate of once or twice
a day, each ex-conjugant producing a clone.

As fission continues day after day, it is found that the adult sizes are
changing in each clone. In the clone descended from A, the individuals
of the successive generations grow smaller; in the clone descended from

Figure 177. Changes in mean size of
the descendants of the two members, A
and B, of an unequal pair, in another ive 4 I
cross. At the upper left are shown the
6

relative sizes immediately after conju- A ; 4
gation. Reading from left to right are

shown in the three rows (from above

downward) the successive sizes of the
descendants of the two at intervals of : a i
two days, till at the end of twenty-four

days (lower right) the two have reached
a common small size that is not greatly
different from the original size of B.

(Based on the measurements of De
Garis, 1935.)
24

B, they grow successively larger (Fig. 176). The average sizes of the
two races approach one another. This continues for twenty-two days,
including about the same number of generations. By that time the indi-
viduals in both clones have reached a size that is approximately midway
between the original size of race A and that of race B. At that point,
the changes in size cease; this intermediate size remains constant in the
two clones until there is another conjugation. The two clones having
come to a common size, now form a single race of uniform adult size.

Thus for a long period, twenty-two days, in this case, the size in the
descendants is affected both by the cytoplasm and by the nucleus, but
finally the size is controlled entirely by the nuclear constitution. During
the intervening period the two clones differ in size, and this can be
due only to the difference in their cytoplasm, since they are alike in their
nuclei. The large cytoplasmic body of A is reduced only slowly and
gradually to the new size, and while this is occurring, potentially mil-

764 INHERITANCE

lions of new individuals are produced, all with body size partly de-
pendent on the cytoplasmic constitution. Similarly, the small body of B
is raised to the new size only slowly and gradually through many genera-
tions. The gradual change in size to an intermediate condition, which is
finally the same in both the clones, can be due only to the fact that after
conjugation the two ex-conjugants and their descendant clones are alike
in their nuclei. The nucleus gradually alters the cytoplasm, bringing the

Figure 178. Different ultimate mean
sizes (numbered 1, 2, 3, 4 in each case)
reached by descendants of different pairs
from crosses of the same two races (A
and B, in each case). The results are
shown for four different crosses. To be
read as follows: at the upper left A and
B shows the mean sizes of two races
that were crossed. Four pairs of this
cross gave four stocks of the ultimate
mean sizes shown at 1, 2, 3, 4. Of the
next cross to the right four pairs were
similarly obtained, of the two others but
three. (Based on the measurements of
De Garis, 1935.)

size in both clones ultimately to that which is characteristic for the nu-
clear constitution.

Many such crosses between large and small races were made by De
Garis. In all cases the cytoplasm affects the size for many generations,
but finally the descendants of the two ex-conjugants come to a common
size, corresponding to the common nuclear constitution. The final size is,
however, not always midway between the sizes of the two conjugants of
the pair. In some cases it is much nearer to that of one of the two parental
clones than to that of the other. Such a case is illustrated in Figure 177.
In this, two clones were crossed, in which the mean lengths were re-
spectively 203 microns (clone A) and 81 microns (clone B). After the
gradual change in size through some 24 generations, the ultimate size
reached was much nearer to that of the smaller parent, B. Such results
were seen in many of De Garis’s crosses (see Table 20). The ultimate

INHERITANCE 765

size obviously depends on what nuclear (chromosomal) combination
is present in the two parents after conjugation.

Repeated crosses between the same two clones give different final re-
sults in different cases, just as the same pair of parents in multicellular
organisms produce in different cases offspring that differ in hereditary
constitution. Table 20 shows the different final sizes reached by the de-
scendants in repeated examples of a number of different crosses. Figure

TABLE 20: LENGTHS IN MICRONS OF THE Two RACES CROSSED (DESIG-
NATED A AND B IN EACH CASE) WITH THE RESULTING FINAL LENGTHS
OF THE OFFSPRING, Paramecium caudatum; IN CASES IN WHICH SEVERAL
DIFFERENT Pairs WERE OBTAINED FROM THE SAME Cross, THEIR DE-
SCENDANTS ARE NUMBERED 1, 2, 3, 4 (DATA FROM DE Garis, 1935)

ae Mean Lengths of the Descendants of the Different Pairs

I 2 3 4
203 X 81 143 191 9 =e
191 X 175 64 139 ae ase
217 X 75 170 133 170 215
172 X 172 165 132 183 137
TST OL 114 119
201 X 73 69 7
207 X 75 133 200
201 X 198 165
198 X 73 142
152) OG 152 145
198 X 108 204
ToS eS ve

178, drawn to scale, shows graphically the relative sizes of descendants
of different pairs in certain crosses.

The fact that different pairs of conjugants from the crossing of the
same two clones give descendants of different sizes indicates that in these
Protozoa, as in Metazoa, recombination of the chromosomal materials
occurs, giving different combinations in different cases. It shows also
that clones of P. caudatum must in many cases be heterozygotic for size
factors; otherwise different results would not be produced from different
pairs of the same cross.

The fact that the descendants of the two ex-conjugants of any pair
yield descendants that are finally of sensibly the same size shows that

766 INHERITANCE

reduction of the chromosomes, so far as factors for size are concerned,
must occur at the first or the second of the maturation divisions, not at
the third. The fact that they are of the same final size shows that the two
descendant clones have the same factors for size. This can occur only if
the third maturation division, producing the two pronuclei, is non-
reductional. In that case the two pronuclei of an individual are neces-
sarily alike, and only when this is so will the nuclear combinations be
the same in the two ex-conjugants. If reduction occurred at the third
maturation division, the two pronuclei would often be diverse, leading
to different final sizes.

In summary, it may be said that the final size of the descendants of the
ex-conjugants is determined by the nuclear constitution, as is shown by
the fact that the final sizes are the same for the clones derived from the
two conjugants of a pair. But for a long time, for many vegetative
generations, the nature of the cytoplasm affects the size of the descend-
ants; descendants with cytoplasm of different constitution remain for
long periods diverse. The longest period observed by De Garis during
which the diversity of cytoplasm persisted was thirty-six days, which
would mean about the same number of vegetative generations.

A similar differential effect of the cytoplasm is seen in the inheritance
of other characteristics. Sonneborn and Lynch (1934), before the work
of De Garis on size, observed such a cytoplasmic effect in crosses of clones
that differed much in the rate of multiplication. If before conjugation
one of the clones multiplies rapidly, the other slowly, after conjugation
the clone that receives the cytoplasm from the rapid race continues for
a time to multiply rapidly, while the other, receiving its cytoplasm from
the slow race, continues to multiply slowly. This effect of the diverse
cytoplasms continues for about ten vegetative generations. But during
that time the difference in fission rate for the two ex-conjugant clones
gradually disappears, till at the end of the period the rate of fission is
the same in the two. For example, the two clones N 21 and B were
crossed by Sonneborn and Lynch. The mean daily fission rate in N 21
was 0.67, while in B the fission rate was 1.97, three times as great as in
N 21. During the first five days after conjugation, the daily rate for the
clones that had received cytoplasm from the slow race N 21 was 1.10;
for those that had received cytoplasm from the fast race, B, the daily
rate was 2.00. In the second five-day period the rates were respectively

INHERITANCE 767

1.6 and 1.8. After an interval of twelve days the rates were taken again.
They were now the same in the two ex-conjugant clones; during seven-
teen days they stood for both at 2.0 to 2.5 fissions daily. Other crosses
between fast and slow clones showed similar conditions.

The effect of the cytoplasmic constitution in thus delaying the as-
sumption of the final characteristics resulting from the nuclear consti-
tution is commonly known as the “‘cytoplasmic lag.”

A cytoplasmic lag of a similar sort is at times seen in the inheritance
of mating type at endomixis in exceptional clones of P. awrelia, as de-
scribed by Kimball (1939). Normally in both P. aurelia and P. bursaria
no such lag is evident, unless it be in the fact that there is a period of
immaturity during which no mating reaction occurs. The cases described
by Kimball are in certain clones of variety 1 of P. avrelia. These clones
were of the mating type II. At endomixis some of these are transformed
to type I, as before set forth. But for a few generations after the forma-
tion of the new nucleus, they remain of type II, and will still conjugate
with individuals of type I. After these few fissions, however, the new
nucleus asserts itself and the members of the clone transform to type I;
they now conjugate only with type II. The animals thus, after the forma-
tion of the new nucleus, remain for a time of the mating type appropriate
to the cytoplasmic constitution only; then transform to correspond to the
new nuclear constitution.

These phenomena illuminate the rdle of the cytoplasm in inheritance.
At every fission the volume of the cytoplasm present in any individual
is reduced one half; then the original volume is restored by new growth.
Thus, in the case of the ex-conjugant of the large race A of Figure 176,
the descendants of which are gradually diminished in size after conjuga-
tion, the size of the individual is reduced at the first fission after conju-
gation to one half the original size, so that if growth did not occur the
body would in two or three generations be reduced to the final size.
But owing to the properties of the cytoplasm of that race A, growth oc-
curs after the first fission to practically the original racial size. At every
succeeding fission the original cytoplasm is again diluted one half, so
that after ten generations, it has been diluted to less than one-thousandth
part, the remainder being new cytoplasm produced by growth. Yet after
ten generations the nature of the original cytoplasm still has a marked
effect on size. The original cytoplasm must therefore have to some extent

768 INHERITANCE

the power of reproducing itself in its distinctive nature, at the time that
growth occurs. In this respect it partakes of the character of genetic ma-
terial, since it shows the two distinctive features of that material: it affects
the characteristics of the individuals, and it reproduces itself in some
degree true to type. But in time it is made over by the new nucleus.
These facts as to the differential effect of the cytoplasm on inherited
characteristics in crosses furnish a basis in normal genetics for the idea
of Jollos, set forth in a previous section, that inherited environmental
modifications may have their seat in the cytoplasm. These, like the size
due to the nature of the cytoplasm in the crosses made by De Garis, are
inherited for a number of generations, but finally fade away. But there
is so great a difference in the time, the number of generations, during
which the inheritance continues, in the two cases, that it raises doubts
as to the fundamental similarity of the two. In the crosses, the inherited
cytoplasmic effect continues in different cases for ten, twenty, thirty
generations, the extreme limit observed being thirty-six generations. By
the end of such a period, the cytoplasm has been made over by the
nucleus, so that it is thereafter the constitution of the nucleus that de-
termines the characteristics. But such experimental modifications as
acclimatization are inherited for hundreds of generations. If they are
merely modifications of the cytoplasm, it might be anticipated that long
before so many generations had passed the cytoplasm would have been
made over by the nucleus, so that its modifications would have disap-
peared. Yet of course it is not certain that the time required for the
nucleus to dominate the cytoplasm would be subject to similar limits in
all cases. Here much remains to be discovered. The question whether
inherited environmental modifications are exclusively cytoplasmic, or
whether they affect the chromosomal materials of the nucleus, must be
left open for the present. Decision of this question appears practicable
by experimental breeding. What is required is to induce environmental
modifications in a clone, then to cross this clone with another which
lacks the modification. In conjugation only nuclei with their chromosomal
materials are transferred from one clone to the other. If the modifications
have affected the nuclei, they should be transferred by conjugation from
the modified clone to the unmodified one. But if they affect only the
cytoplasm, they will not thus be transferred. The prospects for success-
fully carrying through such experiments have been greatly increased by

INHERITANCE 769

the recent discovery of diverse mating types in these organisms. This
discovery renders it possible to make any desired crosses as readily in
these organisms as in fruit flies or in rats.

In general terms, the relations of nucleus and cytoplasm to inheritance,
revealed in crosses in the ciliates, may be expressed as follows. The
primary source of diversities in inherited characters lies in the nucleus.
But the nucleus by known material interchanges impresses its constitu-
tion on the cytoplasm. The cytoplasm retains the constitution so im-
pressed for a considerable time, during which it assimilates and repro-
duces true to its impressed character. It may do this after removal from
contact with the nucleus to which its present constitution is due, and
even for a time in the presence of another nucleus of different consti-
tution. During this period, cytoplasmic inheritance may occur in vegeta-
tive reproduction. The new cells produced show the characteristics due
to this cytoplasmic constitution impressed earlier by a nucleus that is no
longer present. But in time the new nucleus asserts itself, impressing its
own constitution on the cytoplasm. Such cycles are repeated as often as
the nucleus is changed by conjugation.

LITERATURE CITED

An extensive bibliography of contributions on the genetics of Protozoa prior to
1929 will be found in the author’s Genetics of the Protozoa (1929). The present list
includes only articles referred to in the foregoing chapter.

Calkins, G. N. 1919. Uroleptus mobilis Engelm. II. Renewal of vitality
through conjugation. J. exp. Zool., 29: 121-56.

Dallinger, W. H. 1887. The president’s address. J. R. micr. Soc., 1: 185-99.

De Garis, C. F. 1935. Heritable effects of conjugation between free individuals
and double monsters in diverse races of Paramecium. J. exp. Zool., 71:
209-56.
Diller, W. F. 1936. Nuclear reorganization processes in Paramecium aurelia,
with descriptions of autogamy and “hemixis.” J. Morph., 59: 11-67.
Dobell, C. 1924. The chromosome cycle of the sporozoa considered in relation
to the chromosome theory of heredity. Cellule, 35: 169-92.

Erdmann, R. 1927. Endomixis bei Paramecium bursaria. S. B. Ges. Naturf. Fr.
Berl, 19252 24-25.

Hammerling, J. 1929. Dauermodifikationen. Handbuch der Vererbungswissen-
schaft (Baur u. Hartmann), Bd. 1, Liefrg. II: 1-69.

Jennings, H. S. 1916. Heredity, variation and the results of selection in the

uniparental reproduction of Défflugia corona. Genetics, 1: 407-534.

1929. Genetics of the Protozoa. Bibliogr. genet., 5: 105-330.

770 INHERITANCE

—— 1938. Sex reaction types of their interrelations in Paramecium bursaria.
Proc. nat. Acad. Sci. Wash., 24: 112-20.

—— 1939a. Genetics of Paramecium bursaria. 1, Mating types and groups,
their interrelations and distribution; mating behavior and self-sterility.
Genetics, 24: 202-33.

— 1939b. Paramecium bursaria: Mating types and groups, mating be-
havior, self-sterility; their development and inheritance. Amer. Nat.,
73: 414-31.

Jennings, H. S., D. Raffel, R. S. Lynch, and T. M. Sonneborn. 1932. The
diverse biotypes produced by conjugation within a clone of Paramecium.
J. exp. Zool., 63: 363-408.

Jollos, V. 1913. Experimentelle Untersuchungen an Infusorien. Biol. Zbl.,
335 222-56.

— 1920. Experimentelle Vererbungsstudien an Infusorien. Z. indukt.

Abstamm.- u. VererbLehre., 25: 77-97.

1921. Experimentelle Protistenstudien. I. Untersuchungen tber Vari-

abilitat und Vererbung bei Infusorien. Arch. Protistenk., 43: 1-222.

1924. Untersuchungen tiber Variabilitét und Vererbung bei Arcellen.

Biol. Zbl., 44: 194-208.

— 1934. Dauermodifikationen und Mutationen bei Protozoen. Arch.
Protistenk., 83: 197-219.

Kimball, R. F. 1937. The inheritance of sex at endomixis in Paramecium
aurelia. Proc. nat. Acad. Soc. Wash., 23: 469-74.

1939. A delayed change of phenotype following a change of genotype

in Paramecium aurelia. Genetics, 24: 49-58.

Maupas, E. 1889. La Rajeunissement karyogamique chez les ciliés. Arch. zool.
exp. Sen.,, (2))5 7 lao ie

Moewus, F. 1932. Untersuchungen iiber die Sexualitat und Entwicklung von
Chlorophyceen. Arch. Protistenk., 80: 467-526.

—— 1934a. Uber Subheterézie bet Chlamydomonas eugametos. Arch. Pro-
tistenk., 83: 98-109.

— 1934b. Uber Dauermodifikationen bei Chlamydomonaden. Arch. Pro-
tistenk., 83: 220-40.

— 1935a. Uber den Einfluss ausserer Faktoren auf die Geschlechtsbestim-
mung bei Protosiphon. Biol. Zbl., 55: 293-309.

— 1935b. Uber die Vererbung des Geschlechts bei Polytoma pascheri und
bei Polytoma uvella. Z. indukt. Abstamm.- u. VererbLehre., 69: 374-417.

—— 1935c. Die Vererbung des Geschlects bei verschiedenen Rassen von
Protosiphon botryoides. Arch. Protistenk., 86: 1-157.

— 1936, Faktorenaustausch, insbesondere der Realisatoren bei Chlamyo-
monas-Kreuzungen. Ber. dtsch. Bot. Ges., 54: (45)-(57).

—— 1937a. Die allgemeinen Grundlagen der Sexualitat. Biologe, 6: 145-51.

—— 1937b. Methodik und Nachtrige zu den Kreuzungen zwischen Poly-

INHERITANCE 771

toma-Arten und Zwischen Protosiphon-Rassen. Z. indukt. Abstamm.-

u. VererbLehre., 73: 63-107.

1938. Vererbung des Geschlechts bei Chlamydomonas eugametos und

verwandten Arten. Biol. Zbl., 58: 516-36.

Neuschloss, S. 1919, 1920. Untersuchungen tiber die Gew6hnung an Gifte.
I, II, Ill. Pfliig. Arch. ges. Physiol., 176: 223-35; 178: 61-79.

Philip, N., and J. B. S. Haldane. 1939. Relative sexuality in unicellular algae.
Nature, 143: 334.

Raffel, D. 1930. The effects of conjugation within a clone of Paramecium
aurelia. Biol. Bull., 58: 293-312.

Reynolds, B. D. 1923. Inheritance of double characteristics in Arcella polypora
Penard. Genetics, 8: 477-93.

Sonneborn, T. M. 1936. Factors determining conjugation in Paramecium
aurelia. 1. The cyclical factor: the recency of nuclear reorganization.
Genetics, 21: 503-14.

— 1937. Sex inheritance and sex determination in Paramecium aurelia.
Proc. nat. Acad. Sci. Wash., 23: 378-85.

— 1938a. Sex behavior, sex determination and the inheritance of sex in
fission and conjugation in Paramecium aurelia. Genetics, 23: 168-69.
1938b. Mating types, toxic interactions and heredity in Paramecium

aurelia, Science, 88: 503.

1938c. Mating types in Paramecium aurelia; diverse conditions for mat-
ing in different stocks; occurrence, number and interrelations of the
types. Proc. Amer. phil. Soc., 79: 411-34.

—— 1939. Paramecium aurelia: Mating types and groups, lethal interactions,
determination, inheritance, relation to natural selection. Amer. Nat., 73:
390-413.

Sonneborn, T. M., and R. S. Lynch. 1934. Hybridization and segregation in
Paramecium aurelia. J. exp. Zool., 67: 1-72.

Taliaferro, W. H. 1929. The immunology of parasitic infections. New York
and London.

Tartar, V., and T. T. Chen. 1940. Preliminary studies on mating reactions of
enucleate fragments of Paramecium bursaria, Science, 91: 246-47.

CHAPTER XVI

THE PROTOZOA IN CONNECTION WITH
MORPHOGENETIC PROBLEMS

FrRANcIS M. SUMMERS

THE QUEST for underlying causes of organic unity or individuality has
been for many years one of the most intriguing and, at the same time,
one of the most evasive problems in experimental biology. That an im-
mense amount of work, involving a large variety of organisms, has pro-
duced little more than a background for what may be the ultimate
solution is neither surprising nor discouraging. If analytical research
cannot now detail all of the vital processes, then what progress should
be expected in the full appreciation of the total relations between them?

One of the principal approaches to the evaluation of factors which
condition growth and development is through studies of regeneration.
This method consists essentially in disturbing the normal trend of de-
velopment in order to obtain unusual or exaggerated expressions of one
or more of the growth-conditioning or integrating factors. The fact that
a fragment of an organism is frequently capable of regenerating a com-
plete organism the total characteristics of which are homologous with
those of the original is convincing evidence that some kind of funda-
mental organization characterizes the fragment and is responsible for the
individuality of the new organism derived from it. Conversely, regional
specialization or localization in the regenerating fragment presupposes
the existence of overlying control mechanisms, i.e., the operation of
factors or a set of energy transformations which restrict, direct, or deter-
mine the complete developmental freedom of its various parts or regions.
If some such hypothesis of energy changes and translocations serves to
fix our experimental point of view, then the many fragmentary accounts
and dangling issues herein reported may serve as a challenge to those
who are acquiring the newer instruments and techniques provided by the
sciences.

In common with other organisms, the Protozoa exhibit such features

MORPHOGENESIS TI

as polarity and symmetry, which, according to Child (1920), appear to
be largely independent of specific differences in the protoplasmic con-
stitution. Should not the Protozoa, either as cells or organisms, present
vety suitable material for studying the potentialities of the protoplasmic
constitution in a somewhat less complex setting than that which charac-
terizes the higher invertebrates? Furthermore, does the disadvantage of
the operative techniques involved with Protozoa offset the advantage of
being able to disregard some of the physiological and mechanical rela-
tionships between the cells of compact tissues? The answers to these
and other questions relative to the significance of Protozoa in connection
with problems in regeneration, or their merits as material for investi-
gating these problems, are to be attempted in this section.

It is the purpose of this section to organize under special headings the
unassembled work having to do with regeneration phenomena in
Protozoa. Microdissection or micrurgical studies which have as an ob-
jective the elucidation of the physical properties of protoplasm, the
functions of fibrillae, membranes, and so forth, are to be treated only
insofar as they have a bearing upon the topics under consideration.

PHYSIOLOGICAL , REGENERATION

One aspect of regeneration pertains to those functions by which cells
or organisms are able to maintain a certain structural and functional in-
tegrity, in spite of wear-and-tear processes of the normal life cycle. The
maintenance functions are commonly referred to as physiological re-
generation and they appear to have much in common with reparative
regeneration.

As applied to Protozoa, particularly the Infusoria, the term has an
added significance because it should be taken to include not only the
gradual and continuous energy changes within the protoplasm, but also
the grosser reorganizational changes encountered during periods of di-
vision, conjugation, endomixis, and encystment. In ciliates and flagel-
lates generally, the structures observed to be affected include particularly
the nuclei, the external motor organellae, and their associated fibrils.

It is beyond the scope of this section to consider those repair phe-
nomena which follow the normal vegetative or cyclical variations in
Protozoa. Of especial interest in this connection, however, is the “spon-
taneous” dedifferentiation and redifferentiation described for Bursaria

774 MORPHOGENESIS

by Lund (1917). Slightly starved Bwrsaria frequently undergo exten-
sive reorganizational changes distinct from those which accompany divi-
sion, conjugation, and so forth. According to Lund, this distinctive type
of physiological regeneration has no apparent extrinsic cause. That such
is the case seems improbable. Hetherington (1932) cultivated Stentor
coeruleus under mote carefully controlled conditions and was unable to
find cases of spontaneous regeneration except when the environment
was unfavorable. Starvation, wide variations in oxygen tension and or-
ganic content of the medium are apt to initiate these reorganizational
changes. Hetherington maintains that physiological regeneration occurs
very infrequently, if at all, and that it has never been demonstrated in
a known medium. Both Lund and Hetherington are inclined to employ
the term physiological regeneration in a special sense, not including the
cyclical changes already mentioned.

Periodic reorganizations during periods of partial or complete starva-
tion in Stylonychia mytilis were recently reported by Dembowska (1938).
In conductivity water Stylonychia lives for fourteen to nineteen days.
During this time the organisms undergo repeated processes of complete
body reorganization by renewing the entire ciliature and by nuclear re-
organization. The reorganizations are increasingly frequent up to a cer-
tain point and are unaccompanied by manifestations of division.

SOME OF THE FACTORS IN REGENERATION

1. EXTERNAL ENVIRONMENT

Calkins (1911b) and especially Peebles (1912) noted a definite cor-
relation between the regenerative behavior of Paramecium caudatum and
the periods of depression to which their cultures were subject. During
such periods pieces cut from organisms, which under more favorable
conditions exhibited a greater capacity to regenerate, were unable to
regenerate or to divide. These authors concluded that when paramecia
are starved or are undergoing periods of depression from other causes,
the division rate is greatly diminished and the reparative activities greatly
reduced or altogether lost.

Diminutive but perfectly formed regenerates were obtained when
Sokoloff (1923) cut slightly starved Bursaria into several parts. These
regenerates were proportionate in size to the pieces from which they
were derived. In other words, within reasonable limits, inanition in-

MORPHOGENESIS WT

hibits growth but does not impair the ability to regenerate; the latter
remains the same as for normally fed specimens. Extreme starvation
leads to a state of depression in which fragments attempt but never
achieve full structural restitution. The cases of physiological reorganiza-
tion, as reported by Lund (1917) and Hetherington (1932) for Bur-
saria and by Dembowska (1938) for Stylonychia, confirm the validity
of Sokoloff’s conclusion.

Chejfec (1932), following Balbiani (1893), Calkins (1911b), and
Peebles (1912), accepted the possibility of a low potential for regenera-
tion in Paramecium caudatum. He attempted to increase the regenerative
power in this species by altering the environment in several ways. The
experimental organisms, placed under conditions of starvation or in
acidified media (initial pH 4.5-6.0), regenerated more readily than the
control group grown in ordinary hay infusion.

A variety of alkaloids such as morphine, strychnine sulphate, and so
forth, when administered in sublethal concentrations to Blepharisma
undulans, cause the pink-colored pellicle to be discarded (Nadler, 1929).
Animals so treated readily regenerate new pellicles if returned to the
customary medium. If they are left in the alkaloid-containing medium,
the pellicle does not reappear. Naked but otherwise normal organisms
were maintained in this fashion for 110 days by Nadler.

Since reparative processes are set in motion by unfavorable alterations
of an organism or the medium in which it lives, almost the entire sub-
ject of regeneration could be exhaustively but not effectively treated
under this heading. The many other pertinent publications are discussed
throughout the chapter.

2. CYCLICAL VARIATIONS

Division cycle-—The experiments of Calkins (1911a) on Uronychia
transfuga wete designed to test whether or not Protozoa are capable of
regenerating with equal facility during the various phases of cell division.
It appears that prior to that time regeneration was generally assumed to
be independent of cyclical phenomena. Operations performed on Uro-
nychia shortly after cell division showed the regenerative capacity of the
individual to be feebly developed. The presence of both macronucleus
and micronucleus was essential for such cases of regeneration as did
occur at this period. At the mid-interphase, eight to sixteen hours after

776 MORPHOGENESIS

division, the tendency to regenerate was slightly greater than before.
These fragments sometimes regenerated in the absence of micronuclear
material. The highest percentage of successful regenerates was obtained
as the cells entered upon the division period. When Uronychia was cut
in the early prophase, either transversely or obliquely in a region anterior
to the presumptive division plane, three complete individuals were pro-
duced (Fig. 179). Three individuals instead of two resulted in such
cases, because the predetermined division plane was not appreciably

Figure 179. Regeneration in Uronychia transfuga. A, transection made anterior to the
division plane in mid-division phase (B, C, and D show the results after twenty-four
hours) ; B, small amicronucleate individual derived from the anterior fragment; C and D,
anterior and posterior daughters produced when the large posterior fragment divided.
(From Calkins, 1911a.)

altered by the type of cut just described. The small amicronucleate an-
terior piece regenerated a diminutive organism whose morphological
features were normal. Fission occurred in the large posterior fragment
soon after the healing of the cut surface. A small anterior regenerated
daughter and a normal posterior daughter were the products, both of
which were normal as regards nuclear apparatus. The regenerative power
diminished in the mid-division phases, inasmuch as one or another of
the three cells frequently failed to regulate. As the division process
drew to a close, reparative regeneration reached its lowest ebb; those
which did regenerate were low in vitality and short-lived. In regard to

MORPHOGENESIS 777

the progressive development of regenerative power from one division
to the next, Calkins’s experiences with Uronychia have been repeated and
reaffirmed by D. B. Young (1922) and Dembowska (1926). Similar
results were also reported by M. E. Reynolds (1932) for an amicro-
nucleate race of Oxytricha fallax.

To the contrary, in Spathidium there appears to be no correlation be-
tween the degree of regeneration and the division cycle. Before, during,
and after fission, small pieces excised from either extremity disintegrate
immediately or regenerate without dividing. Large nucleated fragments
nearly always regenerate, irrespective of the stage at which they are
taken (Moore, 1924).

Peebles (1912) cut Paramecium caudatum at two different division
stages without discovering any marked differences in the regenerative
ability. Both halves of those which were transected in the division plane,
at a time when the macronucleus was elongated and the body slightly
constricted, usually survived to produce normal descendants. Traumatic
effects were reduced in operations made during the later stages. Peebles
also made note of the fact that while the power of regeneration is present
in cells obtained from two to five hours after separation, approximately
90 percent of the operated individuals died as a result of injury. This
was attributed to lowered viscosity in the cytoplasm of growing para-
mecia, such that an injury to the ectosarc allows the endoplasm to escape.
The surviving organisms regenerated as readily as those cut in the late
interphase.

Taking exception to Calkins’s statement that the “power of regenera-
tion” varies in different stages of the division cycle, Tartar (1939)
restates the problem in terms of the observed data, without reference to
the “‘power’’ of regeneration. Calkins’s experiments
reveal that specimens of Uronychia transfuga ate able to regenerate mor-
phologically without the presence of the micronucleus when the transection
removes this structure before or during division, and not when it is removed
after division. The fate of the amicronucleate fragments was apparently not
followed long enough to determine to what extent subsequent division is
possible in the absence of the micronucleus, but it is probable from the work
of others (e.g. Moore, 1924) that division would not have taken place
without the micronucleus. It is for this reason that my restatement of Calkins’

results restricts the restoration to morphological regeneration. A corollary of
the statement is that when a fragment contains both nuclei, it regenerates

778 MORPHOGENESIS

regardless of the time of cutting during the division cycle. Calkins presented
exceptions to this corollary: fragments from cells cut immediately after division
which contained both types of nuclei did not regenerate. A more complete
history of fragments than he presented would, however, be necessary to
ascertain whether their failure was due to a decrease in the “power of regenera-
tion” or merely to unsuccessful recovery from the operation. These cases seem
to indicate that just after division animals may fail to regenerate even though
they contain the full nuclear complement; but such failures were certainly
the exception and not the rule (four cases out of twenty-two), so that demon-
stration of a fundamental decrease in the ‘power of regeneration’”’ during
this period, exclusive of the subtraction of one of the nuclei, was not in-
tended [p. 199}.

From data including only fifty operations made during and after
division in one race of Paramecium caudatum, of which twenty-four suc-
cessfully regenerated and subsequently divided, Tartar concluded that
the division cycle in this species does not influence regeneration. even
when both nuclei are present.

Evidence of progressive physiological differentiation between divi-
sional periods, other than the increasing ability to regenerate, is the
determination of the division plane. Lewin (1910), Calkins (1911b),
and Peebles (1912) have repeatedly demonstrated its occurrence in P.
caudatum,; other amply described cases are those of Uronychia (Calkins,
1911a; D. B. Young, 1922; Dembowska, 1926) and Oxytricha (M. E.
Reynolds, 1932). All of these species may be transected above or below
the mid-region sometime prior to cell division, without injury to this
definitive region. As already described for Uronychia, the larger piece
divides unequally through what was originally the mid-region of the
intact cell. Peebles identified the division plane as early as 2.5 hours
after fission. She thought that several division planes develop in vegeta-
tive cells when fission has been delayed for a time. This would account
for the fact that several divisions follow in rapid succession when one of
the extremities is cut away from such an individual.

Conjugation —Four giant races of Paramecium caudatum wete fe-
ported by Calkins (1911b) as having different regeneration potentials.
The incidence of regeneration in three of the races, those having very
restricted regenerative powers, was found to be greater in ex-conjugants,
or in cells that were operated during conjugation, than in the ordinary
vegetative individuals.

The merotomy studies on Urole ptus mobilis (Calkins, 1921) proved

MORPHOGENESIS 779

that conjugating pairs may be cut apart without perceptibly altering the
trend of events, which, once started by conjugation stimuli, continue to
completion. The regeneration requirements, when superimposed upon
the internal readjustments already in progress as a consequence of the
sexual processes, appear only to prolong redifferentiation in the ex-con-
jugants. Calkins dissected away the apical protoplasmic junction con-
taining the migratory pronuclei and thereby eliminated the amphinucleus
in both conjugants; nevertheless the resulting cells regenerated complete-
ly and with full restoration of their vegetative and reproductive powers.
Although cytological details are not supplied in this article, as seen in
the living material the reorganization processes without exception fol-
lowed the same general sequences as outlined for the normal ex-con-
jugants (Calkins, 1919).

In P. caudatum, anterior or posterior cut-offs made during conjuga-
tion result in a large number of fatalities. Those which survive regener-
ate slowly and eventually divide (Peebles, 1912).

Cases of autogamy following the separation of conjugants were re-
cently described by Poljansky (1938) for Bursaria. The processes of
sexual differentiation continue in the majority of individuals derived
from pairs split apart four to six hours after the onset of conjugation.
In these instances autogamy supplants heterogamy; the two native pro-
nuclei of each cell fuse to produce the amphinucleus, from which the
nuclear apparatus of the reorganized cell originates.

A somewhat different situation prevails in Spathidium. Tests for re-
generative ability were made at three different phases of the conjugation
process by Moore (1924). The fragments obtained while maturation is
in progress, but before the exchange of pronuclei, do not regenerate.
Those obtained immediately after fertilization regenerate fully, pro-
vided the amphinucleus is included. Ex-conjugant pieces containing only
the degenerating macronuclei and the maturation by-products achieve a
partial restoration of form, but they subsequently dedifferentiate and
never divide.

Encystment.—Slightly starved, pre-cystic individuals of Spathidium
spathula are capable of regulation in a fair percentage of cases. The
usual consequence of cutting at this stage is immediate dedifferentiation
and encystment. Interestingly enough, the external form of a regenerator
may be restored before encystment, but nevertheless this cell dedifferenti-
ates and then encysts in order to complete the nuclear reorganization

780 MORPHOGENESIS

already in progress. Young excysted specimens have a lowered regenera-
tive capacity because physiological restoration is incomplete; with in-
creasing age, the normal vegetative existence is resumed and the re-
generative capacity is restored to the normal level (Moore, 1924).

Recently Garnjobst (1937) described an interesting case of what she
called regeneration cysts in Sty/onethes. Anterior and posterior halves of
bisected organisms round up and secrete a cyst wall about themselves.
Regeneration occurs within the cysts. Excystment is spontaneous, re-
leasing minute but perfectly formed individuals. Garnjobst maintained
these excysted regenerates until they formed reproductive cysts, the nor-
mal condition for binary fission in the species.

3. RACIAL DIFFERENCES

The amount of literature on genetics and regeneration is pitifully
small. Perhaps this is due, in part, to the difficulty of making clear-cut
distinctions between the total inherent capacities and the strictly extrinsic
factors which are responsible for variations in the expression of these
capacities.

In contrast with Balbiani’s (1893) conclusion that Paramecium
caudatum does not regenerate as do other Protozoa, Calkins (1911b)
found that the power of regeneration varied in different giant races of
P. caudatum, Of these races for which data are given, one produced
regenerates in approximately one percent of the cases; another race
produced 10 percent; and a third produced 30 percent. Mention is made
of a fourth race, which showed 100 percent regeneration (data not
given). Peebles (1912) likewise reported four races of P. caudatum
which, according to her conclusions, showed wide variations in regenera-
tive power. Regeneration in the different races varied from 23 percent
to 67 percent for anterior cut-offs and from 25 percent to 100 percent
for posterior sections (her Table 3, p. 164). In view of her generaliza-
tions that ‘Paramecium taken from a pure line will regenerate in ninety
cases out of a hundred if the cytoplasm is in a viscid state and the ant-
mals are well-fed” (p. 165) and that “The power to regenerate is not
so much a characteristic of the race as it is an indication of the vitality of
the individual cell” (p. 165), the data tabulated in percentages only do
not distinguish the racial from the individual differences.

M. E. Reynolds (1932) described experiments with a non-regenerat-

MORPHOGENESIS 781

ing species (undescribed) of Explotes. The fact that E. patella regener-
ated very well under the same conditions led her to the conclusion that
“under identical conditions there is a difference in the regenerative
power of these two races of Euplotes” (p. 353). It should be noted that
none of the undescribed species survived the operations; all distinte-
grated during or immediately after the operation.

Of particular merit is the work of Tartar (1939), in which rigid
criteria were set up for distinguishing between recovery and regenera-
tion and between form restoration and complete regeneration. Twenty-
five races, representing seven species, were used in the experiments to
determine whether there are racial or specific differences in the regenera-
tive ability of Paramecium. A total of 865 anterior transections were
performed, in 509 of which the organisms survived. Morphological and
complete physiological regeneration occurred in 98 percent of the sur-
vivals. Having established from minor operations the working hypothesis
that ‘‘any Paramecium able to recover from the injury of cutting is able
to regenerate completely” (p. 196), Tartar performed operations of a
more serious character—excisions involving the major portion of the
posterior end—upon five races of P. caudatum. Complete regeneration
occurred in 93 percent of the 121 survivals. According to these results,
the ability of Paramecium to regenerate is much greater than previous
investigations have shown and, furthermore, the regional differences in
individuals of the same species are relatively slight. The final conclu-
sion that there is no racial nor species variation in regeneration of cells,
even after quite severe cuts, may be subject to further qualification. Al-
though the compiled data show no large order variations in the incidence
of regeneration among the different species or races of Paramecium,
there remains the possibility of genetic differences in the capacity for
regeneration after repeated injuries or in the rate of regeneration.

4, DEGREE OF INJURY AND REORGANIZATION

In many Protozoa the extent of cytoplasmic dedifferentiation varies
with the degree of injury; but is does not necessarily follow that a small
or even a large operation will be followed by complete resorption and
reintegration of the cell structures. In holotrichs, Loxophyllum for exam-
ple (Holmes, 1907), the mode of restoration may be simple and direct,
with the alteration of uninjured, preéxisting parts reduced to a minimum.

782 MORPHOGENESIS

Differences in the degree of reorganization in anterior and posterior
halves of Spathidium spathula are clearly described by Moore (1924).
In this species the alterations prior to restoration do not follow the same
course, but vary with the size of the fragment and with the condition of
the organism at the time of cutting. She observed no instance in which
the cytostome remained entirely unchanged throughout the course of
regeneration in anterior halves. Shortly after the cut surface heals, an-
terior pieces begin to round out at the apex. The assumption of spherical
form extends to the oral region, the oral parts become less distinct, the
neck disappears completely, and finally only an indication of the oral
lips is apparent. Occasionally dedifferentiation proceeds further, the frag-
ment forming a complete sphere with all traces of the oral apparatus
totally obliterated before redifferentiation sets in. The posterior halves
require a longer time for the restoration of form, although they contain
none of the original oral parts to be resorbed and remodeled.

Heterotrichs are not strikingly different from holotrichs in regard to
the extent of dedifferentiation following injury. The greater the relative
size of the regenerator in such forms as Spirostomum (Sokoloff, 1923),
the greater its regenerative capacity, and the sooner are such parts re-
stored to complete, full-size individuals. According to Moore (1924),
the oral structures in anterior pieces of Blepharisma do not dedifferenti-
ate unless they are injured when the cell is operated upon. A somewhat
similar condition obtains in Bursaria (Lund, 1917). In this heterotrich
the An/age of the gullet in reorganizing individuals may be torn slightly
without provoking complete dedifferentiation. The injured region per-
sists as an abnormal part of the gullet, which is normal in all other
respects. This appears to be a clear-cut demonstration of embryonic
localization as found in metazoan embryogeny, a definite spatial corre-
spondence between the undifferentiated and the fully formed parts. In
Bursaria, however, there is considerable variation in the degree of injury
required to produce more drastic regenerative measures. If sufficiently
great, an injury to the gullet rudiment causes complete dedifferentiation,
such that all visible traces of the rudimentary structure disappear before
redifferentiation begins. The regenerative processes in hypotrichs tend
to obey an all-or-none rule for, once initiated by a requisite degree of
injury, the dedifferentiation proceeds to completion. It 1s noteworthy,
moreover, that minimal injuries to certain of the motor organelles will

MORPHOGENESIS 783

provoke reorganizational activities as far-reaching as those which occur
at the time of division.
Taylor and Farber (1924) removed small drops of endoplasm from

Figure 180. Regeneration in Uronychia uncinata. A, shallow marginal incision which
did not injure any of the motor organelles, the healing of the wound was not accom-
panied by cell reorganization; B, deep incision not involving motor organelles, fol-
lowed by complete reorganization; C, excision involving the large posterior cirri, fol-
lowed by complete reorganization; D, excision of anterior portion, including several
membranelles, followed by complete reorganization. (From Taylor, 1928.)

Euplotes patella without producing serious structural changes through
dedifferentiation, although death invariably occurred within two to three
days, if the micronucleus was taken out. In this and a good many other
species, shallow marginal incisions, or wounds from small excisions, heal

784 MORPHOGENESIS

almost immediately, whereas large-scale operations involving some of
the kinetic organelles are apt to incite breakdown changes that lead to
the resorption of the entire ciliary apparatus (Fig. 180). Extreme cases
were reported by Dembowska (Stylonychia, 1925; Uronychia and Sty-
lonychia, 1926) and Taylor (Uronychia, 1928) in which the removal
of a single cirrus or even severe injury to its basal plate is sufficient to set
in motion the entire regenerative functions. External portions of the
giant citrt on Uronychia may be excised with none of these consequences
(Taylor).

For those hypotrichs studied by Dembowska (1926) and M. E. Rey-
nolds (1932), the duration of the regenerative process is independent
of the degree of injury, requiring from three to five hours for complete
restitution in all cases. The time interval between cutting and the initia-
tion of reorganization varies for different genera. Generally, the greater
the injury, the shorter the interval.

Tittler’s (1938) experiments on Uroleptus mobilis were designed to
test whether or not ciliates injured or mutilated by high-tension currents
undergo reorganization and regeneration comparable to that which fol-
lows other types of mutilation. The induction current caused most of
the organisms to migrate toward the cathode of the break shocks. Ap-
proximately 75 percent of the exposed organisms were vacuolated or
deformed at the posterior end, i.e., the extremity nearest the anode. One-
minute exposures sufficed to produce many fragments with posterior de-
ficiencies and a few with anterior injuries. Regeneration, accompanied by
a complete de- and redifferentiation of cortical organelles and macro-
nuclear reorganization, was the rule. Most of the reconstituted individuals
were ready to divide within thirty-four hours after treatment. The de-
stroyed micronuclei were replaced from those remaining. Nuclear clefts
appeared in the intact macronuclei; the latter fused into a single mass
before constricting into the eight parts which characterize the normal
individual.

5. THE SIZE FACTOR

Surprisingly large portions of the cytoplasm may be removed from
most Protozoa without permanently impairing the vital processes; under
favorable growth conditions, complete regeneration occurs within a rela-
tively short time. Moreover, the regenerative capacity can be tested fur-
ther by repeated excisions, so that the reparative processes are made to

MORPHOGENESIS 785

function almost continuously (cf. Hartmann, 1928). Is there a minimal
protoplasmic mass of definite size within which the organization of the
species can find latent expression—i.e., what is the smallest fragment
capable of regeneration?

After shaking Stentor into fragments, Lillie (1896) learned that the
smallest nucleated piece of S. polymorphus to become a perfect form was
equal to a sphere of approximately 80 y in diameter. Since the average
diameter of the normal forms was 230 uy, the smallest piece was about
one twenty-seventh of the original. The same approximate proportions
were obtained for S. coeruleus. For the latter species, Morgan (1901)
obtained minimum values which were even less than those of Lillie.
The smallest regenerates produced were one sixty-fourth of the volume
of large normal stentors. According to Morgan, the conclusion that a
Piece one twenty-seventh or even one sixty-fourth of the entire animal
can produce a new individual gives only a general idea of the relative
size of the minimum reorganization volume. More depends upon the
size of the normal individual than on that of the smallest piece, for
normal size limits vary considerably; a large normal individual may
contain eight times the volume of a small normal cell. The absolute size
of the smallest fragment is of greater significance than its relative size.

Sokoloff (1923, 1924) was mainly interested in the minimum volume
necessary for regeneration and the rate of regeneration in pieces cut from
different regions. For Spirostomum the minimum volume was between
one fifty-third and one sixty-ninth of the initial volume, and was not the
same for pieces taken from different regions. In general, he found dif-
ferent rates in pieces of different sizes; within certain limits, the larger
the piece the greater the rate. A comparison of his results with four genera
is given in the accompanying table (Table 21). In a type like Spiro-
stomum, the substance of which is qualitatively different as regards ability
to regenerate, the minimum volume and rate of regeneration varied ac-
cording to the region from which it was taken. In other Infusoria
(Dileptus, Bursaria, and Frontonia) the substance of the body appeared
to be qualitatively similar throughout (Sokoloff, 1923).

Moore (1924) obtained successful regenerates from Spathidium frag-
ments as small as 1.3 percent of the original volume. The minimum re-
organization mass in Chaos diffluens was determined to be one eightieth
of the normal volume (Phelps, 1926).

The proportionality of the regenerated structures to the size of the

786 MORPHOGENESIS

parts from which they regenerate has been emphasized by Morgan
(1901), Sokoloff (1923), and Dembowska (1926). The latter noted
that even the most minute parts of a small Uronychia regenerate were of
proportionate size. The small regenerators retained none of the large,
original cirri. The new cirri developed from corresponding Anlagen
and grew only until proportionate size was attained.

The quest for minimum reorganization volume in Protozoa originated
with those embryologists who were interested in the totipotency of
early blastomeres and gastrula fragments. Perhaps it has been partly
successful in demonstrating that a specific organization can be contained

TABLE 21: TABLE OF MINIMUM VOLUMES NECESSARY FOR REGENERATION
(Sokoloff, 1923)

Limits or REGENERATIVE

AVERAGE LIMIT
Capacity or DIFFERENT PARTS

Type oF REGENERATIVE
Capacity Front Middle | Hind Part
Spirostomum ambiguum Ehrbg. 1/61 of volume 1/53 1/68 1/67

(1/53 to 1/69)

Dileptus anser O.F.M. 1/72 of volume 1/70 1/72 1/73
(1/70 to 1/75)

Bursaria truncatella O.F.M. 1/35 of volume 1/36 == 1/34
(1/30 to 1/45)

Frontonia leucas Ehrbg. 1/5 of volume

(1/4 to 1/5)

within bits of protoplasm much smaller than previously shown for
separated blastomeres, and that the disadvantages of small volumes for
the mechanical processes involved in cleavage, gastrulation, and so forth,
are not necessarily the reasons for developmental failure in small blasto-
meres. The knowledge that minute fractional parts of a protozoan are
capable of regenerating also has a practical value in experimental work.
The assignment of a certain experimentally determined value as either
the absolute or the relative minimal mass does not greatly enhance the
theoretical implications of these works. We learn that one fifty-third or
one eightieth of the protoplasm of a cell of a given species regenerates
under favorable conditions, but, as Lillie (1896) aptly remarked, this

MORPHOGENESIS 787

does not imply that fifty or eighty diminutive regenerators can be ob-
tained from the original cell. In uninucleate types, only one such frag-
ment may be obtained. It is also apparent that parts of the original
derived organization, cortical organelles, oral structures, and so forth
are not requisite for the survival and regeneration of the smallest frag-
ments. The requirements are primarily qualitative: a representative bit
of the basic protoplasmic organization. The matter of obtaining a viable
nucleus or fragment of the macronucleus has had much to do with the
values so far determined.

6. THE NUCLEI IN REGENERATION

More than a century after Résel (1755) distinguished ecto- and endo-
plasm in amoebae by surgical methods, the works of Brandt (1877),
Nussbaum (1884), and Gruber (1886) inaugurated an era of active
interest in protozoan physiology. Although not in general agreement as
to the sensory and reactive capacities of enucleated fragments, these
investigators, together with Balbiani (1888), Verworn (1888, 1892),
Hofer (1890), Lillie (1896), Prowazek (1904), Popoff (1907), and
many others, showed that both nucleus and a certain amount of cyto-
plasm are essential for the continuation of the vegetative, reproductive,
and reparative activities of the cell. The consensus of these early publica-
tions is aptly stated by Minchin (1912): ‘‘Non-nucleated fragments may
continue to live for a certain time; in the case of amoeba such frag-
ments may emit pseudopodia, the contractile vacuole continues to pulsate,
and acts of ingestion and digestion that have begun may continue; but the
power of initiating the capture and digestion of food ceases, consequent-
ly all growth is at an end, and sooner or later all non-nucleated bodies
dieratk ” \(p.-210))..

There still exists a diversity of opinion regarding the essential nature
of the nucleus or dimorphic nuclei in regenerative or regulative func-
tions. Apropos of the latter, Moore (1924) places a greater premium
than most upon ultimate physiological recovery:

In general previous investigators have centered their attention upon morpho-
logical regeneration alone, and have considered the restoration of external
organelles as sufficient evidence of a completion of the process. Since form
regulation in the Protozoa is of little value if not accompanied by the ability
to continue normal existence, it would appear that a more valuable definition

788 MORPHOGENESIS

of regeneration is one which includes restoration of function as well as of
structure [p. 250}.

Notwithstanding the evident truth that maintenance and perpetuation
are ends to which all of the vital functions are directed, our criteria of
recovery need not be so inclusive, if the practical objectives of the ex-
perimental laboratory are to be realized. To those interested in the
mechanisms of form determination, the ultimate fate of a regenerate is
of secondary importance. Pieces from starved Protozoa do not grow, but
they are frequently capable of regenerating new individuals of propor-
tionate size. Amicronucleate fragments of some ciliates are sometimes
capable of regenerating without being able to maintain the redifferenti-
ated state or to divide. The success of regenerative processes in such
instances is limited only to the extent to which they are correlated with
the general maintenance functions.

According to Stolé (1910), cytoplasmic bits of Amoeba may live for
as much as thirty days and are able to prehend, ingest, digest, and as-
similate food. In agreement with Verworn, Lynch (1919) found evi-
dences of all the usual catabolic activities, but his enucleated amoebulae
showed no symptoms of growth, regeneration, or division. A few years
later, Phelps (1926) concluded that merozoa from Amoeba do not
carry on any of the fundamental body processes except locomotion.
Nevertheless, she did note an increase in the number of crystals within
the fragments which, according to her own criteria, denotes metabolic
change. Others have reported only dissociated movements in cytoplasmic
fragments of Amoeba (Willis, 1916; Mast and Root, 1916). Quite a
number of merotomists have recorded instances of fusion between enucle-
ated bits of cytoplasm with the parent cell in various Foraminifera,
Heliozoa, and testate rhizopods (see p. 793). In Actinophrys sol the
fragments fuse with one another (Looper, 1928).

The survival of enucleated ciliate fragments, even for short periods,
is not the general rule. There appear to be only few instances recorded
in the literature in which such fragments show any tendency to re-
differentiate. Gruber (1886) reported that redifferentiation occurs in
enucleated fragments derived from dividing stentors, provided a de-
veloping peristome is included in each piece. Cytoplasmic portions of
Ble pharisma ate sometimes capable of form regeneration, but since they
are unable to feed involution and cystolysis soon occur (Moore, 1924).

MORPHOGENESIS 789

Garnjobst (1937) found evidence of secretory activity on the part of
enucleate fragments of Sty/onethes. Cyst formation after injury is the rule
in this species. In one case a cyst wall was produced by a fragment which
contained no nuclei.

The possibility of regeneration in Infusoria is not precluded by the
absence of either micronucleus or macronucleus. It is difficult, however,
to formulate any satisfactory conclusion at this time, inasmuch as ob-
servations on different species often present contrasting results. In spite
of the accumulated literature, the respective roles of each of the di-
morphic nuclei remain uncertain.

Gruber (1886) regarded the micronuclei in Stentor as of secondary
importance, since no regeneration occurred until a macronucleus dif-
ferentiated from one of the amphinuclear products. Lewin (1910)
agreed with Gruber and did not believe the micronucleus necessary for
growth or regeneration in Paramecium caudatum. On the contrary, Cal-
kins (1911b), Peebles (1912), and Schwartz (1934) obtained re-
generates in this species only when both types of nuclei were present.

In Stevens’s (1903) experiments on Lichnophora auerbachii, a species
with a beaded macronucleus and one micronucleus, regeneration was
limited to the production of a few oral cilia (77 situ), new peristomes,
and relatively small segments of the attachment disc. She showed that
only pieces including the attachment disc, the neck, a quarter section of
the oral disc, and representatives of both nuclei were capable of re-
generating ‘‘fairly normal” individuals. Isolated oral discs (amicro-
nucleate) and basal discs (with micronuclei) never regenerated, al-
though they survived for several days. More recently, Balamuth (MSS,
1939) studied regeneration in L. macfarlandi and found its regenerative
capacity to be greater than Stevens showed, since isolated amicronucle-
ate oral discs definitely replaced injured adoral zones. Missing basal
discs were not regenerated in L. macfarland?, but it was pointed out that
the two daughter basal discs form in this genus only by division of the
corresponding parent structure.

Calkins (1911a) and D. B. Young (1922) described regeneration
in amicronucleate fragments of Uronychia when the operations were
made very late in the interdivisional period. The regenerates usually be-
came abnormal after three to four days; they apparently starved to death
for, according to Young, no food was ingested or assimilated. The frag-

790 MORPHOGENESIS

ments were also incapable of dividing. In the case of U. setigera, nor-
mally with one micronucleus, only one of the two halves of a bisected
cell was able to divide, although form regeneration occurred in both. In
U. binucleata, with two micronuclei, both halves were successful in
regeneration and division. It appears from these observations that while
form regeneration is possible in amicronucleate Uronychia fragments
under special conditions, the micronucleus is indispensable for ultimate
physiological regeneration. Dembowska (1926) was unable to find evi-
dences of regeneration in amicronucleate pieces of either U. setigera or
U. transfuga.

Euplotes patella does not live longer than a few days or divide more
than twice, in the absence of the micronucleus. Using a mercury micro-
pipette, Taylor and Farber (1924) demonstrated that portions of the
cytoplasm or macronucleus could be removed without serious conse-
quences, whereas the removal of the micronucleus with only a small vol-
ume of the surrounding cytoplasm ultimately proved fatal. In several
of their experiments on E. patella, the micronuclei were completely with-
drawn from the organism and then immediately replaced. The animals —
so treated subsequently gave rise to vigorous cultures. They concluded
that the micronucleus plays more than a germinal réle in the life his-
tory of Explotes.

A majority of the publications bearing on regeneration in ciliate frag-
ments contain casual references to the nonviability of amacronucleate
pieces, but very few have treated the matter at length.

The vegetative individuals of Blepharisma undulans (Moore, 1924)
are incapable of regenerating in the absence of macronuclear material.
However, if cuts are made during the early phases of division, such
Pieces sometimes regenerate. Moore found that regeneration was fol-
lowed by immediate dedifferentiation in all of these cases; the dedif-
ferentiated fragments ultimately disappeared without dividing, in spite
of the fact that some of them were known to contain as many as six
micronuclei. According to the evidence presented, there appears to be
no doubt that the restoration of external organelles may proceed in the
absence of the macronucleus /f the initial steps in the division process
are under way at the time of cutting. From a posterior fragment possess-
ing only a part of the developing membranelles, an organism of nearly
perfect form may arise. The peristome, adoral zone, and undulating

MORPHOGENESIS vou

membranelles differentiate, but a new mouth fails to develop and the
degenerative changes immediately appear.

Fortner (1933) expressed the single, large macronucleus from the
body of gastrostyla-like hypotrichs (species?) by gently compressing the
organism between cover glass and slide. Such operations were followed
by a diminution in general mobility and contractile-vacuole pulse rate and
by the persistence of food vacuoles. Schwartz (1935) concluded that
amacronucleate pieces of Stentor coeruleus containing only micronuclei
are like totally anucleate specimens as to regenerative power, digestion,
and length of life. The amacronucleate pieces merely regulate external
form, without undergoing the sequence of changes involved in physiologi-
cal reorganization.

In physiological regeneration following conjugation, autogamy,
endomixis, or analogous processes, the dimorphic nuclei have a common
origin from the synkaryon or from some micronucleus-like body, but
there is no convincing evidence that the micronuclei of strictly trophic
individuals are able to replace artificially removed macronuclei. It 1s
equally true that macronuclei do not give rise to new micronuclei. Numer-
ous attempts to create amicronucleate races operatively have not met
with success. The operated individuals survive no more than a few days
and do not regenerate new micronuclei. The origin of amicronucleate
races of Spathidium spathula (Moody, 1912), Oxytricha hymenostoma
(Dawson, 1919), O. fallax (Woodruff, 1921; M. E. Reynolds, 1932),
Paramecium caudatum (Lewin, 1910; Landis, 1920; Woodruff, 1921;
Schwartz, 1934), Didimium nasutum (Thon, 1905; Patten, 1921),
Urostyla grandis (Woodruff, 1921; Tittler, 1935), and possibly others,
appears to be the result of anomalous metagamic or post-endomictic dif-
ferentiation.

Small fragments of the macronucleus of ciliates usually are able to
reconstitute the entire macronuclear system. In Stentor, for example, one
or two of the macronuclear nodules ultimately regenerate the long
moniliform chain. Schwartz (1935) thought that the process of re-
organization in this species serves to regulate the size relationships of
the different organelles. The surface relations of nucleus and cytoplasm
are adjusted by fusion, division, or deformation of the macronuclear
segments.

As far as regeneration is concerned, the macronucleus appears to be

¥92 MORPHOGENESIS

qualitatively homogeneous. Full regeneration is frequently possible when
only a small piece of the macronucleus is present. The rate of regenera-
tion in Bursaria is independent of the size of the macronuclear fragment
included. The two halves of an individual, one containing a small and
the other a very large portion of the macronucleus, usually regenerate
in about the same length of time (Lund, 1917).

It is well established that micronuclei are capable of regenerating
other micronuclei. A unimicronucleate section from a multimicronucleate
species eventually regains the characteristic number, provided other con-
ditions favor regeneration. There has been no confirmation of Lewin’s
(1912) experiments on Sty/onychia in which, after merotomy, the micro-
nuclei increased in number beyond that characteristic of the normal races.
The effect of merotomy upon micronuclear number in Pleurotricha was
investigated by Hewitt (1914), who concluded that merotomy does not
permanently alter the numerical relationship between micronuclei and
macronuclei. If at least a representative of each is present in the frag-
ment, the normal nuclear complement occurs in the immediate regener-
ate and in its descendants.

The question arises as to whether or not the inability of an expert-
mentally amicronucleated fragment to regenerate physiologically is like-
wise characteristic of the fragments derived from viable amicronucleate
races. The contribution of M. E. Reynolds (1932) definitely answers
the question: ‘““The regeneration process in the fragments from the
amicronucleate Oxytricha is similar to that which occurs in pieces from
micronucleate Oxytricha which contain both types of nuclei” (p. 357).
The absence of the micronucleus as a structural entity does not alter the
course of regeneration. As in the normally constituted races, regeneration
is accompanied by a protoplasmic reorganization involving the dedif-
ferentiation of old and the redifferentiation of new ciliary fields from
migrating An/agen. The extent and time of regeneration, as well as the
total number of regenerates obtained from the peculiar race of O. fallax,
compare favorably with the normal strains. In view of the fact that ex-
perimentally amicronucleate fragments do not regenerate in the com-
plete sense, we are obliged to assume that whatever the constitution of
the usual micronucleus, it is somehow represented in the amicronucleate
races. Following Woodruff’s (1921) interpretation, Reynolds regarded

MORPHOGENESIS ie

the nuclear mass of the amicronucleate race as an undifferentiated prod-
uct of the amphinucleus.

BEHAVIOR OF FRAGMENTS: GRAFTING AND REINCORPORATION

Balbiani (1888) and Verworn (1889) were first to remark that parts
of ciliates move in the general manner of the entire animals. In his
work on Stylonychia, Oxytricha, and Paramecium, Jennings (1901)
emphasized body shape and the activity of heavy oral cilia as determining
factors in the characteristic swimming movements, whereas Ludwig
(1929) ascribed the spiral progression of ciliates to a more vigorous
activity of the aboral rather than the oral cilia. Bullington (1925) and
Horton (1935) discovered that the spiral movements do not neces-
sarily relate to the configuration of the oral groove. It appears that por-
tions of Infusoria in which oral cilia are lacking react in a fashion much
the same as that of the whole organisms; and, further, that the spiral
motions are not entirely dependent upon body shape or the activity of
oral cilia, but are produced by the coérdinated activity of the body cilia
in general (Horton).

The responsiveness of ciliate fragments to mechanical and chemical
stimuli was investigated in some detail by Jennings and Jamieson
(1902). They found that if the fragments were not too small or irregu-
lar in form, the motor and sensory capacities compared favorably with
those of the intact individuals. On the contrary, Alverdes (1922) re-
ported some degree of sensory localization, wherein only the anterior
halves of Paramecium and Stentor were responsive to chemical stimula-
tion. More recently, Horton (1935) ascertained the sensitivity of the
anterior and posterior halves of P. caudatum to weak acid stimulation.
The results are in general agreement with the earlier work of Jennings
(1901). Unlike his predecessors, Horton noted that the posterior halves
of this species are somewhat more sensitive to weak acid stimuli than
are the anterior halves.

The anastomosis of protoplasmic streamers in myxopods and the re-
combination phenomena in Foraminifera (Verworn, 1892; Jensen,
1896) was known to pioneer students of the Protozoa. In recent years
numerous other workers have discovered that excised, anucleate frag-
ments of many types of Sarcodina will recombine with the parental cell

794 MORPHOGENESIS

mass. This fact is even more striking in virtue of the active part taken
by healthy fragments to effect the union. Kepner and Reynolds (1923)
reported more than one hundred experiments with several species of
Difflugia, in which isolated pseudopodial fragments would again enter
into the protoplasmic structure of the cell (Fig. 181). Fusion in Difflugia
is species specific, occurs along the mid-region rather than at the ends
of extended pseudopods, and is limited to the fusion between fragments
and nucleate cells. The fusion of anucleate fragments with each other

Figure 181. Reincorporation in Difflugia pyriformis. The small figures show form
changes and directional movements of a fragment left by the sudden retraction of a
pseudopod (stippled). The mode of fusion between the fragment and a second pseudopod
is shown below. (From Kepner and B. D. Reynolds, 1923.)

was not observed, even when they were placed in contact. Healthy pieces
were reappropriated after separation by distances as great as 1.5 mm.;
even those of ectoplasmic composition appeared to move and orient with
respect to the parent cell. Autoplastic and homoplastic fusions between
individuals of Actinosphaerium eichorni, under conditions of slight
compression, were made by Howland (1928). Permanent fusions were
made between two medullary surfaces and between cortical and medul-
lary surfaces. Axopodial fragments united with the individual at any
point along an unsevered axopodium or with the cortical surface of the

MORPHOGENESIS 123

cell. Okada (1930) published fusion experiments on Actinosphaerium
and Arcella, which gave results comparable with those described by
Howland.

The reincorporation phenomena in some of the Sarcodina appear to
offer a fertile field for studying the physical and chemical factors in-
volved in the union of cells or fragments. As yet the attention of very
few investigators employing Protozoa has been directed toward this end.
Miller (1932) studied cytoplasmic reappropriation in Arcella discoides
under the influence of different hydrogen-ion concentrations, as well as
under the influence of a low-voltage dc current. Within the limits of
pH 5.0 to pH 7.6, the rates of contact and fusion were not perceptibly
altered, although in media having a pH lower than 5.0 or greater than
7.6 the reappropriation reactions were retarded. Likewise the low-voltage
currents (0.3 to 2.1 microamps) failed to accelerate either contact or
fusion. Miller regarded reappropriation as wholly beyond the control of
pH or direct currents within the ranges tested. It occurred to Brehme
(1933) that the various Sarcodina in which reappropriation has been
reported are forms with relatively viscous protoplasm. She therefore
attempted fusion experiments with Amoeba proteus, a type with less
viscous protoplasm than those previously examined. Since A. proteus did
not reappropriate its own or foreign fragments, she was able to conclude
that increased viscosity may be an important factor in the fusion process.

Richard Hertwig’s (1903, 1908) karyoplasmic relationship hypothesis
has strongly influenced the more recent investigations of nuclear and
cytoplasmic phenomena. Taylor and Farber (1924) excised varying
quantities of the cytosome of Explotes patella with a micropipette. Con-
trary to the results expected in accordance with Hertwig’s rule, the
operated individuals continued to divide in the vigorous, normal fashion.
In Chaos diffluens the initiation of division was believed by Phelps
(1926) to be a direct result of increasing the volume of cytoplasm,
whereas the removal of cytoplasm retards growth, and presumably divi-
sion, owing to the intervention of reconstruction processes. Similarly,
Causin (1931) maintained that the excision of fragments in Stentor
disturbs the K/P equilibrium and that this, in turn, initiates reorganiza-
tional activities which culminate in division or, more frequently, in
physiological regeneration. During periods of physiological regenera-
tion in S. coeruleus, the number of macronuclear segments is regulated

796 MORPHOGENESIS

to correspond with the size of the resulting cell, instead of showing any
constant relation with the number of segments prior to the onset of
reorganization. If all but one or two of the macronuclear segments are
surgically removed, those remaining increase their surface values by
elongation or deformation. In other instances the requisite equilibrium
of surface relations between cytoplasm and nucleus is attained by nuclear
coalescence or fragmentation (Schwartz, 1935). Previously Burnside
(1929) made an unsuccessful attempt to set up divers biotypes by micro-
dissection methods. In some of his experiments the quantity of nuclear
material in one individual was two to three times greater than in another.
Regulatory processes ensued, such that individuals with little nuclear
material increased that amount; and individuals with large proportions
of nucleus to cytoplasm decreased that proportion within a few genera-
tions.

Looper (1928) took advantage of the reincorporation phenomenon in
Actinophrys sol as an approach to the K/P problem. By mechanical
means, several individuals may be fused into temporary syncytia, and
non-nucleated individuals may be fused with each other or with intact
individuals. In this way he altered K/P either by increasing or decreas-
ing the cytoplasmic volume. The division rate in this species was ac-
celerated and the amount of nuclear material ultimately increased when
fragments of cytoplasm were added to the cytosome. Removal of cyto-
plasm retarded the division rate. The same method was employed by
Burch (1930) in studying the possible rdle of the karyoplasmic rela-
tionship as an inciting cause of cell division in pedigreed races of Arcella
vulgaris and A. rotunda. Following Hegnet’s (1920) hypothesis that
additional cytoplasm outside the sphere of influence of a single nucleus
in rhizopods may stimulate that nucleus to divide, Burch made daily
additions to or reductions in the volume of cytoplasm for different lines
of his pedigreed strains. Trauma alone was found to have little effect
upon the daily division rate. In general, the division rate varied directly
with volumetric alterations of the cytoplasm, but these variations were
not proportional to the volumes of cytoplasm gained or lost. As Wood-
ruff (1905), Gregory (1909), Conklin (1912), Moody (1912), and
others have suggested, Burch assumed that other intrinsic factors affect
the division rate to a great degree. Our inability to distinguish between
cause and effect further clouds the dying issue of the Kernplasmaver-
hdltnis theory.

MORPHOGENESIS 797
REGENERATION AND DIVISION

The lines of investigation summarized by Calkins (1934) and in
part by Dixie Young (1939) predicate an increase of vitality through
reorganizational processes in division, encystment, endomixis, and conju-
gation. The de- and redifferentiation incident to the physiological (‘‘spon-
taneous’) and reparative types of regeneration provide other opportuni-
ties for the renovation of derived structures. If we are correct in the

Figure 182. Divisional and physiological reorganization in Uronychia. Sister cells
operated on four hours after division. Five hours after injury, the cell cut as shown in A
regenerated by dividing. Physiological reorganization without division occurred in B
after the same time interval. (Modified from Dembowska, 1926.)

belief that nuclear and cytoplasmic reorganization stimulates vitality,
then regeneration in the strict sense includes not only the replacement
of a part but also some degree of rejuvenescence of the whole cell. Is it
a priori a beneficial process? At least it is one which can be provoked
artificially.

To what extent are reproduction and regeneration dependent upon the
same fundamental activities of the cell? Gruber (1886), Morgan
(1901), Dembowska (1926), Causin (1931), and M. E. Reynolds
(1932) have remarked that the initial steps in division and regenera-
tion are comparable. Since the ends achieved by these processes are

798 MORPHOGENESIS

dissimilar, it would be of interest to discover the conditions under which
differentiation leads only to restoration or to division.

Dembowska (1926) made an interesting discovery while working
with Uronychia. Four hours after division, two sister cells were operated
on in different regions. The individual cut as shown in Fig. 182 A di-
vided five hours later, giving rise to two normal individuals. After the
same interval of time the individual cut anteriorly (Fig. 182 B) began
to regenerate in the usual fashion; it produced a single new individual.
Dembowska suggested that the type of operation determines the mode
of reorganization.

A similar condition obtained in M. E. Reynold’s (1932) experi-
ments on Oxytricha fallax. Amicronucleate individuals were able to
regenerate when cut late in the interphase, from approximately five
hours after one division until the beginning of the next. The majority
of the variously operated organisms reorganized by division, rather than
by the ordinary mode of restoration. In this species injury hastened the
divisional process. Reynolds thinks that once the division processes are
under way, they are completed in spite of moderate surgical disturb-
ances.

Injuries sustained shortly before the onset of division are not repaired
by divisional reorganization in some species. In Paramecium caudatum
injured cells need not regenerate to divide, and may or may not re-
generate before dividing (Calkins, 1911b). A truncated Paramecium
frequently gives rise to a truncated and a perfectly formed daughter. The
former occasionally divides again before regenerating (Fig. 183).

It is also true that regeneration may suppress or supplant division in
certain species. Hartmann (1924), for example, maintained Amoeba
polypoda in an undivided state for long periods by repeatedly cutting
away portions of the cytoplasm. Each time that growth reached the point
of an impending division, he removed cytoplasmic fragments amounting
to as much as one-third of the total cell volume. In one experiment A.
polypoda was operated on 21 times within 25 days. In this manner divi-
sion was held in abeyance while the control strains divided 11 times.
Another experiment continued for 42 days, during which the operated
individual regenerated 32 times, as against 15 divisions for the controls.
Phelps’s (1926) experiences with Chaos diffluens were not in agree-
ment with those of Hartmann. Successive excisions of one-fourth of the

MORPHOGENESIS 799

cytoplasm for at least 32 days did not prevent division in C. diffluens.
Within 24 hours after each cut, the regenerative processes were complete
and division occurred. Hartmann’s initial attempts to substitute regenera-
tion for division in Amoeba proteus met with little success. The animals
lived for not more than 15 days. Later, however, he was able to contest

ior part of a Paramecium
transected anterior to the

division plane may divide
several times before form
restoration occurs in all of

| its descendants. (Modified
from Peebles, 1912.)

Phelps’s results by successfully culturing A. proteus through a long
series of regenerations (Hartmann, 1928). One such series (protocol 3)
was discontinued after a four-month period, during which 130 rfe-
generations replaced the normal division process. In an equivalent period
the control series produced approximately 65 generations. It is Hart-
mann’s conviction that Amoeba can be maintained in a healthy undivided
state for infinite periods by repeated operations.

The question of “artificial imrnortality” through regeneration was

/ Figure 183. Diagram
~ showing delayed regenera-
tion in Paramecium cauda-
tum. The nucleated poster-

800 MORPHOGENESIS

further investigated by Bauer and Granowskaja (1934b) in Oxytricha.
Operations made subsequent to division, but before the end of the growth
period, hastened the next division. When operations were made a short
time before the appearance of division symptoms, regeneration sup-
planted division. Repeated operations of this kind had the effect of
shortening the interdivisional period and, as a consequence, the succes-
sively operated oxytrichas progressively diminished in size until death
occurred. The cytological and some of the physiological aspects of re-
organization are given in a preceding publication (Bauer and Granow-
skaja, 1934a). Luntz (1936) suppressed division in Stylonychia sp. by
subjecting these organisms to weak electric currents (0.95-1.0 ma and
2.0 a, voltage not stated) for approximately one hour each day. Divi-
sion was averted for a period equal to twenty-seven to twenty-eight con-
trol generations, but each application of the current was followed by a
transitory diminution in cell size.

The substitution of regeneration for division is suggestive, but does
not prove that the vitality or longevity of the undivided cell is thereby
increased. It is known that Protozoa which, for some reason, fail to
divide for relatively long periods become morbid. Agonal symptoms
and death are the usual accompaniments of the depression. But we have
not yet succeeded in experimentally inhibiting division in a cell by means
other than surgery without inducing adverse changes in its physiological
behavior. It is significant, nevertheless, that physiological reorganiza-
tion has been detected in a variety of species after mechanical injury,
exposure to irritants, or during periods of starvation. The internal
changes induced by these conditions appear to be restorative rather than
adaptive.

More information is necessary before we can be confident of the
conditions governing the mode of injury repair, whether by divisional
or physiological reorganization. The nuclear and cytoplasmic alterations
accompanying division have been studied extensively. On the cytological
side, the reorganization processes in ciliates are most profound in the
hypotrichs; and, as has been mentioned already, relatively small injuries
often provoke a complete cycle of de- and redifferentiation. In the less
specialized types, the internal changes at division are less striking. Ac-
cording to many observations, some of the parental organelles are passed
unchanged to one or the other of the two daughters. It is in the hypotrichs

MORPHOGENESIS 801

that injuries are apt to be repaired during division. In the types in which
dedifferentiation, preparatory to division, is less extensive, there appears
to be a greater independence between regeneration and division. Whether
or not the imminence of division reduces the possibility of independent
physiological reorganization has yet to be determined.

POLARITY CHANGES AND PROTOPLASMIC STREAMING

The development of temporary heteromorphic individuals in Bursaria
during physiological regeneration or in regenerating halves was first
described by Lund (1917). The suppression of secondary axes and the
reversal of polarity in the weaker member of the heteromorph occurred
as a consequence of the dedifferentiation and reorganization of that re-
gion. In some instances the secondary axes were so feebly developed that
a local reversed ciliary action gave the only clue to their existence.

The operations on Mastigina hyale by Becker (1928) have a more
direct bearing on the mechanisms of polar organization. In normally
creeping animals, the nucleus occupies a vacuolar space at the extreme
anterior tip, and the endoplasm shows a type of fountain streaming,
toward the anterior end in the center and posteriorly at the cell periphery.
When individuals are divided into anterior and posterior halves, only
the anterior half continues to move as before; motility and streaming
are upset in the non-nucleated half, pseudopod formation is interrupted,
and death soon occurs. The importance of the anterior extremity in the
determination of polarity is demonstrated by the fact that decapitated
cells behave just as posterior halves. If the anterior tip, together with the
affixed nucleus, is pulled posteriorly with the surface gel, streaming and
locomotion cease momentarily and then resume with reference to the
new position of the shifted polar cap. But if only the nucleus is dis-
lodged from its vacuole and expressed into the flowing endoplasm
without severe injury to neighboring parts, the original polarity is un-
disturbed, which suggests that the nucleus alone is not responsible for
the axiate pattern. Becker assumed that the resistance of the anterior
peripheral layer to internal pressure is reduced by the physical presence
of the ‘‘foreign body” in the gel. The kinetics of movement in Mastzgina
ate interpreted in the following way: “‘it is the entire anterior tip of
permanently gelled protoplasm which prevents gelation of the ‘endo-
plasm’ immediately behind it, and which by imperfect continuity with

802 MORPHOGENESIS

the outer gelled layer of protoplasm of the intermediate zone creates a
circular zone of weakened elasticity or lowered resistance to the internal
pressure of the plasmasol” (p. 113). The differentiated anterior region
is therefore a prerequisite for normal streaming.

Fragments of Paramecium caudatum were examined by Hosoi (1937)
in an effort to identify some of the forces involved in cyclosis. The
organisms were narcotized with iso-propyl alcohol (Bills, 1922) and
transversely sectioned at intervals along the primary axis. The type of
protoplasmic streaming concerned with the formation and release of
food vacuoles, the Schlundfadenstrémung (Bozler, 1924), was evident
only in pieces possessing a large portion of the gullet, whereas cyclosis
in the strict sense occurred in all of the fragments within a few moments
after operation. Hosoi found that the Schlundfadenstrémung and the
cyclical currents were largely independent phenomena, and that the
nuclei play no direct part in effecting either of these movements. His
suggestion that some special substances are attracted on the ecto-endo-
plasmic interface, which serve as the generating force of the streaming
movements, awaits further amplification.

PHYSIOLOGICAL GRADIENTS

Outstandingly important in morphogenetic studies was Child’s dis-
covery of physiological gradients in various animal types. After consider-
able experience with metazoan forms, Child (1914) directed his atten-
tion to Protozoa (Paramecium, Stentor, Stylonychia, Vorticella, and
Carchesium), in which polarity is well defined. In these first experi-
ments the direct susceptibility or resistance method was used, with several
dilutions of KCN as the reagent in most cases. All of the forms showed
the greatest susceptibility in the apical region, although local regions of
still higher metabolic rate, such as the vacuolar regions in Paramecium,
were sometimes found. These experiments brought out the fact that a
close parallelism exists between the magnitude of the gradient and the
general morphological and physiological differentiation of the cell.
Peebles (1912) had previously noticed that anterior cuts cause greater
physiological disturbances than posterior cuts in Paramecium.

The KCN susceptibility experiments were extended to amoeboid
forms by Hyman (1917). Unlike the clearly polarized types, Amoeba
was shown to have no permanent axial organization. A susceptibility

MORPHOGENESIS 803

gradient arises in Amoeba before a pseudopodium appears; it is greatest
at the tip of the developing pseudopodia, and greater in the more re-
cently produced than in the older ones. The bearing of axial gradients
upon the physiology of amoeboid movement is discussed at length in
Hyman’s report.

Apparently the differential susceptibility of various regions in P.
caudatum is not primarily dependent upon qualitative differences in the
protoplasmic constitution nor upon the precise mode of action of the
physical or chemical agent used to demonstrate it (Child and Deviney,

Figure 184. Successive stages in the regulation of the mouth in a piece of Spirostomum
with the mouth at the anterior end. A, the operated individual; B, changes after one day—
the rounding off of the cut surface; C, twenty-four hours later—disappearance of the
original mouth and the development of a shallow peristomial depression on the side;
D, appearance of the new mouth four days after the injury. (From Seyd, 1936.)

1926). For example ultra-violet radiation has a differential cytolytic
action, which is identical with that of chemical poisons (Child and
Deviney, 1926; Monod, 1933). It is believed that the environmental
agent alters in degree the physiological activities, with the differential
depending upon quantitative differences in the physiological condition.
Permeability and cytolytic gradients are therefore to be regarded as
manifestations, rather than causes, of an underlying physiological
gradient.

The significance of physiological gradients in connection with the
regulation of specific organelles in Protozoa has yet to be made clear.
A more recent article devoted to the determinative action of the physio-
logical gradients is that of Seyd (1936) on Spirostomum ambiguum. A

804 MORPHOGENESIS

new contractile vacuole develops from the long vacuolar feeding canal
near the posterior end of an anterior body section, but never at the for-
ward end of a posterior section. The new vacuole appears only at the
posterior end of a piece cut from the mid-body region. After a deep
transverse cut in the mid-region, a vacuole appears near the cut end of
the anterior half; but when the wound closes the new vacuole disap-
pears, as the posterior part of the feeding canal fuses with it. In long
anterior pieces, in short anterior and posterior pieces, and in cut-out
sections, a new mouth develops at the appropriate position; those cut
in such a manner that the old mouth occupies an odd position will re-
generate a new mouth in the correct position, and the old one is re-
sorbed (Fig. 184). The specificity and location of these regenerated
structures in Spirostomum are attributed to the determining action of
the physiological gradients.

REGENERATION IN COLONIAL FORMS

The foregoing pages have dealt with different aspects of regeneration
as it occurs in wounded cells or in their dismembered parts. Whereas this
phase of morphogenetic investigation revolves about the cell, its funda-
mental organization, or the relations between different regions of the
same cell, an equally fruitful line of inquiry concerns the relationships
of one cell to another in true or temporarily colonial Protozoa.

Twenty-five years ago Runyan and Torrey (1914) became interested
in the determination problem in Vorticella sp. when they discovered
that, after division, the migratory cell always forms from the lateral
daughter. The cleavage plane in this peritrich coincides with the longi-
tudinal cell axis. But instead of bisecting the aboral or attached end of
the constricting cell, the plane deviates from the mid-line enough to
disrupt the stalk connections of one (the lateral) daughter. This is the
presumptive migrant or “ciliospore.’’ The other cell retains its continuity
with the stalk and remains behind to repeat the division process at a
later time. Metamorphosis of the lateral cell does not begin until the
protoplasmic junction between the two cells is reduced to a small thread.
These observations led Runyan and Torrey to suppose that the posterior
girdlet of cilia, the scopula, and other features of the migrant, appear
only after the cell is physiologically isolated from the stalk; and, further-
more, that such isolation does not exist until the organic connection

MORPHOGENESIS 805

between the sister cells becomes very slender. They attributed this
behavior to dominant influence of the stalk upon the expression of cilio-
spore potentialities. Their investigations also included experiments
wherein solitary cells were dislodged from their contractile stalks. Thus
freed, the cells metamorphosed into typical migrants within a two-hour
period. Subsequent reattachment to the substrate was followed by im-
mediate resorption of the locomotor organelles.

Autonomy cannot be ascribed to the stalk portion of peritrichs, despite
the fact that the contractile core, or spasmoneme, is composed of living
protoplasm. When the cell or cells are stripped from the stalk, either
by natural or artificial means, no further activity is manifested; it re-
mains inert and lifeless. Consequently, it seems unsafe to assume that the
stalk, or peduncle, plays more than a passive part in the differentiation
of the cell from which it develops. Changes in the external environ-
ment alone are capable of inaugurating the metamorphic processes in
recently affixed ex-migrants. A migrant of Zoothamnium or Carchesium,
for example, may settle upon the substrate, metamorphose into a typical
trumpet-shaped cell, and secrete a section of the peduncle, then sud-
denly reacquire migrant characteristics and relinquish its peduncle in
order to establish itself in a more favorable location.

Peritrichs of the genus Zoothamnium present a branching type of
colonial organization in which the spatially separated cells show a rela-
tively high degree of integration. One of the large marine species, Z.
alternans, has proved to be especially suitable for morphogenetic studies,
in virtue of its development according to a definitely determined pattern,
as first described by Claparéde and Lachmann (1858). Various aspects
of growth and differentiation in this form have been investigated recently
by Fauré-Fremiet (1930) and Summers (1938a, 1938b).

Asexual reproduction is the general rule in Z. alternans. Cells at pre-
determined nodes along the axis of a colony differentiate as ciliospores.
One by one, these mature migrants break away from the parent colony
and afhx themselves elsewhere, to start new colonies. In this respect
Zoothamnium resembles Vorticella. Unlike the latter, however, its
asexual migrants are endowed with far greater developmental potentialt-
ties. An affixed ciliospore loses its aboral cilia, acquires the conical vor-
ticellid form, and begins to elaborate the primary stalk, a process inter-
rupted periodically by unequal division. The successive divisions of the

806 MORPHOGENESIS

transformed ciliospore produce, each time, an apical cell of the next
generation and a smaller lateral daughter, the initial cell of the presump-
tive branch at that node. The initial branch cells are strictly alternate

Figure 185. A relatively
mature colony of Zootham-
nium alternans showing the

BSL IS alternate arrangement of

3a Ar © (Pp OA branches and cells. The
Nex WOH 0 Ree |

WE a \Aeee apical cell of the nine-

~ SS = O teenth generation (TM. 19)

> val 3 represents the growing

\l ANY oe point of the primary axis.

> Dice, © The numerous common nu-

Fw ) aD tritive cells of each branch

Ae 1S ey are division products of the

& Av VA <= terminal cell of that branch.

Re FY On th th branch, 10g”

=A. n the seventh branch, 10g

(= SK, 5g? 59° 7g? Ar designates the terminal

a4 2) branch cell of the tenth

generation. The lateral cell
of the first branch genera-
ion (Gis, IAD) fr its

La A) : :
CA ml Sm v7) two immediate descendants
S law (e.g., 1G" and 1G’) repre-
( KY a sent the potential cilio-
Q eN ey, spores; several stages in the
RSs: i 2a? differentiation of these cells
Ol y j on several of the branches
Tees a Je are shown. (From Summers,

1938a.)

in position along the primary axis, lying alternately on the right and
left sides of the axis at successive nodes. Cells of the branch strain de-
scend from the initial branch cell by a series of equal divisions. Here
again each division results in a median and a lateral individual, with the

MORPHOGENESIS 807

former remaining in the terminal position on the laterally growing
branch axis. Thus the primary developmental functions of a growing
colony are almost exclusively limited to the terminal cells of the primary
and branch axes (Fig. 185).

A painstaking cytological analysis of normal development in this
species led Fauré-Fremiet (1930) to postulate that the two daughters
resulting from the division of one initial cell are never equivalent as
to their developmental potentialities. He assumed that quantitatively
differential divisions of the apical cell series restrict the subsequent power
of division in the branch strains; and, similarly, the qualitatively differ-
ential nature of the first division of the initial branch cells effects a
segregation of potencies for ciliospore formation. Such a hypothesis of
embryonic segregation by division apparently covers the facts of normal
development. A terminal branch cell, for example, produces fewer gen-
erations than the apical cell (see Fig. 185). And, for the most part,
a ciliospore differentiates only from the lateral cell of the first branch
generation or from its two immediate descendants on certain of the
branches.

The facts derived from regeneration studies are not in accord with
Fauré-Fremiet’s assumptions. Common branch cells above or lateral
to the supposedly differential divisions retain, for a time at least, po-
tentialities for regenerating large portions of the colony. An apical cell,
terminal branch cells, common nutritive cells, ciliospores, and sometimes
gamonts differentiate at appropriate positions on the regenerate. Further-
more, the ciliospore-forming cells can be induced to differentiate as new
apical cells, which continue axial development according to the normal
pattern (Fig. 186).

If the well-defined apical cell is removed, the terminal cell of the
topmost branch usually differentiates as a new apical cell, the subse-
quent development of which is identical with that of the original.
But if the apical cell and the first terminal branch cell are destroyed,
the functions of the former are assumed by either the subterminal cell
of the topmost branch or the terminal cell of the penultimate branch,
more frequently the latter. A variety of operations performed by the
writer (1938b) upon large and small colonies show that subordinate
cells—terminal branch cells or merely the common nutritive cells, the
complete developmental potentialities of which are never otherwise

808 MORPHOGENESIS

expressed—can be induced to assume the dominant generative functions.
The regenerative behavior following simple, compound, or successive
operations is another illustration of what Child (1929) referred to as
physiological correlation: the relations of dominance, or control and
subordination between parts. The single apical cell of Zoothamnium
colonies exercises the controlling influence over growth and differentia-
tion in subadjacent cells.

Bsarean

Figure 186. A, a seventy-two-hour regenerate produced from a lateral cell of the first
branch generation (a cell which ordinarily represents the presumptive ciliospore) ; B,
schematic diagram of the apical portion of the colony at the time of cutting (see arrow) ;
C, a similar diagram of the regenerating colony seventy-two hours later, or as shown in A.
(From Summers, 1938b.)

Regional coérdination, according to Child, depends primarily upon
quantitative rather than specific differences in the protoplasmic condi-
tion of the dominant region. Evidence that this is not necessarily the

MORPHOGENESIS 809

case comes from another phase of development in Zoothamnium. In the
sexual process, the apical cell becomes the sessile macrogamont. The
fusion of a free-swimming microgamont with the sexually differentiated
apical cell arrests axial development for several days, pending the origin
of a new apical cell from one of the ex-conjugants. In the meantime,
all of the cells on three or four of the youngest (uppermost) branches
begin to divide precociously. The terminal branch cells are aroused
to unusual mitotic activity, producing twice as many generations as when
they comprise a part of the vegetative colony. The common branch cells
are likewise activated to produce secondary and even tertiary branches.
This precocious development never occurs when the apical cell presides
over a vegetative colony. Neither does it occur as a result of decapita-
tion—when the apical cell is destroyed. The phenomenon appears to be
initiated by qualitative changes in the co6drdinating mechanism, which
arise in consequence of reorganizational activities in the single apical
cell.

The growth relations are likewise altered by conjugation in Z. arbus-
cula (Furssenko, 1929). Each of the several primary axes in this species
bears an apical cell which becomes the macrogamont during the sexual
period. Conjugation on one axis stops further apical extension of that
branch until two new vegetative axes spring from the two “stem cells”
of the fourth ex-conjugant generation. One daughter cell from each of
the first four generations differentiates into a very large macrozodid
(immature ciliospore). A single conjugant therefore produces two grow-
ing points and a cluster of from four to six bulbous macrozodids. As
is the case in Z. alternans, several of the small secondary branches below
the conjugant-bearing node develop to the proportions of primary axes.
Under the influence of a vegetative apical cell, these branches do not
hypertrophy.

Furssenko accounts for the changed relations between apical and
subordinate regions in terms of local variations in the food-energy re-
quirements. In the light of the above observations, he supposed that
the cluster of huge non-feeding macrozodids at the tip of the stalk, to-
gether with the two developing apical cells, have energy needs in excess
of the apical requirements in non-conjugating colonies. Multiplication
of the actively feeding cells on neighboring branches presumably occurs,
in order to compensate for the unusual metabolic needs at the apex.

810 MORPHOGENESIS

In Z. alternans, however, hypertrophy of the inferior branch cells begins
before the conjugant undergoes its first division. It is therefore doubt-
ful whether any increase in the energy requirements, coincidental with
the conversion of an apical cell into an exconjugant, is adequate to
account for the far-reaching alterations of the normal growth pattern.
It is more than likely that the combined energy demands of the ac-
tively dividing cells on subordinate branches exceed those of the single
conjugant or its first few non-feeding descendants. It is probable, then,
that the flux would be directed away from the cell or cells in the
apical position.

Figure 187. A, branch C of a colony fifty-six hours after injury to the neuromuscular
cord (drawn from above) (the original colony of six branches was pinched in the mid-
region, isolating ABC from DEF and the apical cell; the terminal cell on branch C dif-
ferentiated into a new apical cell, which produced two new branches as illustrated) ; B,
schematic representation of branch C, as drawn in A.

We are thus confronted with two divergent interpretations relative
to the specificity of form-regulating factors in Zoothamnium. The evi-
dence presented by Summers suggests that qualitative physiological
changes in one cell play a greater part in the development of neighbor-
ing cells than heretofore suspected. Furssenko’s hypothesis, on the other
hand, emphasizes the directive influence of metabolic fluctuations. It is
therefore consistent with the metabolic-gradient theory, inasmuch as the
nutritive factors may be quantitative and continuous.

The stalk structure of Z. alternans, when viewed in section, somewhat
resembles a sheathed nerve fiber. There is an elastic surface membrane,
a thick cortical region of hyaline, gelatinous material, and a core of
protoplasm, the neuromuscular cord. The latter is continuous from
branch to branch and from cell to cell. Unlike the axis cylinder of a

MORPHOGENESIS 811

nerve fiber, a break in the neuromuscular cord does not cause degeneration
in either of its separated parts. The motor reactions of the cells joined
by the distal segment of the cord are well integrated, but independent
of those connected by the proximal portion. The cord alone, rather
than the entire stalk structure, appears to be the medium through which
regenerative functions are codrdinated. Preliminary experiments have
shown that a local injury to the protoplasmic portion of the stalk physio-
logically divides a colony. The distal part, with its original apical
cell, continues to develop as before, whereas one of the cells on the
proximal (basal) portion differentiates into an apical cell the activity of
which produces another dominant growth axis (Fig. 187). How the
single apical cell regulates developmental functions in the distantly
separated cells, through the agency of the slender protoplasmic thread,
remains as one of the more important problems that invite attention to
these colonial Protozoa.

We are far from having more than an elementary notion of what
goes on within differentiating cells, but it is probable that the recent
developments in other more specific phases of cell physiology presage
a period of keener interest in the age-old problems of growth and form
determination. Most of the publications on regeneration now extant
are important in substance, but lack the specificity of detail which
characterizes the newer contributions on nutrition, respiration, and so
forth. They also indicate (1) the advisability of more carefully planned
pre- and post-operative culture techniques, wherein a better evaluation
of extrinsic factors is possible; and (2) the value of critical distinction
between the failure of cells to survive an experimental procedure and
their failure to regenerate.

LITERATURE CITED

Alverdes, Friederick von. 1922. Zur Lokalisation des chemischen und therm-
ischen Sinnes bei Paramecium und Stentor, Zool. Anz., 55: 19-21.
Balamuth, W. 1939. Studies on regeneration in Protozoa. I. Cytology and

regeneration of Lichnophora macfarlandi. Univ. Cal. Library, MS, 1939.
Balbiani, E. G. 1888. Recherches expérimentales sur la mérotomie des In-
fusoires ciliés. Rec. zool. suisse, 5: 1-72.
—— 1891. Sur la régénérations successives du peristome comme characteré
d’age chez les Stentors et sur la rdle du noyau dans ce phénoméne. Zool.
Anz., 14: 312-16.

812 MORPHOGENESIS

—— 1892. Nouvelle Recherches expérimentales sur la mérotomie des In-
fusoires ciliés. Ann. Microg., 4: 369-449.

1893. Mérotomie des infusoires ciliés. Ann. Microg., 5: 49-84.

Bauer, E. S., and A. M. Granowskaja. 1934a. Die Rekonstruktion des Kerns
und die Atmungsprozesse bei Hypotricha im Ergebnis operativer Ein-
wirkungen auf das Protoplasma und ihre Abhangigkeit vom Alter. Biol.
Zbl., 3, 457 (Berichte ti. wiss. Biol., 33: 282).

— 1934b. Abhangigkeit der experimentellen “‘individuellen Unsterblich-
keit’’ vom Alter. Biol. Zbl., 3: 609 (Berichte ti. wiss. Biol., 33, 282).

Becker, E. 1928. Streaming and polarity in Mastigina hyale. Biol. Bull., 54:
109-16.

Bills, C. E. 1922. Inhibition of locomotion in Paramecium and observations
on certain structures and internal activities. Biol. Bull., 42: 7-13.

Bozler, E. 1924. Uber die Morphologie der Ernahrungsorganelle und die
Physiologie der Nahrungsaufnahme bei Paramecium caudatum Ehtbg.
Arch. Protistenk., 49: 163-215.

Brandt, K. 1877. Uber Actinosphaerium eichornii. Dissertation, Halle.

Brehme, K. S. 1933. An investigation of the question of cytoplasmic fusion in
Amoeba proteus. Arch, Protistenk., 79: 303-10.

Bullington, W. E. 1925. A study of spiral movement in the ciliate infusoria.
Arch. Protistenk., 50: 219-74.

Burch, P. R. 1930. The effect on the division rates of Arcella vulgaris and A.
rotunda 1. of the injury caused by the excision of cytoplasm; 2. of the
loss of cytoplasm; 3. of the gain of cytoplasm. Arch. Protistenk., 71:
307-22.

Burnside, L. H. 1929. Relation of body size to nuclear size in Stentor coeru-
leus. J. exp. Zool., 54: 473-83.

Calkins, G. N. 1911a. Regeneration and cell division in Uronychia. J. exp.
Zool., 10: 95-116.

— 1911b. Effects produced by cutting Paramecium cells. Biol. Bull., 21:
36-72.

— 1919. Uroleptus mobilis, Engelm. 1. History of the nuclei during divi-
sion and conjugation. J. exp. Zool., 27: 293-357.

—— 1921. Uroleptus mobilis, Engelm. IV. Effect of cutting during conjuga-
tion. J. exp. Zool., 34: 449-70.

—— 1934, Factors controlling longevity in protozoan protoplasm. Biol. Bull.,
67: 410-31.

Causin, M. 1931. Régénération du Stentor coeruleus. Arch. Anat. mict.27:
107-25.

Chejfec, M. 1932. Regulacja i regeneracja u Paramecium caudatum. Acta Biol.
exp; 75, 115-34.

Child, C. M. 1914. The axial gradient in ciliate infusoria. Biol. Bull., 26:
36-54,

MORPHOGENESIS 813

1919. Demonstration of the axial gradients by means of potassium

permanganate. Biol. Bull., 36: 133-47.

— 1920. Some considerations concerning the nature and origin of physio-
logical gradients. Biol. Bull., 39: 147-87.

—— 1929. Physiological dominance and physiological isolation in develop-
ment and reconstitution. Roux Arch. EntwMech. Organ., 117: 21-66.
Child, C. M., and E. Deviney. 1926, Contributions to the physiology of

Paramecium caudatum. J. exp. Zool., 43: 257-312.

Claparéde, E., and J. Lachmann. 1858-60. Etudes sur les Infusoires et les
Rhizopods. Mém, Inst. nat. genev., 5-7.

Conklin, E. G. 1912. Cell size nad nuclear size. J. exp. Zool., 12: 1-98.

Dawson, J. A. 1919. An experimental study of an amicronucleate Oxytricha.
J. exp. Zool., 29: 473-508.

Dembowska, W. S. 1925. Studien iiber die Regeneration von Stylonychia
mytilus, Arch. mikr. Anat., 104: 185-209.

— 1926, Studies on the regeneration of Protozoa. J. exp. Zool., 43: 485-
504.

— 1938. K6rperreorganisation von Stylonychia mytilus beim Hungern.
Arch. Protistenk., 91: 89-105.

Fauré-Fremiet, E. 1930. Growth and differentiation of the colonies of
Zoothamnium alternans (Clap. and Lachm.). Biol. Bull., 58: 28-51.
Fortner, H. 1933. Uber Kernresektion bei einem Hypotrichen (nov. spec.?).

Arch. Protistenk., 81: 284-307.

Furssenko, A. 1929. Lebenscyclus und Morphologie von Zoothamnium ar-
buscula Ehrenberg. Arch, Protistenk., 67: 376-500.

Garnjobst, L. 1937. A comparative study of protoplasmic reorganization in
two hypotrichous ciliates, Stylonethes sterkii and Euplotes taylori, with
special reference to cystment. Arch. Protistenk., 89: 317-80.

Gregory, L. H. 1909. Observations on the life history of Tillina magna.
J. exp. Zool., 6: 383-431.

Gruber, A. 1885. Uber kunstliche Teilung bei Infusorien. Biol. Zbl., 4: 717-
D2:

—— 1886. Beitrage zur Kenntniss der Physiologie und Biologie der Pro-
tozden. Ber. naturf. Ges. Freiburg i. B., 1: 1-33.

Hartmann, M. 1924. Der Ersatz der Fortpflanzung von Amében durch fort-
gesetzte Regenerationen. Weitere Versuche zum Todproblem. Arch.
Protistenk., 49: 447-64.

—— 1928. Uber experimentelle Unsterblichkeit von Protozoen-Individuen.
Ersatz der Fortpflanzung von Amoeba proteus durch fortgesetzte Re-
generationen. Zool. Jahrb., Abt. allg. Zool. Physiol. Tiere, 45: 973-87.

Hegner, R. W. 1920. The relations between nuclear number, chromatin mass,

and shell characteristics in four species of the genus Arcella. J. exp. Zool.,

503 1-95;

814 MORPHOGENESIS

Hertwig, R. 1903. Uber Korrelation von Zell-und Kerngrésse und ihre Be-
deutung fiir die geschlechtliche Differenzierung und die Teilung der
Zelle. Biol. Zbl., 23: 49-62; 108-19.

1908. Uber neue Probleme der Zellenlehre. Arch. Zellforsch., 1: 1-33.

Hetherington, A. 1932. On the absence of physiological regeneration in
Stentor coeruleus. Arch. Protistenk., 77: 58-63.

Hewitt, J. H. 1914. Regeneration of Plewrotricha after merotomy with refer-
ence especially to the number of micronuclei and the occurrence of un-
inucleated cells. Biol. Bull., 27: 169-76.

Hofer, B. 1890. Experimentelle Untersuchungen tber den Einfluss des Kerns
auf das Protoplasma. Jena. Z. Naturw., 24: 105-76.

Holmes, S. J. 1907. The behavior of Loxophyllum and its relation to regenera-
tion. J. exp. Zool., 4: 399-430.

Horton, F. M. 1935. On the reactions of isolated parts of Paramecium cauda-
jum, |. exp. Biola 2213-16.

Hosoi, T. 1937. Protoplasmic streaming in isolated pieces of Paramecium.
J. Fac. Sci. Tokyo Univ., Zool., 4: 299-305.

Howland, R. B. 1928. Grafting and reincorporation in Actinosphaerium
eichornit Ehr. Biol. Bull., 54: 279-88.

Hyman, L. H. 1917. Metabolic gradients in Amoeba and their relation to the
mechanism of amoeboid movement. J. exp. Zool., 24: 55-99.

Ishikawa, H. 1912. Wundheilungs- und Regenerationsvorgange bei In-
fusorien, Roux Arch. EntwMech. Organ. 35: 1-29.

Jennings, H. S. 1901. On the significance of the spiral swimming of organ-
isms. Amer. Nat., 35: 369-78.

Jennings, H. S., and C. Jamieson. 1902. Studies on reactions to stimuli in
unicellular organisms. Biol. Bull., 3: 225-34.

Jensen, P. 1896. Uber individuelle physiologische Unterschiede zwischen
Zellen der gleichen Art. Pflig. Arch. ges. Physiol., 62: 172-200.

Kepner, W. A., and B. D. Reynolds. 1923. Reactions of cell bodies and
pseudopodial fragments in D/fflugia. Biol. Bull., 44: 22-46.

Landis, E. M. 1920. An amicronucleate race of Paramecium caudatum. Amet.
Nat., 54: 453-57.

LeDantec, F. 1897. La Régénération du micronucleus chez quelques infusoires
Giliéss |G Re Acad=SciPariswe2snoi5 2.

Lewin, K. R. 1910. Nuclear relations of Paramecium caudatum during the
asexual period. Camb. Phil. Soc. Proc., 16: 39-41.

— 1912. The behavior of the infusorian micronucleus in regeneration. Proc.
roy. Soc., B, 84: 332-44.

Lillie, F. R. 1896. On the smallest parts of Stentor capable of regeneration.
J. Morph., 12: 239-49.

Looper, J. B. 1928. Cytoplasmic fusion in Actinophrys sol, with special ref-
erence to the karyoplasmic ratio. J. exp. Zool., 50: 31-49.

MORPHOGENESIS 815

Ludwig, W. 1929. Untersuchungen tiber die Schraubenbahnen neiderer Or-
ganismen. Z. f. vergl. Physiol., 9: 734-801.

Lund, E. J. 1917. Reversibility of morphogenetic processes in Bursaria. J.
exp. Zool., 24: 1-19.

Luntz, A. 1936. Unsterblichkeit von Protozoenindividuen, erhalten durch
periodische Reizungen. Arch. Protistenk., 88: 23-26.

Lynch, V. 1919. The function of the nucleus of the living cell. Amer. J.
Physiol., 48: 258-83.

Mast, S. O., and F. M. Root. 1916. Observations on Amoeba feeding on
rotifers, nematodes and ciliates, and their bearing on the surface-tension
theory. J. exp. Zool., 21: 33-49.

Maupas, M. E. 1888. Recherches expérimentelles sur la multiplication des
infusoires ciliés. Arch. zool. exp. gén., II, 6: 165-277.

Miller, E. De Witt. 1932. Reappropriation of cystoplasmic fragments. Arch.
Protistenk., 78: 635-45.

Minchin, E. A. 1912. An introduction to the study of the Protozoa. London.

Monod, J. 1933. Mise en évidence du gradient axial chez les Infusoires ciliés
par photolyse a l’aide des rayons ultraviolets. C. R. Acad. Sci. Paris,
196: 212-14.

Moody, J. E. 1912. Observations on the life history of two rare ciliates,
Spathidium spathula and Actinobolus radians. J. Morph., 23: 349-99.

Moore, E. L. 1924. Regeneration at various phases in the life history of
Spathidium spathula and Blepharisma undulans. J. exp. Zool., 39: 249-
316.

Morea, L. 1935. Régénération chez Spirostomum ambiguum. C. R. Soc. Biol.
Paris, 119: 235-37.

Morgan, T. H. 1901. Regeneration of proportionate structures in Stentor.
Biol. Bull. 2: 311-28.

Nadler, J. E. 1929. Notes on the loss and regeneration of the pellicle in
Blepharisma undulans. Biol. Bull. 56: 327-30.

Nussbaum, M. 1884. Uber spontane und kiinstliche Zelltheilung. Verh. Nat-
uth. Ver, Bonne 259:

Okada, Y6 K. 1930. Transplantationsversuche an Protozoen. Arch. Protistenk.,
69: 39-94.

Patten, M. 1921. The life history of an amicronucleate race of Didinium nasu-
tum. Proc. Soc. Exp. Biol. N.Y., 18: 188-89.

Peebles, F. 1912. Regeneration and regulation in Paramecium caudatum.
Biol. Bull., 23: 154-70.

Phelps, L. A. 1926. Experimental analysis of factors concerned in division
in Ameba. Trans. Amer. micr. Soc., 45: 133-45.

Poljansky, G. 1938. Die Rekonstruktion des Kernapparates der Bursaria trun-
catella bei experimentellen Trennung der konjugierenden Paare. Biol.
Zbl., 7: 123 (Berichte ii. d. wiss. Biol., 48: 608).

816 MORPHOGENESIS

Popoff, M. 1907. Depression der Protozoenzelle und der Geschlechtszelle der
Metazoen. Arch. Protistenk., (Suppl.) 1: 43-82.

Prowazek, S. 1904. Beitrage zur Kenntnis der Regeneration und Biologie der
Protozoen. Arch. Protistenk., 3: 44-59.

Reynolds, B. D. 1924. Interaction of protoplasmic masses in relation to the
study of heredity and environment in Arcella polypora. Biol. Bull., 46:
106-40.

Reynolds, M. E. 1932. Regeneration in an amicronucleate infusorian. J. exp.
Zool., 62: 327-61.

Rosel von Rosenhof, A. S. 1755. Insekten Belustigung. 3 vols., Nurnberg.

Runyan, E. M., and H. B. Torrey. 1914. Regulation in Vorticella. Biol.
Bull., 27: 343-45.

Schwartz, V. 1934. Versuche tiber Regeneration und Kerndimorphismus der
Ciliaten. Nachr. Ges. wiss. Gottingen, math.-physik., N.F. 1: 143-55.

— 1935. Versuche tber Regeneration und Kerndimorphismus bei Stevtor
coeruleus Ehrbg. Arch. Protistenk., 85: 100-39.

Seyd, E. L. 1936. Studies on the regulation of Spirostomum ambiguum. Arch.
Protistenk., 86: 454-70.

Sokoloff, B. 1922. Le Noyau est-il indispensable a la régénération des Proto-
zoaires? C. R. Soc. Biol. Paris, 87: 1144-47.

— 1923. Hunger and regeneration. J. R. micr. Soc., London, 1923, 183-
39:

—— 1924. Das Regenerationsproblem bei Protozoen. Arch. Protistenk., 47:
143-252.

Stevens, N. M. 1903. Further studies on the ciliate infusoria, Lichnophora and
Boveria. Arch. Protistenk., 3: 1-43.

Stol¢, A. 1910. Uber kernlosen Individuen und kernlose Teile von Amoeba
proteus. Roux Arch. EntwMech. Organ., 29: 152-68.

Summers, F. M. 1938a. Some aspects of normal development in the colonial
ciliate Zoothamnium alternans. Biol. Bull., 74: 117-29.

— 1938b. Form regulation in Zoothamnium alternans, Biol. Bull., 74:
130-54.

Tartar, V. 1939. The so-called racial variation in the power of regeneration in
Paramecium. J. exp. Zool., 81: 181-208.

Taylor, C. V. 1928. Protoplasmic reorganization in Uronychia uncinata, sp.
nov., during binary fission and regeneration. Physiol. Zo6l., 1: 1-25.

Taylor, C. V., and W. P. Farber. 1924. Fatal effects of the removal of the
micronucleus in Evplotes. Univ. Cal. Publ. Zool., 26: 131-43.

Thon, K. 1905. Uber den feineren Bau von Didinium nasutum O. F. M. Arch.
Protistenk., 5: 281-321.

Tittler, I. A. 1935. Division, encystment, and conjugation in Urostyla grandis.

Cellule, 44: 189-218.

1938. Regeneration and reorganization in Uroleptus mobilis follow-

ing injury by induced electric currents. Biol. Bull., 75: 533-41.

MORPHOGENESIS 817

Verworn, Max. 1889. General Physiology. 2d ed., London.

1892. Die physiologische Bedeutung des Zellkerns. Pfliig. Arch. ges.

Physiol., 51: 1-118.

Willis, H. S. 1916. The influence of the nucleus on the behavior of Amoeba.
Biol. Bull., 30: 253-70.

Woodruff, L. L. 1905. An experimental study of the life history of hypo-
trichous infusoria. J. exp. Zool., 2: 585-632.

—— 1913. Cell size, nuclear size and the nucleocytoplasmic relation dur-
ing the life of a pedigreed race of Oxytricha fallax. J. exp. Zool., 15:
1-22.

—— 1921. Micronucleate and amicronucleate races of infusoria. J. exp. Zool.,
34: 329-37.

Young, D. B. 1922. A contribution to the morphology and physiology of the
genus Uronychia. J. exp. Zool., 36: 353-90.

—— 1926. Nuclear regeneration in Stylonychia mytilus. Biol. Bull., 51:
163-65.

Young, Dixie. 1939. Macronuclear reorganization in Blepharisma undulans.
J. Morph., 64: 297-353.

CHAPTER XVII

CERTAIN ASPECTS OF PATHOGENICITY
OF PROTOZOA

ELERY R. BECKER

IT Is CUSTOMARY to recognize three functional categories of parasitic
Protozoa: (1) commensals, which neither harm nor abet the host; (2)
symbionts (= symbiotes), which aid the host; and (3) true parasites
or pathogenes, which disarrange the host organism to a greater or less
degree. This practice may be defended on academic grounds, since it
serves to clarify concepts and to attract students’ interest to animal
microérganisms and the rdles they play in the lives of other animals and
plants, but it is in reality highly artificial. The ensuing discussion will
be developed principally about this point, with the deliberate intention
of provoking wide consideration of the subject, particularly as regards
the “pathogenic” aspects of parasite activity, as was done with the
subject of host-specificity of parasites a number of years ago (Becker,
1933). Such terms as commensalism, symbiosis (symbiotism), and
pathogenicity can represent no more than an expression of the state of
adjustment between two separately functioning entities, the host and the
parasite, co€éxisting in one of the most intimate relationships, and as
such are subject to analysis.

PROBLEMS OF VIRULENCE AND PATHOGENICITY

The functional categories have no counterparts in the zodlogical
scheme: that is to say, there are no classes, orders, or families which
have as their distinguishing character that they are pathogenic or other-
wise. The statement applies also to genera, for, as a matter of fact, we
recognize ‘‘pathogenic”’ and ‘“‘non-pathogenic’” members of Trypanosoma,
Trichomonas, Entamoeba, and other genera. The situation is seen, at
the outstart, to limit itself almost entirely to a consideration of ‘“‘patho-
genic species,”’ but it is actually still more complicated than that. There
is indisputable evidence that many species of pathogenic Protozoa are

PATHOGENICITY 819

made up of a number of strains. Entamoeba histolytica, for example, is
believed to be a composite of many races differing both in cyst size (see
Dobell and Jepps, 1918) and virulence (see Meleney and Frye, 1935, pp.
431-32). The evidence for the latter is indisputable, especially since the
appearance of the work of Meleney and Frye (1933, 1935), although
Craig (1936) is still skeptical regarding the existence of avirulent
strains. The latter point can be conceded for the present (though it is
still a live issue), without impugning the significance of observations
on human cases and experimental infection in kittens and puppies point-
ing to the existence of strains of low virulence, medium virulence, and
high virulence.

Meleney and Frye adopted a standardized procedure. Recognizing the
doubtful validity of experiments performed on too few animals and
conducted without due allowance for variability of individual re-
sponse, they made it a practice to test each strain in a large series of
kittens of standard size. Each strain was isolated in culture, young trans-
plants were used for inoculations, and inoculations were made directly
into the caecum after laparotomy incision. Furthermore, the history of
the human patient was known, and there were records regarding the
character of the community and the prevalence of amoebiasis in the com-
munity in which each patient resided. The criteria of pathogenicity were
success or failure in infecting, extent and intensity of lesions produced,
and duration of the infection. The results of the experiment showed
conclusively that certain strains of Entamoeba histolytica of human
origin exhibited more “‘pathogenic activity” in kittens than other strains.

Furthermore, by correlating the experimental data with field observa-
tions, they were led to the following conclusion regarding the relative
pathogenicity of the strains for man: “The more pathogenic strains
(i.e., in kittens), whether they were obtained from acute cases of
amoebic dysentery or from so-called ‘healthy carriers’ were associated
geographically and epidemiologically with acute dysentery, whereas the
less pathogenic strains were associated both individually and epidemi-
ologically with very little evidence of acute dysentery.”

AMOEBIC DYSENTERY AND BACTERIAL COMPLICATIONS

The problem of virulence has had its reflection in matters of specificity.
Brumpt (1925) described Entamoeba dispar as an amoeba of the

820 PATHOGENICITY

histolytica-type dwelling in man, incapable of producing symptoms of
dysentery in its human host, and producing no definite macroscopic
ulcerations in the cat, but capable of penetrating the intestinal wall of the
latter animal so far as the muscularis mucosae. Simi¢, in several papers,
has corroborated Brumpts’s claim for the validity of E. dispar, but his last
(1935) paper presents the strong argument that E. dispar infection in
dogs lasts only from 6 to 8 days, while E. /Azstolytica infection lasts
from 60 to 120 days. E. dispar infection in dogs is quite benign, while
E. histolytica produces characteristic amoebic ulcerations, and amoebae
with ingested red cells may be found in the stools of the infected dog.
Wenyon (1936), however, in commenting on the strong case built
up by Simié (1935) states:

It still seems futile to attempt to separate E. dispar from E. histolytica on
the grounds of pathogenicity. It seems that all the differences described can be
more reasonably accounted for by the supposition that races of E. histolytica
of varying virulence occur and that hosts vary in their susceptibility to the
one species.

Meleney and Frye (1935) likewise prefer to consider E. dispar as a
strain of E. histolytica, possessing a low degree of pathogenicity. Even
the least virulent strains encountered by the latter authors, though not
producing clinical symptoms in the persons in whom they had their
origin, were capable of producing lesions in some kittens. Hence the
skepticism of these authors regarding totally avirulent strains of E. hzs-
tolytica.

Is virulence-level retained by a strain of E. histolytica, or is it sub-
ject to modifying factors, such as attenuating effects of artificial culture
media or exaltation by animal passage? Meleney and Frye (1933) first
noted the contrast in pathogenic activity in kittens between “A”’ strains
of low virulence from the hill country, where symptomless carriers and
persons with mild symptoms were the rule, and “‘B” strains of high
virulence from severe cases of amoebic dysentery in the bottom lands,
where acute cases were much more common than in the hills. Later
(1935), they were able to report, after adequate testing in kittens, that
two “A” strains and two “B” strains had retained their respective patho-
genic indices after a period of three years of artificial cultivation. The
highly virulent strains did not decline in pathogenicity. But what about
the effect of animal passage? Meleney and Frye (1936) state that their

PATHOGENICITY 821

efforts to step up the virulence of less pathogenic strains by serial passage
through kittens and dogs have always failed at the first transfer, but
Cleveland and Sanders (1930) have made some experiments bearing on
this point, except that they ascribe the effects observed to bacteria rather
than to amoebae. Using for the first passage in kittens a strain that had
been carried on in culture for from 460 to 540 days, they found that only
2 out of 26 animals became infected in the first passage, 5 out of 5 in
the second, 3 out of 7 in the third, and 2 out of 2 in the fourth. They
conclude: ‘‘An increase in the percentage of animals that became infected
with passage is demonstrated in these experiments, but this may be due,
as in the liver passages, to an increase in virulence by the bacteria rather
than the amoebae.” The need for further work on the possibility of exalt-
ing the virulence of less pathogenic strains by animal passage is appar-
ent, but efforts along this line, in order to obtain results of significance,
will first have to eliminate the effects of bacteria accompanying the
Protozoa.

The comments of Cleveland and Sanders regarding bacteria suggest
the next point, the effect of bacteria on pathogenicity of E. histolytica.
Their criterion for virulence was principally infectivity for the liver in
kittens, when inoculated with a hypodermic needle directly into this
organ. Pure cultures in liver-infusion, agar-horse serum saline medium
lost most of their ability to establish infection in the liver after a year
or more. Such a strain was reduced to an infectivity of 20 percent in the
first passage. The infectivity increased, however, with succeeding pas-
sages, until by the sixth passage it amounted to 73 percent. Was the ap-
parent increase in virulence to be attributed to the amoebae or to the
bacteria accompanying them? Which had lost virulence during the year
of life in the artificial media?

An attempt to settle the issue was made in a crisscross experiment.
Bacteria from the fifth passage were inoculated with the culture amoeba
that had not been passed; and, conversely, the passed amoebae were
inoculated with bacteria that had not been passed, the latter being the
nonpathogenic Bacillus brevis. The experiments showed that fifth-pas-
sage bacteria increased the virulence of amoebae in culture for a year,
and that amoebae were not able to maintain themselves in the liver of the
cat unless accompanied by bacteria capable of damaging the liver. Thus
Cleveland and Sanders concluded that it was the bacteria accompanying

822 PATHOGENICITY

E. histolytica in culture, and not the amoeba, which lost virulence during
the year in the artificial medium and regained virulence after repeated
liver passage. They add the precautionary remark that failure to infect
livers with cultures containing only nonpathogenic bacteria might not
be repeated with more virulent strains of E. histolytica.

Frye and Meleney (1933) attacked the same problem, using severity
of intestinal infection as the criterion. They, too, tried a crisscross tech-
nique, interchanging the bacteria in a culture of proved high virulence
with those in a culture of proved low virulence. The interchange did not
materially alter the incidence or severity of infection of the two strains
of amoebae; hence their conclusion that the difference in pathogenicity
of the two cultures was really due to the amoebae themselves. Thus
differences in pathogenicity of strains of E. Aistolytica claimed by Mel-
eney and Frye was shown to be due to inherent qualities of the proto-
zoon, and not to accompanying microdrganisms. Since they had previously
not been able to detect any alteration of pathogenicity in artificial me-
dium, the question of alteration of virulence of bacteria in such a medium
does not enter in.

MALARIA: Plasmodium vivax

It is inescapable that there are strains of intestinal Protozoa differing
in virulence, but what is the situation regarding the pathogenic blood
Protozoa? Since the behavior of the trypanosomes in animal passage 1s
complicated by differences in behavior of “‘passage’’ and ‘‘relapse”’
strains, the author prefers to evade discussing this subject. Human
malaria, however, lends itself more readily to discussion, as becomes
evident after reading the chapter entitled “The Complexity of the Ma-
laria Parasite” in Hackett (1937). Malaria therapy in general paralysis
(paresis) has made it possible to determine definitely whether there are
strains of the human malarias differing in morphology, pathogenicity, or
other behavior, and the facts learned have been rather surprising.

Plasmodium vivax is the species commonly employed in malaria ther-
apy. Using infected Anopheles for inoculation, Boyd and Stratman-
Thomas (1933a) showed that during an attack of malaria induced by a
particular strain of this species, a patient acquires a ‘“‘tolerance’’ which
makes him refractory to reinoculation with that strain, but not with a
different strain of the same species. They concluded that a person in-

PATHOGENICITY 823

fected with benign tertian malaria acquires a homologous but not a
heterologous tolerance to P. vivax. Later, Boyd, Stratman-Thomas, and
Muench (1934) discovered that superinfections with heterologous
strains appear to result in clinical attacks of milder intensity than the
original attacks. Manwell and Goldstein (1939) have discovered a
similar situation in P. circum flexum infection in birds. Using six strains,
they concluded that immunity was strain specific rather than species
specific, although all strains conferred at least partial protection against
the others. It should be added that certain strains of P. vivax do have the
ability to immunize (or premunize) the patient toward certain other
strains.

VARIABILITY IN STRAINS AND IN Host RESPONSE

Morphological differences between strains of P. vivax have been ob-
served. Two strains of this species widely used in Europe for malaria
therapy are the so-called Dutch and Madagascar strains. Buck (1935)
has found that the Dutch strain consistently exhibits between twelve
and thirteen merozoites in both mosquito-inoculated and blood-inocu-
lated malaria, while the Madagascar strain exhibits between seventeen
and eighteen. The incubation period of the former is twenty-one days,
while that of the latter is but twelve days. Whether there is a relationship
between merozoite number and incubation period in these cases is some-
what of a problem, especially since the discovery of extracellular schi-
zogony of malaria organisms in the internal organs.

Strains of P. vivax likewise differ exceedingly in pathogenicity. Some
strains are too low in virulence to be useful in malaria therapy of gen-
eral paralysis. The Dutch strain referred to above is said by Hackett
(1937) to give higher fever, to be less susceptible to treatment with
salvarsan, and to be less virulent than the Madagascar strain. Further-
more, it often produced no immediate attack, but in 40 percent of the
cases went into a long latency of several months, a phenomenon that
occurred with the Madagascar strain in only 6 percent of the cases.

There appear to be likewise multiple strains of the other human
malarias, viz., P. falciparum and P. malariae (see Hackett, 1937; Boyd
and Kitchen, 1937).

Every case of parasitism exhibits three aspects—the parasite, the host,
and the effect of the impinging of the one on the other. It has been

824 PATHOGENICITY

shown that parasitic Protozoa differ not only in the response they evoke
from the host according to their standing as species, but also according
to strain properties. There is likewise abundant evidence that individual
hosts differ in their response to the same strain of parasitic protozo6n.
The latter is in reality a statistical concept. It has been a general experi-
ence that when an attribute of an unselected group of individuals was
measured, the plotted measurements fell into the well-known frequency
distribution curve, either normal or skewed. The writer knows of no
data which have been plotted to demonstrate that quantitative data on
either individual resistance or susceptibility to adverse effects of parasit-
ism could be presented in a similar sort of graph, but there are many
facts, to support such a supposition.

COCCIDIOSIS IN POULTRY

For several years the writer (see Becker and Waters, 1938, 1939b)
has been testing the effect of the ration on the course of caecal coccidiosis
in chicks. While, in general, fatality was used as the criterion for com-
paring the effects of two rations, it has been possible to make a number
of additional hitherto-unpublished observations bearing on the variability
of host response to the disease. In one lot of thirty-three White Leg-
horn chicks experimentally infected with the same dosage, there were
three deaths by eight o’clock in the morning of the fifth day, and five
more during the remainder of that day. The next day nine succumbed,
making a total of seventeen. Nine others were noted to be in an ex-
tremely precarious condition, missed succumbing only by a narrow mar-
gin, but recovered to a considerable degree. Five others were observed
to be severely affected, but continued to move about and eat some feed
during the entire ordeal. One was quite active throughout, though its
comb paled significantly. One, a cockerel, continued to eat and move
about with undiminished vigor, and its comb did not pale perceptibly,
though the droppings were streaked slightly with blood. Similar observa-
tions have been common, and justify the assertion that fowls differ sig-
nificantly in the morbidity they exhibit in response to uniform dosage
with the same strain of Coccidinm.

The literature is replete with evidence that similar variability of host
response exists in the case of other protozoan infections. Walker and
Sellards (1913) early distinguished between ‘“‘contact”’ carriers of En-

PATHOGENICITY 825

tamoeba histolytica (who did not develop dysentery) and ‘‘convalescent”’
carriers (who have suffered with dysentery, but have become convales-
cent). In fact, they passed a strain of the amoeba from a convalescent
carrier serially through three other men, two of whom became contact
carriers, i.e., did not develop dysentery, and one of whom became a
victim of an acute attack of amoebic dysentery. Meleney and Frye, in
the experiments previously mentioned, found that kittens inoculated
with the same strain differed as to whether or not they became infected,
as to the extent and severity of the lesions in the colon, and as to the pe-
riod of survival of the diseased kittens.

Individuals differ also in the degree of resistance offered to the multi-
plication of the malaria parasite in their blood and tissues, and in their
reaction to parasite density. The existence of racial tolerance or resist-
ance of Negroes to inoculation with Plasmodium vivax was pointed out
by Boyd and Stratman-Thomas (1933b), though it was by no means
absolute. The same authors later (1934) reported their finding that
Caucasians appear to be universally susceptible. Wilson (1936, quoted
by Hackett) made the observation that Bantu babies in Tanganyika Ter-
ritory were all infected with the three species of human malaria by the
fifth month of life, and commented as follows:

One of the striking features of this period of acute infestation, lasting about
eighteen months, is the difference in degree of infestation in different in-
dividuals. These babies were constantly being reinfected by fresh invasions

of sporozoites. The difference cannot therefore be due to variations in the
parasites, but rather to a variation in individual resistance.

Hackett (1937) discusses the variability in the incubation period ex-
hibited by different individuals, and states that in some cases there were
as few as one parasite per cubic millimeter at the onset of symptoms, while
in others there were 900.

Thus it is evident that the clinical aspects of protozoan infections
may differ, owing to inherent basic characters of both the parasite and
the host. The reaction of the host may be governed further by another
factor that we shall designate the physiological state. Admittedly very
little is known concerning the relationship between the physiological
state and pathogenicity, but one would conclude a priori that a far-reach-
ing relationship should prevail here. As a striking concrete example,
nursling rats usually succumb to the long-supposed ‘non-pathogenic’

826 PATHOGENICITY

Trypanosoma lewisi, while older rats undergo a response to this micro-
organism, in behavior of leucocytes and monocytes, that confers on them
sufficient resistance for survival.

NUTRITION AND RESISTANCE

Nutrition may have a far-reaching effect on physiological state, and
indirectly on resistance. The following hitherto-unpublished experiment
is useful in illustrating the point. Forty young rats of about fifty grams’
average weight were divided into two equal groups. One group was
fed the following mixture (parts by weight): Beet sugar, 67; casein,
unextracted, 10; normal salt mixture, 3; lard, 3; cod liver oil, 2; bright
green alfalfa meal, 15. The other group was fed the same mixture, ex-
cept that alfalfa meal was replaced with whole oats ground to a fine
flour. After two weeks on these rations, the lot receiving the ground
oats had made slightly greater weight gains than the other. On the fif-
teenth to the eighteenth days each rat was fed 10,000 recently sporulated
oocysts of Ezmerza nieschulzi, a coccidium that develops in enormous
numbers in the mucosa of the small intestine. On the sixth day the al-
falfa-fed rats were obviously affected with diarrhoea, while the oat-fed
animals were not showing distress. Strangely enough, on the seventh day
the alfalfa-fed lot appeared to be recovering, with formed stools and
return of appetite, but the oat-feds were off their feed and passing liquid
stools. On the eighth and ninth days, 16 out of 20 of the latter died, a
marked contrast to what happened in the alfalfa-fed lot all of which re-
covered. The result was rather surprising, in view of the biological assays
of Becker and Derbyshire (1937, 1938) and Becker and Waters
(1939a), which showed that alfalfa meal in the ration in some manner or
other stimulated the development of several times as many odcysts of
Eimeria nieschulzi in its rat host as either oat hulls or hulled oats.

What is the explanation of the observed effects? The early develop-
ment of diarrhoea in the alfalfa-feds appears to have been due to the
preponderance of the parasite population, but there is a possibility that
it lies in the superior accessory food factors of alfalfa meal. The follow-
ing experiment suggests that vitamin B may have had something to do
with it. Twenty young rats were fed the following ration: beet sugar,
71; soy-bean oil meal, expeller process, 10; casein, commercial medium
fineness, 10; normal salt mixture, 4; lard, 3; cod-liver oil, 2. Another

PATHOGENICITY 827

lot of 20 was fed the same mixture with 10 micrograms of thiamin
chloride (vitamin B) per rat daily. The second lot made much greater
weight gain during the next ten days than the first. On the tenth to the
fifteenth days each rat was inoculated with daily doses of 6,000 sporu-
lated odcysts of E. nieschulzi. Twelve rats out of 20 in the first lot
succumbed to the infection, and the remaining 8 all lost weight upon
recovery. The recipients of vitamin B all lived and, by the time the in-
fection had cleared up, all had gained in weight.

Thus it is evident that the physiological state may be of prime im-
portance in determining whether or not an animal survives an infection.
In one state it may show few or no outwardly visible symptoms, while
in another it may be seriously affected, or even succumb.

CONCLUSIONS

Such terms as commensalism and true parasitism lose their significance
when a comprehensive analysis is made of the circumstances surround-
ing an infection with any particular protozoan species. Pathogenicity in
the generally accepted sense is a matter of degree, subject in the first
place not only to the species, but also to the strain, of the microorganism
concerned in the infection. The degree of pathogenicity exhibited by a
particular strain in its host may vary from nil to fatal termination,
depending upon the inherent defense mechanisms and the other condi-
tions affecting the resistance of the host. The effectiveness of this resist-
ance, in turn, may vary according to changes in the physiological state
of the host. These considerations are of fundamental importance to the
investigator who conducts researches on the reaction of any host to the
invasion of a protozoan parasite.

LITERATURE CITED

Becker, E. R. 1933. Host-specificity and specificity of animal parasites.
Amer. J. lrop.- Med 13)505-23)

Becker, E. R., and R. C. Derbyshire. 1937. Biological assay of feeding stuffs in
a basal ration for coccidium-growth-promoting substance. I. Procedure,
yellow corn meal, oats, oat hulls, wheat, linseed meal, meat scraps. Iowa
St. Coll. J. Sci., 11 (1938): 311-22. II. Barley, rye, wheat bran, wheat
flour middlings, soy bean meal. Ibid., 12: 211-15.

Becker, E. R., and P. C. Waters. 1938. The influence of the ration on mortality
from caecal coccidiosis in chicks. Iowa St. Coll. J. Sci., 12: 405-14.

—— 1939a. Biological assay of feeding stuffs in a basal ration for coccidium-

828 PATHOGENICITY

growth-promoting substance. III. Dried fish meal, alfalfa meal, white
wheat flour. Iowa St. Coll. J. Sci., 13: 243.

—— 1939b. Dried skim milk and other supplements in the ration during
caecal coccidiosis of chicks. Soc. Exp. Biol. N.Y., 40:439.

Boyd, M. F., and S. F. Kitchen. 1937. The duration of the intrinsic incuba-
tion period in Falciparum malariae in relation to certain factors affecting
the parasites. Amer. J. Trop. Med., 17: 845-48.

Boyd, M. F., and W. K. Stratman-Thomas. 1933a. Studies on benign ter-
tian malaria. I. On the occurrence of acquired tolerance to Plasmodium

vivax, Amer. J. Hyg., 17: 55-59.

1933b. Studies on benign tertian malaria. IV. On the refractoriness

of Negroes to inoculation with Plasmodium vivax. Amer. J. Hyg., 18:

485-89.

— 1934. Studies on benign tertian malaria. V. On the susceptibility of
Caucasians. Amer. J. Hyg., 19: 541-44.

Boyd, M. F., W. K. Stratman-Thomas, and H. Muench. 1934. Studies on be-
nign tertian malaria. VI. On heterologous tolerance. Amer. J. Hyg., 20:
482-87.

Brumpt, E. 1925. Etude sommaire de |’“Entamoeba dispar’ n.sp. Amibe
a kystes quadrinucleés, parasite de "homme. Bull. Acad. Med., Paris,
94: 943.

Buck, A. de. 1935. Ein morphologischer Unterschied zwischen zwei Plasmo-
dium vivax-Stammen. Arch. Schiffs- u. Tropenhyg., 39: 342-45.

Cleveland, L. R. and E. P. Sanders. 1930. The virulence of a pure line and
several strains of Entamoeba histolytica for the liver of cats and the
relation of bacteria, cultivation, and liver passage to virulence. Amer.
i: Tyg 12569-6005»

Craig, C. F. 1936. Some unsolved problems in the parasitology of amebiasis.
Parasitology, 22: 1.

Dobell, C., and M. W. Jepps. 1918. A study of the diverse races of Entamoeba
histolytica. Parasitology, 10: 320-51.

Frye, W. W., and H. E. Meleney. 1933. Studies of Endamoeba histolytica and
other intestinal Protozoa in Tennessee. VI. The influence of the bacterial
flora in cultures of E. Aiéstolytica on the pathogenicity of the Amoebae.
Amer. J. Hyg., 18: 543-54.

Hackett, L. W. 1937. Malaria in Europe. London.

Manwell, R. D., and F. Goldstein. 1939. Strain immunity in avian malaria.
Amer, J. Hyg. sec. Cy 30315222:

Meleney, H. E., and W. W. Frye. 1933. Studies of Endamoeba histolytica
and other intestinal Protozoa in Tennessee. V. A comparison of five
strains of E. histolytica with reference to their pathogenicity for kittens.
Amet:.JaHyg.,'17: 637-55.

— 1935. Studies of Endamoeba histolytica and other intestinal Protozoa

PATHOGENICITY 829

in Tennessee. IX. Further observations on the pathogenicity of certain
strains of E. Aistolytica for kittens. Amer. J. Hyg., 21: 422-37.
1936. The pathogenicity of Endamoeba histolytica. Trans. R. Soc. trop.

Med. Hyg. 29: 369.

Simi¢, T. 1933. L’Infection du chien par l’Entamoeba dispar Brumpt. Ann.
Parasit. hum. corp., 11: 117-28.

— 1935. Infection expérimentale du chat et du chien par Entamoeba
dispar et Entamoeba dysenteriae. Ann. Parasit. hum. corp., 13: 345-50.

Walker, E. L., and A. W. Sellards. 1913. Experimental entamoebic dysentery.
Philipp. J. Sa. (B., Trop. Med:), 6: 259.

Wenyon, C. M. (“C. M. W.”) 1936. Trop. Dis. Bull., 33:534.

CHAPTER XV iil

THE IMMUNOLOGY OF THE PARASITIC PROTOZOA

WILLIAM H. TALIAFERRO

THE CENTRAL theme of the science of immunology is the study of the
defense mechanisms of the host against the invasion of parasitic or-
ganisms or against the introduction of their products or of other 1n-
animate materials. In the present chapter emphasis is placed almost
entirely on the defense mechanisms against living parasites. A complete
analysis of these mechanisms involves such widely diverse subjects as the
origin, nature, and developmental potencies of the cells and tissues of the
host, the physiological action and chemical nature of the humoral forces
marshaled by the host in defense, the activity of the invading parasite,
the chemical nature of the products of the parasite which stimulate the
immune processes in the host, and the effects of the various immune proc-
esses on the parasite. As protozoan immunity is just one aspect of the
general field of immunology, most of the general principles of immunity
can be applied directly to the protozoan parasites. Work on protozoan
immunity itself, however, has been restricted more or less to the biologi-
cal aspects, such as the study of the cellular and the serological mecha-
nisms of the host and the effects of resistance on the parasite, with very
little emphasis on chemical phases.

THE PHYSICAL BASES OF IMMUNITY

Immunity or resistance, in the broad sense, denotes various mecha-
nisms of the host which counteract the invasion and the activities of a
parasite. It may be manifested as hindrances to the action of invasion,
as conditions arising in the body of the host adverse to the parasite, as
efforts on the part of the host to make good the deleterious effects of the
parasite (as evidenced by the hyperactivity of hematopoietic organs after
the destruction of red cells in malaria), or the production of antitoxins
in those infections in which toxins are formed. It may be natural (in-
nate) or acquired. Natural immunity is generally correlated with non-

IMMUNOLOGY 831

specific factors which are incompatible with or unfitted to the life of the
parasite in the unimmunized host. The specificity of parasites for various
hosts (Chapter XVII) is largely an expression of natural immunity.
Acquired immunity, on the other hand, denotes the various conditions
arising in a host as a result of infection or other immunizing procedure
and is generally thought of as resulting in large measure from the pro-
duction of antibodies in the host.

Immunity is the reciprocal of virulence, which in this sense is an ex-
pression of the ability of the parasite to invade and parasitize the host.
Both immunity and virulence are relative and represent the resultant of
the invasive activities of the parasite and the defense activities of the
host; they may, therefore, vary in degree from zero to 100 percent.

THE CELLS INVOLVED IN IMMUNITY

The defense of the vertebrate body against invading parasites, or
even against inanimate foreign material introduced parenterally, appears
to be taken care of predominantly by some of the cells of the connective
tissue and is a specialized or accentuated aspect of their normal functions.
The connective tissue has manifold normal functions, such as respiration,
intermediate metabolism, storage, and mechanical support and in its
widespread distribution throughout the body consists of the blood and
lymph, cartilage, bone, the reticular (blood-forming) tissue of the
myeloid and lymphatic organs, and loose and dense connective (includ-
ing adipose) tissues associated with the skin, omentum, liver, lung, and
so forth. The cells of this tissue arise embryonically from the mesen-
chyme and may be either fixed or free. Those of the blood and lymph
and of the reticular and loose connective tissues are chiefly concerned in
defense.

The terminology of the connective tissue cells is complicated by
the frequent use of several names for the same cell. This condition has
arisen (1) because connective tissue is so widespread and involves so
many organs that it has been studied by histologists, hematologists,
pathologists, and so forth, some of whom have not correlated the
knowledge in fields other than their own; and (2) because investigators
have disagreed as to the nature and developmental potencies of various
cells. In the following brief review we have defined only those cells of
the connective tissue which are known to be involved in defense against

832 IMMUNOLOGY

the infections to be described herein and have followed in the main
Maximow’s views with regard to the origin and potencies of the various
cells. We have simplified and used uniform terms wherever possible.

The role of the various connective tissue cells in immunity is shown
by direct histological studies and by other experimental work. Thus
histological studies of defense reactions have demonstrated directly that
some cells remove parasites and various types of debris by phagocytosis;
that others wall off nonremovable objects and repair damage by filling
in cavities, regenerating certain tissues, and so forth; and that still others,
such as the eosinophils, show a definite pattern of behavior and seem
correlated with certain phases of immunity, although their exact function
is still uncertain. On the other hand, removal or impairment by splenec-
tomy, blockading procedures, and the like, of an appreciable portion
of the connective tissue cells have furnished evidence of the rdle of
phagocytes in the immunity of certain infections and of the rdle of the
macrophages in the production of antibodies.

In the successful carrying out of these studies, certain technical difh-
culties have to be recognized and overcome. To study cellular details
and especially to see transitional forms, migrating cells, and so forth,
early and closely spaced stages in an infection should be studied, fresh
material should be used, and this should be adequately fixed and stained
by a satisfactory technique. One of these techniques involves fixing in
Helly-Maximow’s Zenker formol, preferably embedding in celloidin,
staining with dilute Delafield’s hematoxylin, and counterstaining with
eosin azure II. In impairing the macrophage system, the time when
splenectomy and blockade are performed is important, inasmuch as
impairment is partially made good by the host in time. Furthermore,
splenectomy is more effective in impairing the macrophage system in
certain infections in which the spleen is especially active and in certain
laboratory animals having a high spleen weight-body weight ratio. Thus
the most conclusive results may be expected when certain blood infec-
tions are studied in dogs, rats, and mice splenectomized and blockaded
as rapidly and thoroughly as possible; whereas inconclusive or negative
results may be expected from inadequate blockade, splenectomy a week
or more before infection. In fact, if impairment is slight, the system
may even be stimulated to greater activity.

A, Predominantly Fixed Connective Tissue cells ——From the strictly

IMMUNOLOGY 833

functional aspect of immunity, the predominantly fixed cells of the
reticular and loose connective tissues may be divided into two great
groups: (1) fixed and free macrophages (including the reticular cells),
and (2) the fibroblasts of connective tissue and the endothelial cells
lining the ordinary blood vessels.

The term macrophage is essentially a physiological designation for
almost any large mononuclear connective-tissue cell which is or may
become phagocytic. Under macrophages are classified a group of fixed
mesenchymal cells, which retain many embryonic characters and a wide
range of potencies for development. The concept that the connective
tissue of the adult body possesses fixed cells retaining mesenchymal or
embryonic potencies for development is largely due to Marchand (1924,
review) and Maximow (1927a, review). There are three chief cate-
gories: (1) Pericytes (Maximow) which are fixed, undifferentiated,
outstretched cells in the adventitia of all of the small blood vessels of
loose connective tissue throughout the body; (2) reticular cells, which,
together with fibers, form the stroma of all reticular (myeloid and
lymphatic) tissues; (3) littoral cells (Siegmund), which line the sinuses
or sinusoids of the reticular tissues, the liver, hypophysis, and adrenal
(Pls. 1 and 2). Where phagocytic in the liver, they are generally desig-
nated Kupffer cells (Pl. 1, Fig. 1; Pl. 2, Fig. 1). The cells lining the
sinuses of the reticular tissues are actually reticular cells. The littoral
cells are often called endothelial cells or cells of the special endothelium,
but this is unfortunate because the littoral cells have wide developmental
potencies, whereas the ordinary endothelial cells lining the blood vessels
have restricted developmental potencies.

There is general agreement that under proper stimuli the cells of
these three categories can divide by mitosis, can become phagocytic,
can develop into fibroblasts, or can develop into practically any other
type of cell of the blood or connective tissue. From the standpoint of the
present discussion, it is important that they can become phagocytic either
in their fixed position (fixed macrophages) or after rounding up and
becoming free (free macrophages). It is not definitely known, however,
whether, while engorged, they temporarily or permanently lose their
mesenchymal potencies. There may be a difference, for example, between
the primitive outstretched reticular cell and the same cell after it has
become free and phagocytic.

834 IMMUNOLOGY

In addition to the cells which are generally admitted to retain mesen-
chymal potencies, free cells occur in the loose connective tissue, which
we have called macrophages and which are variously known as histio-
cytes, clasmatocytes, rhagiocrine cells, or resting wandering cells. Just
as in the case of the phagocytic mesenchymal cells, there is no unani-
mity of opinion as to whether these free cells retain all hematopoietic
functions, but in any case they can become phagocytic without morpho-
logical change, can reproduce by mitotic division, and can transform
into fibroblasts.

Many other macrophages occur throughout the body, the develop-
mental capacities of which have not been adequately studied. Thus the
stroma cells of the /amina propria of the intestine probably have develop-
mental potencies identical with those of the reticular cells.

As would be expected, macrophages in different locations and before
and after becoming phagocytic vary somewhat in structure with regard
to the amount of their cytoplasm, the size and shape of their nucleus,
and the amount and size of the chromatin granules and nucleoli in their
nucleus. They generally possess, however, well defined cytoplasm and a
large, vesicular, often indented nucleus, in which are found fine chro-
matin granules and a few small nucleoli (see reticular cell and macro-
phagennPli 3, Fig mir

Fibroblasts of loose connective tissue have outstretched, ill-defined
cytoplasm and a large, regularly oval, vesicular nucleus containing dust-
like chromatin granules and small nucleoli. They can divide by mitosis,
are instrumental in repair and in walling off foreign material, but are
rarely phagocytic and do not generally develop into other cells (except
in bone and cartilage).

Endothelial cells line the larger blood vessels and capillaries. (The
term as herein used, does not include the littoral cells lining the sinuses
and sinusoids of the reticular tissues and elsewhere, which have wide
developmental potencies). The endothelial cells can divide by mitosis,
can form endothelium of new blood vessels, and can develop into fibro-
blasts, but are rarely phagocytic and do not generally develop into other
cells.

B. Free Blood and Connective Tissue Cells.—In accordance with
common usage, cells of the blood and lymph are classified according

IMMUNOLOGY 835

to whether they are of myeloid or lymphoid origin. The lymphoid
cells of the blood and the cells of the lymph consist of various-sized
lymphocytes, which together with monocytes are termed agranulocytes.
The myeloid cells of the blood are the various granulocytes (heterophils
or polymorphonuclears, eosinophils, and basophils), the erythrocytes,
and the platelets. Some authors classify monocytes as lymphoid and
others as myeloid cells, but they are classified in this chapter as both,
since we believe that they arise from lymphocytes of the lymphatic tissue
and from hemocytoblasts (equivalent to lymphocytes) of the bone
marrow.

The heterophils are functional in immunity by virtue of their obvious
phagocytic activities and probably because of their secretion of enzymes.
They are end cells, however, which do not reproduce or develop into
other cells. Lymphocytes and presumably monocytes, on the other hand,
can divide mitotically (Pl. 4, Fig. 3) and both lymphocytes and mono-
cytes can develop into macrophages, with all of their developmental
potencies. Lymphocytes possess basophil cytoplasm and a relatively large,
deeply staining, often indented nucleus, with large acidophil nucleoli
Pl. 3, Fig. 1. The monocytes may be the same size as, but in most cases
are larger than the medium lymphocytes, their cystoplasm is less basophil
and is increased in amount, and their nucleus is more vesicular, more
deeply indented with smaller chromatin granules and smaller and more
numerous nucleoli (cf. monocytoid lymphocyte in Pl. 3, Fig. 1). As the
lymphocytes and monocytes transform into macrophages, they show in-
creased amounts of cytoplasm, their nuclei gradually take on macrophage
characteristics, and they become phagocytic (polyblasts 1-5, in Pl. 3,
Fig. 2). These intermediate forms, together with lymphocytes, mono-
cytes, and macrophages, are grouped under the term lymphoid-macro-
phage system.

There is general agreement that free lymphoid cells, more or less
similar to lymphocytes, occur in varying numbers under various physio-
logical and pathological conditions in the reticular tissues and the loose
connective tissue, and that in such sites they act as “stem” cells of
lymphoid and myeloid cells. The nature, classification, and even exact
morphology of these different stem cells are subject to such controversy
that they are termed by various authors lymphocytes, hemocytoblasts,

836 IMMUNOLOGY

lymphoblasts, myeloblasts, monoblasts, and so forth, according to the
particular theory of blood formation held by the author (see Bloom,
1938). We have adopted essentially the unitarian viewpoint of Maxi-
mow and have called all free mesenchymal stern cells, with wide poten-
cies for development, hemocytoblasts in the bone marrow and lympho-
cytes in all other locations. Lymphocytes and hemocytoblasts are identical
morphologically and probably in their developmental potencies. Under
physiological conditions, lymphocytes in lymphatic tissue give rise only
to lymphocytes (Pl. 4), and hemocytoblasts in bone marrow give rise
only to myeloid cells (erythroblasts, myelocytes, and so forth), but un-
der abnormal stimuli they may exhibit their full potencies for develop-
ment. In general, these free stem cells are self-perpetuating, but they
may arise from the fixed mesenchymal cells of the preceding section.

C. So-called Systems of Cells —The foregoing classification of cells
should be brought into line with the so-called systems of cells frequently
used by various authors. Modern concepts of the cellular basis of im-
munity have been largely based on studies of inflammation. Credit should
be given to Metschnikoff (1892) for insisting upon the essential role of
the mesenchymal cells in inflammation and to Cohnheim, Ziegler, Mar-
chand (1924, review), and Maximow (1927a, 1927b, review), among
others, for studying the histogenesis of the local inflammatory reactions.
Metschnikoff (1892 and 1905, among other studies) laid the whole
foundation for the modern concept of the defense function of fixed
and mobile cells of the connective tissue by phagocytosis. His concept
was essentially physiological. He distinguished (1) microphages, herein
designated heterophils; and (2), macrophages, which are identical with
macrophages as herein defined, except that he included the phagocytic
microglial cells of the brain, which are possibly of mesenchymal origin.
The modern understanding of macrophages is based largely upon the
studies of vital staining and the storage of colloidal dyes, chiefly by
Renaut, Maximow, Goldman, Tschaschin, Kiyono, and Aschoff. The
Gefadsswandzellen of the Marchand-Herzog school (see Marchand,
1924) include pericytes and perivascular macrophages (adventitial cells)
which are supposed to arise from the endothelium of developing vessels.
Aschoff’s (1924, review) reticulo-endothelial system, broadly defined,
consists of the macrophages as we have outlined them.

It has unfortunately been assumed by most writers that the increase

IMMUNOLOGY 837

of macrophages associated with immunity, 1.e., “hyperplasia of the
reticulo-endothelial system,” is due to the proliferation of macrophages
or cells of the reticulo-endothelial system. This is an admitted source,
but detailed studies of a wide variety indicate that most of the new
macrophages arise from lymphocytes, with or without the intervention
of a monocyte stage (see Pl. 4). In order to include both macrophages
and all of their precursors under one term, which would indicate the
cytogenesis of macrophages from agranulocytes (lymphocytes and
monocytes) as well as from reticulo-endothelial cells, W. H. Taliaferro
and Mulligan (1937) proposed the term, lymphoid-macrophage system.
This term includes the mononuclear exudate cells, or Maximow’s poly-
blasts, which form the cellular exudate in inflammation.

ANTIBODIES AND ANTIGENS INVOLVED IN IMMUNITY

Infective organisms, derivatives of them, or other foreign, colloidal,
protein materials can generally act as antigens. When an antigen is intro-
duced parenterally into an animal, it calls forth a substance in the blood
of the animal, known as an antibody, which will react with the antigen
specifically 72 vivo and generally im vitro and is passively transferable.
Such an antibody is often termed an immune antibody, to differentiate it
from natural antibodies, which sometimes exist in blood without im-
munization. Serum from the blood of an animal containing an antibody
is known as antiserum. Some antibodies or antiserums, in addition to
reacting with their specific complete antigens, may also react with iso-
lated carbohydrate or lipoid parts of the antigen i” vitro. These sub-
stances have been differentiated from true antigens by the terms haptenes
or partial antigens, since they generally do not stimulate the production
of antibodies 72 vivo. Both complete antigens and haptenes have been
isolated in high states of purity. Antibodies result from antigenic stimu-
lation and are metabolic products of cells. Thus the amount of circulat-
ing antibody is often decreased by removing the spleen, which is rich
in cells of the lymphoid-macrophage system, or by filling the macro-
phages along the blood stream with colloidal or particulate matter.
Furthermore, antibodies undoubtedly represent definite substances which
are closely associated with the globulin fraction of the serum. It is, how-
ever, impossible to say at present whether they are actually globulins
and, if they are globulins, whether they are new globulins or the regular

838 IMMUNOLOGY

serum globulins slightly modified (see Wells, 1929; Marrack, 1938).
Antibodies are variously named according to the effect produced when
mixed with antigen. The antibody is a precipitin if it produces a pre-
cipitate on mixing with a soluble antigen (precipitinogen). It is an
agglutinin if it induces clumping or agglutination of cellular antigens
(agglutinogens), such as Bacteria, Protozoa or blood cells. It is an
opsonin if it sensitizes the antigen and makes it more readily ingested
by phagocytes. It is a lysin if it sensitizes cellular antigens so that, on the
addition of a thermolabile component of normal serum known as com-
plement or alexin, the cell undergoes death and lysis, during which many
of its internal substances diffuse through the cell membrane. In both of
the foregoing processes, the antigen is first sensitized by antibody. After
such a preparation, it is then lysed by intracellular enzymes (phagocyto-
sis) or by extracellular enzymes (lysis) (see Wells, 1929). The antibody
is known as an antitoxin if it neutralizes the biological action of a toxic
antigen (exotoxin). Definite antitoxins and exotoxins have not been
demonstrated in protozoan infections (see W. H. Taliaferro, 1929).
An increasing number of immunologists accept the unitarian view-
point that the introduction into the body of a single antigen results in
the formation of a single antibody, which is an agglutinin, precipitin, and
so forth, according to the nature of the antigen or the particular method
of testing. This does not mean that a complex cell will not contain many
different antigens. Furthermore, a given antibody in a specific infection
may act as one type of antibody, and not as another, because of the posi-
tion of various antigens on or within the cell (see Topley, 1935).
Finally, an antibody-like substance is known as ablastin if it inhibits
the reproduction of organisms when mixed én vivo. So far, it has been
demonstrated only for certain nonpathogenic trypanosomes. Like other
antibodies, it is associated with the globulin fraction of serum and is
passively transferable, but differs from them in that it has no im vitro
affinity for its specific antigen. In the latter respect, it appears to re-
semble certain nonabsorbable antibodies reported in bacterial, virus,
and worm infections.
The rdle of antibodies is studied by #7 vivo protective (passive trans-
fer) and curative tests and by /z vitro studies involving various serologi-

IMMUNOLOGY 839

cal tests. Protective and curative tests differ only as regards the time of
injecting the serum and organisms. In protective tests the serum is 1n-
jected at the same time (or not more than a day before or after) the
organisms are injected, whereas, in curative tests the serum is injected
some time after the organisms have been injected and generally when
they can be found in some particular part of the body. The effect of the
latter may obviously be more variable, since the organisms already have
a start in the body and may be more difficult to check.

THE CELLULAR AND HUMORAL ASPECTS OF IMMUNITY

The reader is referred to Maximow (1927b) for a general descrip-
tion of the histogenesis of the inflammatory and defense reactions, to
Aschoff (1924), Jungeblut (1930), Gay (1931), and Jaffé (1931, and
1938) for a general consideration of the function of cells and, in par-
ticular, of macrophages; and to Linton (1929), W. H. Taliaferro (1929
and 1934), and W. H. Taliaferro and Mulligan (1937) for a specific
consideration of the role of cells in protozoan immunity.

The way in which the cells of the connective tissue, in particular the
granulocytes and the cells of the lymphoid-macrophage system, are in-
volved in local defense can be seen during the inflammation which fol-
lows the introduction of foreign material into connective tissue of the
skin. The heterophils migrate early from the blood vessels. Their number
and activity depend upon the nature of the inflammatory stimulus and
whether it is sterile or septic. They are generally not numerous in proto-
zoan infections and soon disappear when the inflammatory material is
bacteriologically sterile. Under sepsis, however, they continue to migrate
from the blood vessels and to combat the invading organisms in many
visible ways—by active phagocytosis and digestion, by the secretion of
bactericidal and proteolytic ferments, and the like. They represent an
important first line of defense since they are the most easily mobilized
cells, but their functions are limited since they generally disintegrate
within a few days, ordinarily are recruited only from the blood stream,
i.e., do not multiply 7 sétv, and cannot develop into other cells of the
area.

The cells of the lymphoid-macrophage system are the most important

840 IMMUNOLOGY

cells in local defense. The lymphocytes and the monocytes migrate from
the blood vessels, as do the heterophils; but unlike the heterophils they
are long-lived, may multiply in the tissues, may develop into macro-
phages with phagocytic potencies, and from macrophages may progres-
sively develop into fibroblasts with reparative functions. As macro-
phages, they, together with the macrophages previously present in the
area, actively phagocytose and digest certain invading organisms, remove
cellular and other debris, and after the acquisition of immunity probably
elaborate antibodies which aid in phagocytosis. When large bodies are
present, the macrophages may fuse to form foreign body giant cells;
when microdrganisms are indigestible, they may form giant cells around
them, such as the epithelioid cells of the tubercle; or, when large areas
are necrotic, they may surround the area, become transformed into fibro-
blasts, and effectively wall it off. The fibroblasts, both those of the local
area and those arising from macrophages, react slowly and probably
play an active part only in the later stages of local inflammation during
regenerative and reparative processes, the formation of scar tissue, and
the walling off of foreign bodies.

Several other cells may come into play, generally during late stages
in the defense reaction. Of these, the eosinophils seem to play a part in
the detoxification of foreign proteins and their disintegration products
and are particularly prominent after the body has become sensitized to
the proteins. Like the heterophils, they do not multiply and cannot de-
velop into other cells of the area. Some investigators believe that the
plasma cells are also associated with the detoxification of foreign ma-
terials. They are not phagocytic, do not seem to have any developmental
potencies, and proliferate rarely, if at all. The exact function of the
basophils is unknown.

Ordinarily, when the stimulant is distributed over a large part of the
body, the reaction is designated as a general defense reaction, in contra-
distinction to the local defense reaction just described, but as a matter
of fact such distribution usually signifies that the stimulant is in the
blood stream and is combated by macrophages of organs most closely
associated with the blood, such as the spleen, liver, and bone marrow.
In some cases, as in malaria, these general reactions can actually be con-
sidered local ones in strategically placed organs (see W. H. Taliaferro,

IMMUNOLOGY 841

1934). The same types of cells are involved, and the extent to which
they are involved depends, as in other sites, upon the nature of the
foreign material or infectious agent. The heterophils are often mobilized
first, and the lymphoid-macrophage series shows the most pronounced
histological changes, with the macrophages seeming to bear the brunt of
the activity. Fibroblasts rarely come into contact with foreign material
in the blood and are rarely active. Endothelial cells, although they come
into contact with hematogenous material, show extremely little histologi-
cal change or phagocytic activity.

The foregoing account of defense reactions, involving the disposal of
foreign material and tissue debris with eventual repair, is characteristic
when either antigenic or nonantigenic materials are introduced into a
normal animal, but certain quantitative differences are noted when anti-
genic materials, including parasites, are introduced into an immune
animal. These differences are chiefly due to antibodies and are specific.
When antigens are introduced into the immune body, they are generally
localized by agglutination if they are cells, or by precipitation if they are
in solution, and are made more readily phagocytable by opsonification.
Such localization and opsonification are particularly well seen in local
reactions in the loose connective tissue. They are often limited to organs
such as the spleen, liver, and bone marrow in general reactions, as is
well-illustrated in malaria. In trypanosomiasis, these phases have not
been completely studied, but ablastin (the reproduction-inhibiting anti-
body) at least does not involve either localization or phagocytosis. When
antigen and antibody meet in the tissues of an immune animal, not only
do localization and opsonification of the antigen occur, but there is
generally a much heightened inflammatory reaction (hypersensitivity,
sometimes evidenced by a local reaction—the so-called skin test—when
suitable amounts of antigen are injected intradermally). Provided this
heightened inflammation is not so intense as to overwhelm the body,
it represents a speeding up of the whole cellular response in the immune
animal. In addition to the specific action of antibodies after the acquisi-
tion of immunity, a residual increase in the number of cells of the
lymphoid-macrophage system sometimes is seen at strategic sites, which
results in a much more rapid mobilization of macrophages during im-
munity. This is well illustrated in the spleen in malaria.

842 IMMUNOLOGY

ROLE OF IMMUNE PROCESSES IN THE DEVELOPMENT
OF PROTOZOAN INFECTIONS

GENERAL METHODS

The detailed considerations in the succeeding sections deal largely
with the rdle of antibodies and cells in modifying the course of infection,
together with such allied subjects as recovery, relapse, and immunity to
super- and reinfection. The Protozoa offer the advantage of being large
enough so that one can ascertain the effect of these processes in a way
that is impossible with the smaller bacterial and virus invaders. This
analysis has been further facilitated by selecting certain plasmodia and
trypanosome infections in which practically all stages in the life cycle
of the parasite are accessible for study (1.e., are more or less evenly
distributed in the peripheral blood and are not localized in the deeper
tissues ) .

The course of these infections can be roughly indicated by changes
in the number of organisms. Since, however, the number curve is the
resultant of the number of parasites produced by reproduction and the
number of organisms which die or are actually destroyed, the only
deductions that can be drawn from it are that reproduction is going on
if the numbers increase, although the rate may be actually decreasing;
and that the rate of reproduction is being inhibited, or that the parasites
are being killed, or that both activities may be operating, if the numbers
remain constant or decrease. The rate of reproduction, if ascertained,
however, in conjunction with number counts throughout an infection,
will adequately indicate whether the host acquires a defensive mecha-
nism, and, if so, whether it is directed toward inhibiting reproduction of
the parasites, or destroying the parasites after they are formed, or both.

All valid measures of the rate of reproduction so far devised, which
are independent of the number of organisms killed, depend upon some
measurement of size (since organisms usually grow before they repro-
duce) or upon some determination of division forms. A direct measure
can be devised for the plasmodia, which divide more or less synchro-
nously; but an indirect measure has to be resorted to for the trypanosomes,
since they neither divide synchronously nor can their fission rate be
ascertained directly as can be done among the free-living forms.
The particular criteria used are of course more or less arbitrarily selected

IMMUNOLOGY 843

and, to be satisfactory, necessitate a nice adjustment between the validity
of the criteria selected and the time required to make measurements.

The most convenient measure of the rate of reproduction of the
plasmodia so far devised consists in ascertaining the length of the asexual
cycle directly (i.e., the time it takes for a young merozoite to become a
mature schizont and divide into the next generation of young mero-
zoites), in conjunction with the number of merozoites produced. Thus
the percentage of segmenters are computed in samples of 50 to 100
parasites from stained blood smears made every 4 to 12 hours whenever
parasites can be found. A regularly recurring percentage of segmenters,
considered arbitrarily, for example in P. brasilianum, to have 5 or more
nuclei by W. H. Taliaferro and L. G. Taliaferro (1934a), indicates a
constant rate of reproduction, provided the number of merozoites pro-
duced by each segmenter remains approximately constant. The most
satisfactory measure of the rate of reproduction among the trypanosomes
consists in comparing the percentage of division forms in samples of
50 to 100 forms from stained blood smears made every 6 to 24 hours
throughout an infection. Among the pathogenic trypanosomes, in which
dividing forms are numerous, division forms may simply be considered
as those with some duplication of parts (see Krijgsman, 1933), but
among the nonpathogenic Trypanosoma lewisi and T. duttoni, in which
actual dividing forms are rare, division forms are considered to be
dividing forms plus short young forms 25 1 or less in length (see W. H.
Taliaferro and Pavlinova, 1936). The higher the percentage of division
forms among the trypanosomes is, the higher the rate of reproduction.
Valid measures have also been devised for malaria by L. G. Taliaferro
(1925), G. H. Boyd (1929a), Lourie (1934), and Mulligan (1935);
and for trypanosomes by Robertson (1912), Krijgsman (1933), and
W. H. Taliaferro and L. G. Taliaferro (1922). The last authors’ coefh-
cient of variation method depends upon the fact that, within certain
limits, the variability in total length increases proportionately as the
young and growing forms resulting from reproduction increase.

MALARIA

This analysis applies only to those malarial species which parasitize
erythrocytes and not to such species as Plasmodium elongatum (Huff and
Bloom, 1935), which undoubtedly infect other blood and connective

844 IMMUNOLOGY

tissue cells. We have omitted from discussion the so-called exo-erythro-
cytic and fixed tissue cell stages described in the life cycles of some
plasmodia because of the lack of agreement which now exists among
malariologists as to their nature (Boyd and Coggeshall, 1938, review).

The course of untreated malarial infections has been most thoroughly
studied in avian malaria. This work commenced with the careful statis-
tical studies of the Sergents (1918) and was extended by Ben Harel
1923), L. G. Taliaferro (1925), G. H. Boyd (1929a), Hartman
(1927), Gingrich (1932), Lourie (1934), and others. Treated cases
will not be considered in this analysis because treatment itself has been
shown greatly to affect the length of the asexual cycle and the number
of merozoites produced (G. H. Boyd, 1933; G. H. Boyd and Allen,
1934; Lourie, 1934; and Boyd and Dunn, 1939).

Infections with P. cathemerium in canaries are extremely stereotyped
and therefore afford an excellent base line for considering the so-called
benign infections, which tend to recover and which constitute the ma-
jority of malarial infections. When a few parasites are injected into a
bird, an incubation period follows during which no parasites can be
detected in the peripheral blood. As soon as parasites appear, they
increase from day to day at a constant rate (the intersporulation death
of parasites will be taken up later), according to a geometrical progres-
sion, until sometimes as many as half of the red blood cells are infected
(acute rise of infection). At this point, if the bird does not succumb to
the infection, recovery is initiated and is manifested by the rapid disap-
pearance of many of the parasites from the peripheral blood (crisis).
Following the crisis, parasites remain few in number, but may fluctuate
to some extent (developed infection). Sooner or later, the number of
parasites is reduced to a level at which they can no longer be detected
in peripheral blood films; but a few persist, since transfers of large
amounts of blood will infect other birds. This latent period may last
for several years, but it may be interrupted periodically by spontaneous
or induced relapses (much rarer in P. cathemerium than in many other
species), which are similar to, though generally quantitatively less than
the acute rise of the initial infection, and which are terminated by a
crisis. Occasionally, such a relapse may be fatal. By Hegner’s (1926)
terminology such an infection is divided into prepatent (incubation),
patent (acute rise, crisis, and developed infection), and subpatent

IMMUNOLOGY 845

(latent) periods, with second and third patent periods representing first
and second relapses and crises.

When this infection was analyzed, it was found that the basic rate
of reproduction remains comparatively constant, whenever parasites are

WINMonkey 119

——O— No of Parasites

= —e— % of Segmeriors
C7 ~-*-— Temperature
a)
fad
a>
ay S
z |=
orgs lace
0s 50 a:
54%
ve) = be
1034 -<604 2 | =
Se E
iS 8 ~ = E
= = @ — a
P10-} 407 -2 80
&. 5 = [: |
= oe
Ba
@ 21 4
@
=
= |
QT 0- 0
Aa ie . a 4 %
ate

FIGURE 188. The changes in number of Plasmodium brasilianum and the percentage
of segmenters during the acute rise and crisis of the infection in Central American monkey
119. A natural parasiticidal immunity is operative, as evidenced by the inter- and
intrasporulation death of parasites; and as acquired immunity is developed at the crisis,
further parasiticidal effects are operative, as evidenced by the tremendous death of
parasites. The rate of reproduction is temporarily affected, as evidenced by the irregular
percentage of segmenters. Had the animal lived, reproduction would have resumed its
normal rate, as ascertained from the study of other monkeys similarly infected. (From
W. H. Taliaferro, 1932.)

demonstrable in the blood, since the schizonts produce between ten and
fifteen and a half progeny (merozoites) continuously and produce them
every twenty-four hours. (This statement is relatively true, since there
is no prolonged inhibition of reproduction. Temporary deviations and
fluctuations do occur, however. Thus, Boyd and Allen [1934] and Boyd
[1939] found that the number of merozoites produced decreases as the

846 IMMUNOLOGY

crisis approaches and then rises thereafter.) Furthermore, since parasites
reproduce at a high rate whenever they are found, it seems reasonable
to assume that they reproduce at a high rate during latency, when they
cannot be found in sufficient numbers to study. This assumption is in
accord with the view held by Ross (1910, review), Bignami (1910),
James (1913), and others.

Essentially the same results were obtained from the studies of P.
brasilianum in Panamanian monkeys, except that during the crisis of
the initial infection (W. H. Taliaferro and L. G. Taliaferro, 1934a)
one asexual cycle may deviate or be retarded for a day or two. This
parasite shows a quartan periodicity. Thus in Figure 188 the percentage
of segmenters sharply increases and decreases every third day (4/28,
5/1, 5/4) until at the time the number crises is reached (5/7) the
percentage of segmenters does not rise as high as before and does not
decrease as precipitously. The other so-called benign malarias seem to be
similar, as far as the data on the following species go: P. cynomolgi,
in both Szlenus rhesus and S. irus; P. knowlesz, in S. irus (Sinton and
Mulligan, 1933a, 1933b; Mulligan, 1935); and P. vivax and P. malariae
in man. The rapidly fatal infection of P. knowles in S. rhesus is similar
to the acute rise of benign infections without a crisis. These statements
do not mean that temporary derangements of the cycle may not occur
during the crisis, as in P. brasilianum,; or after treatment with quinine,
as in P. cathemerium (previously cited); or after changes in host habits,
as has been shown to occur in both the latter species (L. G. Taliaferro,
1928; G. H. Boyd, 1929b; W. H. Taliaferro and L. G. Taliaferro,
1934b; and Stauber, 1939).

Since there is no prolonged inhibition of reproduction, the number
curve can be interpreted chiefly in terms of parasiticidal effects. In other
words, the number of parasites after each asexual cycle should increase
by the number of progeny in each mature schizont (minus the number
of merozoites which develop into sexual forms), provided no death of
parasites occurs. The geometrical rate at which P. cathemerium increases
does not, however, account for all the progeny produced. They evidently
die at all stages of growth and segmentation (L. G. Taliaferro, 1925;
Hartman, 1927; Hegner and Eskridge, 1938) and die at a greater rate
during the latter part of the acute infection than during the early part
(Boyd, 1939). Hegner and Hewitt (1938) and Hewitt (1938) sug-

IMMUNOLOGY 847

gested that their death is due to the destruction of multiple infected
red cells.

It is interesting that in 1888 Golgi noted that malaria would always
be progressive until pernicious symptoms were evident, if the parasites
arriving at maturity every two days in tertian or every three days in
quartan malaria should complete their life cycle. W. H. Taliaferro and
L. G. Taliaferro (1934a) found that out of an average of 9 progeny
produced by P. brasilianum, never more than 1.5 complete their develop-
ment and of the 7.5 which fail to complete their development, about 6
fail to get into new cells and 1.5 die or are killed during intracorpuscular
growth. Thus, in Figure 188 on the morning of May 1 there were 60
parasites per 10,000 red blood cells. That evening after sporulation,
there were 221, an increase of 3.66 times instead of 9 times. During
the subsequent 2.5-day period of growth and division, about two-thirds
of these were destroyed, so that on the morning of May 4 there were
only 83 per 10,000 red cells. Similar data are furnished by Brug (1934)
and W. H. Taliaferro and Mulligan (1937), who worked on infec-
tions with P. knowlesi and P. cynomolgi; and by Pijper and Russell
(1925, quoted by Sinton ef a/., 1931), by Rudolf and Ramsay (1927),
by Sinton et al. (1931), and by Lowe (1934), all of whom worked on
one or both of the tertian and quartan malarias of man. Knowles and
Das Gupta (1930) believed that the destruction of parasites takes place
only during the free merozoite stage. Examination of their tables, how-
ever, shows that the infection they studied was made up of several
broods of parasites, the sporulation of one of which would obscure a
decrease of parasites during the intrasporulation period of another brood.

The constant rate of death of large numbers of parasites during the
initial part of the infection is a manifestation of natural immunity and
represents the suitability of the hosts’ blood to the malarial organism.
The crisis, on the other hand, represents the beginning of the immune
reaction, when more progeny die than survive and the infection therefore
declines. Thus the crisis in Figure 188 takes place after the sporulation
of May 4 and between May 5 and May 7, as shown not only by the
conspicuous death of parasites during the intersporulation period of
May 5 and May 6, but by the relatively slight increase of parasites during
the sporulation on May 7. This infection, which was the most acute
encountered, caused the death of the monkey on May 9. The temperature

848 IMMUNOLOGY

curve is extraneous to the present discussion, but is interesting because
of the stiletto-like peaks it shows at each sporulation. Some work on the
infection after the crisis has been done on human malaria by Bohm
(1918), Knowles and Das Gupta (1930), and Sinton ef al. (1931).

Throughout the developed infection, an equilibrium is established
between the number of parasites killed and the number produced. Later,
the defensive factors are usually successful in suppressing the parasites
arising by reproduction, so that latency ensues. Latency may last for
years, but may be interrupted by relapses. Although there is no unanimity
of opinion on the mechanism of relapse, the best evidence indicates that
it represents simply the removal of the defensive factors, so that the
parasites, which are continuously reproducing at a constant rate, reaccu-
mulate in the blood. The relapse may be fatal, or the defensive factors
may again materialize and successfully suppress it. Accordingly, the
severity of the relapse depends upon its extent and upon the length of
time the defensive factors are removed. These statements are substanti-
ated by work on P. cathemerium and P. brasilianum, references for which
have already been given.

Koch (1899) believed that immunity persisted after complete re-
covery, but Wasielewski (1901) suggested, and subsequent workers
have supported the conclusion, that parasites remain in small numbers
for longer periods than was at first supposed and that this latent infection
accounts for the long continued immunity (see Thomson, 1933, review;
Chopra and Mukherjee, 1936; Sergent, 1936). More recently, Nauck
and Malamos (1935) and Coggeshall (1938) have shown that a certain
amount of immunity is retained after complete cure of P. knowlesi.

Acquired immunity to malaria can be demonstrated not only by the
crisis, recovery from initial infection, and recovery from relapse, but by
superinfection. Such immunity is usually species-specific or even strain-
specific in human, simian, and avian infections. A series of investigators,
beginning with Koch (1899), have worked on this aspect of the subject
(for literature and for especial work, see W. H. Taliaferro and L. G.
Taliaferro, 1929a, 1934c; Gingrich, 1932; Mulligan and Sinton, 1935;
M. F. Boyd ef al., 1936; Manwell, 1938; Redmond, 1939; and Manwell
and Goldstein, 1939). The work on human malaria has been recorded
chiefly as a result of the use of therapeutic infections in the treatment
of paresis (see Mulligan and Sinton, 19332). W. H. Taliaferro and

IMMUNOLOGY 849

L. G. Taliaferro (1929a, 1934c) intravenously superinfected birds and
monkeys with such large numbers of parasites that they could study the
superinfection quantitatively. They found that in immune animals the
parasites, after reinfection, begin to be removed at once, with the same
effectiveness that they are removed at the time of the crisis in initially
infected animals, and that in monkeys the asexual cycle occasionally ex-
hibits a transitory delay, accompanied by the production of fewer mero-
zoites. In other words, superinfection, since it occurs in an effectively
immune animal instead of in an uninfected animal, has no incubation
period or acute rise, but begins at once with a crisis and proceeds imme-
diately to latency.

The problem now arises: what is the mechanism whereby the plas-
modia are removed throughout the course of the infection? The death
of all of the parasites, whether it takes place before, during, or after the
crisis (i.e., during natural or acquired immunity), is associated with
phagocytosis by macrophages, chiefly of the spleen, liver, and bone
marrow. Other cells play insignificant rdles. Furthermore, the macro-
phages phagocytose free parasites, parasitized red blood cells, and resi-
dues of parasites such as malarial pigment and uninfected red cells,
which are probably injured by the infection. Malarial pigment is the most
indigestible part of the parasite red-cell complex, since it is often found
in macrophages months after all other vestiges of the parasites have
disappeared. Large monocytes containing malarial pigment may be found
in the peripheral blood, especially during the crisis and thereafter. A
review of the literature covering the considerable amount of work done
on this aspect of the subject will be found in W. H. Taliaferro and
Mulligan (1937) and involves direct evidence from necropsy findings
and indirect evidence from splenomegaly (see Stratman-Thomas, 1935;
Coggeshall, 1937; Afridi, 1938) and splenectomy and blockading
procedures.

Although phagocytosis has been known for years to occur in malarial
infections, Cannon and W. H. Taliaferro (1931) and W. H. Taliaferro
and Cannon (1936) were the first to study infections and superinfec-
tions at closely spaced intervals and to correlate phagocytosis in detail
with the course of the infection and with immunity. The rate of phago-
cytosis during natural immunity, i.e., before the crisis, increases as the
number of parasites increases, but is always comparatively sluggish (PI.

850 IMMUNOLOGY

1, Figs. 1 and 2). When the parasites disappear from the peripheral
blood at the time of the crisis, they are not at first phagocytosed, but are
filtered out and concentrated in the Billroth cords of the spleen (Pl.#1,
Fig. 3). This initial manifestation of acquired immunity probably repre-
sents a localization of the parasites due to an antibody, which may be an
agglutination, as demonstrated 77 vitro in P. knowlesi by Eaton (1938),
or an attachment of parasites to the macrophages, as found in cultures
of P. falciparum by McLay (1922). In a few hours or days the con-
centrated parasites are ingested in great numbers by the macrophages
(Pl. 2, Figs. 1 and 2), owing undoubtedly to an opsonification of the
parasites and parasitized red cells by antibody. This antibody probably
accounts for the protective property of serum taken from latent infections
in avian malaria (Findlay and Brown, 1934), in human malaria (Soti-
riadés, 1917; Kauders, 1927; Neumann, 1933; and Lorando and Soti-
riadés, 1937), and in simian malaria (Coggeshall and Kumm, 1938;
Coggeshall and Eaton, 1938). The fact that protective antibodies have
not always been found (W. H. Taliaferro and L. G. Taliaferro, 1929b
and 1934b) led W. H. Taliaferro and Cannon (1936) to suggest that
antibodies are produced locally in sufficient quantities to be operative in
the specific organs 7m stu, but not in sufficient quantities to be easily
demonstrated in the peripheral blood. A day or two after concentration in
the Billroth cords, the parasites are quickly digested except for the ma-

CAPTION FOR PLATE ON FACING PAGE

Plate 1. Sluggish phagocytosis of Plasmodium brasilianum by macrophages in control
of the American monkeys during the acute rise of the malarial infection (Figs. 1 and 2)
and the concentration of P. brasilianum in Billroth cords of the spleen at the initiation
of the crisis (Fig. 3). & 1450. (From W. H. Taliaferro and P. R. Cannon, “Cellular
Reactions during Primary Infections and Superinfections of Plasmodium Brasilianum and
Panamanian Monkeys,” Journal of Infectious Diseases, LIX [ July-August, 1936], 72-
125.)

Figure 1. A Kupffer cell in the liver containing a parasite, an unparasitized erythrocyte,
and four small masses of malarial pigment. Acute rise of infection.

Figure 2. Two macrophages in a Billroth cord in the red pulp of the spleen, containing
a small number of residues of parasites, red cells, and malarial pigment. Acute rise of
infection.

Figure 3. Large numbers of parasitized erythrocytes concentrated in Billroth cords of
the spleen. Note that parasites are absent from the venous sinus. Also note that the
macrophages in the Billroth cords show the sluggish phagocytosis characteristic of the
acute rise and that the littoral cells lining the sinus are not phagocytic. Initiation of
crisis of infection.

ue oS @

Billroth cord Venous Sinus Billroth cord

PLATE |

Par Mac M Lom

5 tym Mae Pig

PLATE il

IMMUNOLOGY 851

larial pigment (PI. 2, Figs. 3-5, which are from monkey 119; see also
Fig. 188). The pigment is digested within a few months, as may be seen
by a study of animals in which relapses or superinfections do not inter-
vene. The various tissues gradually return to their normal histological
appearance, but as long as immunity lasts they retain their ability to
react more quickly than the tissues of nonimmune animals.

These data raise the interesting immunological question as to whether
the death of the parasites before the crisis and that during or after the
crisis are due to the same factors. In other words, is acquired immunity
simply an enhancement of the high-grade natural immunity present from
the beginning, or is it due to an entirely new set of factors superimposed
on the natural condition? Certain facts tend to indicate that the two
mechanisms are essentially different. In the first place, during natural
immunity, all evidence indicates that phagocytosis is nonspecific, and
there is some evidence that only those parasites are phagocytosed which
are moribund or otherwise abnormal (Hartman, 1927; Gingrich, 1934;
Hegner and Hewitt, 1938; Hewitt, 1938). In the second place, Gingrich
found that the injection of large numbers of red cells breaks down

CAPTION FOR PLATE ON FACING PAGE

Plate 2. Intense phagocytosis of Plasmodium brasilianum by macrophages in Central
American monkeys at the height of the crisis of the malarial infection (Figs. 1 and 2)
and malarial pigment, the residue of parasites after the intense phagocytosis, in the
macrophages shortly thereafter (Figs. 3-5). X 1450. (From W. H. Taliaferro and P. R.
Cannon, “Cellular Reactions during Primary Infections and Superinfections of Plasmodium
Brasilianum and Panamanian Monkeys,” Journal of Infectious Diseases, LIX [July-
August, 1936], 72-125.)

Figure 1. A Kupffer cell in the liver containing many recognizable parasitized and
unparasitized red cells, and a monocyte (Mon.) containing malarial pigment. Crisis of
infection.

Figure 2. Two macrophages in the Billroth cord of the spleen containing many recog-
nizable parasitized and unparasitized red cells. Crisis of infection.

Figure 3. A Kupffer cell in the liver containing malarial pigment. Two days after the
crisis.

Figure 4. Two macrophages in the bone marrow containing malarial pigment. Two
days after the crisis.

Figure 5. Four macrophages in the Billroth cord of the spleen containing malarial
pigment. Two days after the crisis. Figures 3, 4 and 5 were all taken from monkey 119
(see text Figure 188). Note that the Kupffer cell of the liver contains intermediate
amounts of malarial pigment between that found in the macrophages of the spleen and
of the bone marrow.

852 IMMUNOLOGY

acquired resistance to P. cathemerium, but, even when pushed to the
maximum, does not affect natural resistance. This work, however, is open
to the possible criticism that the large amounts of pigment in the macro-
phages after the crisis, which are not there before the development of
immunity, may simply augment the blockading doses to produce the
observed effect. In the third place, as pointed out previously, accumulat-
ing evidence indicates that the greatly superior mechanism for disposing
of parasites associated with acquired immunity is highly specific and is
probably associated with an antibody.

The limitation of phagocytosis to the macrophages of the spleen, liver,
and bone marrow is probably a question of opportunity, as such macro-
phages are advantageously placed where they can remove material from
the blood stream. The so-called general immunity in malaria, therefore,
is actually a local reaction in strategically placed organs (W. H. Talia-
ferro, 1934). It should be noted, however, that in overwhelming infec-
tions the parasites may be so numerous that secondary complications, such
as stasis of the blood, clogging of the capillaries, and hemorrhage may
occur in various organs. In such an event the macrophages of the brain,
lungs, suprarenals, and kidneys, as weil as those of the bone marrow,
liver, and spleen, may actively phagocytose malarial material.

CAPTION FOR PLATE ON FACING PAGE

Plate 3. Portions of a venous sinus and Billroth cords in the spleen of an uninfected
rhesus monkey (Fig. 1) and comparable portions from a rhesus monkey during the late
acute rise of an infection with Plasmodium cynomolgi (Fig. 2). X 1400. (From W. H.
Taliaferro and H. W. Mulligan, “Histopathology of Malaria with Special Reference to
the Function and Origin of the Macrophages in Defence,” Indian Medical Research
Memoirs, No. 29 [May, 1937}, pp. 1-138.)

Figure 1. The normal constituents of the venous sinus are chiefly the cells of the
circulating blood (lymphocytes, granulocytes, and red cells), and those of the Billroth
cords are also cells of the circulating blood with a greater proportion of large lymphocytes
and, in addition, reticular cells with indeterminate cytoplasm and slightly phagocytic
macrophages.

Figure 2. The additional constituents of the venous sinus and of the Billroth cords in
the spleen of a monkey during the late acute rise of an infection of P. cynomolgi are
parasitized red cells and many transitional cells (polyblasts) between lymphocytes and
macrophages, containing malarial pigment. The progressive hypertrophy of lymphocytes
into macrophages is shown by nuclear changes and by increased amounts of cytoplasm in
the lymphocytes (see especially Med Lym 1, which also contains two small granules of
malarial pigment) and by further nuclear changes, increased size, and increased phagocytic
activity in the polyblasts (see especially polyblasts 1 through 5). Throughout this tran-
sitional series, phagocytosis increases approximately with the size of the cell.

Monocytoid lymphocyte Sinus Plasma cell Large lymphocyte Sinall lymphocyte

Heterophil Macro ye
Dew put de

7 BEY oS ®) 5, 24
: Sar Vy
So a OA
fy 7. 22,
, re
; OT hex has
~~ ri Og SP
y a ~ @ f oe
z has « Sg a
I eminent tie Ee -
ss

Large lymphocyte G Polybl.S Parasite Polybl.L Polybl.2 Dotybl.5 5 Doty. 4
PLATE III

Transtttonal
Zone

Marginal
one of
Small
lymphocytes

Secondary
nodule

Large
lymphocyte

~ = £ |
Rettcular cell Reticular Mitoses in Macrophage
cell A Lymphocytes with tnclusions

PLATE IV

IMMUNOLOGY 853

The fact has been stressed that the greatly enhanced phagocytic activity
of individual macrophages during acquired immunity is specific and is
probably related to antibodies. In addition, there is a nonspecific increase
of macrophages at strategic points, particularly in the spleen and the
bone marrow, due to their cytogenesis from lymphocytes through poly-
blast stages and from histogenous macrophages. The latter source is
acknowledged by Bruetsch (1927, 1932a, 1932b) and others, and both
sources have been demonstrated by W. H. Taliaferro and Cannon
(1936) and W. H. Taliaferro and Mulligan (1937). In fact, the chief
source of new macrophages is from lymphocytes (Pl. 3). The lympho-
cytes themselves arise by mitotic proliferation (Pl. 4). This is a promi-
nent part of the so-called lymphoid hyperplasia of the spleen and occa-
sionally of other organs, if the malarial infection is long drawn out.

In passing it may be mentioned briefly that the parasites destroy red
blood cells in large quantities, flood the blood plasma with foreign mat-
ter such as corpuscular debris, free malarial parasites and malarial pig-
ment, sometimes block the capillaries and damage various tissues, espe-
cially the spleen and liver. All these losses and destructions are made
good by various nonspecific hyperplastic and reparative activities of the
host. The pathological and regenerative changes have been extensively
studied, especially in human malaria (see W. H. Taliaferro and Mulli-
gan, 1937, for a review of the literature).

The foregoing results indicate that acquired immunity against malaria
largely involves parasiticidal effects, with no pronounced inhibition of
the rate of reproduction for extended lengths of time. The parasiticidal
effects can be correlated with phagocytosis. Phagocytosis is sluggish dur-

CAPTION FOR PLATE ON FACING PAGE

Plate 4. A nodule in the white pulp of the spleen during lymphoid hyperplasia as-
sociated with the late acute rise of Plasmodium cynomolgi in a rhesus monkey. (From
W. H. Taliaferro and H. W. Mulligan, “Histopathology of Malaria with Special
Reference to the Function and Origin of the Macrophages in Defence,” Indian Medical
Research Memoirs, No. 29 [May, 1937}, pp. 1-138.)

Figure 1. This activated splenic nodule due to malaria is slightly enlarged, shows a
pronounced transitional zone and a markedly active secondary nodule, in which occur
swollen phagocytic reticular cells and mitoses, among many lymphocytes and a few reticu-
lar cells. & 180.

Figure 2. A detail of the upper portion of the secondary nodule shown in Figure 1
which consists mainly of medium lymphocytes and swollen, slightly phagocytic reticular
cells. Reticular cell A is beginning to mobilize. X 1015.

Figure 3. A detail of the lower portion of the secondary nodule shown in Figure 1,
which consists mainly of lymphocytes many of which are dividing. x 655.

854 IMMUNOLOGY

ing the acute rise of the infection and is greatly enhanced at the time of
the crisis, when immunity, which is probably associated with the elabora-
tion of antibodies, is developed. Thereafter the infection progressively
subsides, unless the immunity is lowered. If the immunity is lowered,
the parasites, because their reproduction has not been inhibited, reac-
cumulate in the blood until immunity again develops and becomes
operative.

LEISHMANIASIS

The course of kala azar cannot be studied and analyzed as was done
in the case of malaria because its causative agent, Lezshmania donovani,
is not accessible for study; but it is of interest here because L. donovani
lives in the macrophages themselves, as has been shown by Christophers
(1904), Meleney (1925), Hu and Cash (1927), and others. It not
only lives in the macrophages of the spleen, liver, bone marrow, and
intestinal wall, and, in extreme cases, the macrophages of almost all
organs and tissues, but proliferation of the macrophages constitutes the
chief characteristic of the disease. For a review of the literature, see C. J.
Watson, 1928; Linton, 1929. See the former reference also for a seem-
ingly similar condition in the little-known histoplasmosis. The parasites,
therefore, instead of being digested, find the cytoplasm of the phagocytes
a suitable medium in which to grow and multiply. Splenectomy should
be particularly illuminating in trying to decide whether the macrophage
system is valuable, imperfect as it is, as the only defense the body has;
or is deleterious, as being the most suitable location for the parasites.
Some work on kala azar has been done (see Laveran, 1917), but further
systematic experimental work on animals should prove valuable. The
fact that the disease is so often fatal indicates that reproduction of the
parasites is continuous, as in malaria. Immunity, nevertheless, is devel-
oped in approximately 10 percent of the infections, but it is not
apparent whether the suppression of the infection is predominantly due
to an increase in the ability of the macrophages to digest the parasites or
to an inhibition of reproduction of the parasites.

Oriental sore, a cutaneous leishmaniasis caused by L. tropica, on the
other hand, usually spontaneously heals and confers an immunity to
reinfection. Sections of the skin at the site of the sores often show pro-
nounced local accumulations of macrophages. As in the case of the small

IMMUNOLOGY 855

percentage of human beings recovering from kala azar, it 1s impossible
to decide from the available data whether suppression of the infection
is predominantly due to a destruction of the parasites or to an inhibition
of their reproduction. The fact that immunity is more or less generalized
indicates, however, that some humoral principle is involved.

NONLETHAL INFECTION WITH THE Trypanosoma lewist GROUP OF
TRYPANOSOMES

The trypanosomes differ from the plasmodia in that they live in the
blood stream and do not infect the red blood cells. Some are pathogenic.
Others are nonpathogenic. Among the latter is a large group of trypano-
somes which produce nonlethal infections in rodents, are morphologi-
cally identical or similar to T. /ew7si of the rat, and are differentiated
almost entirely by their specificity for their rodent hosts. Of these, T.
lewisi of the rat and T. duttoni of the mouse have been extensively
studied and furnish the basis for the conclusions in the following discus-
sion.

The number curve of T. /ew7si in the rat when a few parasites are
injected, as shown by Steffan (1921), W. H. Taliaferro and L. G.
Taliaferro (1922), W. H. Taliaferro (1924), and Coventry (1925),
starts, as does a malarial infection, with an incubation period and an
acute rise, until the trypanosomes may reach 300,000 or more per cu.
mm. Then there is a crisis between the eighth and the fourteenth days,
during which most of the parasites are destroyed. Those that remain
continue to live in the blood for some time (varying from several weeks
to several months), until they are removed either gradually or suddenly.
Thereafter they are not found in the blood, and relapses seldom occur,
but a few may persist, as ascertained by relapses which sometimes ensue
after splenectomy and blockade with India ink, or after other conditions
which lower the immunity of the host. Whether a few always persist
cannot be determined from the available data. The rat, however, 1s
immune to reinfection for long periods, as was first shown by Kanthack
et al. (1898). Fatal infections of T. /ewzsi in young rats were first
reported by Jiirgens in 1902 (see also W. H. Brown, 1914; Herrick and
Cross, 1936; Duca, 1939; Culbertson and Wotton, 1939). They are
often complicated by a concomitant occurrence of either or both of the

856 IMMUNOLOGY

following: a subnormal condition of the rats or infections, such as
Bartonella or paratyphoid. In any case Culbertson and Wotton (1939)
have found that fatal infections develop in rats in which the content of
ablastin is low.

Early investigators (Rabinowitsch and Kempner, 1899; von Wasie-
lewski and Senn, 1900; especially Laveran and Mesnil, 1901; MacNeal,

50:

Rat 105

S a
Coefficient of variation. % of Division Forms

Incubation period

_-
_
_-=-—@=— — —

Thousands of trypanosomes per cmm. of blood

Day of Infection

FIGURE 189. The changes in number of Trypanosoma lewisi and the coefficient of
variation and percentage of division forms during the course of infection in rat 105.
As acquired immunity develops, the rate of reproduction is inhibited by ablastin, as
evidenced by the low coefficient of variation and low percentage of division forms be-
ginning at location 1, and the parasites are killed by trypanolysins operative at locations
2 and 3. Whether, in addition, natural immunity operates has not been ascertained.
(From W. H. Taliaferro, 1924; division forms added.)

1904; W. H. Brown, 1915) were convinced by their microscopical
studies that T. Jewis7 reproduces only during the first few days in the rat,
after which the trypanosomes live in the blood as nonreproducing adults.
This conclusion has been substantiated by W. H. Taliaferro (1924),
Coventry (1925), and Regendanz and Kikuth (1927). Thus in Figure
189 the coefficient of variation of the total length of the trypanosomes
and the percentage of division forms, each of which, as will be recalled,
measure the rate of reproduction, are high when trypanosomes appear

IMMUNOLOGY 857

in the blood on the fourth day after infection and then drop precipitously
until on the tenth day of the infection they reach a low level (at location
1, in Figure 189), from which they do not thereafter deviate. Provided
such a large number of adult trypanosomes are injected intravenously
that they appear in the blood and can be studied immediately, the coeffi-
cient is low on the first day and rises precipitously, as may be seen in
control rat 980 in Figure 190. The inhibition of reproduction, as will
be shown later, is due to the development of an acquired immunity
involving an antibody which has been called ablastin (W. H. Taliaferro,
1924, 1932). The rate of reproduction of T. /ew7s7 is similarly retarded
and inhibited when grown in an abnormal host, the guinea pig, as
ascertained by Coventry (1929). Essentially the same results were found
for T. duttoni in the mouse, except that the rate of reproduction is never
as high and is not as completely inhibited, according to W. H. Taliaferro
and Pavlinova (1936) and W. H. Taliaferro (1938). Hence the
trypanosomes are never as numerous during the acute rise and may
increase slightly in numbers during the first part of the developed infec-
tion. T’. zowensis in the striped ground squirrel, as described by Rouda-
bush and Becker (1934), closely parallels the development of T. duttonz.
Since T. nabiasi in its natural host, the rabbit, increases in numbers only
during the first few days of the infection and thereafter does not show
division forms, as reported by Kroé (1936), the rate of reproduction
of this trypanosome may also be inhibited.

The question arises: Is there a natural immunity during the acute rise
of these infections? For it must be realized that in spite of the rise in
numbers a constant percentage of the parasites formed may be being
killed as was demonstrated in malaria. There are two ways of demon-
strating natural immunity. The first applies to the death of the organisms
and can be used only in such infections as malaria, in which it can be
demonstrated that all of the progeny formed do not survive. This is
impossible in the trypanosome infections, in which reproduction is not
synchronous and in which no method of ascertaining the total number
of progeny produced has so far been devised. The second method applies
not only to the death of the organisms, but also to the rate of reproduc-
tion, and involves various procedures such as comparisons of the same
species in various hosts and splenectomy combined with blockade. Posi-
tive experiments of this kind will indicate the existence of a natural im-

858 IMMUNOLOGY

munity, but, since natural immunity may be due to many factors, the
only conclusion that is warranted from negative experiments is that the
experimental method used did not disclose any natural immunity. With
these facts in mind, we may conclude the following: Guinea pigs seem
to possess a high natural parasiticidal immunity to T. /ew7s7, since T.
lewisi hardly increases in numbers at all in spite of the fact that it goes
through the same reproductive cycle as it does in the rat. Mice seem
to possess a high natural ablastic immunity to T. duttoni, since T. duttoni
in splenectomized and blockaded mice reproduce at a higher rate than
in normal mice. Rats have not been shown to possess any natural im-
munity against T. Jew7s7.

The drop in numbers at the crisis (at location 2, in Fig. 189) repre-
sents the acquisition of a trypanocidal response on the part of the host,
in addition to the ablastic effect, since if only the latter were present the
numbers would remain constant. Also, the disappearance of the trypano-
somes at the end of the infection (at location 3, in Fig. 189) represents
another acquisition of the same type of immune response. The comple-
mentary action of the ablastic and trypanocidal effects not only effec-
tively suppresses the infection, but also prevents relapses and reinfections
for long periods (at least 325 days). Thus a few trypanosomes left from
the initial infection or introduced by reinfection may be killed at once,
or, if they are not killed at once, their reproduction is inhibited until
they are killed. These statements hold for T. duttoni and, as far as they
have been tested, for T. nabiasi (previous citations).

The three effects of immunity which operate at points 1, 2 and 3 in
Figure 189 are all due to humoral antibodies, as tested by passive transfer
experiments. They are associated with the globulin fraction of serum,
are acquired as a result of specific infection or specific immunization, and
are decreased in amount or delayed in time of appearance by splenectomy
and blockade. They differ in the following ways: The titer of the three
varies independently, as far as can be tested. The trypanocidal effects
are due to typical lysins which may, however, act as opsonins 77 vivo.
The trypanolysin which terminates the infection kills either adult or
dividing trypanosomes taken at any time during the course of infection,
whereas that which causes the first number crisis kills only those try-
panosomes which have just appeared in a rat’s blood. The parasites that
survive the first number crisis are either basically nonsusceptible to this

IMMUNOLOGY 859

antibody or acquire a resistance to it. So far, the reproduction-inhibiting
properties of ablastin have not been demonstrated 7 vitro, but suitable
amounts of serum, containing ablastin together with adult trypanosomes,
when tested in vivo in a rat allow the trypanosomes to live, but prevent
them from reproducing. (Adult trypanosomes have to be used for this

Control Rat 980

650 5 =
600 4 | | }
1!
\
550 | 4 4 Us
slit
30 7 5 4 sl
J
450 | x | él 1
sl s
$
Fae 4 | 4 A
= 2
~390{ | | Ey
S !
5300; 304 Seed Rat 972 | | Pr. ef
“a I
E I
525 251 4 Experimental Rat 977 4 I
3 lg 2 I
2004.8 204 2] | !
= is z
3 = =
= 150) 2 15 3 | i
2 |5 = 7 | 2 = i
<= Ss = = =
= 104 10) = 12 Js u
2 = 2 /
S 2
507 pS
| 3 n | Caottiaent Variation | \
S erin | a
O24 6° 8S ) OR 2 eB ti ON Pesce aCe Meni ma4

Days after Injection

FIGURE 190. The demonstration of ablastin against Trypanosoma lewisi by passive
transfer. The rate of reproduction of the trypanosomes, as shown by the constant coeffi-
cient of variation, is completely inhibited in experimental rat 977, which received 2 cc.
of serum and a large number of adult trypanosomes from seed rat 972; whereas the rate
of reproduction goes through the normal cycle in control rat 977, which received a
similar number of adult trypanosomes, but no serum, from seed rat 972. (From W. H.
Taliaferro, 1924.)

test since a curtailment of the reproductive activity of dividing trypano-
somes, which is all that could be expected, is difficult to demonstrate with
certainty.) Thus the difference between the coefficient of variation curve
in experimental rat 977, which was given ablastic serum plus adult
trypanosomes, and in control rat 980, which was given normal serum
plus adult trypanosomes, is seen in Figure 190. The number curve re-

860 IMMUNOLOGY

mains constant in experimental rat 977 because the trypanosomes are not
reproducing. As was indicated above, ablastin is not absorbed by the
trypanosomes 7” vitro, whereas the trypanocidal antibodies are. There is
thus no lasting union of trypanosomes and ablastin, nor is there a sensiti-
zation of trypanosomes by ablastin as there is with the trypanolysins.
Moreover, if smaller and smaller doses of trypanocidal antibody are
given, a point is reached at which the trypanosomes are not killed, but
their reproduction remains unaltered. A group reaction can be demon-
strated between T. duttoni and T. Jewisi and their ablastins 72 vivo, and
between T’. Jewisi and antiduttoni trypanocidal antibody 7m vivo and in
vitro; but whether the reaction of anti/ew7s7 trypanocidal antibody against
T. duttoni in vivo and im vitro is a true group reaction of an immune antt-
lewis? antibody is not evident because normal rat serum is also trypano-
cidal to T. duttoni. These statements are based on a series of investiga-
tions involving either or both of the following: zm vitro work for the
trypanocidal effects and 7 vivo passive transfer experiments for all three
effects by W. H. Taliaferro and coworkers (vide infra), Regendanz and
Kikuth (1927), Perla and Marmorston-Gottesman (1930) and co-
workers, W. H. Taliaferro, Cannon, and Goodloe (1931), and W. H.
Taliaferro (1932). Culbertson (1938) has shown that the immunity to
T. lewisi is passed through the mother’s placenta and milk to young rats
where it persists for several weeks. Later, Culbertson and Wotton
(1939) found that the young rats do not appear to produce ablastin as
promptly or as well as older rats.

Various procedures designed to lower the macrophage function, such
as splenectomy, especially if combined with India-ink blockade or some
infection such as Bartonella which affects the macrophage system, de-
crease the strength and delay the appearance of ablastin and the terminal
trypanolytic antibody (Regendanz and Kikuth, 1927; Perla and Mar-
morston-Gottesman, 1930; Regendanz, 1932; and W. H. Taliaferro,
Cannon, and Goodloe, 1931). This is an effect on acquired immunity.

The next question which arises is whether the macrophages or other
phagocytic cells assist in passive immunity or in the action of ablastin
or the trypanolysins in the body. The work of W. H. Taliaferro (1938)
indicates the following: Splenectomy and blockade have no effect on the
passive transfer of ablastin, but the following interesting secondary
effect results: The passive transfer of ablastin lasts only for a few days.

IMMUNOLOGY 861

Thereafter it is not adequately augmented and supplemented by an active
ablastic immunity in splenectomized and blockaded animals, as it is in
normal rats, because the active immunity is slow in developing and
decreased in amount. Splenectomy and blockade definitely decrease the
effectiveness of the trypanolysins. It would seem that such an effect
could be adequately explained by a decrease in the amount of comple-
ment which would prevent the lysis of sensitized trypanosomes, or by a
decrease of macrophages which would prevent the removal of opsonized
parasites. The fact that previously sensitized trypanosomes are as readily
removed in splenectomized and blockaded animals as in normal rats
seems to negate both of these suppositions, unless the sensitized trypano-
somes are agglutinated and removed mechanically—a possibility which
because of technical difficulties has not yet been ruled out. A more likely
explanation is that there is an interference with the union of antigen
and antibody.

It has already been indicated that a lysin differs from an opsonin only
in that the terminal lysis and death of sensitized organisms may be
effected by extra- rather than intracellular enzymes. Just as in the
indirect studies discussed in the preceding paragraph, however, direct
studies on phagocytosis have failed to indicate whether phagocytosis or
lysis is more important in acquired immunity. Laveran and Mesnil
(1901) considered that the parasites are actively phagocytosed, and
Roudsky (1911) and Delanoé (1912), studying the acquired immunity
of mice to T. /ewis7, came to the same conclusion. Regendanz and Kikuth
(1927) believed that the parasites are phagocytosed in a nonspecific
way. MacNeal (1904), Manteufel (1909), W. H. Taliaferro (1924),
and Coventry (1929), on the other hand, considered that they are lysed.
In studying the tissues for evidence of phagocytosis, we are handicapped
by the fact that no easily recognizable vestiges of trypanosomes, such as
malarial pigment, remain in macrophages for any appreciable length of
time. The fact that pigment by itself may be phagocytosed does not
invalidate this statement, since the whole complex, consisting of red
cell, parasite, and pigment, is often recognized intact in macrophages.

To sum up: acquired immunity against nonpathogenic trypanosomes
primarily involves ablastin and trypanolysins, the first of which prevents
the trypanosomes from undergoing growth and cell division, and the
second of which kill the trypanosomes. They are both humoral anti-

862 IMMUNOLOGY

bodies which are associated with the globulin fraction of immune serum,
are passively transferable, and are probably a product of the lymphoid-
macrophage system; but whereas ablastin possesses no 7m vitro affinity
for the trypanosomes, the trypanolysins are typical antibodies (ambo-
ceptor) and can sensitize the trypanosomes 7m vitro. Furthermore, the
macrophage system does not appear to intervene in the passive transfer
of ablastin, but in some way functions in the union of antigen and antt-
body during the passive transfer of the trypanolysins.

CONTINUOUS FATAL TRYPANOSOMIASIS IN THE MOUSE AND SOMETIMES
IN THE RAT

Most species of trypanosomes appear to be nonpathogenic, and there
is a growing mass of evidence that even many of the pathogenic forms

Millions of Trypanosomes per cmm. of dbod
m3 w

ES —— Fe
Coefficient of Variation
(ae

<— Ineclion ip

cme illons of
_——

<— Dead

vonatuy Ja} sheg
y
pit )
UOIPLID\ J
JUdI91JJ005

FIGURE 191. The changes in number of Trypanosoma rhodesiense and the coefficient
of variation during the course of infection in a mouse. No acquired immunity is de-
veloped in the mouse, since the number of trypanosomes more or less steadily increase
and their rate of reproduction, as evidenced by the high coefficient of variation, is not
inhibited. (Redrawn from W. H. Taliaferro and L. G. Taliaferro, 1922.

in man and domesticated animals may be nonpathogenic in their natural
hosts (cf. Duke, 1936). Little is known of the course of their infection
in their natural hosts, but they have been extensively studied in labo-
ratory animals, in which they are all pathogenic. The well-known patho-
genic trypanosomes, which produce disease in man and domestic ani-
mals, are T. gambiense, T. rhodesiense, T. brucei, T. congolense, T.
vivax, T. evansi, T. equinum, and T. eguiperdum. When injected into
mice, the parasites almost invariably appear in the blood after a short
incubation period, and increase in number more or less steadily until
the death of the host. This type of infection is composed of an incuba-
tion period and acute rise, with no crisis or developed infection. It
sometimes occurs in the rat. Its continuous nature was clearly pointed

IMMUNOLOGY 863

out by Massaglia (1907) and is illustrated in Figure 191 by T. rho-
desiense in the mouse.

Whether there is a natural immunity against the trypanosomes in
the mouse cannot be answered at present, because of insufficient data.
On the one hand, W. H. Taliaferro, Johnson and Cannon (unpublished
work, see W. H. Taliaferro, 1929) reported no effect of splenectomy
on mice infected with T. equinum, and on the other hand Schwetz
(1934) and Russeff (1935) found a slight effect of splenectomy in
mice infected with T. congolense and T. equiperdum respectively.

With regard to acquired immunity, most data are in accord in show-
ing that the mouse does not develop any appreciable amount. As may
be seen in Figure 191, the rate of reproduction of the parasites remains
constant and fairly high (the C.V. varies between 8.9 and 10.5 per-
cent) and the parasites progressively increase in number until the death
of the host (see also control mouse, infected with T. eguimum, in
Figure 193, which is drawn on a semilogarithmic scale).

In 1933 Krijgsman showed that the rate of increase of T. evans
in the mouse and rat is not uniform during the acute rise, but that two
periods of high rates of increase, approximately between eighteen and
thirty-two hours and sixty and sixty-six hours, alternate with three pe-
riods of lower rates of increase at the beginning, middle, and end of
the infection. He believed that the terminal low rate of increase is due
to a destruction of parasites, as evidenced by the occurrence of more
degenerating forms, but that the earlier low rates are due to a partial
inhibition of reproduction. He reached this conclusion because he found
no increase in degenerating trypanosomes in the blood and in spite of
the fact that he found no diminution in the percentage of division
forms. He visualized the mechanism of this partial inhibition of re-
production as a uniform retardation of all stages of the cycle of
growth and cell division. There is no doubt that theoretically such
a uniform lengthening of all stages would give a partial inhibition of
reproduction, without affecting either the percentage of division forms
or the coefficient of variation. Nevertheless, the existence of such a
mechanism is doubtful, in view of the fact that in T. /ew7s7, as well as
in cells in general, a retardation of cell division is characterized by an
increased length of the resting stage (the so-called adult stage of T.
lewis’), and not by a gradual slowing down of the whole process with

864 IMMUNOLOGY

uniform increases in the length of each stage. Furthermore, Krijgs-
man’s inability to find degenerating stages in the blood early in the
infection and his finding them during the terminal phases is not con-
clusive evidence that trypanosomes are not dying during the early low
rates of increase. It is very likely that the macrophages remove such
forms less quickly during the latter part than during the early part of
the infection, because they have become partially blockaded. The inter-
pretation of the varying rates of increase of T. evansi in the mouse, as
found by Krijgsman, will therefore have to await further analysis. From
an enormous mass of work on the mouse as a carrier of so-called pas-
sage strains, however, it appears that no trypanolysin usually develops
in the mouse which kills most of them and toward which the residue
become antigenically resistant, as has been demonstrated in the infec-
tions to be described in the following section.

We may accordingly conclude that the course of the infection in the
mouse and sometimes in the rat most closely approximates the simplest
type of infection, which increases as a geometrical progression and in
which little, if any, immunity is acquired of either an ablastic or trypano-

cidal type.

INTERMITTENT FATAL TRYPANOSOMIASIS IN VARIOUS LABORATORY
ANIMALS

When the same pathogenic trypanosomes considered in the preced-
ing section are grown in the guinea pig, the infection is typically char-
acterized by an incubation period, followed by alternate increases (the
first is an acute rise and the succeeding ones are relapses) and decreases
(crises) in the parasite population until the animal dies. Besides the
guinea pig, this kind of infection is observed in rabbits, dogs, cats, and
occasionally in rats infected with these same trypanosomes, in man
infected with T. rhodesiense and sometimes in mice infected with T.
congolense. Sometimes the initial acute rise and crisis do not develop,
as is shown in Figure 192. In other animals, such as sheep, the entire
infection is of such low grade that trypanosomes are rarely found and
then only in thick film. These statements are based on work by Ross
and D. Thompson (1910, 1911), J. G. Thomson (1912), W. H.
Taliaferro and L. G. Taliaferro (1922), Knowles and Das Gupta
(1928), Davis (1931), Krijgsman (1933), Browning ef al. (1934),

IMMUNOLOGY 865

and others. The fact that animals often die when trypanosomes are
scarce in their blood has been explained as being due to sugar depletion,
asphyxiation, toxins, and so forth (see von Brand, 1938, review). A
possibility that has not been adequately discussed in the literature is
that death shortly after a crisis may be due to the severity of the im-
mune reaction, comparable to that seen in overwhelming hypersensi-
tivity (cf. graph of malaria, Figure 188).

In analyzing such infections, W. H. Taliaferro and L. G. Taliaferro
(1922) found that the basic rate of reproduction remains relatively

nm
wn

: bs

5 5

Zl > fK

Ss £# \

= S.¢ et

E | Fal ce. 52 ! N

S = = 35 len, ! 4

Fol Bol = oe SN a 3 \~ g

ss oc

Bt ear EE aI oe
= LSS a

G. Ss | Ss) Ay | K |
&

Days after Injection

FIGURE 192. The changes in number of Trypanosoma rhodesiense and the coefficient
of variation during the course of infection in a guinea pig. As acquired immunity de-
velops, the parasites are killed by trypanolysins operative during the long chronic period
and at the two crises thereafter, but the rate of reproduction is not inhibited, as evi-
denced by the high coefficient of variation. (Redrawn from W. H. Taliaferro and L. G.
Taliaferro, 1922.)

constant whenever parasites can be found in the blood in sufficient num-
bers for study. Thus in Figure 192 the coefficient of variation remains
between 8.5 and 12.0 percent from twenty-three through forty-three
days after infection. Knowles and Das Gupta (1928) corroborated
this finding for T. evansi in the rat, and Davis (1931) for T. rho-
desiense in the cat. There is some evidence that these trypanosomes,
when originally isolated in Africa, exhibit infections essentially simi-
lar, except that superimposed on the reproductive activity seen in
Figure 192 are intermittent periods of heightened reproductive activity.
Thus Robertson (1912) reported that periods of active reproduction of
T. gambiense in the monkey alternate with periods of less active repro-
duction. Since reproduction continues at a relatively high rate, even
during the periods of less active reproduction, and is never completely

866 IMMUNOLOGY

inhibited, these infections may be considered with the constantly repro-
ducing experimental infections discussed in this section.

Little is known about natural immunity because, as pointed out in
the consideration of the T. Jews? group of trypanosomes, the rate of
reproduction of the trypanosomes cannot be measured directly and few
attempts have been made to raise the rate of reproduction (see T. duttoni
in the mouse) or to increase the percentage of surviving trypanosomes.
Nieschulz and Bos (1931), however, reported a slightly shorter incuba-
tion period in dogs infected with T. evansi as the result of splenectomy.

Acquired immunity, which develops later and which is superim-
posed on any natural immunity which may be present, is entirely para-
siticidal, with no evidence of an inhibition of reproduction. When the
infection shows a typical acute rise in numbers, the first manifesta-
tion of acquired immunity is a crisis (see guinea pig, in Figure 193).
Thereafter, one or several relapses and crises follow. When a pro-
longed chronic low-grade infection ensues, with no typical acute rise
as in Figure 192, it is probable that parasiticidal effects of acquired
immunity, similar to those produced at the two typical crises later, hold
the numbers down. Since all the animals die, the acquired immunity
is obviously ineffective. In other words, once the parasites are intro-
duced into the host, they reproduce during the entire infection, and
although at intervals most of those that have accumulated in the blood
are destroyed, the few that escape destruction repopulate the blood
again and again until the host dies.

An extensive series of 72 vivo and in vitro investigations (see Laveran
and Mesnil, 1912; W. H. Taliaferro, 1929, for reviews of the pioneer
work) indicate (1) that the periodic destructions of the trypanosomes
are due to typical trypanolysins which can be demonstrated 7v vivo and
in vitro and which are acquired by the host as a result of specific infec-
tion or specific immunization; (2) that the trypanosomes reaccumulate
during the relapses not because the trypanolysins disappear, but be-
cause the parasites have become resistant to them; and (3) that the
relapse trypanosomes differ antigenically and that the majority of them
are subsequently killed by a new trypanolysin. In all of this work, the
strains of trypanosome used and their continuous maintenance are of
paramount importance. The experimental infection is started with what
is known as the passage strain and is usually maintained in mice by

IMMUNOLOGY 867

serial transfer, since it has been found that strains remain immunologi-
cally unchanged for long periods in mice. The trypanosomes which re-
populate the blood after each trypanolytic crisis are immunologically
different from the passage strain and are resistant to the lysin produc-
ing the crisis. They are known as relapse strains and also have to be
maintained in mice by serial transfer. Thereafter, as many relapse strains
as are studied have to be extracted and maintained separately and con-

Guinea Dig

(experinjental)

—_

00000

Injected ip with 3cc. of

Immune Serurn

Crists inf hr

=
oS
Ss

Number of Trypanosomes per cmm. of Blood

= Killed for Immune Serum

100

YO @ A 8 8 [0 fre PV) i a Gr El ip) 1 Ta @)
Days after Injection

FIGURE 193. The demonstration of a trypanolysin against a passage strain of Trypano-
soma equinum by passive transfer. An artificial crisis is produced in the trypanosome
infection in the experimental mouse, which received 0.3 cc. of serum taken from the seed
guinea pig after its infection had undergone a crisis, whereas the infection in the control
mouse which was not given serum proceeded uniformly until the mouse died. (From
W.H. Taliaferro and T. L. Johnson, 1926.)

tinuously until the investigation is terminated. Reference to Figure 193
will make this clearer. Both the guinea pig and the two mice were in-
fected at appropriate intervals with a passage strain. Serum taken from
the guinea pig at the time of the naturally occurring crisis produced an
artificial crisis when injected into the experimental mouse, but would
have been ineffective had the experimental mouse been infected with
the relapse strain, which would have repopulated the blood of the
guinea pig after the crisis. (The artificial crisis produced by the immune
serum in this so-called curative test is very temporary.) A brief résumé
of the investigations follows.

868 IMMUNOLOGY

Rouget (1896) first found that the serum of rabbits and dogs, which
had been infected with T. eqguiperdum and had become cachetic, exerted
a protective action, in a dose of 0.3 cc., on mice infected with the pass-
age strain as measured by the survival time of the mice. The fact that
immune serum is protective led Laveran and Mesnil (1901) to hope
that serotherapy might possibly be developed against pathogenic trypano-
somiasis, but so far this hope has not materialized. Schilling (1902)
was the first to recognize the phenomenon of trypanolysis in vitro.
Rodet and Vallet (1906) studied the lysins systematically, and Massag-
lia (1907) showed that the trypanosomes which repopulate the bléod
after each trypanolytic crisis are immunologically different from the
original strain and are resistant to the lysin producing the crisis. Thus
serum from an infected guinea pig before a crisis is only slightly lytic,
whereas that during and after the crisis is strongly lytic to the original
strain of trypanosomes, but has no deleterious effect on the trypano-
somes reappearing after the crisis. Levaditi and Mutermilch (1909)
reported that the lysis is a complement-amboceptor reaction (i.e., in-
volves a heat labile component of serum and the heat stable antibody),
and Leger and Ringenbach (1911 and 1912) found a group specificity
between trypanolytic immune serums and different species of pathogenic
trypanosomes.

W.H. Taliaferro and T. L. Johnson (1926), in a study of the pro-
duction of artificial crises (Figure 193) by immune serum against T.
equinum in mice, found that zones of inhibition may occur. T. L. John-
son (1929), in a continuation of this work, found that the production
of the artificial crisis, with resulting lengthening of life in the mouse,
is dependent not only upon the amount of immune serum, but upon
the absolute number of parasites present and upon the strain of para-
site used. For example, when a given serum was injected into mice
whose blood showed one to five parasites of a particular strain per
microscopic field, it caused lysis of the trypanosomes uniformly in all
doses greater than the minimal effective dose; when injected into mice
the blood of which contained from ten to twenty-eight parasites of the
same strain per microscopic field, it caused alternate zones of lysis and
non-lysis (zone phenomenon); whereas when injected into mice whose
blood showed fifty parasites per microscopic field, no lysis occurred,
no matter what dose of serum was given. Moreover, Johnson was able

IMMUNOLOGY 869

to subject this strain to immune serum and to secure a relapse strain
which showed the zone phenomenon with one to five parasites per
field. Such data give a basis for the interpretation of the variable and
often contradictory results obtained by investigators doing only a
few tests.

The relapse strains can be differentiated from the passage strain not
only by their resistance to lysins, but by their behavior in other serologi-
cal tests, such as the Rieckenberg blood platelet test (Rieckenberg,
1917; see also Brussin and Kalajev, 1931). They also differ antigeni-
cally and therefore stimulate different immune mechanisms, as is
shown, for example, by cross-immunity tests.

The difference in antigenic constitution of various strains was origi-
nally studied by Ehrlich and his coworkers in infections in mice in
which artificial crises were produced by incomplete cures with drugs.
Of the earlier papers, that of Ritz (1914) is particularly interesting.
He incompletely cured a mouse twenty times, during which seventeen
immunologically different relapse strains were produced, as tested by
cross-immunity in mice after cure. Some of these strains were identical
with those of another mouse which had been incompletely cured nine-
teen times, during which nine immunologically different strains had been
produced. The immunological variations may be inherited, but in time
may be lost. Ritz (1916) also showed that the strains arising naturally
in the rabbit could be differentiated by the same methods. In the suc-
ceeding years, more or less similar studies have been made with both
antibody and drug-induced relapse strains. Recently, Lourie and O’Con-
nor (1937), in an 7” vitro study of relapse strains after drug treatment,
obtained twenty-two relapse strains of which thirteen were immuno-
logically distinct. In addition, they ascertained that certain strains tended
to occur more frequently than others, that a strain may be a combina-
tion of two or several strains, and that individual strains may disappear
from such compound strains.

The acquisition of this antibody resistance by the trypanosomes, with
a concomitant antigenic change, is an interesting case of an environ-
mentally induced persistent modification which is inherited for many
asexual generations, sometimes through 400 mouse passages. It seems
to be similar to the acquisition of drug resistance by free-living Proto-
zoa. It can be produced not only im vivo, but also in vitro. It is al-

870 IMMUNOLOGY

ways associated with the destruction of many organisms, and hence
involves a selection, but the selection is effective within a clone, i.e.,
within the progeny of a single trypanosome. At the present time, how-
ever, it 1s impossible to decide whether such persistent modifications
are due to changes in gene constitution, or, if they are not, whether they
may eventually lead to such changes (see Robertson, 1929; W. H. Talia-
ferro and Huff, 1940; and, in part, Dobell, 1912). Nevertheless, they
are of extreme importance in allowing the parasite to overcome the
defensive processes of the host and are probably largely responsible for
the continued survival of the parasite.

The lymphoid-macrophage system and particularly the macrophages
along the blood stream, appear to be involved in immunity, as indicated
by enlargement and histological changes in the spleen (Laveran, 1908;
Van den Branden, 1935; and others) and by the decreased length of
life of splenectomized animals infected with various trypanosomes (see
Davis, 1931, for most of the work prior to 1931; Nieschulz and Wawo-
Roentoe, 1930; Nieschulz and Bos, 1931; Russeff, 1935). Negative
results, as might be expected from the complexity of the problem as
explained previously, have also been reported by some of the earlier
workers (see Davis, 1931), and also by Davis (1931) and Browning
et al. (1934), whereas increased length of life was noted in partially
blockaded rats infected with T. eguiperdum by Kolmer et al. (1933).
Data on splenectomized and blockaded animals which were treated are
omitted from consideration because the treatment itself may affect the
course of infection. |

Whether the trypanocidal antibody acts within the body as a trypano-
lysin, or as an opsonin with resulting phagocytosis, or both, has been
variously answered. Some authors have maintained that one or the other
is the sole method of defense; some that they share equal honors; and
some that, although lysis is the fundamental mechanism, phagocytosis
is responsible for clearing up the debris, and so forth. No one can
doubt the occurrence of phagocytosis after its careful description by
sO many competent observers (Neporojny and Yakimoff, 1904; Sauer-
beck, 1905; Yakimoff, 1908; Mesnil and Brimont, 1909; Levaditi and
Mutermilch, 1910). On the other hand, W. H. Taliaferro and T. L.
Johnson (1926) reported the finding of disintegrating trypanosomes

IMMUNOLOGY 871

in the blood during experimental crises, which they interpreted as
stages in lysis. This question can probably be answered as was the simi-
lar question with regard to the trypanolysins in T. /ew/s7 infections.
The trypanosomes become sensitized by antibody, and the process may
be completed by lysis (extracellular enzymes) on one hand, or by diges-
tion within phagocytes (intracellular enzymes) on the other hand. Which
occurs may depend to a certain extent on the strength of the antibody.

Besides the cellular basis for the production of the trypanocidal anti-
body and the codperation of phagocytes in removing sensitized para-
sites, Kuhn (1938) has shown a peculiar role of the lymphoid-macro-
phage system in passive transfer of anti 7. equiperdum protective serum
to mice. Thus immune serum, which is effective in protecting normal
mice in doses of 0.4 cc. per 20 gm. body weight, gives only partial pro-
tection to splenectomized mice, blockaded with India ink, in doses as
high as 1.7 cc. per 20 gm. body weight. Suitable experiments indicate
(as in previous work with T. /ewzs7) that this finding is due neither to
the lowering of complement nor to the removal of phagocytic cells
which might be necessary in removing opsonized parasites, but rather
to the prevention of antibody uniting with trypanosomes. An interest-
ing, but confusing element in these experiments was that unilateral
nephrectomy or ureterotomy was accompanied by a slight reduction in
the protective titer of the serum.

A comparison of the resistance acquired by hosts against pathogenic
and against nonpathogenic trypanosomes is very illuminating. In the
first case, the host acquires practically no resistance (mouse) or it pe-
riodically forms trypanolysins (guinea pig, dog, etc.) which hardly
ever effectively rid the animal of infection because a few of the patho-
gens generally become resistant and repopulate the blood again and
again until the host dies. In the second case, the host first produces an
antibody which inhibits cell division of the parasites and then periodi-
cally forms trypanolysins which get rid of the nonreproducing parasites.

PRACTICAL APPLICATIONS OF IMMUNE REACTIONS

By far the most extensive literature on the immunology of the para-
sitic Protozoa deals with experiments fundamentally planned in the hope
of achieving some practical method of preventing, curing, or diagnosing

872 IMMUNOLOGY

infections. This work has yielded many facts of great interest, but ac-
tual practical applications have been limited.

ARTIFICIAL IMMUNIZATION

The earlier literature on this subject has been critically reviewed by
W. H. Taliaferro (1929), to which publication the reader is referred
for details. Only a few of the more successful examples are cited.

The greatest success with artificial immunization has been attained
in Babesia infections of cattle and consists of inducing in young healthy
animals a low-grade or latent infection which is frequently controlled
with drugs. During this latent infection, the animal possesses a solid
immunity to superinfection, similar to the condition in malaria. Like
malaria, however, the host’s defenses may weaken and permit severe
and even fatal relapses.

Mention has been made of the fact that one attack of oriental sore
in man generally confers a lasting immunity. As the natural sores oc-
cur on the face or other exposed portions of the body and leave dis-
figuring scars, it has been the practice in many endemic centers for
centuries to inoculate children on unexposed portions of the body. In
a sense this is the crudest type of immunization, in that the highly viru-
lent virus is employed to induce the ordinary disease. The use of at-
tenuated organisms has not met with particular success.

Several investigators have been able to immunize laboratory animals
with dead trypanosome vaccines. So far, however, such vaccines have
not been extensively applied in a practical way and the outlook is
not favorable. Among other difficulties, the attainment of an ade-
quately polyvalent vaccine can hardly be hoped for, owing to the exist-
ence of so many immunologically different strains of trypanosomes.

IMMUNOLOGICAL REACTIONS USED IN DIAGNOSIS

Considerably more success has followed the practical application of
immunological reactions in diagnosis than in immunization, but even
here the success has been limited. This is due in part to the technical
difficulty of perfecting the tests, especially when only weak reactions
ensue, and in part to the fact that they have to be as satisfactory as or
better than the demonstration of the parasites, which has been rendered
remarkably delicate in certain blood infections, notably malaria and

IMMUNOLOGY 873

trypanosomiasis, by the use of stained thick-blood films (see Barber,
1936). From the great mass of literature only the tests which have
been perfected or show considerable promise will be mentioned. De-
tailed protocols and specific methods of procedure can be found in
W.H. Taliaferro (1929) or in some of the more recent articles.

A. Specific Immunological Reactions—The reactions between antigen
(either complete or haptene) and antibody are so specific that, within
certain limits, the presence of a suspected antigen can be ascertained
with a known antibody, or, vice versa, a suspected antibody can be
verified with a known antigen. Both have been used in diagnosis. When
the invading organism liberates some antigen, either whole or partial
(haptene) into the blood, sputum, urine, and so forth, the antigen may
be detected and identified by its reaction with a high titer immune serum,
generally prepared in the laboratory. Sometimes, if the organism iso-
lated from an infected host cannot be fully identified by morphological
criteria, it may be further classified in this way (see section on im-
munological methods of classification). Or, if the invading organism
during infection stimulates the formation of a specific antibody in the
blood, it may be identified by its reaction with a known antigen which
is prepared from the organism in the laboratory. In the Protozoa only
the last type of reaction has been extensively used.

The specific complement fixation test is one of the most highly stand-
ardized laboratory tests. It is based on the fact that antibody will combine
with antigen, and the resultant sensitized antigen will then combine fur-
ther with complement (a heat labile component of serum), but neither
antigen nor antibody will combine with complement alone. In practice,
serum suspected of containing an antibody is first heated at 56° C. for
twenty minutes to inactivate the complement which it also contains and
then is added in varying proportions to a known antigen. To such mix-
tures, known quantities of complement (generally fresh guinea-pig
serum) are subsequently added. The actual fixation of complement gives
no visible sign, but is tested by adding to the system at this point a
suspension of red blood cells which have been previously sensitized with
their specific lysin (sheep cells and antisheep lysin are generally used).
Obviously, if the complement was previously fixed, there will not be
enough left to lyse the sensitized cells. In terms of the original test,
if the red blood cells undergo lysis and their hemoglobin colors the

874 IMMUNOLOGY

solution, the suspected antibody was not present (test negative); if the
cells remain entire and unlysed and the supernatant clear, the suspected
antibody was present in the serum (test positive). From the foregoing
brief résumé, it is obvious that this test demands careful preparation
and standardization of the component parts for its successful execution.
It differs from the nonspecific complement fixation or Wassermann test
widely used in syphilis only in that the test antigen is derived from the
immunizing organism or antigen. The test antigen for the Wassermann
test, on the other hand, involves the use of a lipoid extracted from
normal tissue, such as beef heart.

The specific complement fixation test has been most satisfactorily
standardized in amoebiasis and in dourine of horses. The successful
cultivation of Endamoeba histolytica, the causative agent of amoebic
dysentery, made amoebae available in sufficient quantities to provide a
suitable antigen, and since the work of Craig in 1927 the complement
fixation test for amoebiasis has been intensively studied (see Craig,
1937; Meleney and Frye, 1937; Paulson and Andrews, 1938). The con-
sensus of opinion seems to be that the test has to be carefully carried
out to be dependable and that at best it can be used only as an adjunct
to fecal diagnosis.

The sum total of published work through 1910 indicated that com-
plement-fixing antibodies could be demonstrated in various trypano-
somiases under controlled conditions, but there was little to indicate
that they could be used for diagnosis. From 1911 onward, however,
the test was perfected and used extensively for the diagnosis of dourine
caused by Trypanosoma equiperdum in horses and mules. It was stand-
ardized mainly through the efforts of Mohler, Eichhorn, and Buck
(1913), E. A. Watson (1920), who used an aqueous antigen; and
Dahmen (1922), who used both aqueous and alcoholic extracts. Ac-
cording to Watson, the test is often positive before symptoms are ap-
parent and during latent stages, and in practice no animal should be
considered free of the disease unless negative two months after a last
exposure. C. M. Johnson and Kelser (1937) concluded that the test
is distinctly valuable in revealing active cases of Chagas’s disease.

Little success has attended workers using specific complement fixation
in malaria and the leishmaniases, especially kala azar, owing perhaps
to the low titer of serums from infected persons and the difficulty of

IMMUNOLOGY 875

obtaining antigens. Recently Coggeshall and Eaton (1938) have re-
ported good results in simian malaria with an aqueous antigen obtained
from heavily infected blood or spleen.

The red-cell adhesion test grew out of Rieckenberg’s (1917) blood
platelet test. As used by Duke and Wallace (1930), it involves the
addition of one drop of a citrated trypanosome suspension to one drop
of equal parts of blood from the suspected animal and 2-percent sodium
citrate. If the blood comes from an infected animal, red blood cells
(occasionally also blood platelets) adhere to the trypanosomes within
ten to fifteen minutes. In 1931 Wallace and Wormall concluded that
complement is necessary, and H. C. Brown and Broom (1938) found
that the concentration of trypanosomes should be between 3,000 and
100,000 per cu. mm. and the red cells between 300,000 and 1,250,000.
This test compared favorably with specific complement fixation, when
untreated horses infected with T. h7ppicum were tested by W. H. and
L. G. Taliaferro (1934d).

B. Nonspecific Serological Reactions.—Infection often results in defi-
nite changes in serum which can be detected by various physical and
chemical means and which, although not specific in the immunological
sense, are characteristic enough to be useful in diagnosis. Even when
the same changes occur in several infections, they may still be used in
conjunction with other criteria or if the infections have different geo-
graphical distributions.

Several miscellaneous tests have been devised for kala azar which are
associated with an increase in the euglobulins of the serum. They in-
clude the serum-globulin test of Brahmachari, the aldehyde test of
Napier, and the urea-stibamine test of Chopra, Gupta, and David. These
tests have been modified and combined by these same and other work-
ers. In general, upon the addition of distilled water, formaldehyde, or
urea-stibamine in proper proportions to serum from a person infected
with kala azar, the mixture becomes characteristically opaque, owing to
the formation of a precipitate within a comparatively short time. These
tests appear to be extremely useful and Menon ef al. (1936) advocate
testing a serum by both the aldehyde and the urea-stibamine test (see
Menon ef al., 1936, and Chorine, 1937, for the literature on this
subject).

Some of these tests may also be of value in trypanosomiasis (see

876 IMMUNOLOGY

Hope-Gill, 1938), especially in areas in which kala azar is absent.

In 1927 Henry described certain serological tests for the diagnosis
of malaria, based on the observation that the serums of malarial sub-
jects flocculate in solutions of metharsenate of iron (ferroflocculation
test) and of melanin pigment (Henry’s test, or the melanoflocculation
test). As a reagent for the Henry test, which was shown by later work
to be more sensitive than the ferroflocculation test, Henry (1934) used
the filtered supernatant from a suspension of finely ground choroid
tissue of ox eye in distilled water. This material after formalin had
been added and it had been kept on ice for at least several hours, is
added in proper proportions to the serum to be tested, and flocculation
is looked for after a half hour or more, preferably by means of the
photometer of Vernes, Bricq, and Yvonne. Many subsequent papers on
this test have been ably reviewed by Greig, Von Rooyen, and Hendry
(1934), Trensz (1936), Villain and Dupoux (1936), de Alda Calleja
(1936), Vaucel and Hoang-Tich-Try (1936), and Proske and Watson
(1939). The upshot of this work seems to indicate that the test may
serve as an adjunct to the search for malarial parasites in diagnosing
malaria, but that its use is restricted to laboratories equipped with a
photometer and to areas in which kala azar, certain types of leprosy,
and certain other diseases are not common. Since it has been shown to
be due to an increase of the euglobulin fraction, which flocculates upon
dilution with distilled water or weak salt solutions, Proske and Watson
(1939) have developed the protein-tyrosin reaction, which is a quanti-
tative chemical estimation of the euglobins of the serum.

IMMUNOLOGICAL REACTIONS IN RELATION TO CLASSIFICATION

Various immunological reactions, since they are frequently spectes-
specific, have been used to check and extend other biological classifi-
cations. In other words, the more closely two species are related, the
stronger, in general, is the group reaction between them. This specificity
seems to depend on the basic structure of the antigens and haptenes,
which react specifically with immune serum iz vitro. It also probably
depends upon the quantitative proportions of the various antigens con-
tained in a particular organism (see Wells, 1929). Immunological re-
actions can therefore be employed to compare chemical structure with

IMMUNOLOGY 877

anatomical structure. The reactions have to be studied, however, to see
if, on the one hand, they vary too much within what is a generally recog-
nized species, or if, on the other hand, they do not differentiate suffi-
ciently among large groups. The extreme specificity within a species
may be exemplified by the diversification of a single cell strain of
trypanosomes, through the mediation of immune serums or drugs, into
a large number of strains which will remain immunologically distinct
for long periods.

In a sense most of the work on the serology of parasites can be used
in classification. For example, an investigator, in attempting to dis-
cover a serological test for a given infection, generally considers at
once the specificity of the reaction by ascertaining to what extent group
reactions with other species exist. On the whole, however, the study of
the immunological relationships of organisms can best be attained by
using antiserums from artificially immunized laboratory animals. By
this method animals such as rabbits, in which antibodies are readily
produced, can be immunized until high titer antiserums are obtained.

Immunological methods have been employed extensively to estab-
lish the identity or nonidentity of various proposed species of Lezsh-
mania, which are morphologically identical, and their relationship to
certain insect and plant herpetomonads which resemble the cultural
forms of Leishmania. This work is fairly consistent in showing that the
members of the genus Leishmania are a closely related group and are
entirely distinct from the genus Herpetomonas (Noguchi, 1926; Wage-
ner and Koch, 1926; Zdrodowski, 1931).

Since trypanosomes, like the Leishmanias are frequently morphologi-
cally indistinguishable from one another, various immunological tests,
as well as biological criteria, have been employed to distinguish them.
The in vivo cross-immunity test has been most extensively used (Braun
and Teichmann, 1912; Laveran, 1917; Kroé, 1925, 1926; Schilling and
Neumann, 1932), but 2” vitro tests have also been used, such as com-
plement fixation by Robinson (1926), the phenomenon of “‘attachment’”’
by several authors (see Levaditi and Mutermilch, 1911) and 7m vitro
trypanolysis by Leger and Ringenbach (1912) and others. In evaluat-
ing the results of these methods, it appears that they need to be re-
worked, because of the advance in modern technique and because au-

878 IMMUNOLOGY

thorities such as Wenyon (1926) believe that many of the species for-
merly recognized as distinct should be combined (cf. Becker, Chapter
XVII).

The work on piroplasms, although lacking in conclusiveness, has at
least served to direct the attention of systematists to the problems of
classification (Theiler, 1912; Stockman and Wragg, 1914; du Toit,
1919; Brumpt, 1920).

In malaria cross-immunity tests have been extensively employed
(Manwell, 1938), whereas in amoebiasis complement fixation tests
have occasionally been used (Menendez, 1932).

LITERATURE CITED

Afridi, M. K. 1938. Observations on extra-abdominal spleen in monkeys
infected with P. cynomolgi and P. knowlesi. J. Malar. Inst. India., 1:
sa)

Alda Calleja, M. de. 1936. Estado actual de los estudios sobre sueroflocula-
cion en el paludismo. Med. Paises Calidos, 9: 203-36.

Aschoff, L. 1924. Das reticulo-endotheliale system. Ergebn. inn. Med. Kin-
derheilk., 26: 1-118.

Barber, M. A. 1936. The time required for the examination of thick blood
films in malaria studies, and the use of polychromatophilia as an index
of anemia. Amer. J. Hyg., 24: 25-31.

Ben Harel, S. 1923. Studies of bird malaria in relation to the mechanism of re-
lapse. Amer. J. Hyg., 3: 652-85.

Bignami, A. 1910. Sulla patogenesi delle recidive nelle febbri malariche. Atti
Soc. Studi Malar. 11: 731-45. Translated by W. M. James: Sth. med.
J. Nashville, 1913 (Feb.).

Bloom, W. 1938. Lymphocytes and monocytes: theories of hematopoiesis.
Downey: Handbook of Hematology, 1: 373-436.

Bohm. 1918. Hamatologische Studien bei Malaria. Arch. Schiffs- u. Tro-
penhyg., 22: 49-55.

Boyd, G. H. 1929a. Induced variations in the asexual cycle of Plasmodium
cathemerium. Amer. J. Hyg., 9: 181-87.

—— 1929b. Experimental modification of the reproductive activity of Plas-
modium cathemerium. J. exp. Zool., 54: 111-26.

—— 1933. Effect of quinine upon reproduction of the avian malaria parasite,
Plasmodium cathemerium. J. Parasit., 20: 139-40.

—— 1939. A study of the rate of reproduction of the avian malaria para-
site, Plasmodium cathemerium. Amer. J. Hyg., 29 (Sect. C): 119-29.

Boyd, G. H., and L. H. Allen. 1934. Adult size in relation to reproduction
of the avian malaria parasite, Plasmodium cathemerium. Amer. J. Hyg.,
20: 73-83.

IMMUNOLOGY 879

Boyd, G. H., and M. Dunn. 1939. Effects of quinine and plasmochin admin-
istration upon parasite reproduction and destruction in avian malaria.
Amer js Hye:.300(SechG@) ee i7-

Boyd, M. F., and L. T. Coggeshall. 1938. A résumé of studies on the host-
parasite relation in malaria. Trans. Third Inter. Congress Trop. Med.
and Malaria, 2:292-311.

Boyd, M. F., W. K. Stratman-Thomas, and S. F. Kitchen. 1936, On the
duration of acquired homologous immunity to Plasmodium vivax, Amet.
Jnttop: Meds 162 31 1215;

Brand, T. von. 1938. The metabolism of pathogenic trypanosomes and the
carbohydrate metabolism of their hosts. Quart. Rev. Biol., 13:41-50.

Braun, N., and E. Teichmann. 1912. Versuche zur Immunisierung gegen
Trypanosoma. Jena.

Brown, H. C., and J. C. Broom. 1938. Studies in trypanosomiasis. H. Ob-
servations on the red cell adhesion test. Trans. R. Soc. trop. Med. Hyg.,
32:209-22.

Brown, W. H. 1914. A note on the pathogenicity of T. Jew7s7. J. exp. Med.,
19: 406-10.

—— 1915. Concerning changes in the biological properties of Trypano-
soma lewisi produced by experimental means, with especial reference to
virulence. J. exp. Med., 21: 345-64.

Browning, C. H., D. F. Cappell, and R. Gulbransen. 1934. Experimental
infection with Trypanosoma congolense in mice: the effect of splenec-
tomy. J. Path. Bact., 39: 65-74.

Bruetsch, W. L. 1927. Ein Beitrag zur Wirkungsweise der Impfmalaria auf
den histopathologischen Prozess bei progressiver Paralyse. Z. ges. Neurol.
Psychiat., 110: 713-28.

—— 1932a. The histopathology of therapeutic (tertian) malaria. Amer.
J. Psychiat., 12: 19-65.

— 1932b. Activation of the mesenchyme with therapeutic malaria. J.
nerv. ment., Dis., 78: 209-19.

Brug, S. L. 1934. Observations on monkey malaria. Riv. di Malariol., 13: 3-
25.

Brumpt, E. 1920. Les Piroplasmes des bovidés et leurs hétes vecteurs. Bull.
Soc. Path. exot., 13: 416-60.

Brussin, A. M., and A. W. Kalajev. 1931. Die Bedeutung des Komplements
and der Blut-plattchen fiir die Feststellung der Thrombozotobarine. Z.
ImmunForsch., 70: 497-521.

Cannon, P. R., and W. H. Taliaferro. 1931. Acquired immunity in avian
malaria. III. Cellular reactions in infection and superinfection. J. prev.
Med. Lond., 5: 37-64.

Chopra, R. N., and S. N. Mukherjee. 1936. The trend of immunity studies
in malaria. Indian med. Gaz., 71: 34-39.

880 IMMUNOLOGY

Chorine, V. 1937. Les Réactions sérologiques dues aux euglobulines. Ann.
Inst. Pasteur, 58: 78-124.

Christophers, S. R. 1904. A preliminary report on a parasite found in persons
suffering from enlargement of the spleen in India. Sci. Mem. Offrs. Med.
san. Dept. Gov. India, N.S., 8: 1-17.

Coggeshall, L. T. 1937. Splenomegaly in experimental monkey malaria. Amer.
J. trop. Med., 17: 605-17.

—— 1938. The cure of Plasmodium knowlesi malaria in Rhesus monkeys
with sulphanilamide and their susceptibility to reinfection. Amer. J.
trop. Med., 18: 715-21.

Coggeshall, L. T., and M. D. Eaton. 1938. The complement fixation reaction
in monkey malaria. J. exp. Med., 67: 871-81.

—— 1938. The quantitative relationship between immune serum and infec-
tive dose of parasites as demonstrated by the protection test in monkey
malaria. J. exp. Med., 68: 29-38.

Coggeshall, L. T., and H. K. Kumm. 1938. Effect of repeated superinfection
upon the potency of immune serum of monkeys harboring chronic in-
fections of Plasmodium knowlesi. J. exp. Med., 68: 17-27.

Coventry, F. A. 1925. The reaction product which inhibits reproduction of
the trypanosomes in infections with Trypanosoma lew7si, with reference
to its change in titer throughout the course of infection. Amer. J. Hyg.,
5: 127-44.

—— 1929, Experimental infections with Trypanosoma lewisi in the guinea-
pig. Amer. J. Hyg., 9: 247-59.

Craig, C. F. 1937. Observations upon the practical value of the complement-
fixation test in the diagnosis of amebiasis. Amer. J. publ. Hlth., 27:
689-93.

Culbertson, J. T. 1938. Natural transmission of immunity against Trypano-
soma lewisi from mother rats to their offspring. J. Parasit., 24: 65-82.

Culbertson, J. T., and R. M. Wotton. 1939. Production of ablastin in rats
of different age groups after infection with Trypanosoma lewisi, Amer.
j. Hye730 ‘Geer @)ealoi=i3°

Dahmen, H. 1922. Die Serodiagnostik der Beschalseuche. Arch. wiss. prakt.
Tierheilk., 47: 319-53.

Davis, L. J. 1931. Experimental feline trypanosomiasis with especial reference
to the effect of splenectomy. Ann. trop. Med. Parasit., 25: 79-90.

Delanoé, P. 1912. L’Importance de la phagocytose dans l’immunité de la
souris a l’égard de quelques flagelles. Ann. Inst. Pasteur, 26: 172-203.

Dobell, C. 1912. Some recent work on mutation in microorganisms. J. Genet.,
2: 201-20.

Duca, C. J. 1939. Studies on age resistance against trypanosome infections:
II. The resistance of rats of different age groups to Trypanosoma lewisi,
and the blood response of rats infected with this parasite. Amer. J. Hyg.,
29: \(Sects.G), 25-32.

IMMUNOLOGY 881

Duke, H. L. 1936. Recent observations on the biology of the trypanosomes of
man in Africa, Trans. R. Soc. trop. Med. Hyg., 30: 275-96.

Duke, H. L., and J. M. Wallace. 1930. ‘Red cell adhesion” in trypanosomiasis
of man and animals. Parasitology, 22: 414-56.

Du Toit, P. J. 1919. Experimentelle Studien tber die Pferdepiroplasmose.
Arch. Schiffs- u. Tropenhyg., 23: 121-35.

Eaton, M. D. 1938. The agglutination of Plasmodium knowlesi by immune
serum. J. exp. Med., 67:857-70.

Ehrlich, P., and K. Shiga. 1904, Farbentherapeutische Versuche bei Trypano-
somenerkrankung. Berl. klin. Wschr., 41: 329-32, 362-65.

Findlay, G. M., and H. C. Brown, 1934. The relation of the electric charge
of the red cells to phagocytosis in avian malaria. Brit. J. exp. Path.,
15: 148-53.

Gay, F. P. 1931. Tissue resistance and immunity. J. Amer. med. Ass., 97:
195-99.

Gingrich, W. 1932. Immunity to superinfection and cross-immunity in
malarial infections of birds. J. prev. Med. Lond., 6: 197-246.

—— 1934. The effect of an increased burden of phagocytosis upon natural
and acquired immunity to bird malaria. J. Parasit., 20: 332-33.

Golgi, C. 1888. Il fagocitismo nell’infezione malarica. Rif. med., 4.

Greig, E. D. W., C. E. van Rooyen, and E. B. Hendry. 1934. Observations on
the melano-precipitation serological reaction in malaria. Trans. R. Soc.
trop. Med. Hyg., 28: 175-92.

Hartman, E. 1927. Certain interrelations between Plasmodium praecox and
its host. Amer. J. Hyg., 7: 407-32.

Hegner, R. W. 1926. The biology of host-parasite relationships among Pro-
tozoa living in man. Quart. Rev. Biol., 1: 393-418.

Hegner, R., and L. Eskridge. 1938. Mortality of merozoites in infections
with Plasmodium cathemerium in canaries. Amer. J. Hyg., 28: 299-316.

Hegner, R., and R. Hewitt. 1938. The influence of young red cells on in-
fections of Plasmodium cathemerium in birds. Amer. J. Hyg., 27:
417-36.

Henry, X. 1934. Seroflokkulation bei Malaria. Technik und anwendung in
der Praxis. Arch. Schiffs- u. Tropenhyg., 38: 93-100.

Herrick, C. A., and S. X. Cross. 1936. The development of natural and
artificial resistance of young rats to the pathogenic effects of the parasite
Trypanosoma lewisi, J. Parasit., 22: 126-29.

Hewitt, R. 1938. Multiple-infected red cells in avian malaria. Amer. J. Hyg.,
28: 321-44.

Hope-Gill, C. W. 1938. A study of the reaction rate of the serum-formalin
reaction in Trypanosoma gambiense sleeping sickness. Trans. R. Soc.
trop. Med. Hyg., 31: 507-16.

882 IMMUNOLOGY

Hu, C. H., and J. R. Cash. 1927. Considerations of the relationship of the
reticulo-endothelial system to kala-azar. Proc. Soc. Exp. Biol. N.Y.,
24: 469-72.

Huff, C. G., and W. Bloom. 1935. A malarial parasite infecting all blood
and blood-forming cells of birds. J. infect. Dis., 57: 315-36.

Jaffé, R. 1931. The recticulo-endothelial system in immunity. Physiol. Rev.,
11°27 7-32ik

—— 1938. The reticulo-endothelial system. J77 Downey: Handbook of Hema-
tology, 2: 973-1272.

James, W. M. 1913. Notes on the etiology of relapse in malarial infections.
Jainfect: Dist al29777-325-

Johnson, C. M., and R. A. Kelser. 1937. The incidence of Chagas’ disease
in Panama as determined by the complement-fixation test. Amer. J. trop.
Med., 17: 385-92.

Johnson, T. L. 1929. In vivo trypanolysis with especial reference to ‘‘zones of
inhibition,” relapse phenomena and immunological specificity. Amer. J.
Hyg., 9: 260-82.

Jungeblut, C. W. 1930. Die Palanan des retikulo-endothelialen Systems
fiir die Infektion und Immunitat. Eiebat Hyg. Bakt., 11: 1-67.

Jurgens, R. J. 1902. Beitrag zur Biologie der Rattentrypanosomen. Arch.
Hyg. Berl., 42: 265-88.

Kanthack, A. A., H. E. Durham, and W. F. H. Blandford. 1898. On nagana,
or tsetse-fly disease. Proc. roy. Soc., 64: 100-18.

Kauders, O. 1927. Immunititsstudien bei Impfmalaria. Zbl. Bakt., I, Orig.,
104: 158-60.

Knowles, R., and B. M. Das Gupta. 1928. Laboratory studies in surra.

Indian J. med. Res., 15: 997-1058.

1930. Studies in untreated malaria. Indian med. Gaz., 65: 301-10.

Koch, R. 1899. Uber die Entwickelung der Malariaparasiten. Z. Hyg. In-
fektKr., 32: 1-24.

Kolmer, J. A., and J. F. Schamberg, with the assistance of A. Rule and B.
Madden. 1933. The influence of reticuloendothelial “blockade” and
splenectomy upon experimental trypanosomiasis and syphilis and the
chemotherapeutic properties of arsphenamine and neoarsphenamine.
Amer. J. Syph., 17: 176-87.

Krijgsman, B. S. 1933. Biologische Untersuchungen ber das System: Wirt-
stier-Parasit. 1 & 2 Teil: die Entwicklung von Trypanosoma evansi in
Maus und Ratte. Z. Parasitenk. 5: 592-678.

Kroo, H. 1925. Beitrag zur Immunbiologie der Trypanosomen. Ueber Stam-
meinheit und Arteinheit des Trypanosomo brucei. Z. Hyg. InfektKr.,
105: 247-53.

—— 1926. Weiterer Beitrag zur Immunbiologie der Trypanosomen. Zur
Kritik des Kreuzinokulationsverfahrens als immunbiologische Methode
der Artabgrenzung. Z. Hyg. InfektKr., 106: 77-82.

IMMUNOLOGY 883

—— 1936. Die spontane, apathogene Tryanosomeninfektion der Kaninchen.
Z. ImmunForsch., 88: 117-28.

Kuhn, L. R. 1938. The effect of splenectomy and blockade on the protective
titer of antiserum against Trypanosoma equiperdum. J. infect. Dis., 63:
217-24.

Laveran, A. 1908. Sur quelques altérations de la rate chez les cobayes in-

fectés de trypanosomes. Bul. Soc. Path. exot., 1: 393-98.

1917. Leishmanioses. Paris.

Laveran, A., and F. Mesnil. 1901. Recherches morphologiques et expéri-
mentales sur le trypanosome des rats (Tr. Jewss7 Kent.). Ann. Inst.
Pasteur, 15: 673-715.

——— 1912. Trypanosomes et trypanosomiases. Paris.

Leger, A., and J. Ringenbach. 1911. Sur la spécificité de la propriété try-
panolytique des sérums des animaux trypanosomiés. C. R. Soc. Biol. Paris,
70: 343-45.

—— 1912. Sur la spécificité de la propriété trypanolytique des sérums des
animaux trypanosomiés. C. R. Soc. Biol. Paris, 72: 267-69.

Levaditi, C., and S. Mutermilch. 1909. Recherches sur la méthode de Bordet
et Gengou appliquée a l'étude des trypanosomiases. Z. ImmunForsch.,
Onige 225702222.

—— 1910. I. Mécanisme de la phagocytose. C. R. Soc. Biol. Paris, 68: 1079-
81.

—— 1911. Le Diagnostic de la maladie du sommeil par |’examen des pro-
priétés attachantes du sérum. C. R. Acad. Sci. Paris, 153: 166.

Linton, R. W. 1929. The reticulo-endothelial system in protozoan infections.
Arch. Pathol., 8: 488-501.

Lorando, N., and D. Sotiriades. 1937. Treatment of malaria with immune
Blood. Trans. R. Soc. trop. Med. Hyg., 31: 227-34.

Lourie, E. M. 1934. Studies on chemotherapy in bird malaria. Ann. trop. Med.
Parasit.,. 287) 151-69), 255-77 95 13-23.

Lourie, E. M., and R. J. O'Connor. 1937. A study of Trypanosoma rhodest-
ense relapse strains in vitro. Ann. trop. Med. Parasit., 31: 319-40.
Lowe, J. 1934. Studies in untreated malaria. Numerical studies of the para-

sites in relation to the fever. Rec. Malar. Surv. India, 4: 223-41.

McLay, K. 1922. Malaria in Macedonia, 1915-1919. Part III. Haematological
investigations on malaria in Macedonia. Jour. R. Army med. Cps., 38:
93-105.

MacNeal, W. J. 1904. The life history of Trypanosoma lewisi and Trypano-
soma brucei. J. infect. Dis., 1: 517-43.

Manteufel, P. 1909. Studien ttber die Trypanosomiasis der Ratten mit Be-
riicksichtigung der Ubertragung unternatiirlichen Verhiltnissen und der
Immunitat. Arb. GesundhAmt. Berl., 33: 46-83.

Manwell, R. D. 1938. Reciprocal immunity in the avian malarias. Amer. J.
Hyg., 27: 196-211.

884 IMMUNOLOGY

Manwell, R. D., and F. Goldstein. 1939. Strain immunity in avian malaria.
Amer); Hyg 0.sec. Cs 15-22%

Marchand, F. 1924. Die Ortlichen reacktiven Vorginge in Krehl und March-
and: Handb. d. Allgemein. Pathol., 4: 78-649.

Marrack, J. R. 1938. The chemistry of antigens and antibodies. Special Rep.
Ser. Med. Res. Counc., No. 230.

Massaglia, M. A. 1907. Des causes des crises trypanolytiques et des rechutes
qui les suivent. C. R. Acad. Sci, Paris, 145: 687-89.

Maximow, A. 1927a. Bindegewebe und blutbildende Gewebe. In Handb.
Mikrosk. Anatomie d. Menschen, 2:232-583. Berlin.

—— 1927b. Morphology of the mesenchymal reactions, Arch. Pathol. Lab.
Med., 4: 557-606.

Meleney, H. E. 1925. The histopathology of kala-azar in the hamster, monkey
and man. Amer. J. Path., 1: 147-67.

Meleney, H. E., and W. W. Frye. 1937. Practical value and significance of
the complement-fixation reaction in amebiasis. Amer. J. publ. Hlth., 27:
505-10.

Menendez, P. E. 1932. Serological relationships of Extamoeba histolytica.
Amer. J. Hyg., 15: 785-808.

Menon, T. B., D. R. Annamalai, and T. K. Krishnaswami. 1936. The value
of the aldehyde and stiburea tests in the diagnosis of kala-azar. J. trop.
Med. Hyg., 36: 92-95.

Mesnil, F., and E. Brimont. 1909. Sur les propriétés protectrices du sérum
des animaux trypanosomiés: Races résistantes 4 ces sérums. Ann. Inst.
Pasteur, 23: 129-54.

Metschnikoff, E. 1892. Lecons sur la pathologie comparée de l’inflammation.
Paris.

—— 1905. Immunity in infective diseases (Trans. of French book of 1901).
Cambridge.

Mohler, J., A. Eichhorn, and J. Buck. 1913. The diagnosis of dourine by
complement fixation. J. agric., Res., 1: 99-107.

Mulligan, H. W. 1935. Descriptions of two species of monkey Plasmodium
isolated from Szlenus irus. Arch. Protistenk., 84: 285-314.

Mulligan, H. W., and S. A. Sinton. 1933. Studies in immunity in malaria.
Rec. Malar. Surv. India, 3:529-68; 809-39.

Nauck, E. G., and B. Malamos. 1935. Uber Immunitat bei Affenmalaria. Z.
ImmunForsch, 84: 337-58.

Neporojny, S. D., and W. L. Yakimoff. 1904. Uber einige pathologischana-
tomische Verainderungen bei experimentellen Trypanosomosen. Zbl.
Bakt., Ref. 35: 467-68.

Neumann, H. 1933. Der Nachweis des parasitiziden Antikérpers bet der Ma-
laria des Menschen. Riv. Malariol., 12: 319-34.

IMMUNOLOGY 885

Nieschulz, O., and A. Bos. 1931. Uber den Einfluss der Milz auf den In-
fektionsverlauf von Surra bei Hunden. Dtsch, tierarztl. Wschr. 39:488-
89.

Nieschulz, O., and F. K. Wawo-Roentoe. 1930. Uber den Einfluss der Milzexs-
tirpation bei Infektionen mit Trypanosoma gambiense und Schizotry-
panum cruzt. Z. ImmunForsch., 65: 312-17.

Noguchi, H. 1926. Comparative studies of herpetomonads and leishmanias.
J. exp. Med., 44: 327-37.

Paulson, M., and J. Andrews. 1938. Complement fixation in amebiasis. A
comparative evaluation in clinical practice. Arch. intern. Med., 61:562-
78.

Perla, D., and J. Marmorston-Gottesman. 1930. Further studies on T. lew7si
infection in albino rats. J. exp. Med., 52: 601-16.

Proske, H. O., and R. B. Watson. 1939. The protein tyrosin reaction. Publ.
Hlth. Rep. Wash., 54: 158-72.

Rabinowitsch, L., and W. Kempner. 1899. Beitrag zur Kenntniss der Blut-
parasiten, speciell der Rattentrypanosomen. Z. Hyg. InfaktKr., 30: 251-
94,

Redmond, W. B. 1939. The cross-immune relationship of various strains
of Plasmodium cathemerium and P. relictum. J. infect. Dis. 64: 273-87.

Regendanz, P. 1932. Uber die Immunititsvorgange bei der Infektion der Rat-
ten mit Trypanosoma lewisi. Z. ImmunForsch., 76: 437-45.

Regendanz, P., and W. Kikuth. 1927. Uber die Bedeutung der Milz fiir die
Bildung des vermehrungshindernden Reaktionsproduktes (Taliaferro)
und dessen Wirkung auf den Infektionsverlauf der Ratten-Trypano-
somiasis (Tryp. lewrs7). Zbl. Bakt., Abt. 1. Orig. 103: 271-79.

Rieckenberg, P. 1917. Eine neue Immunititsreaktion bei experimenteller Try-
panosomen-Infektion: die Blutplattchenprobe. Z. ImmunForsch., 26:
53-64.

Ritz, H. 1914. Uber Rezidive bei experimenteller Trypanosomiasis. Dtsch.
med. Wschr., 40: 1355-58.

—— 1916, Uber Rezidive bei experimenteller Trypanosomiasis. Arch. Schiffs-
u. Tropenhyg., 20: 397-420.

Robertson, M. 1912. Notes on the polymorphism of Trypanosoma gambiense
in the blood and its relation to the exogenous cycle in Glossina palpalis.
Rept. Sleep. Sickn. Comm. roy. Soc., 13: 94-110.

— 1929. The action of acriflavine upon Bodo caudatus. Parasitology, 21:
375-416.

Robinson, E. M. 1926. Serological investigations into some diseases of do-
mesticated animals in South Africa caused by trypanosomes, Eleventh
and Twelfth Rept. vet. Res. S. Afr. (Sept.), pp. 9-25.

Rodet, A., and G. Vallet. 1906. Contribution 4 l'étude des trypanosomiasis.
Arch, Méd. exp., 18: 450-94.

886 IMMUNOLOGY

Ross, R. 1910. The prevention of malaria. New York.

Ross, R., and D. Thomson. 1910. A case of sleeping sickness studied by pre-
cise enumerative methods: regular periodical increase of the parasites
disclosed» Prog toy. Soc., BY 8237414-15:

—— 1911. A case of sleeping-sickness studied by precise enumerative meth-
ods. Further observations. Proc. roy. Soc., B, 83: 187-205.

Roudabush, R. L., and E. R. Becker. 1934. The development of Trypanosoma
iowensis in the blood of the striped ground squirrel, C/tellus tridecem-
lineatus. Iowa St. Coll. J. Sct., 8: 533-35.

Roudsky, D. 1911. Mécanisme de l'Immunité naturelle de la souris vis-a-vis
du Trypanosoma lewisi Kent. C. R. Soc. Biol. Paris, 70: 693-94.

Rouget, J. 1896. Contribution a l’étude du trypanosome des mammiferes.
Ann. Inst. Pasteur, 10: 716-28.

Rudolph, G. de M., and J. C. Ramsay. 1927. Enumeration of parasites in
therapeutic malaria. J. trop. Med. Hyg., 30: 1-8.

Russeff, C. 1935. Der Einfluss der Milzexstirpation auf den Verlauf der
Dourineinfektion bei verschiedenen Versuchstieren. Z. ImmunForsch.,
84: 295-99.

Sauerbeck, E. 1905. Beitrag zur pathologischen Histologie der experimentellen
Trypanosomen-Infection (mit Trypanosoma brucez). Z. Hyg. InfektKr.,
52; 3ie8G:

Schilling, C. 1902. Bericht uber die Surra-Krankheit der Pferde und Rinder
im Schutzgebiete Togo. Zbl. Bakt. I. Orig. 31: 452-59.

Schilling, C., and H. Neumann. 1932. Zur Methodik der immunologischen
Differenzierungsmethoden von Trypanosomenstémmen. Arch. Schiffs- u.
Tropenhyg., 36: 214-29.

Schwetz, J. 1934. L'Influence de la splénectomie sur |’evolution des trypano-
somes pathogénes (Tr. gambiense et Tr. congolense) chez les rats et
les souris. Bull. Soc. Path. exot., 27: 253-60.

Sergent, Ed. 1936. Immunité ou prémunition dans les maladies a hémocyto-
zoaires (paludismes, piroplasmoses). Arch. Inst. Pasteur Algé., 14: 413-
LG.

Sergent, Ed., and Et. Sergent. 1918. Sur le paludisme des oiseaux du au
Plasmodium relictum (vel Proteosoma). Ann. Inst. Pasteur, 32: 382-88.

Sinton, J. A., Harbhagwan, and J. Singh. 1931. The numerical prevalence of
parasites in relation to fever in chronic benign tertian malaria. Indian
J. med. Res., 18: 871-79.

Sinton, J. A., and H. W. Mulligan. 1933a. Mixed infections in the malaria
of the lower monkeys. I. Mixed infections as the cause of apparent varia-
tions in the morphology and pathogenicity of simian Plasmodia. Rec.
Malar. Surv. India, 3:719-67.

— 1933b. Mixed infections in the malaria of the lower monkeys. II. The
probable occurrence of mixed infections in some of the older records
of monkey malaria. Rec. Malar. Surv. India, 3: 769-808.

IMMUNOLOGY 887

Sotiriadés, D. 1917. Essais de sérothérapie dans la malaria. Grece méd., 19:
27-28.

Stauber, L. A. 1939. Factors influencing the asexual periodicity of avian
imalanias. §) Patasit., 25) 95-116:

Steffan, P. 1921. Beobachtungen tiber den Verlauf der kiinstlichen Infektion
der Ratte mit Trypanosoma lewisi. Arch. Schiffs- u. Tropenhyg., 25: 241-
47.

Stockman, S., and W. G. Wragg. 1914. Cross immunisation with Piroplasma
bigeminum and Piroplasma divergens. J. comp. Path., 27: 151-55.
Stratman-Thomas, W. K. 1935. Studies on benign tertian malaria. Amer. J.

Phyo 22 561-63.

Taliaferro, L. G. 1925. Infection and resistance in bird malaria, with special
reference to periodicity and rate of reproduction of the parasite. Amer.
ye lye. 5: 742-89.

—— 1928. Return to normal of the asexual cycle in bird malaria after re-
tardation by low temperatures in vitro, J. prev. Med. 2: 525-40.

Taliaferro, W. H. 1924. A reaction product in infections with Trypanosoma
lewisi which inhibits the reproduction of the trypanosomes. J. exp. Med.,
393, T7190:

—— 1929. The immunology of parasitic infections. New York.

— 1932. Trypanocidal and reproduction-inhibiting antibodies to Trypano-
soma lewisi in rats and rabbits. Amer. J. Hyg., 16: 32-84.

1934. Some cellular bases for immune reactions in parasitic infections.

J. Parasit. 20: 149-61.

—— 1938. The effects of splenectomy and blockade on the passive transfer
of antibodies against Trypanosoma lewisi. J. infect. Dis., 62: 98-111.

Taliaferro, W. H., and P. R. Cannon. 1936. The cellular reactions during
primary infections and superinfections of Plasmodium brasilianum in
Panamanian monkeys. J. infect. Dis., 59: 72-125.

Taliaferro, W. H., P. R. Cannon, and S. Goodloe. 1931. The resistance of
rats to infection with Trypanosoma lewisi as affected by splenectomy.
mimer. > Hye. 14: 1-37,

Taliaferro, W. H., and C. G. Huff. 1939. The genetics of the parasitic pro-
tozoa. (In press.)

Taliaferro, W. H., and T. L. Johnson. 1926. Zone phenomena in in vivo
trypanolysis and the therapeutic value of trypanolytic sera. J. prev. Med.
i 85-123.

Taliaferro, W. H., and H. W. Mulligan. 1937. The histopathology of ma-
laria with special reference to the function and origin of the macrophages
in defence. Indian med. Res. Mem., No. 29. Pp. 138.

Taliaferro, W. H., and Y. Pavlinova. 1936. The course of infection of Trypan-

osoma duttoni in normal and in splenectomized and blockaded mice. J.
Parasit., 22: 20-41.

888 IMMUNOLOGY

Taliaferro, W. H., and L. G. Taliaferro. 1922. The resistance of different
hosts to experimental trypanosome infections, with especial reference
to a new method of measuring this resistance. Amer. J. Hyg., 2: 264-319.

— 1929a. Acquired immunity in avian malaria. I. Immunity to superinfec-
tion. J. prev. Med. 3: 197-208.

—— 1929b. Acquired immunity in avian malaria. I]. The absence of protec-
tive antibodies in immunity to superinfection. J. prev. Med. 3: 209-23.

—— 1934a. Morphology, periodicity and course of infection of Plasmodium
brasilianum in Panamanian monkeys. Amer. J. Hyg., 20: 1-49.

— 1934b. Alteration in the time of sporulation of Plasmodium brasilianum
in monkeys by reversal of light and dark. Amer. J. Hyg., 20: 50-59.
—— 1934c. Superinfection and protective experiments with Plasmodium

brasilianum in monkeys. Amer. J. Hyg., 20: 60-72.

—— 1934d. Complement fixation, precipitin, adhesion, mercuric chloride
and Wassermann tests in equine trypanosomiasis of Panama (murrina).
J. Immunol., 26: 193-213.

Theiler, A. 1912. Weitere Unterschungen tiber die Anaplasmosis der Rinder
und deren Schutzimpfung. Z. InfektKr. Haustiere, 11: 193-207.

Thomson, J. G. 1912. Enumerative studies on T. bruce7 in rats and guinea-
pigs, and a comparison with T. rhodesiense and T. gambiense. Ann.
trop. Med. Parasit., 5: 531-36.

— 1933. Immunity in malaria. Trans. R. Soc. trop. Med. Hyg., 26: 483-
514.

Topley, W. W. C. 1935. An outline of immunity. Baltimore.

Trensz, F. 1936. La Valeur pratique de la mélanofloculation de Henry. Arch.
Inst. Pasteur Algér., 14: 353-90.

Van den Branden, F. 1935. Sur le rapport du poids de la rate ou du foie au
poids du corps, chez des rats blancs (variété albinos de Mus decumanus)
non infectés, ainsi que chez les animaux de méme espéce, préalablement
infectés de Trypanosoma congolense ou de Trypanosoma brucei, puis
guéris ou non guéris par traitement. C. R. Soc. Biol. Paris, 119: 529-30.

Vaucel, M., and Hoang-Tich-Try. 1936. Reactions de malaria-floculation au
Tonkin. Bull. Soc. méd.- chir. Indochine, 14: 1101-14.

Villain, G., and R. Dupoux. 1936. Contribution 4 l'étude serologique du
paludisme. Utilisation d’une mélanine artificielle: la M. A. floculation.
Arch. Inst. Pasteur Afr. N., 25: 469-551.

Wagener, E. H., and D. A. Koch. 1926. The biological relationships of
Leishmania and certain herpetomonads. Univ. Cal. Publ. Zool., 28:
365-88.

Wallace, J. M., and A. Wormall. 1931. Red-cell adhesion in trypanosomiasis
of man and other animals. I]. Some experiments on the mechanism of
the reaction. Parasitology, 23: 346-59.

Wasielewski, T. K. W. N. von. 1901. Uber die Verbreitung und kiinstliche
Ubertragung der Vogelmalaria. Arch. Hyg. Berl., 41: 68-84.

IMMUNOLOGY 889

Wasielewski, T. K. W. N. von, and G. Senn. 1900. Beitrage zur Kenntniss
der Flagellaten des Rattenblutes. Z. Hyg. InfektKr. 33: 44-72.

Watson, C. J. 1928. The pathology of histoplasmosis (Darling) with special
reference to the origin of the phagocytic cells. Folia haemat., 37: 70-93.

Watson, E. A. 1920. Dourine in Canada, 1904-1920: History, research and
suppression. Canada, Dept. Agric. Health of Animals Branch.

Wells, H. G. 1929. The chemical aspects of immunity. 2d ed., New York.

Wenyon, C. M. 1926. Protozoology. 2 vols., New York.

Yakimoff, W. L. 1908. Contribution aux altérations du sang des animaux
atteints de trypanosomiases expérimentales. Arch. Sci. biol. St. Pétersb.,
13: 243-76.

Zdrodowski, P. 1931. Sur la sérologie comparée du groupe de Leishmanies
d’origine humaine et canine. Bull. Soc. Path. exot., 24: 37-41.

CHAPTER XIX

RELATIONSHIPS BETWEEN CERTAIN PROTOZOA
AND OTHER ANIMALS

HAROLD Kirby, JR.*

IN THE LITERATURE in which consideration is given to close rela-
tionships between organisms of different species the effort is often made
to group the associations discussed under definite categories. The cate-
gories are defined, and it is shown in what manner and to what extent
each separate association can be referred to its proper position. A reader
of this literature soon becomes sensible of the lack of agreement in al-
most every major particular. Unlike names are given to the categories,
definitions are dissimilar, there is difference of opinion or lack of exact
information on the nature of the relationship itself, and the impossibility
of making unequivocal distinctions is apparent in many instances. In
order to make an advance toward harmony of opinion, the bionomics
of many groups of living things must be taken into consideration. The
author must here confine his discussion to a statement of what he re-
gards as a satisfactory denomination of the types of relationship be-
tween Protozoa and other organisms, including other Protozoa, with
which they are intimately associated.

At the outset, it is apparent that a comprehensive term is necessary
to designate all types of relationship between Protozoa and their hosts,
whether the Protozoa are epibiotic or endobiotic, whether they live at
the expense of their hosts or aid them in some way. Such a term would
be applicable also in the instances in which it is uncertain in what sub-
division an association belongs. The choice should be between two exist-
ing terms, parasitism and symbiosis, the latter of which has etymo-
logically exactly the meaning desired. Both words have been used in
the general sense. The word parasite, however, has by universal agree-
ment been used to designate an organism that lives at its host’s expense,

* Assistance rendered by personnel of Work Projects Administration, Official Project
number 65-1-08-113, Unit C1, is acknowledged.

PROTOZOA AND OTHER ANIMALS 891

obtaining nutriment from the living substance of the latter, depriving it
of useful substance, or exerting other harmful influence upon it. In the
interests of exactitude, a word should, if possible, express a single defi-
nite idea; and therefore it seems undesirable to use parasitism also in
a general sense if it can be avoided.

There is much justification for applying the term symbiosis to the
general relationship under consideration, although many authors have
given it a restricted applicability to mutually advantageous associations
only. The word in this restricted meaning has, in fact, acquired what-
ever sanction general usage confers. Most textbooks in biology and
zoology, as well as protozodlogy and parasitology, so define it, the
oldest one noted being T. J. Parker’s (1893); and Hegner (1926b)
restricted the meaning further, stating that in symbiosis life apart is
impossible. J. A. Thomson (1934; with Geddes, 1931; also in the
Encyclopaedia Britannica fourteenth ed.) contended that symbiosis
is a mutually beneficial internal relationship, and that externally mutual-
istic relationships are commensalism. To Haupt (1932) symbiosis
includes what others consider commensalism, but does not include
parasitism; the same sense is implicit in some dictionary definitions.
The extended meaning of the word has been found in only two general
biology texts (McFarland, 1913; Eikenberry and Waldron, 1930);
it has that meaning also in the article on symbiosis in the New Interna-
tional Encyclopaedia (1925). Most important, however, is the report of
Hertig, Taliaferro, and B. Schwartz (1937).

The sense in which the word was employed by its originator, A. de
Bary (1879), is of decisive importance. The three members of the
Committee on Terminology of the American Society of Parasitologists,
as well as W. Schwartz (1935), appear to have been the only ones
among recent authors to understand de Bary. It has been widely stated
that he meant symbiosis to designate mutually beneficial relationships
(by Caullery, 1922; Hegner, 1926b; as well as by the others cited in
the Committee’s report). The Committee gave quotations from de
Bary showing clearly that he used symbiosis as a collective term, the
subdivisions of which include parasitism and mutualism; he recog-
nized two main categories, antagonistic and mutualistic symbiosis. The
results of the writer's examination of de Bary’s paper are in complete
agreement with their interpretation; there is no ambiguity in de Bary’s

892 PROTOZOA AND OTHER ANIMALS

usage. Hertwig (1883) had a similar understanding of the meaning
of the word; parasitism and mutualism, he stated, are types of symbiosis.
As the Committee pointed out, he changed his usage later.

In recent literature other opinions that the words should be used in
this original sense have appeared. W. Schwartz (1935) took that atti-
tude, although he would restrict symbiosis to the relationship in which
there is physiological dependence of one partner on another. Cleve-
land (1926) remarked that it would be much better to use it in the gen-
eral sense, if the change could be made.

Van Beneden (1876) referred to certain associated animals as mutual-
ists, before the term symbiosis had been coined. The word conveys the
idea of reciprocal benefit, although the examples he described indicate
a vague concept on his part of the relationships concerned, and none of
them would now be regarded as mutualistic. (He discussed among
mutualists parasitic copepods, opalinids, endozoic rotifers, and even
Vaginicola on Gammarus). He recognized the three types of associa-
tion: commensalism, mutuality, and parasitism. If symbiosis is used
in the broad sense, reciprocal relationships should be termed mutualism
or mutualistic symbiosis.

Protozoa that live in natural cavities of the body, such as the mantle
cavities of molluscs and the lumen of the alimentary canal, but do not
nourish themselves at the expense of the host, have been termed in-
quilines (Caullery, 1922; Grassé, 1935). All inquilines are commensals,
but not all commensals are inquilines. There are also ectozoic com-
mensals, or ectocommensals; endocommensalism is equivalent to in-
quilinism. In a sense, inquilines, like ectozoic symbionts, have not in-
vaded the body itself. They occupy cavities open to the outside.

Endozoic Protozoa which invade the interior of the body proper,
living intracellularly, among tissue cells, or in blood or coelomic cavi-
ties, are all parasitic. The conditions of their nutrition necessarily involve
strict dependence on the host. Ectozoa and inquilines, which usually are
commensals, may become parasites when in their nutritive processes
they develop one or another means of using the substance of the host,
generally by attack upon, or extraction of substance directly from, the
epithelial cells. They are also called parasites when they consume enough
material that would otherwise be used by the host to make a difference
to it; or when in some way not connected with nutrition they injure the

PROTOZOA AND OTHER ANIMALS 893

host without invading its substance. There are few instances among
Protozoa in which injurious effects of this type have been proved.

There are only two ways in which a protozo6n can directly benefit
a larger animal. It may contribute its own body to be used in the nutri-
tion of the host; but such a relationship would not constitute mutualism
unless some essential substance not otherwise obtained is thus supplied.
Another way is action upon the food materials such as to make a sub-
stance usable that otherwise would not be. The latter is the situation in
the only proved instance of significant mutualism between Protozoa
and their hosts—that between flagellates and certain termites, as well
as Cryptocercus punctulatus. Another, indirect, benefit might be con-
ferred by aid in controlling an injurious organism or substance, but no
instance of that is known with certainty.

In all its ramifications, the problem of the symbiotic relationships
between Protozoa and other animals is far too large for concise treat-
ment. It has been considered in various textbooks of protozodlogy, par-
ticularly in the chapter on “Ecology, Commensalism, and Parasitism’”’
in Calkins (1933) and in Doflein-Reichenow ( (1927-29). Protozoan
relationships are discussed, together with associations in other groups
of animals, in Caullery (1922) and Grassé (1935). An important gen-
eral article on the subject is that by Wenrich (1935); and a general dis-
cussion was published by Fantham (1936). Various aspects have been
discussed by Hegner (1924, 1926a, 1926b, 1926c, 1928, 1937), by
Metcalf (1923, 1929), by Cleveland (1926, 1934), by contributors to
Hegner and Andrews (1930), by Becker (1932, 1933), and by Kirby
(i937)?

In undertaking to make a contribution to the subject, the author recog-
nized two possible paths of approach. Either he could attempt to make
a comprehensive scrutiny of the entire expanse of pertinent information,
or he could explore in as much detail as possible certain chosen fields
of inquiry. The former approach would lead to a generalized account,
with selected and perhaps original illustrations; and it would in large
part reiterate existing, readily accessible, sometimes commonplace con-
cepts. The latter course, although less exhaustive, permits selection for
more detailed consideration of certain representative topics; that is the
course which has in the main been followed here.

In the ecology of symbiotic relationships, an introductory chapter is

894 PROTOZOA AND OTHER ANIMALS

provided by accidental and facultative parasitism. Among Protozoa this
is best exemplified by certain holotrichous ciliates. As the subject de-
velops, instances in which certain genera contain both free-living and
symbiotic species come under consideration, together with examples of
closely related genera in the two types of habitat. The situation may be
illustrated by certain euglenid and some polymastigote flagellates, and
again by certain holotrichous ciliates. In certain groups of Protozoa, in-
structive series in degree of adaptation and types of relationship appear.
One of the most fruitful of these series, in which the range is from
incidental commensalism to strict parasitism, is found in the Thig-
motricha. Host relationships of a variety of types, which bring out also
the adaptation of life cycles to the conditions of symbiotic existence,
are found among certain holotrichs of the marine Crustacea, Conidio-
phrys pilisuctor, and the Apostomea. There is more or less morpho-
logical alteration and adaptation in the symbiotic holotrichs considered
above, particularly in the Thigmotricha; and this is also well brought
out in the development of attachment structures in Ptychostomidae,
Astomata, peritrichs, and certain flagellates of termites.

Animals of certain specific groups are characterized by protozoan
faunules of particular types. That does not express only the common-
place fact that examination of particular hosts will reveal particular
symbionts, but makes the point that related hosts often have faunules
of similar composition. In this matter of distribution of symbionts
among species of hosts is incorporated the problem of specificity in
symbiosis, the quality of a symbiont of being restricted to certain hosts.
This quality is known as host-specificity. The incorrectness of the term
“host-parasite specificity” has been commented on by Hertig, Talia-
ferro, and Schwartz (1937). Faunules of two groups of animals are
discussed here in their composition, host-specificity, and distributional
characteristics: the ciliates of sea urchins, in which there is in part rela-
tionship to free-living species; and the Protozoa of termites and Crypto-
cercus, in which the symbiotic relationship has reached a maximum de-
velopment.

Finally, physiological host relationships are discussed in detail in
two groups of Protozoa: Ophryoscolecidae in ruminants, and flagellates
in termites and Cryptocercus. The former relationship, though long sus-
pected of being mutualism, is probably simple commensalism; there is

PROTOZOA AND OTHER ANIMALS 895

general agreement on the mutualistic character of the latter. To com-
plete this series, relationships of strict parasitism should be discussed
from the standpoint of the physiology of the parasite and the effect on
the host. The absence of this is readily compensated for, however, in
the abundant literature of parasitology; and examples of parasitism are
described in almost all groups considered in this chapter.

As is apparent, the material is grouped under certain headings;
but in every section data bearing on various topics in symbiotic rela-
tionships will be found. To group all facts under specific topics would
involve much dislocation in other respects; and it has seemed prefer-
able to preserve systematic continuity to a considerable extent.

ACCIDENTAL AND FACULTATIVE PARASITISM

In connection with the origin of host relationships, it is of interest
to consider instances in which organisms can develop in both free-
living and symbiotic habitats. Accidental and facultative parasitism,
therefore, come up for primary consideration. Facultative parasites, as
opposed to obligate parasites, are able to live either associated with
hosts or not, but parasitism is a natural occurrence in the bionomics of
the species. Accidental parasitism is that of a naturally free-living
species, which happens through some accident to become parasitic.
Although the two types are not identical, they are obviously closely
related and categorical separations are not attempted in the discussion.

Mercier and Poisson (1923) pointed out that those forms that are
most ubiquitous and are preadapted to varied modes of nutrition have
the best chance of surviving in the new medium into which they are intro-
duced accidentally. For such forms Giard (1880) used the term “‘in-
choate parasitism”; Giard had reference to the incomplete and tempo-
rary parasitism illustrated by the occurrence of geophilids in the nasal
cavities of man.

These conditions are met by the ciliate Glaucoma pyriformis. Under
natural conditions G. pyriformis ingests bacteria. Hetherington (1933)
stated that it is one of the commonest fresh-water Protozoa, appearing
in the early stages of the usual infusions of hay, wheat, or lettuce if
they are inoculated with pond water. (At that time he named the ciliate
Col pidium cam pylum, but later [1936] reported that it is G. pyriformzs.)

896 PROTOZOA AND OTHER ANIMALS

In 1923 Lwoff reported that he had succeeded in growing “Col pidium
colpoda’’ in pure culture, with neither living nor dead microdrganisms,
in a medium of peptone broth. He later revised the identification to
Glaucoma piriformis, and in 1932 considered it to be exceptional and
unique in its utilization of dissolved nutrient material only. Since then
some other ciliates have been maintained in sterile, non-particulate cul-
ture; but the number is limited, and some have turned out to be actually
G. pyriformis (Hetherington, 1936).

It has been shown that G. pyriformis, when introduced into the
hemocoele of certain insects, multiplies rapidly and exhibits marked
pathogenic potentialities. Lwoff (1924) inoculated the ciliate from
pure culture into about thirty caterpillars of Galleria mellonella, all of
which succumbed to the infection in from eight to fifteen days. Shortly
before death, the blood contained no more leucocytes, but only great
numbers of ciliates invading all parts of the body. The ciliates nourished
themselves phagocytically at least in part, and contained many globules
of fat from the fat bodies of the caterpillar.

Janda and Jirovec (1937) injected bacteria-free cultures of G. pyri-
forms into the body cavities of various invertebrates and vertebrates, and
also brought them into contact with artificially produced wounds. At-
tempts to infect annelids, molluscs, crustacea, fish, and amphibia failed,
but many insects were successfully inoculated. The ciliates multiplied
so rapidly as almost completely to fill the hemolymph in a few days.
The fatty tissues especially were destroyed, the ciliates became larger
than normal, and the infected insects usually died in a few days. Infec-
tion through wounds was achieved only in the aquatic larvae of Aeschna
cyanea. Infection by mouth did not occur. Glaucoma that had been para-
sitic for some time when returned to the water survived and multi-
plied normally.

It appears, then, that insects’ blood is a favorable medium in which
G. pyriformis may grow, and that the tissues often provide no protec-
tion against the organism once it has entered. One would expect that
occasionally so common a ciliate might enter an aquatic insect through
an external wound or a damaged gut wall, and multiply in the same
way with disastrous consequences to the host. That has, indeed, been
found to take place.

It is possible, as Wenyon (1926) suggested, that Lambornella

PROTOZOA AND OTHER ANIMALS 897

stegomyiae (Fig. 194), found by Lamborn (1921) in mosquito larvae
(Stegomyia scutellaris) in an earthenware pot in the Malay States, may
actually be this species. All the infected larvae died in a few days; the
ciliates escaped while the host was still living or soon after death.
Keilin (1921), who described the species from formalin-preserved ma-

?
iD

: OD OTRAS

Figure 194. Posterior end of larva of Aédes (Stegomyia) scutellaris parasitized by
ciliates, Lambornella stegomyiae Keilin (=Glaucoma pyriformis ?). (After Keilin,
1921.)

terial, regarded the ciliate as a true parasite, and others have agreed
with him, apparently largely because of the epizoic character of the
supposed cysts which Keilin found studding the external surface of
one mosquito larva. There is no proof that these were cysts of the
ciliate—one may, in fact, be justified in thinking it improbable that
they were. If they were not, there seems to be no reason why Codreanu

898 PROTOZOA AND OTHER ANIMALS

(1930), Lwoff (1932), and others should except it from the list of acci-
dental parasites. The ciliates found by MacArthur (1922) in larvae of
Theobaldia annulata and studied also by Wenyon (1926) showed but
little difference in habit or appearance from Lambornella; and, con-
sidering the inadequacy of Keilin’s material, may well have been the
same. Wenyon concluded they were Glaucoma pyriformis.

Next came the report by Treillard and Lwoff (1924) of the finding
of ciliates corresponding to G. pyriformis in larvae of Chironomus
plumosus bought at a market and probably obtained in the vicinity of
Paris. Of 300 larvae, 13 were parasitized. The ciliates multipled ac-
tively, causing death of the host in about eight days. In the cytoplasm
were granules of yellow pigment, probably derived from hemoglobin.
In 5 of the hosts conjugation was in progress, with all ciliates in any
one larva at about the same stage.

From another chironomid, Culicoides peregrinus in India, Ghosh
(1925) reported Balantidium knowlesu. The ciliates were numerous in
the “coelomic cavity’’; there is no statement as to whether the host was
a larva or adult, or how many hosts there were. Though Grassé and
Boissezon (1929) proposed the new genus Leptoglena for this very
inadequately described ciliate, and it seemed to Lwoff (1932) and
Codreanu (1930) to be a Glaucoma, it is impossible to recognize it
from the description as any one of a considerable number of ciliates.
No doubt it belongs in the list of accidental or facultative parasites.

The same is true of Twrchiniella culicis, a new genus and species,
described, from sections only, by Grassé and Boissezon (1929). The
ciliates occurred in the hemocoele of an adult female Cu/ex. Boissezon
(1930) suggested that adults may die on the surface of the water and
the ciliates may escape and infect larvae; in the original paper it was
considered that the parasites lived in larvae, and occurrence in the adult
was an impasse. It must, in fact, be rare in adults if the ciliates are as
pathogenic as G. pyriformis in other hosts is known to be, for an in-
fected larva would then seldom transform into an adult. Codreanu
(1930) and Lwoff (1932) considered this ciliate to be Glaucoma.

Glaucoma or Glaucoma-like ciliates have been found also in other
endozoic habitats than the hemocoele of aquatic larvae of Nemocera.
G. parasiticum was observed by Penard (1922) in the gills of Gam-
marus pulex, not only on the surface but also in the interior, where it

PROTOZOA AND OTHER ANIMALS 899

consumes the soft parts of the parenchyma and blood cells. Penard con-
sidered that it may be a temporary parasite only, and is closely related
to G. “pyriforme.”

There is one record of a similar ciliate in the tissues of a vertebrate.
Epstein (1926) studied an infection of very young fish, Abrams brama
L., with Glaucoma, probably G. pyriformzs according to Lwoff (1932).
Two to three percent were naturally infected in an aquarium at a lake
near Moscow. The infection began with the yolk sac, which the ciliates
reached through the gut. They then entered the heart and spread
throughout the vascular system. In two or three days the hosts suc-
cumbed, with all except the resistant parts consumed. The ciliates oc-
curred in great abundance in the canal of the spinal cord.

Related ethologically to the invasion of the bodies of aquatic larvae
of Nemocera by G. pyriformis or related ciliates is the occurrence of
the common marine ciliate, Uronema marinum Duj., in the coelom
of a sipunculid. Madsen (1931) mentioned the observation by Mrs.
E. Wesenberg-Lund of masses of ciliates in several Halicryptus spinulo-
sus that had been kept in Copenhagen for several months without food.
After some days the sipunculids died, and the ciliates lived longer in
the cadavers. He regarded this invasion as following a bacterial infec-
tion, Uronema feeding on the bacteria, but did not suggest how the
ciliates may have entered Halicryptus.

Accidental parasitism similar to that of G. pyriformzs is the relation-
ship of Anophrys sarcophaga to crabs, noted by Cattaneo (1888) and
studied exhaustively by Poisson (1929, 1930). This marine ciliate
normally lives in decomposing animal matter. Under certain circum-
stances it invades the hemocoele of Carcinus maenas, but natural in-
fection is rare. Cattaneo found it in one of 300; Poisson in 7 of more
than 3,000 at the biological station of Roscoff. The ciliates multiply ac-
tively in the blood, consuming the amoebocytes, and when these are
exhausted feeding on plasma. When the host dies, the ciliates devour
bacteria and fragments of tissue, surviving for some hours until de-
composition is advanced, when they encyst or die.

Artificial transmission was easily accomplished. Of 25 Carcinus maenas
inoculated, 20 died within 7 days, usually with a massive infection. Five
crabs survived and soon lost the ciliates. Attempts were made to inocu-
late 7 other crabs of the genera Cancer, Portunus, Maia, and Eupagurus.

900 PROTOZOA AND OTHER ANIMALS

Of these only Portunus depurator developed a heavy infection and died.
Some were naturally immune, the serum agglutinating and destroying
the ciliates. In others the serum was not toxic 7m vitro, but the ciliates
were arrested in certain lymphatic spaces, killed, and phagocytized.

Accidental parasitism of a nymph of the hemipteran Nepa cinerea
was reported by Mercier and Poisson (1923). A species of Colpoda had
invaded the body, probably through a wound in the integument, and
produced a tumor the size of a pinhead on the lateroventral surface of
the metathorax. The tumor extended part inside and part outside of
the body, and in it ciliates were numerous. Large ones contained nu-
merous inclusions, especially phagocytized amoebocytes. There were
also very small ones with no inclusions; these were believed to be nour-
ishing themselves by absorption of dissolved substances. Though locally
destructive, the parasite did not prevent growth of the nymph up to the
imaginal molt, when it was killed by the observers.

Instances of accidental parasitism by Protozoa have been noted in
sea urchins. Lucas (1934), in examining Bermuda sea urchins, en-
countered transient Protozoa in the body fluids. She stated that these
“were normally free-living forms, which probably gained entrance
through the water-vascular system, and gave no evidence of coloniza-
tion.’ André (1910) reported Explotes charon in certain abundance
in the perivisceral fluid of the sea urchin Echinus esculentis, as well as
on the surface of the host. Accidental invasion of the body cavity of
these marine echinoderms is apparently not infrequent.

Warren (1932) studied at Pietermaritzburg, Natal, a ciliate which
possibly, according to his account, was a facultative parasite, in the
common garden slug Agriolimax agrestis. He considered it to belong
to a new genus and species, Paraglaucoma limacis. Kahl (1926) had
already established a genus Paraglaucoma for P. rostrata, found in moss
in Germany; later he found the species in moss from California and
Wisconsin. Apparently Warren knew nothing of Kahl’s work, but the
two ciliates appear similar. The length (60-80 y) as reported by Kahl
(1931) is greater than that usual in Warren’s form (40 1 in the free-
living form; mean lengths 41-63 in the parasitic form); but in 1926
Kahl had reported the length as 45-55 yp. Warren did not report the
posterior bristle which Kahl observed. The species also resembles
Glaucoma maupasi Kahl, 1926, the ciliate Maupas (1883) described
as G. pyriformis.

PROTOZOA AND OTHER ANIMALS 901

Warren found the ciliate swarming in certain fecal deposits, and
then determined that they live in the lumen of the liver tubules, some
at times passing into the stomach and being discharged in ‘fecal cham-
bers of mucus.” The incidence of infection varied from 50 to 87 per-
cent at different times of the year, and in one slug 18,000 ciliates were
present. What seemed to be the same ciliate was found in the “greenish
incrustation of earthy matter underneath bricks and flower pots.” This
ciliate seemed to have no injurious effect on the slugs, even when present
in large numbers.

Reynolds (1936) observed ciliates in freshly passed feces of the
same species of slug in Virginia. He determined these as Colpoda stezni,
but as he gave no illustration or description, and even made the state-
ment that the parasitic stage of this (holotrich) resembles (the hetero-
trich) Balantidium more closely than it does its own free-living stage,
we may not unreasonably consider the systematic status to be unsettled.
He determined in sections that the ciliates may be widely distributed in
the tissues of the body, and were most abundant in the respiratory
chamber and the anterior and posterior ends of the alimentary tract. In
one region more than 94 percent of the slugs were infected, in another
25 percent. Infection occurred by ingestion, presumably, of the free-
living ciliates in the soil, where C. ste7n7 was also found. Unlike Warren,
Reynolds considered that many slugs are killed by the ciliate, and even
suggested that the ciliates may be useful in combating molluscan pests.
Warren had also examined sections, but did not find invasion of the
tissues other than the liver tubules. It is likely that the extensive invasion
noted by Reynolds would be more harmful to the slugs.

Probably the ciliate described by van den Berghe (1934) as Glaw-
coma paedophthora n. sp. belongs in this group of facultative parasites.
At any rate, it seems to be a form that has been directly adapted from
a free-living habitat to parasitism in the egg masses of Planorbis and
Physopsis. At Elizabethville, Belgian Congo, van den Berghe found the
ciliates in certain eggs, generally two or three in an egg mass, number-
ing from four or five to a great many. They were not found in the
genital organs of the snails, and were abundant in the water of the
aquarium. Infection of all eggs in a dish took place quickly if ciliates
from an infected egg were introduced into the water. In the egg, mul-
tiplication from a few ciliates to an intense infection occurred within
twelve hours. The embryo was killed by the parasites and within twenty-

902 PROTOZOA AND OTHER ANIMALS

four hours had disappeared, the eggshell bursting and the ciliates escap-
ing. Though the author stated decisively that the ciliate belongs to the
genus Glaucoma, the description and figure do not prove that system-
atic position.

Along with the adaptation of free-living Protozoa to a symbiotic
environment, there should be considered a number of instances of a
secondary type of infection of associates in the same hosts. These have
been referred to as facultative parasites, from the standpoint of the
secondary hosts. Facultative parasitism of Heterocineta janickii on the
oligochaete Chaetogaster limnaez, which occurs with the hypocomid
ciliate in the mantle cavity of snails, is described below (p. 940). Theil-
er and Farber (1932, 1936) found Trichomonas muris present with
considerable frequency in oxyurid nematodes in white mice, and divi-
sion took place in the intestine of the worms. They even found tricho-
monads in nematodes when the flagellates could not be demonstrated
elsewhere in the mice. J. G. Thomson (1925) found Gvardia present
in abundance in all of hundreds of nematode worms, V zanella sp., from
a specimen of the South American rodent Viscacia viscacia. Although
he found no trophozoites or cysts of Gzardia elsewhere in the intestine
of the rodent, he observed the flagellate from the nematode to be mor-
phologically identical with G. viscaciae Lavier. Graham (1935) found
Giardia in nematodes, probably Cooperia oncophora, from a bull; but
was unable to find the flagellates in the intestine of the bull. A com-
parison with G. bovis Fantham would be of interest. As species of Gzar-
dia are otherwise exclusively parasites of vertebrates, it is likely that the
nematodes with Gvardia had, like those with Trichomonas, been sec-
ondarily infected with the mammalian flagellates. Flagellates can evi-
dently survive for long and even multiply in the worms, so that their
presence in them without simultaneous occurrence in the lumen of the
vertebrate intestine is not significant.

SYSTEMATICALLY RELATED FREE-LIVING AND
SYMBIOTIC PROTOZOA

MASTIGOPHORA

In addition to the existence of accidental and facultative parasitism, it
is significant in connection with the origin of symbiotic relationships

PROTOZOA AND OTHER ANIMALS 903

that certain genera contain both free-living and symbiotic species, or
that the two types of habitat are occupied by members of closely related
genera. The organisms have become closely adapted to their biotic en-
vironment, but have not undergone extensive modification. That does
not necessarily imply recent adaptation, since stability of characteristics
would equally well explain it; but it does indicate a direct origin from
free-living types.

There are some epibiotic euglenids, including species of Ascoglena
and Colacium, Euglena cyclopicola described by Gicklhorn (1925), and
Euglena parasitica described by Sokoloff (1933). The last species ad-
hered by the anterior end to all of numerous colonies of Vo/vox in a
tank in Mexico City, and was not found free in the water. In the
green color, stigma, and other structures, except for lack of a flagellum,
this is a typical Euglena. It is not certain, however, whether the relation-
ship is more than occasional phoresy. E. cyclo picola is normally epibiotic,
occurring on Cyclops strennuus and species of Daphnia. Epibiotic eug-
lenids have been observed on plankton Crustacea in reservoirs in the
vicinity of Berkeley, California.

The euglenids, Exglenamorpha hegneri, E. pellucida, and Hegneria
leptodactyli, are obligate inquilines of amphibia. They have never been
found free-living. Species of Evglena and Phacus, with normal green
color and activity, have, however, been found living in frog tadpoles
(Alexeieff, 1912; Hegner, 1923; Wenrich, 1924a). This is merely a
survival of free-living forms in the intestine, and Alexeieff may be un-
justified in terming it facultative parasitism. A colorless euglenid of the
genus Menoidium was found living in the intestine of one specimen of
Spirobolus marginatus by Wenrich (1935); and it occurred free-living
in damp Sphagnum in the aquarium jar. He reported no observations on
how long this flagellate might survive in the host. Evglena gracilis fed
to the millipeds could be in part recovered alive in one or two days.

Euglenamorpha hegneri Wenrich was observed by Hegner (1922)
and described by Wenrich (1923, 1924a) and Hegner (1923) from
tadpoles of frogs and toads and from H)/a in the North Atlantic states.
The typical form has green chloroplasts, a red stigma, and three flagella.
In 0.6 percent salt solution it survived for weeks in a hanging drop,
and multiplied at first, but continued cultivation was not achieved. A
colorless form, distinguished as the variety pellucida by Wenrich, 1s also

904 PROTOZOA AND OTHER ANIMALS

present in tadpoles. This differs from the type in several respects, the
most important being the lack of a stigma and the presence of from two
to six flagella. Most frequently there are from four to six flagella. Ac-
cording to Wenrich, six is the doubled number, three new ones growing
out very early in preparation for division. In other numbers above three,
there are various stages of outgrowth. Division of a flagellate with four
flagella results in daughter flagellates with two.

Brumpt and Lavier (1924) considered Wenrich’s colorless variety to
be a separate species, and Wenrich (1935) seemed inclined to the same
opinion. Brumpt and Lavier described a similar colorless form with no
stigma, from tadpoles of Leptodactylus ocellatus at Sao Paulo, Brazil, as
Hegneria leptodactyli. That flagellate has seven flagella ordinarily, but
may have only six. The authors did not mention the presence of an
accompanying green form with fewer flagella, and Wenrich (1935)
stated that he found the colorless flagellate in some hosts, unaccompanied
by the green one. The six-flagellated forms of Hegneria seem to resemble
very Closely the six-flagellated forms of Evglenamorpha hegneri vat. pel-
lucida, so that it may be necessary to revise the taxonomy of the flagel-
lates.

One 1s tempted to find, in this interesting series of forms, as Wenrich
has brought out, adaptation to the conditions of an endobiotic habitat in
loss of chloroplasts and increase of the number of flagella.

Endozoic colorless euglenid flagellates of the Astasia type have often
been found, especially in Turbellaria, but also in rotifers, Gastrotricha,
fresh-water nematodes, fresh-water oligochaetes, nudibranch eggs, and
copepods. They usually are in vigorous metabolic movement, and gen-
erally lack a flagellum when in the host.

Haswell (1892) found them abundant in parenchymal cells in all
specimens examined of a rhabdocoele turbellarian in Sydney. A flagellum
was present in many but not in most cases. No stigma is mentioned. In
1907 Haswell described a similar euglenid in many specimens of an-
other rhabdocoele, within cells of the digestive epithelium and in the
spaces between the gut and the body wall. In the host, it was motion-
less or executed slow movements, but was more active when freed. No
flagellum was present until two hours or more after the organisms were
freed from the host. They were kept alive outside of the host for several
days, but no euglenids were found normally free in the water.

PROTOZOA AND OTHER ANIMALS 905

Playfair (1921), who made his studies in the vicinity of Sydney also,
stated that on one occasion he found half a dozen specimens of Astasza
margaritifera Schmarda within the tissues of a turbellarian. This species
he also found in the water of ponds, not in a free-swimming form and
very often lacking a flagellum. This is the only species of Astasza that he
reported in the survey of Australian fresh-water flagellates, and his iden-
tification is not convincing proof that the form in Turbellaria is the
fresh-water species named.

Astasia captiva was described by Beauchamp (1911) from the rhabdo-
coele Catenula lemnae in France. In one pond almost all individuals were
infected, while in another a mile away the flagellates occurred in a small
percentage only. In some there were only one or two to a chain of zodids,
whereas in others the flagellates were very abundant. They were in con-
tinual movement in the “pseudocoele,”’ between the parietal cells. A
flagellum was present sometimes even on flagellates in the tissue, but
most of the organisms lacked that structure. A colorless rudiment of a
stigma, which was invisible in life, was seen frequently in stained prep-
arations. Beauchamp stated that no euglenid was seen in other species
of rhabdocoeles, including the common Stenostomum leucops. Howland
(1928) identified as Astasia captiva an actively metabolic euglenoid
flagellate, without flagellum or stigma, which she observed in Stentor
coeruleus and Spirostomum ambiguum.

S. R. Hall (1931) found euglenids rarely in the mesenchyme of an-
other species of Stenostomum and in S. predatorium in Virginia, where
Kepner and Carter (1931) doubted the existence of S. Jewcops. While
the flagellate was in the host, the flagellum did not extend beyond the
edge of the body; but when it was liberated into water the flagellum
soon grew out, metabolic movement ceased, and the organism swam
rapidly. The euglenids could be kept alive in spring water for three or
four days, but attempts to cultivate them failed. When infected hosts
were added to a culture of the rhabdocoeles, practically all became in-
fected within a week. In one instance, when an infected worm was de-
voured by another, several flagellates were observed to pass through the
wall of the enteron into the mesenchyme, where they multiplied. There
was no apparent effect on the host except in instances in which two or
three hundred were present; then the rhabdocoeles became sluggish and
bloated, ruptured with liberation of the flagellates, and died.

906 PROTOZOA AND OTHER ANIMALS

Because of the presence of a red stigma and bifurcation of the root of
the flagellum, Hall assigned this flagellate to the genus Euglena, al-
though it is colorless, naming it E. Jeucops.

Nieschulz (1922) examined large numbers of the fresh-water nema-
tode Trilobus gracilis, in the hope of finding Herpetomonas (— Lepto-
monas) bitschli. This was not found, but he reported Astas7a from
some specimens, usually only one or two in a host. There was no stigma
and no flagellum. He did not state in what part of the body the parasites
occurred.

In the rotifer Hydatina senta, Astasia has been reported on three occa-
sions. Leydig (1857) observed it in the alimentary tract of almost all of
the hundreds of rotifers that were examined. Metabolic movements were
very active, a red stigma was present, and no flagellum was mentioned.
Hudson and Gosse (1889) wrote: ‘‘H. senta, too, suffers from an in-
ternal parasite. It... swims up and down its host’s stomach by jerking
the contents of its body constantly backwards and forwards.’ Their fig-
ures show no flagella, and one, in color, shows a red stigma. Valkanov
(1928), without reference to other observers, named the organism he
found parasitic in the intestine of the same species of rotifer, A. /y-
datinae.

In the intestine of gastrotrichs, Astasia-like inquilines were reported
by Voigt (1904). He found them in some specimens of a gastrotrich
that he later (1909) named Chaetonotus ploenensis, and was unable,
despite careful search, to find free-living examples of Astasza in the
material. Remane (1936, p. 231) stated that he found the same species
in the intestine of another species of Chaetonotus.

Astasia doridis was found by Zerling (1933) to be rather abundant
in some eggs of the egg masses of the nudibranch Doris tuberculatus at
Wimeraux. When heavily parasitized, larvae were destroyed. The para-
sites lacked flagella and stigmas and showed intense euglenoid activity.
Freed from the eggs, they lived many days with no change in morphology
and behavior. The flagellate was not found in the genital tract of the
adult. Zerling believed it probable, nevertheless, that adult molluscs are
infected by the parasites liberated into sea water at the hatching of in-
fected larvae, and that they transmit the parasites to their egg masses.
This is the only published record of a euglenid parasite in a marine host.

Codreanu and Codreanu (1928) found a considerable percentage of

PROTOZOA AND OTHER ANIMALS 907

the fresh-water oligochaete Chaetogaster diastrophus Gruith in the vicin-
ity of Bucharest infected by a euglenid parasite that they named Astasia
chaetogastris. The flagellates multiplied rapidly in the coelom, and the
infection was always fatal in from eight to thirteen days. When freed
into water, metabolic movement lessened and a flagellum developed.
Both forms had a stigma, and the free form as well as the parasitic one
was capable of division. This euglenid is more pathogenic than any
other described. One is reminded of the invasion of the hemocoele of
insect larvae by Glaucoma pyriformis.

Foulke (1884) wrote concerning the fresh-water sabellid Manayunkia
speciosa: “Several individuals of Manayunkia were observed to be
preyed upon, while still living, by large monads, embedded in one or
more of the segments, which were sometimes excavated to a considerable
degree.” It is possible that in this statement there is reference to a situa-
tion analogous to that of Astasia chaetogastris.

Finally, in copepods, occurs Astasia mobilis, which was the first en-
dozoic euglenid to be observed (Rehberg, 1882). Alexeieff (1912)
studied it in Cyclops, finding it not only in the lumen of the intestine but
also twice in the eggs. It sometimes had a flagellum, and a stigma was
described. The metabolic activity and some features of the structure of
this organism have suggested to some sporozoan affinities. By Labbé
(1899), for example, it was included in the genus Monocystis. Alexeieft
discussed the possible euglenid origin of Sporozoa, and Stein (1848)
had long before remarked upon the apparent relationship between
euglenids and Monocystzs.

Jahn and McKibben (1937) studied a colorless, stigma-bearing
euglenid flagellate whose habitat is given as putrid leaf infusion. They
found the root of the flegellum to be bifurcated, as in Euglenidae;
whereas in Astasiidae, according to Hall and Jahn (1929), it is not
bifurcated. The new genus Khawkinea was established by Jahn and
McKibben for flagellates whose characteristics agree with those of
Euglena except that they are permanently colorless; and they assigned
to this genus not only their new species, K. /alli, but also the free-living
form that had been known as Astasia ocellata Khawkine, A. captiva
Beauchamp, A. mobilis Alexeieff, A. chaetogastris Codreanu and Codre-
anu, and E. /eucops Hall.

In the question of the relationship of free-living and endozoic Pro-

908 PROTOZOA AND OTHER ANIMALS

tozoa, the flagellate recently discovered in pond water by Bishop (1935,
1936) in England, and by Lavier (1936c) in France is of much inter-
est. Bishop found it on four different occasions in the course of thirteen
months, in a small pond with thick, black mud and much decaying or-
ganic matter; and Lavier found it in samples from two separate places.
Many of its characteristics are those of a trichomonad and, as Lavier
pointed out, it seems to be the only free-living member of the Tricho-
monadidae. There is a slender axostyle, which often is extended into a
pointed, posterior projection of the cytosome, or itself projects from the
body. Sometimes the flagellate anchors itself to an object by the end of
the axostyle. There are three anterior flagella and a trailing flagellum
that usually adheres to the body, forming an undulating membrane, but
that sometimes, according to Lavier, is free. The nucleus is trichomonad
in position, structure, and division, and there is a well-defined para-
desmose. Its manner of progression, which differs from that of other
free-living forms (Lavier), impressed Bishop with its similarity to the
movement of Trichomonas. Bishop (1935) wrote of it under the name
“Thichomonas” Keilini n.sp.

The flagellate differs from Trztrichomonas in the absence of a costa.
Lavier assigned it to the genus Eutrichomastix, which resembles T17-
chomonas in all respects except the lack of the costa and undulating
membrane. Although it has been shown that the trailing flagellum of
Eutrichomastix may adhere to the body under certain conditions, the
usual presence of an undulating membrane in the pond flagellate differ-
entiates it from that genus. Neither Bishop nor Lavier made any men-
tion of the parabasal body or of an attempt to demonstrate it. If this
structure, so characteristic of Monocercomonas (Eutrichomastix) and
Trichomonas, is present, it would leave no doubt of the trichomonad
affinities of the organism; if absent, the flagellate would not show so
close a relationship to endozoic forms. Bishop (1939) proposed the new
genus Pseudotrichomonas for the organism.

It is not possible to state that in this organism there is evidence of
the origin of trichomonads, which are widespread and evidently have
been adapted for a great period of time to endozoic existence. It may be
a survival of an ancestral type; on the other hand, there is the possibility,
which Bishop considered, that it might be a parasite of some cold-
blooded host that had survived and multiplied in the water. Rosenberg

PROTOZOA AND OTHER ANIMALS 909

(1936) found that Tr7trzchomonas augusta sometimes survived in salt
solution, on slides ringed with vaseline, for nearly a year. Cleveland
(1928b) was able to cultivate indefinitely T. fecal7s in water with feces
or tissue, in hay infusion, and in other ways, at temperatures from —3°
C. to 37° C.; and this, although it was supposed to have been derived
from a warm-blooded host, man. Cleveland also maintained T. augusta
in tap water with feces. He did not report on the ability of T. batra-
chorum, which Wenrich (1935) stated is morphologically indistinguish-
able from T. fecalis, to grow under the conditions supplied. It would
not be surprising if flagellates that have such marked ability to survive
and even to multiply outside of the host, under such simple conditions,
might find natural circumstances occasionally favorable to outside main-
tenance of life. They might, at times, be found by collectors. This does
not apply immediately to the studies of the species P. kezlini, however,
as no endozoic flagellate just like it is now known, and Bishop (1936)
found that it would not live in tadpoles; but it raises a general question.

Hollande (1939) described as a free-living trichomonad the new
genus and species Coelotrichomastix convexas. The flagellate was found
in liquid manure. It has four flagella, one of them trailing and said to
border an undulating membrane in a deep groove of the body; but
there is no costa. There is a unique axostyle, ribbon-like in its posterior
part and located superficially near the groove, anteriorly expanded to a
hemispherical cupule covering a considerable part of the large nucleus.
All parts of the axostyle are covered by small siderophile granules. A
very small bacilliform parabasal body was reported. In considering the
characteristics of Coelotrichomastix, Hollande failed to comment on the
striking similarity in many respects that exists between it and certain
flagellates that have been assigned to the genus Tetramitus. This cannot
fail to impress the reader of the accounts by Klebs (1893), Bunting
(1926), Bunting and Wenrich (1929), and Kirby (1932a). In those
papers, furthermore, especially the second and third, one will find
facts that suggest the possibility of a different interpretation of certain
unexpected characteristics described by Hollande. In assigning ‘Trzcho-
mastix’’ salina, originally described by Entz (1904), to Coelotricho-
mastix, Hollande made no comment on the writer’s account of what
seemed to be the same flagellate under the name Tetramitus salinus
(Entz).

910 PROTOZOA AND OTHER ANIMALS

Trepomonas agilis, the only species that has been described in that
genus, is a common flagellate associated with decaying organic matter in
fresh water, and has been found in a coprozoic habitat in human feces
(Wenyon and Broughton-Alcock, 1924). The writer on several occa-
sions found Trepomonas in salt-marsh pools, associated with marine
flagellates and ciliates. Whether it was T. agzl7s was not determined.
Flagellates of the genus have also become adapted to an endobiotic
habitat in fish, amphibia, and reptiles. Alexeieff (1910) observed Tre-
pomonas in Box salpa; and Lavier (1936b) found T. agzlzs once in that
fish, where, he stated, it is doubtless an accidental saprozoite. According
to Alexeieff (1909) and Lavier (1935), the endozoic Tre pomonas com-
mon in amphibia is probably T. ag7/7s. Lavier found the flagellate rather
constantly in tadpoles of Rana temporaria, R. esculenta, and Alytes ob-
stetricans, and in one adult Triton. He discussed it as an interesting pos-
sibility of parasitism in a flagellate normally living free, and possibly
finding the endozoic habitat more favorable than the free-living. Das
Gupta (1935) found a species of Tvepomonas, usually in small num-
bers, in the caeca of three different species of turtles: Terrapene major,
Kinosternon hippocrepis, and Chelydra serpentina. A cytological study
of the flagellate has recently been made by Bishop (1937).

The genus Hexamita includes both free-living and endozoic species.
The former are common in fresh water and infusions with decaying or-
ganic matter; they also occur in salt water; and a species resembling H.
inflatus, and only 11 y long, has been observed by the writer in a salt-
marsh pool with decaying algae and a salinity of fifty parts per thou-
sand. The type of habitat of the ‘‘trichomonad” named Pseudotricho-
monas keilini by Bishop (1939) and of Trepomonas and Tetranutus 1s
similar to that of Hexamita; Lavier reported them all from one sample
taken in France.

Urophagus and Octomastix are considered by most protozodlogists to
be synonymous with Hexamita. This was the opinion of Lavier (1936a),
who also rejected Octomitus; but he proposed two new genera, Sprro-
nucleus and Syndyomita (the latter of which is of the original Octomitus
type) for morphological types of Hexamzta-like flagellates in amphibia.
Lavier retained the name Hexamita for the common form in amphibia,
the type of which, among those he considers, is most like that of free-
living Hexamita. This, H. intestinalis, has undergone little modification.
The others differ from the free-living type, according to him; but there

PROTOZOA AND OTHER ANIMALS 911

seems to be a similarity in type of Spzronucleus to H. rostrata (?), as
figured by Wenrich (1935) from the outside of a dead fresh-water snail.
At least until a systematic review of the whole group of Hexamzta-like
forms is made, it appears to be necessary, for the sake of clarity, to use
only the one genus name.

The endozoic species of Hexamita are many and are found in a wide
variety of hosts. Though given species are restricted to single or related
hosts, the tendency to give different names to those in different hosts,
without adequate comparison with other described species, has been
manifest.

Certes (1882) found Hexamita frequently in the stomach of oysters
from certain localities on the coast of France. Though he considered
this to be H. snflata, he regarded it as a normal, reproducing inhabitant
of the stomach; and the identification is in no way positive. In other
invertebrates, Hexamita has been recorded from the reproductive organs
of the trematode Deropristis inflata in marine eels, but not in the in-
testine of the eel (Hunninen and Wichterman, 1936); from the cock-
roaches Blatta orientalis (Bishop, 1933), Periplaneta americana, and
Cryptocercus punctulatus (Cleveland, 1934); from the horse-leech
Haemo pis sanguisugae (Bishop, 1932, 1933); from the milliped Spzro-
bolus marginatus (Wenrich, 1935); from the larvae of T7pula (Mac-
kinnon, 1912; Geiman, 1932); and from Twbifex (Ryckeghem, 1928).

These all occur in the gut, except for the trematode form, as noted,
and the one in Twbifex. The latter, furthermore, is the only one in in-
vertebrates to which probable pathogenicity has been ascribed. Hexamzta
tubifici was encountered at intervals in the course of fifteen years in the
body cavity of Twbifex kept in culture in the laboratory in Louvain.
Worms that lost their power of activity, appeared whitish, and died
were found to have a more or less intense infection with Hexamzita.
Ryckeghem considered the question as to whether the flagellate is a
parasite or a free-living form invading decomposing tissue. He con-
cluded that the former relationship exists, for he found it in living
worms in apparently healthy tissue, decomposing chironomid larvae
were not invaded by the flagellate, and it was encountered in different
collections at long intervals. Each time it was a source of trouble.

Hexamita species occur in vertebrates of all classes. Fry and young
fingerlings of trout and salmon in hatcheries in the United States were
found by Moore (1922, 1923a, 1923b, 1924) and Davis (1923, 1926)

O12 PROTOZOA AND OTHER ANIMALS

to be extensively infected, especially in the anterior part of the intestine;
and they believed the flagellate to be severely pathogenic and to constitute
a serious menace to the success of trout culture. The flagellates have been
found also in European trout (Moroff, 1903; Schmidt, 1920) and in
the fan-tailed darter (Etheostoma flabellare) (Davis, 1926). Davis and
Moore did not prove that the flagellates were not secondary in diseased
fish, as Schmidt believed.

Lavier (1936b) examined 33 species of marine fish and found six
species of Hexamita, five of them new, in seven of these. He remarked
that the morphology of Hexamzta is much varied if one does not think
in general terms, and that an attentive study enables one to recognize
clear and constant morphological differences.

Hexamita is commonly found in the intestine of amphibia, and has
been reported from the intestine of turtles and tortoises, as well as from
the bladder of Emys orbicularis (Grassé, 1924) and from the stomach,
oesophagus, and small intestine of the snake Natr7x t2grina (Matubayasi,
1937). It occasionally invades the blood of amphibia (Lavier and Gal-
liard, 1925; and others) and tortoises (Plimmer, 1912) through a dam-
aged intestinal wall.

Among birds, Hexamita occurs in pigeons (Noller and Buttgereit,
1923), ducks (Anas boschas, Kotlan, 1923), turkeys (Hinshaw, Mc-
Neil, and Kofoid, 1938), and various wild birds in Brazil (Cunha and
Muniz, 1922, 1927).

Of mammals, rodents especially have been found infected with
Hexamita. In addition to rats, mice, ground-squirrels, and woodchucks
(Crouch, 1934), the South American hystrichoid rodent Myopotamus
coipus |—Myocastor coypus (Molina) | contains a species (Artigas and
Pacheco, 1932).

Hexamita has also been reported in primates, including man (Cunha
and Muniz, 1929; Wenrich, 1933; Chatterji, Das, and Mitra, 1928;
Perekropoff and Stepanoff, 1931, 1932). Dobell (1935), discussing all
these records except the third, believed that diplozoic forms of Entero-
monas, which “‘are very frequently found in feces, in intestinal contents,
and in cultures,” were misidentified. As regards Wenrich’s record, how-
ever, from Macacus rhesus, this is improbable when one considers his
extensive knowledge of the genus as well as the exactness of his de-
scription and figures.

PROTOZOA AND OTHER ANIMALS 913

From this survey of the distribution of members of the genus Hex-
amita, it is apparent that the flagellates are as widespread in animals as are
members of the strictly endozoic genus Trichomonas. In their case, how-
ever, flagellates equally or more closely related than most of the endozoic
forms to the ancestral type are common free-living forms. The endozoic
forins, nevertheless, are for the most part as strictly adapted to their
habitat as trichomonads.

There is no evidence, except possibly in certain species in Amphibia
and invertebrates, that the obligate symbionts have been recently adapted
from facultatively endozoic forms; any more than that Trichomonas can
be supposed to have recently so originated. There is little evidence of
parallelism in phylogenetic development in members of these two
genera and their hosts (Wenrich, 1935).

In most instances Hexamita has been regarded as a commensal in its
hosts. A possible exception in an invertebrate host is Hexamita tubifici.
In the body cavity of the aquatic annelid the flagellates may be fatal to
the host, in a manner comparable to the effect of Glaucoma in dipteran
larvae and Astasia in Chaetogaster. Hinshaw, McNeil, and Kofoid
(1938b), on the basis of experimental data which they obtained, sug-
gested a possible relationship between a condition of enteritis in young
turkeys and a heavy infection of Hexamita that occurred in the affected
part of the small intestine. They also reviewed reports of possible rela-
tionship in other vertebrates between pathological conditions and the
occurrence of Hexamita.

HOLOTRICHA

Among holotrichous ciliates, all types of biotic relationship exist, so
that the group is especially favorable for study of the development of
symbiosis and host-specificity. In this section will be considered holotrich
groups in which free-living and symbiotic species are closely related.

In some instances it seems that there has been no more than survival
of ordinarily free-living forms in or on a host, where certain conditions
of nutrition or protection favored the occurrence of the associate. Per-
haps the occurrence of Coleps hirtus on the rhabdocoele Vortex sexden-
tatus aS a common epizoon, as recorded by Graff (1882), is a relation-
ship of this type.

The relationship of Enchelys difflugiarum Penard to Difflugia acumt-

914 PROTOZOA AND OTHER ANIMALS

nata (Penard, 1922), and that of E. nebulosa Entz to Cothurnia is ap-
parently obligatory predatism; this relationship, of course, is comparable
to parasitism, as, if the host were a metazoan and were only partially
destroyed by the attacks of the ciliate, we would doubtless consider the
latter a true parasite.

Haematophagus megapterae Woodcock and Lodge and Metacystis
megapterae Kahl are commensals on the bristles of the whale Megaptera
nodosa (Kahl, 1930).

A number of pleurostomatous and hypostomatous gymnostomes have
become associated with animal hosts. In the former group there is
Amphileptus claparédei Stein, ‘parasitic on the stalks of colonial Vor-
ticellidae” (Kahl, 1933); and A. carchesii Stein in a similar situation.
Edmondson (1906) reported that after feeding upon a zodid the latter
species (discussed by him as A. me/eagris Ehr.) attaches itself to a stalk.
He found that many were present on Carchesium polypinum, clasping
the stalks by a deeply cleft posterior end. In addition to these more or
less predatory species, there is Lionotus branchiarum (Wenrich) Kahl,
described by Wenrich (1924b) as A. branchiarum. It is a true parasite
on the gills of the tadpoles of several species of Rana, where it lives in a
capsule under the cuticular membrane and occasionally detaches and en-
gulfs gill cells. Wenrich (1935) discussed the possibility that A.
branchiarum is transitional between a predatory and parasitic status.
There is a predaceous, free-swimming phase on the surface of the gills
by which other Protozoa may be devoured. Three species of Lionotus,
L. impatiens Penard, L. aselli Kahl, and L. hirundi Penard are com-
mensal among the gills of Ase//ws; and one, L. agilis Penard, occurs on
the ventral surface, among the legs, and on the egg masses of Cyclops.
The pleurostome genus Branchioecetes Kahl is very closely related to
Loxophyllum, in which Svec (1897) and Penard (1922) put the spe-
cies. The two species B. aselli (Svec) and B. gammari (Penard) are
commensals on their hosts, to which they adhere by thigmotactic cilia.

Commensal hypostomes belong to the genera Trochilia and especially
Chilodonella. Trochilia (Dysteropsis) minuta (Roux) has been found
free-living as well as commensal with Cyclops, Gammarus, and Asellus
(Penard, 1922). Commensalism is widespread in Chilodonella, several
species of which are apparently obligatory commensals on fish, others on
certain rotifers, amphipods, and isopods. The species on the gills of fish

PROTOZOA AND OTHER ANIMALS 915

have often been thought to exert direct or indirect pathogenic action,
but proof that they are more than ectocommensals is lacking. Kidder
and Summers (1935) distinguished several species on the carapaces of
three species of Orchestiidae from beaches in the region of Woods Hole;
they noted that no similar ciliates were found free in the sand or sea-
weeds, and the commensals lived only a short time when separated from
their hosts. C. capucinus Penard, 1922, and C. granulata Penard, 1922,
are commensal on Asellas and Gammarus, and C. porcellionis occurs in
the gill cavities of the terrestrial isopod Porcellio sp. (Dogiel and Furs-
senko, 1921). In aquatic hosts, transmission would take place through
the water; in C. porcellionis it must be through survival, at least for a
short period, in moist soil.

A number of hymenostomes of the new genus Allosphaerium were

also described by Kidder and Summers (1935) from the one species of
Talorchestia and two of Orchestia that they examined at Woods Hole,
Massachusetts. They remarked, concerning the ectocommensal holotrichs
of amphipods and isopods, that the external characteristics are singularly
well adapted to the environment.
They are all small flat forms and possess ventrally placed thigmotactic cilia
(Chilodonella, Trochilia, Allosphaerium). When one considers the forces,
mainly in the form of water currents, to which they must be subjected and
which would tend to effect their removal from the carapace of their various
hosts, it is seen that the flatness of their bodies and the adhesive powers of
their ventral cilia are of absolute necessity. Existing under the same conditions,
it is perhaps not surprising that representatives of two orders of ciliates ex-
hibit convergence to such a degree as to render them practically indistinguish-
able one from the other except under extreme magnifications.

Genera with free-living Trichostomata and Hymenostomata include
only a few commensal and parasitic species, but there are numerous
genera all members of which are associated with animal hosts. Frontonza
branchiostomae was found in abundance at Banyuls-sur-Mer by Cod-
reanu (1928) in the atria of most specimens of Branchiostoma lanceo-
latum exceeding 3 cm. in length. The genus Glaucoma has been dis-
cussed at length under facultative parasitism. Uronema rabaudi was be-
lieved by Cépéde (1910) to be a coelomic parasite of Acartia clausi and
Clausia elongata, in the empty carapaces of which it was observed. With-
out free-living congeners, but similar enough to Uronema to have been
put in that genus by Biitschli (1889) and Cuenot (1891), is PAilaster

916 PROTOZOA AND OTHER ANIMALS

digitiformis, which was described by Fabre-Domergue (1885) in mucus
on the body of Asterzas glacialis, multiplying abundantly on damaged
and disintegrating starfish, but disappearing with death of the host.

The genus O phryoglena comprises large holotrichous ciliates of which
some species are free-living and others endozoic. Kahl (1931) listed
eleven of the former and five of the latter. Within the genus there is a
range from free-living habits, often with predatism, through commensal-
ism to strict parasitism in close relationship to the developmental cycle
of the host.

The free-living species O. flava, according to Penard (1922), is vo-
racious and usually preys upon animals larger than itself, including
rotifers, small worms, and small Crustacea, especially Cyclops. It passes
under the carapace of Cyclops and consumes the living animal, the soft
parts of which are converted into food balls in the cytoplasm.

Ophryoglena maligna, described by Penard (1922), preys upon O.
flava as a parasite. It invades the cytoplasm, in which the number is one
to four or more, and devours the host little by little until it is empty.
The ciliates were also found free in the water, but Penard believed that
before long they would attach themselves to O. flava.

The three species that have been found in the intestine of Turbellaria
appear to be commensals. These are O. parasitica, reported by André
(1909) from 11 of 234 Dendrocoelum lacteum; O. pyriformis found
infrequently by Rossolimo (1926) in Sorocoelis maculosa and Planaria
nigrofasciata at Lake Baikal; and O. intestinalis from a large turbellarian
of the genus Dicotylus at Lake Baikal. It was shown that the last two
species cannot survive long in the water.

Truly parasitic, however, are the species reported by Lichtenstein
(1921) and Codreanu (1930, 1934) from May-fly naiads. The former
found the parasites in the schizocoele and gonads of Baetis sp. near
Montpellier; the latter in five Ephemerida from the Alps and the Car-
pathians. Codreanu believed that parasitism by these ciliates may occur
widely in Ephemeroptera. In young Réthrogena the ciliates occur as cysts,
division taking place within the cyst, but in Baetis they are not encysted
at any time. When the reproductive organs develop in the females, most
or all of the ciliates invade the ovaries, the contents of which they ulti-
mately destroy. The May flies nevertheless become adults and in the act
of what would normally be egg-laying, ciliates are deposited in the water

PROTOZOA AND OTHER ANIMALS 917,

instead of eggs. Only the female hosts are able to propagate the infec-
tion. Codreanu (1934) remarked that this is the only sufficiently de-
fined case of true parasitism of the schizocoele of insects by ciliates. The
species found in Baetzs by Lichtenstein was named by him O. collini; that
studied in Baetzs by Codreanu (1930) was, he stated, probably the
same.

Haas (1933) noted the similarity between the oral apparatus of the
swarmers of Ichthyophthirius multifiliis Fouq. and that of O phryoglena;
Kahl (1935), in consequence, placed that important parasite of fish in
the family Ophryoglenidae. Commensalism and parasitism being so well
developed in O phryoglena, although along with free-living habits, there
are clear ethological relationships between it and Ichthyophthirius.

Pleuronema anodontae, the only commensal species of that genus, was
reported by Kahl (1926) in small crushed mussels. He stated later
(1931) that it is infrequent in Anodonta, but occurs regularly in Sphae-
rium species. (Perhaps one should investigate the possibility that this
may be one of the Ancistrumidae, not Plezronema.) Very close to
Pleuronema is Pleurocoptes hydractiniae Wallengren, an ectocommensal
on the hydromedusan Hydractinia echinata.

DISTRIBUTIONAL Host RELATIONSHIPS AND HosT-SPECIFICITY
IN REPRESENTATIVE SYMBIOTIC FAUNULES

GENERAL CONSIDERATIONS

There are some generic groups of Protozoa that have a rather wide
distribution among animals; these groups are represented by species in
hosts widely separated systematically. That is true, for instance, of Hex-
amita and Trichomonas among polymastigotes, of Endolimax among
endamoebae, of Nosema and Eimeria among sporozoa, of Nyctotherus,
Balantidium, and urceolarids among ciliates. These examples have been
discussed by Wenrich (1935). The genus Trypanosoma is represented
in a very large number of vertebrates of all classes, but is limited to them,
as is also Grardia. (The occurrence of Giardia in nematodes is faculta-
tive; see p. 902.) A more or less closely restricted host distribution 1s,
however, characteristic of many generic, familial, and even higher groups
of Protozoa. Entodiniomorphina occur only in certain herbivorous mam-
mals, chiefly ruminants and Equidae; opalinids are most likely to have
anurous amphibian hosts, although a few have been found in Urodeles,

918 PROTOZOA AND OTHER ANIMALS

fish, and reptiles; hypocomids (except the small genus Hypocoma) are
parasites of certain groups of molluscs; Astomata are mainly inhabitants
of annelids, to which most genera are limited; hypermastigote flagellates
occur only in termites and roaches; and certain groups of polymastigotes
are restricted to certain groups of termites (p. 923).

The problem of host-specificity is ordinarily approached from the
standpoint of the individual species; that is, the degree in which it 1s
limited to a particular host species. In strict host-specificity, the host ts
rigorously determined; there is only one host for a species of symbiont.
As has been pointed out by Grassé (1935) and Wenrich (1935), strict
host-specificity is not a general phenomenon. Surveys of lists of species
and their hosts often bring out many instances in which there 1s only
one host for a species, for example, in the genera Giardia, Babesia,
Plasmodium, Haemo proteus, Leucocytozoén, and Eimeria. But such data
cannot be taken at face value, because the apparent strict host-specificity
may be based on insufficient search for the organism in other hosts, or
on a tendency of taxonomists to differentiate species on insufficient
grounds. More intense study in certain groups, as Trypanosoma and
Devescovininae in termites, has shown less rigorous limitation than at
first seemed to exist. More commonly, host-specificity is relative. The lim-
itation is to more or less related animals; and it depends, as Becker
(1933) and Wenrich (1935) have pointed out, on the characteristics
of the symbiotic environment, the opportunities for transmission, and
the evolutionary tendencies of the Protozoa. The phenomenon is of the
same nature as that of the geographical distribution of free-living or-
ganisms, though of course it is more complex.

It is a commonplace that given animals have characteristic protozoan
faunules; this phenomenon is of particular interest when there are
faunules of particular types peculiar to major groups. In instances of
the highest development of this tendency, it can be predicted what types
of Protozoa will be found in unexamined hosts. One may be reasonably
certain of finding Opalinidae in anuran species, Ophryoscolecidae in
ruminants, and certain types of polymastigotes and hypermastigotes in
all termites other than Termitidae .

Questions of distributional host relationships and host-specificity will
now be considered in greater detail in certain representative symbiotic
faunules. Two faunules have been selected for this purpose: ciliates of

PROTOZOA AND OTHER ANIMALS O19

sea urchins, and flagellates in termites and roaches. The former is inter-
esting also from the standpoint of the relationship of inquilines and
free-living forms; the sea-urchin intestine is one of the least specialized
environments of its type. The flagellates, however, occupy one of the
most specialized of symbiotic habitats. Not only are the circumstances
under which they live and are transmitted exceptional, but the hind-gut
of the host has actually undergone structural modification to accom-
modate them.

CILIATES OF SEA URCHINS

Faunules of ciliates occur in the greater number of sea urchins that
have been examined, but there are some without any. Uyemura (1934),
giving positive reports from eight species of sea urchins of Japan, found
none in Brissus agassizi, At Amoy, Nie (1934) found none in Tem-
nopleurus toreumaticus. Of the species at Yaku Island, Japan, Yagiu
(1935) found two uninfected: Colobocentrotus mertensiu and Cidaris
(Goniocidaris) biserialis. Powers (1935) found no faunule in Ezcidaris
tribuloides at Tortugas, nor were ciliates present in members of the
genus Arbacia at Beaufort, Woods Hole, and Naples (1933a), in spite
of association with infected species. Why a few species possess no
faunules, while so many have ciliates in abundance, is an interesting ques-
tion.

The intestinal faunules of sea urchins consist mostly of ciliates, which,
in whatever part of the world the host occurs, are members of a number
of characteristic genera. Most of them are holotrichs; outside of this
group are a few species of Metopus and one of Strombilidium, hetero-
trichs with many free-living congeners. There are sometimes as many
as twelve distinct species in eight genera in Strongylocentrotus pur-
puratus (Lynch, 1929); and Yagiu (1933, 1934) found twelve species
in Anthocidaris crassispina. On the other hand, from S. franciscanus in
Japan, Yagiu (1935) reported only Conchophthirus striatus. Four or
five species is perhaps the average infection. The occurrence of amoebae,
Chilomastix echinorum (Powers, 1935), nematodes, and rhabdocoeles
(Syndesmis) is much less prominent than that of ciliates.

In given hosts there is variability in the occurrence of different species
of ciliates. Some occur in abundance almost or quite universally; others
have a lower incidence, some being of rare occurrence. Plagiopyla minuta

920 PROTOZOA AND OTHER ANIMALS

Powers (1933a) occurred in only about 10 percent of Strongylocentrotus
drobachiensis, and then there were not more than twelve in a host;
whereas some ciliates have been found in all sea urchins of the species.
Most often the incidence is not 100 percent.

Powers (1933a) pointed out the existence of two groups of ciliates in
sea urchins. One group contains diverse species with many free-living
congeners, which he regarded as chance or vagrant forms that were en-
gulfed with food and survived; the other consists of obligatorily endozoic
species. The members of the first group are ‘apparently free-living and
only occasionally or accidentally associated with their host.’’ There is,
however, no evidence in the literature that many, if any, of the intestinal
ciliates in sea urchins are accidentally introduced free-living forms.
Though there are species belonging to genera of which most members
are free-living, that in itself is no indication that they are not obligatory
inquilines.

Colpidium echini Russo, found also by Powers (1933a) in all spect-
mens of Strongylocentrotus lividus examined at Naples, probably, ac-
cording to Kahl (1934), is not a Colpidium. Uronema socialis, described
by Powers (1933a) from S. drébachiensis, was later renamed by him
(1935) Cyclidium stercoris. Kahl (1934) doubted the generic assign-
ment of Colpoda fragilis, described by Powers (1933a) from Tox-
opneustes variegatus of Beaufort, North Carolina. These forms, which
Powers mentioned in the occasional associate group, together with
Plagiopyla, may be obligatory commensals. The Ezplotes sp. found by
Powers (1933a) in the gut and outside of S. drébachiensis is possibly
an accidental invader. He also reported Trichodina from the sea urchin
and in seaweed.

Cyclidium stercoris, which occurs in great abundance in S. drébachien-
575, will live and reproduce in sea water (Powers, 1933a); but it is not
known that it does so under natural conditions. ““Colpoda” fragilis, on
the other hand, is very sensitive to changes in its environment. Many of
the ciliates can survive for more or less prolonged periods outside of the
host. Entodiscus borealis, one of the strictly endozoic forms, was kept in
sea water from fifteen to twenty-three days (Powers, 1933b).

Species of Cyclidium occur also in sea urchins of China (Nie, 1934)
and Japan (Yagiu, 1933, 1934). Several species of Anophrys have been
reported from various echinoids. There is only one free-living species

PROTOZOA AND OTHER ANIMALS 921

of Anophrys, A. sarcophaga Cohn, which has been discussed above as a
facultative parasite of crabs. Kahl (1934) suggested a relationship of
certain of these ciliates to Philaster digitiformis, which occurs, as men-
tioned above, on the body of starfish. He was doubtful about the cor-
rectness of their position in the genus Anophyrs. There is confusion
about the taxonomy of many sea-urchin ciliates.

Genera that are restricted to sea urchins, and may be supposed to have
evolved in the shelter of these hosts, are Lechriopyla Lynch, Entorhipi-
dium Lynch, Entodiscus Madsen, Bigggaria Kahl, Madsenia Kahl. Cryp-
tochilidium Schouteden, in part included in Biggaria, has a species in
the annelid Phascolosoma vulgaris.

Lechriopyla mystax Lynch, commensal in the Pacific Coast sea urchins
Strongylocentrotus franciscanus and S. purpuratus, is closely related to
Plagiopyla. It is markedly thigmotactic: “Although almost continuously
in movement [it] adheres almost constantly to surfaces. The large
peristomal groove seems to act as a sucker” (Lynch, 1930). Lechriopyla
apparently has diverged from Plagopyla in relation to its obligatory
endocommensalism, but there are no profound alterations.

Four species of Extorhipidium were distinguished by Lynch (1929)
in Strongylocentrotus pur puratus in California. None of these flattened,
fan-shaped trichostomes was present in S. franciscanus from the same
localities, so there seemed to be marked host-specificity. Uyemura
(1934), however, found one of the same species in another sea urchin
of Japan, and described a new species, E. fwkuii, which occurs in five
hosts of four genera.

Related to Entorhipidium is Entodiscus, represented by E. borealis
(Hentschel) from several different hosts of the North Atlantic and
Japan; and E. sabulonis Powers found in all individuals examined of
two species of C/ypeaster at Tortugas. E. borealis is present in great
abundance and, with its greatly flattened form, probably in appearance
and occurrence suggests Opalina in Amphibia. According to Powers
(1933b), besides swimming about in the lumen of the intestine, it ad-
heres by the ventral side to the intestinal mucosa. The food vacuoles, he
stated, contain rods, probably bacteria, and objects resembling nuclei of
epithelial cells. At that time Powers thought that the ciliate might attack
the intestinal mucosa, secreting cytolytic enzymes, thus being definitely
parasitic; but later (1935) he did not stress this ill-founded conclusion.

922 PROTOZOA AND OTHER ANIMALS

Powers (1933a) discussed the possibility that Cryptochilidium echini
(Maupas), abundant and universal in Strongylocentrotus lividus at
Naples, is a true parasite. In several instances the body was found partly
embedded in the intestinal mucosa. As he probably recognized later,
this observation does not constitute adequate proof for his conclusion.
The genus Cryptochilidium, together with Biggaria, Kahl’s genus for
some of the forms described as Cryptochilidium, is well represented in
sea urchins of all regions.

Metopus histophagus Powers, as the species name indicates, contains
in its food vacuoles epithelial cells from the intestine of its host (Powers,
1935); but it was not observed to cause lesions, and probably simply
ingests cellular debris, as does M. circumlabens (Lucas, 1934). The
species occurs only in C/ypeaster subdepressus of Tortugas. The species
M. circumlabens Biggar occurs in a number of hosts at Bermuda, Tor-
tugas, Amoy, and Japan, but several other species seem to have a limited
host-specificity.

Questions of host-specificity and geographical distribution of the
ciliates have been discussed by Powers (1935, 1937). He remarked that
there 1s little evidence of rigid host-specificity. Species differ in that re-
spect. Yagiu (1935) found Cryptochilidium echini and Anophrys
elongata in all but one of the host species, examined by him at Yaku
Island, which contained any ciliates; and Powers (1935) found Crypto-
chilidium bermudense (—Biggaria bermudense) and Anophrys elongata
in all sea urchins at Tortugas that were infected with ciliates. Nor are
those ciliates limited to those regions; they have been found in various
localities. There are some ciliates that have been found in only one or
a few hosts, these being sometimes in one region only but also some-
times in widely separated localities. There is nothing, however, which
leads us to expect that, with the accumulation of more data, most or all
of them will not be known to be in various hosts in various parts of the
world. There is no limitation to certain genera or other taxonomic groups
of sea urchins, as would occur in evolutionary development of assocta-
tions with strict specificity. Though no experimental work has been
done, it seems likely that cross infection would ordinarily be easy to
accomplish; nevertheless it is noteworthy that given species have char-
acteristic faunules, and there are a few sea urchins with no faunules,
facts that call for experimental investigation of the host relationships.

PROTOZOA AND OTHER ANIMALS 923

Another problem that calls for further investigation is the type of
faunule in the same host species in different localities, data on which
are meager. Strongylocentrotus franciscanus in California harbors
Lechriopyla mystax, as well as ciliates of four other genera (Lynch, 1929,
1930); from S. franciscanus at Yaku Island, Japan, Yagiu (1935) re-
ported only Conchophthirus striatus; and S. franciscanus examined by
Powers (1936, 1937) at Acapulco, Mexico, was found to harbor “en-
tirely different ciliates” from those on the coast of California. As regards
similarity of faunules, there is the presence of Entodiscus borealis and
Madsenia indomita in Strongylocentrotus drobachiensis from both Swe-
den and the Bay of Fundy.

PROTOZOA OF TERMITES AND THE ROACH Cry ptocercus

Flagellates have undergone no more spectacular development than is
exemplified in the faunules now existing in certain termites and in
Cryptocercus. Elsewhere in that class of Protozoa, in fact, there is noth-
ing that is comparable to it. Many groups of the Polymastigida and all
but two species of the Hypermastigida have been found only in those
insects. There are also a few Protozoa of more ordinary types. Such are
among flagellates Trichomonas and related forms, Retortamonas, Mono-
cercomonas, Monocercomonoides, Hexamita, and Chilomastix; flagel-
lates of these types occur only rather sparingly in higher termites and,
except for Trichomonas, in most roaches. There are also Nyctotherus,
Balantidium, amoebae, gregarines, and coccidia. But, in insects ancestral
to modern termites and roaches, flagellates originating in the Monocer-
comonas, Monocercomonoides, and Trichomonas type have undergone
a remarkable evolution, giving us the main polymastigote components
of the faunules that today exist in lower termites and Cryptocercus. Hy-
permastigotes doubtless developed from polymastigotes, but their origin
has not been traced.

A table of the classification of termites, giving the approximate num-
ber of species and the number examined, is given by Kirby (1937).
About a quarter of the 1,600 termites are in the four lower families:
Mastotermitidae, Hodotermitidae, Kalotermitidae, and Rhinotermitidae;
three-quarters are in Termitidae. Flagellate infections in Termitidae are
sparse, and the species are small and of common types. In certain Ter-
mitidae, faunules have developed consisting mainly of amoebae, which

924 PROTOZOA AND OTHER ANIMALS

are almost completely lacking in lower termites. In lower termites there
have been recognized, in examinations of less than a third of the known
species, 30 genera with 133 species of polymastigotes, and 18 genera
with 63 species of hypermastigotes; and certainly thorough study will
reveal many more genera and species even in that third. In Cryptocercus
punctulatus alone, Cleveland et al. (1934) found 9 genera of hyper-
mastigotes with 20 species (only one genus and no species of which
occur in termites); and 5 polymastigotes in 3 genera, including Hexamita
and Monocercomonoides.

Every termite species in the lower families, so far as has been learned,
has a flagellate faunule; individual termites lack the Protozoa only in
certain phases of the life history, as when they are very young, imme-
diately preceding and following a molt, and in certain functional repro-
ductive stages. For the most part, any termite of a species, wherever
obtained, will be found to have the same group of flagellate species.
Sometimes one or more flagellates are absent, but uniformity in com-
position of the faunules is the rule. This fact is an aid in termite sys-
tematics. Identical faunules do occur in different termite species of cer-
tain groups; the fact that the faunules are identical does not necessarily
indicate that the hosts belong to the same species. There are often more
or less well-marked differences, and this is a strong indication for specific
differentiation of the hosts. The flagellates often provide a ready means
of distinguishing nymphs in regions where both the termites and their
faunules are known.

Individual faunules of flagellates in termites may comprise from two
to ten or, occasionally, more species. Often a genus is represented in a
host by more than one species. In Zootermopsis angusticollis and Z.
nevadensis there are three species of Trichonympha (Kirby, 1932b).
Nine of sixty-seven hosts of Devescovina contain two species. The genus
Foaina is represented by two species in thirty-four, and by three species
in three of eighty-three hosts. In Cryptocercus punctulatus Cleveland
et al. (1934) differentiated seven species of Trichonympha, four of Bar-
bulanympha, three of Leptospironympha, and three of Saccinobaculus.
Koidzumi (1921) distinguished six species of Dinenympha in Reticu-
litermes 5 peratus.

The degree of host-specificity varies in different genera and species.
Many species are known from one host only, but as more flagellate

PROTOZOA AND OTHER ANIMALS 925

faunules become known the tendency probably will be relatively to re-
duce this number. Many species are known from several or many hosts.
Trichonym pha agilis probably occurs in all species of Retzculitermes, but
has not been found in other termites. Stawrojoenina is widespread in
Kalotermes sensu lato, and there are few if any differences between
species of different hosts. Of twenty species of the genus Devescovina,
only nine have been found in but one host each. On the other hand,
there are species with many hosts widely separated geographically. D.
glabra has been identified in eighteen termites from Africa, Madagascar,
Java, and Sumatra; D. lemniscata has seventeen hosts in Central and
South America, the West Indies, Australia, the Pacific Islands, Africa,
Madagascar, Java, and India. A unique, elaborately organized deves-
covinid, when first found in a Ceylon termite, was thought to be a
strictly host-specific form; but it has since been found also in a
termite from Australia. The small, simply organized Tricercomitus,
which occurs in most if not all species of Kalotermes sensu lato, appar-
ently is one species, T. divergens, in all those in which it has been
studied. Another species exists in Zootermo psis.

Many species of termite flagellates in all groups have a present host
distribution which indicates greater stability in characteristics than ex-
isted in the same period of time in the insects. Speciation has occurred
in the hosts without having taken place in certain of the symbionts. That
there are other termite flagellates which have evolved into different spe-
cies in single hosts is probable; but we cannot designate any one as cer-
tainly rigidly host-specific. Although there are many one-host forms, the
situation is such that finding any one of them in a termite, even in
another part of the world, would not be astonishing. Even although there
is only a single extant host, it would in no instance be unlikely that
formerly existing species not directly ancestral served as hosts of the
flagellate.

But whether a flagellate species occurs in one or in several host species
of termites is far from being the question of greatest interest in host
distribution. It has, in fact, little significance for general considerations.
More important is the fact that there is limitation of certain flagellate
types to certain groups of termites. That is true mainly among poly-
mastigotes. There are also some very widely distributed flagellate types,
but that only adds to the significance of the instances of strict limitation.

926 PROTOZOA AND OTHER ANIMALS

Trichomonas, as is not unexpected, is one of the most widely dis-
tributed forms, occurring in termites of all families, including Ter-
mitidae. Trichonympha, although absent from Mastotermes and
Termitidae, not only has a wide distribution among other termites but
occurs also in Cryptocercus punctulatus. The genus has been found rep-
resented in forty-five termites of ten genera or subgenera in three fam-
ilies; and among those that have been studied for detailed characteristics
fourteen species have been distinguished. Various Holomastigotidae in
termites are related to hypermastigotes of this family in Cryptocercus,
although no genus is the same. Several genera are distributed widely in
termites, the situation being comparable to that of Trichonym pha.

In the distribution of the polymastigote family Pyrsonymphidae, there
is a high degree of correlation with the systematic relationships of the
hosts. Cleveland ef a/. (1934) extended this family (as Dinenymphidae )
to include other forms than Pyrsonympha and Dinenympha, on the
basis of the type of division figure and structural similarities. There are
three subfamilies: Saccinobaculinae, in which the flagella are free and
there is no attachment organelle; Oxymonadinae, in which the flagella
are free and there is an attachment organelle, the rostellum, developed
to a high degree; and the Pyrsonymphinae, in which there is a slightly
developed attachment organelle and the flagella are adherent to the sur-
face of the body for most of its length. Saccinobaculinae have been found
only in Cryptocercus punctulatus; Oxymonadinae are known only from
Kalotermes sensu lato, in which group they occur in most species;
Pyrsonymphinae seem to be restricted to the genus Reticulitermes. It
seems possible that evolutionary development of the groups has taken
place within the confines of the host groups concerned; although it is
unsafe to state that the distribution of the flagellates may not be wider
than we now know it to be.

The polymastigote subfamily Devescovininae, of which Monocer-
comonas (Eutrichomastix) appears to be an ancestral type, is represented
in all but five or six of ninety-seven species of Kalotermes sensu lato that
have been examined. There has been a most elaborate evolutionary de-
velopment in the group; but devescovinids also occur in Mastotermitidae
and Hodotermitidae. They appear to be absent, however, from Rhino-
termitidae and Termitidae.

The polymastigote family Calonymphidae is of particular interest to

PROTOZOA AND OTHER ANIMALS 927

the evolutionist, and it appears to have affinities in common with the
Devescovininae (Kirby, 1939). It is restricted, except for one enigmatic
form that may not belong in the group, to the genus Kalotermes sensu
lato.

Amoebae rarely occur in lower termites, but among the Termitidae
they are not infrequent. Small amoebae were present in almost all species
of Amitermes from the United States, Africa, and Madagascar that
were examined by the writer; and many larger amoebae, some with un-
usual nuclear characteristics, were found consistently in Central Ameri-
can and African species of Mzrotermes and in African termites of the
Cubitermes group (Kirby, 1927; Henderson, MS). It is likely that fur-
ther study of these amoebae will yield results significant for problems
of host-specificity.

Figure 195. One-day-old nymph of Kalotermes flavicollis, receiving proctodaeal food
from the female termite, showing the manner in which infection with flagellates takes
place. (After Goetsch, 1936.)

Transmission of the flagellates of termites takes place in the active
state (see Andrews, 1930). There is no evidence for true encystation,
though observations by Trager (1934) and Duboscq and Grassé (1934)
indicate a possibility of this in some small polymastigotes. Flagellates
of most species disappear prior to each molt except the last. Infection,
then, must take place not only at the beginning, but following each molt
in the growth period. Refaunation takes place when termites, either nat-
urally or experimentally defaunated, are left in contact with normally
faunated individuals. Experimentally, termites can be infected by placing
flagellate-containing material on the mouth parts. Under natural condi-
tions, except for cannibalism, flagellate-containing material can or-
dinarily be obtained only directly from the anal opening of another
termite, as the Protozoa do not survive long after deposition. Proctodaeal
feeding is a common habit among termites. Goetsch (1936) has de-

928 PROTOZOA AND OTHER ANIMALS

scribed the early infection of young nymphs of Kalotermes flavicollis by
direct application of the mouth parts, accompanied by sucking, to the
end of the abdomen of the dedlates (Fig. 195).

Certain small polymastigotes are often retained through the molting
period (Kirby, 1930; Child, 1934). In Zootermo psis this is true of the
minute forms Tricercomitus and Hexamastix. At the last molt the situa-
tion differs from that in the preceding molts. Child (MS) reported
that in the last molt of Zootermopsis, although the number of flagellates
is greatly reduced, all species are carried through from the seventh
instar nymph to the winged imago. Cross (MS) and May (MS) have
confirmed this fact in Kalotermes minor and Zootermopsis; the shed
intima of the nymph, still containing Protozoa, is retained within the
gut; and the Protozoa later escape into the lumen of the imago’s intestine.

In Cryptocercus the Protozoa are not lost at the time of molting; but
then, and only then, most of them form either well-defined cysts (in
Trichonympha and Macros pironympha) or resistant stages (Cleveland,
et al., 1934). Flagellates are present, often in great numbers, in pellets
passed in the first few days after ecdysis. Cleveland found that some
pellets, passed immediately after molting, consisted mostly of Protozoa
in encysted or resistant form. Reinfection of defaunated roaches took
place when they were placed with molting roaches; but not, except occa-
sionally with smaller polymastigotes, by association with other infected
roaches. Proctodaeal feeding, then, does not have the same role in trans-
mission in Cryptocercus as in termites. Cleveland could not find out the
exact manner in which infection is first acquired, but thought it probable
that it is by association with molting individuals. Once acquired, the
faunule persists until the death of the roach.

It is probable that cross infection has not been a significant factor in
determining the present distribution of flagellates in termites, below the
Termitidae at least. Furthermore, the unique characteristics of almost all
the flagellates, which have no close relatives except in roaches, indicate
that the faunules do not to any great extent include acquisitions from
other arthropods or other animals. The present distribution of the flagel-
lates, the absence of resistant stages, and the isolated habits of termites
support this opinion. If it is sustained by further studies the flagellates
of termites will be shown to be easily the leading group of animals for
correlative studies in phylogeny of symbionts and their hosts.

PROTOZOA AND OTHER ANIMALS 929

If it could be shown that there is resistance to cross infection, such
that flagellates introduced experimentally from a natural host species
into another one would not survive, this thesis would of course be sup-
ported. The writer (1937), however, stated that there seems to be no
resistance to cross infection, basing this opinion on experiments by Light
and Sanford (1927, 1928) and Cleveland et al. (1934). No expert-
ments yet reported, however, have been continued long enough to war-
rant any definite conclusion. Unpublished experiments by Dropkin,
furthermore, showed that Protozoa of Reticulitermes flavipes, Kalo-
termes schwartzi, and K. jouteli could not establish a physiological rela-
tionship with Zootermopsis sufficient to permit survival of the termite
for more than fifty days in the absence of the normal faunule.

Although there has been evolutionary development of the flagellates
within termites of groups that exist today, many of the types doubtless
go back to ancestral insects. The genus Trichonympha, being found in
both termites and Cryptocercus, may be supposed to have passed into both
these insects from ancestral protoblattids (Kirby, 1937). The distribu-
tion of Trichonympha in termites alone would indicate its antiquity and
stability (Kirby, 1932b). The existence of representatives of other hyper-
mastigote groups in Cryptocercus indicates the very ancient differentia-
tion of those flagellate types. By loss of members of the faunules here
and there, together with continued but less drastic evolutionary changes,
the present composition of the faunules may have originated. The flagel-
lates were probably present in ancestors of Termitidae, but were, in the
course of differentiation of those insects, dropped out. The origin of
the amoebae needs to be explained; possibly they were acquired later.

ADAPTIVE Host RELATIONSHIPS IN MORPHOLOGY
AND LIFE HISTORY

GENERAL CONSIDERATIONS

Structural modifications in animals that live in association with hosts
take two general forms. There are morphological changes in direct
adaptation to the requirements of the habitat; and there are changes un-
related directly to that habitat, but made possible by various factors in
it. In the former group, among Protozoa, is the development of or-
ganelles of fixation, though this development is not restricted to Protozoa
that live in close relationship with other animals. Special adaptations

930 PROTOZOA AND OTHER ANIMALS

may appear for nutrition. Probably also in that category is the increase
of the number of flagella and the development of undulating membranes
and axostyles in certain groups of flagellates. In the latter category are
the reduction or loss of cilia, the reduction or loss of mouth structure,
the elaborate development of the parabasal apparatus and other or-
ganelles in certain polymastigote flagellates, the complex characteristics

pai?

\ Aare

Figure 196. Streblomastix strix attached to the lining of the hind-gut of Zootermopsis
angusticollis. (After Kofoid and Swezy, 1919.)

of many hypermastigotes, the elaborate morphological specialization of
Ophryoscolecidae.

Organelles of fixation appear among flagellates in epibiotic dinoflagel-
lates (Chatton, 1920; Steuer, 1928); in Streblomastix strix, which often
is attached (Fig. 196) to the wall of the hind-gut of its termite host, by
a holdfast (Kofoid and Swezy, 1919; Kidder, 1929); in Pyrsonympha
and Dinenym pha, which occur free in the gut lumen of Retzcwlitermes or
attached by a small, simple, anterior knob (Koidzumi, 1921); and in
Oxymonadinae. In the last group the holdfast, which is applied to the
intima of the termite gut, is at the end of a rostellum, which may reach
a relatively great length and often contains many fibrils (Kirby, 1928;

PROTOZOA AND OTHER ANIMALS 931

(Ge immer eee 3
aN mea
“yy ia:

Sim eet.

See
IN.
own 1 Motes
em ii
aes
& Rte e £5
Kenn

AIS
APTN SSSR
Wes

NS
ON NS

Z, ZB Zz de mh
iy
aM eral piri

Figure 197. Fixation mechanisms in peritrichs. A, Ellobiophrya donacis, with ring
formed by two posterior limbs applied at the ends; B, Ellobiophrya suspended from the
bridges uniting the gill filaments of Donax vittatus; C, section of Trichodina pediculus
on the ectoderm of Hydra; D, Cyclochaeta (Urceolaria) korschelti from Chiton mar-
ginatus. (A, B, after Chatton and Lwoff, 1929; C, D, after Zick, 1928.)

Cleveland, 1935). Giardia adheres by a sucking disc to the wall of the
small intestine.

In some ectoparasitic dinoflagellates, the organelle of fixation 1s pro-
longed by rhizoids into the tissues of the host, and apparently nutriment
is absorbed by this mechanism. In the polymastigotes in termites, fixation
is only to the gut intima; there is no relationship to the epithelial cells.

932 PROTOZOA AND OTHER ANIMALS

In gregarines, the epimerite, which often is elaborately developed with
hooks or other appendages and inserted into the cell, may serve also
for absorption (see Watson, 1915).

As regards ciliates, development of fixation habits and structures in
the holotrichous groups of Thigmotricha, Ptychostomidae, and Astomata
is discussed below. There is among heterotrichs a well-developed fixa-
tion apparatus in Licnophora (Stevens, 1901; Balamuth, MS). Urceola-
tids have an elaborately organized scopula, a cup-like apparatus

Bo
5

eH
Abas: tt ee
iii

Figure 198. Fixation apparatus of Cyclochaeta (Urceolaria) korschelti, A, seen from
above, showing radially arranged ribs bent downward in hook-like points and ring com-
posed of overlapping, sickle-shaped individual pieces; B, cross section. (After Zick,
1928.)

supported by radially arranged ribs and a ring of denticles (Fig. 197D;
Fig. 198) (see for description and illustrations, Zick, 1928, on Cyclo-
chaeta (Urceolaria) korschelti; Fulton, 1923, on Trichodina pedt-
culus). As has been described in T. pediculus on Hydra and Tricho-
dinopsis paradoxa in Cyclostoma elegans, the epithelial cells of the host
may be elevated into this sucker (Fig. 197C). A unique attachment
mechanism is that of Ellobiophrya donacis, an inquiline of the gill
cavity of a lamellibranch. The posterior end of the body of this peritrich

PROTOZOA AND OTHER ANIMALS 2 Be,

is prolonged into two limbs hollowed into cups at the ends, the homo-
logues of halves of the scopula (Fig. 197A). The two cups are sealed
firmly together, so that the limbs form a closed ring by which the
ciliate is suspended (Fig. 197B) from the framework of the gills (Chat-
ton and Lwoff, 1923b, 1929).

In Protozoa that live in association with other animals as hosts, the
developmental cycle must be adjusted to the requirements of the habitat.
This is so arranged as to insure transmission of the organism from one
host to another; production of a sufficient number of infective forms so
that the likelihood of some reaching a place where development can
continue is not too small; protection of the organism, if necessary, in the
period when it is out of its host; and often correlation with the life cycle
and habits of the host, so that escape from one host and infection of an-
other can take place. The situation is most complex in heteroxenous
forms, in which the life cycle is shared between two different species of
animals, and is correlated with the bionomics of each of them. Cyclic
adaptation has perhaps achieved its most perfect development when
there is a regular and direct transmission to the next generation through
infection of the eggs or embryos. There are not many instances in Pro-
tozoa of this last method, which is so perfectly exemplified among the
cyclic endosymbionts of insects (Buchner, 1930; W. Schwartz, 1935).
Nosema bombycis invades the eggs of silkworms, but this may be in-
cidental and is not the only method of transmission. Other instances
occur among heteroxenous Sporozoa in the invertebrate host in Babesia
and Karyolys7s (see also Lavier, 1925).

The developmental cycle and methods of transmission have been con-
sidered widely in textbooks and in a general way by many authors, in-
cluding Hegner (1924) and Grassé (1935). The situation in certain
epibiotic Protozoa is also of considerable interest; and in that connection
accounts are given below of the holotrichs Conidiophrys pilisuctor and
apostomatous ciliates.

THIGMOTRICHA

Chatton and Lwoff (1922c) proposed the name Thigmotricha for a
group (suborder) of holotrichs including the families Ancistridae, Hy-
pocomidae, and Sphenophryidae. Most of these ciliates are inquilines,
commensals, or parasites on the gills or palps of molluscs, though some

954 PROTOZOA AND OTHER ANIMALS

occur on Protozoa or other invertebrates. They are provided with thigmo-
tactic cilia; and they show a series in the evolution of thigmotacticism,
in the course of which there is developed a penetrative and absorptive
organelle, and in the regression of body ciliature. Chatton and Lwoff
(1923a) described Thigmophyra, which was later (1926) placed in
a fourth family, Thigmophryidae. Thigmophrya, it was stated, closely
resembles Conchophthirus; but, unlike Conchophthirus, it possesses a
well-defined thigmotactic area identical with that of other Thigmotricha.
Kahl (1934) summarized the characteristics of the suborder Thigmo-
tricha and included in it the Conchophthiridae, which he had formerly
(1931) treated in the suborder Trichostomata, and which Chatton and
Lwoff (see 1937) evidently did not intend to include in their group.

Though Calkins (1933) considered adaptations to parasitism in the
thigmotrichs as a group, he included most of the genera in the Tricho-
stomata, including the Ancistrumidae which Kahl (1931) had put in
the Hymenostomata. Calkins, on the other hand, separated Hemzspeira
from other Ancistrumidae, putting it in the Hymenostomata. Whether
the Thigmotricha constitute a homogeneous group may be questionable
(Fauré-Fremiet, 1924, p. 7); but for consideration of the ethological
relationships and adaptations to symbiotic existence, the object of interest
in this account, it is convenient to treat them together.

Most Conchophthiridae occur in the mantle cavity of Pelecypoda,
both marine and fresh-water species. Andreula antedonis (André) Kahl
(Concho phthirus antedonis André) occurs abundantly in the alimen-
tary canal of a crinoid echinoderm; and Uyemura (1934) described as C.
striatus a ciliate in the intestine of several sea urchins of Japan. Myxo-
phyllum steenstrupii (Stein) lives in the slime covering the body of a
variety of land pulmonates. The species of Morgania Kahl and all ex-
cept the one species of Conchophthirus Stein (the original spelling by
Stein, 1861, not “Conchophthirius” as given by Strand, 1928) men-
tioned above are restricted to bivalves.

The most detailed studies of the genus Concho phthirus are contained
_ in several articles published in 1933-34 by Kidder and by Raabe. Uye-
mura (1935) found three species in great abundance in a fresh-water
mussel of Japan, Anodonta lauta. There is no doubt that in all parts of
the world certain lamellibranchs will be found abundantly infected with
these commensals. Only a beginning has been made in their study, as

PROTOZOA AND OTHER ANIMALS 935

in that of all the Thigmotricha, insofar as a knowledge of geographical
distribution and host-specificity is concerned.

In the mantle cavity of the hosts, some species are not localized,
whereas others are. Kidder (1934a) found C. curtus and C. magna on
all exposed surfaces and also swimming freely in the mantle fluids;
C. anodontae, on the other hand, he found to be invariably localized
on the nonciliated surface of the palps. The cilia of the flat left side
(left if, with Kahl and Raabe, we consider the flattening to be lateral;
according to De Morgan and Kidder, it is dorsoventral, and the attach-
ment is by cilia of the ventral surface) are thigmotactic. The thigmotactic
area usually covers the whole broad side, but in C. dsscophorus there 1s
more specialized adhesive apparatus, a circular, sharply outlined area,
which occupies only part of the left side, is markedly concave, and 1s
provided with differentiated cilia (Raabe, 1934b). C. discophorus swims
slowly, and often fastens itself firmly by the thigmotactic region. C.
mytili (Fig. 199A) also swims about or clings firmly to surfaces (Kid-
der, 1933a). C. anodontae on Elliptio complanatus seems to be most
markedly thigmotactic (Kidder, 1934a), remaining quiet, attached to
the surface of the palp.

Kidder (1933a) found the food vacuoles of C. mytili (Morganta
mytili, according to Kahl, 1934) to contain plankton organisms, includ-
ing algae, and sperm cells of the host. C. caryoclada (Morgania caryo-
clada, according to Kahl) contained mostly algae (Kidder, 1933d).
Other species contained algae, bacteria, and sloughed-off epithelial cells.
The relationship appears to be simple commensalism, but Kidder
(1934a), finding only well-preserved epithelial cells in the food vacuoles
of C. magna, was “‘a little in doubt as to its purely commensal rdle.”’

Kidder (1934a) remarked that there is a fair degree of host-
specificity. In nature certain species are characteristic of certain molluscs;
and the faunules may differ, even though in nature the hosts are very
closely associated. Rarely there are as many as three species in one host.
A number of species have been found in only one or a few related
hosts, but this may be a consequence of the relatively few examinations.
A cosmopolitan distribution is characteristic of such species as C. curtus,
reported from various fresh-water clams in Europe, the eastern United
States, and Japan. Morgania mytili is a commensal of Mytilus edulis in
various localities on both sides of the North Atlantic.

936 PROTOZOA AND OTHER ANIMALS

In the family Thigmophryidae, Thigmophrya bivalviorum, which
occurs on the gills of the marine pelecypods Mactra solida and Tapes
pullastra, has a thigmotactic region reduced to an elliptical area in the
anterior fifth of the body (Chatton and Lwoff, 1923a). The movements
of the cilia of this area are not synchronous with those of the rest of
the body. The ciliate swims in the mantle cavity or fixes itself to the gills.

The family Ancistrumidae is large and diverse. In general, the ciliates
are more sedentary than those previously considered in the order Thigmo-
tricha and the thigmotactic area is still more restricted. Although the
most frequent habitat is the mantle cavity of Pelecypoda, other molluscs
as well as members of other phyla of invertebrates serve as hosts for
species of the family. Probably, however, the original hosts were Pelecy-
poda.

The two principal genera are Ancistruma Strand, 1928 (given incor-
rectly as 1926 by Kahl and Kidder) (Fig. 199B, C) and Boverza
Stevens, 1901, but there are many others: Eupoterion MacLennan and
Connell, Ancistrina Cheissin, Ancistrella Cheissin, Plagiospira Issel
(Fig. 199D), Ancistrospira Chatton and Lwoff, Proboveria Chatton and
Lwoff, Tzarella Cheissin, Hemispeira Fabre-Domergue (Fig. 200C),
Hemis peiro psis Konig (Fig. 200A, B). Kahl (1934) put into the fam-
ily, though doubtfully, two ciliates parasitic in Littorina, Protophrya
ovicola Kofoid and Isselina intermedia Cépéde.

The Ancistrumidae possess more or less conspicuous peristomal cilia;
often these rows constitute a prominent fringe. In typical forms the
organisms adhere to the surfaces on which they live by the thigmotactic
cilia in a tuft at the anterior end. Ancistrella choanomphthali, however,
adheres to the gills by its entire concave, ventral surface (Cheissin, 1931).
In the Ancistrumidae an evolutionary series is apparent in the shifting
posteriorly of the mouth and of the peristome, which becomes spiraled.
Chatton and Lwoff (1936b) suggested that the Ancistrumidae constitute
so extraordinarily homogeneous a family that we may consider that there
is only one genus, subdivided into subgenera, an opinion that expresses
the homogeneity, though possibly the conclusion that there should be
only one genus is not sound taxonomically. These authors remarked that
the characteristics separating the genera or subgenera are purely quanti-
tative, consisting of more and more accentuated retrogradation of the
mouth and prostomal ciliary lines from the anterior half of the body
to the posterior end (Fig. 200K-N).

PROTOZOA AND OTHER ANIMALS D5

Other habitats than the mantle cavity of Pelecypoda have been adopted
by various species of Ancistruma and Boveria, as well as by members of
other genera. Thus Issel (1903) found A. cyclidioides on certain chitons
and gasteropods (Natica heraea) as well as on Pelecypoda; and he de-
sctibed A. barbatum solely from gasteropods of the genera Fusus and
Murex. Adaptation to these hosts is, as was stated above, probably second-
ary. In a similar manner, one species of Boveria, the type species B. sub-
cylindrica Stevens, is attached to the membrane of the respiratory tree of
the holothurian Stichopus californicus (Stevens, 1901). So similar to
this, however, that it has been classified as a variety of the same species,
B. s. var. concharum Issel, is a Boveria that occurs on the gills of ten of
fourteen Pelecypoda that harbor Ancistrumidae at Naples (Issel, 1903).
B. labialis lives in the respiratory trees of holothurians as well as on the
gills of a clam (Ikeda and Ozaki, 1918).

Expoterion pernix, which has many characteristics of a species of
Ancistruma, inhabits the intestine of the limpet Acmaea persona (Mac-
Lennan and Connell, 1931). The aberrant Hemispeira asteriasi Fabre-
Domergue (1888) and Hemispeiropsis antedonis (Cuenot, 1891) occur
on echinoderms, the former on the dermal branchiae of a starfish, and
the latter on the pinnules of a crinoid (Cuenot, 1894; Konig, 1894).
Protophrya ovicola Kofoid occurs upon the surface of the egg capsules
in the brood sac of the gasteropod Littorina rudis; and Isselina intermedia
is found in the mantle cavity of Littorina obstusata. The two latter
species, at least Protophrya, are more truly parasitic than other Ancis-
trumidae, and they have undergone some retrogressive changes.

For the most part, Ancistrumidae feed on bacteria, diatoms, and
other material extracted from the currents of water. Issel (1903) noted
that two bivalves constantly rich in the ciliates, Capsa fragilis and Tellina
exigua, live under conditions most suitable for offering their inquilines
copious food. They occur in calm, muddy water, rich in organic sub-
stances. The diet of plankton organisms may be supplemented by
sloughed-off epithelial cells, as noted by Stevens (1901) in Boveria
subcylindrica and Pickard (1927) in B. teredinidi. The account by Ikeda
and Ozaki (1918) of tissue invasion by B. Jabialis is not acceptable
without corroboration. The changes said to be undergone by the encysted
ciliate within the tissue are bizarre.

Protophrya ovicola in the brood sac of Littorina has a destructive
chemical action upon the eggs (Kofoid, 1903) and a teratogenic action

938 PROTOZOA AND OTHER ANIMALS

on the embryo (Cépéde, 1910). The parasites do not act directly on the
embryos, but on the medium, which exerts an injurious effect on embryos
in the early stages. Abnormal embryos result, in which the shell is more
or less unrolled; not only may the shell be misshapen, but the cells that
secrete the shell may fail to function normally.

The Hypocomidae (Fig. 199E-1) are true parasites, and occur mostly
in marine and fresh-water bivalves and snails. There is no mouth, but the
anterior end is provided with a short retractile tentacle. Normally the
ciliates are attached to the gills or skin of the mollusc, the tentacle being
embedded in an epithelial cell. The parasites obtain nutriment by ex-
tracting the contents of the cells to which they are attached, the tentacle
combining suctorial functions with those of attachment. The tentacle
continues in a tubular structure, extending more or less deeply into the
cytoplasm. In many Hypocomidae a fine inner canal has been observed
extending from the apex of the tentacle into the deeper cytoplasm. Ac-
cording to Chatton and Lwoff (1922c), this adherent organelle is de-
rived from structures of Ancistrumidae, where it is indicated in Ancis-
truma mytili and is well developed in A. cyclidioides.

In relation to the attached parasitic condition of hypocomid ciliates
are the regression of the mouth and peristomal ciliature and the reduc-
tion of the general ciliature. The former structures have for the most
part already disappeared. There is no mouth, but Chatton and Lwoff
(1924) stated that in some genera there are residual segments of the
adoral ciliary zone.

In reduction of the general ciliature there is in Hypocomidae a well-
integrated series. The body of Hypocomagalma dreissenae Jarocki and
Raabe, 1932, is covered with cilia except for a small ventroterminal area.
Ancistrocoma pelseneeri Chatton and Lwoff, as figured by Raabe
(1934a), has a larger cilia-free area, occupying a large part of one side
of the body, and an anterolateral peristomal fringe. Perhaps the less
firm fixation of these forms is also a phylogenetically primitive character.
Raabe stated that Ancistrocoma adheres rather weakly to the gills of its
host and separates readily. Hypocomagalma swims more rapidly than
some other ciliates of the group. Reduction of the ciliature continues
through Hypocomides Chatton and Lwoff and Hypocoma Gruber, in
which it occupies the inner area of the ventral surface of the body. In
Heterocineta (—Hypocomatomorpha) unionidarum Jatocki and Raabe

ie eee

ine ee

Sapte 285 Ua
AER;

.
¥
=

‘
Ue
3
s
,
i
ii
+3,

Ctr

<< wumuatlunnduuyy, ¢ m,.

ne!

Figure 199. Thigmotricha. A, Morgania (Conchophthirus) mytili from Mytilus edulis ;
B, C, Ancistruma mytili from Mytilus edulis, B, dorsal view, C, lateral view, tuft of
straight tactile cilia near anterior end; D, Plagiospira crinita from Loripes lacteus; E, F,
Hypocomina carinata from Mytilus edulis; G-1, Hypocoma parasitica: G, lateral view,
tentacle and tubular structure below, H, ventral view, I, two hypocomids attached to
Zoothamnium. (A, after Kidder, 1933a; B, C, after Kidder, 1933c D, after Issel, 1903;
E, F, after Raabe, 1934a; G-I, after Plate, 1888.)

940 PROTOZOA AND OTHER ANIMALS

and Hypocomatidium 5 phaerii Jarocki and Raabe, from the gills of fresh-
water mussels, reduction is far advanced to a thigmotactic zone restricted
almost entirely to the anterior half of the ventral side. Cilia are not lost
altogether in known Hypocomidae.

Syringo pharynx pterotracheae, which lives on the gills of the heteropod
Pterotrachea coronata either swimming free or fixed to epithelial cells
by the rostrum (Collin, 1914), was included by Kahl (1934) among
the parasitic gymnostomes. Probably, however, this is a hypocomid
ciliate, one with a general body ciliature like Anczstrocoma and Hypo-
comagalma. Parachaenia myae, described from Mya arenaria by Kofoid
and Bush (1936), may also be a hypocomid, although attachment to the
cells of the gills of its host was not described. Chatton and Lwoff’s
statement (1926, p. 351) about the prolongation of the anterior indi-
vidual in a spur covering the dorsal anterior region of the posterior
individual in binary fission of Ancistrocoma pelseneeri is in exact agree-
ment with the division process of Parachaenia myae. Another point of
agreement with Ancrstrocoma is the type of conjugation. In the shape of
the ciliates, the unique attachment of the conjugants by the posterior
ends, and the shape and arrangement of the nuclei of P. myae, there 1s
almost complete agreement with A. pelseneeri as figured by Raabe
(1934a).

Among the many genera of Hypocomidae, not all of which can even
be named here, the number of which we may expect will be markedly
reduced with further study, only the species of Hypocoma (Fig. 199G-1)
do not occur on bivalves or snails. They are parasites of Protozoa, and
are discussed elsewhere in this book (p. 1083).

Hypocomidae are host-specific in marked degree. They are obligatory
parasites on certain individual or closely related molluscs, and do not
readily infect other molluscs. Jarocki (1935) found that Heterocineta
janicki, placed free-swimming into an aquarium with various molluscs,
would attach only for periods of from fifteen to eighteen hours in the
absence of its natural host Physa fontimalis. When various molluscs, 1n-
cluding Bithynia tentaculata, were put together in an aquarium, Hetero-
cineta krzysiki, though abundant on the body of Brthynia, did not infect
any other species. Sometimes two hypocomids are present on the same
host, and Raabe (1934a) noted that there seems to be a tendency to
inhibition by one parasite of the development of the other.

Figure 200. Thigmotricha. A, B, Hemispeiropsis comatulae from Antedon (Comatula)
mediterranea; C, Hemispeira asteriasi from Asterias glacialis ; D-H, Sphenophrya dosiniae
from Dosinia exoleta: D, adult, E, budding individual, F, young individual, G, longi-
tudinal section of Sphenophrya on branchial filament, H, transverse section showing ciliate
in furrow between branchial filaments; I, J, Gargarius gargarius (Rhynchophrya cristal-
lina) from Mytilus edulis ; K-N, series of diagrams showing retrogradation of peristome
in Ancistrumidae: K, Ancistruma, L, Proboveria, M, Boveria, N, Hemispeira. (A, B,
after Konig, 1894; C, after Wallengren, 1895; D-H, after Chatton and Lwoff, 1921;
I, J, after Raabe, 1935; K-N, after Chatton, 1936.)

942 PROTOZOA AND OTHER ANIMALS

In connection with host-specificity among hypocomids, Jarocki’s ob-
servation (1934, 1935) that Heferocineta janicki is also a facultative
parasite on the oligochaete Chaetogaster limnaei is of considerable in-
terest. The oligochaete is usually present as an inquiline in the mantle
cavity of Physa fontinalis and other snails. The hypocomids are almost
always present on the snails; and they also infect almost all the oligo-
chaetes, attaching to various parts of the body and inserting the suctorial
tentacles into the hypodermal cells. Parasite-free worms quickly became
infected if brought into contact with ciliates either in or out of the
mantle cavity. Parasite-free Physa became infected if parasitized olli-
gochaetes were introduced into the mantle cavity. The worms pass
freely from one host to another, and thus facilitate the spread of the
infection. Chaetogaster limnae/ in other snails became facultatively para-
sitized by their specific hypocomids; but Heferocineta species could not
be introduced into unnatural hosts on the oligochaetes. To two other
species of Chaetogaster, Heterocineta janickii became attached tempo-
rarily, but soon dropped off.

The Sphenophryidae (Fig. 200D-H) ) all occur on the gills of marine
lamellibranchs. They are sedentary, immobile, and nonciliated in the
adult phase. They are not true parasites; the relationship as defined by
Chatton and Lwoff (1921) is “inquilinism complicated by phoresy.”
The ciliates are rather large, mostly flattened laterally, and adhere to the
surface by a long ventral edge (Fig. 200G). Sometimes they adhere
in a very precise and constant position, as Sphenophrya dosiniae in the
furrows separating adjacent branchial filaments of Dosinia exoleta (Fig.
200H). There is no mouth opening, and the ciliates apparently feed
osmotically; but Mjassnikowa (1930a) found evidence that S. sphaeri
ingests cells of the gill epithelium. She may, however, have misinter-
preted the nature of certain cytoplasmic spherules. Reproduction is by
development of motile buds. Cilia develop from the infraciliature, which
consists of a few rows of granules that are present in the vegetative indi-
vidual.

An unusual sphenophryid is Gargarius gargarius, described by Chat-
ton and Lwoff (1934a) from Mytilus edulis at Roscoft. Rhynchophrya
cristallina from M. edulis in the Baltic Sea, of which a more complete
account was later given by Raabe (1935), is evidently the same ciliate
(Fig. 2001, J). Along one surface are two longitudinal, parallel, comb-

PROTOZOA AND OTHER ANIMALS 943

like structures, producing a plaited surface suggesting Aspidogaster to
Raabe. At the anterior end is a beak-like process, which is embedded in
the cells of a filament, whereas the plaited surface adheres to another
filament. Nutrition is osmotic.

PTYCHOSTOMIDAE

The holotrichous ciliates of the family Ptychostomidae are considered
by some to be related to the Thigmotricha, if indeed they do not belong
in that group. Beers (1938b), following Jarocki (1934), gave prefer-
ence to Hysterocinetidae Diesing, 1866, as having priority over Ptycho-
stomidae Cheissin, 1932. There is, however, no general recognition of
priority in family names, but these are based on the name of the type
genus. In this instance it seems that Ptychostomum Stein, 1860, the first
described, best known, and largest genus, should not be supplanted as
the type by Hysterocineta Diesing, 1866. Rossolimo (1925) suggested
that the Thigmotricha and Ptychostomum represent two parallel evolu-
tionary series, derived from the same group of free-living organisms, but
adapted in somewhat different ways to attachment and the requirements
of sedentary life.

The family Ptychostomidae now includes some eighteen species, of
which nearly half are from Lake Baikal. The ciliates occur in the in-
testine of fresh-water oligochaetes, except for Hysterocineta ezseniae de-
scribed by Beers (1938b) from a terrestrial oligochaete, and three species
from the intestine of gasteropods. In oligochaetes, Ptychostomidae are
associated with astomatous ciliates. Cheissin (1932) remarked that there
is a tendency for Astomata to be located more anteriorly in the intestine,
whereas Ptychostomidae occur in the posterior part. Beers found that 90
percent of H. eiseniae are localized in the third quarter of the gut of
Eisenia loénnbergi; and an astomatous ciliate occurs more anteriorly.
Cheissin (1928) stated that “Lado psis” (—Hysterocineta) benedictiae is
found in the mantle cavity (?) of Benedictia baikalensis; later (1932)
he wrote that that ciliate occurs mostly in the intestine and enters the
mantle cavity seemingly only accidentally.

It may be expected that study of fresh-water oligochaetes in various
parts of the world will greatly increase the size of the group. There may
then be a tendency to greater subdivision, but at present there are only
two genera, Ptychostomum and Hysterocineta, Lada Vejdovsky being a

944 PROTOZOA AND OTHER ANIMALS

synonym of the former and Ladopsis Cheissin of the latter. According
to Jarocki (1934), the points of distinction are the shape of the macro-
nucleus, the size and position of the micronucleus, and the position of
the contractile vacuole. Whether such points are sufficient for distinction
of genera is questionable.

The power of attachment, achieved by a sucker-like organelle, is
marked (Fig. 201D). Miyashita (1927) found that Ptychostomum
tanishi, when observed in the dissected-out gut, was in part attached to
the inner surface of the intestine and in part swam freely in the fluid.

Figure 201. Ptychostomidae. A, Ptychostomum rossolimoi from Limnodrilus newaensis ;
B, skeletal fibrils of the sucker area of that species; C, Ptychostomum chattoni from
Lumbriculus variegatus ; D, Pt. chattoni adherent by sucker to the intestinal wall of the
annelid. (A, B, after Studitsky, 1930; C, D, after Rossolimo, 1925.)

When put into water, the ciliates eventually attached themselves to sur-
faces. Heidenreich (1935) described strong attachment by the sucker
in P. rhynchelmis, folds of the intestinal wall being drawn into it. Beers,
however, remarked that in H. e/seniae the sucker appears to be only
weakly functional, and most specimens were swimming freely in the
lumen of the intestine. In relation to the sedentary position, there is more
or less marked dorsoventral flattening of the elongated body.

According to Studitsky (1930), the first step in the development of
the fixation apparatus is represented by the horseshoe-shaped, non-
ciliated area in the anterior part of the ventral surface of Ptychostomum
Saenuridis, in which no skeletal structures have been described. The next

PROTOZOA AND OTHER ANIMALS 945

step is the strengthening of this area by skeletal fibrils, consisting, first,
of a set of longitudinal fibrils, and second, of a set of fibrils crossing
these. In further development there is differentiation and strengthening
of the two systems. The skeletal fibrils form an irregular network in the
floor of the sucker. The sucker itself is a simple concavity in P. rossolimoz
(Fig. 201A, B) and some other species; sometimes there are a few rows
of cilia on an elevated area in its floor. In other species, probably more
advanced in the evolutionary series, the border of the sucker is a lip-
like elevation. In some forms the sucker is circular (P. tanishi), but
often it is pointed posteriorly (P. chatton7, Fig. 201C) or has an opening
(P. wrzesniewskii) or indentation (P. elongata). In H. eiseniae it is V-
shaped.

Cheissin (1932) described myonemes in addition to the skeletal
fibrils. Heidenreich (1935) stated that the fibrils of the sucker, as de-
scribed by authors, are contractile, thus being not skeletal structures but
myonemes. Beers, however, found no myonemes in the species he studied,
and concluded that all the fibrils have a supporting function. P. rhyn-
chelmis has, Heidenreich stated, unlike other species, a skeleton in the
sucker. The sucker is bordered by two sickle-formed skeletal bows, form-
ing a ring open posteriorly, and each sickle is prolonged posteriorly in a
handle. The two handles form a canal, the neck of which is surrounded
by three or four myoneme bands.

In Ptychostomidae the oral apparatus is situated at what has been
regarded as the posterior end. Beers described a shallow, transverse peris-
tomal groove, bordered by lips bearing cilia, and a small cytostome
leading into a short, tubular cytopharynx. He found no food vacuoles
and no ingestion of ink particles, and consequently concluded that the
mouth is non-functional. In this group of ciliates the feeding apparatus
is in process of reduction. It functions in some species, possibly together
with saprozoic nutrition; in others it has, though still present, little rdle
to play in nutrition.

ASTOMATA

The suborder Astomata is systematically heterogeneous, lacking phylo-
genetic unity, as Kahl (1934) remarked. Cépéde (1910) himself noted
that fact. The group includes many forms that lack complete descrip-
tions. There is no systematic unity to be obtained by bringing together

946 PROTOZOA AND OTHER ANIMALS

forms according to the negative characteristic of absence of a cytostome,
as ciliates of quite divergent relationships may have suffered regression
of that structure. Since a cytostome is not absent in free-living ciliates,
even of the most primitive type, it seems most likely that there has been
regression, rather than that lack of it is a primitive condition. Further-
more, we have to take account of the fact that oral structures may oc-
casionally have been overlooked.

Cépéde (1910) removed the opalinids from the group, and since then
several other genera have been excluded. The Ptychostomidae have
gone; in them the mouth structures are not absent, as at first supposed.
Protophrya appears to have affinities with the Thigmotricha. Chatton and
Lwoff (1935) stated that Metaphrya sagittae from the coelomic cavity
of Sagitta sp. is an apostome; and Kofoidella eleutheriae may also be a
foettingeriid. The description of Kofosdella, from the gastrovascular
canals of a medusa, is too inadequate for systematic purposes; but so far
as it goes relationship to Pericaryon, from the gastrovascular canals of
Cestus veneris, is not excluded. The macronucleus is described as com-
pact and central, and quite variable in size. The macronucleus of Peri-
caryon cesticola 1s reticular and peripheral; but it is not impossible that
Cépéde, who stated that the supposed macronucleus of Kofo/della could
be demonstrated (by Maupas) only after treatment with acetic acid,
was referring instead to the trophic mass.

The greater part of the Astomata inhabit the intestine of Oligochaeta.
In the table of distribution given by Cheissin (1930), records are given
of 69 species in the intestine of Oligochaeta, and of 41 elsewhere. Among
the latter, omitting Kofoidella and Chromidina, the affinities of which
are doubtful, there are 36 species, and 12 of them occur in polychaetes
and in the coelom and gonads of oligochaetes. Heidenreich (1935)
added 11 species in the intestine of oligochaetes, and Beers (1938a)
added one. With about 75 percent of the known species in the intestine
of oligochaetes and polychaetes, and the affinities of many of those found
elsewhere doubtful, we may correctly consider the Astomata to have a
close ecological relationship to that group of animal hosts.

Cheissin (1930), examining invertebrates of Lake Baikal for Asto-
mata, found none in many Turbellaria, molluscs, and polychaetes, and
only a few amphipods had Ano plophrya in the body cavity. Of 24 species
of oligochaetes examined, all the Lumbriculidae and most of the others

PROTOZOA AND OTHER ANIMALS 947

had one or more species of Astomata; three-quarters of 2,062 individ-
uals were infected. Some hosts have several species of the ciliates; in
one there are as many as 7, but all may not be present at the same time.
Heidenreich (1935) examined worms, mostly oligochaetes and turbel-
larians, collected in the vicinity of Breslau. He noted that very few
ciliates are found in worms from flowing water, presumably because
of the fact that cysts are carried away.

There is a certain amount of host-specificity in the group. Hoplo-
phryidae and Intoschellinidae appear to be restricted to annelids. Many
species have been described from one host only, but some occur in many
hosts. Cheissin (1930) found Radiophrya hoplites Rossolimo and Mes-
nilella rostrata Rossolimo in most of the Lumbriculidae; the former oc-
curred only in worms of that family, the latter was found also in an
enchytraeid. There has been a high degree of differentiation of species
and genera in the Astomata, although there are comparatively few char-
acteristics in which that differentiation can be exhibited.

There are two large groups of the Astomata from annelids, those with-
out and those with skeletal structures. The former constitute the family
Anoplophryidae; the latter were put by Heidenreich (1935) into the
two families Hoplitophryidae and Intoshellinidae. The skeletal struc-
tures are differentiations of the ectoplasm or endoplasm, or of both, in
the form of resistant, refractile, and stainable rods, hooks, rays, or fibrils.
They are completely renewed at division. According to the scheme of de-
velopment outlined by Heidenreich, the simplest form is a small, ecto-
plasmic, skeletal plate with a short tooth that scarcely extends from the
pellicle (Evmonodontophrya kijenskiji, Fig. 202K). The plate elon-
gates to the rod-like spicule characteristic of Hoplitophrya, in some
species of which there is a point projecting from the anterior end of the
body. In H. fissispiculata (Ch.) (==Protoradio phrya fissis piculata Cheis-
sin) the spicule is divided in a narrow-angled cleft in its posteriar part.
This is an approach toward the V-shaped ectoplasmic skeletal element
of Radiophrya (Fig. 202 C). The latter is usually provided with an
apical point, which projects from the body surface (Fig. 202, J). Some-
times a second element, a hook or tooth attached to the central, basal part
of the arrowhead and projecting free of the body, is present. This
pointed tooth typically projects backward at an angle. In R. hoplites,
Cheissin (1930) observed that the tooth is capable of movement. There

ot lo,
7h \ aaa
4
|
4 C
4
yg
rome
Waar
LS ATG

Figure 202. Skeletal structures and attachment organelles in Astomata. A, B, Metara-
diophrya asymmetrica from the oligochaete Essenia lonnbergi; C, Radiophrya tubificis
from Tubifex tubifex; D, Mrazekiella costata (anterior end) from Rhynacodrilus coccin-
eus; E, Buchneriella criodrili from Griodrilus lacuum (the sphere around the projecting
spine is normally formed in the tissue cell to which the ciliate attaches) ; F, Maupasella
nova from Essenia foetida and species of Lumbricus; G, Mesnilella fastigata from Enchy-
traeus mobbii; H, Mesnilella maritui from oligochaetes; 1, Intoschellina poljanskyi from
Limnodrilus arenarius; J, Radiophrya lumbriculi from Styloscolex sp. and Lamprodrilus
sp., attachment apparatus; K, Eum monodontophrya kijenskiji from Tubifex inflatus.
(A, B, after Beers, 1938a; C, after Rossolimo and Perzewa, 1929; D, E, F, after Heiden-
reich, 1935; G, after Cépéde, 1910; H-K, after Cheissin, 1930.)

PROTOZOA AND OTHER ANIMALS 949

is a third skeletal element in Radsophrya; more or less numerous ecto-
plasmic skeletal strands attached along the entire inside of the V and
extending posteriorly on the ventral region of the body. These spread
laterally and, although usually restricted to the anterior part, may reach
almost to the posterior end. Skeletal elements of similar type are present
in Mrazekiella (Fig. 202D) and Metaradio phrya.

The attachment organelle of Metaradiophrya asymmetrica, described
by Beers (1938a) from the terrestrial oligochaete Ezsenza lonnbergi in
North Carolina, consists of a shaft embedded in the ectoplasm of the
anterior part of the body and a stout projecting hook, which Beers found
to be immovable (Fig. 202A, B). The left half of the V-shaped element,
such as is present in Radio phrya, is lacking. From the attachment organ-
elle, skeletal fibrils radiate in the ventral ectoplasm, very close to the
surface, the principal group originating near the base of the hook, fol-
lowed by an area of the shaft devoid of fibers, then a group of a few
short fibers at the posterior end. The asymmetrical arrangement of the
fibrils, which is contrasted with the bilaterally symmetrical systems of
M. falcifera and M. lumbrici, is the source of the specific name.

In other forms the skeleton is completely or partly endoplasmic. In
Mau pasella (Fig. 202F) the side arms of the V-shaped element have
become reduced and the point has developed into a prominent, project-
ing, pointed organelle, that serves for fixation. The longitudinal rays
have become endoplasmic. Related to Mamupasella is Buchneriella crio-
dvili Heidenreich, which has a particularly well-developed movable
spine (Fig. 202E). This penetrates into cells of the intestinal epithelium,
anchoring the ciliate firmly. In many ciliates torn from attachment, the
end of the spine was surrounded by a globule of differentiated host tissue.

In Mesnilella the V-formed element is lacking, and the longitudinal
rays are endoplasmic and often reduced in number. A series may be ar-
ranged from a many-rayed condition (Fig. 202H) to that in which there
is only one spicule, reaching almost the full length of the body (Fig.
202G).

Intoschellina has a different type of skeletal apparatus (Fig. 2021).
It is an open ring in the ectoplasm surrounding the apex of the body.
From this ring three short spines project above the body surface an-
teriorly, and three extend in the ectoplasm posteriorly. Two of the

posterior spines are short; one, located at one end of the ring, is rela-
tively long.

950 PROTOZOA AND OTHER ANIMALS

A more or less marked concavity is present on one side near the
anterior end of many species of the Astomata mentioned above. This
concavity, often supported by skeletal fibrils, may fit easily on the convex
surface of the intestinal folds. It is not differentiated as a true sucker,
however. The projecting spines and hooks of the skeletal apparatus of
many forms serve definitely for attachment. These are adaptations to the
requirements of the habitat, but it is a question whether the skeletal
apparatus as a whole can be considered to be strictly a fixation apparatus.

In the Astomata of the family Haptophryidae, there is a true sucker.
If there is a systematic unity in the family, the wide separation of the
two groups of hosts, Turbellaria and Amphibia, is noteworthy. The
species that have spicules, Lachmannella without and Steinella with an
anterior acetabulum-like concavity, occur only in various Turbellaria,
and since there are no complete and modern descriptions, comparison
with other Haptophryidae is difficult to make. The several species of
Haptophrya are better known, especially H. michiganensis Woodhead,
1928, as described by Bush (1933, 1934). H. gigantea has been found
in certain European and Algerian frogs and toads; and H. michiganensis
in several American salamanders and one frog. Rankin (1937) reported
the latter species from 5 of 19 species of North Carolina salamanders,
in incidence of 6.3-21.4 percent; and Hazard (1937) found it once in
Plethodon cinereus in Ohio, which species Rankin had reported nega-
tive. Hazard also found the ciliate in 20 percent of Rana sylvatica in
Ohio. There may be some difference in infection in the same host species
in different geographic regions. Cépéde (1910) noted that R. esculenta
harbors H. gigantea in Algeria, but lacks it in Northern France. Rankin
found what he considered to be H. gigantea, together with H. michi-
ganensis, in a few of the many Plethodon glutinosus studied. Meyer
(1938) reported H. virginiensis, a new species, in R. palustris.

The occurrence in a turbellarian of a species often put into the same
genus, Haptophrya, is of interest from the standpoint of host-specificity.
The species planariarum occurs in various marine and fresh-water Tur-
bellaria (Cépéde, 1910), principally in Planaria torva. Bishop (1926)
found it in 70 percent of that triclad at Cambridge. Finding certain
differences from the forms in vertebrates, she kept it in the genus Sze-
boldiellina; but Cheissin (1930), followed by Bush (1934), did not
recognize any generic differences. Speculation on the origin of this di-
versity of hosts would, with our present information, be vain.

5 j \ Oa an >,

Figure 203. Anoplophrya (Collinia) circulans in Asellus aquaticus, A, B, large indi-
viduals showing nuclei and pulsating vacuoles; C, terminal portion of antenna broken
at end, ciliates enclosed in blood vessel and escaping into water, on contact with which
some disintegrate; D, thoracic leg containing ciliates; E, segment of the basal part of an
antenna, ciliates carried in opposite directions in the currents of blood. (After Balbiani,

1885.)

952 PROTOZOA AND OTHER ANIMALS

Astomatous ciliates that occur in other hosts than annelids, except the
Haptophryidae and Chromidinidae, were placed by Cheissin (1930) in
the family Anoplophryidae. Heidenreich (1935) separated many of
these from that family, without giving them other systematic assignment.
So separated by him were the species of the genus Collinia Cépéde, which
occur in the hemocoele of amphipods and isopods. According to Cheis-
sin (1930) and Summers and Kidder (1936), Collinia is a synonym of
Anoplophrya; so that members of that genus occupy very diverse situa-
tions. There are several species of the ciliates which are evidently not
uncommon in asellids and gammarids. Summers and Kidder believed
that there is a relatively strong host-specificity.

When Balbiani (1885) described Anoplophrya circulans (Fig. 203),
he stated that it was the first example of a ciliate living in the blood
of its host (Asellas aquaticus) and circulating with the corpuscles. When
the ciliates become too crowded to pass through orifices they consti-
tute an obstruction that impedes the circulation. Here and there they
pass out through orifices perforating the walls of the arteries, and return
with the current to the heart. Only a few continue to the ends of the
arteries. As the oxygen is used up in a dead isopod, the ciliates slow
down and die; and they ordinarily perish quickly in fresh water. Some,
however, survive and encyst on plants or on the legs and antennae of
Asellus, later escaping from the cyst and becoming active for a time in
the water.

The species of Dogzelella are tissue parasites which occur in the pa-
renchyma of the mollusc Sphaerium corneum and the rhabdocoeles
Stenostomum leucops and Castrada sp. in Russia (Poljanskij, 1925).
Poljanskij did not refer to Fuhrmann’s statement (1894, p. 223) that
numerous holotrichs occurred in the parenchyma of two individuals of
S. leucops near Basel; but he believed that ““Holophrya virginia’ de-
scribed by Kepner and Carroll (1923) from the same rhabdocoele in
Virginia is Dogzelella. The ciliates seem to have no unfavorable effect
upon Sphaerium corneum, even in a moderately heavy infection, but with
excessive multiplication the host-parasite balance is disturbed and the
molluscs perish from mechanical injury. Rarely, the ciliates may infect
the developing embryos in the brood chamber. The forms in rhabdo-
coeles are apparently harmless to the host. .

Cé pédella hepatica occurs in the hepatic caecum of Sphaerium corneum

PROTOZOA AND OTHER ANIMALS 953

in France. An organelle of fixation, a slightly concave plate to which a
cone of myonemes is related, is developed at the anterior extremity.
The ciliate may penetrate into the hepatic cells. The parasitized cell
undergoes degenerative vacuolization, which extends to neighboring
cells (Cépéde and Poyarkoff, 1909). Cysts have been found in the
liver (Poyarkoff, 1909); these may persist in the outer medium and in-
fect a new molluscan host.

Another tissue parasite is Orchitophrya stellarum Cépéde, a rare
ciliate which was found in 3 of more than 6,000 Asteracanthion rubens
(Cépéde, 1910). The infected sea stars were all males, and the ciliates
occurred in the gonads, among the reproductive cells. Cépéde found
that the parasites were well adapted to life in the sea water, underwent
no pathological changes, and survived for a long time. In a putrefying
genital gland, removed from the starfish, the ciliates lived well after
a day and multiplied. In the host, the parasites bring about what Cépéde
termed partial castration. The ciliate absorbs material in the gonad and
transforms the contents by so doing and by adding its waste products;
and it also brings about mechanically detachment and degeneration of
certain sexual cells. Is Orchitophrya an obligate parasite, or is it an acci-
dentally invading free-living type, in which Cépéde overlooked the
mouth structures? Consideration of instances of accidental parasitism
among holotrichs (Glaucoma, Anophrys), as well as of the great in-
frequency of the occurrence of Orchitophrya and its ready adaptation to
sea water, suggest that the latter may be true.

Conidio phrys

One of the most complete accounts of the life history and host rela-
tionships of an epibiotic ciliate, which is probably a trichostomatous
holotrich, is that of Conidiophrys pilisuctor Chatton and Lwoff, 1934
(Fig. 204). In its profound modification in relation to its mode of life,
it is approached by no other member of its suborder, and, in fact, by
few other ciliates. C. prlisuctor occurs on the secretory hairs, frequently
on the thoracic appendages, of a number of freshwater amphipods, espe-
cially Corophinm acherusicum, in France. A second species, C. guttipotor
Chatton and Lwoff, 1936, is attached to the hairs of Sphaeroma serratum.
These ciliates were placed in a new family of Trichostomata, named Pili-
suctoridae, by Chatton and Lwoff (1934b), though the International

954 PROTOZOA AND OTHER ANIMALS

Rules of Zodlogical Nomenclature demand Conidiophryidae. A complete
account of Conidiophrys was given by these authors in their second
article (1936a).

In a manner suggesting the case of Sacculina, the determination of
the systematic position of Conidiophrys is possible only through study of
its early development.

The form attached to the hairs (Fig. 204A) is immobile, nonciliated
(though an infraciliature is present), and is enclosed in a shell-like
pellicle which has no opening and beyond the body proper closely en-
cases the hair (Fig. 204B). The cucurbitoid trophont undergoes several
transverse divisions within the capsule, toward its distal end, producing
normally two or three (Fig. 204C), or sometimes as many as six tomites.
One specimen was observed with eleven tomites and a twelfth forming,
but the distal seven were degenerate (Fig. 204D). When completely
formed, the tomite, the longitudinal axis of which is transverse to the
longitudinal axis of the trophont, is provided with cilia, with a cytostome
Opening on the ventral surface, and with a relatively long, incurved,
ciliated cytopharynx (Fig. 204F). Tomites are liberated periodically
and have a very short period of free-swimming existence.

When the cytostome comes in contact with the end of a secretory hair,
this is drawn in and the tomite becomes impaled obliquely on it (Fig.
204F). The form rapidly changes to that of a tear drop and the cilia are
lost (Fig. 204G). Growth to the typical trophont proceeds. Chatton
and Lwoff maintained that Conidiophrys is not nourished by diffusion
from the surrounding water, but depends on the fluid secretion that
enters it through the pores at the end of the secretory hairs. Dependence
of the trophic form (trophont) upon the host is thus absolute.

In discussing the multiplicative polarity of Conidiophrys, fission being
localized at the distal pole, Chatton and Lwoff (1936a) speculated con-
cerning a possible trophic or humoral influence emanating from the
host. Instances of inhibition of division, complete or partial, under the
influence of parasitic nutrition are given among parasitic dinoflagellates,
apostomatous ciliates, and other Protozoa. (The authors did not com-
ment, however, on the absence of any indication of such inhibition in a
great number of endozoic forms, a fact which is an impediment to the
acceptance of their theoretical explanation.) In Conidiophrys inhibition
is exhibited in the removal of the zone of multiplication to a distance

eo 2° LEY Peay. *” econ
LLG ES

Sate os
a a ee 4 ee YS

Figure 204. Conidiophrys pilisuctor on Corophium acherusicum. A, trophonts on
appendage of the amphipod host, attached to hairs; B, trophont at beginning of re-
productive period; C, trophont that has formed two tomites, and third forming; D, large
trophont with eleven tomites, and a twelfth forming, the distal seven degenerated;
E, unattached tomite; F, tomite impaled on a hair by its cytostome; G, young trophont,
cilia lost. (After Chatton and Lwoff, 1936a.)

956 PROTOZOA AND OTHER ANIMALS

from the pole of communication with the host. When this influence is
reduced on the commencement of molting, supernumerary tomites may
be produced. There are, Chatton and Lwoff stated, many examples of
parasites certain phases of the development of which are conditioned by
the molt or sexual maturity of their host. The influence may be chemi-
cal, absorbed substances preventing a denaturation of proteins, which
may be the essence of cell division. The existence, in trophoepibiotic
ciliates, of a trophohumoral gradient of inhibition, susceptible to analysis
and analogous to other types of biological gradients, is suggested.

APOSTOMEA

Though certain ciliates that are now included in the suborder
Apostomea have been known for a long time, it is only recently that
the group has become well known. Chatton and Lwoff (1935) published
an outstanding memoir on the Apostomea, which, they stated, is only
the first of three parts. This first part is a monographic study of the
genera and species. In the suborder, according to this account, there are
two families, by far the more important of which is the Foettingeriidae,
with thirteen genera and twenty-six species. In the Opalinopsidae there
are only two genera. Chatton and Lwoff expressed doubt that one of
these, Opalinopsis, really is an apostome; and the other genus, Chrom-
dina, was included by Cheissin (1930) in the Astomata. Kudo (1939)
listed the Opalinopsidae in the Astomata.

The active, growing, vegetative phase of a foettingeriid ciliate 1s the
trophont. The ciliature is in dextral spirals. In the process of growth the
basal granules are spaced without multiplying. At the end of the period
of growth the organism may encyst, the cilia are lost, and the infracilia-
ture undergoes detorsion, the lines becoming meridional. This phase 1s
called the protomont. It passes into the multiplicative phase, or tomont,
which produces by transverse fission a variable number of tomites. The
tomite is a small free-swimming ciliate. The ciliary rows are more or
less meridional, with a tendency to turn in a spiral. There is a thigmo-
tactic ciliary field, consisting of the parabuccal ciliature. Chatton and
Lwoff maintained that the tomite represents the free-living, ancestral
type. In twenty-two of the twenty-six species, and possibly in the others
also, the tomite becomes fixed to the body surface of a crustacean, and
transforms into an encysted phase, the phoront. In the phoront there

PROTOZOA AND OTHER ANIMALS oy,

is renewed multiplication of the basal granules and torsion of the ciliary
lines, leading to the characteristics of the trophont. In the active phases
of most species there is a more or less rudimentary, ventrally placed
mouth, which is surrounded by a characteristic rosette; sometimes the
mouth and rosette are lacking.

Almost all the apostomes occur on or in marine animals. Chatton and
Lwoff (1935) assigned to the genus Gymnodinioides three species from
fresh-water Crustacea, two of which were described by Penard (1922)
as Larvulina, commensals on Gammarus, the third by Miyashita (1933)
as Hyalospira, from Japanese shrimps.

Among the apostomes are the only ciliates with heteroxenous cycles,
cycles that alternate as regularly as those in many Sporozoa, though there
is no obligatory sexuality.

In one group of apostomes, the phoront occurs on copepods, fixed to
the integument; and excystation with subsequent development occurs,
normally when the host is wounded or is ingested by a predator. The
ciliates, however, do not remain long enough in the predator for it to
be regarded as a second host. The predators involved are mostly co-
elenterates. The hydroid Cladonema radiatum appears to be a very special
site for the trophont of Spirophrya sub parasitica, the phoront of which
is fixed to the integument of the benthonic copepod Idya furcata. When
the copepod is ingested, Spzrophrya excysts and grows rapidly in its
remains, accumulating fluid or tissue material in a central vacuole. The
trophont does not encyst within the predator, but is expelled with the
residues of digestion. Encystation takes place on the carcass of the cope-
pod, in the environment, or on the stalk of Cladonema, producing a
tomont. This divides into a number of tomites, which may live free for
a few days, and eventually degenerate or become fixed to Idya. The
phoronts of apostomes of this group will excyst when the copepod mols,
but subsequent development is not normal (see Kudo, 1939, Fig. 257).

In a second group of apostomes there are encysted phoronts on Crus-
tacea, excystation occurs at the molt, and the trophonts develop in the
exuvial fluid. Species are associated with a great variety of Crustacea,
including Entomostraca, balanids, copepods, and many Malacostraca.
The widely distributed genus Gymnodinioides belongs in this group.
Polyspira is another genus. P. delagei is phoretic on the gill leaves of
pagurids (Eupagurus bernhardus). Excystation occurs at the molt, and

Phoront :
ae. Em ply pho retic_ cyst

\ Youn sanguicolous
: trophont or
Bee hypertrophont

for hypertorndnt)

Tomont T >

intomie =.

eee petpal

Grown exuvicolous oy PL AVS akg)
Rents HOP nor ee. ee ae le sont ee ee

Figure 205. Synophrya hypertrophica. A, diagram of cycle of development as a para-
site of Portunus or Carcinus; B-D, parasitized branchial lamellae of Portunus holsatus,
showing different types of reactional cysts. (After Chatton and Lwoff, 1935.)

PROTOZOA AND OTHER ANIMALS 59

the young trophonts grow in the fluid contained in the discarded exo-
skeleton. The proteins accumulate in a violet trophic mass, giving the
color (which in other genera may be orange, red, and so forth) so
characteristic of ciliates of the family Foettingeriidae. Unlike many:
apostomes, the trophont does not become encysted. Linear palintomy
occurs in the motile stage, producing from eight to sixty-four daughter
tomonts, which metamorphose into tomites. In addition to the natural
host, the tomites will become fixed on the gills of Portunus holsatus, on
which development proceeds normally.

In a third group of apostomes, in which there 1s only the genus
Synophrya, the trophont is at one stage parasitic in the tissues of the
crustacean to which the phoront is attached. Synophrya hypertrophica
(Fig. 205) is phoretic on Portunus depuratus, and also on other species
of Portunus and Carcinus maenas. The sanguicolous trophonts are in-
ternal parasites in the branchial sinus of Portunus or the subcutaneous
sinus of Carcinus. They are large, mouthless, immobile, irregularly lobed
masses under the integument, enclosed in a double envelope. The re-
sulting lesions of Carcinus appears as brown or black spots 1-4 mm. in
diameter, found chiefly on the dorsal surface of the carapace. They oc-
cur in a high percentage of crabs less than two centimeters in diameter,
but not on large crabs. At ecdysis, tomites are produced which disperse
in the molted exoskeleton and develop into exuvicolous trophonts. When
growth is completed, these encyst as tomonts, each of which produces a
number of tomites. The tomites fix themselves and become phoretic
cysts on the integument of the gills or branchial cavity of the crab. The
parasites then migrate from the cyst into the underlying tissue.

A fourth type of cycle is that of Foettingeria actiniarum, which is
heteroxenous. It was first known as an inquiline in the gastrovascular
cavity of sea anemones, some species of which are almost always in-
fected. It has been found in various sea anemones on the coast of France,
but it was not found in three species at Woods Hole. Chatton and
Lwoff (1935, p. 313) listed ten host species. The ciliates are chymo-
trophic. They enter the digestive mass when the coelenterate feeds and
there find their sustenance. The ciliates eventually leave the host and
encyst, the tomont undergoes palintomy, and the tomites become fixed
to a crustacean. The host-specificity of the phoronts is almost nil; the

960 PROTOZOA AND OTHER ANIMALS

list of hosts given by Chatton and Lwoff (1935, p. 371) includes cope-
pods, ostracods, amphipods, caprellids, the isopod Sphaeroma, and the
decapod Carcinus. When the crustacean is ingested by a sea anemone,
the phoronts excyst and become young trophonts.

Apostomes of the genus Phtorophrya are hyperparasites on other
apostomes. The phoront is fixed on the phoront of the host species, and
the parasite introduces itself into the body of the other ciliate. It grows
rapidly and soon comes to occupy a cyst otherwise empty. Tomites are
eventually produced; these leave the empty cyst of the host and swim
actively in search of another host phoront.

Rose (1933, 1934) reported two unnamed ciliates, considered by him
to be Foettingeriidae, parasitic in the oil drop in the oleocyst of the
siphonophore Galeolaria quadrivalvis. He thought it probable that the
cysts are attached to pelagic copepods.

Apostomatous ciliates have been found in the digestive cavity of
certain ophiurans and the ctenophore Cestws veneris. Pericaryon cesticola
is unusual among Foettingeriidae in adhering firmly to the walls of the
gastrovascular cavity of its host. It has an apical stylet, which seems to
be an organelle of fixation.

Sexual processes have been described in a number of Foettingeriidae.
Conjugation is contingent, as in other ciliates, and is of a common type
throughout the family. The trophonts conjugate and remain associated
during the formation of tomites. At the end of the series of fissions,
meiosis Occurs, pronuclei migrate, and the tomites separate.

While phoresy on Crustacea is known or presumed to occur in all the
Foettingertidae, except in Phtorophrya, the host phoront of which occurs
on Crustacea, it is unknown in the Opalinopsidae. The vermiform,
elongated (up to 1,200 1), vegetative forms of Chromidina elgans are
fixed to the renal cells of cephalopods by an apparently retractile apical
papilla. There is no mouth. Multiplication is by simultaneous or succes-
sive fissions, producing chains of daughter individuals. The tomite has
a buccal ciliature and a buccal orifice, but no rosette. It is believed that
a crab may be involved in the cycle. O palinopsis occurs in the liver and
intestine of cephalopods, and one species has been found in the liver
of the pelagic gasteropod Carinaria mediterranea.

PROTOZOA AND OTHER ANIMALS 961

PHYSIOLOGICAL. Host RELATIONSHIPS ILLUSTRATIVE OF
MUTUALISM AND COMMENSALISM

FLAGELLATES OF TERMITES AND Cryptocercus

Before discussing the relationship between the xylophagous flagel-
lates and their wood-eating termite or roach hosts, it is desirable to give
consideration to the problem of nutrition in some other invertebrates
that ingest material consisting largely of cellulose.

The most abundant single constituent of wood ts cellulose, which aver-
ages in general between about 54 and 64 percent (Pringsheim, 1932
after Schorger). Among other important carbohydrates are hemicellu-
loses, which Pringsheim stated is a poorly defined collective name for
polysaccharides. A small amount of starch may be present in wood,
about 3 to 4 percent, or less; and a certain amount of sugar (Schorger).
Lignin is a noncarbohydrate incrustation substance in wood and makes up
from about 23 to 28 percent of its bulk (Pringsheim). There are also in
wood ash, less than one percent; proteins, a little under one percent,
according to Pringsheim, in fir, pine, oak, and beech; fats; waxes; resins;
and other substances. Straw and hay have about 30 to 35 percent cellu-
lose, about 20 to 30 percent lignin, 3 to 10 percent protein, and 20 to
30 percent starch.

The animals that ingest these materials may use one or more of the
constituents, and that is not necessarily cellulose. The larva of the goat
moth Cossus cossus, though ingesting wood, does not affect the cellu-
lose (Ripper, 1930). It has no cellulase and contains no symbiotic micro-
Organisms. Ripper found that the carbohydrate used is supplied at least
in part by soluble sugars, perhaps also by hemicelluloses. Mansour and
Mansour-Bek (1934a) concluded that larvae of the cerambycid Xystro-
cerca globosa, with no cellulase and no microdrganisms, derive their
sustenance from the relatively high content of sugars and starch in the
wood attacked (10.4 percent). Data bearing on the fact that some wood-
eating insects seem to make no use of cellulose, but depend on the
starch and sugars in the wood, being limited therefore to certain
kinds of wood rich in these substances, were discussed by Mansour and
Mansour-Bek (1934b). Ullmann (1932) stated that the carbohydrate
requirements of invertebrates are met chiefly by sugars and hemicellu-
loses.

962 PROTOZOA AND OTHER ANIMALS

On the other hand, it has been found that certain termites can sur-
vive indefinitely on cotton cellulose or a cellulose-lignin complex (Cleve-
land, 1925b); and larvae of the rose beetle Potosia cuprea lived for
more than six months on filter paper (Werner, 1926). According to
Dore and Miller (1923), the wood that is ingested by Teredo navalis
loses in the alimentary tract 80 percent of its cellulose, as well as from
15 to 56 percent of the hemicellulose, but the amount of lignin is not
reduced. Digestion of cellulose undoubtedly occurs in the alimentary
tract of many beetles, as, for example, the anobiid Xestobium rufovil-
losum (Campbell, 1929; Ripper, 1930) and the cerambycids Hylo-
trupes bajulus (Falck, 1930), Stromacium fulvum, and Macrotoma pal-
mata (Mansour and Mansour-Bek, 1933, 1934a). The wood eaten by
this last species was found to have very little soluble sugar and starch
(0.47 percent). There are many other instances of cellulose digestion
among vertebrates and invertebrates. Yonge (1925) published a review
of cellulose digestion in invertebrates, but his statement that no cellu-
lase has been found in Insecta is not true today.

Xystrocerca globosa is reported to have a strong amylase, as well as
maltase and saccharase, enabling it to make use of the starches and
sugars in wood (Mansour and Mansour-Bek, 1934a). Tissue-produced
cellulase has been demonstrated in a number of gasteropods and insects.
Among xylophagous insects, cellulase appears to be produced by the
digestive epithelium of certain cerambycid and anobiid larvae.

Most cellulose decomposition in nature is brought about by bacteria,
filamentous fungi, and certain Protozoa. In many animals that make ultt-
mate use of cellulose in nutrition, the material is first acted on by micro-
organisms living in the alimentary tract. This is the only method of
cellulose breakdown in vertebrates, and it is true also of the process
in many invertebrates. Herbivorous mammals harbor bacteria capable
of acting on cellulose. Bacillus cellulosam fermentens was isolated by
Werner (1926) from larvae of Potosia cuprea, which feed mainly on
spruce and pine needles. Bacteria in the intestine of the lamellicorn
beetles Oryctus nasicornis and Osmoderma eremita are able to break
down cellulose (Wiedemann, 1930). Cleveland et al. (1934) found
evidence that symbiotic bacteria are the agents of cellulose decomposi-
tion in the xylophagous roach Panesthia javanica.

In the above-mentioned animals, bacteria dwell in the lumen of the

PROTOZOA AND OTHER ANIMALS 963

gut. Very widespread in wood-eating insects, but by no means restricted
to hosts of that group, are the intracellular symbionts studied intensively
by Buchner and his associates, as well as by many others (see Buchner,
1930). These bacteria or yeast-like fungi live with their hosts in “cyclic
endosymbiosis,” being regularly transmitted to the next generation.
Cyclic endosymbionts exist in the termite Mastotermes (Jucci, 1932;
Koch, 1938a, 1938b). Buchner believed that these symbionts might play
a rdle in the digestive processes of the host, but this opinion, lacking
experimental proof, has not been generally adopted (Mansour and
Mansour-Bek, 1934b; Schwartz, 1935).

Protozoa are present in many of these insects, and sometimes them-
selves derive nutriment from cellulose-rich materials. Beetle larvae fre-
quently harbor a moderate number of small flagellates (Polymastix,
Monocercomonoides) which feed on bacteria (Wiedemann, 1930). As
stated above (p. 916), a limited number of small, non-xylophagous
flagellates (mainly Trichomonas) and occasionally Nyctotherus are pres-
ent in many termites of the family Termitidae (Kirby, 1932b, 1937).
Some of these higher termites, especially species of Mérotermes and
Cubitermes, harbor large amoebae which ingest wood or other cellulose-
rich material on which the termite feeds (Kirby, 1927; Henderson,
MS). The wood-feeding roach Panesthia javanica contains two small
flagellates, Monocercomonoides and Hexamita; large xylophagous
amoebae; smaller amoebae; and a number of ciliates (Kidder, 1937).
Mutualistic symbiosis, however, finds its best illustration, so far as
Protozoa are concerned, in the abundant and diverse xylophagous flagel-
lates of termites other than Termitidae and of Cryptocercus punctulatus.

According to some investigators, Protozoa and other organisms of
the gut may serve the host as a supplementary food source. Wiedemann’s
observation of cellulose-decomposing bacteria in lamellicorn larvae was
mentioned above. He believed that the breakdown products are en-
tirely used in the metabolism of other bacteria. The bacteria multiply
rapidly in the large intestine, where they live in association with the
small flagellates. The mid-gut, he found, secretes protease which is in-
active in the alkaline medium there, but in the hind-gut, where bac-
terial acids accumulate, it digests the bacteria and flagellates. Mansour
and Mansour-Bek (1934a, 1934b) and Mansour (1936) have dis-
cussed the possibility that the flagellates in termites do not benefit the

964 PROTOZOA AND OTHER ANIMALS

hosts in nutritive processes except that, multiplying and being digested
continually, they are a direct and supplementary food source for the
insects. This seems unlikely, however. The flagellates in termites
multiply rapidly for a few days after a molt following which there has
been a new infection; then there is little division, and they are destroyed,
usually, on the approach of the next molt. What use the host might
make of the disintegration products at that time is entirely unknown,
but certainly there is no evidence that the Protozoa could be available
at any other period as a supplementary food source accounting for gen-
eral nutrition (see p. 968).

Cyclic endosymbionts seem to be necessary for normal development
of the host in some instances. Aschner and Ries (1933) and Aschner
(1932, 1934) succeeded in freeing Pediculus of the symbionts that
normally inhabit the mycetome, and found that without them larvae
died sooner or later. The harmful effects of the absence of symbionts in
Pediculus were reduced by rectal injection of yeast extract. Koch (1933a,
1933b) obtained symbiont-free larvae of the anobiid S/todrepa panicea
and found that they would not develop normally unless yeast was added
to the food. (Koch, however, also reported freeing the saw-tooth grain
beetle Orzyaephilus surinamensis of symbionts in the mycetomes by in-
cubation [1933b, 1936]; and absence of the microérganisms seemed to
be without detrimental effect.) It has been suggested that the symbionts
are sources of vitamins or growth factors. It is possible, in the light of
these facts, that certain symbiotic Protozoa may be necessary to the life
of the host, without participating in the digestive processes or serving
as a food source important in bulk.

We now come to a consideration of the demonstration—one of the
outstanding advances in modern protozodlogy, though not yet complete
—that wood-eating flagellates in termites and Cryptocercus are neces-
sary for the survival of their hosts in making the products of decomposi-
tion of cellulose available for the nutrition of the insects. This has justly
received very wide attention, so it is unnecessary to recount all details
of the demonstration here (see Cleveland 1924, 1926, 1928a, 1934).

Termites feed primarily upon wood. This is especially true of the
members of the families Mastotermitidae, Kalotermitidae, and Rhino-
termitidae. Many Termitidae attack wood also; others bring into their
nests dried grass, ingest soil and extract from it the nutrient materials

PROTOZOA AND OTHER ANIMALS 965

it contains, or devour leaves; in fact, almost all types of vegetable mat-
ter are utilized by certain members of the group. Hodotermitidae forage
for grass and herbs, even eat straw from unbaked bricks; some on the
Karroo collect twigs. Kalotermitidae and Rhinotermitidae can live on
paper; even, as stated above, cotton cellulose and a lignin-cellulose com-
plex (see Cleveland, 1924, 1925b). The wood-boring roach Cry ptocercus
punctulatus eats the wood of fallen timber, well-decayed or sound.

Cleveland (1923) pointed out that, in the groups of termites that
use a uniform diet of wood, all species examined had rich faunas of
Protozoa; and this has been confirmed by studies by the writer of more
than a hundred additional species. In Termitidae, with varied food
habits, such faunas are absent, though there are some Protozoa in many
species (Kirby, 1937). Cleveland ef al. (1934) remarked that the
correlation of wood feeding and intestinal flagellates is not so close as
he at first supposed, since there are some Termitidae that eat wood and
have no (xylophagous) flagellates.

We know very little of the actual nutrient substances among the
varied materials taken in by termites as a group. Matter that has passed
through the digestive tract is used extensively by higher termites in
building mounds, fungus gardens, and carton nests. Cohen (1933) and
Holdaway (1933) analyzed mound material of Ewtermes exitiosus,
which contains no Protozoa and feeds on wood. They found cellulose
to be much reduced, though some passes out undigested, whereas lig-
nin is unaffected. These results agree with those reported by Oshima
(1919) after analyses of wood and nest material of Coptotermes formo-
sanus, which does contain xylophagous flagellates. Oshima concluded
that the principal food of that termite is cellulose and that there is no
decrease of lignin. In termites of still another group, Zootermo psis,
Hungate (1936, 1938) found essentially the same thing by analyses of
uneaten wood and pellets.

Tissue-produced cellulase is absent from Cryptocercus punctulatus
(Trager, 1932), Kalotermes flavicollis (Montalenti, 1932), and Zoo-
termopsis angusticollis (Hungate, 1938). Probably none is present in
any Kalotermitidae or Rhinotermitidae, though, in the light of the
situation with wood-boring beetles, one should not generalize from
limited data. Termitidae have not been investigated for cellulase. Man-
sour and Mansour-Bek (1934a) suggested that some termites may be

966 PROTOZOA AND OTHER ANIMALS

found to have cellulase, and it is in that higher group that one might
be most likely to find the enzyme.

The literature on this subject, so far as the writer has determined,
contains no discussion of the hemicellulase lichenase, which Ullmann
(1932) reported to occur in all the insects, including roaches, and the
snails that he tested; and which Oppenheimer (1925) stated is wide-
spread in invertebrates. Montalenti (1932) wrote that in the fore-gut
of K. flavicollis he found a trace of amylase, which was probably pres-
ent in the salivary secretion; in the mid-gut, amylase and invertase, as
well as a protease that acted only in acid, though the mid-gut is basic;
and in the hind-gut, amylase and invertase probably derived from the
mid-gut. Hungate (1938) found amylase in an extract of the fore-gut,
and protease in the mid-gut of Zootermopsis angusticollis. On the basis
of these findings, it should be possible for the termites to hydrolyze
starch; to invert sucrose; to digest the small amount of protein in wood
and possibly also some of their own microdrganisms, when the resistance
of the latter to the enzyme has been overcome.

As remarked above, bacteria in many cellulose-utilizing animals are
necessary for the preliminary breakdown of cellulose. Cleveland et al.
(1934) suggested the possibility that some Termitidae may profit from
the presence of bacteria in the same way. But in those termites that have
been examined for cellulose-decomposing bacteria, it appears that the
latter cannot account for cellulose digestion. A few positive results have
been obtained. Dickman (1931) found them in one of six nitrate-
cellulose tubes inoculated with gut contents of Reticulitermes flavipes,
and Tetrault and Weis (1937) obtained some from the same termite;
but Cleveland (1924) failed in many and varied attempts to isolate
cellulose-decomposing bacteria or other fungi from R. flavipes. Beck-
with and Rose (1929), using termites of six genera, including one of
the Termitidae, obtained cellulose-digesting bacteria in some instances,
but not at all in two species. Their results, however, are subject to
criticism (Dickman, 1931; Hungate, 1936). Hungate (1936) was un-
successful in efforts to show cellulose decomposition by bacteria from
the gut of Z. nevadensis, and concluded that bacteria in the alimentary
tract are of no importance in the digestion of cellulose. A possible ex-
planation of the occasional positive tests is found in Cleveland’s dis-
covery that in Cryptocercus punctulatus, feeding on its normal diet of

PROTOZOA AND OTHER ANIMALS 967

wood, it is usually possible to obtain cellulose-digesting bacteria in cul-
ture from all regions of the alimentary canal, especially the fore-gut.
These disappear in time when roaches are fed on paper, and he believed
that they are forms living in the wood and accidentally ingested by
the insects.

Numerous fungi were isolated by Hendee (1933) from wood in-
habited by Zootermopsis angusticollis, Reticulitermes hesperus, and
Kalotermes minor. Dickman (1931) obtained cellulose-digesting or-
ganisms, both bacteria and molds, from material attacked by termites,
probably R. flavipes and Zootermopsis sp. Cellulose-decomposing molds
were found by Hungate (1936) in burrows and pellets of Zootermop-
sis. He concluded, after analyses of sawdust acted on by external or-
ganisms and material that had passed through the termites (possibly
several times), that cellulose decomposition by bacteria and molds in
the wood of the colony is negligible in comparison with that digested
in the termites. That fungous action can render cellulose usable by
termites is shown, however, by an observation of Cleveland’s (1924).
Termites deprived of Protozoa died soon on a cellulose diet, but lived
indefinitely when a cellulose-decomposing fungus accidentally developed
in certain vials.

The flora of spirochetes and other bacteria in the gut of termites, and
this applies also to Termitidae, is considerable. They live free in the
lumen, attached to certain Protozoa, or attached to the lining of the
walls. Spirochetes do not grow on the usual laboratory media (Dick-
man, 1931). The possibility that they may participate in digestion of
cellulose and hemicellulose in termites was admitted by Cleveland
(1928a). In Cryptocercus, however, Cleveland killed the Protozoa,
leaving the spirochetes, by contrifuging; and cellulase disappeared in
twenty-four hours. The enzyme was not found after defaunated roaches
were reinfected with bacteria and spirochetes.

Excepting certain castes and brief phases of development, all termites
except Termitidae have great numbers of flagellates in the hind-gut.
The vestibule, large intestine, and caecum become voluminous organs to
accommodate these symbionts. Hungate (1939) estimated that the gut
contents containing the Protozoa amount to from a seventh to a fourth
of the total weight of Zootermopsis angusticollis. Katzin and Kirby
(1939) found the gut contents to be about a third of the weight of

968 PROTOZOA AND OTHER ANIMALS

nymphs of Z. angusticollis and Z. nevadensis, and about a sixth of the
weight of soldiers. In this fluid gut contents the Protozoa are about as
thick as they could possibly be. Hungate, by centrifuging, showed that
about half consists of fluid, half of organisms. The organisms are in
mass mostly Protozoa, but there are also a great many bacteria and
spirochetes. Lund (1930) estimated the number of Trichonympha,
Streblomastix, and Trichomonas in Zootermopsis as 54,000; but obvi-
ously this would vary greatly with the size of the termite.

In Cryptocercus punctulates the colon is enlarged to a relatively
greater degree than in termites, becoming ‘‘an immense thin-walled bag
completely filled with Protozoa” (Cleveland ef al., 1934). There are
probably millions of flagellates in a single full-grown roach.

Most species of these flagellates ingest particles of wood. None of
them possesses cytostomes. Ingestion is through the surface of the body.
In Trichonympha wood ingestion has been described by Swezy (1923),
Cleveland (1925a), and Emik (MS). Ordinarily most of the
wood in the faunated portions of the hind-gut is enclosed in the cyto-
plasm of the flagellates. Cleveland (1924) stated that in Reticulitermes
flavipes nearly all the particles of wood are taken into the Protozoa,
whereas in Zootermopsis he found many particles free in the lumen of
the gut.

Bacteria and other flagellates are sometimes ingested by Tricho-
nympha collaris (Kirby, 1932b) and other flagellates of termites. This
predatory habit is more frequent in some species than in others; and
ingestion of other organisms occurs more frequently under the con-
ditions of filter-paper feeding. Yamasaki (1937b) observed Dine-
nympha in many T. agilis after oxygenation. Wood is, however, the
chief and usually the only material taken into holozoic forms. Lund
(1930) noted that when Zoofermopsis was fed on cornstarch, many
Trichonympha and Trichomonas ingested starch grains. Trichomonas
and Hexamastix in Zootermopsis ate, according to Cleveland, able to
use starch. Grains of rice starch were taken in by three of the hyper-
mastigotes in Cryptocercus, and had some food value for them; and
Monocercomonoides in the roach could make full use of starch (Cleve-
land et al. 1934).

Some flagellates in termites are saprozoic and do not take in solid
particles. That is true of Streblomastix in Zootermopsis, of Hoplo-

PROTOZOA AND OTHER ANIMALS 969

nym pha in Kalotermes hubbardi, and probably of some forms of Dzne-
nympha in Reticulitermes. It is also true of certain very small flagellates.

That the flagellates possess an enzyme capable of acting on the cellu-
lose of the ingested wood has been clearly demonstrated by a number
of investigators. Trager (1932, 1934) proved that Trchomonas termop-
sidis produces cellulase. He maintained the flagellate in culture for sev-
eral years in the presence of only one species of bacteria, which was not
capable of fermenting cellulose or cellobiose. The addition of finely di-
vided cellulose to the medium was necessary, and Trichomonas did not
live when that was replaced by other polysaccharides. An extract of the
ground bodies of the flagellates, concentrated from cultures, acted on
cellulose. Emik (MS) obtained fairly pure concentrations of
Trichonym pha from Zootermopsis by gravity filtration. Extracts of these
concentrates were able to digest certain preparations of cellulose as
shown by osazone tests, demonstrating crystals of glucosazone and
cellobiosazone. Emik concluded that two enzymes were present, derived
from Trichonympha: cellulase, hydrolyzing cellulose to cellobiose; and
cellobiase, hydrolyzing cellobiose to glucose.

It is not difficult to show the action of cellulase in the contents of the
hind-gut, and, in view of the absence of tissue-produced cellulase and
the virtual absence of cellulose-digesting bacteria or fungi, the Protozoa
must be its source. Both cellulase and cellobiase were found there by
Trager (1932). Cleveland et al. (1934) and Hungate (1938) so
identified cellulase in flagellates of Cryptocercus punctulatus and Zoo-
termo psis angusticollis.

Substance stained brown or reddish brown by iodine dissolved in
potassium iodide, and assumed, as is customary, to be glycogen, has
been found in many of these xylophagous flagellates. The earliest demon-
stration, which was discussed critically by Cleveland (1924), was made
in Trichonympha agilis by Buscalioni and Comes (1910). Kirby
(1932b) mentioned iodine-staining granules in T. campanula. Yama-
saki (1937a, 1937b) described abundant glycogen deposits in the
species of Trichonym pha, Teratonym pha, Holomastigotes, Pyrsonym pha,
Dinenympha, Pseudotrichonympha, Holomastigotoides, and Spiro-
trichonym pha in Japanese termites, preparing the material by staining
in Ehrlich’s hematoxylin and Best’s carmine after fixation in 90-percent
alcohol. Diminution of the glycogen in T. agi/is under conditions of

970 PROTOZOA AND OTHER ANIMALS

incubation, starvation, and oxygenation was studied by Yamasaki
(1937b). Kirby (1931) stained the axostyle of Trichomonas termop-
sidis brown in Lugol’s solution; and stated that this may be taken, as
Alexeieff pointed out in the case of Tritrichomonas augusta, to be indic-
ative of the possible presence of glycogen in the axostyle. In the light
of these results, it seems likely that carbohydrate is stored as glycogen
in these Protozoa. (Cleveland ef al. [1934], however, considered it
possible that the substance colored by iodine may not be glycogen, but
a breakdown product of cellulose which gives the same reaction as glyco-
gen. See also page 981.)

Since only in the bodies of the flagellates can cellulose be digested,
and termites live and develop normally when only cellulose is eaten,
the rdle of the symbionts is evident. According to Hungate (1938),
about one-third of the total material removed from wood, adding that
acted on in the fore- and mid-gut to the soluble materials present, can
be obtained without the aid of the Protozoa. It is possible that materials
adequate for nutrition of the insect may be obtained in the diet with-
out the Protozoa, as Cleveland (1924) found by feeding humus and
fungus-decomposed cellulose. Presumably sufficiently rotted wood would
also be adequate; Cleveland (1930) stated that defaunated Cryptocer-
cus, which dies in two or three weeks on partially decayed wood or
cellulose, will live two or three months on completely decayed wood.
Cleveland has conclusively demonstrated that continued survival of de-
faunated termites and Cryptocercus is impossible on a natural diet of
wood. Hungate’s third, therefore, could not provide all necessary sub-
stances, it appears; as, if it did, the amount could be multiplied merely
by the ingestion of more wood, or further use of that which ordinarily
passes to the hind-gut for use of the Protozoa in faunated individuals.

Experiments in feeding various cellulose-free carbohydrates to
termites have been made by Montalenti (1927) and Lund (1930); and
to Cryptocercus by Cleveland (1930, 1934). Montalenti kept Kalo-
termes flavicollis alive for several months on soluble starch, alone or
mixed with glucose, though the hypermastigotes soon disappeared and
the polymastigotes greatly diminished in number. He concluded that the
termite could live a long time, if not indefinitely, on soluble carbo-
hydrates without Protozoa, but no other worker has confirmed this.
Lund’s studies were made to determine the effect of various diets on the

PROTOZOA AND OTHER ANIMALS 971

Protozoa of Zootermopsis, not the maximum period of survival of the
termites. Cornstarch caused death of the Protozoa and the termites after
twenty-three days; the starch was apparently in granular form. (In com-
parison with Montalenti’s results, light may be thrown on the discrepancy
by the statement of Ullmann (1932) that invertebrates are unable to use
the starch of the plant food, but that soluble cooked starch is very well
digested by all animals.) Lund used a variety of carbohydrates, on most
of which the maximum survival of both Protozoa and termites did not
greatly exceed the effects of starvation; and on some they died more
quickly. On inulin, dextrin, and lactose Trichomonas and Streblomastix
were living in the last termites reported at forty-eight, forty-four, and
sixty-five days. Cleveland (1925c) found that Trichomonas (accom-
panied by Streblomastix) can keep the host alive from forty to fifty
days longer than when no Protozoa are present, but “very few if any
[termites ] were able to live indefinitely.’” The hypermastigotes are most
important in the mutualistic symbiosis in Zootermopsis.

Cleveland et al. (1934) studied the effects of various diets on Cry pio-
cercus punctulatus and its Protozoa, using various cellulose-free carbo-
hydrates, peptone, gelatin, and glycogen. On no substance did any ex-
cept the smaller polymastigotes survive very long; nor did the roaches
live more than a few days longer than when water alone was given.
These authors found that dextrose is of more food value than the other
substances, and considered it likely that a diet including dextrose might
be found upon which the insects could live for a long time, if not in-
definitely, without Protozoa.

Dextrose prolonged the survival period also of defaunated Reticw-
litermes flavipes (Cleveland, 1924). Trager (1932) demonstrated dex-
trose in the presence of extract from the hind-gut contents of Cry pto-
cercus. Cleveland et al. (1934) suggested that dextrose, produced from
cellulose by the action of cellulase and cellobiase in the cytoplasm of
the flagellates, insofar as it is not used in their metabolism or stored as
glycogen, diffuses from their bodies. Hungate (1939), however, identi-
fied acetic acid, carbon dioxide, and hydrogen as metabolic products of
the Protozoa; and thought it likely that most of the sugar resulting from
their digestive processes undergoes anaérobic dissimilation by the Proto-
zoa. According to this view, the termites would make use of the acetic
acid.

OFZ PROTOZOA AND OTHER ANIMALS

A further problem arises in the absorption of the substances released
from the flagellates by the termite or roach tissues. Either absorption
must take place through the chitinous layer of the hind-gut, or fluid
must be passed forward into the mid-gut. The problem has been dis-
cussed by Buchner (1930) and Cleveland ef a/. (1934). There are dif-
ferences of opinion as to whether absorption in the hind-gut is possible,
and some authors are inclined to the view that it is. Buchner is one of
those. Abbott (1926) found that the hind-gut of Periplaneta aus-
tralasiae is permeable to dextrose. Cleveland ef a/. took the opposite
view, as a result of osmotic experiments on the colon of Cryptocercus,
which showed it to be impermeable to dextrose and water. The peri-
trophic membrane also seemed to be largely impermeable to dextrose.
The iliac valve controls the passage of materials between the mid-gut
and the hind-gut, and when the mid-gut is severed it permits no ma-
terial to flow out from the hind-gut. Cleveland concluded that fluid con-
taining dextrose passes forward at times through the iliac valve into the
space between the peritrophic membrane and the wall of the mid-gut.

A problem in the metabolism of xylophagous animals is the source
of nitrogen. The small amount of protein present in wood, and the
larger amount in straw and hay (important in the case of Hodotermes
and some higher termites), may account for the nitrogen metabolism
of the Protozoa; but in the absence of action on cellulose outside of the
Protozoa it might not be directly available to the termite. Bacteria and
molds ingested with the wood might account for some, but that would
probably be very little. Pierantoni (1937) hypothesized a fixation of
nitrogen by bacteria in the gut, and Green and Breazeale (1937) re-
ported the isolation of nitrogen-fixing bacteria from an unidentified
species of Kalotermes. Wiedemann (1930) stated that bacteria in
certain lamellicorn larvae can use inorganic nitrogen, and the host satis-
fies its nitrogen need by digesting these microérganisms. Use by termites
and Cryptocercus of some dead Protozoa or pieces of cytoplasm from
their bodies (Cleveland ef a/., 1934), while it could not account for any
important part of general nutrition (p. 961), might be significant in
providing nitrogen.

We have seen in the foregoing discussion that many animals ingest
substances of which cellulose is an important constituent. In the diges-
tive tract of some of them cellulose is broken down, whereas certain

PROTOZOA AND OTHER ANIMALS Die

others make use only of other constituents of the ingested material.
These two nutritional variants may be found in members of the same
group, as in Cerambycidae. The decomposition of cellulose may, in some
invertebrates, be accomplished by means of a tissue-produced cellulase;
in other, even related, forms it may require the action of symbiotic bac-
teria. It is possible, though the truth of the hypothesis remains to be
shown, that endobiotic bacteria and Protozoa may, in some instances,
benefit their hosts as a supplementary food source. There 1s a hypo-
thesis also that certain symbiotic microérganisms are a source of vitamins
or growth factors, or play a rdle in nitrogen economy.

In all termites below Termitidae, except in certain functioning repro-
ductive castes, and in the roach Cryptocercus punctulatus, xylophagous
flagellates are exceedingly abundant in a specially enlarged part of the
hind-gut. These flagellates possess cellulase and cellobiase, and reduce
cellulose taken into their cytoplasm to dextrose. The insects possess no
tissue-produced cellulase, and few if any cellulose-decomposing bac-
teria or other fungi are present. These insects cannot live for long on
their usual diet or on cellulose without the flagellates, which presumably
release part of the dextrose or its dissimilation products for the use of
the host. This may be passed forward into the mid-gut to be absorbed,
or perhaps may be absorbed in the hind-gut. To what extent the nitrogen
needs of termites may be provided for by occasional digestion of Proto-
zoa or fragments of the cytoplasm, or by symbiotic nitrogen-fixing bac-
teria, remains to be determined.

CILIATES OF RUMINANTS

Among the most notable endozoic faunules of Protozoa are the ciliates
in ruminants and certain other herbivorous mammals. There are some
holotrichs, sparsely represented among the species of ciliates in rumi-
nants, but constituting an important and diversified part of the faunules
of the caecum and colon of the horse (Hsiung, 1930). There are also
a few flagellates and amoebae, but the most characteristic forms belong
to the Entodiniomorphina. Of the two families of this suborder of
highly organized spirotrichs, Ophryoscolecidae occur chiefly in rumi-
nants (see Dogiel, 1927; Kofoid and MacLennan, 1930, 1932, 1933;
Wertheim, 1935); and Cycloposthiidae are best known in the horse
(see Hsiung, 1930). The latter family is represented also in a number

974 PROTOZOA AND OTHER ANIMALS

of other mammals, including the tapir, rhinoceros, chimpanzee, gorilla
(Reichenow, 1920), and elephant (Kofoid, 1935).

Ciliates in ruminants, except for certain species less constant in oc-
currence (as Buxtonella sulcata Jameson, in the caecum of cattle) are
localized in the rumen and reticulum. Their relative abundance in the
rumen and reticulum is approximately equal (Dogiel and Fedorowa,
1929; Wertheim, 1934a); and they are distributed throughout the con-
tents. Dogiel and Fedorowa found that the ciliates are somewhat more
abundant in the central part than at the periphery, but that the difference
is not very great. Distribution is sufficiently uniform so that counts of
a small sample from the rumen have been used for an estimate of the
total population.

Poor nutrition of the ruminant can cause a rapid reduction in the
number of ciliates, and this may be responsible for low counts, in slaugh-
terhouse animals, of under 100,000 per cc. (Dogiel and Fedorowa,
1929), under 200,000 per cc. (Wertheim, 1934a), and under 400,000
per cc. (Winogradowa-Fedorowa and Winogradoff, 1929). Under
conditions of normal nutrition, many counts above 500,000 per cc. have
been obtained. Mangold (1929, 1933) stated that in sheep and goats
the normal number remains with much constancy at about 1,000,000
per cc.; and Mowry and Becker (1930) agreed with this as regards goats.
Under certain conditions, the population may be much denser than this.
By experimental feeding Mowry and Becker obtained a count of nearly
7,600,000 of Entodinium and Diplodinium alone. Ferber (1928) esti-
mated that at 900,000 per cc. a gram of rumen contents would contain
about one-twentieth of a gram of ciliates.

The total number of ciliates in an individual ruminant is enormous.
Calculating from a volume of material in the rumen and reticulum of
goats of from 2.8 to 5.2 liters, and a ciliate count of from 121,000 to
391,000 per cc., Winogradowa-Fedorowa and Winogradoff (1929) estt-
mated a population of from 471,000,000 to 1,548,000,000; for the
normal condition these figures should probably be multiplied by about
three. In an ox with from 56 to 87 liters and from 70,000 to 117,000
ciliates per cc., there would be nearly 10,000,000,000; and probably the
population may be at least five times as dense as that.

The ciliates are consistently absent from suckling animals, but, as
soon as a diet of plant food begins, the faunule of the rumen and reticu-
lum develops.

PROTOZOA AND OTHER ANIMALS a7

The kind of food taken has a striking influence on the ciliate popula-
tion. Green plant material was regarded as of fundamental importance
by Trier (1926), with emphasis on the chlorophyll content. Weineck
(1934) expressed agreement with this view, but Westphal (1934b)
denied that there is proof of chlorophyll need. In experiments by
Mowry and Becker (1930), green fodder alone maintained a low popu-
lation. Hay and water alone also maintained a low population, which
was more than doubled when cornstarch was added. A much greater
increase occurred when a grain mixture, consisting of ground corn,
ground oats, wheat bran, and linseed oil meal, was given with hay.
There is a limit to this increase, however. Although the densest popula-
tion of all was developed on grain alone, there was soon a very great
decrease in the number of ciliates. As was pointed out by Mangold
(1929), some coarse food is essential. Hay with cornstarch and either
plant or animal protein, instead of with grain, also maintained a high
level of population density. Apparently in the grain both the starch and
the protein constituents are stimulating factors, although the Mangold
school has maintained that the protein alone is determinative.

Other factors influencing the ciliate population of the rumen that
have been discussed are density of the contents and pH. Mowry and
Becker (1930) could not corroborate the findings of Dogiel and
Fedorowa (1929) and Ferber (1929b) that thick rumen contents con-
tain relatively more ciliates than thin fluid contents. Mowry and Becker
found the average pH in the rumen of goats to be 7.7, with two-thirds
from 7.6 to 7.8 and extremes of from 6.7 to 8.2. Within these limits,
there seemed to be no notable changes in the ciliate population that
could be attributed to the pH itself. Mangold and Usuelli (1930) found
the pH of fresh rumen contents of sheep to be from 7.5 to 7.8.

When the ciliates pass into the omasum, abomasum, and intestine,
they are destroyed. As nutriment passes posteriorly from the reticulum,
a vast number of ciliates must go with it; it is difficult to conceive of
any mechanism by which they could be kept back. The population could
be maintained only by an adequate rate of multiplication, going on con-
tinuously. The rapid disappearance of the ciliates on starvation of the
host probably could not be explained simply by starvation, followed
by death of the ciliates in the rumen and reticulum; it is more likely that
the rate of reproduction declines, and the passage of ciliates into the third
stomach rapidly reduces the population.

976 PROTOZOA AND OTHER ANIMALS

Several attempts have been made, with varying results, to estimate
the reproductive rate by counting dividing ciliates. No adequate deter-
mination has been made of the rate of reproduction in a day, a calcu-
lation which cannot be based only on the amount of fission seen on one
occasion. Rate of reproduction in culture, furthermore, at least in the
absence of completely satisfactory culture methods, is not necessarily
the same as that in the rumen. Mowry and Becker (1930) found in
goats usually less than 0.5 percent of dividing forms, and never as many
as one percent. Ferber and Winogradowa-Fedorowa (1929) found in
a ram on different occasions from 0.9 to 15 percent in division, the aver-
age being 7 percent. Examinations were made twice a day, but they
failed to note, as Mowry and Becker pointed out, that from observation
of 7 percent dividing forms in two samples per day, it does not follow
that 7 percent of the ciliates are dividing in a day. The rate of multipli-
cation would probably be much higher than that. Westphal (1934a)
found that in culture of certain forms each ciliate divided an average of
once in fourteen hours, and the population became 3-fold in a day. It
reached in more dilute medium a rate of 5.8-fold in twenty-four hours.
Dogiel and Winogradowa-Fedorowa (1930) published a report that
from 50 to 90 percent of the ciliates were observed in division in goats
under normal conditions of nutrition, and from 12 to 50 percent in
slaughterhouse oxen. Westphal (1934a) calculated that there must be
daily at least a quadrupling of the number of ciliates.

Rumen ciliates live in a chemically complex and delicately balanced
environment, and 7 vitro culture has been a difficult problem. Becker
and Talbott (1927) and earlier workers failed to obtain more than
limited survival. Knoth (1928) obtained longer survival in a medium
of rumen fluid, with controlled pH, and with partly anaérobic condi-
tions provided by a mixture of carbon dioxide and methane, the maxt-
mum being the life of Extodinium, with daily change to fresh solution,
for five days. Margolin (1930), in media of hay infusion with rice
starch and filter paper acted on by cellulose-decomposing bacteria, the
pH being kept at 6.8, reported maintenance of cultures for twenty-four
days; but others have been unable to use his methods successfully
(Becker, 1932; Westphal, 1934a). Westphal (1934a, 1934b) reported
real success with a medium of rumen fluid kept under anaérobic con-
ditions, with urea and starch added. There was active multiplication in

PROTOZOA AND OTHER ANIMALS O77,

the cultures, which, with daily renewal of medium, were kept several
weeks, and, he stated, might be continued indefinitely. Entodinium lived
particularly well.

The rumen ciliates, so far as is known, do not form cysts. Tropho-
zoites have been found in the mouth fluids (Becker and Hsiung, 1929),
and ruminants have been infected by giving this material with the food
(Mangold and Radeff, 1930). Natural transmission is by contact, in
common feeding, in which there is a certain period when the tropho-
zoites ate exposed to the external environment (Becker and Hsiung,
1929; Mangold and Radeff, 1930; Strelkow, Poljansky, and Issakowa-
Keo, 1933). Their ability to withstand external conditions is therefore
of crucial significance. The holotrichs and Extodimium are more re-
sistant than the larger Ophryoscolecidae. Strelkow ef al. reported that
after six hours at room temperature all ciliates were still active, and
many survived longer. At 0° C. all continued normal activity for an
hour. On dilution of the rumen fluid, they survived for various periods
of from one to thirty-two hours; and most were alive after six hours in
material two-thirds evaporated. They are thus clearly able to live long
enough on feed or in water to infect other animals using the same con-
tainers. The interesting report of Fantham (1922) that “species of
Entodinium and Diplodinium may be found on wet grass and in aque-
ous washings of fresh grass and even of dried grass (fodder) from
sheep runs and pasturage’”’ leaves the reader desirous of details con-
cerning his observations.

It has been found to be a simple matter to bring about elimination of
the ciliates from the rumen and the reticulum. Modern investigators
have used three defaunation treatments. Mangold and his coworkers
have found starvation alone to be satisfactory. The ciliates may ap-
parently be absent after only three or four days, but Dogiel and Wino-
gradowa-Fedorowa (1930) found six to seven days without water
necessary for complete defaunation. They found preferable, however,
partial feeding with dry food and water and a liter of milk daily. Milk
feeding was used by Falaschini (1935). On the basis of studies made
on material kept in the thermostat, Mangold and Usuelli (1930) con-
cluded that the increased acidity induced in the rumen contents 1s re-
sponsible for the incompatibility of milk and ciliates. The best method
of defaunation, according to Strelkow, Poljansky, and Issakowa-Keo

ss
Si a
ees ore |
a SASS) "
x x
[es |

TERE Ot ee

Fes( |
ay
SS |

Bs
SS

Figure 206. Ingestion of plant material by Ophryoscolecidae, A, a piece of grass with
three fiber bundles, which are beset with Diplodinium gracile: B, long cellulose fiber
rolled up in D, gracile; C, large fragment of grass in Elytroplastron bubalidis ; D, a piece
of grass with two D. gracile and four Opisthotrichum janus. The former have ingested
the ends of fibers, the latter ingests fine detritus from the surface; E, E. bubalidis, which
has partly ingested a large piece of grass. (After Dogiel, 1925.)

PROTOZOA AND OTHER ANIMALS Se)

(1933), who compared it with milk feeding, is the one discovered by
Becker (1929). Becker starved his animals for three days, then gave
two doses, twenty-four hours apart, of fifty cc. of 2-percent copper sul-
phate, passed through a rubber tube into the rumen. Strelkow ef al.
shortened the starvation period to one day and gave three doses of
copper sulphate, thus shortening the defaunation period to three days.

The ciliates take up plant fragments in the rumen, and Trier (1926)
stated that they are apparently exclusively plant feeders. Bacteria, flagel-
lates, amoebae, and ciliates may also be ingested, however. According
to Dogiel (1925), no Ophryoscolecidae are entirely predatory, as plant
debris is always to be found in the plasma. Kofoid and MacLennan
(1930, 1932, 1933) and Kofoid and Christenson (1934) recorded the
food contents of most of the species they studied, and showed clearly
that food habits differ. Some appear to feed only on bacteria and other
Protozoa, especially small flagellates. Others use various combinations of
plant and animal material, some only plant material, and bacteria with
plant debris are ingested by a large proportion. Some species are more in-
clined than others to be predatory on ciliates. Entodinium vorax, accord-
ing to Dogiel (1925), almost always contains the remains of one or more
smaller Entodinium.

The plant material is often in relatively small particles, but some
ophryoscolecids take in large pieces that may distort the body. Dogiel
(1925) described how Diplodinium gracile may seem actually to tear
away fibers (Fig. 206A, B); Opisthotrichum janus may bite from the
surface of a plant piece the remains of ruptured tissue (Fig. 206D);
and D. bubalidis, D. medium, and D. maggii may devour large irregular
or flat grass pieces (Fig. 206C), but not fibers. Ostracodinium sp. can
take in and roll up large cellulose fibers (Weineck, 1934).

Green plant fragments are taken in preference to non-green ones,
according to Usuelli (1930b), who offered a choice by feeding hay and
barley. A third to a half of the green fragments were in about half of
the ciliates; whereas less than 10 percent of them took in non-green
fragments, all but a few of which remained free in the lumen. For
this selectivity, Usuelli contended, the softer, smoother characteristics
of the green plant pieces are responsible.

When available, starch grains are ingested avidly by the ciliates, both
in the rumen and in the thermostat. Four hours after giving a sheep

980 PROTOZOA AND OTHER ANIMALS

fifty grams of cornstarch, 76 percent of the ciliates had taken in grains;
and in from four to six hours 87 percent took in rice starch (Usuelli,
1930a). If the amount of starch is not excessive, most of it is eventually
taken into the Protozoa. The size of the grains is a factor in ingestion.

Figure 207. Ingestion and digestion of starch in Eudiplodinium medium. A, before
feeding in culture; B, 2.5 minutes after feeding, starch grains in cytoplasm; C, 12
minutes after feeding, cytoplasm filled with starch; D, 2.5 hours after feeding, abundant
deposits of glycogen; E, 16 hours after feeding, some residues of starch, glycogen deposits
in certain areas. (After Westphal, 1934a.)

For example, fewer ciliates take in potato starch, the diameter of many
of the grains of which exceeds 100 1.

In material kept in the thermostat, it has been found that fat droplets
in milk will be ingested (Ferber, 1928); but ingestion of accessible ma-
terial is not indiscriminate (Westphal, 1934a).

PROTOZOA AND OTHER ANIMALS 981

That there is digestion of the starch has been well established (Trier,
1926; Westphal, 1934a, 1934b). Figures from Westphal (1934a) are
reproduced here (Fig. 207) showing successive stages in the rapid in-
gestion by Exdiplodinium medium in culture of rice starch and the dis-
solution of this. Although there seems to be no good reason for denying
starch-splitting ability to the ciliates themselves, Ullmann (1932) con-
sidered it possible that starch-digesting bacteria, taken in with the food
and continuing their action in the digestive vacuoles, are responsible.

As starch is digested, glycogen (paraglycogen) accumulates in the
cytoplasm in granular form. The reserve material is stored in the ecto-
plasm, in the region of the gullet and the rectal tube, and sometimes
also in the endoplasm. It has been asserted by many that, in addition to
the other storage areas, the skeletal plates contain deposits of glycogen
(Schulze, 1924; Trier, 1926; Weineck, 1934; Westphal, 1934a,
1934b; MacLennan, 1934). Dogiel disagreed with this concept; and
Brown (MS), by a series of chemical tests, solubility tests with sub-
stances that would have been expected to extract glycogen, and enzy-
matic reactions, found no evidence that the contents of the skeletal
prisms is glycogen. Brown pointed out that the results of iodine reac-
tions are insufficient in themselves to identify glycogen, as other sub-
stances stain in the same way.

Glycogen may be built up from simpler carbohydrates, appearing
after feeding with dextrose (Trier, 1926; Weineck, 1934) and lactose
(Trier).

When, after deposits of glycogen have accumulated, the ciliates re-
main without food, the reserve is used up in cell metabolism. Trier
found that within forty-eight hours after ingestion of the starch most
of the accumulated glycogen had disappeared.

There is disagreement as to whether the ciliates can use cellulose,
though certainly a quantity is ingested. Much quoted has been the state-
ment by Dogiel (1925) that, in the endoplasm of the Ophryoscolecidae,
cellulose pieces undergo no morphological change and leave by the
anus, still with sharp margins and no wrinkling or swelling. The state-
ment was evidently based on observations on Diplodinium maggii and
D. medium, which ingest large particles (Dogiel and Fedorowa, 1925).
Westphal (1934b) reported that he had confirmed this account of
ejection of large particles, but Weineck (1934) wrote that it has very

982 PROTOZOA AND OTHER ANIMALS

seldom been seen, and that ejected pieces are those with excessively
heavy membranes. Reichenow (in Doflein, 1927-29) did not agree
that it provides evidence against the use of cellulose, stating that Pro-
tozoa, especially when in unfavorable circumstances, may give up nutti-
ment useful to them. Usuelli (1930b) saw no microscopic indications of
corrosion of ingested fibers, and commented that in any case the green
plant fibers, that are the ones chiefly used, contain relatively little cellu-
lose. Westphal (1934a, 1934b) denied cellulose digestion in ophryo-
scolecids, and showed that, in spite of the presence of cellulose and
chlorophyll, cultures died out in the absence of starch. According to
Mangold, the colorless pieces persist for a long time, even for four days,
and during that time digestible substances are extracted.

Earlier opinions that there is digestion of cellulose have been sum-
marized by Becker, J. A. Schulz, and Emmerson (1930). P. Schulze
(1924, 1927) and Trier (1926) believed that cellulose particles are
reduced chemically and structurally. Recently, Weineck (1934) ob-
tained positive evidence, including observations on corrosive changes
and loss of the original double reactivity in polarized light.

No cellulose-splitting enzymes have been isolated from the bodies of
ophryoscolecids; that would be difficult in the presence of so many
cellulose-decomposing bacteria. It was the suggestion of Trier (1926),
and the opinion of Mangold (1929, 1933) and Westphal (1934a),
that bacteria taken in with plant fragments are responsible for what
cellulose decomposition has been observed within the ciliates. It is, of
course, well known that the main rdle, at least, in cellulose-splitting in
ruminants is performed by bacteria (Schieblich, 1929, 1932). If the
ciliate had a cellulase, according to Mangold (1929), more intensive
cellulose decomposition would be observed than has been possible.

Doflein (Reichenow ed., 1927-1929) stated that there is no instance
of fat digestion in Protozoa. Ferber (1928) observed, in successive
rumen samples, that milk-fat droplets ingested iz vivo underwent de-
formation and eventually disappeared, but he recognized the probability
that bacteria, ingested also, were responsible for this breakdown of
fat. Weineck, in experimental feeding with lipoids, found that no fat
was ingested, but he observed a small amount in the ciliates that probably
had been taken up from plant materials.

Inasmuch as the ciliate population can be increased, up to a limit,

PROTOZOA AND OTHER ANIMALS 983

by the addition of protein to the diet (Ferber, 1928; Mowry and Becker,
1930), and declines when protein is deficient, it is evident that, as
Mangold (1929) stated, the ciliates have an important protein need.
They obtain the protein ordinarily from the plant food. Mangold (1929,
1933) thought it unlikely that ingested bacteria or other Protozoa could
sufficiently provide for this need. It is normally supplied by the addition
of grain. Whether or not the cellulose of the plant food ts fully utilized,
the starch and protein of the plant cell plasma supply energy and ma-
terials for the activity and growth of the Protozoa.

Many investigators who have concerned themselves with the ciliates
of ruminants have sought an answer to the question of their possible
value to their hosts. The literature was reviewed by Becker, Schulz,
and Emmerson (1930); and the subject has been discussed by Becker
(1932) and Mangold (1933). Of the various opinions advanced,
Zurn’s belief that the ciliates could cause injury has been found entirely
untenable. They are present in every ruminant in good condition; and
Ferber (1929b) pointed out that the number of ciliates may serve as
a guide to the host’s well-being. Favorable conditions of nutrition and
optimum physiological activity of the host at the same time favor a
large ciliate population; and under the best conditions there may be
approximately a doubling of the average density, to 2,000,000 per cc.
Unfavorable conditions are rapidly reflected in a decline of that popula-
tion.

Some, beginning with the first observers of the ruminant faunule
(Gruby and Delafond, 1843), have supposed that the relationship is
one of mutualistic symbiosis, the ciliates being in one way or another
beneficial to their hosts. It is not disputed that great numbers of the
ciliates are digested, but mutualism does not follow from that, unless
some special contribution is made to the economy of the host.

There is still no general agreement as to whether or not the ciliates
can break down cellulose, but opinions have been widely published
that they aid their hosts in cellulose decomposition. Reichenow (1920)
observed morphological changes in ingested cellulose fragments in
Troglodytella; and suggested that the significance of this ciliate to its
primate hosts, as well as that of Ophryoscolecidae in ruminants, lies in
the use of cellulose in constructing their own easily digestible bodies,
which serve as animal nutriment for the mammals. In Doflein (Reiche-

984 PROTOZOA AND OTHER ANIMALS

now ed., 1927-1929) he expressed the same opinion, and stated that the
physiological rdle of these ciliates is evidently similar to that of termite
flagellates, admitting, however, that they are not so important to the
life of the host as these, because ruminants have other aids in cellulose
decomposition. Usuelli (1930b) remarked that the softer green plant
parts, which are those chiefly ingested, contain relatively little cellulose;
so that even if intracellular digestion of this did take place, it would have
little quantitative significance in the decomposition of cellulose in the
rumen.

Mangold (1929, 1933) and his coworkers have emphasized the rdéle
of the ciliates in protein economy, reasoning that their bodies contain
a significant part of the nitrogen available to the host, and that they
derive this from plant protein, and presupposing that the ruminants
can make better use of this animal protein than they can directly of plant
protein. The last fact is certainly fundamental to their thesis, but Becker
(1932) remarked that there is no proof of it. Becker, Schulz, and
Emmerson (1930) found that goats digested slightly more protein
when the ciliate population was present; but the difference was so small
as to have little significance without further studies.

According to analyses by C. Schwarz (1925) of the rumen contents
of slaughterhouse cattle, 20 percent of the nitrogen is in the ciliates
and 11.7 percent in bacteria. Ferber (1928) found the ciliate nitrogen
in sheep and goats, with a population of from 837 to 2079 ciliates
per cubic millimeter, to be from 10.27 to 20.33 percent of the total,
averaging about 15 percent. Ferber and Winogradowa-Fedorowa (1929)
calculated that with a population of 900,000 ciliates per gram and with
the total nitrogen 0.166 percent, there would be, in a 3-kilogram
rumen content, 150 grams of ciliates. These would contain about 4.7
grams of protein, and the estimate would be nearly doubled by use of the
figures of Mangold and Schmitt-Krahmer (1927) for the total nitrogen.
There is ciliate protein also in the reticulum, but this amounts to only
a fraction of that in the rumen.

There are no exact estimates of the amount of ciliate protoplasm di-
gested in a day. Ferber and Winogradowa-Fedorowa (1929), on the
basis of a fallacious estimate of a 7-percent daily division (see p. 973),
calculated that 2 percent of a sheep’s daily protein requirement of thirty
grams might be met by the ciliates. In this estimate the higher figures

PROTOZOA AND OTHER ANIMALS 985

for total nitrogen were used. Mangold (1933) accepted the estimate that
the ciliates provide about one percent of the protein used daily and,
under certain circumstances, from 2 to 3 percent. C. Schwarz (1925),
however, thought it probable that the greater part of the protein
requirements are met by the microdrganisms; and Dogiel and Wino-
gradowa-Fedorowa (1930), as well as Westphal (1934a), considered
the daily reproductive rate to be much higher than 7 percent.

The conclusion that the ciliates are mutualists, participating in the
protein economy of the host, was deduced by Ferber (1928, 1929a)
from the observed fact that the population density increases at the time
of growth, reproduction, and lactation. He himself, like Mowry and
Becker (1930), attributed the increase to the increased nutrition and
especially to larger protein supply; but, as the latter authors pointed out,
the facts do not warrant the deduction.

Ferber pursuing the ideas advanced by C. Schwarz (1925), sug-
gested that the rdle of the ciliates may be transformation of the protein
in the plant food into easily digestible animal protein, and that in
times of increased protein need by the host, this activity is enhanced
by additions to the ciliate population. The result, of course, would be
an increased ratio of ciliate nitrogen to total nitrogen. Becker, Schulz,
and Emmerson (1930) remarked that although it is an observed fact
that such protein transformations take place, and ciliates are eventually
digested, it is doubtful if this substantially benefits the host.

Mangold (1933) recognized usefulness to the host, in the mechant-
cal rdle of the ciliates in mixture and trituration of the rumen contents,
a role which had been suggested by Bundle (1895), Scheunert (1909),
and Dogiel (1925). Conclusive evidence that there is any essential aid
to the digestive processes in the mechanical activities of the ciliate 1s,
however, lacking.

It has been found that cattle will develop and reproduce normally on
a diet that will produce symptoms of vitamin B (B,) deficiency in other
animals. It has therefore been believed that vitamins of the B complex
are synthesized within the alimentary tract of ruminants, and the micro-
organisms have been investigated in this connection. Bechdel, Honey-
well, Dutcher, and Knutsen (1928) found evidence of synthesis of the
B complex by bacteria, as others have also reported. Manusardi (1933)
investigated the possibility of synthesis of the vitamin (antiberiberic)

986 PROTOZOA AND OTHER ANIMALS

by ciliates. Separating by filtration the ciliate, bacterial, and food frac-
tions, he fed each to pigeons, together with polished rice. He concluded
that it 1s extremely improbable that the ciliate fauna has the capacity
of synthesizing vitamin B. The bacterial fraction proved to be the most
antiberiberic.

Becker was the first to carry on the obvious experiments which should
be made in investigating the significance of the ciliates to their hosts.
That is defaunation, which led to such dramatic results in Cleveland’s
work with termites. If the ciliates perform any necessary function, the
effect should become apparent in animals deprived of them. Becker,
Schulz, and Emmerson (1930) defaunated four goats, and for periods
of two and three weeks made detailed analyses of their use of nutrients.
The goats were then reinfected, and after ten days analyses were con-
tinued for the same period as before. The presence or absence of ciliates
was accompanied by little difference in the coefficients of digestibility.
There were no differences of practical significance in the digestion of
cellulose, and the goats with ciliates used only slightly more protein
than those without. Winogradow, Winogradowa-Fedorowa, and Wer-
eninow (1930) had found that raw fiber was 12.8 percent better digested
in a normally faunated than in a ciliate-free ram.

Becker and Everett (1930) compared during nineteen weeks the
growth of seven lambs with ciliates and seven without, the Protozoa
having been removed by giving some lambs copper sulphate with milk.
They found that the defaunated lambs actually grew a little more rapidly
than the others.

Poljansky and Strelkow (1935) made observations on growth of four
pairs of twin goats for ten months, beginning at the age of from one
to two and a half months. The goats were isolated from the parents so
early that they did not become naturally infected. One member of each
pair was given a ciliate population. In this experiment, also, the ciliate-
free goats of three pairs grew a little faster than the others; in one pair
the goat with ciliates gained more.

Falaschini (1935) compared for a period of fourteen months the
growth of four lambs. Two were defaunated by a milk diet after six
months, then in five weeks reinfected with ciliates. The other two were
defaunated after eight months, then continued on a normal diet with-
out ciliates. The growth curves of the four lambs corresponded.

PROTOZOA AND OTHER ANIMALS 987

These experiments demonstrate conclusively that, at least in a period
of a year or so, the host suffers no apparent detriment from lack of
ciliates. The Protozoa perform no necessary role in nutrition, nor are
their services necessary to aid the host mechanically or in keeping down
the bacterial flora. When present, they may be a source of certain
incidental benefits, but apparently the relationship can still best be de-
fined in the words of Doflein (1916). He regarded it as ordinary
commensalism, any value that the ciliates might have to the host being
minimal and incidental.

LITERATURE CITED

Abbott, R. L. 1926. Contributions to the physiology of digestion in the
Australian roach, Periplaneta australastae Fab. J. exp. Zool., 44: 219-54.

Alexeieff, A. 1909. Les Flagellés parasites de l’intestin des batraciens in-
digénes. C. R. Soc. Biol. Paris, 67: 199-201.

— 1910. Sur les Flagellés intestinaux des poissons marins. (Note preé-
liminaire.) Arch. zool. exp. gén., (5) 6: N. et R., 5-20.

— 1912. Le Parasitisme des Eugléniens et la phylogénie des Sporozoaires
sensu stricto, Arch. zool. exp. gén., (5) 10: N. et R., 73-88.

André, E. 1909. Sur un nouvel Infusoire parasite des Dendrocoeles
(Ophryoglena parasitica n. sp.). Rev. suisse Zool., 17: 273-80.

— 1910. Sur quelques Infusoires marins parasites et commensaux. Rev.
suisse Zool., 18: 173-87.
Andrews, B. J. 1930. Method and rate of protozoan refaunation in the termite
Termopsis angusticollis Hagen. Univ. Cal. Publ. Zool., 33: 449-70.
Artigas, P. de T., and S. Pacheco. 1932. Sobre um flagellado parasito do
Myopotamus coipus. Octomitus myopotami n. sp. Ann. Fac. Med. S.
Paulo, 8: 79-81.

Aschner, M. 1932. Experimentelle Untersuchungen tiber die Symbiose der
Kleiderlaus. Naturwissenschaften, 27: 501-5.

— 1934. Studies on the symbiosis of the body louse. I. Elimination of the
symbionts by centrifugalisation of the eggs. Parasitology, 26: 309-14.

Aschner, M., and E. Ries. 1933. Das Verhalten der Kleiderlaus bei Aus-
schaltung ihrer Symbionten. Eine experimentelle Symbiosestudie. Z.
Morph. Okol. Tiere, 26: 529-90.

Balamuth, W. 1939. Studies on regeneration in Protozoa. I. Cytology and
regeneration of Licnophora macfarlandi. MS, Univ. Cal. Library.

Balbiani, E. G. 1885. Sur un Infusoire cilié parasite du sang de I’Aselle
aquatique (Anoplophrya circulans). Rec. zool. suisse., 2: 277-303.

Bary, A. de. 1879. Die Erscheinung der Symbiose. Strassburg.

Beauchamp. P. de. 1911. Astasia captiva n. sp. Euglénien parasite de Catenula
lemnae Ant. Dug. Arch. zool. exp. gén. (5) 6: N. et R., 52-58.

988 PROTOZOA AND OTHER ANIMALS

Bechdel, S. I., H. E. Soneywell, R A. Dutcher, and M. H. Knutsen. 1928.
Synthesis of vitamin B in the rumen of the cow. J. biol. Chem., 80:
231-38.

Becker, E. R. 1929. Methods of rendering the rumen and reticulum of
ruminants free from their normal infusorian fauna. Proc. nat. Acad. Sci.
Wash., 15: 435-38.

—— 1932. The present status of problems relating to the ciliates of
ruminants and Equidae. Quart. Rev. Biol., 7: 282-97.

— 1933. Host-specificity and specificity of animal parasites. Amer. J. trop.
Med., 13: 505-23.

Becker, E. R., and R. C. Everett. 1930. Comparative growths of normal
and Infusoria-free lambs. Amer. J. Hyg. 11: 362-70.

Becker, E. R., and T. S. Hsiung. 1929. The method by which ruminants
acquire their fauna of Infusoria, and remarks concerning experiments
on the host-specificity of these Protozoa. Proc. nat. Acad. Sci. Wash.,
15: 684-90.

Becker, E. R., J. A. Schulz, and M. A. Emmerson. 1930. Experiments on the
physiological relationships between the stomach Infusoria of ruminants
and their hosts, with a bibliography. Iowa St. Coll. J. Sci., 4: 215-51.

Becker, E. R., and M. Talbott. 1927. The protozoan fauna of the rumen and
reticulum of American cattle. Iowa St. Coll. J. Sci., 1: 345-71.

Beckwith, T. D., and E. J. Rose. 1929. Cellulose digestion by organisms from
the termite gut. Proc. Soc. exp. Biol. N.Y., 27: 4-6.

Beers, C. D. 1938a. Structure and division in the astomatous ciliate Meta-
radiophrya asymmetrica n. sp. J. Elisha Mitchell sci. Soc., 54: 111-25.

—— 1938b. Hysterocineta eiseniae n. sp., an endoparasitic ciliate from the
earthworm Evsenia lénnbergi. Arch. Protistenk., 91: 516-25.

Beneden, P. J. van. 1876. Animal parasites and messmates. New York. Also
German and French editions.

Berghe, L. van den. 1934. Sur un ciliate parasite de pontes de mollusques
d’eau douce Glaucoma paedophthora, n. sp. C. R. Soc. Biol. Paris, 115:
1423-26.

Bishop, A. 1926. Notes upon Sieboldiellina planariarum (Siebold), a ciliate
parasite of Planaria torva. Parasitology, 18: 187-94.

—— 1932. Entamoeba aulastomi Néller. Cultivation, morphology, and
method of division; and cultivation of Hexamita sp. Parasitology, 24:
225-322

—— 1933. The morphology and division of Hexamita gigas n. sp. (Flagel-
lata). Parasitology, 25: 163-70.

—— 1935. Observations upon a “Trichomonas” from pond water. Para-
sitology, 27: 246-56.

—— 1936. Further observations upon a “Trichomonas” from pond water.
Parasitology, 28: 443-45.

PROTOZOA AND OTHER ANIMALS 989

— 1937. The method of division of Trepomonas agilis in culture. Para-
sitology, 29: 413-18.

—— 1939. A note upon the systematic position of “Trichomonas” keilini
(Bishop, 1935). Parasitology, 31: 469-72.

Boissezon, P. de. 1930. Contribution a l’étude de la biologie et de I’histo-
physiologie de Culex pipiens L. Arch. zool. exp. gén., 70: 281-431.

Brown, W. H. 1937. Nature and function of the skeletal platelets in the
Ophryoscolecidae. MS, Univ. Cal. Library.

Brumpt, E., and G. Lavier. 1924. Un Nouvel Euglénien Polyflagellé parasite
du tetard de Leptodactylus ocellatus au Brésil. Ann. Parasit. hum.
comp.,) 2: 248-52:

Buchner, P. 1930. Tier und Pflanze in Symbiose. Berlin.

Bunting, M. 1926. Studiees on the life cycle of Tetramitus rostratus Perty.
J. Morph., 42: 23-81.

Bunting, M., and D. H. Wenrich. 1929. Binary fission in the amoeboid and
flagellate phases of Tetramitus rostratus. J. Morph., 47: 37-87.

Bundle, A. 1895. Ciliate Infusorien im Coécum des Pferdes. Z. wiss. Zool.,
60: 284-350.

Buscalioni, L., and S. Comes. 1910. La digestione delle membrane vegetali
per opera dei Flagellati contenuti nell’ intestino dei Termitidi e il
problema della simbiosi. Atti Accad. gioenia, (5) 3: mem. 17, 1-16.

Bush, M. 1933. The morphology of the ciliate Haptophrya michiganensis
Woodhead and its relation to the other members of the Astomatea. Trans.
Amer, mict. SoG, 5225223-52:

—— 1934. The morphology of Haptophrya michiganensis Woodhead, an
astomatous ciliate from the intestinal tract of Hemidactylium scutatum
(Schlegel). Univ. Cal. Publ. Zool., 39: 251-76.

Butschli, O. 1889. Protozoa. III. Abt. Infusoria und System der Radiolaria
in Bronn: Klassen und Ordnungen des Thier-Reichs. Leipzig.

Calkins, G. N. 1933. The Biology of the Protozoa. 2d ed. Philadelphia.

Campbell, W. G. 1929. The chemical aspect of the destruction of oak wood
by powder-post and death watch beetles-Lyctws spp. and Xestobium sp.
Bio-chem. J., 23: 1290-93.

Cattaneo, G. 1888. Su di un Infusorio ciliato, parassito del sangue del
Carcinus maenas, Zool. Anz., 11: 456-59.

Caullery, M. 1922. Le Parasitisme et la symbiose. Paris.

Cépéde, C. 1910. Récherches sur les Infusoires astomes. Anatomie, biologie,
éthologie, parasitaire, systématique. Arch. zool. exp. gén (5) 3: 341-609.

Cépéde, C., and E. Poyarkoff. 1909. Sur un Infusoire astome Cepedella
hepatica Poyarkoff parasite du foie des Cyclas (S. corneum L.). Bull.
sci. Fr. Belg., 43: 463-75.

Certes, A. 1882. Sur les parasites intestinaux de I’huitre. C. R. Acad. Sci.
Paris, 95: 463-65.

990 PROTOZOA AND OTHER ANIMALS

Chatterji, G. C., K. N. Das, and A. N. Mitra. 1928. On an Octomitus n. sp.
found in the intestinal contents of Hylobates hoolock. J. Dep. Sci.
Calcutta Univ., 9: 21-24.

Chatton, E. 1920. Les Péridiniens parasites. Morphologie, reproduction,
éthologie. Arch. zool. exp. gén., 59: 1-475.

— 1936. Les Migrateurs horizontalement polarises de certains Péritriches.
De leur signification. Mém. Mus. Hist. nat. Belg., 3: 913-40.

Chatton, E., and A. Lwoff. 1921. Sur une famille nouvelle d’Acinétiens, les
Sphenophryidae adaptés aux branchies des mollusques acéphales. C. R.
Acad. Sci. Paris, 173: 1495-97.

—— 1922a. Sur |’évolution des Infusoires des Lamellibranches. Relations
des Hypocomidés avec les Ancistridés. Le genre Hypocomides n. gen.
CR. Acad. VSciePatis, 175: 787-90.

— 1922b. Sur l’évolution des Infusoires des Lamellibranches. Le Genre
Pelecyophrya, intermédiaire entre les Hypocomidés et les Sphénophryides.
Bourgeonnement et Conjugasion. C. R. Acad. Sci. Paris, 175: 915-17.

— 1922c. Sur l’évolution des Infusoires des Lamellibranches. Relations
des Sphénophryidés avec les Hypocomidés. C. R. Acad. Sci. Paris, 175:
1444-47,

— 1923a. Sur l’évolution des Infusoires des Lamellibranches. Les formes
primitives du phylum des Thigmotriches; le genre Thigmophrya. C. R.
Acad. Sci. Paris, 177: 81-83.

— 1923b. Un Cas remarquable d’adaptation: Ellobiophrya donacis n. g.,

n. sp., Péritriche inquilin des branches de Donax vittatus (Lamelli-

branche). C. R. Soc. Biol. Paris, 88: 749-52.

1924. Sur |’évolution des Infusoires des Lamellibranches: Morphologie
comparée des Hypocomidés. Les nouveaux genres Hypocomina et Hypo-
comella. C. R. Acad. Sci. Paris, 178: 1928-30.

— 1926. Diagnoses de Ciliés thigmotriches nouveaux. Bull. Soc. zool. Fr.,
D3 45-528

— 1929. Contribution a l'étude de l’adaptation. Ellobiophrya donacis Ch.
et Lw. Péritriche vivant sur les branchies de |’Acéphale Donax vittatus
da Costa. Bull. biol., 63: 321-49.

—— 1934a. Sur un cilié thigmotriche nouveau: Gargarius gargarius n. gen.,
n. sp., de Mytilus edulis. Bull. Soc. zool. Fr., 59: 375-76.

— 1934b. Sur un Infusoire parasite des poils sécréteurs des Crustacés
Edriophthalmes et la famille nouvelle des Pilisuctoridae. C. R. Acad.
Sci. Paris, 199: 696-99.

—— 1935. Les Ciliés apostomes. Morphologie, cytologie, éthologie, évolu-
tion, systématique. Premiére partie. Apercu historique et général. Etude
monographique des genres et des espéces. Arch. zool. exp. gén., 77:
1-453.

—— 1936a. Les Pilisuctoridae Ch. et Lw. Ciliés parasites des poils sécréteurs
des Crustacés Edriophthalmes. Polarité, orientation, et desmodexie chez
les Infusoires. Bull. biol., 70: 86-144.

PROTOZOA AND OTHER ANIMALS 991

1936b. Les Remaniements et la continuité des cinétome au cours de la

scission chez les Thigmotriches Ancistrumidés. Arch. zool. exp. gén., 78:
N. et R., 84-91.

— 1937. Sur l’enkystement d’un Thigmotriche Ancistrumidé: Proboveria
loripedis Chatton et Lwoff, et les phénomeénes qui l’accompagnent. C. R.
Soc. Biol. Paris, 124: 807-10.

Cheissin, E. [E. Chejsin}. 1928. Vorlaufige Mitteilung tiber einige parasitische
Infusorien des Bajkal-Sees. C. R. Acad. Sci. U.R.S.S., 1928 (A) : 295-99.

— 1930. Morphologische und systematische Studien uber Astomata aus
dem Baikalsee. Arch. Protistenk., 70: 531-618.

—— 1931. Infusorien Ancistridae und Boveridae aus dem Baikalsee. Arch.
Protistenk., 73: 280-304.

— 1932. Sur la morphologie et la classification des Infusoires parasitiques
de la famille des Ptychostomidae. Trav. Sta. limnol. Lac Bajkal, 2:
29-59.

Child, H. J. 1933. The anatomy and histology of the digestive tract and
associated organs in Zootermopsis nevadensis (Hagen), with some obser-
vations on morphogenesis, behavior during ecdysis, and the protozoan
fauna. MS, Univ. Cal. Library.

—— 1934. The internal anatomy of termites and the histology of the diges-
tive tract 7 Kofoid et al., Termites and termite control. Berkeley, Cal.

Cleveland, L. R. 1923. Correlation between the food and morphology of
termites and the presence of intestinal Protozoa. Amer. J. Hyg., 3:
444-61.

—— 1924, The physiological and symbiotic relationships between the in-
testinal Protozoa of termites and their host, with special reference to
Reticulitermes flavipes Kollar. Biol. Bull., 46: 178-227.

—— 1925a. The method by which Trichonympha campanula, a protozoén in
the intestine of termites, ingests solid particles of wood for food. Biol.
Bull., 48: 282-88.

—— 1925b. The ability of termites to live perhaps indefinitely on a diet of
pure cellulose. Biol. Bull., 48: 289-93.

—— 1925c. The effects of oxygenation and starvation on the symbiosis be-
tween the termite, Termopsis, and its intestinal flagellates. Biol. Bull.,
48 :309-26.

——1926. Symbiosis among animals with special reference to termites and
their intestinal flagellates. Quart. Rev. Biol., 1: 51-60.

— 1928a. Further observations and experiments on the symbiosis between
termites and their intestinal Protozoa. Biol. Bull. 54: 231-37.

—— 1928b. Tritrichomonas fecalis nov. sp. of man; its ability to grow and
multiply indefinitely in faeces diluted with tap water and in frogs and
tadpoles. Amer. J. Hyg., 8: 232-55.

— 1930. The symbiosis between the wood-feeding roach, Cryptocercus
punctulatus Scudder, and its intestinal flagellates. Anat. Rec., 47: 293-94.

O92 PROTOZOA AND OTHER ANIMALS

—— 1935. The intranuclear achromatic figure of Oxymonas grandis sp.
nov. Biol. Bull., 69: 54-63.

Cleveland, L. R., S. R. Hall, E. P. Sanders, and J. Collier. 1934. The wood-
feeding roach Cryptocercus, its Protozoa, and the symbiosis between
Protozoa and roach. Mem. Amer. Acad. Arts Sci., 17: i-x, 185-342.

Codreanu, M., and R. Codreanu. 1928. Un Nouvel Euglénien (Astastia
Chaetogastris n. sp.) parasite coelomique d’un Oligochete (Chaetogaster
diastrophus Gruith). C. R. Soc. Biol. Paris, 9: 1368-70.

Codreanu, R. 1928. Un Infusoire nouveau (Frontonia branchiostomae n. sp.)
commensal de l’Amphioxus (Branchiostoma lanceolatum Pall). C. R.
Soc. Biol., Paris, 98: 1078-80.

— 1930. Sur la phase interne du cycle évolutif de deux formes
d’Ophryoglena, Infusoires endoparasites des larves d’Ephéméres. C. R.
Acad. Sci. Paris, 190: 1154-57.

— 1934, La Présence d’Ophryoglena, Ciliés endoparasites chez les nymphes
de l’Ephémére Oligoneuria rhenana Imhoff en France. Ann. Protist.,
4: 181-83.

Cohen, W. E. 1933. An analysis of termite (Ewtermes exitiosus) mound ma-
terial. Jour. Counc. sci. industr. Res. Aust., 6: 166-69.

Collin, B. 1914. Notes protistologiques. Arch. zool. exp. gén., 54; N. et R.,
85-97.

Cross, J. B. A study of Oxymonas minor Zeliff from the termite Kalotermes
minor Hagen. Univ. Cal. Publ. Zool. (J press.)

Crouch, H. B. 1934. Observations on Hexamita mormotae n. sp., a protozoan
flagellate from the woodchuck Marmota monax (Linn). Iowa St. Coll.
Je Sei. 82 13-107.

Cuénot, L. 1891. Protozoaries commensaux et parasites des Echinodermes.
Rev. biol. nord Fr., 3: 285-300.

—— 1894. Uber Hemispeiropsis antedonis Cuén., ein an den Comatulen
lebendes Infusorium. Zool. Anz., 17: 316.

Cunha, A. M. da, and J. Muniz. 1922. Octomitus avium n. sp. Brazil-med.,
36: 386-88.

— 1927. Estudo sobre os Flagellados intestinaes das Aves do Brazil. Mem.
Inst. Osw. Cruz, 20 19-337

— 1929. Nota sobre os parasitas intestinaes do Macacus rhesus com a
descripcao de uma nova especie de Octomitus. (Suppl.) Mem. Inst. Osw.
Cruz, 53) 34-37.

Das Gupta, B. M. 1935. The occurrence of a Trepomonas sp. in the caecum
of turtles. J. Parasit., 21: 125-26.

Davis, H. S. 1923. Observations on an intestinal flagellate of the trout. J.
Parasit., 9: 153-60.

—— 1926. Octomitus salmonis, a parasitic flagellate of trout. Bull. U. S.
Bur. Fish., 42: 9-26.

Dickman, A. 1931. Studies on the intestinal flora of termites with reference to
their ability to digest cellulose. Biol, Bull., 61: 85-92.

PROTOZOA AND OTHER ANIMALS 993

Diesing, K. M. 1866. Revision der Prothelminthen. Abtheilung: Amastigen.
I. Amastigen ohne Peristom. S. B. Akad. Wiss. Wien., Math.-naturw.
Cl 22505-79.

Dobell, C. 1935. Researches on the intestinal Protozoa of monkeys and man.
VII. On the Exteromonas of macaques and Embadomonas intestinalis.
Parasitology, 27: 564-92.

Doflein, F. 1916. Lehrbuch der Protozoenkunde 4th ed., Jena.

Doflein, F., and E. Reichenow. 1927-29. Lehrbuch der Protozoenkunde. Sth
ed., Dofleins, Lehrbuch, Jean.

Dogiel, V. A. 1925. Uber die Art der Nahrung und der Nahrungsaufnahme
bei den im Darme der Huftiere parasitierenden Infusorien. Trav. Soc. Nat.
St-Pétersb. (Leningr.) Sect. Zool. et Physiol., 54: 69-93.

—— 1927. Monographie der Familie Ophryoscolecidae. Teil I. Arch.
Protistenk., 59: 1-288.

Dogiel, V. A., and T. Fedorowa. 1925. Uber den Bau und die Funktion des
inneren Skeletts der Ophyroscoleciden. Zool. Anz., 62: 97-107.

—— 1929. Uber die Zahl der Infusorien im Wiederkauermagen. Zbl. Bakt.,
(ip) (12: 135-42.

Dogiel, V. A., and A. V. Furssenko. 1921. Neue ektoparasitische Infusorien
von Landisopoden. Trav. Soc. Nat. St-Pétersb. (Leningr.), 51: 147-58,
199-202.

Dogiel, V. A., and T. Winogradowa-Fedorowa. 1930. Experimentelle unter-
suchungen zur Biologie der Infusorien des Wiederkauermagens. Wiss.
Arch. Landwirtsch. (B)3: 172-88.

Dore, W. H., and R. C. Miller. 1923. The digestion of wood by Teredo
navalis. Univ. Cal. Publ. Zool., 22: 383-400

Duboscq, O., and P. Grassé. 1934. Notes sur les protistes parasites des
termites de France. IX. L’Enkystement des flagellés de Calotermes
flavicollis. Arch. zool. exp. gén., 76: N. et R., 66-72.

Edmondson, C. H. 1906. The Protozoa of Iowa. Proc. Davenport Acad. Sci.,
ft 124.

Eikenberry, W. L., and R. A. Waldron. 1930. Educational biology. Boston.

Emik, L. O. MS,. Studies on the nutrition of Trichonympha. (Univ. Cal.
Library, 1937.) MS,. Ingestion of food by Trichonympha. Trans. Amer.
micr. Soc. (I7 press).

Entz, G. 1904. Die Fauna der kontinentalen Kochsalzwasser. Math. naturw.
Ber. Ung., 19: 89-124.

Epstein, H. 1926. Infektion des Nervensystems von Fischen durch Infusorien.
Arch, russ. protist., 5: 169-80.

Fabre-Domergue, P. 1885. Note sur les Infusoires ciliés de la Baie de
Concarneau. J. Anat. Paris., 21: 554-68.

—— 1888. Etude sur lorganisation des Urcéolaires et sur quelques genres
d'Infusoires voisins de cette famille. J. anat. Paris, 24: 214-60.

Falaschini, A. 1935. Gli infusori dell’apparato digerente degli erbivori sono

994 PROTOZOA AND OTHER ANIMALS

indispensabili per la vita dei loro ospiti? Boll. Lab. Zool. agr. Bachic.
Milano, 4 (II): 151-60.

Falck, R. 1930. Die Scheindestruktion des Koniferenholzes durch die Larven
des Hausbockes (Hytotrupes bajulus L.). Cellulose-chem., 11: 89-91.

Fantham, H. B. 1922. Some parasitic Protozoa found in South Africa. -V S.
Afr. J. Sel.9 1925332239:

— 1936. The evolution of parasitism among the Protozoa. Scientia,
Bologna, 59: 316-24.

Fauré-Fremiet, E. 1924. Contribution a la connaissance des Infusoires plank-
toniques. Bull. biol., (Suppl.), 6: 1-171.

Ferber, K. E. 1928. Die Zahl und Masse der Infusorien im Pansen und ihre
Bedeutung fiir den Eiweissaufbau beim Wiederkauer. Z. Tierz. Ziicht-
Biol., 12: 31-63.

— 1929a. Die Verinderungen der Infusorienzahl im Pansen der Wieder-
kduer im Zusammenhang mit den Verinderungen des Eiweissumsatzes.
Z. Tietz. ZuchtBiol., 15: 375-90.

— 1929b. Uber die optimale Beschaffenheit des Panseninhalts der Wieder-
kaur. Wiss. Arch. Landw., 1: 597-600.

Ferber, K. E., and T. Winogradowa-Fedorowa. 1929. Zahlung und Teilungs-
quote der Infusorien im Pansen der Wiederkauer. Biol. Zbl., 49: 321-28.

Foulke, S. G. 1884. Some notes on Manayunkia speciosa. Proc. Acad. nat. Sci.
Philad., 1884: 48-9.

Fuhrmann, O. 1894. Die Turbellarien der Umgebung von Basel. Rev. suisse
Zool., 2: 215-90.

Fulton, J. F. 1923. Trichodina pediculus and a new closely related species.
Proc. Boston Soc: mat, Hist:, 37: 1-29.

Geiman, Q. M. 1932. The intestinal Protozoa of the larvae of the crane fly,
Tipula abdominalis, J. Parasit., 19: 173.

Ghosh, E. 1925. On a new ciliate, Balantidium knowlesi sp. nov., a coelomic
parasite in Culicoides peregrinus, Parasitology, 17: 189.

Giard, A. 1880. Note sur l’existence temporaire de Myriapodes dans les
fosses nasales de l’homme, suivie de quelques reflexions sur le parasitisme
inchoatif. Bull. Sci. Fr. Belg., 12: 1-11.

Gicklhorn, J. 1925. Notiz tiber Evglena cyclopicola nov. sp. Arch. Protistenk.,
51: 542-48.

Goetsch, W. 1936. Beitrage zur Biologie des Termitenstaates. Z. Morphol.
Okol. Tiere, 31: 490-560.

Graff, L. von. 1882. Monographie der Turbellarien. I. Rhabdocoelida. Leipzig.

Graham, G. L. 1935. Géardia infections in a nematode from cattle. J. Parasit.,
20.1 27-28.

Grassé, P. P. 1924. Octomastix parvus Alex., diplozoaire parasite de la Cistude
d’Europe. C. R. Soc. Biol. Paris, 91: 439-42.

—— 1935. Parasites et parasitisme. Paris.

Grassé, P. P., and P. de Boissezon. 1929. Turchiniella culicis n.g., n. sp.

PROTOZOA AND OTHER ANIMALS 995

Infusoire parasite de I’hemocoele d’un Cw/ex adulte. Bull. Soc. zool. Fr., 54:
187-91.

Green, R. A., and E. L. Breazeale. 1937. Bacteria and the nitrogen metab-
olism of termites. J. Bac., 33: 95-96.

Gruby, and O. Delafond. 1843. Récherches sur des animalcules se développant
en grand nombre dans |’estomac et dans les intestins, pendant la diges-
tion des animaux herbivores et carnivores. C. R. Acad. Sci. Paris, 17:
1304-08.

Haas, G. 1933. Beitrige zur Kenntnis der Cytologie von Ichthyophthirius
multifiliis Foug. Arch, Protistenk., 81: 88-137.

Hall, R. P., and T. L. Jahn. 1929. On the comparative cytology of certain
euglenoid flagellates and the systematic position of the families
Euglenidae Stein and Astasiidae Bitschli. Trans. Amer. micr. Soc., 48:
388-405.

Hall, S. R. 1931. Observations on Euglena leucops, sp. nov., a parasite of
Stenostomum, with special reference to nuclear division. Biol. Bull., 60:
327-44.

Haswell, W. A. 1892. Note on the occurrence of a flagellate infusorian
as an intracellular parasite. Proc. Linn. Soc. N. S. W., (2) 7: 197-99.

1907. Parasitic Euglenae. Zool. Anz., 31: 296-97.

Haupt, A. W. 1932. Fundamentals of biology, 2d. ed., New York.

Hazard, F. O. 1937. Two new host records for the protozoan Haptophrya
michiganensis Woodhead. J. Parasit., 23: 315-16.

Hegner, R. W. 1922. Frog and toad tadpoles as sources of intestinal Pro-
tozoa for teaching purposes. Science, 56: 439-41.

—— 1923. Observations and experiments on Euglenoidina in the digestive
tract of frog and toad tadpoles. Biol. Bull., 45: 162-80.

—— 1924. Parasitism among the Protozoa. Sci. Mon., N.Y., 19: 140-45.

—— 1926a. Host-parasite relationships among human Protozoa. Proc. R.
Soc. Med., 19: 41-44.

—— 1926b. The biology of host-parasite relationships among Protozoa
living in man. Quart. Rev. Biol., 1: 393-418.

—— 1926c. Homologies and analogies between free-living and parasitic
Protozoa. Amer. Nat., 60: 516-25.

—— 1928. The evolutionary significance of the protozoan parasites of

monkeys and man. Quart. Rev. Biol., 3: 225-44.

1937. Parasite reactions to host modifications. J. Parasit., 23: 1-12.

Hegner, R., and J. Andrews (ed.). 1930. Problems and methods of research
in protozoology. New York.

Heidenreich, E. 1935. Untersuchungen an parasitischen Ciliaten aus Anne-
liden. Teil I: Systematik. Arch. Protistenk., 84: 315-92.

Hendee, E. C. 1933. The association of the termites, Kalotermes minor, Reti-
culitermes hesperus, and Zootermopsis angusticollis with fungi. Univ.
Cal. Publ. Zool., 39: 111-34.

996 PROTOZOA AND OTHER ANIMALS

Henderson, J. C. Studies of some amoebae from a termite of the genus
Cubitermes. Univ. Cal. Publ. Zool. (In press.)

Hertig, M., W. H. Taliaferro, and B. Schwartz. 1937. Report of the Com-
mittee on Terminology. J. Parasit., 23: 325-29.

Hertwig, O. 1883. Die Symbiose oder das Genossenschaftsleben im Thier-
reich. Jena.

Hetherington, A. 1933. The culture of some holotrichous ciliates. Arch.
Protistenk., 80: 255-80.

—— 1936. The precise control of growth in a pure culture of a ciliate,
Glaucoma pyriformis. Biol. Bull., 70: 426-40.

Hinshaw, W. R., E. McNeil, and C. A. Kofoid. 1938a. The presence and dis-
tribution of Hexamita sp. in turkeys in California. J. Amer. med. Ass.,
93: 160.

—— 1938b. The relationship of Hexamita sp. to an enteritis of turkey poults.
The Cornell Veterinarian, 28: 281-93.

Holdaway, F. G. 1933. The composition of different regions of mounds of
Eutermes exitiosus Hill. J. Counc. sci. industr. Res. Aust., 6: 160-65.
Hollande, A. 1939. Sur un genre nouveau de Trichomonadide libre: Coelo-

trichomastix convexas nov. gen. nov. sp. Bull. Soc. zool. Fr., 64: 114-19.

Howland, R. B. 1928. A note on Astasia captive Beauch. Science, 68: 37.

Hsiung, T. S. 1930. A monograph on the Protozoa of the large intestine of
the horse. Iowa, St. Coll. J. Sci., 4: 359-423.

Hudson, C. T., and P. H. Gosse. 1889. The Rotifera: or wheel-animalcules,
both British and foreign. London.

Hungate, R. E. 1936. Studies on the nutrition of Zootermopsis. I. The rdle
of bacteria and molds in cellulose decomposition. Zbl. Bakt., (II), 94:
240-49.

— 1938. Studies on the nutrition of Zootermopsis. I. The relative impor-
tance of the termite and the Protozoa in wood digestion. Ecology, 19:
1-25:

— 1939. Experiments on the nutrition of Zootermopsis. UI. The anaerobic
carbohydrate dissimilation by the intestinal Protozoa. Ecology, 20: 230-
45,

Hunninen, A. V., and R. Wichterman. 1936. Hyperparasitism: A species of
Hexamita (Protozoa, Flagellata) found in the reproductive systems of
Dero pristis inflata (Trematoda) from marine eels. J. Parasit., 22: 540.

Ikeda, I., and Y. Ozaki. 1918. Notes on a new Boveria species, Boveria
labialis n. sp. J. Coll. Sci. Tokyo, 40: art. 6, 1-25.

Issel, R. 1903. Ancistridi del Golfo di Napoli. Studio monografico sopra una
nuova famiglia di cigliati commensali di molluschi marini. Mitt. Zool.
Sta. Neapel, 16: 63-108.

Jahn, T. L., and W. R. McKibben. 1937. A colorless euglenoid flagellate,
Khawkinea halli n. gen., n. sp. Trans. Amer. micr. Soc., 56: 48-54.

Janda, V., and O. Jirovec. 1937. Uber kiinstlich hervorgerufenen Parasitismus

PROTOZOA AND OTHER ANIMALS O97

eines freilebenden ciliaten Glaucoma piriformis und Infekionsversuche
mit Exglena gracilis und Spirochaeta biflexa. Mém. Soc. Zool. Tchéco-
slovaque de Prague, 5: 34-57.

Jarocki, J. 1934. Two new hypocomid ciliates, Heterocineta janickii sp. n.
and H. /lwoffi sp. n., ectoparasites of Physa fontinalis (L.) and Viviparus
fasciatus Miller. Mém. Cl. Sci. Acad. polon. math. nat. (B) 1934:
167-87.

— 1935. Studies on ciliates from fresh-water molluscs. I. General remarks
on protozoan parasites of Pulmonata. Transfer experiments with species
of Heterocineta and Chaetogaster limnae?, their additional host. Some
new hypocomid ciliates. Bull. int. Acad. Cracovie, Cl. Sci. math. nat.
(B:II) 1935: 201-30.

Jarocki, J., and Z. Raabe. 1932. Uber drei neue Infusorien-Genera der
Familie Hypocomidae (Ciliata Thigmotricha), Parasiten in Susswasser-
muscheln. Bull. int. Acad. Cracovie, Cl. Sci. math. nat. (B:II) 1932:
29-45.

Jucci, C. 1932. Sulla presenza di batteriociti nel tessuto adiposo dei Termitidi.
Arch. zool. (ital.) Torino, 16: 1422-29.

Kahl, A. 1926. Neue und wenig bekannte Formen der holotrichen und
heterotrichen Ciliaten. Arch. Protistenk., 55: 197-438.

— 1930. Urtiere oder Protozoa I: Wimpertiere oder Ciliata (Infusoria) .
I. Allgemeiner Teil und Prostomata, 77 Dahl: Die Tierwelt Deutschlands,
18 Teil, Jena.

— 1931. Ibid., Il. Holotricha, 21 Teil.

—— Ibid., Il. Spirotricha, 25 Teil.

—— 1933. Ciliata libera et ectocommensalia, 72 Grimpe, A. and E. Wagler:
Die Tierwelt der Nord- und Ostsee. Lief. 23, Teil II]. C3, 29-146.
Leipzig.

—— 1934. Ciliata ectocommensalia et parasitica, 72 Grimpe, G. and E.

Wagler: Die Tierwelt der Nord- und Ostsee, Lief. 26, Teil II C4, 147-83.

Leipsig.

1935. Ibid., IV. Peritricha und Chonotricha, 30 Teil.

Katzin, L. I., and H. Kirby, Jr. 1939. The relative weights of termites and
their Protozoa. J. Parasit., 25: 444-45.

Keilin, D, 1921. On a new ciliate: Lambornella stegomyiae n. g., n. sp.,
parasitic in the body-cavity of the larvae of Stegomyza scutellaris Walker
(Diptera, Nematocera, Culicidae). Parasitology, 13: 216-24.

Kepner, W. A., and R. P. Carroll. 1923. A ciliate endoparasitic in Stenostoma
leucops. J. Parasit., 10: 99-100.

Kepner, W. A., and J. S. Carter. 1931. Ten well-defined new species of
Stenostomum. Zool. Anz., 93: 108-23.

Kidder, G. W. 1929. Streblomastix strix, morphology and mitosis. Univ. Cal.
Publ. Zool., 33: 109-24.

—— 1933a. Studies on Conchophthirius mytili De Morgan. I. Morphology
and division. Arch. Protistenk., 79: 1-24.

998 PROTOZOA AND OTHER ANIMALS

—— 1933b. Studies on Conchophthirius mytili De Morgan. II. Conjugation
and nuclear reorganization. Arch. Protistenk., 79: 25-49.

—— 1933c. On the genus Ancistruma Strand (Ancistrum Maupas). 1. The
structure and division of A. mytilz Quenn. and A. isseli Kahl. Biol. Bull.,
64: 1-20.

—— 1933d. Conchophthirius caryoclada sp. nov. (Protozoa, Ciliata). Biol.

Bull., 65: 175-78.

1933e. On the genus Ancistruma Strand (Ancistrum Maupas). II. The
conjugation and nuclear reorganization of A. isseli Kahl. Arch. Protis-
tenk., 81: 1-18.

—— 1934a. Studies on the ciliates from fresh water mussels. I. The structure
and neuromotor system of Conchophthirius anodontae Stein, C. curtus
Engl., and C. magna sp. nov. Biol. Bull., 66: 69-90.

—— 1934b. Studies on the ciliates from fresh water mussels. II. The nuclei
of Conchophthirius anodontae Stein, C. curtus Engl., and C. magna
Kidder, during binary fission. Biol. Bull., 66: 286-303.

—— 1937. The intestinal Protozoa of the wood-feeding roach Panesthia.
Parasitology, 29: 163-205.

Kidder, G. W., and F. M. Summers. 1935. Taxonomic and cytological studies
on the ciliates associated with the amphipod family Orchestiidae from the
Woods Hole district. I. The stomatous holotrichous ectocommensals.
Biol. Bull., 68: 51-68.

Kirby, H., Jr. 1927. Studies on some amoebae from the termite Mzrotermes,
with notes on some other Protozoa from the Termitidae. Quart. Jour.
mick. Sci. 7 189-222.

— 1928. A species of Proboscidiella ftom Kalotermes (Cryptotermes)
dudleyi Banks, a termite of Central America, with remarks on the oxy-
monad flagellates. Quart. Jour. micr. Sci., 72: 355-86.

— 1930. Trichomonad flagellates from termites. I. Tricercomitus gen. nov.,
and Hexamastix Alexeieff. Univ. Cal. Publ. Zool., 33: 393-444.

— 1931. Trichomonad flagellates from termites. II. Evtrichomastix and the
subfamily Trichomonadinae. Univ. Cal. Publ. Zool., 36: 171-262.

— 1932a. Two Protozoa from brine. Trans. Amer. micr. Soc., 51: 8-15.

— 1932b. Flagellates of the genus Trichonympha in termites. Univ. Cal.
Publ. Zool., 37: 349-476.

—— 1932c. Protozoa in termites of the genus Amzitermes. Parasitology, 24:
289-304.

— 1937. Host-parasite relations in the distribution of Protozoa in termites.
Univ. Cal. Publ. Zool., 41: 189-212.

— 1939, The Templeton Crocker Expedition of the California Academy of
Sciences, 1932, No. 39. Two new flagellates from termites in the genera
Coronympha Kirby, and Metacoronympha Kirby, new genus. Proc. Cal.
Acad. Sci. (4) 22: 207-20.

PROTOZOA AND OTHER ANIMALS 99

Klebs, G. 1893. Flagellatenstudien. Theil I. Z. wiss. Zool., 55: 265-351
(p. 328).

Knoth, M. 1928. Neue Versuche sur Ziichtung der im Pansen von Wieder-
kauern lebenden Ophryoscoleciden (Ciliata). Z. Parasitenk., 1: 262-82.

Koch, A. 1933a. Uber das Verhalten symbiontenfreier Sitodrepalarven. Biol.
Zbl., 53: 199-203.

— 1933b. Uber kiinstlich symbiontenfrei gemachte Insekten. Zool. Ans.,
(Supplb.) 6: 143-50.

—— 1936, Symbiosestudien. I]. Experimentelle Untersuchungen an Oryzae-
philus surinamensis L. (Cucujidae, Coleopt.) Z. Morph. Okol. Tiere,
52-0 137-60.

— 1938a. Die Bakteriensymbiose der Termiten. Zool. Anz. (Supplb.) 11:
81-90.

— 1938b. Symbiosestudien. III. Die intrazellulare Bakteriensymbiose von
Mastotermes darwiniensis Froggatt (Isoptera). Z. Morph. Okol. Tiere,
34: 534-609.

Kofoid, C. A. 1903. On the structure of Protophrya ovicola, a ciliate infu-
sorian from the brood-sac of Littorina rudis Don. Mark Anniversary
Volume, Art. 5, 111-20. New York.

— 1935. On two remarkable ciliate Protozoa from the caecum of the In-
dian elephant. Proc. nat. Acad. Sci. Wash., 21: 501-6.

Kofoid, C. A., and M. Bush. 1936. The life cycle of Parachaenia myae gen.
nov., sp. nov., a ciliate parasitic in Mya arenaria Linn. from San Francisco
Bay, California. Bull. Mus. Hist. nat. Belg., 12 (22) : 1-15.

Kofoid, C. A., and J. F. Christenson. 1934. Ciliates from Bos gaurus H. Smith.
Univ. Cal. Pub. Zool., 39: 341-92.

Kofoid, C. A., and R. F. MacLennan. 1930. Ciliates from Bos indicus Linn.
I. The genus Entodinium Stein. Univ. Cal. Publ. Zool., 33: 471-544.

—— 1932. Ciliates from Bos indicus Linn. II. A revision of Diplodininm
Schuberg. Univ. Cal. Publ. Zool., 37: 53-152.

—— 1933. Ciliates from Bos indicus Linn. III. Epidinium Crawley, Epiplas-
tron gen. nov., and Ophryoscolex Stein. Univ. Cal. Publ. Zool., 39: 1-34.

Kofoid, C. A., and O. Swezy 1919. Studies on the parasites of the termites.
On Streblomastix strix, a polymastigote flagellate with a linear plasmodial
phase. Univ. Cal. Publ. Zool., 20: 1-20.

Kofoid, C. A., ef al. (ed.) 1934. Termites and termite control. Berkeley,
Cal.

Koidzumi, M. 1921. Studies on the intestinal Protozoa found in the termites
of Japan. Parasitology, 13: 255-309.

Konig, A. 1894. Hemispeiropsis comatulae, eine neue Gattung der Urceolari-
den. S.B. Akad. Wiss. Wien, 103: 55-60.

Kotlan, A. 1923. Zur Kenntnis der Darmflagellaten aus der Hausente und
anderen Wasservégeln. Zbl. Bakt., (1) Orig. 90: 24-28.

1000 PROTOZOA AND OTHER ANIMALS

Kudo, R. R. 1939. Protozoology. Springfield.

Labbé, A. 1899. Sporozoa im Schulze, F. E.: Das Tierreich, Lief. 5, 1-180.
Berlin.

Lamborn, W. A. 1921. A protozoon pathogenic to mosquito larvae. Parasitol-
ogy, 43: 213-15.

Lavier, G. 1925. Infections héréditaires par les parasites animaux. Ann. Parasit.
hum. comp., 3: 306-21.

—— 1935. Sur le parasitisme dans I’intestin d’amphibiens, de Flagellés du
genre Trepomonas Duj. C. R. Soc. Biol. Paris, 118: 991-92.

—1936a. Sur la structure des Flagellés du genre Hexamita Duj. C. R.
Soc. Biol, Parise 120%: 1177-80.

—— 1936b. Sur quelques Flagellés intestinaux de poissons marins. Ann.
Parasit. hum. comp., 14: 278-89.

—— 1936c. Sur un Trichomonadideé libre des eaux stagnantes. Ann. Parasit.
hum. comp., 14: 359-68.

Lavier, G., and H. Galliard. 1925. Parasitisme sanguin d’un Hexamitus chez
un crapaud Bufo calamita. Ann. Parasit. hum. comp., 3: 113-15.

Leydig, F. 1857. Uber Hydatina senta. Arch, Anat. Physiol. wiss. Med., 24:
404-16,

Lichtenstein, J. L. 1921. Ophryoglena collini n. sp. parasite coelomique des
larves d’Ephéméres. C. R. Soc. Biol. Paris, 85: 794-96.

Light, S. F., and M. F. Sanford. 1927. Are the protozoan faunae of termites
specific ? Proc. Soc. exp. Biol. N.Y., 25: 95-96.

— 1928. Experimental transfaunation of termites. Univ. Cal. Publ. Zool.,

31: 269-74.
Lucas, M. S. 1934. Ciliates from Bermuda sea urchins. I. Metopws. J. R. micr.
Soc., 54: 79-93.

Lund, E. E. 1930. The effect of diet upon the intestinal fauna of Termopsis.
Univ. Cal. Publ. Zool., 36: 81-96.

Lwoff, A. 1923. Sur la nutrition des Infusoires. C. R. Acad. Sci. Paris, 176:
928-30.

—— 1924, Infection expérimentale 4 Glaucoma piriformis (Infusoire) chez
Galleria mellonella (Lépidoptére). C. R. Acad. Sci. Paris, 178: 1106-08.

— 1929. Milieux de culture et d’entretien pour Glaucoma piriformis
(Cilié) C. R. Soe. Biol. Paris, 100: 635:

—— 1932. Réchérches biochemiques sur la nutrition des Protozoaires, le
pouvoir de synthése. Monogr. Inst. Pasteur.

Lynch, J. E. 1929. Studies on the ciliates from the intestine of Strongy-
locentrotus. I. Entorhipidium gen. nov. Univ. Cal. Publ. Zool., 33: 27-56.

—— 1930. Studies on the ciliates from the intestine of Strongylocentrotus.
Il. Lechriopyla mystax gen. nov., sp. nov. Univ. Cal. Publ. Zool., 33:
307-50.

MacArthur, W. P. 1922. A holotrichous ciliate pathogenic to Theobaldia
annulata Schrank. J. R. Army med. Cps., 38: 83-92.

PROTOZOA AND OTHER ANIMALS 1001

Mackinnon, D. L. 1912. Protists parasitic in the larva of the crane fly, T7pula
sp. Parasitology, 5: 175-89.

MacLennan, R. F. 1934. The morphology of the glycogen reserves in Poly-
plastron. Arch, Protistenk., 81: 412-19.

MacLennan, R. F., and F. H. Connell. 1931. The morphology of Expoterion
pernix gen. nov., sp. nov., a holotrichous ciliate from the intestine of
Acmaea persona Eschscholtz. Univ. Cal. Publ. Zool., 36: 141-56.

Madsen, H. 1931. Bemerkungen iiber einige entozoische und freilebenden
marine Infusorien der Gattungen Uronema, Cyclidium, Cristigera, As pi-
disca und Entodiscus gen. nov. Zool. Anz., 96: 99-112.

Mangold, E. 1929. Die Verdauung der Wiederkauer 7 Handbuch der Ernah-
rung und des Stoffwechsels der landwirtschaftlichen Nutztiere, als Grund-
lagen der Fitterungslehre, 2: 107-237 (Berlin, Springer).

— 1933. Die Infusorien des Pansens und ihre Bedeutung fiir die Ernah-
rung der Wiederkauer. Biederm. Zbl., (A), n.f. 3: 161-87.

Mangold, E., and T. Radeff. 1930. Die Quelle fiir die Infektion des Wieder-
kaiiermagens mit Infusorien. Wiss. Arch. Landw. (B), 4: 173-99.
Mangold, E., and C. Schmitt-Krahmer. 1927. Die Stickstoffverteilung im Pan-
sen der Wiederkauer bei Fiitterung und Hunger und ihre Beziehung zu

den Pansen-Infusorien. Biochem. Z., 191: 411-22.

Mangold, E., and F. Usuelli. 1930. Die schadliche Wirkung der Milch und
der Verinderung der H.- Ionkonzentration auf die Infusorien des Wie-
derkiuermagens. Wiss. Arch. Landw. (B), 3: 189-201.

Mansour, K. 1936. The problem of the nutrition of wood eating insects.
C. R. XIle Int. Cong. Zool., 1: 233-41.

Mansour, K., and J. J. Mansour-Bek. 1933. Zur Frage der Holzverdauung
durch Insektenlarven. Proc. K. Akad. Wetensch., 36: 795-99.

—— 1934a. On the digestion of wood by insects. Jour. exp. Biol., 11: 243-56.

—— 1934b. The digestion of wood by insects and the supposed réle of
microorganisms. Biol. Rev., 9: 363-82.

Manusardi, L. 1933. Gli Infusori ciliati del rumine sintetizzano la vitamina
B? Boll. Lab. Zool. agr. Bachic. Milano, 4(1) : 140-48.

Margolin, S. 1930. Methods for the cultivation of cattle ciliates. Biol. Bull.,
Se ellen

Matubayasi, H. 1937. Studies on parasitic Protozoa in Japan. I. On flagel-
lates parasitic in snakes. Annot. zool. jap., 16: 245-52.

Maupas, E. 1883. Contribution a l'étude morphologique et anatomique des
Infusoires ciliés. Arch. zool. exp. gén. (2) 1: 427-664.

May, E. 1939. The behavior of the intestinal Protozoa of termites at the time
of the last ecdysis. MS, Univ. Cal. Library.

McFarland, J. 1913. Biology general and medical. 2d ed., Philadelphia.

Mercier, L., and R. Poisson. 1923. Un Cas de parasitisme accidentel d’une
Neépe par un Infusoire. C. R. Acad. Sci. Paris, 176; 1838-41.

1002 PROTOZOA AND OTHER ANIMALS

Metcalf, M. M. 1923. The opalinid ciliate infusorians. Bull. U. S. nat. Mus.,
120: 1-484.

—— 1929. Parasites and the aid they give in problems of taxonomy, geo-
graphical distribution, and paleogeography. Smithson. misc. Coll.,
81(8): 1-36.

Meyer, S. L. 1938. Haptophrya virginiensis nov. sp., a protozoan parasite of
the pickerel frog, Rana palustris Le Conte. Anat. Rec., 72, Suppl.:
54-55.

Miyashita, Y. 1927. On a new parasitic ciliate, Lada tanishi n. sp. with pre-
liminary notes on its heterogamic conjugation. Jap. J. Zool., 1: 205-18.

—— 1933. Studies on a freshwater foettingeriid ciliate Hyalospira cardinae
n. g.n. sp. Jap. J. Zool., 4: 439-60.

Mjassnikowa, M. 1930a. Sphenophrya sphaerii, ein neues Infusorium aus
Sphaerium corneum L. Arch. Protistenk., 71: 255-94.

—— 1930b. Uber einen neuen Vertreter der Familie Sphenophryidae aus
Myatruncata L. Arch. Protistenk., 72: 377-89.

Montalenti, G. 1927. Sull’ allevamento dei termiti senza 1 protozoi dell’
ampolla cecale. R.C. Accad. Lincei, (6) 6: 529-32.

— 1932. Gli enzimi digerenti e l’assorbimento delle sostanze solubili nell’
intestino delle termiti. Arch. zool. (ital.) Tonino, 16: 859-64.

— 1934. Un interessante caso di simbiosi: i flagellati dell’ intestino delle
termiti. Rasseg. faunist. Roma, 1: 25-35.

Moore, E. 1922. Octomitus salmonis, a new species of intestinal parasite in
trout. Trans. Amer. Fish, Soc., 52: 74-97.

— 1923a. Diseases of fish in state hatcheries. 12th Ann. Rep. N.Y. St.
Conserv. Comm., 66-79.

— 1923b. A report of progress on the study of trout diseases. Trans. Amer.
Fish, Soc., 53: 74-94.

— 1924. The transmission of Octomitus salmonis in the egg of trout.
Trans. Amer. Fish. Soc., 54: 54-56.

Moroff, T. 1903. Beitrag zur Kenntnis einiger Flagellaten. Arch. Protistenk.,
3: 69-106.

Mowry, H. A., and E. R. Becker. 1930. Experiments on the biology of In-
fusoria inhabiting the rumen of goats. Iowa St. Coll. J. Sci., 5: 35-60.

Nie, Dashu. 1934. Studies of the intestinal ciliates of sea urchin from Amoy.
Rep. Mar. Biol. Ass. China, 3: 81-90.

Nieschulz, O. 1922. Uber eine Astasia—Art aus dem Sitisswassernematoden
Trilobus gracilis Bst. Zool. Anz., 54: 136-38.

Noller, W., and F. Buttgereit. 1923. Uber ein neues parasitisches Protozoén
der Haustaube (Octomutus columbae nov. spec.) Zbl. Bakt., Refer (1), 75.
239-40.

Oppenheimer, C. 1925. Die Fermente und thre Wirkungen. Leipzig.

Oshima, M. 1919. Formosan termites and methods of preventing their dam-
age: Philipp. J: Sa., 15°319-83.

PROTOZOA AND OTHER ANIMALS 1003

Parker, T. J. 1893. Lessons in elementary biology. 2d, ed., London.

Penard, E. 1922. Etudes sur les Infusoires d’eau douce. Généve.

Perekropoff, G. I., and P. I. Stepanoff. 1931. On an intestinal disturbance in
man caused by Octomitus. (In Russian.) J. Mikrobiol., 13: 191-96.

— 1932. Zur Frage der Darmerkrankungen des Menschen, die durch Octo-
mitus bedingt sind. Zbl. Bakt., Orig. (1) 123: 324-30.

Pickard, E. A. 1927. The neuromotor apparatus of Boveria teredinidi Nelson,
a ciliate from the gills of Teredo navalis. Univ. Cal. Publ. Zool., 29:
405-28.

Pierantoni, O. 1934. La digestione della cellulosa e del legno negli animali
e la simbiosi delle termiti. Riv. Fis. Mat. Sci. nat., 9: 57-64.

— 1936. La simbiosi fisiologica nei termitidi xilofagi e nei loro flagellati
intestinali. Arch. zool. (ital.) Torino, 22: 135-71.

—— 1937. Osservazioni sulla simbiosi nei termitidi xilofagi e nei loro flagel-
lati intestinali—II. Defaunazione per digiuno. Arch. zool. (ital.) Torino,
25-207.

Playfair, G. I. 1921. Australian freshwater flagellates. Proc. Linn. Soc. N.S.W.,
BOm9o- 146.

Plimmer, H. G. 1912. On the blood-parasites found in animals in the Zo-
ological Gardens during the four years 1908-1911. Proc. zool. Soc. Lond.,
1912: 406-19.

Poisson, R. 1929. Apropos de I’ Anophrys maggii Cattaneo, Infusoire parasite
du sang du Carcinus maenas L. (Crustacé Décapode) ; Sur son identité
avec l’Anophrys sarcophaga Cohn. C. R. Soc. Biol. Paris, 102: 637-39.

— 1930. Observations sur Anophrys sarcophaga Cohn (=A. maggii Cat-
taneo) Infusoire holotriche marin et sur son parasitisme possible chez
certains Crustacés. Bull. biol., 64: 288-331.

Poljanskij, J. I. 1925. Drei neue parasitische Infusorien aus dem Parenchym
einiger Mollusken und Turbellarien. Arch. Protistenk., 52: 381-93.
Poljansky, G., and A. Strelkow. 1935. Uber die Wirkung der Panseninfu-
sorien auf das Wachtum der Wiederkauer. Trav. Inst. Biol. Peterhof.,

13-14: 68-87.

Powers, P. B. A. 1933a. Studies on the ciliates from sea urchins. I. General
Taxonomy. Biol. Bull., 65: 106-21.

—— 1933b. Studies on the ciliates from sea urchins, II. Entodiscus borealis
(Hentschel), (Protozoa, Ciliata), behavior and morphology. Biol. Bull.,
65: 122-36.

—— 1933c. Studies on the ciliates from Tortugas echinoids. Yearb. Carneg.
Instn., 32: 276-80.

—— 1935. Studies on the ciliates of sea urchins. A general survey of the
infestations occurring in Tortugas echinoids. Publ. Carneg. Instn., 452:
293-326.

—— 1936, Ciliates infesting Acapulco sea urchins. J. Parasit., 22: 541.

1004 PROTOZOA AND OTHER ANIMALS

—— 1937. Studies of the ciliates of sea urchins. Ann. Rep. Tortugas Lab.,
1936-19372 O13:

Poyarkoff, E. 1909. Cepedella hepatica, Cilié astome neuveau, parasite du
foie des Cyclas. C. R. Soc. Biol. Paris, 66: 96-97.

Pringsheim, H. 1932. The chemistry of the monosaccharides and of the poly-
saccharides. New York.

Raabe, Z. 1933. Untersuchungen an einigen Arten des Genus Conchophthirus
Stein. Bull. int. Acad. Cracovie, Cl. Sci. math. nat., (B: II) 1932 (8-
10) : 295-310.

— 1934a. Uber einige an den Kiemen von Mytilus edulis L. und Macoma
balthica (L.) parasitierende Ciliaten-Arten. Ann. Mus. zool. polon.,
10; 289-303.

— 1934b. Weitere Untersuchungen an einigen Arten des Genus Conchoph-
thirus Stein, Mém. Cl. Sci. Acad. polon., math. nat., (B) 6: 221-35.

— 1935. Rhynchophrya cristallina g. n., sp. n. nouvelle forme d’Infusoire
de la famille des Sphaenophryidae Chatton et Lwoff. Bull. Inst. océanogr.
Monaco, 676: 1-6.

Rankin, J. S. 1937. An ecological study of pasasites of some North Caro-
lina salamanders. Ecol. Monogr. 7: 171-269.

Rehberg, H. 1882. Eine neue Gregarine. Lagenella mobilis n. g. et n. sp. Abh.
naturw. Ver. Bremen, 7: 68-71.

Reichenow, E. 1920. Den Wiederkauer-Infusorien verwandte Formen aus
Gorilla und Schimpanse. Arch. Protistenk., 41: 1-33.

Remane, A. 1936. Gastrotricha und Kinorhyncha, Bronn’s Klassen, Bd. 4,
Abt. 2, Buch 1, Teil 2, Lief 2, Leipzig.

Reynolds, B. D. 1936. Colpoda steini, a facultative parasite of the land slug,
Agriolimax agrestis. J. Parasit., 22: 48-53.

Ripper, W. 1930. Zur Frage des Celluloseabbaus bei der Holzverdauung
xylophager Insektenlarven. Z. vergl. Physiol., 13: 314-33.

Rose, M. 1933. Sur un Infusoire Foettingéridé parasite des Siphonophores
C. R. Acad. Sci. Paris, 197: 868-69.

— 1934. Pluralité des espéces de Foettingeridae (Infusoires Apostomes)
parasites des Siphonophores de la baie d’Alger. Bull. Soc. Hist. nat. Afr.
INI 257 149-518

Rosenberg, L. E. 1936, On the viability of Tritrichomonas augusta. Trans.
Amer, micr. Soc., 55: 313-14.

Rossolimo, L. 1925. Infusoires parasites du tube digestif des Oligochétes-
genre Ptychostomum St. Arch. russ. protist., 4: 217-33.

—— 1926. Parasitische Infusorien aus dem Baikalsee. Arch. Protistenk.,
54: 468-509.

Rossolimo, L. L., and T. A. Perzewa. 1929. Zur Kenntnis einiger astomen
Infusorien: Studien an Skelettbildung. Arch. Protistenk., 67: 237-52.

Ryckeghem, J. van 1928. Hexamitus tubifici nov. sp. Ann. Soc. Sci. Brux.
(B), 48, pt. 2, Mémoires: 139-43.

PROTOZOA AND OTHER ANIMALS 1005

Scheunert, A. 1909. Verdauung. IV. Besondezheiten der Verdauung bei
Tieren mit mehrhéligen Magen (Wiederkaduer), Kaltbliitern und Végeln,
in Oppenheimer, C., Handbuch der Biochem. des Menschen und der
Tiere, 3: 2: 152-70. Jena. (Also, 1924, in 2d ed., vol. 5.)

Schieblich, M. 1929. Die Mitwirkung der Bacterien bei der Verdauung im
E. Mangold: Handbuch der Ernahrung und des Stoffwechsels der land-
wirtschaftliche Nutztiere als Grundlagen der Fiitterungslehre, 2: 310-48.
Berlin.

—— 1932. Die Bedeutung der normalen Magendarmflora fiir den Wirtsor-
ganismus. Biederm. Zbl. (A) n. f. 2: 483-501.

Schmidt, W. 1920. Untersuchungen tiber Octomitus intestinalis truttae. Arch.
Protistenk., 40: 253-89.

Schulze, P. 1924. Der Nachweis und die Verbreitung des Chitins mit einem
Anhang tiber das komplizierte Verdauungsystem der Ophryoscoleciden.
Z. Morph. Okol. Tiere, 2: 643-66.

— 1927. Noch einmal die ‘‘Skelettplatten’”” der Ophryoscoleciden. Z.
Morph. Okol. Tiere, 7: 678-89.

Schwartz, W. 1935. Untersuchungen iiber die Symbiose von Tieren mit
Pilzen und Bakterien. IV. Der Stand unserer Kenntnisse von den physio-
logischen Grundlagen der Symbiosen von Tieren mit Pilzen und Bak-
terien. Arch. Mikrobiol., 6: 369-460.

Schwarz, C. 1925. Die ernahrungsphysiologische Bedeutung der Mikroor-
ganismen in den Vormagen der Wiederkauer. Biochem. Z., 156: 130-37.

Sokoloff, D. 1933. Algunas nuevas formas de flagelados del Valle de Mexico.
An. Inst. Biol. Univ. Mex., 4: 197-206.

Stein, F. (Ritter) von. 1848. Uber die Natur der Gregarinen. Arch. Anat.

Physiol. wiss. Med., 1848: 182-223.

1860. Uber die Eintheilung der holotrichen Infusionsthiere und Stellte
einige neue Gattungen und Arten aus dieser Ordnung. $.B. béhm. Ges.
Wiss., Nat.-Math., 1860: 57-63.

—— 1861. Uber ein neues parasitisches Infusionsthier (Ptychostomum Paln-
dinarum) aus dem Darmkanal von Paludinen und uber die mit demsel-
ben zunachst verwandten Infusorienformen. S.B. boéhm. Ges. Wiss.,
1861: 85-90.

Steuer, A. 1928. Uber Ellobiopsis chattoni Caullery 1910, einen ektoparasitis-
chen Flagellaten mariner Copepoden. Arch. Protistenk., 60: 501-10.
Stevens, N. M. 1901. Studies on ciliate Infusoria. Proc. Calif. Acad. Sci. (3),

3: 1-42.

Strand, E. 1928. Miscellanea nomenclatorica zoologica et paleontologica.
Arch. Naturgesch., 92: 30-75.

Strelkow, A., G. Poljansky, and M. Issakowa-Keo. 1933. Uber die Infek-
tionswege der im Pansen und in der Haube der Wiederkduer befindlichen
Infusorien. Arch. Tierernahr. Tierz., 9: 679-97.

Studitsky, A. 1930. Eine neue Art der Gattung Prychostomum Stein (Lada

1006 PROTOZOA AND OTHER ANIMALS

Vejdovsky) Pt. rossolimoi n. sp. Eine systematische Studie. Zool. Anz.,
87: 247-56.

Summers, F. M., and G. W. Kidder. 1936. Taxonomic and cytological studies
on the ciliates associated with the amphipod family Orchestiidae from
the Woods Hole district. II. The coelozoic astomatous parasites. Arch.
Protistenk., 86: 379-403.

Svec, F. 1897. Beitrage zur Kenntnis der Infusorien Bohmens. I. Die ciliaten
Infusorien des unterpocernitzer Teiches. Bull. int. Acad. Prag, Sci. Math.
Nat., 4(2): 29-47.

Swezy, O. 1923. The pseudopodial method of feeding by trichonymphid
flagellates parasitic in wood-eating termites. Univ. Cal. Publ. Zool.,
20:391-400.

Tetrault, P. A., and W. L. Weis. 1937. Cellulose decomposition by a bac-
terial culture from the intestinal tract of termites. J. Bact., 33: 95.

Theiler, H., and S. M. Farber. 1932. Trichomonas muris, parasitic in oxyu-
rids of the white mouse. J. Parasit., 19: 169.

— 1936. Trichomonas muris, parasitic in the oxyurid nematodes, Aspicu-
laris tetraptera and Syphacia obvelata, from white mice. Parasitology,
28: 149-60.

Thomson, J. A. 1934. Biology for Everyman. London.

Thomson, J. A., and P. Geddes. 1931. Life: Outlines of general biology.
London.

Thomson, J. G. 1925. A Gvardia parasitic in a bursate nematode living in
the Viscacha. Protozoology (Suppl., J. Helminth.), No. 1, 1-6.

Trager, W. 1932. A cellulase from the symbiotic intestinal flagellates of ter-
mites and of the roach, Cryptocercus punctulatus. Bio-chem. J. 26:
1762-71.

— 1934. The cultivation of a cellulose-digesting flagellate, Trichomonas
termopsidis, and of certain other termite Protozoa. Biol. Bull., 66: 182-
90.

Treillard, M., and A. Lwoff. 1924. Sur un Infusoire parasite de la cavité
générale des larves de chironomes. Sa sexualité. C. R. Acad. Sci. Paris,
178: 1761-64.

Trier, H. J. 1926. Der Kohlehydratstoffwechsel der Panseninfusorien und
die Bedeutung der griinen Pflanzenteile fiir diese Organismen. Z. vergl.
Physiol., 4: 305-30.

Ullmann, T. 1932. Uber die Einwitkung der Fermente einiger Wirbellosen
auf polymere Kohlenhydrate. Z. vergl. Physiol., 17: 520-36.

Usuelli, F. 1930a. Starkeaufnahme und Glycogenbildung der Panseninfuso-
rien. Wiss. Arch. Landw., 3: 4-19.

— 1930b. Das Verhalten der Panseninfusorien gegeniiber cellulose und
grinen Pflanzenteilen. Wiss. Arch. Landw., 3: 368-82.

PROTOZOA AND OTHER ANIMALS 1007

—— 1930c. Gli infusori Ciliati che vivono nell’apparato digerente degli er-
bivori. Clin. vet. Milano.

Uyemura, M. 1934. Uber einige neue Ciliaten aus dem Darmkanal von japan-
ischen Echinoideen (1). Sci. Rep. Tokyo Bunrika Daig. (B)1: 181-91.

— 1935. Uber drei in der Sisswasser-Muschel (Anodonta lauta v. Mattens)
lebende Ciliaten (Conchophthirus). Sci. Rep. Tokyo Bunrika Daig.
(B) 2: 89-100.

Valkanov, A. 1928. Protistenstudien. II. Notizen tiber die Flagellaten Bul-
gariens. Arch. Protistenk., 63: 419-50.

Voigt, M. 1904. Die Rotatorien und Gastrotrichen der Umgebung von Plon.

Foschber. biol. Sta. Pl6n, 11: 1-180.

1909. Nachtrag zur Gastrotrichen-Fauna Plons. Zool. Anz., 34: 717-22.

Wallengren, H. 1895. Studier 6fver Ciliata infusorier. Arsskr., Lunds Univ.
Sa 7 pp:

Warren, E. 1932. On a ciliate protozoan inhabiting the liver of a slug. Ann.
Natal. Mus., 7: 1-53.

Watson, M. E. 1915. Studies on gregarines. Illinois biol. Monogr., 2: 215-
468.

Weineck, E. 1934. Die Celluloseverdauung bei den Ciliaten des Wieder-
kduermagens. Arch. Protistenk., 82: 169-202.

Wenrich, D. H. 1923. Variations in Evglenamorpha hegneri n. g., n. sp., from
the intestine of tadpoles. Anat. Rec., 24: 370-71.

—— 1924a. Studies on Euglenamorpha hegneri n. g., n. sp., a euglenoid
flagellate found in tadpoles. Biol. Bull., 47: 149-74.

—— 1924b. A new protozoan parasite, Amphileptus branchiarum, n. sp.,
on the gills of tadpoles. Trans. Amer. micr. Soc., 43: 191-99.

—— 1933. A species of Hexamita (Protozoa, Flagellata) from the intestine
of a monkey (Macacus rhesus). J. Parasit., 19: 225-28.

—— 1935. Host-parasite relations between parasitic Protozoa and their
hosts. Proc. Amer. phil. Soc., 75: 605-50.

Wenyon, C. M. 1926, Protozoology, a manual for medical men, veterinarians
and zoologists. New York.

Wenyon, C. M., and W. Broughton Alcock. 1924. A Trepomonas coprozoic
in human faeces. Trans. R. Soc. trop. Med. Hyg., 18: 9.

Werner, E. 1926. Der Erreger der Zelluloseverdauung bei der Rosenkaferlarve
(Potosta cuprea Fbr.) Bacillus cellulosam fermentans n. sp. Zbl. Bakt.,
67 (Il): 297-330.

Wertheim, P. 1934a. Uber die Infusorienfauna im Magen von Bos taurus L.
Ann. Mus. zool. polon., 10: 251-66.

—— 1934b. Warmeversuche mit Wiederkauermageninfusorien. Biol. Zbl.,
4990-402.

—— 1935. Infusorien aus dem Wiederkauermagen vom Gebiete Jugosla-
wiens nebst einer Ubersicht dieser Tierchen vom Balkanhalbinsel-Bereich

1008 PROTOZOA AND OTHER ANIMALS

und ein kurzer Bericht tiber die Pferdedarminfusorien, zugleich Revision
der Familie Ophryoscolecidae. (Croat, German summary). Arhiva vet.,
5: 388-526.

Westphal, A. 1934a. Studien wber Ophryoscoleciden in der Kultur. Z. Parasi-
temkew 7.37 lata

— 1934b. Ein Ziichtungsverfahren fiir die Wiederkauerinfusorien und
dessen Ergebnisse fiir die Frage nach der Bedeutung der Infusorien fiir
den Wirt. Zool. Anz., (Suppl.) 7: 207-10.

Wiedemann, J. F. 1930. Die Zelluloseverdauung bei Lamellicornierlarven.
Z. Morph. Okol. Tiere, 19: 228-58.

Winogradow, M., T. Winogradowa-Fedorowa, and A. Wereninow. 1930.
Zur Frage nach der Einwirkung der Panseininfusorien auf die Verdauug
der Wiederkauer. Zbl. Bakt., II, 81: 230-44.

Winogradowa-Fedorowa, T., and M. P. Winogradoff. 1929. Zahlungsmethode
der Gesamtzahl der im Wiederkauermagen lebenden Infusorien. Zbl.
Bakt., 78 (II) : 246-54.

Woodhead, A. E. 1928. Haptophrya michiganensts sp. nov., a protozoan
parasite of the four-toed salamander. J. Parasit., 14: 177-82.

Woodhead, A. E., and F. Kruidenier. 1936. The probable method of infec-
tion of the four-toed salamander with the protozoén, Haptoyhrya michi-
ganensis. J. Parasit., 22: 107-8.

Yagiu, R. 1933. Studies on the ciliates from the intestine of Amthocidaris
crassispina (A. Agassiz). I. Cyclidium ozakii sp. nov. & Strombilidium
rapulum sp. nov. J. Sci. Hiroshima Univ., (B:I) 2: 211-22.

—— 1934. Studies on the ciliates from the intestine of Anthocidaris crassis-
pina (A. Agassiz). II. Cryptochilidium sigmoides sp. nov. and Crypto-
chilidium minor sp. n. J. Sci. Hiroshima Univ., (B:1) 3: 25-31.

—— 1935. Studies on the ciliates from sea urchins of Yaku Island, with a
description of a new species, Cryptochilidium ozakii sp. nov. J. Sc.
Hiroshima Univ. (B:1)3: 139-47.

Yamasaki, M. 1937a. Studies on the intestinal Protozoa of termites. III. The
distribution of glycogen in the bodies of intestinal flagellates of termites,
Leucotermes (Reticulitermes) speratus and Coptotermes formosanus.
Mem. Coll. Sci. Kyoto, (B)12: 211-24.

—— 1937b. Studies on the intestinal Protozoa of termites IV. Glycogen in
the body of Trichonympha agilis var. japonica under experimental con-
ditions. Mem. Coll. Sci. Kyoto, (B)12: 225-35.

Yonge, C. M. 1925. The digestion of cellulose by invertebrates. Sci. Prog.,
20: 242-48.

Zerling, Mlle. 1933. Une Astasza sans flagelle, Astasia doridis n. sp., parasite
des pontes de nudibranches. C. R. Soc. Biol. Paris, 112: 643-44.

Zick, Karl. 1928. Urceolaria korschelti n. sp., eine neue marine Urceolarine,
nebst einem Uberblick tiber die Urceolarinen. Z. wiss. Zool., 132: 355-
403.

CHAPTER XX

ORGANISMS LIVING ON AND IN PROTOZOA
HAROLD Kirpy, JR.’

PROTOZOA as a group may be hosts of a great variety of other organisms.
Some of these are epibiotic, and in them the relationship ranges from
occasional phoresy to obligatory and constant association. True ectopara-
sitism exists in some epibionts, though often the distinction from preda-
tism is disputable. Protozoa are not so constituted as to be capable of
harboring inquilines; all endobionts are intracellular. Unless autotrophic,
therefore, endobiotic forms are parasites in the sense that they are de-
pendent on their hosts in nutrition. In many instances, however, the
protozoan suffers no apparent detriment from the relationship; and
sometimes the association of host and symbiont is constant. (Symbiosis
is used as a collective term, including commensalism, mutualism, and
parasitism.) It may be, even, that there is mutual advantage; but only
autotrophic forms are in a position to confer the commonest type of
benefit in mutualism, a nutritive one. It has been suggested, although
not demonstrated, that certain intracellular microdrganisms may produce
enzymes that function in the nutritional metabolism of the host. Many
endobionts are more or less destructive parasites, which cause injury
or death; that is true, so far as is known, of all that invade the nucleus.

When the association has an obligatory and constant character, as in
the occurrence of bacteria on the body surface of certain Protozoa, or
of bacteria present in certain areas of the cytoplasm of all or almost
all specimens, the error has often been made of interpreting the sym-
bionts as structures of the host. When they are present only occasionally,
they have sometimes been mistakenly regarded as representing occasional
phases in the life history of the host, that is, reproductive phases. In-
creasing knowledge of the symbionts of the Protozoa has corrected most

* Assistance rendered by personnel of Work Projects Administration, Official Project
number 65-1-08-113, Unit C1, is acknowledged.

1010 PARASITES OF PROTOZOA

of these errors, but there are sometimes greater difficulties in the way
of correct interpretation than might be supposed.

A large number of organisms symbiotic with Protozoa are Schizo-
mycetes or Phycomycetes. In the latter group, Chytridiales are especially
widespread, as parasites of both the cytoplasm and the nucleus. There
are in all the major groups of Protozoa species that are symbiotic with
other Protozoa. Some groups of these, such as the Metchnikovellidae
and endozoic Suctoria, are known only from hosts of this phylum. Only
a few Metazoa occur as parasites in Protozoa. Although the relative size
relationships are sometimes such as to make it possible, parasitization
by these higher forms is not less prevalent than might be expected, in
the light of the infrequency, in general, of intracellular parasitism by
Metazoa.

Cyanellae, chlorellae, and xanthellae have not been included in this
account of symbionts of Protozoa. The last two types, at least, are
widespread in members of many free-living groups, the former in fresh-
water forms, the latter in marine species. Although inhabiting the
cytoplasm, the nutritive processes of these organisms are autotrophic;
and they are not necessarily dependent on the host. Their relationship
to their hosts is often cited as mutualistic, in one way or another. The
problem is in part the same as that of the relationship of similar endo-
symbionts to many invertebrate animals—a problem that has been rfe-
viewed by Buchner (1930). Pascher (1929) discussed the endosym-
biosis of blue-green algae in Pawlinella chromatophora and some other
Protozoa, as well as in algae. Lackey (1936) described ‘blue chromato-
phores” in Paulimella and a number of flagellates, although he failed
to recognize them as resembling Pascher’s cyanellae. Goetsch and Scheur-
ing (1926) discussed the relationship of the alga Chlorella to proto-
zoan and some metazoan hosts. The work of Pringsheim (1928) and
others has sustained the thesis of mutualistic symbiosis between Chlorella
and Protozoa. Most of the xanthellae have been placed in the crypto-
monad genus Chrysidella. Hovasse (1923a) maintained on the basis
of nuclear characteristics that xanthellae are dinoflagellates.

EPIBIOTIC SCHIZOMYCETES
SCHIZOMYCETES ON MASTIGOPHORA
Bacteria attached to the surface, either by one end or applied full
length, occur on many flagellates. They have been known for some

PARASITES OF PROTOZOA 1011

time on Mastigamoeba aspera, small rods besetting the surface having
been described by Schulze (1875) in the original description of this
type species of the genus. He stated that these rods could best be com-
pared with certain bacteria, such as Bacterzum termo. Most of them are
applied full length to the body surface. Of other observers, some recog-
nized the similarity of the rods to bacteria and others opposed this in-
terpretation, but Penard (1905c, 1909) definitely established their bac-
terial nature. The bacteria, he found, vary in number, but there are
few individuals of the species without them. Lauterborn (1916) stated
that a sapropelic flagellate, possibly belonging to the genus Mastv-
gamoeba, possessed a yellow-green mantle of radially adherent chloro-
bacteria, which he named Chlorobacterium symbioticum.

These are the only free-living flagellates, to the writer’s knowledge,
on which bacteria have been reported. Many endozoic flagellates bear
Schizomycetes. Grassé (1926a, 1926b, and elsewhere) has done much
to increase our understanding of those microdrganisms, which many
earlier observers had mistaken for pellicular differentiations, cilia, or
flagella. Duboscq and Grassé (1926, 1927) found short rods adherent
by full length on many specimens of “Devescovina’’ hilli, showed that
these are bacteria, and named them Fusiformis hilli; and they also found
spirochetes, named Treponema hilli, adherent to all parts of the body
sutface. The former report of the rods first established the true nature
of the “‘striations,’ which Foa (1905), Janicki (1915), and Kirby
(1926b) had described on Devescovina.

In many instances the presence of certain microdrganisms is character-
istic of the species, and may indeed be considered, together with its
morphological characteristics, as an aid in taxonomy. Simpler phoretic
relationships, in which the presence of the adherent forms is only
occasional, do, however, exist between microdrganisms and some flagel-
lates. Examples are the rod-like bacteria occasionally adherent, full length,
to Hexamastix claviger (Kirby, 1930); the bacteria sometimes present
on the larger forms of Tricercomitus termopsidis (Kirby, 1930); the
occasional spirals and rods on Emtrichomastix trichopterae, E. colubro-
rum, and Octomitus intestinalis (Grassé, 1926b) ; and the lumen-dwelling
types of bacteria and spirochetes often adherent sporadically to various
termite flagellates. Boeck (1917) stated that rod-shaped bacteria at
times, in certain preparations, covered the body and adhered to the
flagella of Giardia microti.

1012 PARASITES OF PROTOZOA

Fusiformis-like Rods Adherent Full Length—The genus Fusiformis
has the type species F. termitidis Hoelling, 1910. This organism is gen-
erally free in the lumen of the hind-gut of termites. It was originally
reported in Coptotermes sp. of Brazil, has been observed by the writer

Figure 208. Fusiformis-like rods adherent to the surface of flagellates. A, B, Fusiformis
grandis and F. melolonthae on Polymastix melolonthae; C, F. melolonthae. on P.
melolonthae; D, F. legeri on P. legeri; E, F. lophomonadis on Lophomonas striata;
F-J, rod-shaped microédrganisms on Devescovina sp. from Neotermes dalbergiae. (A-E,
after Grassé, one 926b; F-J, original.)

in C. niger of Panama, and was found by Duboscq and Grassé (1926,
1927) in Glyptotermes ividipennis, where it occasionally adheres to
“Devescovina’ hill1, Chromatic granules number from one to sixteen,
according to Hoelling’s account; Hoelling identified these with nuclei.
In the form studied by the writer in C. m7ger, the chromatic granules
are well defined, usually extend the full width of the cell, and in most

PARASITES OF PROTOZOA 1013

cases number from one to four. Fusiformis hilli, although much smaller,
is similar in shape and contains one or two chromatic granules that
occupy the full width. The bacteria were also observed in transverse
fission, the occurrence of which, together with the structure, readily
distinguishes them from pellicular striations.

Ecologically, Fasiformis hilli is closer to F. termitidis than to the
“pellicular striations” on species of true Devescovina. It is abundant
in the lumen of the intestine, as well as on the surface of Crucinympha
hilli, which is not true of the “‘striations.” On the surface it is arranged
in a manner scarcely suggesting striations; it sometimes adheres by one
end, and it is inconstant in occurrence. Kirby (1938b) noted that of 100
Crucinym pha, 78 had no adherent rods, or very few.

Later in the same year that Fuséformis hilli was reported, Grassé
(1926a) described F. grandis and F. melolonthae on Polymastix melo-
lonthae (Fig. 208A, B, C,); F. legeri on Polymastix legeri (Fig.
208D); and F. lophomonadis on Lophomonas striata (Fig. 208E). F.
grandis and F. legeri adhere by one extremity. This article, and the more
extended account by Grassé (1926b), first established the true nature
of the microdrganisms that adhere by full length to Polymastix and
Lophomonas, which had been regarded as pellicular structures.

The writer has observed Fus/formis-like microdrganisms on many
polymastigote flagellates of termites. They are present on all the twenty
species of Devescovina, so constantly that no individual lacks them (Fig.
208A; Fig. 209F-J). On all except D. elongata, the rods appear almost
identical with F. lophomonadis of Lophomonas striata, both in their
morphology and their morphological relationship to the host flagellate.
They are evenly spaced, generally occur over the whole body except
the papilla, and are usually situated closer together than Grassé (1926b,
Pl. 15) indicated for F. /ophomonadis. They always adhere firmly by the
full length, and are not subject to detachment in the ordinary course
of technical manipulations. On smears in which the cytosome of the
flagellate has been much disturbed, they may have been partly or com-
pletely detached, and can then be conveniently studied. Specimens sub-
jected to such treatment may sometimes be bent considerably, as was
noted by Grassé (1926b) in F. melolonthae. Surface microérganisms
were absent from many specimens of Devescovina lemniscata of Neo-
termes insularis that had been fed on filter paper soaked previously in

1014 PARASITES OF PROTOZOA

10-percent acid fuchsin, but were never absent from normal material.
Presumably the treatment had brought about detachment of many of
the bacteria. Generally these slender microdrganisms appear homogene-
ous, but chromatic granules can be demonstrated by suitable methods.

Similar microorganisms have been found by the writer on three
of the five species of Caduceia. On C. nova and C. theobromae (Fig.
209C), they are short, slender, and are confined to a limited, sharply
bounded area at the posterior end of the body (Kirby, 1936, 1938a;

x ww

A Bh
gi ILI Many Wiig
UL ETT vs
(1 YM iy i
(CLL MLL
sua LM WOU MM yyy i

UL l

His)

iis ;

Wag nh
TINTS
x iY

Miah
WW

G14

Figure 209. Adherent microérganisms on flagellates of termites. A, Fus/formis-like
rods on Devescovina sp. from Glyptotermes niger; B, regularly arranged rods on Caduceia
sp. from Neotermes greeni; C, investment of spirochaetes and posteriorly localized rods
on Caduceia theobromae. (A, B, original; C, after Grassé, 1938.)

Grassé, 1937, 1938). On a species of Caduceia from Neotermes greent,
they are abundant on the entire surface, except the papilla; and they
show a tendency to arrangement in transverse bands, between which,
in many specimens, there is regularity in spacing (Fig. 209B).

On other genera of Devescovininae from termites, the striation-like
bacteria are altogether absent. None have been found in Foaina, on
most species of Metadevescovina, on Pseudodevescovina, or on Macro-
trichomonas, except for an occasional, irregular occurrence on the sur-
face, in a manner comparable to the situation in Cracinympha hill. The
absence is the more striking because of their universality on Devescovina
and certain species of Caduceia.

PARASITES OF PROTOZOA 1015

Rods adhere also to certain hypermastigote flagellates. They were
noted by Kirby (1926a) on Staurojoenina assimilis, but were wrongly
regarded as pellicular striations; and Cleveland ef al. (1934) found
them on Barbulanym pha, Rhynchonympha, and Urinympha of Crypto-
cercus punctulatus. Cleveland considered them to be cuticular striations,
but noted their resemblance to the adherent bacteria described by
Duboscq and Grassé (1926, 1927). Grassé (1938) identified them as
bacteria.

Spirochetes and Rods Adherent by One End.—Spirochetes occur
in great abundance in termites, mostly free in the lumen or attached
to the lining of the hind-gut, but also adherent by one extremity to
certain flagellates. It is not known whether this phoresy is obligatory
or occasional from the standpoint of the spirochete, but the former
condition is probable, at least in many instances. The presence of ad-
herent spirochetes is especially characteristic of certain Pyrsonymphinae,
Oxymonadinae, Devescovininae, and Calonymphidae among polymas-
tigotes. Spirochetes are less frequent on hypermastigotes, but do occur
on some genera (Holomastigotoides, Koidzumi, 1921; Spzrotricho-
nym pha, Sutherland, 1933; Dogiel, 1922a; Spirotrichonym phella, Suther-
land, 1933; Rostronympha, Duboscq, Grassé, and Rose, 1937). Cleve-
land et al. (1934) did not report them on either polymastigotes or hyper-
mastigotes of Cryptocercus.

On many of these flagellates, spirochetes are invariably present,
either distributed widely on the surface or localized on very definite
areas of the body. Localization is illustrated by the distribution of
spirochetes on Foaina nucleo flexa and Oxymonas grandis. On the former
flagellate, spirochetes are always present on the anterior and posterior
parts of the body; many of those on the anterior part are arranged in
a row along the surface just over the parabasal filament (Fig. 210C).
Grassé (1938) noted in another species of Foaina (mistakenly named
by him Parajoenia decipiens) that an anterior tuft of spirochetes obeyed
a fixed rule in its distribution. Localization is even more marked in
Oxymonas grandis, which bears a dense group of spirochetes on an
elongated, limited area at the base of the rostellum (Fig. 210A, B).
The rest of the body surface of this flagellate is covered with minute
epibiotic bacilli (Fig. 210A).

Many observers have mistaken spirochetes for flagella or cilia in a

1016 PARASITES OF PROTOZOA

wide range of flagellates of termites; and sometimes the same observer
interpreted them correctly on one flagellate and wrongly on another,
or even reached different conclusions concerning the filaments on differ-
ent parts of the body of the same protozoan. The spirochetes have even

Figure 210. Adherent microorganisms on flagellates of termites. A, B, small rods on
surface and localized spirochaetes at base of rostellum of Oxymonas grandis; C, spiro-
chaetes on the posterior part and on a localized area of the anterior part of Foaina sp.
from Cryptotermes merwei. (Original.)

been responsible for the erroneous classification of some polymastt-
gote flagellates as hypermastigotes. Historical data on the interpretation
of adherent spirochetes has been reviewed by Kirby (1924, 1926a);
Duboscq and Grassé (1927); and Grassé (1938). Cleveland (1928)
discussed their occurrence on flagellates.

The spirochetes range in length from only 2 uy, the minimum for

PARASITES OF PROTOZOA 1017

Treponema hilli of Crucinympha hilli, to 10 y, which is about the
average length of those on species of Devescovina; and up to 20 un, as
in the forms on Metadevescovina cus pidis of Kalotermes minor, or even
40 y in the longer species on Caduceia theobromae (Grassé, 1938). On
Ste phanonym pha sp. from Neotermes insularis, long spirochetes of from
40 to 60 , are adherent (Fig. 211). Those of an investment or group
are often comparatively uniform in length. On Psewdodevescovina uni-

Figure 211. Spirochetes adherent to Stephanonympha sp. from Neotermes insularts.
(Original. )

flagellata, which is completely covered with spirochetes, simulating a
dense coat of cilia, the majority have a length of from 8 to 10 u.
Caduceia theobromae is similarly covered by spirochetes from 4 to 6 y1
long, except for the area occupied by the above-mentioned Fusiformzs-
like rods (Fig. 209C).

The spiral of short spirochetes has only one or two turns. Two turns
were counted in those of average length (9 to 13 1.) on Metadevescovina
cus pidis, while in the longer ones, up to 20 1, there were three or four.

1018 PARASITES OF PROTOZOA

The very long ones on the above-mentioned Stephanonympha had a
much larger number of turns, but none like those have been seen on
Devescovininae.

Normally the spirochetes are in continual, very active flexuous move-
ment. They are not rigid, like Spirillaceae, although some observers
have compared them with spirilla. Their activity has been described by
Koidzumi (1921) on Holomastigotoides hartmanni, Light (1926) on
Metadevescovina debilis, Duboscg and Grassé (1927) on “Devescovina’’
hilli, Kirby (1936) on Pseudodevescovina uniflagellata, and by others.
They can be studied best in living material by dark-field illumination.
Their movements are not synchronized, and are uncodrdinated either in
direction or activity. The difference between this movement and that
of cilia or flagella has impressed all students who have observed it.
As noted by Kirby (1936), they may move at an equally active rate
under the same environmental conditions, on moving flagellates, quiet
flagellates, dead flagellates, and detached balls of cytoplasm. This activ-
ity, together with their form, readily distinguishes them from flagella;
but distinction is less easy in fixed material, in which the form is often
less evident.

The spirochetes do not, in the writer’s experience, detach readily
in preparation of smears. Grassé (1938) stated that certain flagellates
lacking spirochetes may have lost them in consequence of fixation, but
offered no proof that this occurs. Spirochetes of P. wniflagellata were ob-
served to be rubbed off by movement of the large flagellate in close
contact with the cover glass, and severe manipulation might cause their
loss; but that treatment is more drastic than would ordinarily occur in
the preparation of specimens.

The spirochetes can be removed, however, by relatively simple meth-
ods. Light found that treatment with iodine in 70-percent alcohol freed
the bodies of most Metadevescovina debilis of spirochetes. Cleveland
(1928) discovered that all the spirochetes could be removed, both from
the surface of the Protozoa and the lumen of the gut, by feeding the
termites on cellulose thoroughly moistened with 5-percent acid fuch-
sin. That method was used by Sutherland (1933) to remove spirochetes
from Spirotrichonymphella and Stephanonympha; and it has been used
in the study of many Devescovininae by the writer. Feeding Kalotermes
hubbard: for twelve days on filter paper moistened in 5-percent aqueous

PARASITES OF PROTOZOA 1019

acid fuchsin removed the attached spirochetes, and examination by the
dark-field method showed that the tertiary flagella described by Light
(1926) were absent, proving that they also were spirochetes. The
spirochetes of Psewdodevescovina of Neotermes insularis were not re-
moved by this method, however, showing that it cannot be depended
upon as always effective.

Rods of types other than the longitudinally adherent bacteria are
less frequent than spirochetes. They occur occasionally on devescovinids,
adherent by one end; have been found abundant on Proboscidiella kofoid:
(Kirby, 1928), Joenia annectens (Franca, 1918), Oxymonas dimorpha
(Connell, 1930), and Microrhopalodina enflata (Duboscq and Grassé,
1934). Occasionally there are also long filamentous organisms, which
occur, for example, among the spirochetes on Metadevescovina debilis.

Microérganisms adherent by one end to flagellates of termites some-
times seem to be actually embedded in the ectoplasm, or to be associated
with cytoplasmic differentiations. This was described by the writer
(1936) in Pseadodevescovina uniflagellata, and it was noted that the
apparent embedded part may stain more deeply than the rest and appear
thicker (Fig. 212C). In P. ramosa (Kirby, 1938a) and P. punctata
(Grassé, 1938), there are bacteria in the ectoplasm not directly associated
with the spirochetes, but in P. wniflagellata the apparent granules are
not of the same nature. Grassé (1938) made similar observations on
adherent spirochetes of Caduceia theobromae, and interpreted the thick-
ening not as part of the spirochete but as a modification of the cytoplasm
in reaction to the microdrganism. Rounded corpuscles associated with
the point of attachment of spirochetes on Parajoenia grassii wete de-
scribed by Janicki (1915) and by Kirby (1937). These bodies seem to
be cytoplasmic structures, neither part of the spirochetes nor parasites.
A notable instance of a cytoplasmic differentiation, associated with the
point of adherence of a microdrganism, was seen by the writer in a
species of Macrotrichomonas in Procrytotermes sp. from Madagascar.
Rods 2.5-7 4 > Y4-Y% py adhere in large numbers to the posterior part
of the body of almost all specimens. Where each of the rods meets the
body is a deep-staining, cup-shaped structure (Fig. 212D, E). Rods
frequently become detached from these cytoplasmic structures, not
being so firmly adherent as are spirochetes.

It has been observed in several devescovinid flagellates with complete

1020 PARASITES OF PROTOZOA

investment of short spirochetes, that pockets may be formed inward from
the surface, enclosing some spirochetes. This was noted by Kirby (1936)
in Pseudodevescovina uniflagellata, and by Grassé (1938) in Caduceia
theobromae and P. punctata. Grassé noted further that the pockets in
the former species may become closed, so that spirochetes are enclosed

SSSA oer Tg
A RENNIN ay

‘=

ne ed
=

Ly
4
Ly

Figure 212. A, spirochetes adherent to Trichodinopsis paradoxa; B, microérganisms
on the capitulum of the axostyle of Macrotrichomonas pulchra from Glyptotermes dubius ;
C, adherent spirochetes on Pseudodevescovina uniflagellata, with enlargement in ecto-
plasm at point of adherence of each; D, E, rods adherent to Macrotrichomonas sp. from
Procryptotermes sp., showing the cup-shaped structure in the ectoplasm at the end of
each rod. (A, after Cépéde and Willem, 1912; B-E, original.)

in vacuoles in the cytoplasm. He believed also that the external, fusiform
bacilli may at times enter the cytoplasm and be digested; but this opinion
may have been based on the presence of an intracytoplasmic symbiont,
which actually is quite different, as was noted in Caduceia nova and C.
theobromae by the writer (1936, 1938a).

The possible physiological relationship between the adherent spiro-
chetes and their flagellate hosts has been considered by Cleveland (1928)
and Grassé (1938). Cleveland thought at first that they might live in
some sort of mutualistic relationship; but he found that when the spiro-

PARASITES OF PROTOZOA 1021

chetes were removed, no apparent detrimental effect on the Protozoa
developed within three months. Sutherland (1933) found that Stephano-
nympha died after detachment of the spirochetes, while Spirotrichonym-
phella showed no impairment; but other factors may have been respon-
sible for the death of the polymastigote. The writer found a marked
reduction in size of Metadevescovina debilis after spirochete detachment,
but the relationship was not proved. Grassé (1938) concluded that the
relationship with the host is at least not simple phoresy. He discussed
the possibility that diffusing substances attract and nourish the spiro-
chetes, and that localization in certain regions may be related to certain
areas of greatest diffusion, or to the chief phagocytic and absorptive areas.

Schizomycetes on Sarcodina.— Lauterborn (1916) found Amoeba
chlorochlamys, a sapropelic /imax type of rhizopod, to be characterized
by a yellow-green mantle of close-set, radially adherent chlorobacteria.
These bacteria, which he named Chlorobacterium symbioticum, were
rods about 2 ,, long. When the amoeba was inactive, the mantle com-
pletely surrounded it; in activity it opened more or less before advanc-
ing pseudopodia. As stated above, Lauterborn found the same micro-
organism on a colorless, sapropelic flagellate.

It is probable that a similar mantle of bacteria is present on D/-
namoeba mirabilis. Leidy (1879) described the surface of the body as
“bristling with minute spicules or motionless cils.’’ In the majority of
specimens he found a thick investment of hyaline jelly, at the surface
of which were abundant, minute, perpendicular rods, termed by Leidy
“bacteria-like cils.’’ The rods covering the body surface were some-
times absent; and Leidy recorded instances of their disappearance from
individuals that possessed them when first observed.

Schizomycetes on Ciliophora.—Certain schizomycetes occur in speci-
fically phoretic relationship to a number of ciliates and suctorians. The
instance earliest known was the adherence of spirochetes to Trichodi-
nopsis paradoxa, a peritrich in the intestine of Cyclostoma elegans (Fig.
212A). Earlier authors described this as possessing, unlike other peri-
trichs, a general investment of vibratile cilia (Issel, 1906). Issel found
basal granules for the supposed cilia; according to Pellissier (1936),
these are mitochondria disposed under the pellicle. Observations on
microorganisms adherent to Devescovininae (p. 1013) suggest another
explanation. Fauré-Fremiet (1909) noted that the filaments have an

¥O22 PARASITES OF PROTOZOA

undulatory movement unlike that of cilia, have an uneven distribution,
and may quit the host entirely. They differ in staining from true cilia,
and their movement remains unmodified, even though the ciliate be
crushed. Fauré-Fremiet considered them to be spirilla, but later ob-
servers (Cépéde and Willem, 1912; Bach and Quast, 1923; Pellissier,
1936) recognized them as spirochetes. Bach and Quast reported spiro-
chetes also in the gut lumen, but found them present only when Tricho-
dino psis was also present.

Collin (1912) recorded a number of instances of the presence of
adherent mircodrganisms on Suctoria. Short, rod-shaped bacteria, many
of them in division, were shown adherent full length, in a close-set
investment, on Discophrya lyngbyei (Collin, 1912, his Fig. 17). Schizo-
phytes adherent by one end, often obviously simple phoretic micro-
organisms with no closer relationships, were found on the lorica of
Acineta tuberosa and on the tentacles of Choanophrya infundibulifera.
Bacteria were adherent in a gelatinous investment on the surface of
several species of Paracineta.

More recently, especially through the work of Kahl (1928, 1932),
the presence of characteristic types of rod-shaped bacteria on the sur-
face of certain marine ciliates—chiefly sapropelic—has become known.
The rods adhere either by one end, as on Parablepharisma pellitum
(Fig. 213G), P. collare (Fig. 213H), Metopus contortus var. pellitus
(Fig. 213F), and the stalk of Epzstylis barbata (Fig. 213E); or flat, as
on Cristigera vestita (Fig. 213C, D), C. cirrifera (Fig. 213B), Blepha-
visma vestitum, Parablepharisma chlamydophorum (Fig. 2131), and
species of Sonderia. The presence of adherent bacteria is characteristic
of all members of the genus Sonderia. Many of these ciliates are covered
by a gelatinous layer, and it is to this that the bacteria adhere (Fig.
213A). Yagiu (1933) and Powers (1933, 1935) found bacilli con-
stantly adherent longitudinally to the surface of three species of Cyel7-
dium from sea urchins, a different type on each species (Fig. 214). Kirby
(1934) believed the protuberances of Metopus verrucosus (Fig. 213]),
which are irregular in distribution, to consist of groups of vertically ad-
herent bacteria. Kahl questioned this statement, but in view of what
we now know of bacteria on flagellates and ciliates it is not improbable.
Kahl (1933) maintained that the adherent bacteria are advantageous
symbionts, contributing somehow to the nutrition of their hosts; but that

=
=<
|S

Figure 213. Bacteria adherent to ciliates, A, bacteria in the gelatinous investment of
Sonderia pharyngea Kitby (= S. schizostoma Kahl ?); B, surface bacteria on Cristrgera
cirrifera Kahl; C, D, bacteria on Cristigera vestita Kahl; E, bacteria on stalk of Epistylss
barbata Gourret and Roeser; F, vertically adherent bacteria on Metopus contortus vat.
pellitus Kahl; K, on Parablepharisma pellitum Kahl; H, on Parabl. collare Kahl; I, Parabl,
chlamydophorum Kahl, with Ja, longitudinally adherent bacteria; J, vertical rodlets,
possibly adherent bacteria, on Metopus verrucosus (da Cunha) (Spirorhynchus ver-
rucosus da Cunha). (A, J, after Kirby, 1934; B-D, after Kahl, 1928; E, after Kahl, 1933

from Gourret and Roeser; F-I, after Kahl, 1932.)

1024 PARASITES OF PROTOZOA

is only speculation and is, in fact, improbable. Kahl stated that it
seemed to him inconceivable that this symbiosis is without advantage to
the host.

Dogiel (1929) observed Sarcina-like bacterial epiphytes on Didesmis
ovalis. These formed a group of regularly quadrangular form, in definite
number and arrangement, and were located in a preferred place on the
body surface. Other epiphytes, regarded as being probably bacteria, were

t
1400
4a eA)

w
WS
="
G—=
x4

| am

Figure 214. Characteristic bacteria adherent to the pellicle of Cyclidium from the
intestine of sea urchins. A, C. rhabdotectum Powers; B, C. ozakii Yagiu; C, C. stercoris
Powers. (After Powers, 1935.)

found on two species of Diplodinium. These had the form of an elongate
oval body, attached by a stalk to the pellicle. They were found also
free, ophryoscolecids being only an accidental substrate.

ENDOBIOTIC SCHIZOMYCETES

The relationship between Protozoa and bacteria that live in the cyto-
plasm or, less frequently, the nucleus, is closer than that of the surface
forms. The bacteria must obtain all their nutriment from the host, in
the body of which they multiply. The association is sometimes a con-
stant one, the host seldom, or never, being found without the customary
mictodrganisms. These are then probably not detrimental to the host,

PARASITES OF PROTOZOA 1025

but have come to occupy a normal place in the metabolism of the
combination. Other bacteria occur as occasional endobionts, present
in a variable percentage of hosts; often there are certain types that are
more or less likely to occur. Some of these are not noticeably detri-
mental to the maintenance by the host of normal activity. At the other
extreme are some that cause fatal diseases; this is true especially of the
nuclear parasites.

ASSOCIATIONS OF A CONSTANT CHARACTER

Instances in which endobiotic bacteria are always or at least usually
present are known chiefly in flagellates of termites and amoebae of the
genus Pelomyxa. Doubtless there are many other such associations
among Protozoa. Miyashita (1933) found the occurrence of abundant
rod-like bacteria in the cytoplasm of Ptychostomum (Hysterocineta)
bacteriophilum to be characteristic of the ciliate; and similar rods were
seen by Studitsky (1932) in the endoplasm of P. chattoni. Flexuous
rods from 8 to 20 y long were observed by Chatton and Lwoff (1929)
in all specimens of Ellobiophrya donacis, but not in the mantle cavity of
the lamellibranch host of the peritrich. Furthermore, the rods often
showed division and were never corroded; so that these authors con-
cluded that the microdrganism is a specific symbiont.

Schizomycetes in Pelomyxa.—So characteristic are bacteria in Pelomyxa
that Penard (1902) designated the genus as always provided with
symbiotic bacteria. Greeff (1874), who observed them in Pelomyxa
palustris, considered them to be crystals, and he had at first held them
to be seminal threads. F. E. Schulze (1875), though noting their
similarity to bacteria, agreed with Greeff that the rods are peculiar
structures of the Pelomyxa body. Leidy (1879) observed rods in his
Pelomyxa villosa (which species, according to Penard [1902], repre-
sents nothing in reality, the name having been applied to an aggregate
of several species of Pelomyxa), and noticed, as had Greeff, that many
appeared to be transversely striated. Bourne (1891) identified the rods
as bacteria, and Penard (1893) expressed the same opinion as to their
nature.

Penard (1902) described the bacteria as having a length of from
10 to 15 y, or sometimes 20 1; and in one individual there were rods
of from 40 to 50 . Leiner (1924) found the rods varying in length

1026 PARASITES OF PROTOZOA

from 1.5 to 22 y (Fig. 215B). Penard found that all the rods were
divided by equidistant transverse partitions, usually into two or three sec-
tions. Gould (1894) (Fig. 215A) noted their division into from two
to sometimes as many as nine sections; and later (1905), under the name
of Veley, she observed transverse fission of the rods. Veley stated that
when the rods are set free from the cytoplasm, they are capable of in-
dependent movement, of a kind associated with the presence of flagella,

Figure 215. Bacteria (Cladothrix pelomyxae Veley, and a small species) in Pelomyxa
palustris Greeff. (A, after Gould, 1894; B, after Leiner, 1924.)

though none were demonstrated. This movement was at first rapid, and
could readily be distinguished from Brownian movement. Leiner ob-
served no independent movement.

Attempts to cultivate the rods have given inconclusive results. Though
Veley (1905) cultured in sheep’s serum, inoculated from washed
Pelomyxa, rods which she considered to be identical with those in the
cytoplasm, Leiner (1924) concluded that the amoebae cannot be cer-
tainly freed of foreign bacteria, and that the sources of error are too
great.

Veley (1905) named the organism Cladothrix pelomyxae, noting its
resemblance to the two existing species of Sphaerotilus, of which genus,
according to Buchanan (1925), Cladothrix may probably be regarded
as a synonym.

The rods are generally aggregated in proximity to the nuclei and
refractile bodies (Fig. 215A). Penard (1902) noted that in certain

PARASITES OF PROTOZOA 1027

individuals of Pelomyxa vivipara, all the nuclei are enveloped by close-
set bacteria applied to the surface. Leiner stated that in P. palustris bac-
teria may thickly invest the nuclei, especially in animals in which the
refractile bodies are small. Veley observed jointed rods attached to the
refractile bodies, and believed that these afford them a point of attach-
ment without which the cycle would not be completed. She thought it
probable that the refractile bodies, which she considered to be protein
in nature, serve the bacteria as a food supply. Leiner found evidence
that the long rods extract glycogen from the refractile bodies.

Fortner (1934) studied the occurrence of bacteria in different forms of
P. palustris. The large, club-shaped, gray-greenish forms were free of, or
poor in bacteria. The yellow ones contained very numerous small refractile
bodies, with large numbers of bacteria in proximity to these. The small,
spherical or pyriform, milky-white type contained no refractile bodies,
in place of which were the characteristic bacteria in vacuoles. The white
forms he believed to be degenerate, and thought it conceivable that the
whole metamorphosis of Pelomyxa might be conditioned by the bacterial
infection. Leiner (1924) also noted the variability in the number of the
rods and their abundance in yellow animals. He distinguished a second
species of parasite, smaller and less numerous than the other, distributed
in the cytoplasm (Fig. 215B).

Leiner found reason to believe that when the bacteria are excessively
abundant, they become definitely injurious to the host. They may cause
hypertrophy, structural alteration, and eventual dissolution of the nuclei;
and the trophic functions of the cell appear to be disturbed. There is
decreased storage of glycogen. When Pelomyxa dies, the bacteria multi-
ply extraordinarily.

Schizomycetes in Flagellates of Termites ——There are numerous in-
stances of a constant association with intracytoplasmic bacteria among
flagellates of termites, and often the microdrganisms are restricted to
specific areas of the cell. There is not positive proof in all instances
discussed below that these are bacteria; but reaction to certain fixatives,
staining properties, comparison with known cytoplasmic inclusions, and,
frequently, observation of fission stages make it extremely probable.

There is no evidence that the bacteria are harmful to the flagellates,
though it is possible that as regards certain ones some such evidence
may eventually be adduced. They may be referred to as intracellular

1028 PARASITES OF PROTOZOA

symbionts with as much justification as may the microdrganisms in the
bacteriocytes and mycetocytes of certain insects. Pierantoni (1936) pro-
posed a hypothesis concerning the function of bacteria in flagellates of
termites. According to this hypothesis, with which Grassé (1938) ex-
pressed agreement, the bacteria function in the xylophagous nutrition
of the flagellates. The flagellates, which are unique among Protozoa
in their xylophagous habits, are, like so many wood-ingesting animals,
incapable of digesting cellulose, but depend upon symbiotic bacteria.
The bacteria are sometimes localized in “symbiotic organelles,” some-
times diffusely distributed in the cytoplasm. The hypothesis rests on
grounds similar to that of the supposed réle of the intracellular symbionts
in insects—the constant association and frequent localization suggest
the likelihood of a fundamental significance in the relationship. The
weakness of both hypotheses is patent; there is no physiological evi-
dence in their support. Furthermore, the existence of “organelles” of
the type mentioned is exceptional in xylophagous flagellates.

It is probable that the so-called chromidial zone of Joenia annectens
is a bacterial aggregate. It occurs constantly in that hypermastigote,
and has been shown or described by all students of the flagellate (Grassi
and Foa, 1904, 1911; Franga, 1918; Duboscq and Grassé, 1928, 1933,
1934; Cleveland et a/., 1934; Pierantoni, 1936). According to Duboscq
and Grassé, the bodies in this group are rods. The group surrounds the
axostyle posterior to the nucleus, often forming a broad ring. Duboscq
and Grassé (1934) figured an instance in which they form a spherical
group, not encircling the axostyle, in a nondividing flagellate; and ap-
parently in division stages they disperse. In the figures presented by
these authors there are forms of the small rods that might be interpreted
as division stages.

Grassi and Foa (1911) concluded that the area they earlier called
the chromidial zone acts as a phagocytic organ, since in experimental
feeding they found granules of carmine included in it. Duboscq and
Grassé regarded the rods as mitochondria, but their reaction to fixatives
and stains rather suggests bacteria. They are resistant to fixatives after
which true mitochondria are not demonstrable. Pierantoni considered
them to be bacteria, and showed differential staining of them and mito-
chondria in Altmann-Kull.

The writer has observed a comparable aggregate of granules or rods

PARASITES OF PROTOZOA 1029

around the axostyle of Devescovina glabra a short distance posterior
to the parabasal body. It is conspicuous in the species in several hosts,
in some of which it has been found in all specimens from several
colonies. Usually the limits of the group are well defined, and it has the
form of a thick ring around the trunk of the axostyle. The bodies are
generally short rods or granules; in some specimens from one host
the group consisted of long rods. Their properties of fixation and
staining exclude the possibility that they are mitochondria. In division
stages the bacteria are dispersed. Most species of Devescovina have no
such bacterial aggregate.

Pierantoni found no “‘chromidial zone” in Mesojoenia decipiens, but
a similar area was occupied by filamentous bacteria. Kirby (1932a)
described as a “proximo-nuclear parasite’ a group of bacteria, usually
rod-formed or filamentous and often bent or curved (Fig. 219A), located
iN a mass surrounding, or in proximity to the nucleus of all specimens
of Trichonympha campanula and T. collaris. The suggestion was made
that the organism may depend for its nutrition upon immediate proximity
to the source of the nuclear influences upon metabolism.

The peripheral granules located in the outer zone of the ectoplasm
of the flagella-bearing region of T. collaris and T. turkestanica (Kirby,
1932a), which stain with the Feulgen reaction and show stages of appar-
ent division, are probably localized bacterial symbionts. They have not
been found in other species of Trichonympha. Ectoplasmic granules,
considered by the authors to be bacteria and constant in occurrence and
distribution, were described by Kirby (1938a) as immediately under
the surface layer of Psewdodevescovina ramosa, and by Grassé (1938)
as in a similar position in P. punctata. Similar peripheral bacteria occur
in Bullanympha silvestrii (Kirby, 1938b). Grassé found minute bac-
teria more deeply situated under the surface of P. brevirostris. Kirby
(1932a) discussed the similarity between the peripheral granules of
Trichonympha and those described in certain ciliates.

A still more remarkable localization is that of certain bacteria which
occur on the capitulum of the axostyle. Not all granules that occur in
that location are held to be bacteria, but some seem definitely to be.
Rods on the capitulum of Pseudodevescovina uniflagellata were
described by Kirby (1936) as probably bacteria. These were present
in a large percentage of specimens, but not in all; and in only a very

1030 PARASITES OF PROTOZOA

small percentage were they present also in the general cytoplasm, par-
ticularly the ectoplasm. Rod-shaped microérganisms were found on the
capitulum of Macrotrichomonas pulchra from Kalotermes contracti-
cornis (Kirby, 1938a) and have since been observed in that species
from other hosts (Fig. 212B). Their variability in number and the
fact that they do not occur on all specimens, or on any specimens
in certain hosts, are in agreement with the view that they are symbionts
rather than structures of the flagellate.

The greatly expanded capitulum of a large devescovinid flagellate
from Neotermes insularis is constantly encrusted with short, stout rod-
lets which show evidence of fission. In this remarkable flagellate there
are similar rodlets in the peripheral cytoplasm, usually separated by a
narrow or a broad bacteria-free band from the capitular group.

Bacteria distributed generally in the endoplasm of termite flagellates
are frequent and of many types. Some of them are practically constant
in occurrence, as are the slender granule-containing rods in the endo-
plasm of Caduceia nova and C. theobromae (Kirby, 1936; 1938a).
Devescovinids often contain many deep-staining cytoplasmic granules
which may be bacteria. Jirovec (1931b) found two kinds of bacteria
vety abundant in the cytoplasm of every specimen of Trichonympha
serbica he studied. They were not present in T. agzlis, of which T. serbica
is probably a synonym, from termites in Spain; so probably they are
either present or absent in flagellates of different termite colonies. Para-
sites similar to one type (paired cocci) were found by Georgevitch
(1929, 1932) in T. serbica. Pierantoni (1936) found minute bacteria
present in large numbers in the cytoplasm of Trichonym pha minor and
T. agilis, and he reported their occurrence to be constant.

ASSOCIATIONS OF AN OCCASIONAL CHARACTER

Schizomycetes in Mastigophora—Bacteria in the cytoplasm of flagel-
lates of termites have been reported by Kirby (1924) in Dinenympha
fimbriata, by Duboscq and Grassé (1925) in Pyrsonympha vertens, by
Connell (1930) in Oxymonas dimorpha, by Connell (1932) in Gz-
gantomonas lighti, by Powell (1928) in Pyrsonympha major, and by
Kirby (1932a) in Trichonympha. Forms like the crescentic organism
described by Connell have been seen by the writer in occasional speci-
mens of a number of devescovinids. They are not stages of Sphaerita,

PARASITES OF PROTOZOA 1031

as Connell suggested that they might be. Organisms with a peg form,
differentiated sharply into a clear section at the broader end and a
deep-staining section, are frequent in Stephanonympha (Fig. 216A).
These have a general resemblance to the peg-formed parasite (Fig.
219D, E) in Trichonym pha cam panula (Kirby, 1932a), but are larger. A
unique type is the spindle-shaped organism (Fig. 216B), up to 20 uy
in length, with a deeply stainable area at one end, which occurs usually

Figure 216. A, microérganisms in Stephanonympha sp. from Kalotermes jeannelanus ;
B, spindle-shaped organisms in Caduceia sp. from Neotermes greeni, (Original.)

in groups in a species of Caduceia from Neotermes greeni. In most
termites the infection of Caduceia was only from 1 to 2 percent, but
occasionally about half the flagellates were parasitized. With the larger
forms were shorter, slender ones. The smallest forms were usually
gathered into stout spindle-shaped groups, suggesting origin by splitting
of a short, stout spindle. Many other bacterial parasites in termite flagel-
lates have been studied by the writer.

Spirochetes frequently invade the cytoplasm of flagellates of termites,

1032 PARASITES OF PROTOZOA

especially when the insects are on a filter-paper diet, and have been
observed in active movement. Some of these are in vacuoles, but often
they lie directly in the cytoplasm. Their condition and activity suggest
that many of them are not merely ingested as food, but invade the body
as facultative parasites.

Motile intracytoplasmic organisms were reported by Kirby (1932a)
in Trichonympha from Zootermopsis. They were, indeed, recognized
only in consequence of their relatively rapid movements, which left
clear tracks in the granular prenuclear endoplasm. One was seen in a
nucleus. Nothing is known of the detailed structure or the affinities of
these parasites, which vary greatly in size. Abundant, vigorously vibrat-
ing bodies were present in many specimens of Psewdodevescovina uni-
flagellata, among the particles of wood. Sometimes these were sufficiently
numerous to give an appearance of great activity in the cytoplasm. Like
the parasite of Trichonympha, they have not been found in preserved
material.

Few reports have been published of bacteria in flagellates other than
these of termites. Yakimoff (1930) named Micrococcus batrachorum
(s7¢) a coccus-like form which occurred, grouped in masses of irregular
form or isolated, in a small percentage of Trichomonas batrachorum.
Dangeard (1902) named Caryococcus hypertrophicus a bacterial parasite
of the nucleus of Ezglena deses, which caused a disease developing in
great intensity. This was manifested by considerable hypertrophy of
the nucleus, discoloration of plastids, and loss of the power of division.
No figure of Caryococcus exists. As Dangeard had previously described
Nacleophaga, it is probably distinct from that chytrid. Boeck (1917)
found rod-shaped bacteria in the cytoplasm of Giardia microti in certain
preparations. He considered these to be the same as those which oc-
casionally adhered externally, and referred to the relationship as parasit-
ism, leading to deleterious results.

Schizomycetes in Sarcodina—A number of records exist of cocci
and other bacteria in amoebae. Nagler (1910) found a heavy parasitic
infection of amoebae, similar to Amoeba albida Nagler, cultivated on an
agar plate. The cytoplasm was crowded with small, rounded, deep-
staining granules of variable size, considered to be micrococci; and
rod-formed and fusiform bacilli, likewise parasites, also occurred. Even-
tually all the amoebae were destroyed, with swelling and degeneration

PARASITES OF PROTOZOA 1033

of the nuclei. The author regarded the micrococcus as a facultative
parasite, which multiplied in the body after ingestion. Mackinnon
(1914) reported, in Entamoeba minchini (Léschia hartmann?) in tipulid
larvae, a similar organism, which occurred in the nucleus as well as
in the cytoplasm; and she stated that Polymastix melolonthae and Mono-
cercomonas melolonthae are also infected. Micrococcus was reported by
Dangeard (1896) in Sappinia and by Wenyon (1907) in Entamoeba
muris. Granules filling the cytoplasm of Mastigina hylae, resembling
in size, shape, and distribution the bodies called micrococci by others,
were found by Sassuchin (1928b). He mentioned their resemblance
to Chytridiales, but no sporangia were shown.

Cytoplasmic granules like micrococci were described by Alexeieff
(1912) in an amoeba, probably Lecythium sp., and in Tetramitus ros-
tratus. He considered these, however, to be produced by multiplication
of elementary corpuscles, existing as a corona of small granules around
an initial corpuscle. The initial corpuscle was a small spherule with a
central nucleus. He thought this to be a chlamydozoén, and named it
Chlamydozoin biitschlii, Later Alexeieff (1929) described the same
parasite in Monas vulgaris. The evidence, however, that these bodies
in amoebae and flagellates are filtrable viruses and his speculations on
the relationship of Chlamydozoa and Chytridiales are no more convinc-
ing to the reader than his argument that the parasite of cancer is a
chlamydozo6n, which he named (1912) Chlamydozodn perniciosum.
The reports of Alexeieff are considered here because of the possibility
that the granules described are bacterial parasites. The “initial cor-
puscles’’ are inclusions of another nature.

Mercier (1910) found bacterial filaments made up of short rods
in Endamoeba blattae. Eventually they invaded the whole cytoplasm
of a parasitized amoeba, which disintegrated. He also observed granular
bodies, believed to be parasites, in the cytoplasm of cysts.

Bacilli in masses in the cytoplasm of an amoeba, probably Vahl-
kam p fia sp., grown on an agar plate, were considered by Epstein (1935)
to be parasites. There were no indications of digestion of the bacteria,
and the masses increased in volume. In consequence of the infection,
division was supposed to be delayed; the nuclei continuing to multiply,
several nuclei occurred in the hypertrophied bodies. This article does
not carry conviction; the possibility is not excluded that the large

1034 PARASITES OF PROTOZOA

multinucleate bodies occur in consequence of abundant nutrition, and it
is not proved that the bacteria are parasites.

Schizomycetes in Ciliophora and S porozoa.—Bacterial parasites of the
nucleus and the cytoplasm have been observed repeatedly in free-living
ciliates, and some of these have been studied more intensely than any
other bacteria in Protozoa. They are of considerable interest, too, in the
history of protozodlogy, having frequently come to the attention of stu-
dents, in the latter half of the nineteenth century, and having been
variously interpreted.

J. Muller (1856) considered the question of spermatozoa in In-
fusoria, on the basis of observations by himself and his students, Lieber-
kuhn, Claparéde, and Lachmann. They observed undulatory filaments in
the cytoplasm of Stentor (which were parasites, as discussed below),
and found fine curved threads in hypertrophied nuclei (macronuclei) of
Paramecium “aurelia’ (—caudatum) . Miller commented on the presence
of these threads in what Ehrenberg had regarded as a seminal gland,
but did not commit himself as to their real nature. Claparéde and Lach-
mann (1857; 1860-61, p. 259) reported the observation of immobile
rods in the nucleus of Chilodon cucullulus and in Paramecium. Believing
that the nucleus plays an important réle in “embryo formation”
(Sphaerophrya), they advanced the hypothesis that it may at certain
times play the rdle in some individuals of a testis, in others of an ovary
(1857). Stein (1859) observed hypertrophied macronuclei of Para-
mecium “aurelia’ (—caudatum) containing fine, straight rods. He stated
that he was at first inclined to regard individuals with such nuclei as
males, the nucleus functioning as a testis and producing spermatozoa,
while in others it had the rdle of an ovary. Later, he concluded that the
spermatozoa developed in the nucleolus (micronucleus), then penetrated
the nucleus, and he pointed out the analogy with fertilization in Volvox.

Balbiani (1861) observed the rods in nuclei of the same species of
ciliate, and noted, as had the others, that they were immobile. He con-
cluded that they are parasites, which develop in the interior of the
female reproductive organ, and pointed out fundamental differences
between them and the ‘‘spermatic filaments’ enclosed in the “nucleolus”
of this animal. It was then Balbiani’s contention, developed in 1858
(1858a, 1858b), that Infusoria are hermaphroditic sexual animals, the
nucleus (macronucleus) an ovary, and the nucleolus (micronucleus)

PARASITES OF PROTOZOA 1035

a testis (the chromosome filaments having been mistaken for sperma-
tozoa). Engelmann (1862), finding these parasites in nuclei of Para-
mecium caudatum and Blepharisma lateritia, nevertheless continued to
regard them as spermatozoa; but Engelmann (1876) wrote of bacteria
parasitic in nuclei of Stylonychia mytilus. Butschli (1876), severely
criticizing the Balbiani-Stein hypothesis of sexual reproduction, reported
rods in the nucleus (macronucleus) of P. aurelia, and agreed with Balbiani
that they are parasites. He remarked that similar parasites are sometimes
found in the nucleolus (micronucleus) of many Infusoria. A review of the
earlier observations is given by Biitschli (1889, p. 1828).

Balbiani (1893), giving a figure and a brief discussion of parasites
in the macronucleus of Stentor polymorphus, remarked on the fact that
the parasitized animal appeared to be perfectly normal and continued
its ordinary functions. Zodchlorellae were neither more nor less abundant
than usual. The alteration of the nucleus might seem to be equivalent
to its artificial extirpation, as the nuclear substance had completely or
almost completely disappeared. He discussed these facts in connection
with his experimental results in the removal of the macronucleus, and
concluded that they were not in disagreement. A minimal amount of the
nuclear substance might still have been present and sufficed; or the
animal observed might have been in the limited period during which
normal life continues.

Studies of the nuclear parasites of Paramecium and their effects on the
animal were made by Hafkine (1890), Metschnikoff (1892), and
Fiveiskaja (1929); and certain observations on the consequences of
parasitism were reported by Bozler (1924). A similar parasite, studied
in detail by Petschenko (1911), was believed to be cytoplasmic. This
author stated that the possibility of confusing microérganisms which
live in the micronucleus with those that live in the cytoplasm cannot
be denied.

Hafkine distinguished three species of Holospora. H. undulata in-
vades the micronucleus and is spiraled. At the beginning the organism
is a small, fusiform corpuscle. The second species, H. elegans, occurs
also in the micronucleus, but is never associated with the first. The
vegetative stage is fusiform and more elongated and slender than the
others. The third species, H. obtusa, invades the macronucleus. It 1s
not spiraled and the two ends are rounded, instead of both or one

1036 PARASITES OF PROTOZOA

being pointed. According to Hafkine, multiplication of Holospora takes
place in two ways. During development, for a time there is a rapid
transverse division. The spiral form of H. wndulata develops after di-
vision ceases; the other two species remain straight. The dimensions in-
crease in this phase, which was supposed by Hafkine to represent a
transformation into resistant spores. H. undulata in this phase loses its
pale, transparent aspect, and becomes more refractive at one of its
extremities. This modification extends until the whole organism is re-
fractive; sometimes it is divided into three or four parts of different
refractivity. Of the other reproductive process, there are only traces in
H. undulata; but it is more frequent in the other two species. A bud
forms at one of the extremities, and grows into a cell like that from
which it originated. This type of reproduction, according to Hafkine,
makes Holospora transitional between yeasts and Schizomycetes. He
maintained, as did Metschnikoff and Fiveiskaja, that there are grounds
for not placing the parasite among the typical bacteria. Fiveiskaja studied
only H. obtusa in the macronucleus, and did not find any parasitized
micronuclei. She found the parasites to be elongated, straight, or slightly
curved rods from 12 to 30 1 in length by 0.6 to 0.8 y in width, often
showing differential refractivity or stainability. Part, up to half, or all,
of a rod might be dark and the other part or other entire rods clear—
a characteristic that appears in many of the early drawings (Butschli,
1876; Balbiani, 1893).

Petschenko (1911), who studied an organism considered to be a
cytoplasmic parasite, named it Drepanospira miilleri and assigned it to
the Spirillaceae. He remarked that the external aspect of this organism
is the same as that of Holospora undulata and H. elegans. The parasites
develop from a group of curved rods in the cytoplasm to a large ellipsoi-
dal mass, almost filling the body. In the vegetative period, the micro-
organism is a spiral with a nuclear portion near the anterior end, and it
shows helicoidal movement. The karyoplasm separates into granules and
bands, and endospores are said to be developed. There is a resting period
in which the rod is small, curved only once, and the nuclear substance
occupies from half to almost all of the cell. There is said to be no
cell division, reproduction being only by the endospores. Petschenko
stated that the essential difference from Holospora lies in the fact that
Hafkine did not establish in his parasites the presence of nuclear ele-

PARASITES OF PROTOZOA 1037

ments; but it seems possible that the differences in refractivity noted
by Hafkine may have indicated the same structure upon which Pet-
schenko based his interpretation of nuclear organization. The latter author
found micronuclei to be absent in ciliates with Drepanospira; so that,
allowing for differences in detail of observation and interpretation,
it seems not impossible that H. wndulata and D. miilleri are actually the
same.

Hafkine spread the infection by introducing infected paramecia into
cultures. In early infection he noted no anomalies in the ciliates, but
development of the parasites proceeds rapidly and by the next day all the
contents of the infected nucleus are used up. When the parasite fills
up a large part of the ciliate, development of the latter is arrested, and
the same phenomena appear as in insufficient nutrition. Bozler (1924)
and Fiveiskaja (1929) noted the vacuolization of animals in which the
macronucleus was parasitized, the accumulation of fat drops and excre-
tion granules, and the reduction and disarrangement of the trichocysts.
Food-taking, digestion, and defecation continue for a long time, accord-
ing to Fiveiskaja, but the number of food vacuoles formed becomes pro-
gressively fewer. In late stages food currents are absent, there are no food
vacuoles, and the mouth, gullet, and cytopyge may disappear. There is
partial atrophy of the ciliary coat. At first there is no change in the
activity of the pulsating vacuole; later the shape of the canals changes
and the pulsations become slower. Eventually, with large masses of
parasites, the pulsations stop and the ciliate dies.

Petschenko found that the cytoplasmic (or micronuclear) parasite
Dre panos pira miilleri causes the cytoplasm to take on an alveolar char-
acter, and that the macronucleus undergoes degenerative changes and
may fragment and dissolve. He noted that a chemical action on the cell
is indicated, waste products and secretions of the parasite entering the
cytoplasm of the cell. There is intoxicatiog of the cell, acting directly
on the cytoplasm, indirectly on the nucleus. Bozler stated that the possi-
bility that degenerative changes are to be traced back to the influence
of a bacterial toxin is not excluded; and Fiveiskaja thought that the
changes external to the nucleus were influenced by a substance excreted
from the macronuclear parasites, probably a toxin. Destruction of chro-
matin, with consequent disturbance of the normal macronuclear control
of metabolism, together with the mechanical influence of large masses

1038 PARASITES OF PROTOZOA

of parasites, may be sufficient, however, to account for the effect of
Holos pora obtusa.

Calkins (1904) recorded an infection of the macronuclei of 80 per-
cent of Paramecium caudatum, in preparations from one culture, with a
parasite that he named Caryoryctes cytoryctoides. The organisms appeared
as scattered bodies in parts of each nucleus, and have no resemblance to
Holos pora or Nucleophaga.

Bacteria are especially prevalent in the cytoplasm of many ciliates that
live in decaying matter, and Kahl (1930) stated that often these may
be regarded as symbionts (mutualists), rather than parasites. He showed,
for example, in Epalxis antiqguorum, \arge symbiotic bacilli which Penard
(1922) had described as rods. Recently the occurrence and significance
of bacteria in sapropelic ciliates has been the subject of studies by Lieb-
mann (1936a, 1936b, 1937).

Liebmann (1936a) found in Colpidium colpoda chlorobacteria that
appeared to live as facultative symbionts. They were enclosed in vacuoles
occurring more or less abundantly in the cytoplasm under conditions
of anaérobiosis and H,S content of the water, but not in the presence of
oxygen. Their appearance was definitely correlated with the amount of
H,S. Similar chlorobacteria were present in the hay infusion from which
the Col pidium came, and in normal oxygenated water these were ingested
and digested. Under anaérobic conditions with H,S, they remained alive
in the vacuoles; and Liebmann believed that through their assimilating
activity in the presence of light, H,S is reduced in amount, and energy
is contributed to the ciliate. After reserve glycogen is used up, the bac-
terial vacuoles may be attacked by digestive processes, and the ciliates
die soon thereafter.

In many other sapropelic ciliates, Liebmann (1936b, 1937) found,
together with dead bacteria, large numbers of living bacteria. These were
either packed together in parallel arrangement in bundles (Mefopus)
or distributed in the cytoplasm (Chaenia). After a time these symbionts
may be digested, and the loss is made up by taking in new saprophytes.
When this is prevented, and all symbionts are used in nutrition, the
ciliates perish, in spite of filled food vacuoles. Certain living bacteria
are therefore necessary for the ciliates’ continued life, under existing
anaérobic conditions with hydrogen sulphide. In this connection Lieb-
mann suggested that the bacteria split off oxygen, which the ciliates
use.

PARASITES OF PROTOZOA 1039

Hetherington (1932) mentioned an extensive cytoplasmic invasion
of Stentor coeruleus by bacilli, the ciliates losing their bright blue-green
color and some of their capacity for motor response. Pale stentors from
mass cultures are, he stated, often infected with a great number of
bacilli. Numerous instances of physiological regeneration occurred in the
recovery of these animals. The report did not indicate whether the
bacilli were isolated or grouped.

In the cytoplasm of Spirostomum ambiguum, a motile spirillum was
found in large numbers by Takagi (1938). He studied nine ciliates and
found all infected, and considered it probable that all in the culture
were parasitized. One hundred and six were present in one ciliate; in
another, 10 of the 67 were undergoing binary fission. A flagellum was
detected at one end of the parasite, which swam about actively in the
cytoplasm. Takagi stressed the fact that his is the first report of a cyto-
plasmic parasite with active motility in a protozoan. He did not com-
ment, however, on the observations by Miiller, Claparéde and Lachmann,
and Stein, nor on that by Kirby. Miller (1856) mentioned observations
by himself, Lieberkithn, Claparéde and Lachmann of motile threads
in Stentor; and the isolation of these by the last-named observers,
when their motility soon disappeared in the water. Butschli (1889, p.
1831) discussed these observations in his account of parasites of ciliates.
The threads occurred in the vacuoles in bundles, and displayed active
movement. Claparéde and Lachmann (1857) thought their parasitic
nature not improbable, noting their great similarity to certain vibrios.
Butschli, while admitting that the threads might be ingested food, be-
lieved it more likely that they were parasites. These forms differed from
Takagi’s in being in bundles instead of isolated.

Mangenot (1934) found rhodobacteria sufficiently abundant in a
ciliate identified as Spirostomum teres to impart to it a rose color. They
were distributed mostly in the peripheral cytoplasm. He regarded the
relationship between them and their host as parasitic or symbiotic, and
compared the “rhodelle’”’ association to that with chlorellae, xanthellae,
and cyanellae.

Irregular aggregations of minute granules (Fig. 217D) were found
in the cytoplasm of many individuals of Nyctotherus ovalis by Sassuchin
(1928a, 1934). He made various microchemical tests, excluding the
possibility that these were glycogen or glycoprotein granules, chondrio-

1040 PARASITES OF PROTOZOA

somes, or volutin; and he concluded that they were bacterial parasites,
which do not occur in all ciliates. Similar groups of granules were re-
ported by Kirby (1932b) in Nyctotherus silvestrianus.

Bacteria were found by Hesse (1909) in monocystid gregarines from
the seminal vesicles of oligochetes. Each of the species Monocystis lum
briculi, M. agilis, M. striata, Rhynchocystis pilosa, and Stomatophora
coronata had its own peculiar parasite which was unlike those of the
others. Their forms varied, in different species, from ovoid to filamen-
tous. Hesse remarked that the bacterial parasites were uncommon, but
when present attacked most individuals of a species, and often led to the
destruction of the invaded gregarines.

SPHAERITA AND NUCLEOPHAGA

HISTORICAL ACCOUNT AND DISTRIBUTION

In Free-living Protozoa.—Most of the fungi of the order Chytridiales
are parasitic in plants or animals (Fitzpatrick, 1930; Minden, 1915). In
the lower plants they occur mainly on or in algae; and a considerable num-
ber have been found in Phytomastigophora. Though most abundant in
this group of Protozoa, they attack also other free-living forms, especially
Sarcodina and cysts of ciliates (Biitschli, 1889; see also p. 1059), and
many have been encountered in parasitic Protozoa. The chytrids that are
known to be hyperparasitic in Protozoa all belong to the genera S phaerita
and Nucleophaga. These are the chytrids, also, that have most often been
found in free-living species, except for euglenid flagellates.

Carter (1856) described “irregular, botryoidal masses, dividing up
into spherical cells” in “Astasza’”’ (—Peranema). It is likely that he was
observing Sphaerita, and that the enlarged granular nuclei described in
Amoeba radiosa (?) were parasitized by Nuacleophaga. The specimens
of A. verrucosa, “partly filled with spherical ovules in the granuliferous
stage of development,” were probably heavily parasitized by chytrids.
Wallich (1863a) found that a large subspherical, granular mass ap-
peared in each of the specimens of A. villosa in a saucer; and later from
five to a dozen of these masses developed in individual specimens. He
observed extrusion and rupture of these, which he regarded as of the
nature of nuclei. He evidently was describing an increasingly heavy
infestation of a culture of amoebae by Sphaerita. The granulation of the
nucleus described by Carter (1863) in A. princips, accompanied by

PARASITES OF PROTOZOA 1041

the enlargement of the nucleus to between three and four times its
normal diameter, indicates the presence of Nucleophaga. Greeff (1866)
mistook the early plasmodial stages of Nucleophaga for young amoebae
entering the nucleus of A. ferricola.

Stein, who had mistaken parasitic bacteria for reproductive elements
of ciliates (p. 1034), observed Sphaerita-like Chytridiales in a number
of flagellates (1878, 1883). His plates include figures of what are
probably such fungi in Monas guttula, Chlamydomonas alboviridis,
Euglena viridis, Trachelomonas volvocina, T. hispida, Phacus pleuronec-
tes, Tropidocyphus octocostatus, Anisonema grande (A. acinus), Gleno-
dinium pulvisculus, Heterocapsa triquetra, and Dinopyxis laevis. In
many of these he represented the escape of minute, flagellated organisms.
He likewise concluded that these are reproductive elements, the nucleus
undergoing growth and fragmentation, and giving rise thus to endoge-
nous germs reproducing the flagellate. This theory of flagellate reproduc-
tion was accepted by Kent (1880-82), who confirmed the observations
of Stein on Ezglena and other euglenid flagellates. Ryder (1893) com-
pared an “‘endoblast”’ figured in E. viridis, from which flagellate “‘germs”’
were said to escape and become amoeboid forms developing into adult
euglenas, with Stein’s reproductive stage. Discussion of the early errors
of interpretation is given in many of the publications on chytrid para-
sites of Protozoa, particularly those of Dangeard (1886b, 1895), Penard
(1905b), Chatton and Brodsky (1909), and Mattes (1924).

The evidence for Stein’s notion of reproduction did not satisfy Klebs
(1883), who pointed out that the ‘““Keimkugel” was a sporangium of
“Chytridium spec.,” which, he stated, is one of the most frequent para-
sites of Evglena. The problem was studied independently by Dangeard,
and he arrived at the same conclusion (1886a, 1886b). The name Sphae-
rita endogena was given (1886a) to cytoplasmic chytrids in the rhizopods
Nuclearia simplex and a species of Heterophrys, which was later (1886b)
named H. dispersa. The illustrated account of the parasite (1886b) in-
cluded a report of its occurrence in Evglena viridis. Dangeard (1889a),
recorded S. endogena in Phacus pyrum, Trachelomonas volvocina, and
T. hispida; later (1889b) he described it in Euglena sanguinea and P.
alata; and in 1895 he gave a fairly complete and well-illustrated descrip-
tion of the life history of the parasite in Evglena (viridis?). Serbinow
(1907) studied the chytrid in E. viridis and E. sanguinea.

1042 PARASITES OF PROTOZOA

In more recent work, Sphaerita-like Chytridiales in free-living rhizo-
pods and flagellates have been differentiated into several species, but
a comparative account of the differential characteristics is lacking. Chat-
ton and Brodsky (1909) proposed to give the parasite of euglenids, if
separated from S. endogena, the name S. dangeardi; Skvortzow (1927)
briefly designated as S. trachelomonadis a parasite of Trachelomonas teres
var. glabra and T. swirenkoi in Manchuria; Jahn (1933) differentiated
S. phaci from Phacus pleuronectes and P. longicauda, and Gojdics
(1939) reported the same species from Exglena sanguinea. Puymaly
(1927) failed to recognize S. dangeard/, describing the life history of a
chytrid of E. viridis under the name S. endogena. Dangeard (1895),
with no great positiveness, proposed the name Psewdosphaerita euglenae
for a parasite of E. viridis in which, in the formation of the sporangium,
there is fragmentation into islets, and the contour of the sporangium
often becomes irregular and cord-like. Mitchell (1928) suggested assign-
ment to Psewdos phaerita of a parasite, found in species of Evglena, which
showed neither of these characteristics; Jahn (1933) considered at least
those Mitchell described in E. viridis to be S. dangeardi.

The parasites found by Nagler (1911b) in Evglena sanguinea ate
Sphaerita-like; but the form in the cyst, with prominent protuberances,
does not resemble Sphaerita. Mainx (1928) found Sphaerita often in
E. sanguinea and E. viridis; Giinther (1928) reported it in E. geniculata.
Further records of Sphaerita, by Alexeieff (1929), are from Monas
vulgaris and Dimastigamoeba gruberi.

Since Dangeard’s accounts (1886a, 1886b), Sphaerita in free-living
rhizopods has been studied by Chatton and Brodsky (1909) in Amoeba
limax, by Penard (1912) in A. alba, and by Mattes (1924) in A.
5 phaeronucleolus. The last observer described two new species, 5. amoebae
and S$. plasmophaga. The confused and improbable cycle of Allogromia
sp. (Cryptodifflugia sp., according to Doflein, 1909, 1911), outlined by
Prandtl (1907), probably was based on a free-living testacean, certain
small free-living flagellates, ingested Testacea, and an infection of A.
proteus with Sphaerita. Prandtl discussed the observations on supposed
reproduction by Carter (1863), Wallich (1863a), Greeff (1866), and
even those of Stein (1878), which as stated above were based on para-
sitization by chytrids; and he considered that they were really made on
“gamete formation” by Allogromia or other parasitic rhizopods. A para-

PARASITES OF PROTOZOA 1043

site described by Penard (1912) in A. ferricola appears to be a chytrid,
but it is not like typical Sphaerzta.

There seems to be only one record of Sphaerita in a free-living ciliate
—the brief account of Cejp (1935) of the parasite in Paramecium, up
to eleven sporangia occurring in a cell. Bodies like the sporangia of
Sphaerita, but with exit tubes, were shown by Collin (1912) in Acéneta
tuberosa. Chytrids in other Suctoria, found by Claperéde and Lachmann
and by Stein, are mentioned below (p. 1064).

Dangeard (1895) established the genus Nacleophaga for a parasite,
N. amoebae (not amoebaea as Penard, 1905b, and Doflein, 1907, have
it), which he studied in the nucleus of Amoeba verrucosa (A. proteus
according to Penard, 1905b). Gruber (1904) found Nuwcleophaga in
A. viridis, and supposed it to be different from Dangeard’s species, but
according to Penard (1905b) it is probably the same. Penard described
N. amoebae in A. terricola and A. sphaeronucleolus; and Doflein re-
corded the parasite in A. vespertilio Penard. Mattes also found, in the
nuclei of A. terricola and A. sphaeronucleolus, parasites which he named
Sphaerita nucleo phaga. He believed that the forms of Penard and Doflein
belonged to this same species, those of Dangeard and Gruber each being
a different species. Although he did not comment on the relationship of
the genera Sphaerita and Nucleophaga, his treatment of the chytrids
seems to indicate that he regarded the latter as synonymous with the
former. Indeed, no difference exists between the two except the habitat,
and the basis for their separation seems scarcely valid.

In Endozoic Protozoa—Because most of the studies of Chytridiales in
endozoic Protozoa are comparatively recent, there have been few errors
of interpretation. At about the time when Stein was describing germ
balls in euglenid and other flagellates, Leidy (1881) observed what is
clearly Sphaerita in Trichonympha agilis. He “suspected that they are
masses of ova-like bodies or spores,” but discussed them as inclusions
in the endosarc, not as reproductive elements. Casagrandi and Barba-
gallo (1897) described nuclei in E. co// containing small, round bodies,
equal in size, and sometimes so numerous as to fill the entire nucleus;
and they figured several of them in certain vegetative amoebae (Pl. 2,
Fig. 13). These, as suggested by Cragg (1919), were doubtless para-
sites, probably Sphaerita, and not nuclear parasites; Cragg suggested also
that the account by Craig (1911) of vegetative schizogony in this

1044 PARASITES OF PROTOZOA

amoeba was based on parasites. The statement by Craig that the nuclei
were visible in life as “brightly refractile masses of granules’ is in
keeping with the probability that the supposed nuclei were Sphaerita.
Dogiel (1916), finding sporangia of Nucleophaga in Myxomonas poly-
mor pha, thought he was observing chromosomes. The same investigator
noted what are probably Sphaerita and Nucleophaga in Joenia intermedia
and recognized the former as one of the lower fungi, but he hesitated
to interpret the latter as parasitic. Early students of Trichomonas (Wen-
yon, 1907; Kofoid and Swezy, 1915; Kuczynski, 1918; Mayer, 1920;
Wenrich, 1921) showed chytrids without interpreting them correctly.

Sphaerita has been found in many parasitic flagellates, especially those
in termites. Several species have been differentiated. Cunha and Muniz
(1923) gave the name Sphaerita minor to a parasite of Trichomonas
muris and T. gallinorum,; chytrids in Trichomonas vitali from Bufo
marinus (Pinto and Fonseca, 1926), Trichomonas muris, T. caviae, and
Eutrichomastix lacertae (Grassé, 1926b) have been assigned to the same
species. Grassé also stated that parasites, probably Sphaerita, invade the
plasma of Extrichomastix colubrorum. Sphaerita trichomonadis was
described by Crouch (1933) from Trichomonas wenrichi of Marmota
monax, and S$, chilomasticis by Cunha and Muniz (1934) from Chilo-
mastix intestinalis. Sassuchin (1931) found Sphaerita-like parasites in
Chilomastix magna of ground squirrels. In Mastigina hylae, Sassuchin
(1928b) noted parasites that are Sphaerita-like in some respects, but
are not shown grouped in sporangia; perhaps these are cocci.

Among flagellates of termites, Sphaerita, which in no instance has
been given a specific name, has been reported or figured in Joenia inter-
media (Dogiel, 1917), Staurojoenina assimilis (Kirby, 1926a), Meta-
devescovina debilis (Light, 1926), Trichonympha chattoni (Duboscq
and Grassé, 1927), Stephanonympha dogieli (Bernstein, 1928), Coro-
nympha clevelandi (Kirby, 1929), Oxymonas minor (Zeliff, 1930),
Pyrsonympha and Dinenympha (Jirovec, 1931b), Pyrsonym pha elongata
(Georgevitch, 1932), Gigantomonas lighti (Connell, 1932), and sev-
eral species of Trichonympha (Kirby, 1932a). In undescribed poly-
mastigote and some hypermastigote flagellates in the writer's collection,
S phaevita has been found to be extremely prevalent.

The presence of Sphaerita has been indicated in all species of intestinal
amoebae of man: Entamoeba histolytica (Noller, 1921; Lwoff, 1925;

PARASITES OF PROTOZOA 1045

Greenway, 1926; Bacigalupo, 1927, 1928), E. coli (Cragg, 1919; Nol-
ler, 1921; Epstein, 1922; Lwoff, 1925; Bacigalupo, 1927, 1928), Endo-
limax nana (Dobell, 1919; Noller, 1921; possibly Epstein, 1922; Green-
way, 1926; Wenyon, 1926; Bacigalupo, 1927, 1928), lodamoeba
biitschlii (Noller, 1921; Wenrich, 1937), and Dientamoeba fragilis
(Noller, 1921; Wenrich, 1940). Lwoff (1925) thought that the parasite
of Entamoeba dysenteriae (histolytica), E. coli, and Endolimax nana 1s
identical with Sphaerita endogena; but he provided the name S. normeti
for use if it is proved to be a new species.

The chytrid is equally prevalent in other endozoic amoebae. Leger
and Duboscg (1904) stated that an amoeba in Box boops (E. salpae)
is often ravaged by microspheres which lead to its destruction. Wenyon
(1907) noted the parasites in Entamoeba muris as ‘vacuoles containing
cocci’; Kessel (1924) recorded Sphaerita from the same amoeba. Becker
(1926) described S$. endamoebae from Entamoeba citelli, and the same
chytrid was found by Sassuchin, Popoff, Kudrjewzew, and Bogenko
(1930) in this amoeba of ground squirrels in Russia, though without
reference to Becker’s account. A parasite of Hyalolimax cerco pitheci was
named S, parvula by Brumpt and Lavier (1935b). Other records are from
Endamoeba simulans of termites (Kirby, 1927), Entamoeba bobaci of
Marmota bobaci (Yuan-Po, 1928), E. pitheci from Macacus rhesus
(Sassuchin et al., 1930), and Entamoeba sp. of cattle (Jirovec, 1933).
Third-degree parasitism is that of Sphaerita in entamoebae in Ze/lerzella,
reported by Stabler and Chen (1936). In almost all instances, in intes-
tinal amoebae, the parasites have been encountered only in the tropho-
zoites.

Among endozoic ciliates, aside from Chen and Stabler’s (1936) state-
ment that Sphaerita has been found in Zelleriella as well as in its
entamoeba parasites, the chytrid has been reported only in Nyctotherus
and Ophryoscolecidae. Sassuchin (1928a, 1934) found it to be common
in N. ovalis from Periplaneta (Fig. 217C, D). In ciliates of antelopes,
Dogiel (1929) described Sphaerita diplodiniorum in Diplodinium costa-
tum and Ostracodinum gracile. Jitovec (1933) gave the name S. ento-
dinii minor to a chytrid in Entodinium simplex (?), and S. entodinu
major to one in Entodinium longinucleatum. He observed also a Sphae-
vita-like parasite in an undetermined species of Entodinium, and stated
that in other Ophryoscolecidae none of these chytrids were observed.

1046 PARASITES: OF PROTOZOR

Winogradowa (1936) reported Sphaerita, as well as larger, distributed,
probably bacterial, parasites in Entodinium (Fig. 217B).

Discussing Joenia annectens and Mesojoenia decipiens, Grassi and
Foa (1911) mentioned an enormous enlargement of the nucleus by the
presence of a parasite, and reported also a parasite in the cytoplasm.
This probably is the first record of Nucleophaga in a flagellate. Its pres-
ence in Joenia intermedia and Myxomonas polymorpha (== Giganto-
monas herculea), noted by Dogiel (1917, 1916), has been mentioned
above. In Hexamastix termitis, Kirby (1930) showed some parasitized
nuclei, as Duboscq and Grassé (1933, p. 392) pointed out, but failed
to interpret them correctly. The large nuclei with numerous small, unt-
form-sized granules contained Nucleophaga, and the parasite has been
found, on reéxamination of the material. The parasite has been observed
by the writer in many Devescovininae, but not in the smaller species
of Foaina. There seems to be a lower limit in the size of nuclei in which
it can develop. Nuclear parasites of Trichonympha are considered below
(p. 1059). Psewdospora volvocis, a parasite of Volvox with apparent
affinities to the Bistadiidae, has been found infected with intranuclear
chytrids by Roskin (1927) and by Robertson (1905), the latter of
whom misinterpreted the parasite as representing gamete formation by
Pseudos pora.

Nucleophaga has been found in many endozoic Amoebidae, and
several species have been named. Lavier (1935b) reviewed most of the
accounts, with the exception of those of Kirby (1927, 1932b) and
Sassuchin (1931). The earliest observations were made in Endamoeba
blattae (Mercier, 1907, 1910; Janicki, 1909). Tyzzer (1920) found a
nuclear parasite in Pygolimax gregariniformis of chickens and turkeys.
Two amoebae of man are known to be parasitized: Endolimax nana and
lodamoeba biitschli, in which Nucleophaga was first recorded by Noller
(1921). Epstein (1922) named Nuacleophaga hypertrophica a nuclear
parasite of Endolimax nana; in 1935 he stated that he had studied then
(1922) a nuclear infection of both E. nana and I. bitschli. Brug (1926)
independently named a nuclear parasite of the latter amoeba N. /ntest-
nalis; according to Brumpt and Lavier (1935a), that parasite is the same
as the one (Fig. 218F-J) which they also studied in E. nana, and Brug’s
name is a synonym for N. Aypertrophica. Kirby (1927) described an
unnamed Nuacleophaga (Fig. 218A-E) in Endamoeba dis parata of Miro-

PARASITES OF PROTOZOA 1047

termes hispaniolae, and reported it also in E. majestas, E. simulans, and
Endolimax termitis; and Sassuchin (1931) found a chytrid in the nucleus
of Entamoeba citelli. In Entamoeba ranarum, Lavier (1935a, 1935b)
found a parasite described as Nucleophaga ranarum. Although not a
protozoan parasite, and one that is of doubtful affinities, the organism
named Erythrocytonucleophaga ranae by Ivani¢é (1934), which invades
the nuclei of the red blood cells of Rana esculenta, is interesting to con-
sider in this connection.

LIFE HISTORY AND STRUCTURE

Sphaevita—Chytrids of the family Olpidiaceae, to which Sphaerita
and Nucleophaga belong, have a one-celled, intramatrical thallus,
enclosed from an early period by a delicate membrane, amoeboid in
nature, which at maturity changes into a single sporangium or resting
sporangium. The sporangium of Sphaerita lacks elongate discharge tubes,
the spores escaping through an opening or papilla at one or both ends.
The zodspores of chytrids of this family are uniflagellate, according to
Minden (1915), Fitzpatrick (1930), and Gwynne- Vaughan and Barnes
(1937); but in the spores of many forms in Protozoa, either two flagella
or no flagella have been observed.

Sphaerita has often been encountered in only a small percentage of
the host species, but some records report a high incidence. Noller (1921)
found, in certain material, the majority of Endolimax nana and a vety
high percentage of Entamoeba coli infected; and Dobell (1919) saw
several E. nana infections in which a considerable proportion of the
amoebae were parasitized. Sphaerita was present in 80 percent of E. colz
and E, histolytica studied by Lwoff (1925). Both Becker (1924) and
Sassuchin ef al. (1930) found E. cte//7 in certain ground squirrels very
heavily parasitized, and Yuan-Po (1928) reported about 60 percent
infection of Entamoeba bobaci. Almost all Chilomastix in a guinea pig
contained S. chilomasticis (Cunha and Muniz, 1934). In flagellates of
termites, infection varies from light to heavy. In a few instances almost
every individual of certain devescovinids on some slides has been para-
sitized; on the other hand, the parasite may be infrequent or absent
in other host faunules of the same species. Distributional factors would
facilitate the presence of the chytrids in higher incidence in endozoic
than in free-living Protozoa under natural conditions, but an infection

1048 PARASITES OF PROTOZOA

in free-living amoebae in cultures may develop a high incidence in a
short time. Dangeard (1886b) found S. endogena in great abundance
in cultures of its two rhizopod hosts; and Ivanié (1925) stated that
cultures often perish from severe infection. A host may be parasitized
by two or more species (Mattes, 1924; Yuan-Po, 1928), but usually only
one species has been distinguished.

Brumpt and Lavier (1935b) described two different sphaeritas in
two amoebae on the same smears: S. parvula from Hyalolimax cerco-
pitheci, and one with larger spores in an Entamoeba of the minuta type.
This indicates host-specificity; but various amoebae of man, it appears,
contain a common species (p. 1042), and Lwoff (1925) stated that the
chytrids do not seem to manifest a narrow host-specificity.

The earliest stage in the cytoplasm of the host is a small, amoeboid,
uninucleate thallus. Dangeard (1895) described the parasite in Euglena
as at first smaller than the flagellate’s nucleus, with a dense, homoge-
neous cytoplasm and a vesicular nucleus with a large nucleolus. Mitchell
(1928) found the earliest stages to be from 2.5 to 3.5 1, in diameter, with
a vesicular nucleus from 1.3 to 1.5 p in diameter. Early stages of the
parasites in amoebae have been found as small as 1.5 y (Chatton and
Brodsky, 1909) and 2 1 (Mattes, 1924); the latter observer failed to
find a distinct nucleus. Sphaerita endamoebae, according to Becker
(1926), is from 1.9 to 2.5 1 in its early intracytoplasmic stage, with a
fine cell membrane and a relatively large, solid nucleus.

Most accounts describe increase in the size of the cytosome and the
nucleus before the nucleus begins to divide. In Euglena viridis (Dan-
geard, 1895; Mitchell, 1928) the uninucleate thallus may become
larger than the host nucleus, and its nucleus becomes correspondingly
large. Its shape is spheroidal, ellipsoidal, or elongated. The shape in
this phase, together with its size and the presence of vacuoles, is re-
garded as of taxonomic significance by Jahn (1933), who distinguished
E. phaci on such grounds. Other sphaeritas appear to attain no such size
before nuclear multiplication sets in. Sphaerita in Vahlkampfia limax
attains only about triple its diameter before nuclear divisions begin
(Chatton and Brodsky, 1909). In S. endamoebae, according to Becker
(1926), nuclear multiplication keeps pace with growth, and there are
binucleate stages no larger than uninucleate ones. Similar development
has been noted by the writer in Sphaerita (Fig. 217A) in several

PARASITES OF PROTOZOA 1049

species of Devescovininae. There are very few observations on actual
nuclear division; Dangeard (1895) interpreted as this some figures he
observed, but did not see nuclear division in the larger nuclei of the
early stages. Nagler (1911b) reported dumb-bell-shaped figures, as well
as granular fragmentation stages, in the parasite of Evglena sanguinea.
The outcome, in any event, is in typical Sphaerita a multinucleate thallus,
which is converted entire into the sporangium.

Parasites, which in some phases are much like Sphaerita but lack a

088.
eo

soo

Figure 217. A, various stages in development of Sphaerita in Devescovina sp. from
Neotermes tectonae; B, Sphaerita and other microorganisms in Entodinium sp. and
Exudiplodinium sp. from ruminants; C, mature sporangium of Sphaerita from Nyctotherus
ovalis ; D, developmental stages of Sphaerita and aggregations of bacteria in Nyctotherus
ovalis. (A, original; B, after Winogradowa, 1936; C, D, after Sassuchin, 1928a.)

multinucleate structure, have been described, however. Mitchell (1928)
reported a parasite in Euglena caudata, which, after growing to a
relatively large uninucleate body, underwent repeated division of
both nucleus and cytoplasm to form spores. Ivani¢ (1925), describing
in free-living amoebae parasites which appear to be Sphaerita-like, stated
that the uninucleate forms grow and multiply by binary fission before
the plasmodial period begins. When, as is often true in preparations, the
cytoplasm of the parasite is not apparent, division of the nuclei may
be mistaken for division of individuals within a vacuole. Individual

1050 PARASITES OF PROTOZOA

parasites multiplying in this way alone would probably be cocci. That
may account for possible errors, not made in the above examples, but
probably involved in certain accounts of nuclear parasites, as discussed
below.

When the parasite has reached a certain size, growth stops and
sporulation sets in. In Sphaerita amoebae the size when spores are
formed is very variable; sporangia are larger when only a few are pres-
ent in an amoeba (Mattes, 1924). The number of spores produced
is also variable in this species, ranging from less than a hundred to
several hundred. In sporulation the protoplasm simultaneously organizes
into membrane-confined bodies around each nucleus, and the spores
appear as spheroidal or ellipsoidal structures. The membrane of the
sporangium may remain very thin, so as to be scarcely recognizable, as
in Sphaerita in Amoeba alba, where groups of spores showed no trace
of an envelope (Penard, 1912). Sometimes it becomes more distinct
at sporulation; and in the unique case of Sphaerita from Nyctotherus
ovalis, according to Sassuchin (1928a), it becomes 1 1 or more thick
(Fig 2i7@):

The account by Sassuchin ef a/. (1930) and Sassuchin (1934) of
the parasite of Entamoeba citelli is not easy to understand. The parasites
are said to occur either in groups, varying considerably in size, which
resemble sporangia, but around which a membrane was never observed;
or arranged singly in the protoplasm. Though these parasites show a
spore-like character, the authors did not call them spores, nor did they
discuss multiplication. The description by Becker (1926) of Sphae-
rita endamoebae from Entamoeba citelli, to which Sassuchin did not
refer even in his later article, is in essential agreement with the usual con-
cept of the life cycle of Sphaerita.

The parasite of Exglena caudata (probably Sphaerita dangeardt)
may form as many as 500 spores (Mitchell, 1928). Sporangia of S.
endogena contain 100 or more (Dangeard, 1886a). Dogiel (1929)
found only from 30 to 40 spores in the “‘spore balls’’ of S. dzplodiniorum.
In Sphaerita of Monas vulgaris, nuclear divisions preceding spore forma-
tion proceed to stages 16, 32, or sometimes 64 (Alexeieff, 1929). Pinto
and Fonseca (1926) mentioned sporangia of only from 7 to 9 “individ-
uals” in S. minor of Trichomonas vitali; Canha and Muniz (1934)
found from 20 to 30 spores in S. chilomasticis.

PARASITES OF PROTOZOA 1051

There is variability in the number of spores and the size of sporangia
within a species. That sporulation can occur at different stages of growth
was noted by Chatton and Brodsky (1909), in Sphaerita of Vahlkamp fia
limax; sporangia ranged from 20 y in diameter down to small ones,
with few spores, of 4 yy. Mattes (1924) stated that the size at which
sporulation starts in S. amoebae is very variable; and Lwoff (1925), in
Sphaerita of entamoebae of man, found that sporulated parasites are of
different sizes. The size of the sporangium and the number of spores
must not be used indiscriminately for definition of species.

The spores are spherical, ovoidal, or ellipsoidal in shape—most fre-
quently the first. They range in size, in different species, from a diameter
of 0.25 to 0.30 yp (S. parvula, Brumpt and Lavier, 1935b) to elongated
forms of from 2.5 to 3 p (Sphaerita from Euglena; Mitchell, 1928; Puy-
maly, 1927). Yuan-Po (1928) reported spores of from 2.5 to 4 y ina
parasite of Entamoeba bobaci; this is exceptional in Sphaerita of endozoic
Protozoa. In the parasite of Nyctotherus ovalis, the spores measure from
1.5 to 2 y (Sassuchin, 1928a). Dangeard (1886a, 1886b) stated that
the spores of S. endogena ftom amoebae have a size of 1.5 y. There ap-
pears in general to be only a limited variability in the size of mature
spores; but Becker (1926) found that in S. endamoebae, the spores of
which usually were from 1.0 to 1.6 u, some were as small as 0.5 p. The
size and shape of spores is, used discretely, a valuable taxonomic guide.

The spore of S. endogena in the rhizopods Nuaclearia simplex and
Heterophrys dispersa has, according to Dangeard (1886a, 1886b), a
long flagellum (“‘cil’”) placed anteriorly and strongly recurved. Its
movements are very active and jerky, and sometimes there is simple
rotation in one position. When he studied the zodspores of Sphaerita
of Euglena sanguinea (1889b), Dangeard found, in addition to the
posteriorly directed flagellum, a very short one directed anteriorly.
Serbinow (1907) found only one flagellum on the zodspore of Sphae-
vita of Euglena, and thought it possible that Dangeard’s biflagellate
zodpores belonged to some other organism, possibly to the parasite of
Sphaerita, Olpidium sphaeritae Dang. Serbinow described their jerky,
irregular movement. In Sphaerita of E. viridis, Puymaly (1927) also
reported biflagellate zodspores, the larger flagellum directed posteriorly,
as Dangeard noted again in 1895; and he described the movement as
rotation around an axis and rapid, oscillatory swimming. These observa-

1052 PARASITES OF PROTOZOA

tions agree with those of Stein (1878, 1883) on the escape of minute,
flagellated organisms from the so-called germ balls. Cejp (1935) ob-
served two flagella on the zodspores of Sphaerita of Paramecium. Ivani¢
(1925) stated that he repeatedly observed release of the flagellated
swarm sports of the parasite (Sphaerita?) of Amoeba jollosi.

Mattes (1924), however, though he found flagellated zodspores of
Olpidium amoebae of Amoeba sphaeronucleolus, failed to see any
flagella or motility in the spores of two Sphaerita species of the same
amoeba. The same is true of the observations of all other investigators
of the parasite in Protozoa. It appears that Sphaerita in endozoic Protozoa
lack flagellated zoéspores, and that most of those of free-living amoebae
also do.

A central or eccentrically placed nucleus in the spore was reported
by Dangeard (1895) in Sphaerita of Euglena, and by Penard (1912)
in Sphaerita of Amoeba alba; and it was shown by Cejp (1935) in the
parasite of Paramecium. Mitchell (1928) noted a nucleus in the spores
of the chytrid of Exglena sanguinea, and he alone described any detail in
the nuclear structure. In sphaeritas of endozoic Protozoa, the nucleus
has not been found, and there appears to be a thicker spore membrane.
The membrane appears in optical section as a well-defined ring, espe-
cially in spherical spores. In elongated spores there is frequently a stain-
able area at one end, appearing often as a crescentic thickening, as in
Sphaerita (Fig. 217C) in Nyctotherus ovalis (Sassuchin, 1928a, 1934).
This structure was noted also by Becker (1926) in Sphaerita endamoebae,
by Yuan-Po (1928) in the larger species in Entamoeba bobaci, and by
Connell (1932) in Sphaerita of Gigantomonas lighti. It has been ob-
served by the writer in the parasites in a number of flagellates in ter-
mites.

Rupture of the sporangium takes place in the cytoplasm of the host,
then the body of the host may rupture and the spores be released into
the water. In most instances no previously apparent pore or papilla has
been shown. Dangeard (1889b) stated that the zodspores of Sphaerita
in Euglena sanguinea escape by a papilla at one end. Serbinow’s ac-
count (1907) of an elongated or fusiform sporangium in Sphaerita in
E. viridis and E. sanguinea, with a short exit papilla at one or both
ends, does not apply to most forms that have been placed in the genus.
Sphaerita cannot, then, be diagnosed on the basis of this account, as
was done by Minden (1915), without excluding many forms.

PARASITES OF PROTOZOA 1053

There is slight evidence of fusion of spores in Sphaerita, and none
of this carries the conviction of cytological demonstration. Dangeard
(1889b) and Puymaly (1927) reported that in sphaeritas of Euglena
zodspores may touch or adhere, simulating conjugation of gametes, but
that they end by separating. Mattes (1924) found no fusion of spores
of Sphaerita amoebae, Chatton and Brodsky (1909) thought copula-
tion of spores probable, but did not see it. In Sphaerita of Amoeba alba,
Penard (1912) stated that he sometimes encountered the spores in con-
jugation; and fusion was reported in Sphaerita-like parasites of Amoeba
jollosi by Ivanié (1925), as well as in the so-called gametes of Allo-
gromia by Prandtl (1907), which possibly were also Sphaerita.

Dangeard (1889b) reported fixation of zodspores to the wall of
Euglena sanguinea, and penetration into the cytoplasm. Puymaly (1927)
described adherence, loss of flagella, and development of a fine surround-
ing membrane; following which the spore probably emits a fine papilla,
which perforates the flagellate, and empties abruptly into the cytoplasm.
In rhizopods, spores are ingested (Dangeard, 1886a; Chatton and
Brodsky, 1909; Mattes, 1924; Lwoff, 1925); this probably is the general
method of infection of holozoic Protozoa by the non-flagellated spores.

The thallus of Olpidiaceae may develop also into a resting sporangium,
which is ordinarily thicker-walled, and may sometimes bear spines, but
otherwise in structure and development corresponds to the ordinary
sporangium. Spinous cysts or resting sporangia were described by Dan-
geard (1889b) in Sphaerita of Euglena sanguinea; by Serbinow (1907)
in Sphaerita of E. viridis and E. sanguinea; by Skvortzow (1927) in
S. trachelomonadis; and by Mattes (1924) in S. plasmophaga of Amoe-
ba sphaeronucleolus.

Nucleophaga—Nucleophaga has been found by the writer in low
incidence in almost all devescovinid flagellates in termites. Sometimes
the infection is greater. In some material of Endamoeba disparata tt
was from 6 to 12 percent (Kirby, 1927); Brumpt and Lavier (1935a)
found it in 78 percent of trophozoites of E. mana; on one occasion 90
percent of a group of Amoeba sphaeronucleolus were parasitized
(Mattes, 1924); and Gruber (1904) lost an entire culture of A. viridis,
which had been kept ten years, on account of the fungus.

The parasite apparently occurs exclusively in the nucleus. Brumpt and
Lavier found it only twice, among thousands of specimens, in the cyto-

1054 PARASITES OF PROTOZOA

plasm of E. nana; and in those two instances, as they stated, it prob-
ably was not developing there. A given species of Nucleophaga invades
certain hosts, and not others. Brumpt and Lavier failed to find it in
E. dispar, which was associated with heavily parasitized E. nana. Many
related species of devescovinids, however, contain what is probably the
same species; although there is more than one species of Nucleophaga in
that group of flagellates.

In the recent review of studies on Nacleophaga by Lavier (1935b),
it was noted that life-history accounts indicate two modes of develop-
ment. One is in agreement with the life cycle of Sphaerita, as outlined
above. A thallus enlarges and its nuclei multiply, it is converted
into a sporangium, and a spore forms around each nucleus (Dangeard,
1895; Penard, 1905b; Mercier, 1907, 1910; Mattes, 1924; Lavier,
1935b). Nucleophaga of Endamoeba disparata (Fig. 218B-E) 1s con-
sidered to have this type of life history (Kirby, 1927). In the second
type there is no multinucleate structure, and no sporangial membrane,
the individual invading parasite (Fig. 218G-J) multiplying repeatedly
within the nucleus by division (Epstein, 1922; Brug, 1926; Brumpt and
Lavier, 1935a). This is the type of reproduction, evidently, in the nu-
clear parasite of Entamoeba citelli, according to Sassuchin (1931); and
probably such a parasite would not seem to differ essentially from
Caryococcus (Dangeard, 1902). It is not conceivable that there should
be such fundamentally different types of development in members of
the same genus. Either the latter type is nothing but a misinterpretation
of the ordinary chytrid life history, because of failure to see the cytoplasm
of the parasite, or the parasite is not Nucleophaga. The figures of
Nucleophaga supposed to show this second type of reproduction do not
differ essentially from the other accounts—it is probably a matter of
differing interpretations of what is actually one form of development.
The similarity to Sphaerita appears to be too great to separate Nuwcleo-
phaga distantly from that genus; but the relationship of Caryococcus to
this needs further investigation.

In its early phases, in any event, Nacleophaga appears as a group
of granules in the interior of the nucleus, occupying a limited area,
whereas the nuclear structure elsewhere is essentially unchanged. This
group of granules presumably, though it cannot always be ascertained
with certainty, represents the nuclei of a thallus. Nucleophaga amoebae,

Figure 218. Nucleophaga. A-E, Nucleophaga in Endamoeba disparata: A, normal
nucleus of E. disparata Kirby; B, developmental stage of Nucleophaga; C, D, later
stages of Nucleophaga, with residue of chromatin in center; E, surface view of same
stage. F-J, Endolimax nana parasitized by Nucleophaga hypertrophica: F, normal amoeba;
G, beginning of nuclear parasitism; H, I, multiplication of spherules; J, mature spores,
nuclear membrane appears on the point of rupturing. K-O, Nucleophaga in Caduceia
theobromae: K, surface view of parasitized nucleus bulged out at one side; L, mature
sporangium, with nucleus similarly formed; M, detail of spore; N, parasitized, greatly
hypertrophied nucleus; O, normal nucleus, drawn to same scale as K, L, N. (A-E, after
Kirby, 1927; F-J, after Brumpt and Lavier, 1935a; K-O, original.)

1056 PARASITES OF PROTOZOA

described by Dangeard (1895) in a host he considered to be Amoeba
verrucosa, invades the nucleolus, in which it appears at first as a vacuole
enclosing a granule—the former its cytoplasm, the latter its nucleus.
Growth of the parasite is rapid (Mattes, 1924), and as it proceeds
the chromatin is used up. The stainable nuclear material becomes re-
stricted to the periphery in a reticulated structure (as noted by the writer
in Nucleo phaga of devescovinid flagellates), or to the central zone (Fig.
218C, D), as in hyperparasitized Endamoeba dis parata (Kirby, 1927).
Eventually the nuclear material disappears, and the interior is entirely
occupied by the parasite.

The parasitized nucleus hypertrophies considerably (cf. Fig. 218F
and J; O and N), up to several times its original diameter. The para-
site must obtain material for its continued growth by diffusion from the
cytoplasm through the nuclear membrane. Though Mercier (1910) men-
tioned considerable hypertrophy of the nucleus of E. blattae, he showed
spores in nuclei in which there seems to have been little enlarge-
ment. This is unlike the usual situation. Perhaps the very thick membrane
of the nucleus of E. blattae has an influence in restraining the growth
of the parasite. Lavier (1935b) noted precocious spore formation in
Nucleophaga of Entamoeba ranarum, but stated that it generally occurs
when the parasite has attained a large size. In Nucleophaga of devesco-
vinid flagellates, notably that in Caduceia theobromae, expansion of the
nucleus cannot occur equally in all directions, because of its relation-
ship to the axostyle. Instead, it is pushed out on one side, and often has
a bilobed figure (Fig. 218K, L). This figure sometimes is retained
in the mature sporangium; often it fills out. As has been noted also of
Sphaerita, the size of the mature sporangium, as well as the number of
spores, is subject to considerable variation in the same species of Nw-
cleo phaga.

It has been stated that spore formation is in certain forms continuous,
and that there may be present in an individual at a given time mature
spores and granules corresponding to spores not yet formed (Lavier,
1935b). It is more general, however, for the spores to be formed
simultaneously, the entire thallus being converted into the group. Other
granules, which have been seen by the writer among the spores, prob-
ably represent residual or discarded material; there is no evidence for
maturation of later spores.

PARASITES OF PROTOZOA 1057

The spores are very much like those of Sphaerita. Their shape is
spheroidal or ellipsoidal, and the wall stains more intensely than the
contents. Some show a thickening at one side, in crescentic form (Ep-
stein, 1922; Brumpt and Lavier, 1935a; Lavier, 1935b). In the interior,
often no structure is discernible, or one or two granules are seen, or a
central nucleus may be observed. Dangeard (1895) and Epstein (1922)
reported a vesicular nucleus; Brumpt and Lavier, however, failed to
observe a definite nucleus in the parasite of Endolimax nana, studied
by Epstein. In Nucleophaga of Caduceia theobromae, a spheroidal gran-
ule of relatively large size was observed toward one end of the spore
(Fig. 218M). This may be a nucleus. In size, the spores range from
one to 2 py, some being reported as only about one y (Brumpt and
Lavier, 1935a), some 2 y (Lavier, 1935b; Penard, 1905b), others as
having a variability from about one y to 2 y (Mercier, 1910; Mattes,
1924).

No flagella have been observed on spores of Nucleophaga, with the
possible exception of those mentioned in the account by Robertson
(1905). She described what she supposed to be gametogenesis of
Pseudospora volvocis, in a rather complete account of what is probably
the development of Nucleophaga. The ‘‘gametes,” as figured and de-
scribed, are each provided with one flagellum; and they are reported to
fuse, producing a biflagellate zygote.

EFFECT ON HOST
Minden (1915) wrote (translation) :

In the lower plants, mainly algae, which in the widest variety are sought by
parasitic Chytridiales, the injuries are usually so striking that these fungi
ate designated as dangerous parasites of algae. In a short time large cultures
of diatoms, flagellates, and other unicellular organisms may be completely
destroyed; but also filamentous algae die cell by cell. The first indication of
injury is in the discoloration and disorganization of the cell contents . .
finally there remain only granular vestiges.

Infection with Sphaerita may be observed in many Protozoa that
appear entirely normal, but it often ends fatally. The host may some-
times rid itself of the parasite and continue normal life (Dangeard,
1895; Penard, 1912); on the other hand, many observers report death
of the host at the time of sporulation.

1058 PARASITES OF PROTOZOA

Parasitized euglenid flagellates lose their green color, chlorophyll
first being affected and chromatophores degenerating (Dangeard,
1889b, 1895; Puymaly, 1927; Mitchell, 1928; Jahn, 1933). Puymaly
found a decrease in flagellar activity, whereas euglenoid movement con-
tinued to the last moment and became even more energetic. There is
alteration of the nucleus, according to some, though Puymaly stated
that there is none; and the cytoplasm becomes vacuolated. Finally, in
many cases, the flagellate ruptures and zodspores are liberated.

Chatton and Brodsky (1909) found that parasitized amoebae tend to
assume a spherical form with radial pseudopodia, instead of progressing;
and Sassuchin (1928a) noted a progressive slowing of the ciliary
action in Nyctotherus. The pulsating vacuole in these heavily infected
hosts slows or loses its rhythm. Degenerative changes were observed
in the nucleus of parasitized lodamoeba biitschlii by Wenrich (1937);
in that of Entamoeba citelli by Becker (1926); and in the macronucleus
of Nyctotherus by Sassuchin (1938a). When the sporangium ruptures,
or shortly before, the host may perish (Chatton and Brodsky, 1909;
Mattes, 1924; Yuan-Po, 1928; Sassuchin, 1928a), especially if the in-
fection is heavy.

The fact that in amoebae of man the parasites have been found only
in trophozoites has been taken to indicate either that they hinder the
amoebae from encysting or that infected cysts degenerate rapidly (Lwoff,
1925). Lwoff therefore pointed out a possible use of Sphaerita as a means
of biological control, following Noller’s suggestion that it might be
worth while to devote more attention to these natural enemies of
amoebae. If one could transmit the infection to carriers of cysts, Lwoff
stated, there would be a means of diminishing the number of cysts,
this in addition to the inhibition of multiplication. The practicability
of this, however, is doubtful.

The protozoan whose nucleus is parasitized by Nucleophaga con-
tinues activity until the end. Usually there is no apparent change in
protoplasmic activity or in structure, aside from the nucleus, even though
all stainable chromatin material has disappeared. Lavier (1935a) ob-
served increased size and activity in parasitized Entamoeba ranarum, and
remarked that the hyperactivity may be provoked by irritation, and may
constitute a defense reaction on the part of the amoeba. By the time the
parasite reaches the stage of sporulation, however, some changes may

PARASITES OF PROTOZOA 1059

have occurred in the cytoplasm (Dangeard, 1895; Sassuchin, 1931), and
there may have been some hypertrophy of the host’s body (Epstein,
1922). Epstein, indeed, stated that giant amoebae reaching from 10 to
30 times normal size, may result; but that 1s very much more than 1s
usually observed.

When the membrane breaks and the spores escape, the host perishes.
For that reason, even in heavily parasitized groups of Protozoa, individ-
uals with spores dispersed in the cytoplasm are seldom observed.

PARASITES OF THE NUCLEUS OF TRICHONYMPHA

Except for mention (Kirby, 1940) of the parasites described below,
the only report of parasitization of the nucleus of Trichonympha is the
description (Kirby, 1932a) of a form in T. saepiculae (Fig. 219B).
Numerous spheroidal bodies, each apparently subdivided into compart-
ments, filled several nuclei, in which the vestiges of chromatin were con-
fined to the central part. Few specimens of this organism were found,
and its affinities were not discussed, except for the remark that it 1s
unlike Nacleophaga.

An unusually interesting parasitization of the nucleus has been studied
by the writer in Trichonympha in certain termites of Madagascar and
in one from Java. In several series of preparations from Procryptotermes
sp. of Madagascar, a large proportion of the hypermastigotes had parasit-
ized nuclei. Apparently, in the flagellate from this host two different
parasites are involved. One of these has a life history like that of
Nuacleophaga: growth of a multinucleate parasite, using up the chro-
matin, which is restricted to a peripheral reticulum and finally dis-
appears; and formation of numerous spores. The size and structural
detail of the spores distinguish them from those of any described Nuacleo-
phaga, and suggest a possible affinity to the Haplosporidia.

The normal, interkinetic nuclei of these species of Trichonympha
have the chromatin in the form of stout, varicose strands which extend
throughout the intranuclear area. They may extend entirely to the pe-
riphery, but often in the preparations there is a clear outer zone of
variable width. In this zone are minute granules. In some nuclei, which
possibly show the beginning of kinetic changes, the strands tend to be
peripheral, and the central part of the nucleus is occupied by a granular
and reticulo-fibrillar matrix.

1060 PARASITES: OF PROTOZOA

Figure 219. Micro6rganisms in Trichonympha. A, constantly present organism that
forms an aggregate surrounding or near to the nucleus of T. campanula; B, nuclear
parasite of T. saepiculae; C, Sphaerita in T. sphaerica; D, E, peg-formed organisms in
T. campanula,; F, G, fusiform organisms in T. campanula; H, group of parasites (micro-
sporidia?) in T. magna; I, developmental stages of organism shown in H. (After Kirby,
1932a.)

In early stages of invasion by the Nucleophaga-like parasite, a body,
apparently amoeboid, is observed in the process of penetration into the
chromatin mass. In the earliest stages so far found, it is already multi-
nucleate. This becomes located in the interior of the chromatin mass,
and as it grows its nuclei multiply and the chromatin of the Trzchonym-
pha nucleus becomes restricted to a peripheral reticulum (Fig. 220E).

PARASI@ES OF PROTOZOA 1061

By the time the parasite reaches its full size, the nucleus of its host
has become greatly hypertrophied and has left its normal position. A
variable number of spores are produced; in one instance there were
only 17, but usually there are from 150 to 200 or more, located within
an ellipsoidal membrane 25 to 44 22 to 36 y. The individual spore
is ellipsoidal and has a size of 2.5 to 4 y X 2 to 3 y. The spores are
larger in size when their number is smaller.

The structure of the spores (Fig. 220C-E) is the characteristic of
greatest interest in this organism, as nothing like it is known in any
other nuclear parasite, or indeed in any known parasite of Protozoa.
The nucleus is located at one end, and is usually relatively very large,
having a diameter almost equal to the width of the spore. When heavily
stained, or when not well fixed, it appears homogeneous, but in good
preparations it is resolved into closely packed granules. In the cytoplasm
of the spore are a variable number of granules which are relatively large
for cytoplasmic granules. These stain intensely with hematoxylin, and
possibly are volutin, though no tests could be made to support that
view. The cytoplasmic granules are often arranged in an equatorial
ring (Fig. 220D), which appears solid in some preparations. Some-
times there are no granules outside of the nucleus except in this ring;
at the other extreme, the ring constitutes the margin of a solid hemi-
spherical mass of granules, which occupies all the area at its end of
the spore (Fig. 220E). Between these extremes are conditions in which,
in addition to the ring, there are only a few granules at the periphery
of the hemispherical area, or more abundant granules in a peripheral,
semicircular row at right angles to the ring (Fig. 220C).

At the periphery of the mass of mature spores of the hyperparasite
from Procryptotermes sp., there is constantly present a single, apparently
crystalloid body (Fig. 220B). In different parasites this body is rela-
tively uniform in size and shape; and it is generally so located as to
cause a protrusion of the membrane. It has the form of a conventional
diamond, and stains deeply with Heidenhain’s iron-hematoxylin but not
with Delafield’s hematoxylin. It is unlikely that it is to be regarded as
residual chromatin.

What seems to be a second parasite (Fig. 220G, H) of the nucleus
of Trichonympha occurred in from 70 to almost 100 percent of the
hypermastigote in certain preparations from the Madagascar Procrypto-

Figure 220. Nuclear parasites of Trichonympha sp. from Procryptotermes sp. of Mada-
gascar. A-F, Nucleophaga-like parasite, nucleus hypertrophied; G, H, coccoid parasite,
nucleus not hypertrophied. A, developmental stage, probably a multinucleate plasmodium,
chromatin of nucleus restricted to periphery; B, mature sporangium, diamond-shaped
crystalloidal body at periphery of nucleus; C-E, details in structure of spores; F, nucleus
ruptured, with some spores in cytoplasm; G, chromatin masses present with parasites,

many of latter show crescentic stainable area at one side; H, chromatin peripheral, some
dividing forms of parasite. (Original. )

PARASITES OF PROTOZOA 1063

termes. Generally the parasite appears as a mass of spherical bodies,
each about one 1 or less in diameter and often with a stainable crescent
at one side, located in the central part of the nucleus. The spherical
bodies are a good deal smaller than are the nuclei in a plasmodium of
comparable size in the other parasite. The chromatin is usually re-
stricted to the periphery of the host nucleus; but, except for this re-
moval of the central part of the mass, it is little altered; and there
is no marked hypertrophy of the nucleus. In the nuclei of some hosts,
the proportion being greater on certain slides, rounded bodies with a
similar crescentic stainable area are located peripherally, the chromatin
mass being concentrated in the center. The distribution of these periph-
eral bodies is often such that a common embedding cytoplasm appears
unlikely. Further investigation is necessary to establish the nature of
this parasite. It may be a bacterial, coccoid parasite of the nucleus, com-
parable in certain ways, possibly, to Caryococcus, described by Dangeard
(1902).

In several instances multinucleate trichonymphas, with all nuclei
parasitized, have been found. These are the only multinucleate flagel-
lates of this genus that have ever been seen. Cytotomy generally ac-
companies division of the single nucleus, but binucleates occasionally
occur.

PHYCOMYCETES OTHER THAN SPHAERITA AND NUCLEOPHAGA

Chytridiales of a number of genera other than Sphaerita and Nucleo-
phaga have been found parasitic on Protozoa, especially autotrophic
flagellates. Those described up to 1915, in the genera Olpidium, Pseu-
dolpidium, Rhizophidium, Phlyctochytrium, Rhizidiomyces, Saccomyces,
Rhizophlyctis, and Polyphagus, were discussed by Minden. They occur
in or on Exglena, Cryptomonas, Chloromonas, Chroococcus, Gleno-
dinium, Haematococcus, Chlamydomonas, Pandorina, and Volvox. OI-
pidium arcellae is considered doubtful.

Fungus parasites, which are probably Chytridiales, have been found
in cysts of a number of ciliates. Stein (1854) found many cysts of
V orticella microstoma with up to three or four protuberances perforating
the wall and extending a short distance free. Each protuberance was
an extension of a rounded body (Muvtterblase) within the cyst. From the
terminal opening a thin, gelatinous, clear fluid was reported to escape,

1064 PARASITES OF PROTOZOA

forming a globule enclosing about thirty “young,” resembling cer-
tain Monas forms, and, when dispersed, having movements like them.
Similar structures were shown in a cyst of Vorticella nebulifera. At that
time Stein considered this to be a mode of reproduction of Vortzcella.
Cienkowsky (1855b) recorded similar bodies in cysts of Nassula am-
bigua ("N. viridis”), describing the appearance of clear vacuoles in the
cyst contents and the development of “spores,” from many of which a
short process broke through the wall of the cyst and permitted the es-
cape of the swarm spores. Lachmann (1856) mentioned these observa-
tions as showing another kind of reproduction in ciliates. Cohn (1857),
however, remarked on the resemblance of these ‘“‘microgonidia,” with
their flask-formed “‘mother cells,” to the chytrids of many plants. In their
text Claparéde and Lachmann (1860-61) discussed the phenomena as
forms of reproduction by embryos, adding observations of their own on
similar structures in Urnula epistylidis; but in their footnotes they stated
that these were Chytridium. Stein (1859) regarded them as parasites,
comparing them with Saprolegniales and in particular with Pythiwm
entophytum Pringsheim; but their characteristics are suggestive of Ol pid-
zum. Stein recorded similar bodies from cysts of Stylonychia pustulata,
Holosticha (Oxytricha) mystacea, dead Toko phrya (Acineta) lemnarum,
and Metacineta (Acineta) mystacina (observations of 1854). On the
motile bodies escaping from a Vorticella cyst he saw a single flagellum.

Species of Olpidium, which differ from Sphaerita in the elongation
of the exit tube, occur in certain rhizopods and in Suctoria, as well as
in Euglena. O. amoebae was described by Mattes (1924) from Amoeba
Sphaeronucleolus; it is said to parasitize a rotifer also. Gdnnert (1935)
named O. acinetarum a chytrid which destroyed a culture of Lernaeo-
phrya capitata and Podophrya maupasi within a few days. The spores
are relatively large, from 2.5 to 3 1 in diameter, equaling the larger
ones of Sphaerita; and Mattes found a relatively long posterior flagellum.

Rhizophidium and Polyphagus belong to the Rhizidiaceae, in which
there is a restricted mycelium. Rhizophidium beauchampi has recently
been described by Hovasse (1936) in Exdorina illinoisensis. A heavy
infection, exceeding 90 percent, occurred homogeneously in these phyto-
monads in a large lake. The zodspore, which has a single long flagellum,
becomes fixed to the coenobial surface and germinates by the emission
of a tube which penetrates a cell and functions as a sucker. The part

PARASITES OF PROTOZOA 1065

of the tube that remains external to the cell swells and becomes a
sporangium, in which by simultaneous partitioning from 20 to 100 zo0-
spores are produced. Most parasitized colonies had not more than 25 to 28
normal cells, and heavily parasitized colonies may be destroyed.

Poly phagus euglenae, whose structure and life history have been de-
scribed by Nowakowski (1876), Dangeard (1900b), and Wager
(1913), appeared at various times in cultures of Ewglena, which were
destroyed in a few days. Serbinow (1907) at Petersburg, and Skvortzow
(1927) in East Mongolia found it parasitic on Chlamydomonas. The
parasite germinates free in the water, and a single cell, by branched
haustoria, may attack many flagellates. A haustorium perforates the cell
wall, branches, and the cell contents rapidly disintegrate. A sporangium
develops as an outgrowth from the protoplast, and produces a variable,
usually very large, number of uniflagellate zodspores. Polyphagus is one
of the few Chytridiales in which sexual reproduction has been satis-
factorily demonstrated. A zygote is formed by the fusion of two vegeta-
tive cells, and becomes a resting spore, with smooth or spinous mem-
brane.

Skvortzow (1927) reported two other Chytridiales from Eadorina
elegans in Manchuria: Phlyctidium eudorinae n. sp. and Dangeardia |
mamillata. The latter was originally described by Schréder from Pan-
dorina. Phlyctidium is epibiotic, with a haustorium penetrating a cell.
The sporangia of Dangeardia are located in the gelatinous sheath of the
volvocid.

Lagenidium trichophryarum, which belongs near the Chytridiales in
the Ancylistales, was described by Goénnert (1935) in Trichophrya
epistylidis. The parasite, which appeared once in abundance, was fatal
to the suctorian. Lagenidium is rare in Protozoa. Cook’s revision of the
genus (1935), which is in the same number of the Archiv fir Protisten-
kunde as Gonnert’s article, reports no species from them; the habitat ts
filaments of green algae, diatoms, pollen grains, and rhizoids of mosses.

Filamentous appendages on the posterior end of certain large fresh-
water amoebae (Fig. 221) have long been known. Leidy (1879) ob-
served them, and was uncertain as to their nature, regarding them at
first as a bundle of mycelial threads dragged behind Amoeba proteus,
but finally concluding that they were structural elements of the amoebae.
He made the presence of these appendages diagnostic of the new genus

1066 PARASITES OF PROTOZOA

Ouramoeba. Korotneff (1880), encountering an amoeba with similar
posterior prolongations, created for it a new genus, Longicauda. Penard
(1902), as others had already suggested, considered filaments on
Amoeba nobilis to be parasites, and reported observation of appendages
of different types on A. proteus and A. vespertilio. He noted long, fine
filaments also on Pelomyxa tertia. He recounted these observations again
later (1905c), and stated that the fungi probably belong very close to the

Figure 221. Filamentous fungi (Amoebophilus) parasitic on Amoeba proteus (Oura-
moeba vorax Leidy). (After Leidy, 1879.)

Entomophthorales or Saprolegniales, resembling in the former group
Em pusa, in the latter Leptomitus lacteus. Dangeard (1910) studied
filaments on Pelomyxa vorax, and named them Amoebophilus penardi.
He gave the name Amoebophilus caudatus to the parasite described by
Penard on Amoeba nobilis; and A. korotne ffi to that of “Longicauda
amoebina.”’ He thought it possible that they might belong to the Asco-
mycetes. Geitler (1937) studied in A. proteus what is apparently the
same as Penard’s parasite (Amoebophilus caudatus Dangeard), but he
made no reference to Dangeard’s account. Geitler stated that the fungus
probably belongs in the Cladochytriaceae of the Chytridiales.

PARASITES OF PROTOZOA 1067

Geitler found the fungi on a narrowly defined area of the body, the
protruding filaments vertical to the surface of the protoplasm and from
100 to 200 y long. The filaments of a plant are non-septate and arise
from a deep-staining, irregularly lobed vesicle, the haustorium, lo-
cated in the endoplasm of the amoeba at the limits of the ectoplasm.
From the vesicle, which was also noted by Penard, delicate hyphal
threads extend through the ectoplasm to the surface of the body, where
they broaden and continue as extracellular threads. Basal branching 1s
common, and there may also be secondary branching. Infected amoebae
show a polar organization, with the fungi at the posterior end; this
polarity, Geitler concluded, is probably not called forth by the infec-
tion, but was present before.

Filaments seen by Penard (1905c) on Amoeba proteus, the same as
those shown by Leidy on ‘'Ouramoeba botulicauda,’’ were, when of some
length, divided by constrictions into two or more equal parts. The
figure of a filament on A. vespertilio shows constrictions marking
short subdivisions. Dangeard (1910), who observed nuclei in the fila-
ments on Pelomyxa vorax (Amoebophilus penard7), also figured con-
strictions demarcating long sections, which he considered to represent
budding.

The incidence of these parasitic fungi on amoebae is sometimes high.
At one period Geitler found 95 percent of A. proteus infected; later the
incidence declined. Penard (1902) found the fungi on three out of five
A. nobilis, and Dangeard (1910) ona rather large number of Pelomyxa.

A filamentous, cylindrical fungus, 0.75 1 in diameter, was found
by the writer fairly frequently in certain material of Devescovina hawatr-
ensis from Neotermes connexus. A large part or all of the filament was
embedded in the cytoplasm, but characteristically a part projected beyond
the surface. The surface was penetrated at any point.

Fungi which develop in the cytoplasm and then extend projections
beyond the surface were described by Penard (1912) in Amoeba terri-
cola and A. alba. These parasites, which he found usually fatal to the
amoebae, belonged, he thought, in or near the Saprolegniaceae.

A fungus assigned to the Saprolegniales was found by Sand (1899)
infesting more than half the specimens of Acineta tuberosa collected
from the sea at Roscoff. Developing within the cytoplasm, the parasite
soon destroyed the cell and formed isolated spheres in the empty lorica.
These developed into long tubes, wound in the lorica or projecting free.

1068 PARASITES. OF PROTOZOA

One of the tubes terminated in a large, spherical sporangium. Leger and
Duboscq (1909c) reported parasitic fungi which developed a mycelium
in cysts of the gregarine Nina gracilis.

Galleries excavated in the non-protoplasmic parts of calcareous tests
of Foraminifera are the work of an organism behaving somewhat in
the manner of the mycelium of certain fungi, according to Douvillé
(1930). The relationship of this organism to calcareous shells suggests
the habitat of Didymella conchae, an ascomycete which Bonar (1936)
described from the shells of marine gasteropods and barnacles.

PROTOZOA
PHYTOMASTIGOPHORA

An unusual phoretic relationship described by Penard (1904) existed
between a heliozoan and an undetermined species of Chlamydomonas.
He found this organism fixed to the surface of Actinosphaerium etch-
hornii by its two flagella, which were applied by their full length. Often
it was so abundant that the surface of the host was spotted with close-set
organisms, and the heliozoan appeared covered with a green envelope.
When the chlamydomonads were scattered mechanically, they later re-
assembled at the surface of Actinosphaerium. Sokoloff (1933) found
a euglenid flagellate, named Euglena parasitica, adherent in abundance
to the surface of Volvox coenobia in a tank. There was a conical pro-
longation anteriorly by which this adherence was effected; no flagella
were mentioned or figured. Sokoloff did not observe the flagellate in
the free state.

Endozoic, colorless flagellates that probably belong to the genus
Khawkinea have often been found, especially in Turbellaria, but also in
rotifers, Gastrotricha, fresh-water nematodes, fresh-water oligochaetes,
nudibranch eggs, and copepods. In different hosts they occur in the
alimentary canal, in tissues, or in the coelom. Howland (1928) identi-
fied as Astasia captiva, which Beauchamp had described from a rhabdo-
coele (p. 905), an actively metabolic euglenoid flagellate, without
flagellum or stigma, found in the cortical ectoplasm of Stentor coeruleus
and Spirostomum ambiguum. Jahn and McKibben (1927) assigned this
species to their new genus Khawkinea (see p. 907).

Parasitic dinoflagellates occur in Tintinnoinea, in Radiolaria, and in
other dinoflagellates. In the first two groups, as in so many Protozoa,

PARASITES OF PROTOZOA 1069

development of the parasites has been mistaken for a phase of the
cyclical development of the host. In the tintinnids these errors were
first pointed out by Duboscq and Collin (1910); in the Radiolaria by
Chatton (1920b).

Chatton (1920b) established the genus Duboscquella for the para-
site of tintinnids, and recorded D. tintinnicola as occurring in Codonella
galea, Tintinnopsis campanula, and Favella (as Cyttarocylis) ehren-
bergii. In the last ciliate, Duboscq and Collin (1910) observed the
parasite in abundance at Cette. It is a subspherical body which grows
to a large size (100 1.) without apparent inconvenience to the host. Re-
peated division gives rise to a dense mass of gametocytes, each of which,
after ejection from the host, undergoes two divisions inside or outside
of the host, to produce biflagellate gametes. Hofker (1931) found
Duboscquella tintinnicola in Favella ehrenbergii and F. helgolandica.

Although the enigmatic organism described by Campbell (1926) as
Karyoclastis tintinni is apparently not a dinoflagellate, it may be men-
tioned here because of its occurrence in this same group of ciliates.
Campbell found it to be primarily an intranuclear parasite of Tzntin-
nopsis nucula, but, unlike most other described nuclear parasites, it has
a cytoplasmic phase. In the macronucleus the parasites occur as numerous
small bodies, each with a gray-staining mantle, a clear central area, and
within a central granule which undergoes division. The parasites mul-
tiply within the nucleus, then the membrane partially disintegrates,
and the parasites emerge and form a cloud-like mass in the cytoplasm.
Campbell noted that the parasites are distinct in structure from Nzcleo-
phaga and Sphaerita. Further investigation is necessary to elucidate the
complete life cycle and establish the systematic relationships of Karyo-
clastis. Hofker (1931) found a resemblance to Karyoclastis in round
bodies associated in the test, in some instances, with Tintinnopsis fum-
briata; but he recognized the possibility that their occurrence was the
result of a fragmentation phenomenon.

Chatton (1920a, 1920b) pointed out that the so-called anisospores,
or gametes, in Thalassicolla, Sphaerozoum, and Collozoum (Brandt),
the origin of which in the first genus from intracapsular plasmodial
masses was described by Hovasse (1923a), belong not to the radiolarians
but to the parasitic dinoflagellates similar to Syndinium of the pelagic
copepods. Chatton (1923) proposed the genus Merodinium for these

1070 PARASITES OF PROTOZOA

organisms, establishing five species for cytoplasmic parasites of Col-
lozoum, Sphaerozoum, and Myxosphaera, and a sixth species, in the
subgenus Solenodinium, for the intranuclear parasite of Thalassicolla
spumida. The dinoflagellate affinities of these organisms are shown by
the nuclear structure, the mode of mitosis, and the morphological char-
acteristics of the spores. The dinospores are reniform, constricted at the
equator, and have two unequal flagella in typical dinoflagellate arrange-
ment.

Species of Peridinium and related dinoflagellates may be parasitized
by Coccidinium, which, according to Chatton and Biecheler (1934),
resembles coccidia in the vegetative and multiplicative stages, whereas
the spores are typical of dinoflagellates. Chatton and Biecheler (1936)
reported having observed copulation and total fusion of spores of two
types in Coccrdinium mesnili, and considered this to be the first obser-
vation of an indisputable sexual process in an authentic dinoflagellate.

Keppen (1899) described from marine dinoflagellates (Ceratium
tripos, Ceratium fusus, and Ceratocorys horrida) the parasite Hyalosac-
cus cerali1, which he considered to be a parasitic rhizopod. It is impos-
sible to obtain a complete understanding of the structure, life history,
and relationships of the organism from Keppen’s account and illustra-
tions; but certain similarities to Coccidinium are apparent in the structure
and nuclear multiplication of the intracytoplasmic stages. Keppen did
not describe spores. As did the French authors in Coccidinium, Keppen
pointed out a resemblance of Hyalosaccus to coccidia. He considered this
to be the same parasite as that observed by Biitschli (1885) in Ceratzum
fusus.

ZOOMASTIGOPHORA

Chlamydomonads may be attacked by Colpodella pugnax, which 1s
more of a predator than a parasite. Cienkowsky (1865), who first de-
scribed it, found it on Chlamydomonas pulvisculus. Dangeard (1900a)
studied it mainly on C, d7/l7, but remarked that it would attack more or
less all species of the genus. He never, however, observed it on other
Protozoa. The free-swimming Co/podella is colorless, crescentic, about
12 y in length, with a terminal flagellum. It becomes fixed to Chlamy-
domonas, perforates its membrane, and within a few minutes the cyto-
plasm begins to flow into Colpodella. The envelope of Chlamydomonas

PARASITES OF PROTOZOA 1071

is soon emptied, and Co/podella takes on a stouter form and a green
color. The substance of its prey collects in a large digestive vacuole and
is absorbed. In multiplication, the organism rounds up and undergoes
thrice-repeated binary fission within a membrane, from which crescentic
flagellates escape. Thick-walled cysts were described by Dangeard.
Hollande (1938) gave the name Colpodella raymondi to a parasite
found by Raymond (1901) on Chlamydomonas. The parasite, reported

Figure 222. A, B, Bodo perforans Hollande, ectoparasitic on Chilomonas paramaecium ;
C, ectoparasite of Colpoda cucullus. (A, B, after Hollande, 1938; C, after Gonder, 1910.)

Raymond, occurs in one to several spheroidal masses on the surface of
Chlamydomonas. Exceptionally there are more than a hundred; the usual
size appears to be very much less than that of C. pugnax. According to
Raymond, the host appears not to suffer from its presence, at least unless
the infection is very heavy.

An interesting ectozoic organism on Chilomonas paramecium was
named Bodo perforans by Hollande (1938). This flagellate possesses a
long, slender rostrum by which it is fixed in a constant position near
the anterior end of Chilomonas, at the base of the flagella (Fig.
222A, B). It has two unequal flagella, inserted at the base of the rostrum.
Rarely two or three parasites are attached to one host. The rostrum,
according to Hollande, penetrates the cytoplasm shallowly; and he found

1072 PARASITES: OF PROVOZOA

evidence that material may be extracted from the host. Many parasitized
chilomonads had lost their flagella, but otherwise they were apparently
not injured. Bodo perforans was rately seen free from attachment.

Gregarella fabrearum, studied by Chatton and Brachon (1936) and
Chatton and Villeneuve (1937), shows few characteristics that can be
used for taxonomic purposes; yet it was considered by them to be a
much regressed flagellate, representative of a new group of zo6flagellates,
the Apomastigina. It is reported to undergo a cycle of development as
follows: ingestion by Fabrea, fixation to the wall of a vacuole, with
growth and transverse division, discharge from the body and fixation
to the ectoplasm around the cytopyge, where feeding and slower multi-
plication take place. The fixed parasites are claviform with the large
end adherent, are capable of slow changes of shape, contain large
refractile inclusions, and have no flagella or other permanent differentia-
tions.

The parasite on Colpoda cucullus (Fig. 222C), observed in a hay
infusion by Gonder (1910), recalls in some ways certain of these
ectozoic flagellates. The organism has a round or oval figure, with the
narrowed end extended through the pellicle. The nucleus is single,
vesicular, with a large endosome—not an ordinary ciliate type. No fla-
gella or cilia were seen. Gonder was vague about its relationships.

Small mastigamoebae were recorded by Doflein (Lehrbuch, 1909,
and later editions) as not infrequent parasites of Stentor coeruleus; and
he figured an instance of heavy infection. Infected ciliates were faded
and somewhat contracted, and eventually often burst.

Flagellates were found in Craspedophrya rotunda and other Suctoria
by Rieder (1936). The incidence was high in a culture of the first
species and light in four other species. At first only a few Cras pedophrya
were parasitized, but in a few days many were infected. The organisms
were colorless, actively metabolic, from 6 to 11 microns in length, and
had two flagella 1.5 times the body length, of which one was anterior
and the other trailed. They entered the suctorian through the envelope,
sometimes at the thinnest place, as over the brood chamber from which
a swarmer had escaped, but also elsewhere. They were observed swim-
ming about actively within the pellicle, taking up suctorian plasma, and
undergoing binary fission. Eventually the flagellates often rounded up
and lost the flagella; some left through the pellicle, probably in the way

PARASITES OF PROTOZOA 1073

they entered. The host may die and disintegrate, according to Rieder,
even when only one or two parasites are present. He considered the
organism to be a strict endoparasite of Craspedophrya.

A number of enigmatic forms, which at least seem to show certain
flagellate relationships, may be considered here.

Dangeard (1908) gave the name Lecythodytes paradoxus to a parasite
of cysts of Chromulina, which decimated cultures within a few days.
Within the cyst the organism is amoeboid, and grows until it occupies
the whole interior. Division, he stated, results in eight, or less often
four or sixteen zodspores, which escape from the cyst and may infect
another host. The zodspores are elongated and narrowed at the ex-
tremities, each of which, according to Dangeard, terminates in a long
flagellum.

Uncertain is the proper systematic position of Sporomonas infusorium,
which Chatton and Lwoff (1924a) encountered in the marine ciliates
Folliculina elegans, Vorticella sp., and once in Lacrymaria lagenula.
Potts found it in Folliculina ampulla at Woods Hole, Massachusetts. In
the cytoplasm the parasite occurs as a reniform body, provided with a
long flagellum, in active rotation. It increases greatly in size, up to 70 u,
and the flagellum is lost. The parasite is then expelled, and multiplication
takes place only outside. There is rapidly repeated nuclear division and
binary fission without growth (palintomy), resulting in small, virgulate
bodies, each provided with a lateral flagellum. Chatton and Lwoff con-
sidered this organism to be a flagellate, but discussed its resemblance
to Chytridiales. They stated that it differs from that group in multiplying
by palintomy, with rapidly repeated mitosis after growth, instead of by
syntomy following nuclear multiplication accompanying growth. Mitch-
ell (1928), however, described multiplication of the same type as that
in Sporomonas in a chytrid of Euglena caudata; and on other grounds
also it appears that the distinction is not of crucial significance for
classification. The chief differences from chytrid parasites of Protozoa
are the expulsion of the organism from the host before multiplication
occurs and the active motility, by means of a flagellum, of the early
intracytoplasmic growth stages.

Georgevitch (1936a, 1936b) assigned to the genus Lezshmania, as
L. esocis n. sp., a hyperparasite of Myxidium lieberkihni in the urinary
bladder of pike. The intracellular phase is pyriform, with one nucleus

1074 PARASITES: OF) PROTOZOA

and a rod-formed structure called by Georgevitch a blepharoplast.
Growth, nuclear division, and plasmotomy result in rosettes, usually of
eight individuals and a residual body. Longitudinal division also occurs.
Flagellated stages were rarely found. The evidence that the parasite
is Leishmania is unconvincing, and is certainly inadequate as a basis
for extending the distribution of that genus to such an unusual location.

SARCODINA

The genus Psewdospora, which comprises parasites of true algae and
of Volvocidae, has been placed in the Proteomyxa; but there appears
to be good reason for accepting the suggestion of Roskin (1927) that
it belongs rather in the Bistadiidae of the Amoebida. P. volvocis, first
described by Cienkowski (1865), was later reported from Volvox by
Robertson (1905) and Roskin (1927). It seems possible that the
amoebae found by Molisch (1903) and Zacharias (1909) in Volvox
minor (V. aureus) belonged to this genus, though the authors do not
refer to earlier observations on parasites of Volvox, and their work is not
cited in later accounts of Pseudospora. Roskin (1927) described P.
eudorini from Exdorina, in which flagellate Robertson (1905) had te-
ported P. volvocis.

In its free-living state both these species are small, heliozoa-shaped
forms, with immobile or slow-moving pseudopodia. The heliozoan form
becomes amoeboid, with lobose pseudopodia, on contact with the host,
which it enters. Within the coenobium, the amoeboid form engulfs
the cells and undergoes repeated division. After a period, the parasite
comes to the surface of the colony, and there is rapid transition to a form
with two relatively long flagella. The organism may lose the flagella
and become amoeboid, may form cysts and the free heliozoan form, and
the heliozoan type may transform into a flagellate, as well as into an
amoeboid form.

It is generally considered that confusion with parasites is the basis
of the accounts of complicated life cycles in Arcella. The earlier ob-
servations on this testacean were discussed by Dangeard (1910), who
offered convincing evidence that the small amoeboid bodies, supposed
to be produced in numbers by repeated exogenous or simultaneous en-
dogenous budding (Awerinzew, 1906; Elpatiewsky, 1907; Swarczewsky,
1908), and variously interpreted by authors as reproductive phases of

PARASITES OF PROTOZOA 1075

the life cycle, are really parasites. The amoebulae were reported to take
on a heliozoa-like form on becoming free; Dangeard noted a similarity
to Nuclearia. An amoeboid parasite of Arcel/a was reported by Gruber
(1892), and evidently it is quite common, so many have been the ac-
counts of it. Doflein (Reichenow ed., 1927-29) and Deflandre (1928)
were inclined to accept the parasite interpretation of these supposed
reproductive bodies; but Cavallini (1926a, 1926b), without reference
to Dangeard’s paper, reported in Arcella vulgaris and Centropyxis
aculeata division of the protoplasmic body into many small amoebae,
which leave the shell and develop into the mature testaceans. Never-
theless, it is probable that binary fission is, as Deflandre (1928) stated,
the only mode of reproduction that has been satisfactorily demonstrated
in Arcella.

Penard (1912) found what he considered to be small parasitic
amoebae in Amoeba terricola and other species. They were often ob-
served moving actively within the pellicle of dead amoebae, from which
they eventually emerged and moved about freely, feeding on bacteria.
Penard found indication that these are parasites, the development of
which begins in the body of the large amoebae, but proof of this is
lacking.

There is, to the writer’s knowledge, only one record of amoebae
parasitic in a free-living ciliate. Chatton (1910) observed a very small
species living as a true parasite in Trichodina labrorum from the rock-
fish. Opalinid ciliates are, however, not uncommonly parasitized.

The hyperparasites of Opalinidae (Fig. 223A) resemble E. ranarum
(Carini and Reichenow, 1935; Brumpt and Lavier, 1936; Stabler and
Chen, 1936). Those in different opalinids have not been found to show
any taxonomic distinctions; and the systematic name to be used, for
some forms at least, is Entamoeba paulista (Carini); to be used, that is,
if this amoeba is truly an independent species. Carini and Reichenow
were of the opinion that the hyperparasite is either identical with En-
tamoeba ranarum ot is a race or species derived from this. Stabler and
Chen considered the question of the amoeba’s synonymy with E. ranarum
to be still open. Brumpt and Lavier, though recognizing a probable dis-
tinction from E. ranarum, discussed the relationship as paranéoxénie,
in which an intestinal parasite of the amphibian attacks another parasite
that accompanies it, which then becomes a subhost. From the standpoint

1076 PARASITES OF PROTOZOA

of general host-parasite relationships, it seems to the writer unlikely
that the same amoeba both parasitizes opalinids and lives in the in-
testinal lumen; certainly, to date, there is no proof of either possibility.

Published reports seem to indicate a greater prevalence of entamoebae
in Zelleriella than in other Opalinidae, but they have been found also in
Protoopalina, Cepedea, and Opalina (Chen and Stabler, 1936). The
geographical distribution of the amphibian hosts of parasitized Opa-
linidae has been found to be very wide, but Chen and Stabler stated that
“only certain species of anurans and a certain percentage of individuals
of a given species harbor the parasitized opalinids.” In a given host, the
percentage of ciliates containing hyperparasites varies greatly from none
to quite all, as noted by Carini (1933) and Brumpt and Lavier (1936)
in Zelleriella of Paludicola signifera, and by Chen and Stabler (1936)
in Zelleriella of Bufo marinus. The number of amoebae in a ciliate
ranges from one or very few up to a condition in which the cytoplasm
of the host is almost completely filled, more than a hundred being
present. They have been found mainly in the trophozoites, but may
occur also in precystic and encysted ciliates, according to Chen and
Stabler (1936), though Brumpt and Lavier (1936) stated that they had
never found them in the cysts. Chen and Stabler pointed out the im-
portance of the parasite in cysts, in establishing infections in a new
generation of the hosts.

In opalinids, entamoebae occur either as vegetative forms or as cysts.
Stabler and Chen (1936) stated that they apparently feed on the endo-
spherules, and they observed instances of mitotic division. It has been
noted (Stabler, 1933; Carini and Reichenow, 1935; Stabler and Chen,
1936) that the cysts in the ciliates are all uninucleate; the same observers
reported binucleate and quadrinucleate cysts outside of the hosts. It
seems, in general, that all the parasites in one ciliate are in the same
stage of development; but Brumpt and Lavier (1936) gave an uncon-
vincing figure of a Zelleriella said to contain a uninucleate and a bi-
nucleate vegetative form, a precystic form, and a uninucleate cyst.
Furthermore, in their statement that certain cysts contain two or four
nuclei, though most are uninucleate, it is not clear whether or not they
found multinucleate cysts in the opalinid host.

It has been noted that the parasites seem to have no injurious effect on
the host. The fact that parasitized ciliates may yet undergo normal divi-

PARASITES ‘OF PROTOZOA 1077

sion (Brumpt and Lavier, 1936; Stabler and Chen, 1936) is the most
important evidence for this benignity.

No strictly parasitic relationship has been established in any member
of the group Heliozoa proper, but Wetzel (1925) discussed as tempo-
rary parasitism the association between Raphidiocystis infestans and cili-
ates (Fig. 223B). This organism is normally predatory on flagellates
and small Infusoria, but when it attacks larger ciliates, such as Para-
mecium, Colpidium, Glaucoma, Nassula, and Trachelius, it passes,

INNA

Figure 223. A, trophozoites of Entamoeba sp. in Zelleriella (after Stabler and Chen,
1936); B, Raphidiocystis infestans Wetzel on Paramecium (After Wetzel, 1925.)

according to Wetzel, to the parasitic manner of life. The heliozoans
become attached by means of pseudopodia to various parts of the body,
those pseudopodia gradually shorten, and eventually they lie very flat
on the surface. The body of Paramecium may be completely enclosed
by many fused Raphidiocystis, which extract dissolved nutriment until
only a remnant of the ciliate is left—a process which requires from
twenty or thirty minutes to two days—after which the heliozoa disperse.
Wetzel discussed the significance of this type of relationship in the
phylogenetic origin of parasitism as transitional between strict preda-
tism and pure parasitism.

1078 PARASITES OF PROTOZOA

Ivanic (1936) stated that the parasite of Entamoeba histolytica, which
he described as Entamoebophaga hominis, shows affinities to the Myce-
tozoa, but this is hardly apparent from his unconvincing account of the
structure and life history. The earliest stages are reported to occur
within the host cyst, and growth leads to an amoeboid body. At first
uninucleate, this becomes multinucleate. When the cytoplasm of the host
cyst has been largely consumed, the parasite breaks out and carries on
for a time an active, free-living existence in the intestinal lumen, as an
amoeboid organism. In this phase there is nuclear multiplication, binary
fission, growth to a giant, multinucleate plasmodium, and endogenous
budding. Ivani¢ found evidence that the organism was originally a
commensal of the human intestine before it became a parasite of E.
histolytica. This bizarre account probably contains a good deal of mis-
interpretation and confusion of distinct organisms.

SPOROZOA

The sporozoan parasites reported in Protozoa belong for the most
part to the Microsporidia and Haplosporidia. Dogiel (1906) assigned
to the coccidia a parasite, named Hyalosphaera gregarinicola, of a gre-
garine from a holothurian. Caullery and Mesnil (1919) considered this
systematic determination doubtful, but were certain that the parasite 1s
not a Metchnikovella. Dogiel described macrogametes, microgametocytes,
and sporulation, but did not observe schizogony.

Four microsporidian parasites of Protozoa were listed by Kudo
(1924). Three of these are species of Nosema: N. marionis (Thélohan,
1895) Stempell, 1919, in the myxosporidian Ceratomyxa (Le ptotheca)
coris from the gall bladder of Coris julis and C. giofredi; N. balantidu
Lutz and Splendore, 1908, in Balantidium sp. from Bufo marinus; and
N. frenzelinae Leger and Duboscq, 1909, in the gregarine Frenzelina
conformis from Pachygrapsus marmoratus. The last species shows a
certain amount of correlation with the life cycle of the host, in that
sporulation occurs at the moment of encystation of the gregarine. The
gregarines develop normally up to a certain point; then the formation
of gametes does not take place (Leger and Duboscq, 1909a, 1909c). The
fourth species, Perezia lankesteriae, also parasitizes a gregarine, Lankes-
teria ascidiae from Ciona intestinalis (Leger and Duboscq, 1909b).

Microsporidia are probably much more widespread as parasites of

PARASITES OF PROTOZOA 1079

Protozoa than the published accounts indicate. Duboscq and Grassé
(1927) showed certain parasites in “Devescovina’ hill: which they
considered to be possibly Microsporidia. The organism found by Kirby
(1932a) in Trichonympha magna gives certain indications of micro-
sporidian affinities (Fig. 219H, 1). An organism with resemblances
to Nosema, though enigmatic in relationship, has been observed by the
writer in Gigantomonas herculea ftom the termite Hodotermes mos-
sambicus. All the above-mentioned Microsporidia are hyperparasites,
but there is probably at least one recorded instance of their occurrence
in a free-living ciliate. A number of authors have reported “nemato-
cysts” in the large vorticellid Epistylis (Campanella) umbellaria; these
were discovered by Claparéde and Lachmann (1858). They are arranged
in pairs in the ciliate, but are not always present. Fauré-Fremiet (1913),
although having often observed Campanella, found ‘‘nematocysts” only
once. Chatton (1914) suggested that the structures belonged not to the
vorticellid but to a cnidosporidian parasite, and in a recent note Kruger
(1933) supported this view. Kriiger observed in the cytoplasm of the
ciliate granules that he thought might be nuclei of developmental stages
of the parasite.

From the standpoint of host-specificity, the Metchnikovellidae are of
particular interest, for all members of this family, and there are many,
occur in gregarines. The first metchnikovellid was seen by Clapareéde,
but he failed to interpret it correctly, mistaking the cysts for spores of
the gregarine (Caullery and Mesnil, 1919, p. 232.) This group of
Haplosporidia has been studied chiefly by Caullery and Mesnil (1897,
1914, 1919), but contributions have been made also by Awerinzew
(1908), Dogiel (1922b), and Schereschewsky (1924). An account of
the life cycles of two species of Amphiacantha has been prepared by
Stubblefield (MS). MacKinnon and Ray (1931) reported some ob-
servations on species of Mefchnikovella from two species of Polyrhab-
dina at Plymouth; and Ganapati and Atyar (1937) noted the occurrence
of Metchnikovella in Lecudina brasili from a species of Lumbrinereis
at Adyar. In the absence of any description or figure, it is not certain
that this is not a species of Amphiacantha, as found in related gregarines
in Lumbriconerezs elsewhere.

Stubblefield (MS) listed twenty species, including the two new spe-
cies of Amphiacantha recognized by him, in four genera. The largest

1080 PARASITES OF PROTOZOA

genus is Metchnikovella Caullery and Mesnil, with thirteen species;
there are three species of Amphiamblys C. and M., three of Am phia-
cantha C. and M., and one of Caulleryella Dogiel. All the grega-
tines that have been found to contain these hyperparasites occur in
annelids, and all but one in marine polychaetes. This one, Metchnikovella
hesse? Mesnil, 1908, is found in a monocystid gregarine of the terrestrial
oligochaete Fridericia polycheta. The parasitized gregarines belong to
various groups, and, according to Caullery and Mesnil (1919), there is
no parallelism between the structure of the Metchnikovellidae and that
of the gregarine hosts. The host-specificity is apparently on an ethological
rather than a phylogenetic basis. The species of Am phiacantha, however,
have been found in gregarines of the genus Ophiodina (Lecudina) ot
related forms in Lumbriconereis in France and California.

Caullery and Mesnil (1919) stated that when there is an infection,
the greater part of the gregarines of a host are invaded. Stubblefield
(MS) found a high frequency of Amphiacantha in about 20 percent
of the worms collected, almost all of which contained gregarines.

Published literature gives little information about details of the life
cycles of Metchnikovellidae. Caullery and Mesnil (1919) regarded the
individualized, nucleated bodies enclosed by the cyst membrane as spores
(““germes sporaux”’). When the cysts are ingested by an annelid, these
are released in the digestive tract, and penetrate into the cytoplasm of
the gregarines. Growth and nuclear division lead in some instances to
multinucleate plasmodia. In other instances there are numerous indi-
vidual, uninucleate bodies, isolated or arranged in series. Caullery and
Mesnil supposed that cysts develop by the formation of a membrane
around groups of these cells or the plasmodium. The cyst contents 1s
thus either multinucleate or in individualized uninucleate bodies from the
beginning. Such a manner of cyst formation is difficult to understand.

Stubblefield (MS) prepared an account of the life cycle of Amphia-
cantha, which is in closer agreement with that of Haplosporidium.
He found evidence for the penetration of the gregarine by an active
sporozoite; the growth of the sporozoite, followed by schizogony, to
produce trophozoites; the development of the trophozoite into a cyst,
which is at first uninculate; nuclear and cytoplasmic division, to pro-
duce bodies in the cyst (Fig. 224), which he considered to be
gametocytes; the release of these by the rupture of the cyst within the

PARASITES OF PROTOZOA 1081

gregarine, after which they undergo reduction, producing gametes which
fuse; and finally the development of sporozoites from the zygote. Stubble-
field’s observation that cysts rupture within the gregarine host ts in

Figure 224. Cysts of two species of Amphiacantha, metchnikovellids parasitic in the
gregarine Ophiodina elongata from Lumbricoinereis. (After Stubblefield, MS.)

agreement with the statement of Mackinnon and Ray (1931) that the
“spores” of Metchnikovella caulleryi have been seen escaping from the
cysts into the endoplasm of the gregarines.

Caullery and Mesnil stated that Metchnikovellidae seem to have little
pathogenic action on the host, particularly in the vegetative stages. What
injury there is, is mechanical, when infection is heavy. Ganapati and Aiyar
(1937) noted that the entire cytoplasm may be packed with cysts, and

1082 PARASITES; OF) PROTOZOA

the body become much misshapen, the gregarine nucleus seemingly
degenerating. Stubblefield, however, found that more than six cysts
rarely occur in a gregarine. Caullery and Mesnil believed that heavily
parasitized gregarines are incapable of completing their sexual develop-
ment; and Ganapati and Aiyar remarked that parasitized gregarines
were not observed to associate.

The affinities of the Metchnikovellidae are uncertain. They have been
related to fungi (Chatton, 1913, yeasts), to Microsporidia (Schere-
schewsky, 1924), and to Haplosporidia (Awerinzew, 1908). Caullery
and Mesnil (1919), while remarking on a certain similarity in nuclear
structure to Myxomycetes and Chytridiales, concluded that they are
isolated among the lower Protista. Doflein (Reichenow ed., 1927-29)
accepted their allocation to the Haplosporidia, and this position was sup-
ported by Stubblefield (MS).

Caullery and Mesnil (1919) provisionally designated as Bertramza
selenidicola a parasite of a species of Selenidium from certain poly-
chaetes, and reported a related parasite in Selentdium virgula. Other
species of Bertramia are parasites in the body cavities of worms and
rotifers. Another parasite with apparent haplosporidian affinities, but
unlike Metchnikovella, was observed in a species of Polyrhabdina. It
existed as isolated granules and multinucleate masses, the schizonts and
sporonts; and as separate ovoid bodies, not enclosed in a cyst, which
were evidently spores.

Elmassian (1909) found a hyperparasite, Zoomyxa legerz, in Exmeria
rouxi, which causes fatal coccidiosis in tench. He considered this to be
a haplosporidian, but also discussed its similarities to lower Mycetozoa.
It is likely that his account is at least in part incorrect. The parasite 1s
said to occur both on the surface of the epithelium and in the cells of
Eimeria, the intracoccidian parasitism being accidental. There are said
to be several types of schizogony, within coccidia or not; and in this
supposed haplosporidian the author described a sexual cycle with coc-
cidian-like development of microgametes and macrogametes, and the
formation of resistant cysts containing from six to twelve sporozoites.
The parasite has pathogenic effects on the nucleus and cytoplasm of
Eimeria, causing hypertrophy and eventual dissolution of the cell.
Elmassian thought that the effects are brought about by toxic secretions,
which act not only on the coccidia but also on the neighboring fish cells.

PARASITES OF PROTOZOA 1083

CILIOPHORA

Euciliata.—There are few reports of ciliates parasitizing other Proto-
zoa, except for Phtorophrya and Hypocoma. Penard (1904) found a
ciliate, which seemed to resemble Blepharisma, in a large percentage
of the heliozoan Raphidiophrys viridis. The intracytoplasmic forms
showed different degrees of development and lived many days in isolated
Heliozoa. An immobile organism, with a large contractile vacuole but
no cilia or flagella, was found parasitic in three-fourths of a large number
of Pseudodifflugia horrida by Penard (1905a). This, he stated, suggested
the larger ciliate in the heliozoan, but its affinities are uncertain. Hertwig
(1876) reported that a hypotrich bored into the body of Podophrya
gemmipara, in the region in which the body is joined by the stalk, and
destroyed the acinetid.

A number of apostomatous ciliates are parasitic in other Foettin-
geriidae. The most completely known of these is Phtorophrya insidiosa
Chatton, A. Lwoff and M. Lwoff, 1930, which is parasitic on Gymno-
dinioides corophii. The phoront of Phtorophrya is attached to the phoront
of Gymnodinioides, which occurs on Corophium acutum. The body of
the parasite leaves the phoretic cyst and introduces itself into the body of
its host, becoming a parasitic trophont. It grows rapidly, ingesting the
cytoplasm of its host, and soon comes to occupy entirely the otherwise
empty cyst of Gymnodinioides. By division, four to eight small ciliates,
the tomites, are produced. These escape from the host cyst and swim
actively in search of another phoront of Gymnodinioides.

Chatton and Lwoff (1930, 1935) described also the following in-
completely known species of this genus of ciliate parasites: Phtorophrya
mendax in the phoronts of Gymnodinioides inkystans; P. fallax in this
same host species; P. steweri in Vampyrophrya (2) steuert; P. bathy pela-
gica in Vampyrophrya bathy pelagica.

The Hypocomidae, like most other Thigmotricha, occur on bivalves
or snails, except for species of the genus Hypocoma, which are parasitic
on other Protozoa. Hypocoma parasitica (Fig. 199G-1) was found by
Gruber (1884) and Plate (1888) on marine vorticellids, especially
Zoothamnium, on the coast of Italy. Plate recognized a second species,
H. (“‘Acinetoides”) zoothamni. The ciliates occur firmly fixed to the
host, and suck out the contents of the zodids. Hypocoma acinetarum
Collin is ectoparasitic on Suctoria. Collin (1907) found it on various

1084 PARASITES OF PROTOZOA

occasions on Ephelota gemmipara and Acineta papillifera; and Chatton
and A. Lwoff (1924b) encountered it on Trichophrya salparum., In
Ephelota it attacks chiefly the region in which the stalk is attached to the
body. It sucks out plasma, and its presence leads to fragmentation of the
nucleus and degeneration of the whole cytoplasmic mass. The parasite
then detaches and swims to another suctorian. Hypocoma ascidiarum
Collin was found on a tunicate, but probably actually is a parasite of
Trichophrya salparum, and may not be different specifically from H.
acinetarum (Chatton and A. Lwoff, 1924b).

Ectozoic Suctoria—In connection with the relationship between ex-
ternally attached Suctoria and their ciliophoran hosts, we must keep in
mind the fact that phoresy is widespread among Suctoria. Many forms
occur attached to other organisms, and often a species has been found
only on a particular host species. The host is not directly concerned in
the nutritive processes of the suctorian. Ciliophora may, like many Meta-
zoa, serve as hosts for these ectocommensals. Examples of such phoretic
forms are Ophryocephalus capitatum Wailes on species of Ephelota;
Urnula epistylidis Claparéde and Lachmann on Epistylis and other Suc-
toria (see Gonnert, 1935); Tokophrya quadripartita (Cl. and L.) on
Epistylis; Trichophrya epistylidis (Cl. and L.); Metacineta mystacina
(Ehrbg.) on Carchesium,; and Tokophrya carchesu (Cl. and L.) on
Carchesium. Ectocommensalism in such attached forms may be obligatory
or facultative.

Pseudogemma Collin is more closely adapted to an ectoparasitic man-
ner of life on other Suctoria. Reproduction is by internal embryos, and
fixation to the host is by a short, stout peduncle embedded in the cyto-
plasm. Tentacles are absent, and Collin (1912) considered it possible
that the fixation organelle has an absorptive function. Collin listed three
species: P. fraiponti Collin, 1909, on Acineta dirisa; P. pachystyla
Collin, 1912, on Acineta tuberosa; and P. keppeni Collin, 1912, on
Acineta papillifera. The last species is said to have a rounded form and
apparently no pedicle, and its location in its host is sometimes external
and sometimes almost entirely internal. Collin (1912) believed that it
furnishes a natural transition from Psexdogemma to Endosphaera.

The species of Allantosoma occur in the intestine of the horse, an
endozoic habitat which is unique for Suctoria. According to Hsiung
(1928), the species A. dicorniger is strictly a lumen-dweller, and is

PARASITES OF PROTOZOA 1085

not attached to any other organism. The other two species have certain
relationships to ciliates, but apparently that is only occasional. Hsiung
wrote of A. zntestinalis that some were attached to Cycloposthium bipal-
matum and Blepharocorys curvigula; Gassovsky (1919) recorded the
species as occurring in the colon, rarely the caecum, of horses, without
mention of any attachment to ciliates. A. brevicorniger, Hsiung states,
is ‘‘often found attached to the body of the ciliate Paraisotricha colpoidea
by one tentacle.” Apparently these Suctoria prey upon the ciliates, but
are not constantly attached, as obligatory ectoparasites would be.

The only account of a suctorian clearly ectoparasitic on Euciliata is
Chatton and A. Lwoff’s (1927) description of Pottsia infusoriorum. The
chief host is Follzculina ampulla, but it has been found also on Cothurnia
socialis. The parasites may occur in numbers on the body of Folliculina,
within the lorica. There are four tentacles at one end, on the surface in
contact with the host, prolonged rather deeply into the body of the host.
Embryos develop endogenously, swim actively, and become fixed to the
body of Folliculma. On different occasions, from none to 75 percent
of the heterotrichs have been found parasitized with as many as twenty-
two parasites. When the number of Pofts/a is large, the host undergoes
degeneration. The parasites may survive for a time among the remains,
but eventually themselves disintegrate.

Tachyblaston ephelotensis, as described by Martin (1909), has a
curious life cycle, involving both an external phoretic existence with
multiplication, and an intracytoplasmic parasitism, also with multiplica-
tion. It seems not impossible that reinvestigation will show that
two organisms have been confused in this cycle, since it is so unlike
the life histories of other Suctoria. The intracellular phases occur as
rounded bodies in Ephelota gemmipara. There is equal division, fol-
lowed by the formation of buds. Ciliated “‘spores’ escape from the host
and after a brief period of existence become attached to the stalk of
Ephelota, developing a stalked lorica. The fixed form undergoes rapid
budding. Each bud is provided with a single tentacle, with the aid of
which, together with “‘euglenoid changes of shape,” the bud travels up
the stalk to penetrate into the body of Ephelota. The internal parasitic
phase destroys the cytoplasm of the host.

Endozoi Suctoria—The Suctoria that occur as internal parasites of
other Ciliophora, and have a wide variety of hosts, belong to the genera

1086 PARASITES OF PROTOZOA

Sphaerophyra and Endosphaera. In connection with them, it is interesting
to consider the important rdle they have played in the development of
protozodlogy. The Acineta theory and the embryo theory of ciliate devel-
opment held an important place in the thinking of protozodlogists in
the third quarter of the nineteenth century.

Stein’s Acineta theory was in the first instance not related to parasitic
Suctoria. He came (1849, 1854) to the conclusion that free-living
acinetids are the result of metamorphosis of vorticellids, and that they
give rise to embryos from which the vorticella form is again produced.
This embryo production, of course, is the result of the internal budding
process characteristic of acinetids. This theory was successfully attacked
by Cienkowski (1855a), Lachmann (1856), and Claparéde and Lach-
mann (1860-61). Stein later (1859) modified the Acineta theory as it
was originally stated, but still did not admit that acinetids are inde-
pendent organisms. The embryos of various Infusoria, he said, have all
the characteristics of acinetids; and he believed that various higher
Infusoria in their development pass through Acineta-like phases; for
example, that podophryids were developmental phases of Paramecium.

Authors credit Focke (1844) with the first observation of the so-called
motile embryos. He discovered them in Paramecium bursaria, in which
they were soon found by many other observers. They were found also
in a variety of other Euciliata. As late as 1867, Stein could state that
“today no one can doubt that those Infusoria whose reproductive organ-
ization consists of nucleolus and nucleus are in fact hermaphrodites, the
nucleus playing the rdle of a female, the nucleolus of a male sex organ”;
and could maintain that the embryonal spheres were produced from the
nucleus.

Stein’s thesis, however, had already been discredited. Claparéde and
Lachmann (1858-59) had described Sphaerophrya pusilla in water,
associated with numerous oxytrichids; yet they were not firm in their
opinion that Sphaerophrya might not be an embryo of Oxytricha. It was
the view of Balbiani (1860) that the so-called embryos of ciliates were
parasites belonging to the genus Sphaerophrya; and in support of this he
adduced his observations on entry into ciliates, and on the spread of an
infection among Paramecium by the introduction into a sound culture of
a few infected ciliates. Metschnikoff (1864) observed the cycle, from
separation from one Paramecium host through entry into another, and

PARASITES OF PROTOZOA 1087

considered the parasite nature of the so-called embryos to be completely
proved.

Sphaerophrya shows suctorian characteristics in the presence of tenta-
cles. The so-called embryos of certain vorticellids, however, do not have
this characteristic; they are simple spherical or ovoidal bodies with
equatorial bands of cilia. Stein (1867), in his efforts to combat Balbiant,
had only weak arguments against the parasitic nature of the Sphaero-
phyra-type ‘embryos’; but he was firm in his conviction that the “em-
bryos” of vorticellids (Epistylzs plicatilzs) could not be parasitic Infu-
soria. Such a concept, he stated, would be ludicrous. Engelmann (1876),
reviewing the whole question in support of the parasite theory (which
he had vigorously opposed in 1862), reported having observed the
entry of the supposed non-tentaculated embryos of Vortscella microstoma
into that host. Thus he proved the parasitic nature of that organism also,
a view also stated by Biitschli (1876), and gave it the name Endos phaera.

The endozoic forms of Sphaerophrya are but little modified in conse-
quence of parasitism, and the majority of species of the genus are
entirely free-living. S. stentor7s Maupas is free-living or parasitic in
species of Stentor; recently Kalmus (1928) reported it in S. roeselz, The
parasites of other ciliates have all been placed in a second species, which
also is either free-living or endozoic. Biitschli (1889) and Sand (1899)
identified this second species with Claparéde and Lachmann’s free-living
S. pusilla. Collin (1912), however, considered it to be S. so/ Metchnikoff
which also was originally described as a strictly free-living species.
Sand regarded S. so/ as a synonym of S. pusilla.

Species of the genus Sphaerophrya differ from those of Podophrya
in the absence of a stalk. The body is spheroidal or ellipsoidal, and
tentacles radiate from the entire surface. Reproduction is by equal or
unequal fission or by external budding, except in S. stentoris, which 1s
reported to show a transition to internal budding. The free-swimming
forms produced by budding are provided with cilia that are localized
at one extremity, in a girdle, or generally distributed; and they possess
tentacles. This form, in parasitic phases, penetrates the surface of a
ciliate and takes up a position in the cytoplasm, losing cilia and tentacles.
There reproduction by division and budding takes place.

Endosphaera (Fig. 225) has become more closely adapted to para-
sitism. It does not occur as a free-living organism, except briefly in the

1088 PARASITES OF PROTOZOA

motile phase that passes from one host to another; and it has no tentacles
at any time. Endosphaera has been found in vorticellids of the genera
Vorticella, Zoothamnium, Epistylis, Carchesium, Trichodina, and
O pisthonecta. All these have been assigned to the species E. engelmanni
Entz, the most adequate study of which was published by Lynch and
Noble (1931). Génnert (1935) described E. multifilis, reporting tt
from the Suctoria Lernaeophrya capitata Perez, Trichophrya epistylidis
Cl. and L., Tokophryidae, and Dendrosoma,; and from vorticellids.

Figure 225. Opisthonecta henneguyi, parasitized by Endosphaera engelmanni. End.
emb., Endosphaera containing an embryo; dis. emb., embryo being discharged through
birth pore. (After Lynch and Noble, 1931.)

Lynch and Noble found a high incidence of infection in O pssthonecta
henneguyi, with as many as twelve parasites, most of which contained
one or occasionally two or three internal embryos. They found each
parasite to be attached to the pellicle of the host by a short stalk, per-
forated by a canal terminating in a birth bore. The spherical embryo,
provided with three equatorial bands of cilia, was discharged through
this pore. Embryos were observed to attach themselves to the host, and

PARASITES OF PROTOZOA 1089

successive stages of penetration were studied in preparations. The authors
found no evidence that the parasite pushed an extensible pellicle before
it, forming an invaginated chamber in which it dwelt, such as was
described by Balbiani (1860) and Biitschli (1876) in Sphaerophrya.
Endos phaera was observed in cysts of O pisthonecta, which could account
for the survival of the parasite under unfavorable conditions.

The embryos of E. mu/tifiliis GOnnert have five bands of cilia. Génnert
(1935) observed penetration into Lernaeophrya, preceded by the re-
sorption of cilia and the development of a long, mobile, penetrating
protoplasmic process. He observed no canal connecting the internal
parasite to the surface of the host. Exdosphaera lives, he stated, four or
five days, and an embryo may be produced every half hour.

Sphaerophrya and Endosphaera appear to be relatively benign para-
sites, except when present in large numbers. The effect is then evidently
mechanical. Balbiani (1860) remarked that oxytrichids with more than
fifty parasites were greatly swollen and deformed, but that ordinarily the
host seemed to be not at all inconvenienced. Génnert found that Endo-
Sphaera, when present singly, had slight effect on the host, but that the
host often perished from multiple infection.

THE GENUS AMOEBOPHRYA KOEPPEN

Amoebophrya is even more of a zodlogical enigma than is Sticho-
lonche, one of its hosts, which Korotneff (1891) wrote of as a “‘zodlogi-
cal paradox.”” A modern study of the structure and development of the
organism, which would throw light on its affinities, is much to be desired.
The evidence that it is a suctorian, accepted by Koeppen (1894), Bor-
gett (1897), Sand (1899), and Hartog (1909, Cambridge Natural
History), is not convincing. Its assignment to the Mesozoa, made by
Korotneff (1891) and Neresheimer (1904, 1908, and later) and agreed
to by Collin (1912), does little more than emphasize its enigmatic
qualities.

Hertwig (1879) described what he regarded as a very peculiar nuclear
form within the central capsule of three species of acanthometrid Radio-
laria. (Fig. 226D). He stated that he found this body twice in Acantho-
Staurus purpurascens and once each in Acanthometra serrata and A.
claparédei; and he showed what is doubtless the same thing in Am phi-
lonche belonoides. He described this as a large vesicle containing a very

1090 PARASITES OF PROTOZOA

large nucleolus, around the sharpened end of which was a conical struc-
ture, the membrane of which was marked by circular striations.

Fol (1883) found structures, which he described as analogous to
those seen by Hertwig, in the ectoplasm of Sticholonche zanclea. Some of
the Radiolaria contained spherical bodies, of rather complex structure,
which increased in size as the host became older, and contained an ill-
defined “‘spiral body.”’ At maturity, these bodies left the host and were
capable of rapid movement, comparable to that of very active Infusoria.
The free organism had a spiral groove turning from left to right and
was completely covered with short, fine cilia. Other specimens of the
radiolarian contained a mass of globules, which increased in size
and number, finally becoming in volume equal to the rest of the body.
Fol advanced two hypotheses in interpretation of these structures: one,
that the globules are female reproductive elements, while the spiral
body is a sort of spermatophore; the other, which he regarded as also
reasonable, that the structures represent parasites. On the last supposition,
he stated, it would be difficult to explain the fact that the two kinds
of inclusions occur only in different individuals in approximately equal
numbers.

Korotneff (1891), who studied the “spiral body” in Stzcholonche
obtained at Villafranca, concluded that it is a parasite and made the
first suggestion as to its affinities. Believing himself to have demon-
strated an endoderm of a few cellular elements and a cellular ectoderm,
he considered the parasite to be closely related to the orthonectids and
possibly a stage in their development.

The parasites in both Stscholonche and acanthometrids were studied
by Koeppen (1894), who gave them the names Amoebophrya sticho-
lonchae and A. acanthometrae, and who was convinced that the organisms
are acinetids. He stated that he had studied all phases of development
in the same specimen. He based his taxonomic conclusion on supposed
development, in the parasitic phase, of an embryo, the spiral body, by
internal budding; and on the existence of tentacles for a short period
after this embryo became free and lost its cilia. The so-called tentacles
soon disappeared, and the body commenced to vary in form, showing
slow amoeboid movements. There is no proof that the protoplasmic
processes were actually tentacles; evidence is lacking that the behavior was
observed repeatedly under normal conditions; and there are no support-
ing illustrations.

PARASITES OF PROTOZOA 1091

Borgert (1897) found parasitized Sticholonche and acanthometrids
in the Gulf of Naples and prepared the most complete existing account
of the organisms. Although he disagreed with many of Koeppen’s inter-
pretations and found a large number of nuclei in the outer layer, he
nevertheless agreed with him that Amoebophrya is a parasitic suctorian.

Amoebophrya sticholonchae (Fig. 226A-C) is a common parasite
of Sticholonche zanclea in the Mediterranean. Borgert found parasites

/
fore

| (2 Bae

Figure 226. Amoebophrya in Radiolaria. A, Sticholonche zanclea containing A.
sticholonchae; B, longitudinal section of Amoebophrya in Sticholonche, and, in lower
half of figure, section of host and its central capsule; C, A. sticholonchae emerged from
its host; D, A. acanthometrae in Acanthometra serrata. (A-C, after Borgert, 1897; D,
after Hertwig, 1879.)

only in the latter part of March, 1895, though the radiolarian was abun-
dant also before and after that period. In its host (Fig. 226A) it 1s
an approximately spherical body, located on the concave side of the
capsule, transparent, and pale yellowish in color. Within the sphere
is a conical body, the point of which is directed toward the body surface
of the host (Fig. 226B). The outer surface of the cone is marked by
furrows in a close-set, left-wound spiral. The outer surface of the cone
is continuous at its base with the inner surface of the sphere, and the
spiral furrow continues on the latter. The form of the parasite in this
stage has been compared to that of a half-invaginated glove finger. Bor-
gert described a large number of very small nuclei arranged in rows
between the furrows. In younger parasites there were fewer nuclei, and
in an appendix he reported having found a few individuals with single

1092 PARASITES OF “PROTOZOA

large nuclei. No cell boundaries were seen, and the nuclei varied in
size. The outer layer of the body does not have an epithelial structure;
and perhaps the evidence for the nuclear nature of the inclusions is
inconclusive.

The parasites can easily be induced to leave the host. Borgert found
it sufficient to put Stzcholonche in a small amount of water on a slide,
when escape was apparently stimulated by the increase in salinity and
possibly in temperature. At the beginning of the transformation to the
free stage, the tip of the conical part breaks through the surrounding
sphere, and cilia appear and become active. The entire body, having
become everted, emerges and swims actively in the water. Its form 1s
elongated and more or less cylindrical, and it possesses a spiral furrow
in which arise abundant small cilia (Fig. 226C). In the interior is a
cavity, larger in younger specimens, reduced to a tubular form in older
ones, which sometimes is open at the posterior end of the body.

Amoebophrya acanthometrae was found in four acanthometrids by
Hertwig, in two others by Haeckel, and in a seventh species by Borgert.
Borgert stated the probability that the parasite will be found to occur
in all acanthometrids the skeletal structure of which permits. He observed
it only in uninucleate phases of the host. In 1895 at Naples, after
Amoebophrya disappeared from Sticholonche, parasites were found re-
peatedly in acanthometrids.

Unlike the other species, A. acanthometrae occurs within the central
capsule. According to Borgert, it encloses the nucleus of the radiolarian;
but this fact does not discommode the latter. Nuclei are extraordinarily
small (up to 1 to 2 y), and were not observed at all in some, especially
young, specimens. Emergence of the free phase, which is so easy to
observe in A. sticholonchae, happens only occasionally. Apparently the
nucleus of the host is removed in this process. The free form has a
plumper figure than that from Sticholonche, and the cilia are better
developed.

There remain to be considered the groups of small spherules which
occur usually in specimens of Sticholonche without Amoebophrya,
though sometimes, contrary to the opinion of Fol (1883), the two are
found in a single host. Younger stages, according to Borgert, consist of
a spherical protoplasmic mass with a few spherical nuclei. A great
number of small nuclei result from division of these. Eventually these
nuclei become the center of vesicles, which become free in the host

PARASITES OF PROTOZOA 1093

cytoplasm by dissolution of the earlier common plasma mass. Borgert
regarded these bodies as parasites of the radiolaria, unrelated to Amoe-
bophrya. Though probably it is only an analogy, certain features in their
development suggest the life history of Sphaerita.

It appears from statements by Neresheimer (1904, 1908) that Amoe-
bophrya is not restricted entirely to Radiolaria. Doflein, he wrote, showed
him preparations of Noctiluca miliaris in which the parasite was present.

METAZOA

A number of rotifers live attached to other animals as ectocommensals
or ectoparasites. Ehrenberg (1838) found Proales petromyzon (Ehrbg.)
and its eggs attached to the branched vorticellids Epzstylis digitalis, Car-
chesium polypinum, and Zoothamnium geniculatum, and stated that it
devours the vorticellid. Wesenberg-Lund (1929) showed it and its
eggs on Zoothamnium. It is a predator rather than a parasite, but differs
from ordinary predators in its attachment. Hudson and Gosse (1889),
however, found it always free, though often in close association with
Epistylis and Carchesium.

Approaching closer to parasitism are certain species of Proales, which
live in certain algae and Protozoa. P. werneckui (Ehrbg.) occurs rather
commonly in galls on Vaucheria; P. parasita (Ehrbg.) is parasitic in
Volvox; and P. latrunculus Penard invades the heliozoan Acanthocystis
turfacea.

Proales parasita was found by Ehrenberg (1838) and Cohn (1858)
in Volvox. Plate (1886) described Hertwigia volvocicola from Volvox
globator, considering this to be a different species from the preceding.
It is listed as a synonym of P. parasita by Hudson and Gosse (1889),
but Wesenberg-Lund (1929) considered it again as a separate species.
Whether different or not, the habits of the forms are the same. They
swim about within Volvox coenobia and feed on the cells. The males
live only a day or two, remaining entirely within the host in which they
ate hatched. The females may be found within or outside the coenobium.
Eggs are laid in the host, where they hatch and, according to Hudson
and Gosse, the young rotifer either enters a daughter colony and 1s
expelled with it or emerges to swim free. Hudson and Gosse stated that
“Volvox appears to suffer little from the depredations of its ungrateful
guest.”

1094 PARASITES OF PROTOZOA

Penard (1904, 1908-9) has given the most complete account of
Proales latrunculus, certain observations on which had been made by
Archer (1869), Leidy (1879), and Stokes (1884). Penard studied an
epidemic outbreak of the parasite, which eventually carried off most of
a group of the Heliozoa. He stated, however, that it is rare, in the sense
that many Acanthocystis turfacea in various localities may be examined
without encountering it. It is widespread geographically, as indicated
by the records from Switzerland, England, and the United States.

After being introduced into the body of Acanthocystis, probably,
according to Penard, by being ingulfed as prey, the rotifer moves about
actively in the cytoplasm. It devours the zodchlorellae and the substance
of the heliozoan. In two or three hours an egg may be laid, after which
the rotifer may continue to feed and lay a second, smaller egg. The
heliozoan occasionally frees itself of the invader, but usually it perishes
before the end of the first day. After laying its eggs, the rotifer escapes
by an orifice in the then empty envelope of spicules—empty, that is,
except for the few small eggs. The young rotifers develop rapidly,
hatching in two or three days, when they leave by the orifice through
which the parent escaped.

As Penard (1908-9) remarked, these rotifers are not true parasites,
as they are not adapted to continuous existence in their host. They behave
rather as predaceous forms which consume the host from within. One
notes a marked specificity to certain hosts or related hosts in the rotifers
ectozoic on colonial vorticellids and those endozoic in V aucheria, Volvox,
and Acanthocystis.

Ehrenberg (1888) on one occasion found the usually ectozoic P.
petromyzon within Volvox globator,; and Wesenberg-Lund (1929) stated
that Volvox contained also species of Diglena, rotifers that are naturally
free-living.

Living nematode worms have occasionally been encountered in Pro-
tozoa. It is not known whether this ever represents obligatory parasitism,
or is only an invasion by accidentally or facultatively parasitic forms.
Wesenberg-Lund (1929) stated that free-living nematodes have been
found in Volvox, and Schubotz (1908) wrote that Hartmann informed
him of having seen nematodes in that flagellate. Schubotz found as many
as three nematodes in approximately a tenth of Pycnothrix monocystoides
from Procavia capensis. He stated that for entry into this large ciliate,

PARASITES; OF PROTOZOA 1095

the worms use openings in the ectosarc or, in undamaged animals, the
excretion pore. They are then found wholly or partly in the canal system,
whose walls they at times break through. Myers (1938) found nematode
worms in the foraminiferan Rofalza turbinata in an incidence, in the
colder months of the year, of 5 percent.

LITERATURE CITED

Alexeieff, A. 1912. Sur un Chlamydozoaire parasite des Protozoaires. Sur le
Chlamydozoaire du cancer. Arch. zool. exp. gén., (5) 10: N. et R., 101-
KO:

—— 1929. Matériaux pour servir a l'étude des protistes coprozoites. Arch.
zool. exp. gén., 68: 609-98.

Archer, W. 1869. On some freshwater Rhizopoda, new or little known.
Quarts). muicts Sa., n:'s,, 9:°250-7.1.

Awerinzew, S. 1906. Die Siisswasser-Rhizopoden. Trav. Soc. Nat. St.-Pétersb.
(Leningr.), 36: 1-351. (Russian, with German résumé.)

—— 1908. Studien uber parasitische Protozoen. Trav. Soc. Nat. St.-Pétersb.
(Leningr.), 38: v-xii, 1-139.

Bach, F. W., and P. Quest. 1923. Uber Spirochaten im Darme von Cyclostoma
elegans Drap. und thre Beziehungen zu Trichodinopsis paradoxa Clap.
Zbl. Bakt., 90: 457-60.

Bacigalupo, J. 1927. Entameba coli parasitada con Sphaerita. Rev. Soc. argent.
Biol., 3: 694-98.

—— 1928. Entamoeba coli parasitée par une Sphaerita. C. R. Soc. Biol.,
Paris, 98:-170-71.

Balbiani, E. G. 1858a. Note relativ a l’existence d’une génération sexuelle
chez les Infusoires. J. Physiol. Path. Gén., 1: 347-52.

—— 1858b. Recherches sur les organes générateurs et la reproduction des In-
fusoires. C. R. Acad. Sci. Paris, 47: 383-87. Translation in Ann. Mag.
Nat. Hist. (3) 2: 439-43.

—— 1860. Note sur un cas de parasitisme improprement pris pour un mode
de reproduction des Infusoires ciliés. C. R. Acad. Sci. Paris, 51: 319-22.

—— 1861. Recherches sur les phénomeénes sexuels des Infusoires. Arch.
physiol. norm. path., 4: 102-30; 194-220; 465-520.

—— 1893. Nouvelles Recherches expérimentales sur la mérotomie des In-
fusoires ciliés. Ann. micrographie, Paris, 5: 1-25; 49-84; 113-37.

Becker, E. 1926. Endamoeba citelli sp. nov. from the striped ground squirrel
Citellus tridecemlineatus, and the life history of its parasite Sphaerita
endamoebae sp. nov. Biol. Bull., 50: 444-54.

Bernstein, T. 1928. Untersuchungen an Flagellaten aus dem Darmkanal der
Termiten aus Turkestan. Arch. Protistenk., 61: 9-37,

1096 PARASULES OF PROTOZOA

Boeck, W. C. 1917. Mitosis in Giardia microti, Univ. Cal. Publ. Zool., 18:
1-26.

Bonar, L. 1936. An unsual Ascomycete in the shells of marine animals. Univ.
Cal. Publ. Bot. 19: 187-94.

Borgert, A. 1897. Beitrage zur Kenntniss der in Sticholonche zanclea und
Acanthometridenarten vorkommenden Parasiten. Z. wiss. Zool., 63: 141-
86.

Bourne, A. G. 1891. On Pelomyxa viridis, sp. n., and on the vesicular nature
of protoplasm. Quart. Jour. micr. Sci., 32: 357-74.

Bozler, E. 1924. Uber die Morphologie der Ernahrungsorganelle und die
Physiologie der Nahrungsaufnahme bei Paramecium caudatum Ehrbg.
Arch. Protistenk., 49: 163-215.

Brug, S. L. 1926. Nucleophage intestinalis n. sp., patasiet der Kern van
Endolimax williamsi (Prow.) = Endolimax biitschlii (Prow.) Dutch
East Indies Volksgesundheid, 1926: 466-68.

Brumpt, E., and G, Lavier. 1935a. Sur une Nacleophaga parasite d’Endolimax
nana. Ann, Parasit. hum. comp., 13: 439-44.

—— 1935b. Sur un genre nouveau d’amibe parasite Hyalolimax n. g. Ann.
Parasit. hum. comp., 13: 551-58.

—— 1936. Sur l’hyperparasitisme d’Opalines par des amibes. Ann. Parasit.
hum. comp., 14: 349-58.

Buchanan, R. E. 1925. General systematic bacteriology; history, nomenclature,
groups of bacteria. Baltimore.

Buchner, P. 1930. Tier und Pflanze in Symbiose. Berlin.

Biitschli, O. 1876. Studien iiber die ersten Entwicklungsvorginge der Eizelle,
die Zelltheilung und die Conjugation der Infusorien. Abh. senckenb.
naturf. Ges., 10: 213-464.

— 1885. Einige Bemerkungen tiber gewisse Organisationsverhialtnisse der
sog. Cilioflagellaten und der Noctiluca. Morph. Jb., 10: 529-77.

—— 1889. Protozoa. III Abt. Infusoria und System der Radiolaria 7m Bronn:
Klassen und Ordnungen des Thier-Reichs, 1. Leipzig.

Calkins, G. N. 1904. The life-history of Cytoryctes variolae Guarniere. Stud.
Path. Etiol. Variola (Office J. med. Res.) : 136-72.

Campbell, A. S. 1926. The cytology of Tintinnopsis nucula (Fol) Laachmann,
with an account of its neuromotor apparatus, division, and a new intra-
nuclear parasite. Univ. Cal. Publ. Zool., 29: 179-236.

Carini, A. 1933. Parasitisme des Zellerielles par des Microorganismes nouveau
(Brumptina n. g.) Ann, Parasit. hum. comp., 11: 297-300.

Carini, A., and E. Reichenow. 1935. Uber Amébeninfektion in Zelleriellen.
Arch. Protistenk., 84: 175-85.

Carter, H. J. 1856. Notes on the freshwater infusoria of the island of Bombay.
No. 1. Organization. Ann. Mag. Nat. Hist., (2) 18: 115-32; 221-49.
—— 1857. Additional notes on the freshwater infusoria in the island of

Bombay. Ann. Mag. Nat. Hist., (2) 20: 34-41.

PARASITES OF PROTOZOA 1097

— 1863. On Amoeba principes and its reproductive cells, etc. Ann. Mag.
Nat. Hist., (3) 12: 30-54.

Casagrandi, O., and P. Barbagallo. 1897. Entamoeba hominis s. Amoeba coli
(Lésch). Studio biologicoe clinico. Ann. Igiene (sper.), 7: 1-64.
Caullery, M., and F. Mesnil. 1897. Sun un type nouveau (Metchnikovella n.
g.) dorganismes parasites des grégarines. C. R. Soc. Biol. Paris, 49:

960-62.

—— 1905. Recherches sur les Haplosporidies. Arch. zool. exp. gén., (4) 4:
101-81.

— 1914. Sur les Metchnikovellidae et autres Protistes parasites des
grégarines d’Annélides. C. R. Soc. Biol. Paris, 77: 527-32.

— 1919. Metchnikovellidae et autres parasites des Grégarines d’ Annélides.
Ann. Inst. Pasteur, 33: 209-40.

Cavallini, F. 1926a. The asexual cycle in Centropyxis aculeata and its vari-
ability in relation to heredity and environment. J. exp. Zool., 43: 225-43.

— 1926b. The asexual cycle of development in Arcella vulgaris. J. exp.
Zool., 43: 245-55.

Cejp, K. 1935. Sphaerita, parasit Paramecii. Piispévek k poznani houbovych
parasiti Protozoi. Spisy Piirodovédeckou Fakultou Karlovy University,
Praha (Publ. Fac. Sci. Univ. Charles), 141: 3-7.

Cépéde, C., and V. Willem. 1912. Observations sur Trichodinopsis paradoxa.
Bull. Sci. Fr. Belg., 45: 239-48.

Chatton, E. 1910. Protozoaires parasites des branchies des Labres: Amoeba
mucicola Chatton, Trichodina labrorum n. sp. Appendice: parasite des
Trichodines. Arch. zool. exp. gén., (5)5: 239-66.

—— 1913. Coccidiascus n. g., n. sp., levure ascosporée parasite des cellules
intestinales de Drosophila funebris Fabr. C. R. Soc. Biol. Paris, 75:
117-20.

—— 1914. Les Cnidocystes du Péridinien Polykrikos schwartzi Butschli. Arch.
zool. exp. gén., 54: 157-94.

—— 1920a. Les Péridiniens parasites. Morphologie, reproduction, ethologie.
Arch. zool. exp. gén., 59 1-475.

—— 1920b. Existence chez les Radiolaires de Péridiniens parasites con-
siderés comme formes de reproduction de leurs hdtes. C. R. Acad. Sci.
Paris, 170: 413-15.

—— 1923. Les Péridiniens parasites des Radiolaires. C. R. Acad. Sci. Paris,
177: 1246-49,

—— 1934. L’Origine péridinienne des Radiolaires et l’interprétation para-
sitaire de l’anisosporogénése. C. R. Acad. Sci. Paris, 198: 309-12.
Chatton, E., and B. Biecheler. 1934. Les Coccidinidae, Dinoflagelles coccidio-

morphes parasite de Dinoflagelles, et le phylum des Phytodinozoa. C. R.
Acad. Sci. Patis, 199: 252-55.
—— 1936. Documents nouveau relatifs aux Coccidinides (Dinoflagelles

1098 PARASITES ‘OF PROTOZOA

parasites). La sexualite du Coccidinium mesnili n. sp. C. R. Acad. Sci.
Paris, 203: 573-76.

Chatton, E., and S. Brachon. 1936. Sur un Protiste parasite du Cilié Fabrea
salina Henneguy: Gregarella fabrearum n. gen., n. sp., et son evolution.
CR. Acad: Scam Panis= 203) 525-272

Chatton, E., and A. Brodsky. 1909. Le Parasitisme d’une Chytridinée du
gente Sphaerita Dangeard chez Amoeba limax Dujard. Etude compatra-
tive. Arch. Protistenk., 17: 1-18.

Chatton, E., and A. Lwoff. 1924a. Sur un flagellé hypertrophique et palintomi-
que parasite des Infusoires marins: Sporomonas infusorium (n. g., Nn.
sp.) C. R. Soc. Biol. Paris, 91: 186-90.

—— 1924b. Sur l’évolution des Infusoires des Lamellibranches: Morphologie
comparée des Hypocomidés. Les nouveaux genres Hypocomina et Hypo-
comella. C. R. Acad. Sci. Paris, 178: 1928-30.

—— 1927. Pottsia infusoriorum n. g., n. sp., Acinétien parasite des Folli-
culines et des Cothurnies. Bull. Inst. océanogr. Monaco, 489: 1-12.
— 1929, Contribution a l'étude de l’adaptation. Ellobiophrya donacis Ch.
et Lw. Péritriche vivant sur les branchies de I’Acephale Donax vittatus

da Costa. Bull. biol., 63: 321-49.

—— 1935. Les Ciliés apostomes. Morphologie, cytologie, éthologie, évolu-
tion, systématique. Premiere partie: Apercu historique et général. Etude
monographique des genres et des espéces. Arch. zool. exp. gén., 77:
1-453.

Chatton, E., A. Lwoff, and M. Lwoff. 1930. Les Phtorophrya n. g., Ciliés
Foettingertidae, hyperparasites des Gymnodinioides, Foettingertidae
parasites des Crustaces. C. R. Acad. Sci. Paris, 190: 1152-54.

Chatton, E., and S. Villeneuve. 1937. Gregarella fabrearum Chatton et
Brachon Protiste parasite du Cilié Fabrea salina Henneguy. La Notion de
dépolarisation chez les Flagellés et la conception des Apomastigines.
Arch, zool. exp. gén., 78: N. et R., 216-37.

Chen, T. T., and R. M. Stabler. 1935. Further studies on the amoebae parasitic
in opalinid ciliate protozoans. (Abstract). J. Parasit., 21: 428-29.
—— 1936. Further studies on the endamoeba parasitizing opalinid ciliates.

Biol. Bull., 70: 72-77.

Cienkowsky, L. 1855a. Bemerkungen iiber Stein’s Acinetenlehre. Bull. Acad.
Imp. Sci. St.-Pétersb., phys-math., 13: 297-304. Also Quart. J. micr. Sci.,
5: 96-103 (1857).

—— 1855b. Uber Cystenbildung bei Infusorien. Z. wiss. Zool., 6: 301-6.

—— 1865. Beitrage zur Kenntnis der Monaden. Arch. mikr. Anat. 1:
205-52.

Claparéde, E., and J. Lachmann. 1857. Note sur la reproduction des Infusoires.
Ann, Sci. nat., Zool., 4(8): 221-44.

—— 1858-59. Etudes sur les Infusoires et les Rhizopodes. vol. 1, Geneve.
Mém. Inst. nat. genev., 5, 6.

PARASITES OF PROTOZOA 1099

—— 1860-61. Etudes sur les Infusoires et les Rhizopodes. vol. 2, Geneve.
Mem. Inst. nat. genev., 7: 1-291.

Cleveland, L. R. 1928. Further observations and experiments on the symbiosis
between termites and their intestinal Protozoa. Biol. Bull., 54: 231-37.

Cohn, F. 1851. Beitrage zur Entwickelungsgeschichte der Infusorien. Z. wiss.
Zool., 3: 257-79.

— 1857. Uber Fortpflanzung von Nassula elegans Ehr. Z. wiss. Zool., 9:

143-46.

1858. Bemerkungen uber Raderthiere. Z. wiss. Zool., 9: 284-94.

Collin, B. 1907. Note préliminaire sur quelques Acinétiens. Arch. zool. exp.
gén. (4)7: N. et R., 93-103.

— 1912. Etude monographique sur les Acinétiens. II. Morphologie,
Physiologie, Systématique. Arch. zool. exp. gén., 51: 1-457.

Connell, F. H. 1930. The morphology and life cycle of Oxymonas dimorpha
sp. nov., from Neotermes simplicicornis Banks. Univ. Cal. Publ. Zool.,
36: 51-66.

——1932. Gigantomonas lighti sp. nov., a trichomonad flagellate from
Kalotermes (Paraneotermes ) simplicicornis Banks. Univ. Cal. Publ. Zool.,
Sie 155-88.

Cook, W. R. I. 1935. The genus Lagenidium Schenk, with special reference to
L. Rabenhorstii Zopf and L. entophytum Zopf. Arch. Protistenk., 86:
58-89.

Cragg, F. W. 1919. A contribution to our knowledge of Entamoeba coll.
Indian J. med. Res., 6: 462-84.

Craig, C. F. 1911. The parasitic amoebae of man. Philadelphia.

Crouch, H. B. 1933. Four new species of Trichomonas from the woodchuck
(Marmota monax Linn.). J. Parasit., 19: 293-301.

Cunha, A. M. da, and J. Muniz. 1923. Parasitismo de ‘Trichomonas’ pro

“Chyttidacae” do genero “Sphaerita’ Dangeard. Brazil-med., 37: 19-20.

—— 1934. Observations sur un parasite des Flagellés du genre Chilomastix.
@_ RR: SecBiol, Paris, 117: 208-10,

Dangeard, P. A. 1886a. Sur un nouveau genre de Chytridinés parasites des
Rhizopodes et des Flagellates. Bull. Soc. bot. Fr., 33: 240-42.

—— 1886b. Recherches sur les organismes inférieurs. Ann. Sci. nat., (7)
Bot., 4: 241-341.

—— 1889a. Recherches sur les Cryptomonadinae et les Euglenae Botaniste,
fe l33.

—— 1889b. Mémoire sur les Chytridinées. Botaniste, 1: 39-74.

—— 1895. Mémoire sur les parasites du noyau et du protoplasma. Botaniste,
A> 199-248,

— 1896. Contribution a l'étude des Acrasiées. Le Botaniste, 5: 1-20.

1100 PARASITES OF PROTOZOA

—— 1900a. L’Organisation el le développement du Colpodella pugnax.
Botaniste, 7: 5-31.

— 1900b. Recherches sur la structure du Polyphagus euglenae Nowak. et
sa reproduction sexuelle. Botaniste, 7: 213-61

— 1902. Sur le caryophyséme des Eugléniens. C. R. Acad. Sci. Paris, 134:
1365-66.

—— 1908. Sur un nouveau genre, parasite de Chrysomonadinées, le
Lecythodytes paradoxus. C. R. Acad. Sci. Paris, 146: 1159-60.

—— 1910. Etudes sur le développement et la structure des organismes in-
férieurs. Botaniste, 11: 1-311.

Deflandre, G. 1928. Le Genre Arcella Ehrenberg. Morphologie- Biologie.
Essai phylogénétique et systématique. Arch. Protistenk., 64: 152-287.

Dobell, C. 1919, The amoebae living in man. London.

Doflein, F. 1907. Studien zur Naturgeschichte der Protozoen. V. Amdben-
studien, Erster Teil. Arch. Protistenk., Suppl. 1: 250-93.

1909. Lehrbuch der Protozoenkunde, 2d ed., Jena. Also 3d ed., 1911;
4th ed., 1916; 5th ed. (F. Doflein, and E. Reichenow), 1927-29.
Dogiel, V. 1906. Beitrage zur Kenntnis der Gregarinen. I. Cystobia chiridotae
nov. sp. II. Hyalosphaera gregarinicola nov. gen. nova spec. Arch.

Protistenk., 7: 106-30.

—— 1916. Researches on the parasitic Protozoa from the intestine of
termites. I. Tetramitidae. Russk. zool. Zh., 1: 1-54.

—— 1917. Researches on the parasitic protozoa from the intestine of termites.
II. Lophomonadidae. Sci. Res. Zool. Exped. Brit. E. Africa made by V.
Dogiel and I. Sokolow in 1914, no. 10.

—— 1922a. Untersuchungen an parasitischen Protozoan aus dem Darmkanal
der Termiten. III. Trichonymphidae. Arch. russ. Protist., 1: 172-234.

—— 1922b. Sur un nouveau genre de Metchnikovellidae. Ann. Inst. Pasteur,
36: 574-77.

— 1929. Biologische Notizen tiber Darminfusorien der Huftiere. Arch.
russ. protist., 8: 153-62.

Douvillé, N. 1930. Parasitisme ou commensalisme chez les Foraminiféres. Les
canaux chez les Nummulitides, Soc. geol. France, Livre Jubilaire 1830-
1930, 1: 257-62.

Duboscq, O., and B. Collin. 1910. Sur la reproduction sexuée d’un Protiste
parasite des Tintinnides. C. R. Acad. Sci. Paris, 151: 340-41.

Duboscq, O., and Grassé, P. 1925. Notes sur les Protistes parasites des
Termites de France. IV. Appareil de Golgi, mitochondries et vesicules
sous-flagellaites de Pyrsonympha vertens Leidy. C. R. Soc. Biol. Paris,
93: 345-48.

—— 1926. Les Schizophytes de Devescovina hilli n. sp. C. R. Soc. Biol.
Paris, 94: 33-34,

—— 1927. Flagellés et Schizophytes de Calotermes (Glytotermes) iridipen-
nis Frogg. Arch. Zool. exp. gén., 66: 452-96.

PARASITES OF PROTOZOA 1101

—— 1928. Note sur les Protistes parasites des termites de France. L’appareil
parabasal de Joenia annectens Grasst. C. R. Soc. Biol., 99: 1118-20.
— 1933. L’appareil parabasal des Flagellés. Arch zool. exp. gén., 73:

381-621.

—— 1934. Notes sur les Protistes parasites des Termites de France. VII. Sur
les Trimitus de Calotermes flavicollis Rossi. VII. Sur Microrhopalodina
inflata (Grassi). Arch. zool. exp. gén., 75: 615-37.

—— 1937. Les Flagellés de l’Acanthotermes ochraceus Sjost. du Sud-
Algérien. C. R. Acad. Sci. Paris, 205: 574-76.

Duboscq, O., P.-P. Grassé, and M. Rose. 1937. Les Flagellés de l’ Anacantho-
termes ochraceus Sjést. du Sud-Algérien. C. R. Acad. Sci. Paris, 205:
574-76.

Ehrenberg, C. G. 1838. Die Infusionsthierchen als vollkommene Organismen.
Leipzig.

Elmassian, M. 1909. Une Nouvelle Coccidie et un nouveau parasite de la
tanche, Coccidium rouxi nov. spec., Zoomyxa legeri nov. gen., NOV. spec.
Arch. zool. exp. gén., (5) 2: 229-70.

Elpatiewsky, W. 1907. Zur Fortpflanzung von Arcella vulgaris Ehtbg. Arch.
Protistenk., 10: 441-66.

Engelmann, T. W. 1862. Zur Naturgeschichte der Infusionsthiere. Z. wiss.
Zool., 11: 347-93.

— 1876. Uber Entwickelung und Fortpflanzung von Infusorien. Morph.
|DS 12573-635:

Epstein, H. 1922. Uber parasitische Infektion bei Darmamében. Arch. russ.
protist., 1: 46-81.

1935. Bacterial infection in an amoeba. J. R. micr. Soc., 55: 86-94.

Fauré-Fremiet, E. 1909. Sur un cas de symbiose presente par un Infusoire
Gilie Ree soc, Biolk Pariss:67= 113-14)

— 1913. Sur les ‘“‘Nématocystes’” de Polykrikos et de Campanella. C. R.
Soc. Biol. Paris, 75: 366-68.

Fitzpatrick, H. M. 1930. The lower fungi. Phycomycetes. New York.

Fiveiskaja, A. 1929. Einfluss der Kernparasiten der Infusorien auf den Stoff-
wechsel. Arch. Protistenk., 65: 275-98.

Foa, A. 1905. Due nuovi Flagellati parassiti. R. C. Accad. Lincei, (5) 14:
542-46.

Focke, G. W. 1845. Ergebnisse ferneren Untersuchungen dere polygastrischen
Infusorien. Amtlicher Bericht tber' die zweiundzwanzigste Versammlung
deutscher Naturforscher und Artzte in Bremen im September 1844.
Verh. Ges. deutscher Naturf. Artzte, 22: 109-10.

Fol, H. 1883. Sur le Sticholonche zanclea et un nouvel ordre de Rhizopodes.
Mem. Inst. nat. genev., Nr. 15.

Fortner, H. 1934. Untersuchungen an Pelomyxa palustris Greeff. Studien zur
Biologie und Physiologie des Tieres. I. Teil. Arch. Protistenk., 83: 381-
464,

1102 PARASITES°OF PROTOZOA

Franca, C. 1918. Observations sur les Trichonymphides. An. Fac. Med. Porto,
(4) 2: 5-14.

Ganapati, P. N., and R. G. Aiyar. 1937. Life-history of a dicystid gregarine,
Lecudina brasili n. sp., parasitic in the gut of Lumbriconereis sp. Arch.
Protistenk., 89: 113-32.

Gassovsky, G. 1919. On the microfauna of the intestine of the horse. Trav.
Soc. Nat. St.-Pétersbg. (Leningr.), 49: 20-37; 65-69.

Geitler, L. 1937. Uber einen Pilzparasiten auf Amoeba proteus und uber
die polare Organization des Amébenkorpers. Biol. Zbl., 57: 166-75.
Georgevitch, J. 1929. Sur la faune intestinale des Termites de Yougoslavie.

C. R. Soc. Biol. Paris, 103: 325-28.

—— 1932. Recherches sur les Flagellés des Termites de Yougoslavie. Arch.
zool. exp. gén., 74: 81-109.

— 1936a. [Studies on a hyperparasite Le/smania esocis nov. spec.} (In
Serbian.) Ghlas Srpska Kralj. Akad. Belgrade, 172: 127-37.

—— 1936b. Ein neuer Hyperparasit, Leishmania esocis nov. spec. Arch.
Protistenk., 88: 90-92.

Goetsch, W., and L. Scheuring. 1926. Parasitismus und Symbiose der AlI-
gengattung Chlorella. Z. Morph. Okol. Tiere, 7: 220-53.

Gojdics, M. 1939. Some observations on Evglena sanguinea Ehrbg. Trans.
Amer. micr. Soc., 58: 241-48.

Goldschmidt, R. 1907. Lebensgeschichte der Mastigamdben, Mastigella
vitrea n. sp. und Mastigina setosan. sp. Arch. Protistenk., (Suppl.) 1: 83-
168.

Gonder, R. 1910. Ein Parasit von Colpoda cucullus. Arch. Protistenk., 18:
27D:

Gonnert, R. 1935. Uber Systematik, Morphologie, Entwicklungsgeschichte
und Parasiten einiger Dendrosomidae nebst Beschreibung zweier neuer
Suktorien. Arch. Protistenk., 86: 113-54.

Gould, L. J. 1894. Notes on the minute structure of Pelomyxa palustris
(Greef). Quart. J. micr. Sci., 36: 295-306:

Grassé, P. P. 1926a. Sur la nature des cétes cuticulaires des Polymastix et
Lophomonas striata. C. R. Soc. Biol. Paris, 94: 1014-15.

—— 1926b. Contribution a l’étude des Flagellés parasites. Arch. zool. exp.

gén., 65: 345-602.

1937. Sur un Flagellé termiticole “Caduceia theobromae” Franga. C. R.

xii® Cong. Int. Zool.: 1324-29.

— 1938. La Véture schizophytique des Flagellés termiticoles: Parajoenza,
Caduceia et Pseudodevescovina. Bull. Soc. zool. Fr., 63: 110-22.
Grassi, B., and Foa, A. 1904. Ricerche sulla riproduzione dei Flagellati. I.
Processo di divisione delle Joenie e forme affini. Nota preliminare. R. C.

Accad. Lincei, (5) 13, 2 sem.: 241-53.

— 1911. Intorno ai Protozoi dei Termitidi. Nota preliminare. R. C. Accad.

Eincer.(6) e205 Wsem:.:725-4 1c

PARASITES OF PROTOZOA 1103

Greeff, R. 1866. Uber einige in der Erde lebende Amdben und andere Rhizo-
poden. Arch. mikr. Anat., 2: 299-311.

— 1870. Unterschungen tiber den Bau und die Naturgeschichte der
Vorticellen. Arch. Naturgesch., 1: 353-84.

— 1874. Pelomyxa palustris (Pelobius), ein amdbenartiger Organismus
des siissen Wassers. Arch. mikr. Anat., 10: 51-73.

Greenway, D. 1926. Endolimax nana. Arch. argent Enferm. Apar. dig., p.
174.

Gruber, A. 1884. Die Protozoen des Hafens von Genua. Nova Acta K. Leop.
Carol., 46: 475-539.

— 1892. Eine Mittheilung tiber Kernvermehrung und Schwarmerbildung
bei Siisswasserrhizopoden. Ber. naturf. Ges. Freiburg i. B., 6: 114-18.

— 1904. Uber Amoeba viridis Leidy. Zool. Jb., (Suppl.) 7 (Festschr.
Weissmann) : 67-76.

Gunther, F. 1928. Uber den Bau und die Lebensweise der Euglenen, besonders
der Arten E. terricola, geniculata, proxima, sanguinea und lucens nov.
spec. Arch. Protistenk., 60: 511-90.

Gwynne- Vaughan, H. C. I., and B. Barnes. 1937. The structure and develop-
ment of the fungi. 2d ed., Cambridge.

Hafkine, M. W. 1890. Maladies infectieuses des Paramecies. Ann. Inst.
Pasteur, 4: 148-62.

Hertwig, R. 1876. Uber Podophrya gemmipara nebst Bemerkungen zum Bau

und zur systematischen Stellung der Acineten. Gegenbaurs Jb., 1: 20-82.

1879. Der Organismus der Radiolarien. Jena.

Hesse, E. 1909. Contribution a l'étude des Monocystidées des Oligochétes.
ANEGh. ZOO) Expy. gen. (5) 3: 27-30V.

Hetherington, A. 1932. On the absence of physiological regeneration in
Stentor coeruleus. Arch. Protistenk., 77: 58-63.

Hoelling, A. 1910. Die Kernverhiltnisse von Fusiformis termitidis, Arch.
Protistenk, 19: 239-45.

Hofker, J. 1927. The Foraminifera. Siboga Expedition. Leiden.

1931. Studie uber Tintinnoidea. Arch. Protistenk., 75: 315-402.

Hollande, A. 1938. Bodo perforans n, sp. Flagellé nouveau parasite externe
du Chilomonas paramaecium Ehrenb. Arch. zool. exp. gén., 79: N. et R.,
75-81.

Hovasse, R. 1923a. Les Peridiniens intracellulaire — zooxanthelles et Syn-
diniums — chez les Radiolaires coloniaux. Remarques sur la reproduction
des Radiolaires. Bull. Soc. zool. Fr., 48: 247-54.

—— 1923b. Sur les Peridiniens parasites des Radiolaires coloniaux. Bull.
Soce Zool Fre 48293377

—— 1936. Rhizophidium Beauchampi sp. nov., Chytridinée parasite de la
Volvocinée Exdorina (Pleodorina) illinoisensis (Kofoid). Ann. Protist.,
De D=Gils.

Howland, R. B. 1928. A note on Astasia captive Beauch. Science, 68: 37.

1104 PARASITES OF PROTOZOA

Hsiung, Ta-Shih. 1928. Suctoria of the large intestine of the horse: Allan-
tosoma intestinalis Gassovsky, A. discorniger sp. nov. and A. brevi-
corniger sp. nov. Iowa St. Coll. J. Sci., 3: 101-3.

Hudson, C. T., and P. H. Gosse. 1889. The rotifera; or wheel-animalcules,
both British and foreign. London.

Issel, R. 1906. Intorno alla struttura ed alla biologia dell’infusorio Trichodin-
Opsis paradoxa Clap. et Lachm. Ann. Mus. Stor. nat. Genova, (3) 2:
334-57.

Ivani¢, M. 1925. Zur Kenntnis der Agamogonieperiode einiger Amoeben-
parasiten. Zool. Anz., 63: 250-56.

—— 1934, Uber einen Kernparasiten der roten Blutzellen beim griinen
Frosche (Rana esculenta L.), Erythrocytonucleophaga ranae gen. nov.,
spy nov. Zblwbakt. ol “Abt. 1133 a6;

—— 1936. Uber einen Protoplasmakérperparasiten von Entamoeba histoly-
tica Schaudinn (Entamoebophaga hominis gen. nov. spec. nov.). Zbl.
Bakt., I Abt., 138: 48-56.

Jahn, T. D 1929. On certain parasites of Phacus and Beglen: (Abstract) .
Anat. Rec., 44: 249-50.

—— 1933, On certain parasites of Phacus and Euglena; Sphaerita phaci,
sp. nov. Arch. Protistenk., 79: 349-55.

Janicki, C. 1909. Uber Kern und Kernteilung bei Extamoeba blattae Bitschli.
Biol. Zbl., 29: 381-93.

—— 1915. Untersuchungen an parasitischen Flagellaten. HI Teil: Die Gat-
tungen Devescovina, Parajoenia, Stephanonympha, Calonympha. Ueber
den Parabasalapparat. Uber Kernkonstitution und Kernteilung. Z. wiss.
Zool., 112: 573-691.

Jirovec, O. 1931a. Die Silberlinien bei den Pyrsomymphiden. Arch. Pro-
tistenk., 73: 47-55.

—— 1931b. Symbiose von Bakterien und Trichonympha serbica. Zbl. Bakt.,
I Abt., 123: 184-86.

—— 1933. Beobachtungen Uber die Fauna des Rinderpansens. Z. Parasitenk.,
5: 584-91.

Kahl, A. 1928. Die Infusorien (Ciliata) der Oldesloer Salzwasserstellen.
Arch. Hydrobiol., 19: 50-123.

—— 1930. Urtiere oder Protozoa I: Wimpertiere oder Ciliata (Infusoria)
I. Allgemeiner Teil und Prostomata, 7 Dahl: Die Tierwelt Deutsch-
lands, 18 Teil, Jena.

—— 1931. Ibid., II. Holotricha, 21 Teil.

—— 1932. Ibid., Ill. Spirotricha, 25 Teil.

— 1933. Ciliata libera et ectocommensalia, 72 Grimpe, G., und E. Wagler:
Die Tierwelt der Nord-und Ostsee Lief. 23, Teil II. C3, 29-146,
Leipzig.

—— 1935. Ibid., IV. Peritricha und Chonotricha, 30 Teil.

Kalmus, H. 1928. Uber den Bodenfauna der Moldau im Gebiete von Prag.

PARASITES OF PROTOZOA 1105

Ein Jahreszyklus. II. Protozoa, etc. Mit einem Anhang: Okologische
Beobachtungen und Versuche. Int. Rev. Hydrobiol., 19: 349-429.

Kent, W. S. 1880-82. A manual of the Infusoria. London.

Keppen, N. A., see Koeppen, N.

Kessel, J. F. 1923. On the genus Coxncilmania, budding intestinal amoebae
parasitic in man and rodents. Univ. Cal. Pub. Zool., 20: 431-45.

— 1924, The distinguishing characteristics of the parasitic amoebae of
culture rats and mice. Univ. Cal. Publ. Zool., 20: 489-544.

Kirby, H., Jr. 1924. Morphology and mitosis of Dinenympha fimbriata sp.
nov. Univ. Cal. Publ. Zool., 26: 199-220.

—— 1926a. On Staurojoenina assimilis sp. nov., an intestinal flagellate
from the termite, Kalotermes minor Hagen. Univ. Cal. Publ. Zool., 29:
25-102.

—— 1926b. The intestinal flagellates of the termite, Cryptotermes hermsi

Kirby. Univ. Cal. Publ. Zool., 29: 103-20.

1927. Studies on some amoebae from the termite Mirotermes, with
notes on some other Protozoa from the Termitidae. Quart. J. micr., Sci.,
FB N89=222°
— 1928. A species of Proboscidiella ftom Kalotermes (Cryptotermes)

dudleyi Banks, a termite of Central America, with remarks on the oxy-
monad flagellates. Quart. J. micr. Sci., 72: 355-86.

—— 1929. Snyderella and Coronympha, Ewe new genera of puleianelente
flagellates from termites. Univ. Cal. Publ. Zool., 31: 417-32.

1930. Trichomonad flagellates from eenites I. Tricercomitus gen.

nov., and Hexamastix Alexeieff. Univ. Cal. Publ. Zool., 33: 393-444.

1932a. Flagellates of the genus Trichonympha in termites. Univ. Cal.
Publ. Zool., 37: 349-476.

—— 1932b. Protozoa in termites of genus Amitermes. Parasitology, 24:
289-304.

— 1934. Some ciliates from salt marshes in California. Arch. Protistenk.,
82: 114-33.

—— 1936. Two polymastigote flagellates of the genra Pseudodevescovina
and Caduceia. Quart. J. micr. Sci., 79: 309-35.

-—— 1937. The devescovinid flagellate Parajoenia grassii from a Hawaiian
termite. Univ. Cal. Publ. Zool., 41: 213-24.

—— 1938a. The devescovinid flagellates Caduceia theobromae Franga,
Pseudodevescovina ramosa new species, and Macrotrichomonas pulchra
Grassi. Univ. Cal. Publ. Zool., 43: 1-40.

1938b. Polymastigote flagellates of the genus Foaina Janicki, and two

new genera Cracinympha and Bulanympha. Quart. J. micr. Sci., 81: 1-25.

1940. Microorganisms associated with the flagellates of termites. Pp.

407-8 in Thitd International Congress for Microbiology: Report of

Proceedings. New York.

1106 PARASITES OF PROTOZOA

Klebs, G. 1883. Uber die Organisation einiger Flagellaten-Gruppen und ihre
Beziehungen zu Algen und Infusorien. Unters. Bot. Inst. Tubingen, 1:
233-62.

Koeppen, N. [Keppen, N. A.}. 1894. Amoebophrya Sticholonchae nov. gen.
et sp. (“Corps spiral” de Fol) Zool. Anz., 17: 417-24.

—— 1889. Hyalosaccus Ceratit, nov. gen. et sp., parazit Dinoflagellat. Kiev.
obshch. estest. Zap., 16: 89-135.

Kofoid, C. A., and O. Swezy. 1915. Mitosis and multiple fission in tricho-
monad flagellates. Proc. Amer. Acad, Arts. Sci., 51: 289-378.

Koidzumi, M. 1921. Studies on the intestinal Protozoa found in the termites
of Japan. Parasitology, 13: 235-309.

Korotneff, A. 1880. Etudes sur les Rhizopodes. Arch. zool. exp. gén., (1),
8: 467-82.

— 1891. Zoologische Paradoxen. Z. wiss. Zool., 51: 613-28.

Kruger, F. 1933. Epzstylis umbellaria mit ‘Nesselkapseln.” Zool. Anz.
(Suppl.) 6. Verh. dtsch. zool. Ges., 35: 262-63.

Kuczynski, M. H. 1918. Uber die Teilungsvorgange verscheidener Tricho-
monaden und ihre Organization im allgemeinen, Arch. Protistenk., 39:
107-46.

Kudo, R. 1924. A biologic and taxonomic study of the microsporidia. III.
Biol. Monogr., 9: 3-268.

Lachmann, C. F. J. 1856. Uber die Organisation der Infusorien, besonders der
Vorticellen. Arch. Anat. Physiol. wiss. Med., 1856: 340-98.

Lackey, J. B. 1936. Some fresh water Protozoa with blue chromatophores.
Biol. Bull., 71: 492-97.

Lauterborn, R. 1916. Die sapropelische Lebewelt. Ein Beitrag zur Biologie
des Faulschlammes natiirlicher Gewasser. Verh. naturh.-med. Ver.
Heidelberg, n. s., 13: 395-481.

Lavier, G. 1935a. Action, sur la biologie d’une Entamibe, due parasitism
intranucléaire par une Nucleophaga. C. R. Soc. Biol. Paris, 118: 457-59.

—— 1935b. Sur une Nuacleophaga parasite d’Entamoeba ranarum. Ann.
Parasit. hum. comp., 13: 351-61.

Leger, L., and O. Duboscq. 1904. Notes sur les Infusoires endoparasites.
Arch, zool. exp. gén., (4) 2: 337-56.

—— 1909a. Sur une Microsporidie parasite d’une Grégarine. C. R. Acad.
Sci. Paris, 148: 733-34.

—— 1909b. Perezia lankesteriae n. g., n. sp., Microsporidie parasite de

Lankesteria ascidiae (Ray-Lank.). Arch. zool. exp. gén., (5)1: N. et

R., 89-93.

1909c. Etudes sur la sexualité chez les Grégarines. Arch. Protistenk.,

17: 19-134.

Leidy, J. 1879. Fresh-water rhizopods of North America. U. S. Geol. Sur.
Merry Reps.) 12:m12 324:

——— 1881. The parasites of the termites. J. Acad, nat. Sci. Philad., (2)8:
425-47.

PARASITES OF PROTOZOA 1107

Leiner, M. 1924. Das Glycogen in Pelomyxa palustris Greef, mit Beitragen
zur Kenntnis des Tieres. Arch. Protistenk., 47: 253-307.

Liebmann, H. 1936a. Auftreten, Verhalten und Bedeutung der Protozoen bei
der Selbstreinigung stehenden Abwassers. Z. Hyg. InfektKr., 118:
29-63.

—— 1936b. Die Ciliatenfauna der Emscherbrunnen. Z. Hyg. InfektKr., 118:
SDD =i! a6

— 1937. Bakteriensymbiose bei Faulschlammziliaten. Biol. Zbl., 57: 442-
45.

Light, S. F. 1926. On Metadevescovina debilis gen. nov., sp. nov. Univ. Cal.
Publ. Zool., 29: 141-57.

Lutz, A., and A. Splendore. 1908. Uber Pebrine und verwandte Mikro-
sporidien. 2. Mitt. Zbl. Bakt., Abt. I, 46: 311-15.

Lwoff, A. 1925. Chytridinées parasites des Amibes de homme. Possibilité
de leur utilisation comme moyen biologique de lutte contre la dysenterie
amibienne. Bull. Soc. Path. exot., 18: 18-23.

Lynch, J. E., and A. E. Noble. 1931. Notes on the genus Evdosphaera Engel-
man and on its occasional host Opisthonecta henneguyi Fauré-Fremiet.
Univ. Cal. Publ. Zool., 36: 97-114.

Mackinnon, D. L. 1914. Observations on amoebae from the intestine of the
crane-fly larva, Tzpwla sp. Arch, Protistenk., 32: 267-77.

Mackinnon, D. L., and H. N. Ray. 1931. Observations on dicystid gregarines
from marine worms. Quart. J. micr. Sci., 74: 439-66.

Mainx, F. 1928. Beitrage zur Morphologie und Physiologie der Eugleninen.
I. Teil Morphologische Beobachtungen, Methoden und Erfolge der
Reinkultur. Arch. Protistenk., 60: 305-54.

Mangenot, G. 1934. Sur l’association d’une Rhodobactérie et d’un Infusoire.
C. R. Soc. Biol. Paris, 117: 843-47.

Martin, C. H. 1909. Some observations on Acinetaria. I. The ‘“Tinctin-
kérper”’ of Acinetaria and the conjugation of Acineta papillifera. 1. The
life-cycle of Tachyblaston ephelotensis (gen. et spec. nov.), with a possi-
ble identification of Acinetopsis rara, Robin. Quart. J. micr. Sci., 53:
ZDL-89.

Mattes, O. 1924. Uber Chytridineen im Plasma und Kern von Amoeba
sphaeronucleolus and Amoeba terricolo. Arch. Protistenk., 47: 413-30.

Mayer, M. 1920. Zur Cystenbildung von Trichomonas muris. Arch. Pro-
tistenk., 40: 290-93.

Mecznikow, E., see Metschnikoff, E.

Mercier, L. 1907. Un parasite du noyau d’ Amobea blattae Biitschli. C. R. Soc.
Biol. Paris, 62: 1132-34. Also Réun. Biol., Nancy, 1907: 52-54.

—— 1910. Contribution a I’étude de l’Amibe de la Blatte (Entamoeba blattae
Butschli). Arch. Protistenk., 20: 143-75.

Metschnikoff, E [Mecznikow, E.}. 1864. Uber die Gattung Sphaerophrya.
Arch, Anat. Physiol. wiss. Med., 1864: 258-61.

1108 PARASITES OF PROTOZOA

— 1892. Lecons sur la pathologie comparée de l’inflammation. Faite a
l'Institut Pasteur en Avril et Mai, 1891. Paris.

Minden, M. von. 1915. Chytridiineae zz Pilze I. Kryptogamenfl. Mark
Brendenb., 5: 209-422.

Mitchell, J. B., Jr. 1928. Studies on the life history of a parasite of the
Englenidae. Trans. Amer. micr. Soc., 47: 29-41.

Miyashita, Y. 1933. Drei neue parasitische Infusorien aus dem Darme einer
Japanischen Stisswagseroligochate. Annot. zool. jap., 14: 127-31.
Molisch, H. 1903. Amoeben als Parasiten in Volvox. Ber. dtsch. bot. Ges.,

ZU 20223,

Miller, J. 1856. Einige Beobachtungen an Infusorien. Monatsber. preuss.
Akad. Wissensch., 1856: 389-93.

Myers, E. H. 1938. The present state of our knowledge concerning the life
cycle of the Foraminifera. Proc. nat. Acad. Sci. Wash., 24: 10-17.

Nagler, K. 1910. Fakultativ parasitische Micrococcen in Amo6ben. Arch.
Protistenk., 19: 246-54.

— 191la. Studien tber Protozoen aus einem Almtimpel. I. Amoeba hart-
manni n. sp. Anhang: Zur Centriolfrage. Arch. Protistenk., 22: 56-70.

— 1911b. Studien uber Protozoen aus einem Almtiimpel. II. Parasitische
Chytridiaceen in Evglena sanguinea. Arch. Protistenk., 23: 262-68.

Neresheimer, E. 1904. Uber Lohmannella catenata. Z. wiss. Zool., 76: 137-66.

1908. Die Mesozoen. Zool. Zbl., 15: 257-312.

Noller, W. 1921. Uber einige Wenig bekannte Darmprotozoen des Menschen
und ihre nachsten Verwandten. Arch. Schiffs u. Tropenhyg., 25: 35-46.

— 1922. Die wichtigsten parasitischen Protozoen des Menschen und der
Were, Teil, Berlin.

Nowakowski, L. 1876. Beitrag zur Kenntnis der Chytridiaceen. II. Polyphagus
Euglenae. Beitr. Biol. Pfl., ed. by Ferdinand Cohn, 2: 201-19.

Pascher, A. 1929. Studien tiber Symbiosen. I. Uber einige Endosymbiosen von
Blaualgen in Einzellern. Jb. wiss. Bot., 71: 386-462.

Pellissier, M. 1936. Sur certains constituants cytoplasmiques de |’Infusoire
cilié Trichodinopsis paradoxa Clap. et Lach. Arch. zool. exp. gén., 78:
INSiet R 32-36:

Penard, E. 1893. Pelomyxa palustris et quelques autres organismes inférieurs.
Arch. Sci. phys. nat. (3), 29: 165-82.

— 1902. Faune rhizopodique du bassin du Léman. Genéve.

— 1904. Héliozoaires d’eau douce. Genéve.

— 1905a. Sur les Sarcodinés du Loch Ness. Proc. roy. Soc. Edinb., 25:
593-608.

—— 1905b. Observations sur les Amibes a pellicule. Arch. Protistenk., 6:
175-206.

—— 1905c. Notes sur quelques Sarcodinés. Ire partie. Rev. suisse Zool., 13:
585-616.

PARASITES OF PROTOZOA 1109

—— 1908-9. Uber ein bei Acanthocystis turfacea parasitisches Rotatorium.
Mikrokosmos, 2: 135-43.

—— 1909. Sur quelques Mastigamibes des environs de Genéve. Rev. suisse
Zool., 17: 405-39.

—— 1912. Nouvelles Recherches sur les Amibes du groupe Terricola. Arch.

Protistenk., 28: 78-140.

1922. Etudes sur les Infusoires d’eau douce. Geneva.

Petschenko, B. de. 1911. Drepanospira miilleri n. g., n. sp. parasite des Para-
meciums; contribution a l’étude de la structure des Bacteries. Arch.
Protistenk., 22: 248-98.

Pierantoni, O. 1936. La simbiosi fisiologica nei termitidi xilofagi e nei loro
flagellati intestinali. Arch. zool. (ital.) Torino, 22: 135-71.

Pinto, C., and F. da Fonseca. 1926. Trichomonas vitali nova especie. Parasitis-
mo das Trichomonas por Sphaerita minor Cunha et Muniz, 1923, e
relacdo das especies de Sphaeritas conhecidas. (In Portuguese) . Bol. biol.
Fac. Med. S. Paulo, 1926: 34-37.

Plate, L. 1886. Beitrige zur Naturgeschichte der Rotatorien. Jena. Z. Naturw.,
LOE L120.

—— 1887. Uber einige ectoparasitische Rotatorien des Golfes von Neapel.
Mitt. Zool. Sta. Neapel, 7: 234-63.

1888. The genus Acinetoides, g. n., an intermediate form between the
ciliated Infusoria and the Acinetae. Ann. Mag. Nat. Hist., (6) 2: 201-8.
(Translation.) Original in Zool. Jahrb., Abt. Anat. Ont., 3: 135-43
(1888).

Powell, W. N. 1928. On the morphology of Pyrsonympha with a description
of three new species from Reticulitermes hesperus Banks. Univ. Cal. Publ.
Zool., 31: 179-200.

Powers, P. B. A. 1933. Studies on the ciliates from sea-urchins. I. General
Taxonomy. Biol. Bull., 65: 106-21.

—— 1935. Studies on the ciliates of sea-urchins. A general survey of the
infestations occurring in Tortugas echinoids. Publ. Carneg. Instn., 452:
293-326.

Prandtl, H. 1907. Der Entwicklungskreis von Allogromia sp. Arch. Protis-
tenk., 9: 1-21.

Pringsheim, E. G. 1928. Physiologische Untersuchungen an Paramecium
bursaria. Ein Beitrag zur Symbioseforschung. Arch. Protistenk., 64:
289-418.

Puymaly, A. de. 1927. Sur le Sphaerita endogena Dangeard, Chytridiaceé
parasite des Euglénes. Bull. Soc. bot. Fr., 74: 472-76.

Raymond, G. 1901. Sur un Monadinazoosporeé parasite de Chlamydomonas.
Microgr. prép., 9: 128-31.

Rieder, J. 1936. Biologische und dkologische Untersuchungen an Siisswasser-
Suktorien. Arch. Naturgesch., 5: 137-214.

1110 PARASITES OF PROTOZOA

Robertson, M. 1905. Pseudospora volvocis, Cienkowski. Quart. Jour. micr.
Sci., 49: 213-30.

Roskin, G. 1927. Zur Kenntnis der Gattung Psewdospora Cienkowski. Arch.
Protistenk., 59: 350-68.

Ryder, J. A. 1893. The growth of Ezgleno viridis when constrained princi-
pally to two dimensions of space. Contr. zool. Lab. Univ. Pa., 1: 37-50.

Sand, R. 1899. Etude monographique sur le groupe des Infusoires tentaculi-
féres. Ann. Soc. belge Micr., 24: 57-189; 25: 7-205.

Sassuchin, D. N. 1928a. Zur Frage uber die Parasiten der Protozoen. Parasiten
von Nyctotherus ovalis Leidy. Arch. Protistenk., 64: 61-70.

—— 1928b. Zur Frage uber die ecto- und entoparasitischen Protozoen der
Froschkaulquappen. Arch. Protistenk., 64: 71-92.

—— 1931. Zum Studium du Darmprotozoenfauna der Nager im Siid-Osten
RSFSR. I. Darmprotozoen des Citellus pygmaeus Pallas. Arch. Protis-
tenk., 74: 417-28.

—— 1933. Materials on hyperparasitism in Protozoa (in Russian). Rev.
Microbiol. Saratov, 12: 219-27.

—— 1934. Hyperparasitism in Protozoa. Quart. Rev. Biol., 9: 215-24.

Sassuchin, D. N., P. P. Popoff, W. A. Kudrjewzew, and W. P. Bogenko. 1930.
Uber parasitische Infektion bei Darmprotozoen. Arch, Protistenk., 71:
229-34.

Schereschewsky, Helene. 1924. La Famille Metchnikovellidae (C. & M.) et la
plaie qu'elle occupe dans le Systéme des Protistes (Russian with French
summary). Arch. russ. Protist., 3 (3-4): 137-45.

Scherffel, A. 1925a. Endophytische Phycomyceten-Parasiten der Bacillariaceen
und einige neue Monadinen. Ein Beitrag zur Phylogenie der Oomyceten
(Schréter). Arch. Protistenk., 52: 1-141.

— 1925b. Zur Sexualitat der Chytridineen. (Der Beitrage zur Kenntnis der
Chytridineen. Teil I. Arch. Protistenk., 53: 1-58.

—— 1926a. Einiges ber neue oder ungeniigend bekannte Chytidineen. (Der
Beitrage zur Kenntnis der Chytridineen. Teil I]. Arch. Protistenk., 54:
167-260.

— 1926b. Beitrige zur Kenntnis der Chytridineen. Teil HI. Arch. Protis-
tenk., 54: 510-28.

Schouten, G. B. 1937. Nyctotherus ochoterenae n. sp. y Nyctotherus gamarrat
n. sp. Protozoarios parasitos de batracios (Evgystoma ovale bicolor Schn.
e Hyla venulosa Laur.). An. Inst. Biol. Univ. Méx., 8: 387-92.

Schubotz, H. 1908. Pycnothrix monocystoides nov. gen., nov. sp., ein neues
ciliates Infusor aus dem Darm von Procavia (Hyrax) capensis (Pallas).
Denkschr. med-naturw. Ges. Jena, 13: 1-18.

Schulze, F. E. 1875. Rhizopodienstudien. V. Arch, mikr. Anat., 11: 583-96.

Serbinow, J. L. 1907. Beitrage zur Kenntnis der Phycomyceten. Organisation
u. Entwickelungsgeschichte einiger Chytridineen-Pilze (Chytridineae
Schroter). Scripta bot. Petropol, 24: 1-173.

PARASITES OF PROTOZOA 1111

Skvortzow, B. W. 1927. Uber einige Phycomycetes aus China. Arch. Protis-
tenk., 57: 204-6.

Sokoloff, D. 1933. Algunas nuevas formas de flagelados del Valle de Mexico.
An. Inst. Biol. Univ., Mex., 4: 197-206.

Stabler, R. M. 1933. On an amoeba parasitic in Ze/leriella (Protozoa, Ciliata).
J. Parasit., 20: 122.

Stabler, R. M., and T. T. Chen. 1936. Observations on an Endamoeba patra-
sitizing opalinid ciliates. Biol. Bull., 70: 56-71.

Stein, F. (Ritter) von. 1849. Untersuchungen tber die Entwickelung der
Infusorien. Arch. Naturgesch., 15; 1: 92-148.

—— 1854. Die Infusionsthiere auf ihre Entwickelungsgeschichte untersucht.
Leipzig.

— 1859. Der Organismus der Infusionsthiere. I. Abtheilung. Algemeiner
Theil und Naturgeschichte der hypotrichen Infusionsthiere. Leipzig. pp.
96-100.

— 1867. Der Organismus der Infusionsthiere. II. Abtheilung. (1) Dar-
stellung der neuesten Forschungsergebnisse tiber Bau, Fortpflanzung und
Entwickelung der Infusionsthiere. (2) Naturgeschichte der heterotrichen
Infusorien. Leipzig.

— 1878. Der Organismus der Infusionsthiere. III. Abtheilung. Flagella-
ten. Leipzig.

— 1883. Der Organismus der Infusionsthiere. HI. Abtheilung. II. Hialfte
die Naturgeschichte der Arthrodelen Flagellaten. Leipzig.

Stempell, W. 1909. Uber Nosema bombycis Nageli. Arch, Protistenk., 16:
281-358.

— 1918. Uber Leptotheca coris n. sp. und Nosema marionis. Mitt. zool.
Inst. Univ. Minster, 1: 1-6.

— 1919, Untersuchungen tiber Leptotheca coris n. sp. und das in dieser
schmarotzende Nosema marionis Thel. Arch. Protistenk., 40: 113-57.

Stokes, A. C. 1884. A microscopical incident. Microscope, 4: 33-35.

Stubblefield, J. W. The morphology and life history of Amphiacantha ovalis
and Amphiacantha attenuata, two new haplosporidian parasites of gre-
garines. Univ. Cal. Library, MS. 1937.

Studitsky, A. N. 1932. Uber die Morphologie, Cytologie und Systematik von
Ptychostonum Chattoni Rossolimo. Arch. Protistenk., 76: 188-216.
Sutherland, J. L. 1933. Protozoa from Australian termites. Quart. J. micr.

SC, 762, 145-73.

Swarczewsky, B. 1908. Uber die Fortpflanzungerscheinungen bei Arcella vul-
garis Ehrb. Arch. Protistenk., 12: 173-212.

Takagi, S. 1938. On a bacterial parasite with active motility inhabiting the
the internal protoplasm of Spirostomum ambiguum Ehrenb. Annot. zool.
Jap: £7: 170-78.

Thélohan, P. 1895. Recherches sur les Myxosporidies. Bull. Sci. Fr. Belg.,
26: 100-394 (p. 360).

1112 PARASITES OF PROTOZOA

Tyzzer, E. E. 1920. Amoebae of the caeca of the common fowl and of the
tutkey.—Entamoeba gallinarum, sp. n. and Pygolimax gregariniformis,
gen. et spec. nov. J. med. Res., 41: 199-209.

Veley, L. J. 1905. A further contribution to the study of Pelomyxa palustris
(Greeff). J. Linn. Soc. (Zool.), 29: 374-95.

Wager, H. 1913. The life-history and cytology of Polyphagus Euglenae. Ann.
Bot. Lond., 27: 173-202.

Walker, E. L. 1909. Sporulation in the parasitic Ciltata. Arch. Protistenk.,

17: 297-306.
Wallengren, H. 1895. Studier dfver ciliata infusorier. Arsskr. Lunds Univ.,
Ailey

Wallich, G. C. 1863a. Further observations on an undescribed indigenous
amoeba, with notices on remarkable forms of Actinophrys and Difflugza.
Ann. Mag. Nat. Hist., (3) 11: 365-71.

— 1863h. Further observations on Amoeba villosa and other indigenous
thizopods. Ann. Mag. Nat. Hist., (3) 11: 434-53.

Wenrich, D. H. 1921. The structure and division of Trichomonas muris
(Hartmann). J. Morph., 36: 119-55.

—— 1932. The relation of the protozoan flagellate, Retortomonas gryllo-
talpae (Grassi, 1879) Stiles, 1902 to the species of the genus Embado-
monas MacKinnon, 1911. Trans. Amer. micr. Soc., 51: 225-38.

— 1937. Studies on lodamoeba biitschlii with special reference to nuclear
structure. Proc. Amer. phil. Soc., 77: 183-205.

— 1940. Studies on the biology of Dientamoeba fragilis. Pp. 408-9 in
Third International Congress for Microbiology: Report of Proceedings.
New York.

Wenyon, C. M. 1907. Observations on Protozoa in the intestine of mice.

Arch. Protistenk., suppl. 1: 169-201.

1926. Protozoology, a manual for medical men, veterinarians and zo-
ologists. New York.

Wesenberg-Lund, C. 1929. Rotatoria, Rotifera - Radertierchen, 77 Kikenthal,
W. and T. Krumbach: Handbuch der Zoologie. Berlin.

Wetzel, A. 1925. Zur Morphologie und Biologie von Raphidocystis infestans
n. sp. einem temporar auf Ciliaten parasitierenden Heliozoon. Arch.
Protistenk., 53: 135-82.

Winogradowa, T. 1936. Sphaerita, ein Parasit der Wiederkauerinfusorien. Z.
Parasitenk, 8: 356-58.

Yagiu, R. 1933. Studies on the ciliates from the intestine of Azthocidaris
crassispina (A. Agassiz). I. Cyclidium ozakii sp. nov. & Strobilidium
rapulum sp. nov. J. Sci. Hiroshima Univ., (B:1) 2:211-22.

Yakimoff, W. L. 1930. Zur Frage tiber Parasiten bei Protozoa. Arch. Protis-
tenk., 72: 135-38.

eet "eee

PARASITES OF PROTOZOA iI Ie

— 1931. Bolesni domaschnick schirootnich wisiroaemie prosteischmimi
(Protozoa) (in Russian). (Cited by Sassuchin, 1934, without journal.)

Yakimoff, W. L., and A. P. Winnik. 1933. Die Ansteckung der Oocysten
der Kaninchencoccidien durch Bakterien. Arch. Protistenk., 79: 131-32.

Yuan-Po, Li. 1928. Entamoeba bobaci n. sp. des tarabagans (Marmota bobac).
Ann. Parasit. hum. comp., 6: 339-42.

Zacharias, O. 1909. Parasitische AmGben in Volvox minor, Arch. Hydrobiol.,
5: 69-70.

Zeliff, C. C. 1930. A cytological study of Oxymonas, a flagellate, including
the descriptions of new species. Amer. J. Hyg., 11: 714-39.

INDEX

Abbott, 972

Ablastin, 838; involved in immunity
against trypanosomes, 856-62 passim

Accessory vacuoles, see Vacuoles, accessory

Accidental parasitism, defined, 895; see
also Parasitism

Acclimatization, to experimental condi-
tions, 507; and immunity, inherited,
717-21

Acids, effects on consistency, 55, 61;
motor responses to, 333, 342

Actinophrys sol, 600

Actinopoda, sexual reproduction, 598 ff.

Active contraction vs. elastic shortening,
NO, Dil

Activities influencing longevity, 16

Adhesiveness (or stickiness), 71-77; rela-
tion to phagocytosis, 74; in animals
that live in association with hosts,
930 ff., 944, 948, 949

Adolph, 65, 362, 363, 364, 424, 525

Aérobes, why die in absence of oxygen?
394

Aérobic respiration, see Respiration

Afridi, 849

Age changes; sexual immaturity and ma-
turity, 714f., 761

Aggregation, in light, 281, 297; in acid
region, 342

Agranulocytes, 835

Albuminoid reserves, 160, 162

Alda Calleja, M. de, 876

Alexander, 58

Alexeieff, 160, 903, 907, 910, 970, 1033,
1042, 1050

Alkalies, effects on consistency, 55; motor
responses to, 333, 342

Allelocatalytic growth, 533 ff.

Allison, 79

Alsup, 314

Alternating current, responses to, 310-14

Altmann’s bioblast theory, 174, 175

Alverdes, 793

Amberson, 356, 363, 369

American Society of Parasitologists, Com-
mittee on Terminology, 891

Amoeba, properties of protoplasm as ex-
hibited in, 46-50; surface precipitation
reaction, 48; consistency, 51-61 passim;
surface properties, 63-74 passim; spe-
cific gravity, 79, 80, 81; other proper-
ties, 82, 84, 90-97 passim; survey of
functions having granular basis, 168-75
passim; structure, 272; responses to
light, 272-80; to electricity, 305-20,
323; to chemicals, 333-41; dysentery
and bacterial complications, 819-22

Amoebae, parasitic in a free-living ciliate,
1075

Amoebophrya Koeppen, 1089-93

Anaérobes, why are they anaérobes, 390-
94

Anaérobic metabolism, measurement of,
385 f.

Ana€érobiosis, occurrence of glycolysis and,
386-90

Ancestry, relation to conjugation, 615;
necessity of diverse, 696

Ancistridae, 933

Ancistruma, fibrillar system, 228

Ancistrumidae, adaptation, 934-38, 939,
941

André, 900, 916, 934

Andrews, 927

Andrews and von Brand, 365, 368, 369,
485

Anentera, 12

Anesthetics, effect on O2 consumption, 366

Aneurin, or thiamine (vitamin Bi), 490

Angerer, 48, 58

Animals: relationships between certain
Protozoa and other animals, 890-1008
(see also under Relationship)

Anisogamy, term, 584

Anoplophrya circulans, 951, 952

Antibodies and antigens involved in im-
munity, 837-39, 873

Antiserum, 837

Antitoxin, 838

Anus, presence in Infusoria ascertained, 11

Apostomea, 956; adaptation, 957-60

Archer, 1094

1116

Artigas and Pacheco, 912

Aschner, 964

Aschner and Ries, 964

Aschoff, 836, 839

Asexual reproduction, alternation of sex-
ual with, 566 f., 571-73; in alternating
binary and multiple fission, 569-71

Aspidisca, reorganization in, 22 f.

Association, see Relationships

Astomata, 945; adaptation, structure, 946-
53; Conidiophrys, 953-56

Attachment, see Adhesiveness

Autocatalytic growth, 533 ff.

Autogamy (self-fertilization),
654-59; genetic evidence, 757

Avian malaria, 844

Awerinzew, 1074, 1082

606-11,

Bach and Quast, 1022

Bacigalupo, 1045

Bacteria, elimination of, before measure-
ment of respiration, 358; methods for
control or elimination of, 448 ff.; bac-
tericidal agents, 463-67; culture, 467;
amoebic dysentery and bacterial compli-
cations, 819-22; cellulose decomposition
by, 962 ff., 966f.; nitrogen-fixing, 972

Bacteriology, Leeuwenhoek the father of,
11

Baitsell, 528

Balamuth, 789, 932

Balantidium coli and B. suis, fibrillar sys-
tem, 244; B. sushilii, 245

Balbiani, E. G., 775, 780, 787, 793, 951,
952, 1034, 1035, 1036, 1086, 1087,
1089; interpretation of the process of
conjugation, 11, 13, 14

Ball, 66

Bancroft, 281, 287, 320, 321, 323, 324,
327

Barber, 873

Barcroft differential type, 356, 357

Barratt, 362, 366

Barry, M., 13

Barth, 54

Bary, A. de, originator of term symbiosis,
891

Basal apparatus in Paramecium, 195, 198,
200, 204, 226, 227

Basalfibrille, 201, 204, 219

Basal metabolism, measurement, 360

Basophilic granules, 160, 162, 163-65

Bauer and Granowskaja, 800

INDEX

Bayliss, 51, 61, 306, 319

Beams, H. W., and King, R. L., 69, 98;
Some Physical Properties of the Proto-
plasm of the Protozoa (Chap. II), 43-
110

Beauchamp, 905, 907

Bechdel, Honeywell, Dutcher, and Knut-
sen, 985

Becker, Elery R., 85, 801, 818, 878, 893,
918, 979, 983, 984, 1045, 1047, 1048,
1050, 1051, 1052, 1058; Certain As-
pects of Pathogenicity of Protozoa
(Chap. XVII), 818-29

Becker, Elery R., and Derbyshire, R. C.,
826

Becker, Elery R., and Everett, R. C., 986

Becker, Elery R., and Hsiung, T. S., 977

Becker, Elery R., and Talbott, M., 976

Becker, Elery R., and Waters, P. C., 824,
826

Becker, Elery R., Schulz, J. A., and Em-
merson, M. A., 982, 983, 984, 985, 986

Beckwith and Rose, 966

Becquerel, 58

Beers, 91, 117, 119, 527, 528, 529, 540,
543, 616, 653, 657, 943, 944, 945, 946,
948, 949

Bélai, 26, 223, 253, 260, 594, 598, 599,
611

Bélehradek, 57

Bélehradek and Paspa, 94

Benda, 177

Benedict, 481

Ben Harel, 844

Bensley, 86, 118, 119, 120, 181

Berghe, van den, 901

Berkson et al, 518

Bernard, 534

Bernstein, 1044

Beta granules, 406, 435

Bethe, 258

Bibliography, see Literature cited

Bignami, 846

Bills, CG. E.; 802

Biology of the Protozoa, The (Calkins),
3, 616

Biotypes, 712

Biparental reproduction, see Reproduction,
biparental

Bishop, A., 908, 909, 910, 911, 950;
ciliate fibrillar systems, 250, 251

Blaauw, 289

Bles, 72, 77, 125, 167, 368, 384

INDEX

Blockade and splenectomy, 832

Blood cells, classified, 834 ff.

Bloom, 836

Bodine, 56, 542

Bodine and Boell, 374, 378

Bodine and Orr, 357

Boeck, 1011, 1032

Bohm, 848

Boissezon, 898

Bojewa—Petruschewskaja, 411

Bonar, 1068

Bond, 501, 519, 550

Borgert, 153, 1089, 1091, 1092, 1093

Bott, 593

Bourne, A. G. 1025

Bourne, G., 121

Bourne, G., and Allen, R., 121

Boveri, 638

Boveria, fibrillar system, 228, 229

Bowen, 138, 140, 180, 442, 543, quoted,
443

Bowling, 32, 33

Boyd, G. H., 843, 844, 845, 846

, G. H., and Allen, L. H., 844, 845

Boyd, G. H., and Dunn, M., 844

Boyd, M. F., and Coggeshall, L. T., 844

Boyd, M. F., and Kitchen, S. F., 823

Boyd, M. F., and Stratman-Thomas, W. K.,
822, 825

Boyd, M. F., Stratman-Thomas, W. K.,
and Kitchen, S. F., 848

Boyd, M. F., Stratman-Thomas, W. K.,
and Muench, H., 823

Bozler, 802, 1035, 1037

Brahmachari, 875

Brain cells, 836

Brand, von, 151, 157, 159, 162, 361,
385, 387, 388, 389, 390, 485, 865

Brand, von, and Jahn, 361, 387, 392

Brand, von, Regendanz, and Weise, 389

Brandt, 78, 97, 787, 1069

Brauer, 208

Braun and Teichmann, 877

Brehme, 795

Bresslau, 87

Brinley, 51, 53, 55, 59

Broh-Kahn and Mirsky, 374, 381, 388,
391, 392, 394

Brown, D. E. S., and Marsland, D. A., 61

Brown, H. C., and Broom, 875

Brown, M. G., et al., 451, 466

Brown, V. E., 138, 199, 435, 436

Brown, W. H., 855, 856, 981

eh
Browne, 81
Browning et al., 864, 870
Brues, 55
Bruetsch, 853
Brug, 847, 1046, 1054
Brumpt, 586, 623, 819, 820, 878
Brumpt and Lavier, 904, 1045, 1046,

1048, 1051, 1053, 1054, 1055, 1057,
1075, 1076, 1077

Brussin and Kalajev, 869

Buchanan, R. E., 1026

Buchanan, R. E., and Fulmer, E. I., 495,
547

Buchner, 504, 933, 963, 972, 1010

Buck, 823

Budding division, 28

Buder, 287

Bitschli, O., and Schewiakoff, 202, 204

Buffon, 9

Bullington, 793

Bundle, 985

Bunting, 909.

Bunting and Wenrich, 909

Burch, 796

Burge, 384

Burk, 359, 384, 386

Burnside, 530, 796

Burt, 454

Buscalioni and Comes, 969

Buschkiel, 610

Buschkiel and Nerescheimer, 33

Bush, M., 126, 128, 136, 141, 145, 147,
179, 950; ciliate fibrillar systems, 240,
242

Butschli;) ©:, 12, 157, 413.) 566; .915;
1035, 1036, 1039, 1040, 1070, 1087,
1089; interpretation of the process of
conjugation, 13, 14; discovery re carbo-
hydrate granules of gregarines, 111, fi-
brillar system, 201, 202, 208, 210, 213,
217.210: 224.1202

Butts, 53, 54

Cailleau, 478, 384, 485, 486, 492, 493,
500

Cajal, Ramon, y, Santiago, 138, 144

Caldwell, 660

Calkins, Gary N., 16, 18, 21, 28, 29, 30,
32. 33% 344.375. 38) 4551s 85, 86, 112.
117, 161, 431, 529, 647, 649, 666,
699, 714, 893, 934, 1038; General Con-
siderations (Chap. I), 3-42; Biology
of the Protozoa, 3, 616; ciliate fibrillar

bees INDEX :

Calkins, Gary N. (continued)
systems, 256, 257; fertilization, 583,
SOP, SOB, S845 GOs, Gl, GIG, Gl.
618, 624, 627, 630, 632, 633, 637;
morphogenesis, 774, 775, 776, 777,
718; 7195 1805 7895 79715 798

Calkins, G. N., and Bowling, R., 233, 591,
602, 611

Calkins, G. N., and Cull, S. W., 581, 626,
634, 636, 689, 702

Campbell, A. S., 160, 166, 1069; ciliate
fibrillar systems, 253, 254

Campbell, W. G., 962

Candolle, de, 282

Cannon, P. R., and Taliaferro, W. H., 849

Canti, 138

Capillary manometer, 357 f.

Carbohydrates, reserves, 153-59, 173;
granules with definite internal structure,
154; differentiation between glycogen
and paraglycogen, 157

Carini, 1075, 1076

Carini and Reichenow, 1075, 1076

Carlgren, 319, 324, 327

Carlson, 518

Carter, 1040, 1042

Cartesian diver ultramicromanometer, 358

Casagrandi and Barbagallo, 1043

Catalase, 373; detection of, 384

Cattaneo, 899

Cattle, rumen ciliates, 894, 973-87 (see
also under Ciliates of ruminants)

Caullery, 891, 892, 893

Caullery and Mesnil, 1078, 1079, 1080,
1081, 1082

Causin, 795, 797

Cavallini, 1075

Cejp, 1043, 1052

Cell division, longevity influenced by re-
organization through, 16; in flagellates,
25 ff.; material for study or cytology
of, 45; environmental conditions suit-
able for most rapid growth and divi-
sion, 45; division rates of Protozoa
with constant conditions, 527, table,
528; regeneration and, 775-78, 797-801,
division cycle, 775-78

Cell membrane, nature of, 62; permeabil-
ity, 64-66, 69; other than those of
Protozoa, 64

Cell theory, forerunner of, 9; first applied
to the Protozoa, 13

Cells, first use of word, 9; longevity of

derived structures, 38; macronucleus,
38; micronucleus transcends other struc-
tures in ciliate cell, 39; differentia-
tion, 44; comparison between protozoan
and metazoan, 44, 179ff; relative
specific gravity of inclusions and com-
ponents of, 80-82; Golgi bodies in
metazoan, 140, 143; importance of réle
of Golgi apparatus in life history of,
431, 441; link in kinship between all
cells apparently established by
Golgi apparatus, 441, 443; structures
present in all, 432; connective tissue
cells involved in immunity: terminol-
ogy and classification, 831-37; of the
blood and lymph, classified, 834-36; so-
called systems of, used by authors,
836 f.; cellular and humoral aspects of
immunity, 839-41

Cellular concept of living things, disclos-
ure of, 191, difficulties encountered,
191; protoplasmic differentiation con-
trasted with cellular differentiation, 192,
260

Cellulose, constituent of wood, 961; use
of, in nutrition, 961-73; decomposition,
962 ff., 966f., 983; experiments in
feeding cellulose-free | carbohydrates,
970; use by rumen ciliates, 981 f., 983

Centrifuge method, 52; as a research tool,
81

Cépéde, 915, 936, 938, 945, 946, 948,
950, 952, 953

Cépéde and Poyarkoff, 953

Cépéde and Willem, 1020, 1022

Certes, 911

Chakravarty, 158, 163

Chalkley, 45, 57, 59, 67, 73, 83, 84, 85,
121, 340, 518, 523

Chambers, 46, 48, 49, 51, 52, 58, 62, 63,
65, 66, 67, 72, 85, 88, 181, 339, 395,
396

Chambers and Dawson, 76

Chambers and Hale, 58

Chambers and Howland, 54

Chambers and Kempton, 127

Chambers and Pollack, 127

Chambers and Reznikoff, 52, 53, 315

Chapman, 553

Characteristics, inheritance of, 712 f.

Characters, inherited; changes in, in uni-
parental reproduction, 713-31

Chase and Glaser, 59, 340

INDEX

Chatterji, Das, and Mitra, 912

Chatton, E., 587, 930, 941, 1069, 1075,
1079, 1082

Chatton, E., and Biecheler, B., 588, 1070

Chatton, E., and Brachon, S., 1072

Chatton, E., and Brodsky, A., 593, 1041,
1042, 1048, 1051, 1053, 1058

Ghatton, E, and Grasse, P, P., 113, 115

Chatton, E., and Lwoff, A., 75, 1025,
1073, 1083, 1084, 1085; fibrillar com-
plex, 200, 223, 228; relationships, 620,
9351; 933, 934, 936, 938; 940; 941,
942, 946, 953, 954, 955, 956, 957, 958,
959, 960

Chatton, E., and Pénard, C., 621

Chatton, E., and Villeneuve, S., 1072

Chatton, E., Lwoff, A., Lwoff, M., 1083

Chatton, E., Parat, M., and Lwoff, A., 165,
180

Cheissin, 151, 936, 943, 944, 945, 946,
947, 948, 950, 952, 956

Chejfec, 649, 660, 775

Chemicals, responses to, 333-44; rhizo-
pods, 333-41; ciliates, 342-44

Chen and Stabler, 1045, 1076

Child, C. M., 86, 773, 802, 808

Child, C. M., and Deviney, E., 86, 803

Child (MS), 928

Chilodonella, 27, 630f., 632, 633, 634,
635, 637, 638

Chlamydodon, fibrillar system, 229, 231

Chlamydomonas, division, 589f., 613;
kinds of gametic differences in, 589,
667-71; sexuality, 666-87; nature of
physiological differences between gam-
etes in, 671-78; interpretation of sex-
ual phenomena, 678-87; inheritance,
722, 739, 741, 742, 744, 745

Chlorellae, relationship to host, 1010

Cholesterol, 493

Cholodnyj, 344

Chondriome, 170, 432; see also Mito-
chondria

Chondriosomes, 432; permanence,
see also Mitochondria

Chopra and Mukherjee, 848

Chopra, Gupta, and David, 875

Chorine, 875

Christophers, 854

Chromatin, value and importance shown,
39; volutin linked with, 162

Chromatin reaction, in derived structures,
183) 21) tr.

181;

PES

Chromatoidal bodies, 160

Chromidia, the term and its interpretation,
160; associated with mitochondria, 160,
161; term bound up with disproved
theories, 165

Chromidial net a definite morphological
entity, 161

Chromidial origin of nuclei doubted, 594

Chromosomes, individuality retained in all
cell generations, 87; genetic materials
in, 710, 711

Ciacco, 123

Cienkowsky, 1064, 1070, 1074, 1086

Ciliates, reorganization of the macronu-
cleus and other derived structures, 21-
31; waning vitality, 28 ff., reorganiza-
tion by endomixis, 31-36; intracellular
micronuclei forming pronuclei, 33;
longevity of protoplasm, 34; reorgani-
zation by conjugation, 36-39; impor-
tance of micronucleus, 39; adhesiveness,
75 ff., 932, 944, 948, 949; ability to
combine into composite organelles, 76;
color, 83; polarity, 85; contraction, 92,
93; double refraction, 96; segregation
granules, 132 ff., (see entries under Seg-
regation); fibrillar systems, 191-270
(see also under Fibrillar); responses
to light, 295-97; responses to electricity,
321-27; internal processes involved in
responses, 324-27; responses to chemi-
cals, 342-44; conditions necessary for
conjugation, 614, 615 f.; conjugation,
616-39; sexuality in, 621 f., 666, 687-
706; biparental inheritance in diploid
ciliates, 750-58; faunules of, in sea
urchins, 894, 919-23; first to live in
blood of host and circulate with cor-
puscles, 952

Ciliates of ruminants, 894, 973-87; total
number in an individual, 974; repro-
ductive rate, 976; environment, culture,
976; defaunation treatments for elimina-
tion of, 977; food, 979 ff., 985; value
to host, 983-87; rdle of, in protein
economy, 984 f.

Ciliophora, Schizomycetes on, 1021-24;
Schizomyectes in, 1034-40; parasitizing
other Protozoa, 1083-89

Claff, C. L., migration-dilution apparatus,
461-63

Claparéde, 413, 1034, 1039, 1079

Claparéde and Lachmann, 805, 1034,

1120

Claparéde and Lachmann (continued)
1039, 1043, 1064, 1079, 1084, 1086,
1087

ClarkswACnee4s

Clark, N. A., 535

Clark, W. M., 396

Classification, of fresh-water species on an
ecological basis, 5; Millers, the earliest
successful, of Protozoa, 11; cytoplasmic
granules, 177-79; immunological reac-
tions in relation to, 876-78

Cleveland, L. R., 45, 90, 154, 157, 391,
392, 460; 4635 89278935, °909; "911,
931, 962, 964, 965, 966, 967, 968, 969,
970, 971, 1015, 1016, 1018, 1020

Cleveland, L. R., and Sanders, E. P.,
821

Cleveland, L. R., Hall, S. R., Sanders,
E. P., and Collier, Jane, 391, 924, 926,
928, 929, 962, 965, 966, 968, 969,
970, 971, 972, 1015, 1028

Clones, 712; single-type, 756; crosses be-
tween single- and double-type, 757

Coagulation, irreversible, 61

Coccidiosis in poultry, 824-26

Codreanu, M., and Codreanu, R., 906,
907

Codreanu, R., 897, 898, 915, 916, 917

Goe; 532

Coehn, A., and Barrat, W., 324

Coggeshall, 848, 849

Coggeshall and Eaton, 850, 875

Coggeshall and Kumm, 850

Cohen, Barnett, 395

Cohen, R. A., and Gerard, R. W., 381

Cohen, W. E., 965

Cohn, 208, 1064, 1093

Cohnheim, Ziegler, Marchand, 836

Colas-Belcour and Lwoff, A., 485, 486

Collin, B., 623, 627, 940, 1022, 1043,
1083, 1084, 1087, 1089

Colloidal nature of protoplasm, 49 f., 51

Colonial organisms, growth, 526f.; re-
generation in, 804-11

Colonial organisms’ responses, to light,
297-305; shock reaction, 298; kinetic
responses, 299; orientations, 300-303;
wave length and response, 303; rever-
sal in response, 303-5; to electricity,
327-32; orientation, 328; electric
charge, 328; mechanics of response,
329-32

INDEX

Colonies, temporary: formation and pur-
pose, 74

Color of protoplasm, 82 f.

Commensalism, term, 818, 891; inqui-
lines, 892; physiological host relation-
ships illustrative of mutualism and,
961-87

Complement fixation test, 873 f.

Composite motor organelles, separation
into their components, 76

Conchophthirus, reorganization, 21 f.;
fibrillar system, 230, 232, 234

Condensation membrane, 418

Conductivity, fibrillar, 258

Conidiophrys, 953-56

Conjugant meiosis, 624-39; first divi-
sion, stages A, B, 626-31; second,
stage C, 631f.; third, stage D, and
formation of pronuclei, 632-34; stage
E, migration of pronuclei and fertiliza-
tion, 634f.; stages F, G, H, The ex-
conjugants, 635-39

Conjugation, Balbiani’s interpretation of
process of, 11, 13, 14; Miller’s ob-
servations, 11, 13; tests, 31; reorgani-
zation by, 36-39; ciliate, 36 ff., 616-
39; difference between endomixis and,
36; conditions necessary for, in ciliates,
614; defined, 617-23; macronucleus
during, 623f.; sexual differences be-
tween individuals, 688-99; role of en-
vironmental conditions in determining,
700 f.; differences between gamete nu-
clei during, 701 f.; significance of di-
versities between conjugants and be-
tween gamete nuclei, 703-6; regenera-
tive ability, 778 f.

Conklin, 84, 796

Connell, 163, 1019, 1030, 1031, 1044,
1052

Consistency of protoplasm, 50-61; effect
of mechanical agitation, 47, 58; of
water, 53; of salts, 53-55, 61; of acids
and alkalies, 55, 61; of temperature
55-58; of hydrogen-ion concentration,
58; of narcotics, 59; of radiation, 60;
of heavy water, 60; of electric current,
61; hydrostatic pressure, 61; irrevers-
ible coagulation, 61

Contractile vacuoles, see Vacuoles, con-
tractile

Contractility, 90-94; active vs. elastic, 90,
91; Vorticella, 208 ff. 216 ff., 229, 258

INDEX

Cook, 367, 1065

Copeland, 343

Copromonas subtilis, 584, 585 f.

Coprozoic Protozoa, 6

Copulating cells, see Gametes

Copulation, gametic meiosis and fertiliza-
tion, 584-601; Sporozoa, 601-6

Corpse, 28

Councilmania lafleuri (Endamoeba colt),
life cycle, 569, 572

Coventry, 855, 856, 857, 861

Cowdry, 118

Cowdry and Scott, 121, 135, 138

Cragg, 1043, 1045

Craig, 819, 874, 1043, 1044

Crawley, 606

Gross) J: B) 928

Cross-fertilization, differences
self-fertilization and, 606 f.

Crossing over and linkage, 737-40, 743-
45

Crouch, 912, 1044

Crozier, 531

Crozier and Harris, 531

Cryptocercus, Protozoa of termites and,
894, 923-29; relationship between flag-
ellates of termites and, 894, 961-73

Crystals, specific gravity, 80; defined, 179

Cuenot, 915, 937

Culbertson, 860

Culbertson and Wotton, 855, 856, 860

Cultures, use of in study of Protozoa,
14f.; mediums, 15; technique and
significance of control in protozoan,
448-74; ‘“‘pure-mixed”” methods, 448;
problem of sterilization, 449-67; im-
portance of adequate sterility tests,
467 f.; Establishment of sterilized Proto-
zoa in, 468-73; literature cited 473 f.,
507-16; food requirements and other
factors influencing growth of Protozoa
in pure cultures, 475-516; growth in
pure, as a population problem, 495-
99; pedigree isolation culture and life
cycles, 527-31; methods for growing
bacteria-free, pure cultures, 538-44

Cunha, A. M., da, 1023

Cunha, A. M., da, and Muniz, J., 912,
1044, 1047, 1050

Cutler, 154

Cutler and Crump, 523, 524, 535

Cyanellae, relationship to host, 1010

Cyanide, respiratory sensitivity, 376 ff.

between

1121

Cyclic adaptation of Protozoa to hosts,
953

Cyclical variations, and regeneration, 775-
80

Cycloposthiidae in mammals, 973

Cyst, vitality of protozo6n within, 4;
specific gravity, 79

Cytochrome, 373

Cytochrome-cytochrome oxidase system of
hydrogen acceptors, 376-80

Cytokinetic mechanism, 44

Cytology, cytoplasmic granules a special-
ized branch of, 111

Cytology of cell division, material for
study of, 45

Cytomicrosomes, 432; see also mitochon-
dria

Cytoplasm, codrdination between nuclear
and cytoplasmic division processes,
85 f.; effect of, and its relation to nu-
clear constitution, 762-69

Cytoplasmic fission, attachment an im-
portant factor in, 73

Cytoplasmic inclusions, 111-90; mito-
chondria, 112-26; vacuome hypothesis,
126-29; digestive granules, 129-32; seg-
regation granules, 132-38; Golgi bodies,
138-44; excretory granules, 144-50;
lipoid reserves, 150-53; carbohydrate
reserves, 153-59; protein reserve bodies,
160-66; external secretion, 166-68;
granular complex, 168-74; continuity of
cytoplasmic granules, 174-77; classifica-
tion of cytoplasmic granules, 177-79;
comparison with cells of the Metazoa,
179-81; literature cited, 182-90; see also
entries under Granules

Czermak, 208, 217

Dahmen, 874

Dallasia, see Glaucoma frontata

Dallinger, 718

Dangéard, P., 128, 168, 589, 613, 1032,
1033, 1041, 1042, 1043, 1048, 1049,
1050, 1051, 1052, 1053, 1054, 1056,
1057, 1058, 1059, 1063, 1065, 1066,
1067, 1070, 1073, 1074, 1075

Daniel, 369

Daniel and Chalkley, 56, 539

Daniels, M. L., cytoplasmic inclusions, 81,
117, 122, 126, 141, 142, 154, 160, 161,
162, 169,

Darby, 527, 528, 529, 536, 541

1122

Darkness, adaptation to, 291; growth in
relation to light and, 506 f.

Das Gupta, 910

Dauermodifikationen, 720, 730

Daugherty, 59

Davenport, 56, 279

IDEN. Isls Shy Sill, OlZ

Davis, L. J., 864, 865, 870

Dawson, 529, 650, 791

Dawson and Belkin, 74

Day, H. C., contractile vacuole, 147, 407,
410, 418, 420, 422, 424, 428

Death, cause of, 4; in isolation cultures,
39

Debaisieux, 608

Defaunation methods, 977

Deflandre, 1075

De Garis, C. F., 697, 753; inheritance
in Paramecium, 762-69

Degen, 413, 426

Degeneration, inherited: resulting from
unfavorable conditions, 716

Dehorne, 626

Dehydrogenases, 372; inhibition of the
system, 381 f.

Delafield’s hematoxylin, 832

Delage and Herouard, 202

De Lamater, 659

Delanoé, 861

Dellinger, 72, 73, 89, 91

Dembowska, morphogenesis, 774, 775,
777, 778, 784, 786, 790, 797, 798

Density (specific gravity), whole organ-
isms, 77-79; relative, of cell inclusions
and components, 80-82

Derived and fundamental organization, 4

Development, studies of regeneration and,
772

Developmental cycle of parasite and host,
933

Dewey, V. C., 469

Dextrose, value to wood-eating animals,
971

Diagnosis, immunological reactions used
in, 872-76

Dickman, 966, 967

Dierks, K., fibrillar system, 93, 201-4
passim, 218, 219, 220

Diesing, 943

Differentiation, protoplasmic, 44

Difflugia corona, inheritance in, 724-29

Difflugia pyriformis reincorporation, 794

INDEX

Diffraction, X-ray- and ultra-centrifuga-
tion, 97 f.

Digestive function of mitochondria, 123,
124

Digestive granules, 129-32; universality
of, rejected, 172; defined, 178

Dileptus gigas, fibrillar system, 235

Diller; Ws F533; G10, 611, "6385 704;
756, 757; endomixis, 647, 649, 650,
651, 654, 655, 656,657, "658," G59;
661; quoted, 654

Dilution, sterilization by, 451-55; com-
bined with migration, 460-63

Dimitrowa, A., 407, 413, 424, 499, 535

Dioecious races, 741

Diplodinium, fibrillar system, 251-53

Diploid parthenogenesis, 649

Diploids, biparental inheritance in, 750-
58

Direct current, responses to, 306-10

Division, see Cell division; Fission

Diwald, 588, 613

Dixons 356; 3577, 369

Dixon and Bennet-Clark, 317

Dobell, 44, 158, 179, 191, 260, 576, 584,
585, 586, 612, 715, 870, 912, 1045,
1047

Dobell and Jameson, 612

Dobell and Jepps, 819

Doflein, 426, 594, 636, 689, 982, 983,
987, 1042, 1043, 1072, 1075, 1082,
1093

Doflein-Reichenow, 112, 583, 636, 893

Dogiel, 33, 619, 629, 621, 622, 634,
688, 702, 703, 973, 978, 979, 981, 985,
1015, 1024, 1044, 1045, 1046, 1050,
1078, 1079, 1080

Dogiel and Fedorowa, 157, 974, 975, 981

Dogiel and Furssenko, 915

Dogiel and Issakowa-Keo, 70

Dogiel and Winogradowa-Fedorowa, 976,
MT SKS)

Dore and Miller, 962

Double refraction, 95-97

Douvillé, 1068

Doyle, 116, 124, 174

Doyle and Harding, 425

Dropkin, 929

Dubos, 395

Duboscq and Collin, 1069

Duboscq and Grassé, 129f., 138, 154,
155, 158, 927, 1011, 1012, 1015, 1016,

INDEX

1018, 1019, 1028, 1030, 1044, 1046,
1079

Duboscq, Grassé and Rose, 1015

Duca, 855

Dujardin, Felix, 208, 217, 413; quoted,
10, 43; studies by, 11; conclusions on
Protozoa, 12, 43; controversy with
Ehrenberg re Infusoria, 192, 260

Duke, 862

Duke and Wallace, 875

Dunihue, 130, 137, 436, 439

Duryée, 354, 356, 357

Dusi, H., food, etc., influencing growth,
477, 478, 479, 480, 481, 487, 488, 489,
490, 491, 502, 506

Du Toit, 878

Dyestuffs, use in experiments, 66

Dysentery, amoebic: and bacterial com-
plications, 819-22

Eaton, 850

Ecological considerations, 4-8

Ectoparasites, 7

Ectoplasm (plasmagel), 47; elasticity, 87;
contractility, 90, 91

Eddy, 533, 542

Edmondson, 914

Edwards:535 55559) 3i5n355

Efimoff, Nekrassow, and Efimoff, 393

Ehrenberg, C. G., 1093, 1094; studies by,
11, 43, 413; conclusions on Protozoa,
12, 13; controversy with Dujardin re
Infusoria, 192, 260

fibrillar system, 200, 208, 209, 210, 216,
217, 219

Ehrlich and his coworkers, 869

Eikenberry and Waldron, 891

Eimeria schubergi, life cycle, 573-78

Eisenberg, E., 427, 428

Eisenberg-Hamburg, 340

Eksemplarskaja, 151 -

Elasticity, of protoplasm, 87-90; of ir-
reversible structures, 89; shortening vs
active contraction, 90, 91; Vorticella,
ZO9\fF., 217 ff... 258

Electric current, effects of, 61

Electricity, responses to, rhizopods, 305-
20; flagellates, 320; ciliates, 321-27;
colonial organisms, 327-32

Elliott, A. M., 360, 468, 518, 537, 540,
542, 543; food, etc., influencing growth,

LL25

484, 486, 487, 490, 492, 493, 501, 502,
503, 507

Elliott, A. M., and D. F. Johnson, 501

Ellis J. M., 145, 178; fibrillar system,
246

Elmassian, 1082

Elpatiewsky, 594, 595, 1074

Emerson, 362, 369, 371, 378, 388

Emik, 968, 969

Encyclopaedia Britannica, 891

Encystment, regenerative capacity, 779

Encystment test, 31

Endamoeba coli (Councilmania lafleuri),
life cycle, 569, 572

Endomixis, reorganization by, 31-36; dif-
ference between conjugation and, 36;
macronuclear reorganization, 646-48;
endomictic phenomena, 648-54; autog-
amy, 654-59; coinage of name, 657;
periodicity, 659 f.; genetical studies on,
660-62; literature cited, 662-65; segre-
gation of mating types at, 756

Endoparasites, 7

Endoplasm (plasmasol), 47

Endothelial cells, 833, 834

Endozoic Protozoa, defined, 892

Energy, respiration a means of studying,
353 f.; investigations which concern the
source of, 368-72

Engelmann, T. W., 29, 30, 34, 37, 96, 97,
1035, 1087; on conjugation, 14; fibrillar
system, 198, 201, 208-15 passim, 217,
221; motor responses, 273, 281, 282,
289, 305

Enriques, 27, 622, 630, 633, 689, 703

Entamoeba, pathogenicity of, 819-22

Enterodela, 12

Entodiniomorphina, 973

Entodiscus borealis, fibrillar system, 236,
237

Entorhipidium echini, fibrillar system, 236,
239

Entz Get 25

Entz, Geza, fibrillar systems, 208-18 pas-
sim, 258, 259, 909, 914, 1088

Environment, effect upon division and
growth, 45; rdle in determining con-
jugation, 700 f.; inherited degenerative
changes resulting from unfavorable
conditions, 716f.; inherited modifica-
tions in form and structure, 712-23;
variation .. . without obvious action of

1124

Environment (continued)
diverse environments, 723-29; external,
and regenerative behavior, 774 f.

Enzymes, in mitochondria, 120,
synthesis of respiratory, 382-84

Ephrussi, 64

Ephrussi and Neukomm, 340

Ephrussi and Rapkin, 54

“Epibionts,” 7

Epidinium caudatum, fibrillar system, 253

Epistylis, fibrils, 214

Epstein, 899, 1033, 1045, 1046, 1054,
1057, 1069

Erdmann, 154, 523, 524, 526, 530, 649,
659, 660, 758

Erdmann and Woodruff, 649, 657

Erythrocytes, 835

Estabrook, 521, 522, 523, 526

Euciliata, parasitic, 1083 f.

Euglena, responses to light, 280-90

Euglenids, free-living and symbiotic,
903 ff.

Euplotes, reorganization, 23, 24; fibrillar
systems (structural analysis, 204-8, inter-
pretation, 221-24, conclusions, 257 ff.) ;
sexuality, and fertilization, 618, 619,
620, 621, 622, 626, 627, 628, 629,
632, 633, 634, 636, 637, 666, 696,
698, 700, 703

Eupoterion pernix, fibrillar system, 238,
240

Evans, Te G., 357

Everts, 208

Ewles and Speakman, 89

Excretory function of contractile vacuole,
422 ff., 442

Excretory granules, 144-50, 405, 440;
mitochondria associated with, 124; uni-
versality of, rejected, 173; defined, 178

Existence, struggle for, 553 f.

Exogamy, differences between autogamy
and, 606 f.

Extension and retraction, see Contractility

External secretion, 166-68

255

Fabrea salina, fibrillar system, 246

Fabre-Domergue, 916, 936, 937

Facultative parasitism, defined, 895; see
also Parasitism

Falaschini, 977, 986

Falck, 962

Fantham, 893, 977

Farr, 340

INDEX

Fats, see Lipoids

Faunules, distributional host relationships
and _ host-specificity in representative
symbiotic, 894, 917-29; of sea urchins,
894, 919-23; of termites and Crypto-
cercus, 894, 923-29

Fauré-Fremiet, 43, 64, 74, 75, 76, 84,
90, 112, 115, 116, 118 120, 122, 146,
148, 160, 166, 167, 175, 180, 410,
419, 437, 526, 527, 534, 649, 805,
807, 934, 1021, 1022, 1079

Fauré-Fremiet, Léon, Mayer, and Plantefol,
394

Fauré-Fremiet, Mayer, Schaeffer, 127

Favella, fibrillar system, 253

Bennie354.05 57

Fenyvessy, von, and Reiner, respiration,
359) 3625-1366; 37s) 37185 388559.
390

Ferber, 974, 975, 980, 982,
985

Ferber and Winogradowa-Fedorowa, 976
984

Ferment theory of the vacuome, 127

Fermor, 610, 654

Fertilization in Metazoa, 583 f.

Fertilization in Protozoa, 583-645; copu-
lation, 584-606; autogamy, 606-11;
zygotic meiosis, 611-14; significance,
614-17; conjugation, 617-23, 37; mac-
ronucleus during conjugation, 623 f.;
conjugant meiosis, 624-39; literature
cited, 639-45

iat, Sul, Se, 7S)

Feulgen hydrolysis, 18, 21

Fibers, elasticity, 90, contraction, 90, 92

Fibrillar systems in ciliates, 191-270; ex-
amples, 193-228; structural analysis,
193-215, 228-57; Paramecium, 193-
200, 224-28; Holotricha, 193 ff., 228-
44: Heterotricha, 200 ff., 244-51; Sten-
tor, 200-204, 218-21; Hypotricha,
204 ff., 255 ff., Euplotes, 204-8, 221-24;
Vorticella, 208-15, 216-18; functions
interpretation, 215-28, 228-57; Oligo-
tricha, 251-54; conclusions, 257 ff.;
need of future study, 261; literature
cited, 262-70

Fibroblasts, defined, 834

Findlay and Brown, 850

Fine, 532, 542

Finley, 128, 135, 136,
698

983, 984,

137, 168, 438,

INDEX

Fisher, 47

Fission, asexual reproduction in alternat-
ing binary and multiple, 569-71

Fission, cytoplasmic: attachment an im-
portant factor in, 73

Fitzpatrick, 1040, 1047

Fiveiskaja, 153, 1035, 1036, 1037

Fixation mechanisms and habits, 71-77,
930 ff., 944, 948, 949

Fixation of complement test, 873 ff.

Fixed material, nature of, 47

Flabellula, survey of functions having
granular basis, 168-74 passim

Flagella, used as organs of attachment,
74; contraction, 93 f.

Flagellate responses to electricity, 320

Flagellate responses to light, 280-95;
shock reaction and aggregation, 281;
orientation, 282-87; wave length and
stimulating efficiency, 287-90, 295; kin-
etic responses, 290; adaptation to dark,
291; to light, 292-95

Flagellates, reorganization, 24 ff.; division
apparently adequate for, 34; adhesive-
ness, 74, 930 ff.; color, 83; copulation,
586 ff.; sexuality, 666-87; biparental in-
heritance in haploids, 732-40; develop-
ment in faunules of termites and Cryp-
tocercus, 894, 923-29; relationship be-
tween them, 894, 961-73; only free-
living, on which bacteria have been
reported, 1011; of termites, Schizo-
mycetes in, 1013, 1014, 1015, 1027-32

Flather, 423

Flotation, devices aiding, 77

Foa, 1011

Focke, 1086

Foettingeriidae, 956, 959

Fol, 1090, 1092

Folger, H. T., responses to light, 274 ff.

Food, effect on rumen ciliates, 974, 975,
985

Food and feeding habits, use of cultures,
14

Food requirements and other factors in-
fluencing growth of Protozoa in pure
cultures, 475-516; of Protozoa, 476-78;
phototrophic nutrition, 477, 478-82;
heterotrophic nutrition, 477, 482-87;
trophic specialization, 487-89; specific
growth factors, or vitamins, 489-93;
growth stimulants, 493-95; growth in
cultures as a population problem, 495-

1225

97; initial population, 497-99; growth
in relation to waste products, 499 f.;
growth in relation to food concentration,
500 f.; growth in relation to pH of the
medium, 501-3; oxygen relationships,
503 f.; the redox potential, 504f.;
growth in relation to temperature,
505 f.; growth in relation to light and
darkness, 506f.; acclimatization, 507;
literature cited, 507-16; see also Nuttri-
tion

Food vacuole, permeability of, 69-71

Foraminifera, copulation, 596 ff.

Form, inherited environmental modifica-
tions in structure and, 721-23

Forrest, 208

Fortner, 93, 420, 428, 791, 1027

Fosse, 423

Foulke, 907

Fragments, behavior of: grafting and re-
incorporation, 793-97

Franca, 1019, 1028

Frederikse, 59, 84

Free-living and symbiotic Protozoa, sys-
tematically related, 902-17

Frei, 375

Fresh-water Protozoa, classification on an
ecological basis, 5

Frey-Wyssling, 97

Friedemann, 385

Frisch, 150, 168, 428

Frontonia, fibrils, 193, 225

Frosch, 463

Frye and Meleney, 822

Fuhrmann, 952

Fulton, 932

Fundamental and derived organization, 4

Fungi, cellulose decomposition, 962 f.,
967

Furgason, 458

Furssenko, 809, 8i0

Fusiformis-like rods adherent full length,
1012-15

Fusion of cells, 35

Galadjieff, 660

Galileo, 9

Galvanic current, responses to, 306-10

Gamete broods, 33; differentiation, 35 f.

Gamete nuclei, sex differences between,
701f.; significance of diversities be-
tween, and between conjugants, 703-6

1126

Gametes, kinds of differences observed in
Chlamydomonas, 667-71; morphological
differences, 667; functional differences,
667; physiological differences, 668-71;
nature of physiological differences be-
tween, in Chlamydomonas, 671-78

Gametic meiosis and fertilization,
601

Gamogamy, term, 584

Ganapati and Aiyar, 1079, 1081, 1082

Garnjobst, 780, 789

Garrod) Ea) P3535

Gas analysis, 354, 356

Gas bubbles, 77

Gases, evolution of, other than CO:, 367 f.

Gassovsky, 1085

Gastriolar reaction, term, 132

Gastriole, term, 129; digestion associated
with, 172, 178

Gatenby, 138, 139, 145

Gause, G. F., 476; growth, 528, 547, 548,
549) 5505 Salk 5555 4

Gaw, 60

Gay, 839

Geiger, Kligler, and Comaroff, 389

Geiman, 911

Geise and Taylor, 476

Geitler, 705, 1066, 1067

Gel, compared with solid, 50

Gelei, Gabor von, fibrillar system, 198

Gelet Jey vons 169) 1455 146, 47, “409;
419, 423, 439, 649; fibrillar system,
193-99, 202, 204, 206, 219, 220, 224-
28, 258

Generation, spontaneous: history and solu-
tion of problem, 8

Genetic constitution, defined, 710

Genetic materials, essential properties, 710

Genetics of the Pretozoa (Jennings), 710,
TO, FG. Tig. 72152

Georgevitch, 1030, 1044, 1073, 1074

Gerard, 378

Gerard and Hartline, 354, 358

Gerard and Hyman, 376

Gerstein, 528, 529

Ghosh, 898

Giard, 895

Gicklhorn, 903

Giersberg, 53

Giese, 45, 542, 690, 696, 698

Giese and Arkoosh, 690, 696

Gilman, 690, 695, 698, 700

Gingrich, 844, 848, 851

584-

INDEX

Gitter, diagram, 197

Gladstone et al., 539

Glaser, R. W. and Coria, N. A., cultures,
458-60, 463, 472, 478, 481, 484, 485,
486

Glaucoma (Dallasia) frontata, divisions,
31, 32, 35; fibrillar system, 233

Glaucoma pyriformis, parasitism, 895 ff.

Glocker and Reuss, 60

Glutathione, 121, 125, 373

Glycogen, formation of, 155; differenti-
ated from paraglycogen, 157; decrease
and storage, 159; deposits by rumen
ciliates, 981

Glycolysis, measurement of, 385 f.; oc-
currence of anaérobiosis and, 396-90

Génnert, 1064, 1065, 1084, 1088, 1089

Goetsch, 927

Goetsch and Scheuring, 1010

Gojdics, 1042

Goldman, 836

Goldschmidt, 586

Golgi, 847

Golgi apparatus, and contractile vacuole,
431-41, 442; general nature of, 431 ff.;
presence in all cells, 432; usual form,
441, 442; link in kinship between all
cells apparently established, 443; simi-
larity in reaction of protozoan and meta-
zoan, 443

Golgi bodies, 138-44, 145, 147; vacuome
and, different aspects of same thing,
126; structures included, 138; objec-
tive criteria used in identification of,
138 ff.; not universally self-perpetuat-
ing and permanent, 142; only truly
objective criteria, 143; universality of,
rejected, 170, 173, 175; compared with
lipoid bodies, 170; defined, 177; perm-
anence, 181

Gonder, 1071, 1072

Gonium, responses to light, 288, 303

Gordon, 520

Goroschankin, 589

Gould, 1026

Gourret and Roeser, 1023

Gradients, physiological, 802-4

Graff, 913

Grafting and reincorporation: behavior of
fragments, 793-97

Graham, 902

Granules, cytoplasmic, 111-90 (see also
under Cytoplasmic inclusions ) ; morpho-

INDEX wy

logical and functional studies of, often
separated, 112; segregation, 126, 132-
38, 168, 171, 174, 178; segregation of
neutral red by, 127; digestive, 129-32,
172, 178; excretory, 124, 144-50, 173,
178, 405, 440; carbohydrate, 154; baso-
philic, 160, 162, 163-65; metachro-
matic granules, 160, 162, 165; survey
of functions having granular basis, in
group of five Protozoa, 168-74; secre-
tion, 168, 178; number of types, 169;
failure to find general uniformity, 169,
174; comparison on basis of composi-
tion, 171; as permanent organelles and
as temporary components, 174-77; ac-
tive and passive, 175, 177; classifica-
tion, 177-79; comparison with cells of
the Metazoa, 179-81; unknown, 179;
permanence and self-perpetuation, in
Protozoa and Metazoa, 181; basal, in
Paramecium, 195, 198, 200, 204, 226,
227; relationship between contractile
vacuole and, 405 ff., 424, 435, 436,
438 f., 440; beta, 406, 435; osmiophilic,
438 f., 440

Granulocytes, 835

Grassé, 892, 893, 912, 918, 933, 1011,
1012, 1013, 1014, 1015, 1016, 1017,
1018, 1019, 1020, 1021, 1028, 1029,
1044

Grassé and Boissezon, 898

Grassi and Foa, 1028, 1046

Grave and Schmitt, 260, 343

Gray, 46, 548

Greeff, 201, 208, 213, 1025, 1026, 1041,
1042

Greeley, 54, 57, 306, 326

Green and Breazeale, 972

Greenleaf, W. E., 535

Greenway, 1045

Gregarines, gamete brood, 35; Biitschli’s
discovery re carbohydrate granules of,
111; Golgi bodies, 140

Gregory, 541, 543, 628, 629, 631, 796

Grieg, Von Rooyen, and Hendry, 876

Griffin, 23, 205, 221, 647

Griffiths, 422, 424

Gross, 589

Grosse-Allermann, 91

Growth, environmental conditions suitable
for most rapid division and, 45; oxida-
tion-reduction potential vs. respiration
and, 394-96; food requirements and

other factors influencing growth of
Protozoa in pure cultures, 475-516;
specific factors, or vitamins, 489-93;
stimulants, 493-95; in cultures as a
population problem, 495-99; in relation
to waste products, 499 f.; in relation
to food concentration, 500 f.; in relation
to pH of the medium, 501-3; oxygen
relationships, 503f.; in relation to
temperature, 505 f.; in relation to light
and darkness, 506 f.; methods for meas-
urement of, 517-20; individual Protozoa,
520-26; colonial Protozoa, 526f.;
pedigree isolation culture and_ life
cycles, 527-31; protozoan successions:
nonlaboratory, 531 f.; laboratory, 532 f.;
autocatalysis and allelocatalysis, 533-37;
nutrition and, 537-44; population, 544-
52: struggle for existence, 553 f.; litera-
ture cited, 554-64; studies of regenera-
tion and, 772

Growth factor, term, 489

Gruber, A., 78, 82, 787, 788, 789, 797,
1043, 1053, 1075, 1083

Gruber, K., 47

Gruby and Delafond, 983

Ginther, 1042

Guyer, 384

Gwynne-Vaughan and Barnes, 1047

Gymnostomes, association with host, 914

Haas, 917

Habenicht, 315

Hackett, 822,823, 825

Haeckel, 201, 208, 1092

Hammerling, 720

Hafkine, 1035, 1036, 1037

Hahnert, 61, 310, 324

Haldane, 356, 553

Haldane-Henderson, methods. of gas an-
alysis, 356

Hall) RY P. -135,. 139.9143, 6359).9360,
361, 362, 371, 407, 408, 435, 436,
437, 476, 478, 479, 481, 487, 489, 490,
494, 501, 502, 504, 507, 537, 542, 543;
Food Requirements and Other Factors
Influencing Growth of Protozoa in Pure
Cultures (Chap. TX), 475-516

Hall, R. P., and Dunihue, F. W., 130,
437

Hall, R. P., and Elliot, A. M., 484, 492,
493

Hall, R. P., and Jahn, T. L., 907

1128

HallRo PR: andeloefer: JB 31283166,
167, 482, 483, 500, 535

Hall, R. P., and Nigrelli, R. F., 115, 130,
131, 140

Hall, R. P., and Schoenborn, H. W., 477,
478, 479, 482, 490, 498, 499, 543

Hall, R. P., and his associates, 126, 136,
518

Hall, S. R., 905, 906, 907

Halsey, 530

Hamm, Ludwig, spermatozoa discovered
by, 11

Hammett, 543

Hammond, E. C., 537, 548

Hammond, J. C., 227

Hance, 426

Haploids, biparental inheritance in, 732-
40

Haptophrya, accessory bodies from, 179;
fibrillar system, 240, 242

Hardy, 47, 518

Hargitt, G. T., and Fray, W. W., cultures,
452'£., 454, 533, 537

Harrington and Leaming, 277

Hartman, E., 844, 846, 851

Hartmann, M., 587, 594, 746, 785, 798,
799, 1094; sexuality, 666-86 passim,
704, 705

Hartmann, M., and Chagas, C., 25

Hartmann, M., and Nagler, K., 593, 595,
596, 613

Hartog, 426, 1089

Harvey, E. B., 58, 71, 78, 426, 430

Harvey, E. N., 58, 60, 64, 80, 363

Harvey, E. N., and Danielli, J. F., 64

Harvey, E. N., and Marsland, D. A., 63,
80, 95

Haswell, 904

Haupt, 891

Haye, contractile vacuole, 148, 406, 407,
409, 410, 418, 419, 420

Hayes, 113, 119

Hay infusion, 15

Hazard, 950

Heat as a bactericidal agent, 464

Heavy water, effect of, 60

Hegerty, 530

Hegner, 796, 844, 891, 393, 903, 933

Hegner and Andrews, 893

Hegner and Eskridge, 846

Hegner and Hewitt, 846, 851

Heidenhain’s iron-alum-hematoxylin, 419

Heidenreich, 944, 945, 946, 947, 948, 949,
952

INDEX

Heilbrunn, L. V., 309, 344; protoplasm,
tg att Sly SV, De D5 DG, SH, SS), Ga
71, 79, 80, 152; quoted, 111

Heilbrunn, L. V., and Daugherty, K., 53,
54, 60, 153, 319

Heilbrunn, L. V., and Mazia, D., 60

Helly-Maximow’s Zenker formol, 832

Hematochrome, 83

Hemicellulose, ophryoscolecin
as, 157

Hemixis and autogamy, 654 ff.

Hemocytoblasts, 835, 836

Hendee, 967

Henderson, J. C., 927, 963

Henderson, V. E., 59

Henry, Dora P., 569

Henry, X., 876

Hentschel, 921

Herbivorous mammals, ciliates in, 973 f.

Herfs, A., 427, 430

Herrick and Cross, 855

Hertig, M., Taliaferro,
Schwartz, B., 891, 894

Hertwig, O., 892

Hertwig, R., 160, 566, 598, 626, 649, 701,
795, 1083, 1089, 1090, 1091, 1092

Hesse, 1040

Heteroautotrophic nutrition, 477, 482 f.

Heterogamy, term, 584

Heteromesotrophic nutrition, 477, 483

Heterometatrophic nutrition, 447, 483-87

Heterophils, 835

Heterotricha, fibrillar systems, 244-51; see
also Stentor

Heterotrophic nutrition, 477, 482-87

Hetherington, 450, 461, 467, 528, 540,
541, 774, 775, 895, 896, 1039

Hewitt, J. H., 792

Hewitt, L. F., 391, 392, 396

Hewitt, R., 846, 851

Heyningen, van, 376

Higgins, H. T., 249

Hill, John, 11

Hindle, 163

Hinshaw, 569

Hinshaw, McNeil, and Kofoid, 912, 913

H-ion concentration, relation between rate
of locomotion and, 334, 335, 336 ff.,
341

Hirschler, J., 411, 437, 440; cytoplasmic
inclusions, 114, 118, 138, 139, 140, 142,
143, 145

Historical facts, protozoa, 8-14

Hoelling, 1012

identified

W.) H., “and

INDEX

Hofer, 787

Hofker, 1069

Holdaway, 965

Hollande, 909, 1071

Holmes, E., 121, 300

Holmes) S: Ji, 372, 781

Hologamy, term, 584

Holotricha, fibrillar systems, 228-44; free-
living and symbiotic, 913-17; adapta-
tion, 933 ff.; see also Paramecium

Holter, 384

Holter and Doyle, 118, 120, 124, 168, 384

Holter and Kopac, 118, 120, 124, 125,
168

Hooke, Robert, 9

Hope-Gill, 876

Hopkins, D. L., 71, 116, 124, 125, 127,
129, 139, 168, 180, 273, 334, 336, 340,
429

Hopkins, D. L., and Mast, 124

lsloysarnres I. S51 TS}, aUil7, ili), ae,
175, 176

Horning, E. S., and Scott, D. H., 137

Horton, 793

Horvath, 199, 206

Hosoi, 802

Host, variability in strains and in host
response, 823-26; study of defense
mechanisms of, 830 ff.; relationships be-
tween certain Protozoa and, 890-1008
(see also under Relationships)

Host-specificity, term, 894; holotrich
groups, 913 ff.; and distributional host
relationships in representative symbiotic
faunules, 917-29; approach from stand-
point of individual species, 918

Hovasse, 1010, 1064, 1069

Howland, 49, 51, 52, 62, 67, 68, 70, 89,
92, 406, 407, 418, 422, 425, 794, 795,
905, 1068

Howland and Bernstein, 354, 355, 357,
362

Howland and Pollack, 52, 62, 65, 68, 425

Hsiung, 973, 1084, 1085

Hu and Cash, 854

Hudson and Gosse, 906, 1093

Huff and Bloom, 843

Hulpieu, 341

Humoral and cellular aspects of immunity,
839-41

Hungate, 965, 966, 967, 969, 970, 971

Hunninen and Wichterman, 911

Huntington and Winslow, 539

Hutner, 482, 489

1129

Huxley, 525

Huxley and Teissier, 525

Hyaline layer, 49

Hyaloplasm, 50

Hydrogen acceptors, experiments which
concern the cytochrome-cytochrome oxi-
dase system of, 376-80; which concern
other systems, 380 f.

Hydrogen-ion concentration, 58 f.; effect
on growth, 542, table, 540 f.

Hydrostatic pressure effect on consistency,
61; within cell, regulation of, 422,
426 ff., 442

Hyman, 49, 53, 72, 84, 89, 91, 94, 429,
802, 803

Hypocomidae, 933; adaptation, 938-42

Hypotricha, characteristics, 205; fibrillar
systems, 255-57; see also Euplotes

Hypotrichida, reorganization in, 24

Ichthyophthirius, survey of functions hav-
ing granular basis, 168-76 passim; fibril-
lar system, 241

Ikeda and Ozaki, 638, 937

Illumination, responses to, see Light

Imhof tank sewage, 6

Immaturity and maturity, sexual, 714 f.

Immaturity and partial maturity, 761

Immunity and acclimatization, inherited,
717-21

Immunology of the parasitic Protozoa,
830-89; physical bases of immunity,
830-42; cells involved in immunity, 831-
37; antibodies and antigens involved,
837-39, 873; cellular and humoral as-
pects, 839-41; role of immune processes
in the development of protozoan infec-
tions, 842-64; malaria, 843-54; kala
azar, 854 f.; infection with . . . trypano-
somes, 855-62; continuous fatal trypano-
somiasis, 862-64; intermittent fatal try-
panosomiasis, 864-71; practical applica-
tions of immune reactions, 871-76; arti-
ficial immunization, 872; reactions used
in diagnosis, 872-76; reactions in rela-
tions to classification, 876-78; literature
cited, 878-89

Impregnation, Golgi bodies’ relationship
to, 143

Indirekt verbindung System, 194, 195, 199,
226

Infections, from coprozoic forms, 6; rdle
of immune processes in development of
protozoan, 842-64

1130

Inflammation, 836; defense reactions seen
during, 839

Infraciliature, 200, 228, 257

Infusionsthierchen als vollkommene Or-
ganismen, Die (Ehrenberg), 12

Infusoria, term; Miiller’s studies, 11; Eh-
renberg-Dujardin controversy reorgani-
zation of, 192, 260

Inheritance, 710-71; types of reproduction
and, 171; in uniparental reproduction,
711-13; in biparental reproduction, 711,

changes, 714f.; degeneration changes
resulting from unfavorable conditions,
716f.; acclimatization and immunity,
717-21; environmental modifications in
form and structure, 721-23; variation
and, occurring without obvious action
of diverse environments, 723-29; bi-
parental, in haploids: flagellata, 732-40;
sex and sex-linked, 740-50; biparental,
in diploids: ciliata, 750-58; of mating
type in Paramecium aurelia, 753-58; in
Paramecium bursaria, 758-61; effect of
the cytoplasm and its relation to nuclear
constitution, 762-69; literature cited,
769-71

Injury, degree of reorganization and, 781-
84

Inman, Bovie, and Barr, 277

Inquilines, defined, 892; Protozoa not cap-
able of harboring, 1009

Intermediate lipoid body, term, 151, 178

Internal parasites, see Parasites

International Rules of Zoélogical Nomen-
clature, 953

Irreversible coagulation, 61

Isogamy, term, 584

Issel, 936, 937, 939, 1021

Ivani¢, 635, 649, 1047, 1048, 1049, 1052,
1053, 1078

Jacobs, 55, 56

Jacobson, Irene, fibrillar system, 198, 223,
225, 258, 259

Jaffé, 839

Jahn, Theodore Louis, 364, 365, 369, 387,
393, 395, 1042, 1048, 1058; Respira-
tory Metabolism (Chap. VI), 352-403;
food, etc., influencing growth, 481, 484,

INDEX

487, 498, 502, 503, 504, 505, 506, 507;
growth, 528, 534, 536, 538, 539, 540,
547, 548, 549, 552

Jahn, T. L., and McKibben, W. R., 907,
1068

James, 846

Jameson, 576, 586, 604, 612

Janda and Jirovec, 896

Janicki, 26, 27, 1011, 1019, 1046

Jarocki, 940, 942, 943, 944

Jarocki and Raabe, 938, 940

Jay, 362, 370, 377

Jenninesy Ee 'S3 74225) 5852052 Ih 22.
525, 588, 606, 615, 617, 636, 714, 724,
V3, VIS W295 Ws Wa, Wes 1933
physical properties of protoplasm, 72,
75, 76, 83, 88, 91, 92; motor responses,
Dil, 282) 297, 321, 322) 3246 3265542-
sexuality, 689, 690, 691, 692, 694, 695,
696, 698, 700, 701, 704; Inheritance
in Protozoa (Chap. XV), 710-71;
Genetics of the Protozoa, 710, 712, 716,
Flgee72l, 152

Jennings, H. S., and Jamieson, C., 793

Jennings, H. S., and Lashley, K. S., 689

Jenseny Wie s793

Jirovec, 154, 156, 1030, 1044, 1045

Jochims, 95

Johns Hopkins University stock R_ of
Paramecium, 615

Johnson, C. M., and Kelser, R. A., 874

Johnson, D. F., 468, 476, 484, 486, 501,
502, 507, 538

Johnson, H. P., fibrillar systems, 201-4
passim, 219, 258

Johnson, L. P., 81, 83

Johnson, P. L., 592

Johnson, T. L., 868

Johnson, W. H., 471, 476, 498, 528, 536,
540, 541, 542, 548, 551

Johnson, W. H., and Hardin, 499, 536

Jollos, V., 660, 698; inheritance, 718, 719,
720, 721, 723, 730, 768

Jones, E: P., 541,548, 552

Joness Pay Mie 92

Joschida, 79

Joyet-Lavergne, P., cytoplasmic inclusions,
114, 117, 121, 122, 1125, 140, 4ommise
155, 156, 161, 162, 166, 169, 172, 175,
180

Jucci, 963

Jurgens, 855

Jungeblut, 839

INDEX

Kahl, 75, 900, 914, 916, 917, 920, 921,
922, 934, 935, 936, 940, 945, 1022,
1023 1024, 1038

Kala azar, 854 f., 874, 875

Kalmus, 97, 355, 357, 362, 366, 1087

Kamada, 323, 324

Kanda, 79

Kanthack, e¢ al., 855

Katharobic type, 5

Katzin and Kirby, 967

Kauders, 850

Kavanagh and Richards, 534

Kazancev, 163

Kedrowsky, B., cytoplasmic inclusions,
1229126, 127, 128, 129, 1325 133.104,
135, 136, 137, 139, 143, 151, 153, 168,
169, 171, 173, 174, 175, 180

Keilin, 897, 898

Keilin and Hartree, 379

Kempner, 361

Kent, 422, 1041

Kepner, W. A., and Carroll, R. P., 952

Kepner, W. A., and Carter, J. S., 905

Kepner, W. A., and Edwards, J. G., 91

Kepner, W. A., and Reynolds, B. D., 794

Kepner, W. A., and Taliaferro, W. H., 70

Kepner, W. A., and Whitlock, C., 91

Keppen, N. A., see Koeppen, N.

Kessel, 1045

Khainsky, 413

Khawkine, 907

Kidder, George W., 21, 22, 33, 536, 620,
627, 628, 635, 636, 637, 638, 647, 649,
650, 653, 658, 930, 934, 935, 939, 963;
fibrillar system 228, 230, 232, 234;
Technique and Significance of Control
in Protozoan Culture (Chap. VIII),
448-74

Kidder, George W., and Claff, C. L., 647

Kidder, George W., and Dewey, V. C.,
469

Kidder, George W., and Diller, W. F.,
647

Kidder, George W., and Stuart, C. A.,
450, 454, 469, 470, 533, 537

Kidder, George W., and Summers, F. M.,
915

Kidder, George W., Lilly, D. M., and
Claff, C. L., 453, 469

Kimball, R. F., 660, 756, 767; sexuality,
670, 690, 696, 697, 698, 700, 701

Kinetic responses to light, 277-79, 290,
2955, 299

Ltt

King, R. L., contractile vacuole, 68, 69,
408, 409, 410, 419, 420, 438, 439

King, R. L., and Beams, H. W., 67, 68,
70, 71, 81, 88, 98; Some Physical Prop-
erties of the Protoplasm of the Proto-
zoa (Chap. II), 43-110

Kinosita, 323

Kirby, Harold, Jr., 116, 126, 136, 154,
157, 248, 893, 909, 923, 924, 927, 928,
929, 930, 963, 965, 968, 969, 970,
HOM, WOU, WO, WOW “ilolss aWOiles.
1017, 1018, 1019, 1020, 1022, 1023,
1029, 1030, 1031, 1032, 1039, 1040,
1044, 1045, 1046, 1048, 1053, 1054,
1055, 1056, 1959, 1060, 1062, 1079;
Relationships between Certain Protozoa
and Other Animals (Chap. XIX), 890-
1008; Organisms Living on and in Pro-
tozoa (Chap. XX), 1009-1113

Kirkman and Severinghaus, 126, 138, 180,
181

Kitching, J. A., 68, 69, 429

Kite, 51, 52, 62, 67

Kiyono, 836

Klebs, 909, 1041

Klee, 649

Klein, B. M., fibrillar system, 194-200,
206, 224-28

Kligler, Geiger, and Comaroff, 389

Kluyver, 396

Kniep, 684, 705

Knoth, 976

Knowles and Das Gupta, 847, 848, 864,
865

Kober and Graves, 519

Koch, A., 963, 964

Koch, R., 848

Koehler, 324, 325, 326

Koehring, 127, 130, 134

K6lliker, 201, 208

Konig, 936, 937, 941

Koeppen, N. [Keppen, N. A.], 1070,
1089, 1090, 1091

Kofoid, Charles A., 198, 206, 594, 936,
937, 974; Life Cycle of the Protozoa
(Chap. XI), 565-82

Kofoid, Charles A., and Bush, M., 126,
619, 636, 940

Kofoid, Charles A., and Christenson, J. F.,
79

Kofoid, Charles A., and Johnstone, H. G.,
463

1132

Kofoid, Charles A., and MacLennan, R.
F., 973, 979; fiibrillar system, 252

Kofoid, Charles A., and Swezy, O., 25,
572, 930, 1044

Koidzumi, 924, 930, 1015, 1018

Kolatchev method, 435

Kolkwitz, classification of fresh-water
species, 5

Kolmer ef al., 870

Koltzoff, 93, 258, 259

Korotneff, 1066, 1089, 1090

Korr, 381, 395

Korschelt, 203

Koser and Saunders, 492, 494, 537

Kostitzin, 553

Kotlan, 912

Krascheninnikow, 145

Krijgsman, 151, 159, 162, 163, 166, 843,
863, 864

Kroé, 857, 877

Kriger, 1079

Kuczynski, 1044

Kudo, 608, 956, 957, 1078

Kiihne, 208, 217, 305, 315, 323, 332

Kuhn, 871

Kupffer cells, 833, 850”, 851”

Labbé, 907

Lachmann, 208, 213, 413,
1064, 1086

Lackey, 6, 83, 387, 532, 1010

Lamborn, 897

Landis, 636, 702, 791

Langmuir, 64

Lankester, 413

Laurens and Hooker, 303

Lauterborn, 5, 1011, 1021

Laveran, 854, 870, 877

Laveran and Mesnil, 856, 861, 866, 868

Lavier, 908, 910, 912, 933, 1046, 1047,
1054, 1056, 1057, 1058

Lavier and Galliard, 912

Lawrie, 484, 503

Lebedew, 591, 593

Le Breton, 127

Le Calvez, 594, 597, 598

Lechriopyla mastax, fibrillar system, 243

Ledermiiller, Infusoria termed by, 11

Leeuwenhoek, Anton von, 8, 9, 11, 12,
208; contributions to microscopic an-
atomy and to physiology, 9; description
of a protozoén, 10 f.; regarded as the
“Father of Protozodlogy and Bacteriol-

1034, 1039,

INDEX

ogy,” 11; his “little animals” essential
nature remained long obscure, 191

Léger, 603, 604

Leger, A., and Ringenbach, J., 868, 877

Léger, L., and Duboscq, O., 604, 1045,
1068, 1078

Leichsenring, 355, 365, 366, 367

Leidy, 1021, 1025, 1043, 1065,
1067, 1094

Leiner, 155, 158, 1025, 1026, 1027

Leishmaniasis, 854 f., 874, 877

Leontjew, 79

Levaditi and Mutermilch, 868, 870, 877

Lewin, 778, 789, 791, 792

Lewis, 90

Leydig, 210, 906

Lichtenstein, 916, 917

Lieberkiihn, 201, 208, 219, 1034, 1039

Liebmann, 1038

Liesche, 592

Life and vitality, 3 f., 34

Life cycle of the Metazoa, 565-67, 581

Life cycle of the Protozoa, 565-82; con-
ception of, as a characteristic of every
species, 14; pedigree isolation culture
and, 527-31; asexual reproduction in
alternating binary and multiple fission,
569-71; alternation of asexual and sex-
ual reproduction, 571-73; Eimeria schu-
bergi, 573-78; Plasmodium vivax, 577,
578; Paramecium caudatum, 578-81;
literature cited, 581 f.; mating types in
relation to the Maupasian Theory,
699 f.; malarial parasites, 847; adap-
tive host relationships in morphology
and, 929-60

Light, responses to, 272-305; rhizopods,
272-80; flagellates, 280-95; ciliates,
295-97; colonial organisms, 297-305;
growth in relation to darkness and,
506 f.

Light, S. F., 1018, 1019, 1044

Lillie, F. R., 785, 786, 787

Lillie, R. S., 324

Lilly, D. M., control of cultures, 469,
ATlite

Linderstrom-Lang, K., 358

Linear aggregates in protoplasm, 87

Linkage and crossing over, 737-40, 743-
45

Linton, 839, 854

Lipoids, defined, 118”; in mitochondria,
118, 123; interpretation of vacuolar

1066,

INDEX

system as due to concentration of, 148;
reserves, 150-53, 173; boundary be-
tween reserve and active, 151; inter-
mediate lipoid bodies, 151, 178; visible,
152; compared with Golgi bodies, 170

Lison, 118”, 119, 139

Lister, 9, 596

Literature cited, 39-42, 98-110, 182-90,
262-70, 344-51, 397-403, 443-47, 473 f.,
507-16, 554-64, 581 f., 639-45, 662-65,
706-9, 769-71, 811-17, 827-29, 878-89,
987-1008, 1095-1113

“Little animals” of Leeuwenhoek, 10, 191

Littoral cells, 833

Liver cells, plasmosin from, 87

Lloyd, 67, 422

Lloyd and Scarth, 411

Locomotion, relation between rate of, and
H-ion concentration, 334, 335, 336 ff.,
341

Loeb, J., muscle-tonus, or tropism, theory,
282

Loeb, J., and Budgett, S. P., 51, 324, 332

Loeb, J., and Maxwell, S. S., 289

koeb, 1.74

Hoefer, J.B... 525, 538, 540, 5425 543);
food, etc., influencing growth, 476, 477,
478, 480, 481, 482, 484, 485, 486, 487,
495, 501, 502, 507

Loefer, J. B., and Hall, R. P., 481

Longevity, factors influencing, 16-18,
38 f.; division processes inadequate to
account for, 28; of ciliate’s protoplasm,
34

Loofbourow and Dyer, 519

Loomis, 58

Looper, 74, 788, 796

Lophomonas, reorganization in, 26, 27

Lorando and Sotiriades, 850

Lotka, 553

Lourie, 834, 844

Lourie and O’Connor, 869

Lowe, 847

Lucas, K., 324; fibrillar system, 247

Lucas, M. S., 900, 922

Luce, 61, 306

Luck and Sheets, 464

Ludford, 138

Ludloff, 324, 325, 326, 331

Ludwig, W., 77, 78, 425, 793

Lund, E. E., 968, 970, 971; fibrillar sys-
tem, 198 f., 224, 255

Lund, E. J., 84, 86, 774, 775, 782, 792,

LY33

801; respiratory metabolism, 355, 362,
363, 365, 376

Lund, E. J., and Logan, G. A., 71, 78, 332

Luntz, 305, 800

Lutz and Splendore, 1078

Luyet and Gehenio, 58, 67

Lwoff, A., 74, 476, 482, 504, 896, 898,
899, 1044, 1045, 1047, 1048, 1051,
1053, 1058; respiratory metabolism,
360, 362, 366, 367, 371, 377, 378, 379,
382, 383, 384, 388, 390; food, etc., in-
fluencing growth, 477, 479, 480, 483,
484, 485, 486, 491, 493, 503

Lwoff, A., and Dusi, H., food, etc., in-
fluencing growth, 477, 482, 483, 484,
487, 490, 491, 543

Lwoff, A., and Lederer, E., 482, 493

Lwoff, A., and Lwoff, M., 492

Lwoff, A., and Provasoli, L., 483, 485,

Lwoff, A., and Roukhelman, Nadia, 425,
484, 485, 499

Lwoff, M., respiratory metabolism 362,
377, 378, 379, 380, 381, 383, 388; food,
etc., influencing growth, 477, 478, 484,
485, 491, 492, 493

Lwoff, M., and Lwoff, A., 478, 484, 487

Lymph, cells, 834 f.

Lymphocytes, 835, 836

Lymphoid cells, 835 f.

Lymphoid-macrophage system, 835, 837

Lynch, J. E., 113, 114, 119, 126, 136,
145, 752, 788, 919, 921, 923; fibrillar
system, 236, 239, 243

Lynch, J. E., and Noble, A. E., 1088

Lyon; 79; 332

Lysin and opsonin, 858, 861

Mac Arthur, 898

McCay, 544

McClendon, 80, 309, 332

McClung, 384

McCoy, 361

McDonald, fibrillar system, 244

MacDougall, M. S., 27, 542, 622, 623,
630, 631, 632, 633, 634, 635, 637, 638,
647; fibrillar system, 229, 231

McFarland, 891

Mackinnon, D. L., 96, 911, 1033

Mackinnon, D. L., and Vleés, F., 84, 96, 97

Mackinnon, D. L., and Ray, H. N., 1079,
1081

McLay, 850

1134

MacLennan, Ronald F., 113, 114, 115,
LEZ WO, 122 125, 126aeOmIo e132.
135, 136, 137, 139, 140, 141, 142, 143,
145, 146, 147, 148, 149, 150, 151, 154,
155, 156, 157, 163, 164, 167, 169, 174,
175, 180, 525, 981; Cytoplasmic Inclu-
sions (Chap. III), 111-90; fibrillar sys-
tem, 241; contractile vacuole, 409, 411,
421, 424, 436, 439, 443

MacLennan, Ronald F., and Connell, F.
H., 936, 937; fibrillar system, 238, 240

MacLennan Ronald F., and Murer, H. K.,
1198 1205013 1a 749590

Mac Neal, 856, 861

Mc Pherson, Smith, and Banta, 537

Macronucleus, a derived organ, 16; me-
rotomy experiment, 16 ff.; changes with
metabolism, 18 ff.; during reorganiza-
tion, 21 ff., 31 ff., 646-48; during con-
jugation, 623 f.

Macrophages, 835, 836; defined, 833; cells
classified under, 833 f.; structure, 834;
phagocytosis by, during malaria, with
plates, 849 ff.; system valuable or dele-
terious, 854; Le/shmania in, 854

Madsen, 899, 921

Magenthiere (Polygastrica), 12

Maier, 201, 202, 219, 221

Mainx, 481, 684, 705, 1042

Makarov, 59

Malaria, pathogenicity, 822 f.; immunol-
ogy, 843-54; phagocytosis, 849 ff.;
serological tests, 874, 876, 878

Mangenot, 1039

Mangold, 974, 975, 977, 982, 983, 984,
985

Mangold and Radeff, 977

Mangold and Schmitt-Krahmer, 984

Mangold and Usuelli, 975, 977

Mann-Kopsch material, 435

Manometer, standard methods, 354, 356 f.;
micromanometer, 355, 358; capillary,
357 £.

Mansour, 963

Mansour and Mansour-Bek, 961, 962, 963,
965

Manteufel, 861

Manusardi, 985

Manwell, 627, 649, 848, 878

Manwell and Goldstein, 823, 848

Marchand, 833, 836

Margolin, 976

Marrack, 838

INDEX

Marsh, 363

Marsland, 59, 61, 74

Marsland and Brown, 273

Marston, 127

Martin, 1085

Massaglia, 863, 868

Massart, 78

MastauSs Ose 1525 5406n 4185529 Ole
physical properties of protoplasm, 48,
AD. 51, 61. 68, 69, 70.72, 13, 070n184s
85, 90, 91; Motor Response in Unicellu-
lar Animals (Chap. V), 271-351; ort-
entation in Euglena, 283 ff.; conclusions
on responses of Volvox, 297 ff., 327 ff.

Mast, S. O., and Doyle, W. L., 36, 47, 65,
71, 80, 92, 406, 407, 436; cytoplasmic
inclusions, 113-80 passim

Mast, S. O., and Fowler, C., 65

Mast, S. O., and Gover, Mary, 287, 290

Mast, S. O., and Hahnert, W. F., 71

Mast, S. O., and Hawk, Brainard, 291,
292

Mast, S. D., and Hulpieu, H. R., 277

Mast, S. O., and Johnson, P. L., 287, 288,
302

Mast, S. O., and Nadler, J. E., reversal in
ciliary action, 322, 326, 342 ff.

Mast, S. O., and Pace, D. M., 367, 479,
482, 483, 488, 498, 499, 500, 528, 535,
540, 543

Mast, S. O., and Prosser, C. L.,.277, 336,
339, 340

Mast, S. O., and Root, F. M., 91, 788

Mast, S. O., and Stahler, N., 278, 279

Mast, S. O., Pace, D. M., and Mast, L.
Re HO, BAO, BIOS a7!

Mastigina, 85

Mastigophora, free-living and symbiotic,
902-13; Schizomycetes on, 1010-24:
Schizomycetes in, 1030-32

Mattes, 1041, 1042, 1043, 1048, 1050,
1051, 1052, 1053, 1054, 1056, 1057,
1058, 1064

Mattick et al., 520

Matubayasi, 912

Maturity and immaturity, sexual, 714 f.;
partial, 761

Maupas, E., 28, 29, 413, 528, 537, 714,
900, 922, 946, 1087; fibrillar system,
192, 198, 205, 208, 216, 221; fer-
tilization, 605, 614, 618, 621, 624, 626,
634, 636; sexuality, 690, 696, 699, 700,
701

INDEX

Maupasian life cycle, mating types in rela-
tion to, 699 f.

Maximow, 832, 833, 836, 837, 839

May, 928

Mayer, 1044

Mayer’s hemalum, 419

Measurement of growth, methods, 517-20

Mechanical agitation, effect on consistency,
47, 58

Mechanical support, fibrils, 194 ff., 258

Meiosis, gametic, 584-601; zygotic, 611-
14; conjugant, 624-39 (see also under
Conjugant meiosis )

Meldrum, 372

Meleney, 854

Meleney and Frye, pathogenicity, 819, 820,
822, 825, 874

Membranes, cell, 62, 64-66, 69; nuclear,
66 f.; presence or absence of, surround-
ing contractile vacuoles, 67, 413-21,
441; division into two types, 414;
physiological, 414 ff.; morphological, or
permanent, 414, 418, 419, 421

Menendez, 878

Menon ef al., 875

Mercier, 1033, 1046, 1054, 1056, 1057

Mercier and Poisson, 895, 900

Merton, 88, 219, 342

Mesenchymal cells, 833; rdle in inflam-
mation, 836

Mesnil, 1080

Mesnil and Brimont, 870

Mesosaprobic type, 5

Messiatzev, 631

Mestre, 519

Metabolic influence, fibrillar complex, 258

Metabolic waste products, 422

Metabolism, activity and changes with, 18-
21, 35; see also Anaérobic, Basal, and
Respiratory, metabolism

Metachromatic granules, 160, 162, 165

Metallic impregnation methods, in iden-
tification of Golgi bodies, 138

Metalnikov, 653

Metazoa, comparison between metazoan
and protozoan cells, 44, 179-81; Golgi
bodies in cells, 140, 143; differences
between protozoan and metazoan or-
ganization, 191, 260; life cycles of Pro-
tozoa and, 565 ff., 581; analogies in sex
phenomena between Protozoa and,
583 f., 600; as parasites in Protozoa,
1010, 1093-95

135

Metcalf, M. M., 33, 69, 115, 405, 406,
409, 435, 591, 893

Metschnikoff, E., 208, 836, 1035, 1036,
1086

Metopus
247

Meyen, 12, 413

Meyer, A., 219

Meyer, S. L., 950

Meyerhof, quotient, 386, 389

Meyers, E., 160, 161

Meyers Ea Gen 35

Microdissection apparatus in
structure, 52

“Micrographica’”” (Hooke), 9

Micromanometer, 355; Cartesian diver
ultramicromanometer, 358

Micronucleus during reorganization, 28,
29535) fk

Microphages, 836

Microscope, discovery and development of,
5)

Migration, sterilization by, 455-60; com-
bined with dilution, 460-63

Miller, 118, 705

Mills, 540

Milojevic, 604

Milovidov, 161

Minchin, 583, 787

Minden, 1040, 1047, 1052, 1063; quoted,
1057

Minkiewicz, 620

Minnesota, University of, study of Ameba

proteus, 592

Mitchell, 539, 1042, 1048, 1049, 1050,
HOST O52 LOS Sal ONS

Mitochondria, 112-26; identification of,
112, 116; shape, 113; distribution, 115;
supposed universality and permanence
of, 116, 125, 432; composition, 118;
cellular respiration, 121, 122; functions
ascribed to, 122 ff.; not a homogeneous
group, 126; morphological relationship
between paraglycogen and, 155; chro-
midia associated with, 160, 161; uni-
versality of, rejected, 170, 173, 174;
function of carbohydrate storage accom-
plished by, 173; defined, 177; perma-
nence, 181; terms for, 432; relationship
to Golgi apparatus, 432, 437

Mitotic mechanism, 44

Miyashita, Y., 620, 621, 944, 957, 1025

Mizuno, F., 521, 522, 526

circumlabens, fibrillar system,

study of

1136

Mjassnikowa, 942

Moewus, F., sexuality, 666-83, 705; cri-
tique of works of, on Chlamydomonas,
684-87; inheritance, 710, 722, 723, 732,
733, 734, 736, 738, 739, 740, 742, 743,
744, 745, 746, 747, 748, 749, 750

Mohler, Eichhorn, and Buck, 874

Molina, 912

Molisch, 1074

Mond, J., 538, 544, 551

Monkeys, malarial infection and immu-
nity, with plates, 845, 846-54

Monocystis, 601 f.

Monocytes, 835

Monod, 803

Monoecious races, 742

Montalenti, 965, 966, 970, 971

Moody, 791, 796

Moore, A. R., 95, 332

Moore, E. L., 649, 777, 779, 780, 782,
785, 788, 790, 911, 912; quoted, 787

Moore, Imogene, 410, 420, 438

Morea, 540, 541

Morgan, de, 935

Morgan, T. H., 704, 785, 786, 797

Morita and Chambers, 52, 67, 69

Moroff, 912

Morphogenesis, Protozoa in connection
with problems of, 772-817; physiologi-
cal regeneration, 773 f.; some factors
in regeneration, 774-93; behavior of
fragments: grafting and reincorporation,
793-97; regeneration and division, 797-
801; polarity changes and protoplasmic
streaming, 801 f.; physiological gradi-
ents, 802-4; regeneration in colonial
forms, 804-11; literature cited, 811-17

Morphological membranes, 414, 418, 419,
421

Morphology, adaptive host relationships
in life history and, 929-60

Motor organelles, composite:
into their components, 76

Motor response in unicellular animals,
271-351; to light, 272-305; to elec-
tricity, 305-32; to chemicals, 333-44;
literature cited, 344-51

Mottram, 543

Molds, cellulose decomposition, 967

Mowry and Becker, 974, 975, 976, 983,
985

Mueller, 494

Miller, J., 1034, 1039

separation

INDEX

Miller, O. F., studies by, 11, 43; classi-
fication, 11; observation of conjugation,
oboe}

Miller, R. H., 519

Mulligan, 843, 846

Mulligan and Sinton, 848

Mulsow, 601, 602, 611, 689

Munich school of protozodlogists, 566

Muscle, stalk, 208, 216

Muscle-tonus theory, Loeb’s, 282

Mutualism, inclusion in term symbiosis,
891; defined, 892; physiological host
relationships illustrative of commensal-
ism and, 961-87

Myeloid cells, 835 f.

Myers, 594, 597, 1095

Myonemes, 94; of Stentor, 201 ff., 218 ff.,
258, 261

Nadler, 62, 83, 88, 775

Nagler, 27, 1032, 1042, 1049

Nahm, 181

Napier, 875

Narcotics, effects of, 59; responses of
Stentor to, 220

Nassonov, D., contractile vacuole and
Golgi apparatus, 69, 138, 142, 144, 145,
146, 147, 148, 410, 411, 419, 420, 431,
433-35, 438, 439, 443

Nassonov-Bowen theory re Golgi bodies
and secretion, 144

Mauck and Malamos, 848

Naville, fertilization, 576, 602, 607, 608,
609, 611

Necheles, 362

Needham, Joseph, 9, 90, 372, 390, 525

Needham, Joseph, and Boell, E. J., 354,
358

Needham, Joseph, and Needham, D. M.,
51, 52, 62, 396

Nelson, 586, 621, 635

Nematode worms, parasitism, 1094

Neporojny and Yakimoff, 870

Neresheimer, E. R., 33; fibrillar system,
201-4 passim, 218, 219, 220, 225, 591,
1089, 1093

Nernst, 324

Neumann, 850

Neuschloss, 720

Neutral red, segregation of, by cytoplasmic
granules, 127; granules, not identical
with Golgi bodies, 141, 142; in
Amoeba, 170; granule, defined, 178

INDEX

New International Encyclopedia, 891

Nie, 919, 920

Nielsen, 520

Nieschulz, 906

Nieschulz and Bos, 866, 870

Nieschulz and Wawo-Roentoe, 870

Nigrelli, 116, 134, 135, 136

Nigrelli and Hall, 435

Nirenstein, 428

Nitrogen, source of, 972; ciliate, 984

Noble, 576

Noller, 1044, 1045, 1046, 1047

Noller and Buttgereit, 912

Noguchi, 877

Noland, 531, 591, 621, 623, 629, 637, 688

Novy, 371

Novy, Roehm, and Soule, 356

Nowakowski, 1065

Nowikoff, 423

Nuclear and cytoplasmic division proc-
esses, coGrdination between, 85 f.

Nuclear membrane, permeability, 66 f.

Nuclear purification, 21 ff.

Nuclear reorganization processes, 21 ff.,
35 ff.

Nuclei, chromidial origin doubted, 594;
two kinds in ciliates, 687; gamete: dif-
ferentiation, 701, 703; effect of cyto-
plasm and its relation to, 762-69; in
regeneration, 787-93; of Trichonympha,
parasitization, 1059-63

Nucleophaga, and Spaerita, 1040-59; his-
torical account and distribution, 1040-
47; in free-living Protozoa, 1040, 1043;
in endozoic Protozoa, 1046 f.; life his-
tory and structure, 1053-57; effect on
host, 1058 f.

Nussbaum, 787

Nutrition, and growth, 537-44 (see also
Food) ; effect on resistance, 826 f.; of
wood-eating animals, 961-73; effect
upon rumen ciliates, 974, 975, 985

Nyctotherus, fibrillar system, 248-50

Obligate parasites, 895

Oehler, 458, 463, 465, 469, 475
Ogata, 455

Okada, 795

Oligochaeta, host of Astomata, 946
Oligosaprobic type, 5

Oligotricha, fibrillar systems, 251-54
Oligotrichida, sexuality, 688
Oliphant, K., 54, 57, 322, 326, 344

ELS7

Odgamy, term, 584

Opalina, segregation bodies, 132-35; sur-
vey of functions having granular basis,
168-75 passim

Opalinopsidae, 956, 960

Ophryocystis mesnili, 603, 604

Ophryoscolecidae in ruminants, 973, 977-
82 passim

Ophryoscolecin, 157

Oppenheimer, 966

Opsonin and lysin, 858, 861

Optical properties of protoplasm, 82-84

Organic structure, disclosure of cellularity
dependent upon analysis of, 191, 260

Organisms living on and in Protozoa,
1009-1113 (see entries under Parasites
of Protozoa)

Organization, derived and fundamental, 4

Orientation, in light, 279, 282-87, 295,
300-303; electricity, 328

Oshima, 965

Osmic acid methods, in identification of
Golgi bodies, 139, 140

Osmiophilic structures, 144-50, 438 f.,
440

Osmotic pressure within cell, regulation
of, 426 ff.

Osterud, 478, 479, 480, 483

Ostwald, 314

Owen, 13

Owens and Bensley, 139

Oxidase, 373; detection of, 384

Oxidation-reduction potential vs. respira-
tion and growth, 394-96

Oxygen, effect on anaérobes, 390-94; on
a€robes, 394; relationships, 503 f.; ef-
fect upon growth, 503, 538; Oxytricha,
fibrillar system, 255

Pacinotti, 388

Packard, 60

lexvotatioh, (Gs 184 Jay, SA, Sh, 5G, SOs OWS
amoeboid response, 273, 315, 341

Paraglycogen, formation of, 155; morpho-
logical relationship between mitochon-

dria and, 155; differentiated from
glycogen, 157; decrease and _ storage,
159

Paramecium, physical properties of proto-
plasm, 51, 55, 57, 60-82 passim, 86, 88,
91, 97; fiibrillar systems, 193-200, 224-
28, 257 ff.; responses to electricity, 321-
27 passim; to chemicals, 342-44; life

1138

Paramecium (continued)
cycle, 580f.; fertilization, 588, 610,
615, 616, 617, 626, 636; sexuality in
other ciliates and, 687-706; heritable
effects of conjugation, 762-69; regen-
eration, 774 ff., 793, 798, 799, 802

Paramecium aurelia, Yale 33 year old cul-
ture, 615, 653, 654, 659, 660, 661;
Johns Hopkins stock R, 615; autogamy,
654 ff.; inheritance of mating type in,
753-58; see also Paramecium

Paramecium bursaria, sex reactions, 615;
inheritance of mating type in, 758-61

Paramecium caudatum, life cycle, 578-81;
see also Paramecium

Parasites, parasitic and nonparasitic Pro-
tozoa, 6, 892; ectoparasites, 7; endo-
parasites, 7; pathogenic aspects, 818-29;
three functional categories, 818; ma-
larial species, 822f., with plates,
843 ff.; literature cited, 827-29, 878-89;
immunology, 830-89; physical bases of
immunity, 830-42; rdle of immune
processes in the development of proto-
zoan infections, 843-64; agent of kala
azar, 854f.; trypanosome group, 855-
71; intermittent fatal trypanosomiasis
in various laboratory animals, 864-71;
practical applications of immune reac-
tions, 871-76; immunological reactions
in relation to classification, 876-78;
parasitism defined, 890; inclusion in
term symbiosis, 891, 892; accidental and
facultative parasitism, 894, 895-902

Parasites of Protozoa, 1009-1113; epi-
biotic Schizomycetes, 1010-24; endo-
biotic Schizomycetes, 1024-40; Sphaerita
and Nucleophaga, 1040-59; parasites of
the nucleus of Trichonympha, 1059-63;
Phycomycetes other than Sphaerita and
Nucleophaga, 1063-68; Protozoa, 1068-
89; the genus Amoebophrya Koeppen,
1089-93; Metazoa, 1093-95; literature
cited, 1095-1113

Parat, 138, 148, 180

Park, 547, 548

Parker, G. H., fibrillar system, 258

Parker, R. C., 660

Parker, I. J., 891

Parnas, 423

Parpart, A. K., sterility technique, 453 f.

Parsons, 72, 74

Parthenogenesis, diploid, 649

INDEX

Pascher, 93, 590, 613, 671, 679, 1010

Pasteur, Louis, 8, 537

Pathogenicity, certain aspects of, 818-29;
problems of virulence and, 818-23; va-
riability in strains and in host response,
823-26; nutrition and resistance, 826 f.;
literature cited, 827-29; see also Para-
sites

Patten, M., 791

Patten, R., 119

Patten, R., and Beams, H. W., 81

Paulson and Andrews, 874

Pearl, R., 320, 324, 530, 544, 547, 548

Pedigreed series, nuclear behavior in, 654

Pedigree isolation culture and life cycles,
527-31

Peebles, F., 51, 67, 86

Peebles, F., morphogenesis, 774, 775, 777,
Wks WHIDS TO, ses WL, how

Pekarek, 51, 52

Pellicle, elasticity, 87

Pellissier, 120, 1021, 1022

Pelomyxa. Schizomycetes in, 1025-27

Penard, E., 73, 76, 91, 92, 93, 97; Proto-
zoa and other animals, 898, 899, 914,
915, 916, 957; organisms on and in
Protozoa, 1011, 1025, 1026, 1038, 1041,
1042, 1043, 1050, 1052, 1053, 1054,
1057, 1066, 1067, 1068, 1075, 1083,
1094

Peranema tricophorum, responses to light,
290-95

Perekropoff and Stepanoff, 912

Pericytes, cells, 833, 836

Peritrichida, sexuality,
Vorticella

Perla and Marmorstom-Gottesman, 860

Permeability, surface properties, 64-71;
membranes, cells, 64-66, 69; nuclear,
66 f.; vacuoles, contractile, 67-69; food,
69-71; other types, 71

Peroxidase, detection of, 384

Peroxidases, 373

Perty, 413

Peschowsky, 223

Peshpowskaya, 120

Reskett.\Ga ls 53>

Peters, A. W., 529, 542, 543

Peters, J. P., and van Slyke, D. D., 356,
385

Peters, R. A., 362, 377

Petersen, W. A., 498, 535

688; see also

INDEX

Petri-dish method of sterilization by mi-
gration, 457

Petschenko, 1035, 1036, 1037

Pfeiffer, 71, 79, 84, 95

Phagocytosis, relation of adhesion to, 74;
in malarial infections, wth plates,
849 ff.; in infection with trypanosomes,
860

Phelps, A., 495, 497, 498, 501, 504, 528,
529, 536, 541, 542, 548, 550

Phelps, L. A., 785, 788, 795, 798

Philip and Haldane, 687, 750

Phillips, Ro L.; 537

Philpott, 476

Photoautotrophic nutrition, 477, 478-80

Photomesotropic nutrition, 477, 480 f.

Photometatrophic nutrition, 477, 481 f.

Phototrophic nutrition, 477, 478-82

Phycomycetes other than Sphaerita and
Nucleophaga, 1063-68

Physical properties of the protoplasm, 43-
110 (see entries under Protoplasm)

Physiological gradients, 802-4

Physiological membranes, 414 ff.

Physiological regeneration, 773 f.

Phytomastigophora parasites in Protozoa,
1068-70

Pickard, E. A., 937; fibrillar system, 228,
229, 258

Pierantoni, 972, 1028, 1029, 1030

Pigment granules defined, 179

Pijper and Russell, 847

Pinching, ascribed to centripetal pressure,
oT

Pinto and Fonseca, 1044, 1050

IHRE, I, 1 SOA, S/T, KKO

Plant and ciliate conjugation compared,
617

Plasmagel, 47; see also Ectoplasm

Plasmalemma, 47; physical characteristics,
49

Plasmasol, recent name for endoplasm, 47

Plasmodia, infectious, 842; rate of repro-
duction, 842 f.

Plasmodium, malarial parasite, 822 f., with
plates, 843 ff.

Plasmodium vivax, life cycle, 577, 578;
pathogenicity, 822 ff.

Plasmosin, 87

Plate, 939, 1083, 1093

Platelets, 835

PSD

Plating vs. turbidity test, 467

Platt, 79

Playfair, 905

Plimmer, 912

Poisons, effect on O2 consumption, 366

Poisson, 899

Polarity, of protoplasm, 84; physical-
chemical factors involving change in,
85; changes and protoplasmic stream-
ing, 801 f.

Poljanskij, J. L., 952

Poljansky, G., 114, 115, 154, 631, 636,
eS)

Poljansky, G., and Strelkow, A., 986

Pollack, 59

Polygastrica (or Magenthiere), 12

Polysaprobic type, 5

Polytoma, inheritance, 743 f., 746, 747

Popoti Me SS. 52s 52255235 52405255
787

Population, growth in cultures as a prob-
lem of, 495-97; the initial, 497-99;
growth, 544-52

Postgastriole, 129

Poultry, coccidiosis in, 824-26

Powell, 1030

Powers, P. B. A., 919, 920, 921, 922, 923,
1022, 1024; fibrillar system, 236, 237

Poyarkoff, 953

Prandtl, 619, 631, 634, 702, 1042, 1053

Pratje, 588

Pringsheim, E., 590, 1010, 1064

Pringsheim, E. G., food, etc., influencing
growth, 475, 476, 477, 478, 480, 482,
483, 488, 493, 494

Pringsheim, E. G. and Ondracek, K., 671,
679, 687

Pringsheim, H., 961

Pronuclei, formation, 632-34; migration,
634 f.

Proske and Watson, 876

Protein molecules, results of multipolar
character, 86

Proteins, in mitochondria, 118; reserves,
160-66, 171, 174; nomenclature com-
plicated, 160, 165; terms that should be
dropped or restricted, 165; provision of,
by rumen ciliates, 983, 984 f.

Protoplasm, conditions under which ani-
mation maintained, 34; physical proper-
ties, 43-110; Dujardin’s description of,
43; physical properties as exhibited in
Amoeba, 46-50; colloidal nature, 49 f.,

1140

Protoplasm (continued)
51; consistency, 50-61; surface proper-
ties, 61-77; specific gravity or density,
77-82; optical properties, 82-84; struc-
tural properties, 84-98; literature cited,
98-110

Protoporphyrin, 382

Protozoa, discovery of, and other histori-
cal facts, 8-14; importance in solving
question of spontaneous generation and
other problems, 8; Leeuwenhoek’s de-
scription, 10f.; classification, nomen-
clature, 11; differences between proto-
zoan and metazoan organization, 191,
260; relationships between certain
Protozoa and other animals, 890-1008
(see also under Relationships) ; para-
sitic and nonparasitic, 892; ways of
benefiting larger animals, 893; free-
living and symbiotic, systematically re-
lated, 902-17; organisms living on and
in, 1009-1113 (see entries under Para-
sites of Protozoa); parasitizing other
Protozoa, 1068-89; literature cited, see
Literature

Protozoélogy, indebtedness to microscope,
9; Leeuwenhoek the father of, 11

Provasoli, 481, 484, 486, 495

Prowazek, S., 636, 787; fibrillar system,
205: 221-222

Pruthi, 540, 541, 542

Pseudopodia, contractile, 91, 92

Ptychostomidae, family, species, genera,
943; adaptation, 944 f.

Ptychostomum chattoni, fibrillar system,
244

Pitter, 388, 394

Purdy and Butterfield, 458

Puymaly, 1042, 1051, 1053, 1058

Quastel, J. H., 396
Quastel, J. H., and Stephenson, M., 392

Raabe, 934, 935, 938, 939, 940, 941, 942,
943

Rabinowitsch and Kempner, 856

Racial differences, and regeneration, 780 f.

Radiation, effects on consistency, 60; used
for sterilization, 466

Raffel, 752

Rammelmeyer, 154

Rankin, 950

Rapkine, 64

INDEX

Ray, Harendranath, fibrillar system, 245

Ray, J., 282

“Ray-direction theory,” Sachs’s, 282

Raymond, 1071

Reconstruction bands, 23

Red, see Neutral red

Redi, 8; quoted 262

Redmond, 848

Redox potential, 504 f.

Rees, C. W., 343; fibrillar system,
225.1226. 2272521258

Refraction, double, 95-97

Refractive bodies, 170

Refractive index, 83

Regaud, 177

Regendanz, P., 860

Regendanz, P., and Kikuth, W., 856, 860,
861

Regeneration, of macronucleus, 16 ff.;
physiological, 773 f.; factors in, 774-93;
and division, 797-801; in colonial
forms, 804-11; see also Morphogenesis ;
Reproduction

Rehberg, 907

Reich, 484, 486, 498, 500, 501, 535, 538,
549

Reichenow, 161, 162, 163, 164, 165, 179,
253, 638, 974, 982, 983, 1075, 1082

Reidmuller 365, 379, 380, 384

Reincorporation and grafting: behavior of
fragments, 793-97

Reiner, 385

Reiner and Smythe, 389

Reiner, Smythe and Pedlow, 389

Relationships between certain Protozoa
and other animals, 890-1008; symbiosis
the comprehensive term for general rela-
tionship, 891; externally mutualistic,
termed commensalism, 891; protozoan
benefits to larger animals, 893; principal
discussions and articles on, 893; acci-
dental and facultative parasitism, 894,
895-902; systematically related free-
living and symbiotic Protozoa, 902-17;
distributional host relationships and
host-specificity in representative sym-
biotic faunules, 917-29; adaptive host
relationships in morphology and _ life
history, 929-60; physiological host rela-
tionships illustrative of mutualism and
commensalism, 961-87; literature cited,
987-1008

Remane, 906

198,

INDEX

Renaut, 836

Reorganization, through cell division, an
influence in longevity, 16; by endo-
mixis, 31-36; of the macronucleus and
other derived structures in Ciliata, 21-
31; by conjugation, 36-39; degree of
injury and, 781-84

Reorganization bands, 23

Reproduction, asexual and sexual, 566 f.,
571-73; metazoan life cycle, 566;
sexual, in all animals and plants? 568;
asexual, in alternating binary and mul-
tiple fission, 569-71; types of inherit-
ance and, 711; measurement of rate,
842; rate, of rumen ciliates, 976; see
also Regeneration

Reproduction, biparental defined, 711; in-
heritance in, 732-50

Reproduction, uniparental: defined, 711;
inheritance in, 711-13; changes in in-
heritance characters in, 713-31

Reserve bodies, secretion of, 122; defined,
178

Resistance, effect of nutrition on, 826 f.

Respiration, concern of mitochondria with,
121, 122, 125; purposes of studying,
353 f.; measurements, table, 362; in-
vestigations which concern mechanism
of, 372-84

Respiration, aérobic, 354-68; methods of
measuring, 354-58; normal rate, 358-
61; effect on Oz consumption, of O2
tension, 361-64; of COz tension, 364 f.;
of the physiological state, 365; of tem-
perature, 366; of anesthetics and poi-
sons, 366; of nutritive substances and
other materials, 367; evolution of gases
other than COn, 367 f.

Respiratory metabolism, 352-403; pur-
poses of studying respiration, 353 f.;
methods of measuring aérobic respira-
tion, 354-58; aérobic respiration, 358-
68; investigations which concern the
source of energy, 368-72; investigations
which concern the mechanism of respi-
ration, 372-84; measurement of an-
aérobic metabolism and __ glycolysis,
385 f.; occurrence of anaérobiosis and
glycolysis, 386-90; why are anaérobes
anaérobes, and aérobes aérobes?, 390-
94; oxidation-reduction potential vs.
respiration and growth, 394-96; litera-
ture cited, 397-403

1141

Respirometers, 355; sensitivity, tables, 354

Reticular cells, 833

Reticulo-endothelial system, 836

Rey, 125

Reynolds, B. D., 721, 901

Reynolds, M. E., morphogenesis, 777, 778,
780, 784, 791, 792, 797, 798

Reznikoff, 52

Reznikoff and Chambers, 53, 55

Rhizopods, responses to light, 272-80
(shock-reactions, 273-77, kinetic re-
sponses, 277-79, orientation, 279); re-
sponses to electricity, 305-20 (direct
current, 306-10, alternating current, 310-
14, mechanics of response, 314-20) ;
responses to chemicals, 333-41 (rate of
locomotion and H-ion concentration,
334, 335, 336 ff., 341, mechanics of re-
sponse, 338-41)

Rhumbler, 72, 413

Richards, Oscar W., 519, 525, 529, 530,
532, 535, 536, 542, 547, 552; Growth
of the Protozoa (Chap. X), 517-64

Richards, Oscar W., and Dawson, J. A.,
D285 929

Richards, Oscar W., and Jahn, T. L., 519

Richards, Oscar W., and Kavanagh, A. K.,
547

Riddle and Torrey, 423

Rieckenberg, 869, 875

Rieckenberg blood platelet test, 869, 875

Rieder, 1072, 1073

Rigidity, 87; see also Elasticity

Ripper, 961, 962

Ritz, 869

Roach Cryptocercus, Protozoa of termites
and, 894, 923-29; relationship between
flagellates of termites and, 894, 961-73

Robbie, Boell, and Bodine, 376

Robbins, 340, 341

Robertson, M., 530, 843, 865, 870, 1046,
1057, 1074

Robertson, T. B., 497, 499, 533, 534, 535,
536, 548

Robinson, 877

Rodet and Vallet, 868

Rosel, 787

Root, W. S., 356, 364, 370

Ropiness (or thread formation), 94 f.

Roscoff, 899

Rose, 960

Rosenberg, L. E., 167, 908; fibrillar sys-
tem, 248, 249

1142

Rosenhof, Résel von, 43

Roskin, G., 92, 93, 96, 1046, 1074; fibril-
lar system, 202, 204

Roskin, G., and Levinsohn, L., 384

Ross, 846

Ross and Lotka, 554

Ross and Thompson, 864

Rossolimo, L., 916, 943, 944, 947

Rossolimo, L., and Jakimowitch, K., 21 f.

Rossolimo, L., L., and Perzewa, T. A., 948

Rotifers, parasitism, 1093 f.

Rottier, 501, 504, 528, 538

Roudabush and Becker, 857

Roudsky, 861

Rouget, C., 208

Rouget, J., 868

Roux, 531, 914

Rudolf and Ramsey, 847

Ruminants, ciliates of, 894, 973-87 (see
entries under Ciliates of ruminants)

Rumjantzew, 154, 158, 161

Rumjantzew and Suntzowa, 96

Rumjantzew and Wermel, 158, 165

Runyan and Torrey, 804

Russeff, 963, 870

Russell, 519

Ryckeghen, 911

Ryder, 1041

Sachs, “‘ray-direction theory,’ 282

Salts, effect of, on consistency, 53-55, 61;

' motor responses to, 333 ff., 342 ff.; rate
of Amoeba's locomotion in sodium and
calcium salt solutions, 337, table, 338

Sand, 1067, 1087, 1089

Sandon, 6

Sanford, 929

Sappinia (Amoeba) diploidea, 593, 595,
596

“Sapropelic fauna,” 5

“Sarcode,’ 12, 43, 192

Sarcodina, division processes, 28; Schizo-
mycetes on, 1021; Schizomycetes in,
1032-34; parasites in Protozoa, 1074-78

Sassuchin, 153, 1033, 1039, 1044, 1045,
1046, 1047, 1049, 1050, 1051, 1052,
1054, 1058, 1059

Sassuchin, Popoff, Kudrjewzew, and
Bogenko, 1045, 1047, 1050

Satina and Blakeslee, 705

Sauerbeck, 870

Saunders, 75, 541

INDEX

Scarth and Lloyd, 412

Schaeffer, 49, 66, 70, 71, 72, 73, 82, 83,
86, 90, 91, 279

Schaudinn, Fritz, 14, 566, 574, 575, 577,
596, 598, 604

Schellack, 604, 605

Schereschewsky, 1079, 1082

Scheunert, 985

Schewiakoff, 78, 94, 208

Schieblich, 982

Schilling, 868

Schilling and Neumann, 877

Schirch, 163

Schizomycetes, endobiotic, 1024-40; asso-
ciations of a constant character, 1025-
30; in Pelomyxa, 1025-27; in flagellates
of termites, 1027-30; associations of
an occasional character, 1030-40; in
Mastigophora, 1030-32; in Sarcodina,
1032-34; in Ciliophora and Sporozoa,
1034-40

Schizomycetes, epibiotic, 1010-24; on
Mastigophora, 1010-24; on Sarcodina,
1021; on Ciliophora, 1021-24

Schlayer, 362

Schleiden, 9

Schmalhausen
522, 526

Schmidt, 90, 92, 96, 97, 413, 912

Schmitt, 64, 354, 357

Schmitt, Bear, and Clark, 97

Schneider, 623

Schoenborn, 477, 482, 483, 490, 502

Schorger, 961

Schouteden, 921

Schréder, O., 7, 608, 1065; fibrillar sys-
tem, 201, 202, 203, 204, 219, 220

Schuberg, A., fibrillar system, 193, 194,
195, 198, 201-4 passim 219, 224

Schubotz, 1094

Schultz, 73, 89, 92, 94, 96

SchulzewE Es ole 1025

Schulze, P., 157, 981, 982

Schwammerdam, 10

Schwann, 9

Schwartz, V., 789, 791, 796

Schwartz, W., 891, 892, 933, 963

Schwarz, C., 984, 985

Schwetz, 863

Scott, 586

Scott and Horning, 119

Sea urchins, ciliates of, 894, 919-23

and Synagajewska, 521,

INDEX

Secretion, of reserve bodies, 122; Golgi
bodies’ relationship to, 143; external,
166-68

Secretion granules, formation comparable
to segregation granules, 168; defined,
178

Segregation granules, 132-38; term va-
cuome substituted for, 126; morphologi-
cal variations, 133; composition, 134;
formation comparable to secretion
granules, 168; universality of, rejected,
171, 174; defined, 178

Seton 4O, Sil, Ss rh Sik, SO) OS

Selection, results of long-continued, 726-
29

Self-fertilization
654-59

Serbinow, 1041, 1051, 1052, 1053, 1065

Sergent, Ed., 848

Sergent, Ed., and Sergent, Et., 844

Serological reactions, specific, 873 ff.; non-
specific, 875 f.

Serotherapy against trypanosomes, 867 f.

Severtzoff, 463

Sex, an inherent characteristic of organ-
isms? 568; analogies in metazoan and
protozoan phenomena, 583 f.; sex, and
sex-linked, inheritance, 740-50

Sexuality in unicellular organisms, 666-
709; Chlamydomonas, 666-87; Para-
mecium and other ciliates, 687-706; lit-
erature cited, 706-9

Sexual reproduction, in all animals and

(autogamy), 606-11,

plants? 568; alternation of asexual
with, 566 f., 571-73
Seyd, 803

Shapiro, 519, 520

Sharp, R. G., fibrillar system, 206, 251,
2525258

Sharp, R. H., 585

Shettles, L. B., responses to light, 292 ff.

Shock reaction to light, 273-77, 281, 295,
298

Shoup and Boykin, 376

Shumway, 423

Siebold, Th. von, 413; established the
phylum Protozoa, 11; asserted unicel-
lularity of Protozoa, 13, 191

Siegmund, 833

Silverline system, 194, 227

Simic, 820

Simpson, 520

1143

Singh, 81

Sinton et al., 847, 848

Sinton and Mulligan, 846

Size, factor in regeneration, 784-87

Skvortzow, 1042, 1053, 1065

Slater, 387

Smith, 528, 539

Snell, 534

Soil-dwelling Protozoa, ecological consid-
erations, 6

Soil extracts, 493

Sokoloff, B., 774, 775, 782, 785, 786

Sokoloff, D., 903, 1068

Sonneborn, T. M., 588, 611, 615, 616,
654, 660, 661, 677, 690, 693, 696, 699,
700, 701, 704, 714, 752, 753, 754, 755,
756, 757, 758; Sexuality in Unicellular
Organisms (Chap. XIV), 666-709

Sonneborn, T. M., and Cohen, B. M., 615

Sonneborn, T. M., and Lynch, R. S., 697,
766

Sotiriades, 850

Soules 3565 371

Spallanzani, Lazaro, 8, 404

Spasmoneme, 94

Specht, 360, 362, 363, 369, 370, 425

Specific gravity (or density), whole or-
ganisms, 77-79; relative, of cell inclu-
sions and components, 80-82

Specificity in symbiosis, 894

Spek, 54, 82

Spencer, 653

Spermatozoa, discovery, classification, 11

Sphaerita, and Nucleophaga, 1040-59;
historical account and_ distribution,
1040-47; in free-living Protozoa, 1040-
43; in endozoic Protozoa, 1043-46; life
history and structure, 1047-53; effect
on host, 1057 f.

Sphaeromyxa sabrazesi, life cycle, 608 f.

Sphenophryidae, 933; adaptation, 941,
942 f.

Spindle fibers, 90

Spirochetes and rods adherent by one end,
1015-21

Spirostomum ambiguum, fibrillar system,
250, 251

Splenectomy and blockade, 832

Spontaneous generation, history and solu-
tion of problem, 8

Sporozoa, division processes, 28; survey
of functions having granular basis, 168-

1144

Sporozoa (continued)
74 passim; fertilization, 601-6; Schizo-

mycetes in, 1034-40; parasites in
Protozoa, 1078-82

Spurr, 530

Stabler, 1076

Stabler and Chen, 1045, 1075, 1076,
1077

Staining reactions, difficulties with respect
to, in both Protozoa and Metazoa, 180

Stalk, vorticellid, 208 ff., 216 ff., 257,
258, 259

Starch, in wood, 961; digestion by wood-
eating animals, 971; digestion by rumi-
nants, 979 ff.

Statkewitsch, 322, 323, 326, 331

Stauber, 846

Steffan, 855

Stein and Schmidt, 422

Stein, F. von, 213, 218, 413, 637, 646,
907, 914, 934, 943, 1034, 1035, 1039,
1041, 1042, 1043, 1052, 1063, 1064,
1086, 1087; view of conjugation, 13

Steinhaus and Birkeland, 552

Stelluti, Francesco, 9

Stem cells, 835

Stempell, 413, 1078

Stentor, responses to light, 295-97

Stentor fibrillar systems, structural anal-
ysis, 200-204; interpretation, 218-21;
conclusions, 257 ff.

Stephenson, 375

Sterilization, problem of, 449-67; general
material, 449f.; general methods,
450 f.; special methods and manipula-
tions, 451-67; importance of adequate
tests, 467; establishment of sterilized
Protozoa in culture, 468-73

Stern, 384

Steuer, 930

Stevens, 223, 789, 932, 936, 937

Stickiness, see Adhesiveness

Stier, Newton, and Sprince, 519

Stimulants, growth, 493-95

Stimulating efficiency of light, 287-90,
PASN5), PMS 10)

Stockman and Wragg, 878

Stokes, 32, 33, 422, 1094

Stolé, 788

Stone, W. S., and Reynolds, F. H. K.,
455 ff.

Stout, 704

Strains and host response, variability in,
823-26

INDEX

Strand, 934

StranghGner, 649, 650, 652

Strasburger, 289, 412

Stratman-Thomas, 849

Streaming, protoplasmic:
changes, 801 f.

Strelkow, 154, 156, 157, 411

Strelkow, Poljansky, and Issakowa-Keo,
O77 529719.

Structural properties, 84-98; colloids,
49 f.; origin of surface properties, struc-
ture and, 62-64; polarity, 84; elasticity,
87-90; contractility, 90-94; ropiness or
thread formation, 94 f.; double refrac-
tion, 95-97; X-ray diffraction and ultra-
centrifugation, 97 f.; analysis of fibrillar
systems, 193-215; inherited environ-
mental modifications in form and,
721-23 modifications of, in animals
that live in association with hosts,
929 ff.

Struggle for existence, 553 f.

Stuart, Kidder, and Griffin, 467

Stubblefield, 1079, 1080, 1081, 1082

Studitsky, A. N., 270, 944, 1025, 1111;
fibrillar system, 244

Stiitzgitter System, 194, 196, 199, 258

Stump, 73

Subdioecious races, 741 f.

Subramaniam and Ganapati,
142, 143, 145, 175

Subramaniam and Gopala-Aiyar, 143

Suctoria, ectozoic, 1084 f.; endozoic, 1085-
89

Sudanophil material, 1187; as
material, 151; see also Lipoids

Sugars, in wood, 961

Sulfhydryl group, 121

Summers, Francis M., 22, 23, 646, 647,
805, 806, 808, 810; Protozoa in connec-
tion with Morphogenetic Problems
(Chap. XVI), 772-817

Summers, Francis M., and Kidder, G. W.,
623, 624, 952

Surface membrane, 62 ff.

Surface precipitation reaction, 48

Surface properties of protoplasm, 61-77;
structure and origin, 62-64; permeabil-
ity, 64-71; adhesiveness (or stickiness),
71-77, 930 ff., 944, 948, 949

Survival of the fittest, 553 f.

Sutherland, 1015, 1018, 1021

Svec, 914

Swarczewsky, 594, 1074

and__ polarity

13m Ane

reserve

INDEX

Sweet, 528, 536

Swellengrebel, 162

Swezy, 968

Sydney, 905

Symbiosis, 818, 1009; terms of designat-
ing relationship between Prctozoa and
their hosts, 890 (see also under Rela-
tionships) ; defined, 891 f.; main cate-
gories, 891; host-specificity, 894

Symbiotic and free-living Protozoa, sys-
tematically related, 902-17

Synophrya, 958, 959

Szent-Gyorgyi and Banga, 381

Takagi, 1039

Taliaferro, L. G., 843, 844, 846

Taliaferro, William H., 717, 838, 839,
840, 845, 852, 855, 856, 857, 859, 860,
861, 863, 866, 872, 873; Immunology

of the Parasitic Protozoa (Chap.
XVIII), 830-89
Taliaferro, William H., and Cannon,

P. R., 849, 850, 851”, 853

Taliaferro, William H., and Huff, C. G.,
870

Taliaferro, William H.,
T. L., 867, 868, 870

Taliaferro, William H., and Mulligan,
H. W., 837, 839, 847, 849, 852n, 853

Taliaferro, William H., and Pavlinova, Y.,
843, 857

Taliaferro, William H., and Taliaferro,
L. G., 75, 843, 846, 847, 848, 850, 855,
862, 864, 865, 875

Taliaferro, William H., Cannon, P. R.,
and Goodloe, S., 860

Taliaferro, William H., Johnson, T. L.,
and Cannon, P. R., 863

Tang, 361

Tannreuther, 627, 632

Tartar, V., 781; quoted, 777

Tartar, V., and Chen, T. T.,-715

Taylor, C. V., 147, 206, 319, 343, 783,
784; physical properties of protoplasm,
ile 2; (625 167, 68, ‘87, 905" Fibrillar
Systems in Ciliates (Chap. IV), 191-
270; contractile vacuole, 408, 409, 413,
416, 420, 438

Taylor, C. V., and Farber, W. P., 783,
790, 795

Taylor, C. V., and Strickland, A. S. R.,
lez

Taylor, H. S., Swingle, W. W., Eyring,
H., and Frost, A. A., 60

and Johnson,

1145

Tchakhotine, 60

Teissier, 525, 534

Temperature, resistance of Protozoa to
high, 55; effect on consistency, 55-58;
on O: consumption, 366; on growth,
505 f., 539

Tennent, Gardiner, and Smith, 139, 180,
181

Tension, effect on O, consumption, 361-
65

Termites, Protozoa of roach Cryptocercus
and, 894, 923-29; relationship between
flagellates of Cryptocercus and, 894,
961-73; classification, 923; Schizomy-
cetes in, 1012 ff., 1027-32

Terry, 327

Testacea, budding division, 28; adhesive-
ness, 73

Tetrault and Weis, 966

Theiler, 878

Theiler and Farber, 902

Thélohan, 153, 1078

Thiamine, or aneurin (vitamin B), 490

Thiel, van, 162

Thigmotricha, relation to family Ptychos-
tomidae, 943; adaptation, 933-43

Thomson, J. A., 891

Thomson, J. A., and Geddes, P., 891

Thomson, J. G., 848, 864, 902

Thon, 791

Thornton, 53, 56

Thread formation (ropiness), 94 f.

Thunberg, 356

Thunberg-Winterstein principle, 357

Tintinnopsis nucula, fibrillar system, 254

Tippett, 518

Tissue, connective: cells involved in im-
munity, 831-37

Titration methods, 354, 355 f.

Tittler, 649, 784, 791

Topley, 838

Trager, 927, 965, 969, 971

Transparency of protoplasm, 82

Transport concept, 174

Treillard and Lwoff, 898

Trensz, 876

Trichomonas augusta, life cycle, 569, 570

Trichonympha, parasites of the nucleus of
1059-63

Trier, 975, 979, 981, 982

Trophic specializations, 487-89

“Tropism theory,” Loeb’s, 282

Trypanolysins, involved in
against trypanosomes, 861

immunity

1146

Trypanosoma, segregation granules, 134,
136

Trypanosomes, glycolysis rate, 388 ff.;
rate of reproduction, 842; nonlethal in-
fection with the Trypanosoma lewisi
group of, 855-62; nonpathogenicity,
862; best-known pathogenic, 862; im-
munological reactions, 874, 877

Trypanosomiasis, tests, 75; continuous
fatal in mouse and sometimes in rat,
862-64; intermittent fatal, in various
animals, 864-71

Tschaschin, 836

Turner, John P., 24, 471, 619, 620, 621,
626, 628, 633, 634, 637, 647; fibrillar
system, 206-8, 222, 223; Fertilization in
Protozoa (Chap. XII), 583-645

Tuzet, 141

Tyzzer, 1046

Uhlenhuth, 180

Ullmann, 961, 966, 971, 981

Ultracentrifugation and X-ray diffraction,
97 f.

Ultracentrifuge, research with, 81

Ultra-violet radiation, 60

Unger, 533

Uniparental reproduction, see Reproduc-
tion, uniparental

Unknown granules, defined, 179

Uric acid, in contractile vacuole, 422 ff.

Uroleptus halseyi, preparation for divi-
sion, 18, 21; fibrillar system, 256, 257;
division, 637

Uroleptus mobilis, preparation for divi-
sion, 19, 20; vitality, 29-31; conjuga-
tion, 31, 32, 34-38; fertilization, 615,
616, 630, 632, 633

Uronychia, merotomy and
NG fie 75 th 783 he

Usuelli, 979, 980, 982, 984

Uylmura, 919, 921, 934

regeneration,

Vacuolar reaction, term, 132, 176

Vacuolation upon transfer to fresh water,
78

Vacuole, term gastriole substituted for,
129

Vacuoles, accessory (vesicles): relation to
contractile vacuole, 412, 416, 424, 439,
441; contribution of osmiophilic gran-
ules to formation of, 439

Vacuoles, contractile, 404-47; effects of

INDEX

fresh and salt water, 66; permeability,
67-69; membrane surrounding? 67;
color, 83; osmiophilic structures, 144-
50; function, 173, 421-31, 442; origin,
405-13, 441, 442f.; whether perma-
nent or temporary, 410, 419, 441; rela-
tion of accessory vacuoles to, 412, 416,
424, 439, 441; structure, 413-21, 441;
regulation of hydrostatic pressure, 422,
426 ff., 442; and Golgi apparatus, 431-
41, 442; generalizations re processes
associated with, 441-43; outstanding
features, 443; literature cited, 443-47

Vacuoles, food: permeability of, 69-71

Vacuoles, other types: in the cytoplasm,
Wil

Vacuome, hypothesis, 126-29; term sub-
stituted for segregation granule, 126;
ferment theory, 127; digestive granules,
131; a universal cell constituent, 170;
universality of, rejected, 175; present
in all cells? 432; identical with Golgi
apparatus? 432, 436

Valentin, 96

Valkanov, 602, 613, 906

Van Beneden, 892

Van den Branden, 870

Variation and its inheritance occurring
without obvious action of diverse en-
vironments, 723-29

Vaucel and Hoang-Tich-Try, 876

Veley, 1026, 1027

Vernes, Bricq, and Yvonne, 876

Verworn, M., 74, 86, 93, 787, 788, 793;
motor responses, 282, 305, 320, 324,
326, 332

Vesicles, see Vacuoles, accessory

Villian and Dupoux, 876

Virulence, problems of pathogenicity and,
818-23

Viscosity, studies of changes in, 51 ff.

Visscher, J. P., 343, 620; 621, 624;
fibrillar system, 235

Vitality, and life, 3f., 34; waning, in
ciliates, 28 ff.; underlying cause of
waning vitality and death of proto-
plasm in isolation cultures, 39; effect
of conjugation upon, 616

Vitamins, A in the mitochondria, 121,
125; protoporphyrin, 383; specific
growth factors, 489-93; B:, 490 ff., Bz,
492: C, 492; ciliate capacity of syn-
thesizing B, 985

INDEX

Voegtlin and Chalkley, 54

Voigt, 906

Volkonsky, 113, 115, 125, 126, 127, 129,
130, 131, 132, 134, 135, 148, 175, 176,
179

Volterra, 553

Volutin, 160, 161, 162f.; a term with
no standard usage, 163; term should
be dropped or restricted, 165

Volvox, structure, 297, 298; responses to
light, 288, 297-305; responses to elec-
tricity, 327-32

Vorticella, body divisions, 212, 218; fibril-
lar systems, structural analysis, 208-15;
interpretation, 216-18; conclusions,
257 f

Wachendorff, 362, 365, 366, 369

Wagener and Koch, 877

Wager, 285, 1065

Walker, E. L., 463, 464

Walker, E. L., and Sellards, A. W., 824

Walker, H. H., 539

Wallace and Wormall, 875

Wallengren, 24, 941

Wallich, 1040, 1042

Wallin, 118

Wang, 531

Warburg, O., respiratory metabolism, 353,
354, 356, 373, 374, 375, 390

Warburg-Keilin system, 380

Warren, 900, 901

Wasielewski, T. K. W. N. von, 848

Wasielewski, T. K. W. N. von, and Senn,
G, 856

Wassermann test, 874

Waste products, and growth, 499 f.

Water, taking up of, by colloids, 50;
effects on consistency, 53; rate of ex-
change, 65; contractile vacuole regula-
tion of pressure within cell, 426 ff.

Water, heavy, 60

Water fleas, 10

Watson, C. J., 854

Watson, E. A., 874

Watson, M. E., 932

Wave length and stimulating efficiency of
light, 287-90, 295, 297, 303

Weatherby, J, EH. 148,150, 152, 423,
425; Contzactile Vacuole (Chap. VII),
404-47

Weber, 344

Weber and Weber, 319

1147

Weier, 126

Weineck, E., 975, 979, 981, 982

Weineck, I., 157

Weismann, 28

Wells, 838, 876

Wenrich, 93, 410, 419, 893, 903, 904,
SWE, Oilil, Oil, Siler, Gilét ily, Gili.
1044, 1045, 1058

Wenyon, 820, 878, 896, 898, 1033, 1044,
1045

Wenyon and Broughton-Alcock, 910

Wermel, 119, 123, 155, 156

Werner, 962

Wertheim, 973, 974

Weschenfelder, 604, 613, 614

Wesenberg-Lund, C., 1093, 1094

Wesenberg-Lund, Mrs. E., 899

Westphal, 975, 976, 980, 981, 982, 985

Wetzel, 544, 1077

Whole organisms, specific gravity, 77-79

Wipfchterman, 621, 635, 637
iedemann, 962, 963, 972

Willis, 788

Wilson, 160, 162, 825

Wilson and Pollister, 181

Winkler method, 355, 363

Winogradowa, T., 1046, 1049

Winogradow, Winogradowa - Fedorowa,
and Wereninow, 986

Winogradowa-Fedorowa
doff, 974

Winterstein, 59, 356

Wood, constituents, 961

Woodcock and Lodge, 914,

Wood-eating animals, 961-73

Woodhead, 950

Woodruff, Lorande Loss, 34, 476, 498,
A958; 5255 52955230955.) 50, 9505
611, 616, 617, 653, 659, 791, 792,
796; on early microscopes, 9; endomixis
(Chap. XIII), 646-65

Woodruff, Lorande Loss, and Baitsell, G.
Lin SAI S75 S293 Seo

Woodruff, Lorande Loss, and Erdmann,
R., endomixis, 31, 648, 649, 654, 657,
658, 659, 660

Woodruff, Lorande Loss, and Moore, E.
Ik, GBS}

Woodruff, Lorande Loss, and Spencer, H.,
616, 649, 659

Work Projects
890xz, 1009”

Worley, 257

and Winogra-

Administration, 5652,

1148

Wright, 519

Wrisberg, 208

Wrzesniowski, 208, 210, 217, 413
Wurmser, 125, 396

Xanthellae, relationship to host, 1010
X-ray diffraction and ultra-centrifugation,
97 f.

Yagiu, 919, 920, 922, 923, 1022

Yakimoff, 870, 1032

Yale University, race of Paramecium
aurelia, 615, 653, 654, 659, 660, 661

Yamasaki, 154, 156, 157, 159, 968, 969,
970

Yarwood, 576

Yeast, use in cultures, 449, 543

Yellow respiratory pigments, or enzyme,
579

Yocom, H. B., 23, 343, 535, 538; fibril-
lar system, 206, 207, 258

Yonge, 962

Yorke, Adams, and Murgatroyd, 388, 389

Youngs] Di Bes yiiaiiS; 1S,

Young, Dixie, 647, 653, 797

Young, R. A., 419, 420

Young, R. T., 660

INDEX

Yuan-Po, 1045, 1047, 1048, 1051, 1052,
1058

Zacharias, 1074

Zdrodowski, 877

Zederbauer, 588

Zeliff, 1044

Zenker, 413

Zerling, 906

Zhalkovskii, 542

Zhinkin, 153, 154, 157, 159, 388

Zick, 931, 932

Ziegler and Halvorson, 520

Zinger, 123,151) 152; 165

Zodlogical Nomenclature,
Rules of, 954

Zo6mastigophora parasites in Protozoa,
1070-74

Zoothamnium, regeneration, 805 ff.

Zuelzer, M., 426, 595

Zumstein, 463

Zurn, 983

Zweibaum, 153, 362, 365, 689

Zwischenstreifen, 201, 202

Zygote, start of metazoan life cycle with,
566

Zygotic meiosis, 611-14

International

4 pe

Internet Archive Audio

Featured

Top

Images

Featured

Top

Software

Featured

Top

Books

Featured

Top

Video

Featured

Top

Mobile Apps

Browser Extensions

Archive-It Subscription

Save Page Now

Full text of "Protozoa in biological research"

See other formats